Sarah Heilshorn

All Publications

• Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction. Nature materials Ayushman, M., Mikos, G., Tong, X., Sinha, S., Lopez-Fuentes, E., Jones, S., Cai, P. C., Lee, H. P., Morrison, A. J., Spakowitz, A., Heilshorn, S. C., Sweet-Cordero, A., Yang, F. 2024

  Abstract

  Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.

  View details for DOI 10.1038/s41563-024-02038-0

  View details for PubMedID 39487316

  View details for PubMedCentralID 10656994

• Photoactivated growth factor release from bio-orthogonally crosslinked hydrogels for the regeneration of corneal defects. Bioactive materials Kang, N., Seo, Y. A., Jackson, K. J., Jang, K., Song, E., Han, U., Chen, F., Heilshorn, S. C., Myung, D. 2024; 40: 417-429

  Abstract

  In situ-forming hydrogels are an attractive option for corneal regeneration, and the delivery of growth factors from such constructs have the potential to improve re-epithelialization and stromal remodeling. However, challenges persist in controlling the release of therapeutic molecules from hydrogels. Here, an in situ-forming bio-orthogonally crosslinked hydrogel containing growth factors tethered via photocleavable linkages (PC-HACol hydrogel) was developed to accelerate corneal regeneration. Epidermal growth factor (EGF) was conjugated to the hydrogel backbone through photo-cleavable (PC) spacer arms and was released when exposed to mild intensity ultraviolet (UV) light (2-5mW/cm2, 365nm). The PC-HACol hydrogel rapidly gelled within a few minutes when applied to corneal defects, with excellent transparency and biocompatibility. After subsequent exposure to UV irradiation, the hydrogel promoted the proliferation and migration of corneal epithelial cells in vitro. The rate of re-epithelialization was positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea organ culture studies. In an in vivo rat corneal wound healing study, the PC-HACol hydrogel exposed to UV light significantly promoted re-epithelialization, the remodeling of stromal layers, and exhibited significant anti-scarring effects, with minimal alpha-SMA and robust ALDH3A1 expression. Normal differentiation of the regenerated epithelia after healing was evaluated by expression of the corneal epithelial biomarker, CK12. The remodeled cornea exhibited full recovery of corneal thickness and layer number without hyperplasia of the epithelium.

  View details for DOI 10.1016/j.bioactmat.2024.05.045

  View details for PubMedID 39022184

• Porosity dominates over microgel stiffness for promoting chondrogenesis in zwitterionic granular hydrogels. Biomaterials science Asadikorayem, M., Brunel, L. G., Weber, P., Heilshorn, S. C., Zenobi-Wong, M. 2024

  Abstract

  Granular hydrogels comprised of jammed, crosslinked microgels offer great potential as biomaterial scaffolds for cell-based therapies, including for cartilage tissue regeneration. As stiffness and porosity of hydrogels affect the phenotype of encapsulated cells and the extent of tissue regeneration, the design of tunable granular hydrogels to control and optimize these parameters is highly desirable. We hypothesized that chondrogenesis could be modulated using a granular hydrogel platform based on biocompatible, zwitterionic materials with independent intra- and inter-microgel crosslinking mechanisms. Microgels are made with mechanical fragmentation of photocrosslinked zwitterionic carboxybetaine acrylamide (CBAA) and sulfobetaine methacrylate (SBMA) hydrogels, and secondarily crosslinked in the presence of cells using horseradish peroxide (HRP) to produce cell-laden granular hydrogels. We varied the intra-microgel crosslinking density to produce microgels with varied stiffnesses (1-3 kPa) and swelling properties. These microgels, when resuspended at the same weight fraction and secondarily crosslinked, resulted in granular hydrogels with distinct porosities (5-40%) due to differing swelling properties. The greatest extent of chondrogenesis was achieved in scaffolds with the highest microgel stiffness and highest porosity. However, when scaffold porosity was kept constant and just microgel stiffness varied, cell phenotype and chondrogenesis were similar across scaffolds. These results indicate the dominant role of granular scaffold porosity on chondrogenesis, whereas microgel stiffness appears to play a relatively minor role. These observations are in contrast to cells encapsulated within conventional bulk hydrogels, where stiffness has been shown to significantly affect chondrocyte response. In summary, we introduce chemically-defined, zwitterionic biomaterials to fabricate versatile granular hydrogels allowing for tunable scaffold porosity and microgel stiffness to study and influence chondrogenesis.

  View details for DOI 10.1039/d4bm00233d

  View details for PubMedID 39347711

  View details for PubMedCentralID PMC11441418

• In Situ UNIversal Orthogonal Network (UNION) Bioink Deposition for Direct Delivery of Corneal Stromal Stem Cells to Corneal Wounds. bioRxiv : the preprint server for biology Brunel, L. G., Cai, B., Hull, S. M., Han, U., Wungcharoen, T., Fernandes-Cunha, G. M., Seo, Y. A., Johansson, P. K., Heilshorn, S. C., Myung, D. 2024

  Abstract

  The scarcity of human donor corneal graft tissue worldwide available for corneal transplantation necessitates the development of alternative therapeutic strategies for treating patients with corneal blindness. Corneal stromal stem cells (CSSCs) have the potential to address this global shortage by allowing a single donor cornea to treat multiple patients. To directly deliver CSSCs to corneal defects within an engineered biomatrix, we developed a UNIversal Orthogonal Network (UNION) collagen bioink that crosslinks in situ with a bioorthogonal, covalent chemistry. This cell-gel therapy is optically transparent, stable against contraction forces exerted by CSSCs, and permissive to the efficient growth of corneal epithelial cells. Furthermore, CSSCs remain viable within the UNION collagen gel precursor solution under standard storage and transportation conditions. This approach promoted corneal transparency and re-epithelialization in a rabbit anterior lamellar keratoplasty model, indicating that the UNION collagen bioink serves effectively as an in situ -forming, suture-free therapy for delivering CSSCs to corneal wounds. TEASER. Corneal stem cells are delivered within chemically crosslinked collagen as a transparent, regenerative biomaterial therapy.

  View details for DOI 10.1101/2024.09.19.613997

  View details for PubMedID 39386574

• Interpenetrating networks of fibrillar and amorphous collagen promote cell spreading and hydrogel stability. bioRxiv : the preprint server for biology Brunel, L. G., Long, C. M., Christakopoulos, F., Cai, B., Johansson, P. K., Singhal, D., Enejder, A., Myung, D., Heilshorn, S. C. 2024

  Abstract

  Hydrogels composed of collagen, the most abundant protein in the human body, are widely used as scaffolds for tissue engineering due to their ability to support cellular activity. However, collagen hydrogels with encapsulated cells often experience bulk contraction due to cell-generated forces, and conventional strategies to mitigate this undesired deformation often compromise either the fibrillar microstructure or cytocompatibility of the collagen. To support the spreading of encapsulated cells while preserving the structural integrity of the gels, we present an interpenetrating network (IPN) of two distinct collagen networks with different crosslinking mechanisms and microstructures. First, a physically self-assembled collagen network preserves the fibrillar microstructure and enables the spreading of encapsulated human corneal mesenchymal stromal cells. Second, an amorphous collagen network covalently crosslinked with bioorthogonal chemistry fills the voids between fibrils and stabilizes the gel against cell-induced contraction. This collagen IPN balances the biofunctionality of natural collagen with the stability of covalently crosslinked, engineered polymers. Taken together, these data represent a new avenue for maintaining both the fiber-induced spreading of cells and the structural integrity of collagen hydrogels by leveraging an IPN of fibrillar and amorphous collagen networks.Statement of significance: Collagen hydrogels are widely used as scaffolds for tissue engineering due to their support of cellular activity. However, collagen hydrogels often undergo undesired changes in size and shape due to cell-generated forces, and conventional strategies to mitigate this deformation typically compromise either the fibrillar microstructure or cytocompatibility of the collagen. In this study, we introduce an innovative interpenetrating network (IPN) that combines physically self-assembled, fibrillar collagen-ideal for promoting cell adhesion and spreading-with covalently crosslinked, amorphous collagen-ideal for enhancing bulk hydrogel stability. Our IPN design maintains the native fibrillar structure of collagen while significantly improving resistance against cell-induced contraction, providing a promising solution to enhance the performance and reliability of collagen hydrogels for tissue engineering applications.Graphical abstract:

  View details for DOI 10.1101/2024.09.11.612534

  View details for PubMedID 39345483

• Engineered Protein Hydrogels as Biomimetic Cellular Scaffolds. Advanced materials (Deerfield Beach, Fla.) Liu, Y., Gilchrist, A. E., Heilshorn, S. C. 2024: e2407794

  Abstract

  The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein-based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self-assembly or crosslinking, engineered protein-based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain-length, folded structure, and cell-interaction sites. In this review, the modular design of engineered protein-based hydrogels from both a molecular- and network-level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli-responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein-based hydrogels as biomimetic cellular scaffolds is concluded.

  View details for DOI 10.1002/adma.202407794

  View details for PubMedID 39233559

• The lysogenic filamentous Pseudomonas bacteriophage phage Pf slows mucociliary transport. PNAS nexus Burgener, E. B., Cai, P. C., Kratochvil, M. J., Rojas-Hernandez, L. S., Joo, N. S., Gupta, A., Secor, P. R., Heilshorn, S. C., Spakowitz, A. J., Wine, J. J., Bollyky, P. L., Milla, C. E. 2024; 3 (9): pgae390

  Abstract

  Pseudomonas aeruginosa is a major pulmonary pathogen causing chronic pulmonary infections in people with cystic fibrosis (CF). The P. aeruginosa filamentous and lysogenic bacteriophage, Pf phage, is abundant in the airways of many people with CF and has been associated with poor outcomes in a cross-sectional cohort study. Previous studies have identified roles for Pf phage in biofilm formation, specifically forming higher-order birefringent, liquid crystals when in contact with other biopolymers in biofilms. Liquid crystalline biofilms are more adherent and viscous than those without liquid crystals. A key feature of biofilms is to enhance bacterial adherence and resist physical clearance. The effect of Pf phage on mucociliary transport is unknown. We found that primary CF and non-CF nasal epithelial cells cultured at air-liquid interface treated with Pf phage exhibit liquid crystalline structures in the overlying mucus. On these cell cultures, Pf phage entangles cilia but does not affect ciliary beat frequency. In both these in vitro cell cultures and in an ex vivo porcine trachea model, introduction of Pf phage decreases mucociliary transport velocity. Pf phage also blocks the rescue of mucociliary transport by CF transmembrane conductance regulator modulators in CF cultures. Thus, Pf phage may contribute to the pathogenesis of P. aeruginosa-associated CF lung disease via induction of liquid crystalline characteristics to airway secretions, leading to impaired mucociliary transport. Targeting Pf phage may be useful in treatment CF as well as other settings of chronic P. aeruginosa infections.

  View details for DOI 10.1093/pnasnexus/pgae390

  View details for PubMedID 39301510

  View details for PubMedCentralID PMC11412248

• In Situ-Forming, Bioorthogonally Cross-linked, Nanocluster-Reinforced Hydrogel for the Regeneration of Corneal Defects. ACS nano Kang, N. W., Jang, K., Song, E., Han, U., Seo, Y. A., Chen, F., Wungcharoen, T., Heilshorn, S. C., Myung, D. 2024

  Abstract

  Corneal defects can lead to stromal scarring and vision loss, which is currently only treatable with a cadaveric corneal transplant. Although in situ-forming hydrogels have been shown to foster regeneration of the cornea in the setting of stromal defects, the cross-linking, biomechanical, and compositional parameters that optimize healing have not yet been established. This, Corneal defects are also almost universally inflamed, and their rapid closure without fibrosis are critical to preserving vision. Here, an in situ forming, bioorthogonally cross-linked, nanocluster (NC)-reinforced collagen and hyaluronic acid hydrogel (NCColHA hydrogel) with enhanced structural integrity and both pro-regenerative and anti-inflammatory effects was developed and tested within a corneal defect model in vivo. The NCs serve as bioorthogonal nanocross-linkers, providing higher cross-linking density than polymer-based alternatives. The NCs also serve as delivery vehicles for prednisolone (PRD) and the hepatocyte growth factor (HGF). NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties, excellent transparency, and negligible swelling/deswelling behavior. The hydrogel's biocompatibility and capacity to support cell growth were assessed using primary human corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK12, CK14, α-SMA, Tuj-1, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into a transparent stroma by 56 days.

  View details for DOI 10.1021/acsnano.4c02345

  View details for PubMedID 39106436

• FIBRIN-BASED DELIVERY OF HEDGEHOG PATHWAY MODULATORS DURING NERVE REPAIR IN MOUSE MODELS OF NERVE INJURY He, L., Cai, B., Ghorbani, S., Kilian, D., Bekale, L., Heilshorn, S., Pepper, J. WILEY. 2024: S144

  View details for Web of Science ID 001319566000288

• Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids. Nature materials LeSavage, B. L., Zhang, D., Huerta-López, C., Gilchrist, A. E., Krajina, B. A., Karlsson, K., Smith, A. R., Karagyozova, K., Klett, K. C., Huang, M. S., Long, C., Kaber, G., Madl, C. M., Bollyky, P. L., Curtis, C., Kuo, C. J., Heilshorn, S. C. 2024

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

  View details for DOI 10.1038/s41563-024-01908-x

  View details for PubMedID 38965405

  View details for PubMedCentralID 5704175

• Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus. APL bioengineering Cai, P. C., Braunreuther, M., Shih, A., Spakowitz, A. J., Fuller, G. G., Heilshorn, S. C. 2024; 8 (2): 026112

  Abstract

  Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

  View details for DOI 10.1063/5.0187974

  View details for PubMedID 38721267

  View details for PubMedCentralID PMC11078553

• Bio-Orthogonal Chemistry and Hyaluronan Enhance Corneal Restoration of Collagen Gel: 2-Month Response <i>in Vivo</i> Chen, F., Han, U., Seo, Y., Heilshorn, S., Myung, D. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2024

  View details for Web of Science ID 001313316203121

• Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding. Science advances Chen, Q., Cai, P., Chang, T. H., Burgener, E., Kratochvil, M. J., Gupta, A., Hargill, A., Secor, P. R., Nielsen, J. E., Barron, A. E., Milla, C., Heilshorn, S. C., Spakowitz, A., Bollyky, P. L. 2024; 10 (22): eadl5576

  Abstract

  Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

  View details for DOI 10.1126/sciadv.adl5576

  View details for PubMedID 38820163

  View details for PubMedCentralID PMC11141622

• Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model. Acta neuropathologica communications Sinha, S., Huang, M. S., Mikos, G., Bedi, Y., Soto, L., Lensch, S., Ayushman, M., Bintu, L., Bhutani, N., Heilshorn, S. C., Yang, F. 2024; 12 (1): 71

  Abstract

  Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

  View details for DOI 10.1186/s40478-024-01765-4

  View details for PubMedID 38706008

  View details for PubMedCentralID 4161623

• Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding. bioRxiv : the preprint server for biology Chen, Q., Cai, P., Chang, T. H., Burgener, E., Kratochvil, M. J., Gupta, A., Hargil, A., Secor, P. R., Nielsen, J. E., Barron, A. E., Milla, C., Heilshorn, S. C., Spakowitz, A., Bollyky, P. L. 2024

  Abstract

  Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of morbidity and mortality in patients with cystic fibrosis, necessitating treatment with inhaled antibiotics. Pf phage is a filamentous bacteriophage produced by Pa that has been reported to act as a structural element in Pa biofilms. Pf presence has been associated with resistance to antibiotics and poor outcomes in cystic fibrosis, though the underlying mechanisms are unclear. Here, we have investigated how Pf phages and sputum biopolymers impede antibiotic diffusion using human sputum samples and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf phages and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf phages in sputum reduce the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf phages and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

  View details for DOI 10.1101/2024.03.10.584330

  View details for PubMedID 38496625

• Diffusion-Based 3D Bioprinting Strategies. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Cai, B., Kilian, D., Ramos Mejia, D., Rios, R. J., Ali, A., Heilshorn, S. C. 2023: e2306470

  Abstract

  3D bioprinting has enabled the fabrication of tissue-mimetic constructs with freeform designs that include living cells. In the development of new bioprinting techniques, the controlled use of diffusion has become an emerging strategy to tailor the properties and geometry of printed constructs. Specifically, the diffusion of molecules with specialized functions, including crosslinkers, catalysts, growth factors, or viscosity-modulating agents, across the interface of printed constructs will directly affect material properties such as microstructure, stiffness, and biochemistry, all of which can impact cell phenotype. For example, diffusion-induced gelation is employed to generate constructs with multiple materials, dynamic mechanical properties, and perfusable geometries. In general, these diffusion-based bioprinting strategies can be categorized into those based on inward diffusion (i.e., into the printed ink from the surrounding air, solution, or support bath), outward diffusion (i.e., from the printed ink into the surroundings), or diffusion within the printed construct (i.e., from one zone to another). This review provides an overview of recent advances in diffusion-based bioprinting strategies, discusses emerging methods to characterize and predict diffusion in bioprinting, and highlights promising next steps in applying diffusion-based strategies to overcome current limitations in biofabrication.

  View details for DOI 10.1002/advs.202306470

  View details for PubMedID 38145962

• Embedded 3d Bioprinting of Collagen Inks into Microgel Baths to control hydrogel Microstructure and Cell Spreading. Advanced healthcare materials Brunel, L. G., Christakopoulos, F., Kilian, D., Cai, B., Hull, S. M., Myung, D., Heilshorn, S. C. 2023: e2303325

  Abstract

  Microextrusion-based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. We hypothesized that interactions between inks and support baths, which are often composed of granular microgels, could be modulated to control the microscopic structure within these macroscopic-printed constructs. Using printed collagen bioinks crosslinked either through physical self-assembly or bioorthogonal covalent chemistry, we demonstrate that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero-shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity was modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15-50 µm) resulted in large pores (∼40 µm) that allowed human corneal mesenchymal stromal cells to readily spread, while control samples of cast collagen or collagen printed in non-granular support baths did not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/adhm.202303325

  View details for PubMedID 38134346

• Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing. Advanced functional materials Shin, S., Brunel, L. G., Cai, B., Kilian, D., Roth, J. G., Seymour, A. J., Heilshorn, S. C. 2023; 33 (50)

  Abstract

  While the human body has many different examples of perfusable structures with complex geometries, biofabrication methods to replicate this complexity are still lacking. Specifically, the fabrication of self-supporting, branched networks with multiple channel diameters is particularly challenging. Here, we present the Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE-3DP) approach for constructing perfusable networks of interconnected channels with precise control over branching geometries and vessel sizes. To achieve user-specified channel dimensions, this technique leverages the predictable diffusion of crosslinking reaction-initiators released from sacrificial inks printed within a hydrogel precursor. We demonstrate the versatility of GUIDE-3DP to be adapted for use with diverse physicochemical crosslinking mechanisms by designing seven printable material systems. Importantly, GUIDE-3DP allows for the independent tunability of both the inner and outer diameters of the printed channels and the ability to fabricate seamless junctions at branch points. This 3D bioprinting platform is uniquely suited for fabricating lumenized structures with complex shapes characteristic of multiple hollow vessels throughout the body. As an exemplary application, we demonstrate the fabrication of vasculature-like networks lined with endothelial cells. GUIDE-3DP represents an important advance toward the fabrication of self-supporting, physiologically relevant networks with intricate and perfusable geometries.

  View details for DOI 10.1002/adfm.202307435

  View details for PubMedID 38646474

  View details for PubMedCentralID PMC11031202

• Rapid assessment of changes in phage bioactivity using dynamic light scattering. PNAS nexus Dharmaraj, T., Kratochvil, M. J., Pourtois, J. D., Chen, Q., Hajfathalian, M., Hargil, A., Lin, Y. H., Evans, Z., Oromí-Bosch, A., Berry, J. D., McBride, R., Haddock, N. L., Holman, D. R., van Belleghem, J. D., Chang, T. H., Barr, J. J., Lavigne, R., Heilshorn, S. C., Blankenberg, F. G., Bollyky, P. L. 2023; 2 (12): pgad406

  Abstract

  Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

  View details for DOI 10.1093/pnasnexus/pgad406

  View details for PubMedID 38111822

  View details for PubMedCentralID PMC10726995

• A Library of Elastin-like Proteins with Tunable Matrix Ligands for In Vitro 3D Neural Cell Culture. Biomacromolecules Suhar, R. A., Huang, M. S., Navarro, R. S., Aviles Rodriguez, G., Heilshorn, S. C. 2023

  Abstract

  Hydrogels with encapsulated cells have widespread biomedical applications, both as tissue-mimetic 3D cultures in vitro and as tissue-engineered therapies in vivo. Within these hydrogels, the presentation of cell-instructive extracellular matrix (ECM)-derived ligands and matrix stiffness are critical factors known to influence numerous cell behaviors. While individual ECM biopolymers can be blended together to alter the presentation of cell-instructive ligands, this typically results in hydrogels with a range of mechanical properties. Synthetic systems that allow for the facile incorporation and modulation of multiple ligands without modification of matrix mechanics are highly desirable. In the present work, we leverage protein engineering to design a family of xeno-free hydrogels (i.e., devoid of animal-derived components) consisting of recombinant hyaluronan and recombinant elastin-like proteins (ELPs), cross-linked together with dynamic covalent bonds. The ELP components incorporate cell-instructive peptide ligands derived from ECM proteins, including fibronectin (RGD), laminin (IKVAV and YIGSR), collagen (DGEA), and tenascin-C (PLAEIDGIELTY and VFDNFVL). By carefully designing the protein primary sequence, we form 3D hydrogels with defined and tunable concentrations of cell-instructive ligands that have similar matrix mechanics. Utilizing this system, we demonstrate that neurite outgrowth from encapsulated embryonic dorsal root ganglion (DRG) cultures is significantly modified by cell-instructive ligand content. Thus, this library of protein-engineered hydrogels is a cell-compatible system to systematically study cell responses to matrix-derived ligands.

  View details for DOI 10.1021/acs.biomac.3c00941

  View details for PubMedID 37988588

• Custom-engineered hydrogels for delivery of human iPSC-derived neurons into the injured cervical spinal cord. Biomaterials Doulames, V. M., Marquardt, L. M., Hefferon, M. E., Baugh, N. J., Suhar, R. A., Wang, A. T., Dubbin, K. R., Weimann, J. M., Palmer, T. D., Plant, G. W., Heilshorn, S. C. 2023; 305: 122400

  Abstract

  Cervical damage is the most prevalent type of spinal cord injury clinically, although few preclinical research studies focus on this anatomical region of injury. Here we present a combinatorial therapy composed of a custom-engineered, injectable hydrogel and human induced pluripotent stem cell (iPSC)-derived deep cortical neurons. The biomimetic hydrogel has a modular design that includes a protein-engineered component to allow customization of the cell-adhesive peptide sequence and a synthetic polymer component to allow customization of the gel mechanical properties. In vitro studies with encapsulated iPSC-neurons were used to select a bespoke hydrogel formulation that maintains cell viability and promotes neurite extension. Following injection into the injured cervical spinal cord in a rat contusion model, the hydrogel biodegraded over six weeks without causing any adverse reaction. Compared to cell delivery using saline, the hydrogel significantly improved the reproducibility of cell transplantation and integration into the host tissue. Across three metrics of animal behavior, this combinatorial therapy significantly improved sensorimotor function by six weeks post transplantation. Taken together, these findings demonstrate that design of a combinatorial therapy that includes a gel customized for a specific fate-restricted cell type can induce regeneration in the injured cervical spinal cord.

  View details for DOI 10.1016/j.biomaterials.2023.122400

  View details for PubMedID 38134472

• Cell Microencapsulation Within Engineered Hyaluronan Elastin-Like Protein (HELP) Hydrogels. Current protocols Hefferon, M. E., Huang, M. S., Liu, Y., Navarro, R. S., de Paiva Narciso, N., Zhang, D., Aviles-Rodriguez, G., Heilshorn, S. C. 2023; 3 (11): e917

  Abstract

  Three-dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer-based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin-like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Elastin-like protein modification with hydrazine Basic Protocol 2: Nuclear magnetic resonance quantification of elastin-like protein modification with hydrazine Basic Protocol 3: Hyaluronic acid-benzaldehyde synthesis Basic Protocol 4: Nuclear magnetic resonance quantification of hyaluronic acid-benzaldehyde Basic Protocol 5: 3D cell encapsulation in hyaluronan elastin-like protein gels.

  View details for DOI 10.1002/cpz1.917

  View details for PubMedID 37929691

• Transient Competitors to Modulate Dynamic Covalent Cross-Linking of Recombinant Hydrogels CHEMISTRY OF MATERIALS Gilchrist, A. E., Liu, Y., Klett, K., Liu, Y., Ceva, S., Heilshorn, S. C. 2023

  View details for DOI 10.1021/acs.chemmater.3c01575

  View details for Web of Science ID 001096771900001

• Tunable hydrogel viscoelasticity modulates human neural maturation. Science advances Roth, J. G., Huang, M. S., Navarro, R. S., Akram, J. T., LeSavage, B. L., Heilshorn, S. C. 2023; 9 (42): eadh8313

  Abstract

  Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biomechanical signaling cues imparted by the extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in neural maturation gene expression. Together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.

  View details for DOI 10.1126/sciadv.adh8313

  View details for PubMedID 37862423

• 3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels. Biomaterials science Seymour, A. J., Kilian, D., Navarro, R. S., Hull, S. M., Heilshorn, S. C. 2023

  Abstract

  Microgel-based biomaterials have inherent porosity and are often extrudable, making them well-suited for 3D bioprinting applications. Cells are commonly introduced into these granular inks post-printing using cell infiltration. However, due to slow cell migration speeds, this strategy struggles to achieve depth-independent cell distributions within thick 3D printed geometries. To address this, we leverage granular ink modularity by combining two microgels with distinct functions: (1) structural, UV-crosslinkable microgels made from gelatin methacryloyl (GelMA) and (2) sacrificial, cell-laden microgels made from oxidized alginate (AlgOx). We hypothesize that encapsulating cells within sacrificial AlgOx microgels would enable the simultaneous introduction of void space and release of cells at depths unachievable through cell infiltration alone. Blending the microgels in different ratios produces a family of highly printable GelMA : AlgOx microgel inks with void fractions ranging from 0.03 to 0.35. As expected, void fraction influences the morphology of human umbilical vein endothelial cells (HUVEC) within GelMA : AlgOx inks. Crucially, void fraction does not alter the ideal HUVEC distribution seen throughout the depth of 3D printed samples. This work presents a strategy for fabricating constructs with tunable porosity and depth-independent cell distribution, highlighting the promise of microgel-based inks for 3D bioprinting.

  View details for DOI 10.1039/d3bm00721a

  View details for PubMedID 37824082

• 3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels BIOMATERIALS SCIENCE Seymour, A. J., Kilian, D., Navarro, R. S., Hull, S. M., Heilshorn, S. C. 2023

  View details for DOI 10.1039/d3bm00721

  View details for Web of Science ID 001081914900001

• Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing ADVANCED FUNCTIONAL MATERIALS Shin, S., Brunel, L. G., Cai, B., Kilian, D., Roth, J. G., Seymour, A. J., Heilshorn, S. C. 2023

  View details for DOI 10.1002/adfm.202307435

  View details for Web of Science ID 001040672400001

• Spatially controlled construction of assembloids using bioprinting. Nature communications Roth, J. G., Brunel, L. G., Huang, M. S., Liu, Y., Cai, B., Sinha, S., Yang, F., Pașca, S. P., Shin, S., Heilshorn, S. C. 2023; 14 (1): 4346

  Abstract

  The biofabrication of three-dimensional (3D) tissues that recapitulate organ-specific architecture and function would benefit from temporal and spatial control of cell-cell interactions. Bioprinting, while potentially capable of achieving such control, is poorly suited to organoids with conserved cytoarchitectures that are susceptible to plastic deformation. Here, we develop a platform, termed Spatially Patterned Organoid Transfer (SPOT), consisting of an iron-oxide nanoparticle laden hydrogel and magnetized 3D printer to enable the controlled lifting, transport, and deposition of organoids. We identify cellulose nanofibers as both an ideal biomaterial for encasing organoids with magnetic nanoparticles and a shear-thinning, self-healing support hydrogel for maintaining the spatial positioning of organoids to facilitate the generation of assembloids. We leverage SPOT to create precisely arranged assembloids composed of human pluripotent stem cell-derived neural organoids and patient-derived glioma organoids. In doing so, we demonstrate the potential for the SPOT platform to construct assembloids which recapitulate key developmental processes and disease etiologies.

  View details for DOI 10.1038/s41467-023-40006-5

  View details for PubMedID 37468483

  View details for PubMedCentralID PMC10356773

• Rapid assessment of changes in phage bioactivity using dynamic light scattering. bioRxiv : the preprint server for biology Dharmaraj, T., Kratochvil, M. J., Pourtois, J. D., Chen, Q., Hajfathalian, M., Hargil, A., Lin, Y. H., Evans, Z., Oromí-Bosch, A., Berry, J. D., McBride, R., Haddock, N. L., Holman, D. R., van Belleghem, J. D., Chang, T. H., Barr, J. J., Lavigne, R., Heilshorn, S. C., Blankenberg, F. G., Bollyky, P. L. 2023

  Abstract

  Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

  View details for DOI 10.1101/2023.07.02.547396

  View details for PubMedID 37425882

  View details for PubMedCentralID PMC10327207

• Design Parameters for Injectable Biopolymeric Hydrogels with Dynamic Covalent Chemistry Crosslinks. Advanced healthcare materials Narciso, N. d., Navarro, R. S., Gilchrist, A., Trigo, M. L., Rodriguez, G. A., Heilshorn, S. C. 2023: e2301265

  Abstract

  Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self-healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC-crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, we formulated hydrogels comprised of two recombinant biopolymers: (1) a hyaluronic acid (HA) modified with benzaldehyde (HA-BZA) and (2) an elastin-like protein (ELP) modified with hydrazine (ELP-HYD). We synthesized several hydrogel families with distinct HA MW and DoF while keeping the ELP-HYD component constant. The resulting hydrogels had a range of stiffnesses, G' ∼ 10-1000 Pa, and extrudability, which was attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations required lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibited more rapid self-healing. Gel extrusion through a cannula (2-m length, 0.25-mm diameter) demonstrated the potential for minimally-invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC-crosslinked hydrogels and aims to guide future design of injectable hydrogels. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/adhm.202301265

  View details for PubMedID 37389811

• 3D Bioprinting Of Corneal-Cell Laden Inks As Bioengineered Corneal Substitutes Brunel, L. G., Hull, S. M., Fernandes-Cunha, G., Johansson, P. K., Myung, D., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001120356800384

• Collagen Gels Crosslinked by Photoactivation of Riboflavin for the Repair and Regeneration of Corneal Defects. ACS applied bio materials Fernandes-Cunha, G. M., Brunel, L. G., Arboleda, A., Manche, A., Seo, Y. A., Logan, C., Chen, F., Heilshorn, S. C., Myung, D. 2023

  Abstract

  Bioengineered corneal tissue is a promising therapeutic modality for the treatment of corneal blindness as a substitute for cadaveric graft tissue. In this study, we fabricated a collagen gel using ultraviolet-A (UV-A) light and riboflavin as a photosensitizer (PhotoCol-RB) as an in situ-forming matrix to fill corneal wounds and create a cohesive interface between the crosslinked gel and adjacent collagen. The PhotoCol-RB gels supported corneal epithelialization and exhibited higher transparency compared to physically crosslinked collagen. We showed that different riboflavin concentrations yielded gels with different mechanical and biological properties. In vitro experiments using human corneal epithelial cells (hCECs) showed that hCECs are able to proliferate on the gel and express corneal cell markers such as cytokeratin 12 (CK12) and tight junctions (ZO-1). Using an ex vivo burst assay, we also showed that the PhotoCol-RB gels are able to seal corneal perforations. Ex vivo organ culture of the gels filling lamellar keratectomy wounds showed that the epithelium that regenerated over the PhotoCol-RB gels formed a multilayer compared to just a double layer for those that grew over physically cross-linked collagen. These gels can be formed either in situ directly on the wound site to conform to the geometry of a defect, or can be preformed and then applied to the corneal wound. Our results indicate that PhotoCol-RB gels merit further investigation as a way to stabilize and repair deep and perforating corneal wounds.

  View details for DOI 10.1021/acsabm.3c00015

  View details for PubMedID 37126648

• Cell-matrix Interactions Mediate Chemosensitivity In A Tissue Engineered Model Of Osteosarcoma Rao, R. R., Villa-Martin, B. C., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001120356800298

• Magnetized 3D Bioprinting To Construct Multi-Organoid Assembloids Cai, B., Roth, J. G., Brunel, L. G., Huang, M. S., Liu, Y., Pasca, S., Shin, S., Heilshorn, S. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001120356800251

• 3D Bioprinting Of Corneal-Cell Laden Inks As Bioengineered Corneal Substitutes Brunel, L. G., Hull, S. M., Fernandes-Cunha, G., Johansson, P. K., Myung, D., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001120356800116

• Human enteroids as a tool to study conventional and ultra-high dose rate radiation. Integrative biology : quantitative biosciences from nano to macro Klett, K. C., Martin-Villa, B. C., Villarreal, V. S., Melemenidis, S., Viswanathan, V., Manjappa, R., Ashraf, M. R., Soto, L., Lau, B., Dutt, S., Rankin, E. B., Loo, B. W., Heilshorn, S. C. 2023; 15

  Abstract

  Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

  View details for DOI 10.1093/intbio/zyad013

  View details for PubMedID 37874173

• Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing. bioRxiv : the preprint server for biology Shin, S., Brunel, L. G., Cai, B., Kilian, D., Roth, J. G., Seymour, A. J., Heilshorn, S. C. 2023

  Abstract

  While the human body has many different examples of perfusable structures with complex geometries, biofabrication methods to replicate this complexity are still lacking. Specifically, the fabrication of self-supporting, branched networks with multiple channel diameters is particularly challenging. Here, we present the Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE-3DP) approach for constructing perfusable networks of interconnected channels with precise control over branching geometries and vessel sizes. To achieve user-specified channel dimensions, this technique leverages the predictable diffusion of crosslinking reaction-initiators released from sacrificial inks printed within a hydrogel precursor. We demonstrate the versatility of GUIDE-3DP to be adapted for use with diverse physiochemical crosslinking mechanisms by designing seven printable material systems. Importantly, GUIDE-3DP allows for the independent tunability of both the inner and outer diameters of the printed channels and the ability to fabricate seamless junctions at branch points. This 3D bioprinting platform is uniquely suited for fabricating lumenized structures with complex shapes characteristic of multiple hollow vessels throughout the body. As an exemplary application, we demonstrate the fabrication of vasculature-like networks lined with endothelial cells. GUIDE-3DP represents an important advance toward the fabrication of self-supporting, physiologically relevant networks with intricate and perfusable geometries.

  View details for DOI 10.1101/2023.04.02.535250

  View details for PubMedID 37066190

• 3D bioprinting of dynamic hydrogel bioinks enabled by small molecule modulators. Science advances Hull, S. M., Lou, J., Lindsay, C. D., Navarro, R. S., Cai, B., Brunel, L. G., Westerfield, A. D., Xia, Y., Heilshorn, S. C. 2023; 9 (13): eade7880

  Abstract

  Three-dimensional bioprinting has emerged as a promising tool for spatially patterning cells to fabricate models of human tissue. Here, we present an engineered bioink material designed to have viscoelastic mechanical behavior, similar to that of living tissue. This viscoelastic bioink is cross-linked through dynamic covalent bonds, a reversible bond type that allows for cellular remodeling over time. Viscoelastic materials are challenging to use as inks, as one must tune the kinetics of the dynamic cross-links to allow for both extrudability and long-term stability. We overcome this challenge through the use of small molecule catalysts and competitors that temporarily modulate the cross-linking kinetics and degree of network formation. These inks were then used to print a model of breast cancer cell invasion, where the inclusion of dynamic cross-links was found to be required for the formation of invasive protrusions. Together, we demonstrate the power of engineered, dynamic bioinks to recapitulate the native cellular microenvironment for disease modeling.

  View details for DOI 10.1126/sciadv.ade7880

  View details for PubMedID 37000873

• Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function. Journal of biomedical materials research. Part A Shayan, M., Huang, M. S., Navarro, R., Chiang, G., Hu, C., Oropeza, B. P., Johansson, P. K., Suhar, R. A., Foster, A. A., LeSavage, B. L., Zamani, M., Enejder, A., Roth, J. G., Heilshorn, S. C., Huang, N. F. 2023

  Abstract

  Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

  View details for DOI 10.1002/jbm.a.37520

  View details for PubMedID 36861665

• Mobility mediates maturation: Synthetic substrates to enhance neural differentiation. Cell stem cell Roth, J. G., Huang, M. S., Heilshorn, S. C. 2023; 30 (2): 115-117

  Abstract

  The maturation of human induced pluripotent stem cell (hiPSC)-derived neurons in 2D is dependent upon cell attachment, spreading, and pathfinding across a biomaterial substrate. In this issue of Cell Stem Cell, Alvarez etal.1 demonstrate that highly mobile supramolecular scaffolds facilitate long-term hiPSC-derived motor neuron culture, increase maturation-related phenotypes, and recapitulate disease-relevant pathologies.

  View details for DOI 10.1016/j.stem.2023.01.001

  View details for PubMedID 36736286

• Tuning pro-survival effects of human induced pluripotent stem cell-derived exosomes using elastin-like polypeptides. Biomaterials Lee, C., Hunt, D., Roth, J. G., Chiu, C., Suhar, R. A., LeSavage, B. L., Seymour, A. J., Lindsay, C., Krajina, B. A., Chen, Y., Chang, K., Hsieh, I., Chu, P., Wen, M., Heilshorn, S. C. 2022; 291: 121864

  Abstract

  Exosome-based regenerative therapies are potentially easier to manufacture and safer to apply compared to cell-based therapies. However, many questions remain about how to bio-manufacture reproducible and potent exosomes using animal-free reagents. Here we evaluate the hypothesis that designer biomaterial substrates can be used to alter the potency of exosomes secreted by human induced pluripotent stem cells (iPSCs). Two animal-free designer matrices were fabricated based on recombinant elastin-like polypeptides (ELPs): one including a cell-adhesive RGD ligand and a second with a non-adhesive RDG peptide. While iPSCs cultured on these two substrates and Matrigel-coated controls had similar levels of proliferation, the RDG-ELP substrate significantly increased protein expression of stemness markers OCT4 and SOX2 and suppressed spontaneous differentiation compared to those on RGD-ELP. The pro-survival potency of iPSC-derived exosomes was evaluated using three distinct stress tests: serum starvation in murine fibroblasts, hypoxia in human endothelial cells, and hyperosmolarity in canine kidney cells. In all three cases, exosomes produced by iPSCs grown on RDG-ELP substrates had similar pro-survival effects to those produced using iPSCs grown on Matrigel, while use of RGD-ELP substrates led to significantly reduced exosome potency. These data demonstrate that recombinant substrates can be designed for the robust bio-manufacturing of iPSC-derived, pro-survival exosomes.

  View details for DOI 10.1016/j.biomaterials.2022.121864

  View details for PubMedID 36343608

• Rheological Characterization and Theoretical Modeling Establish Molecular Design Rules for Tailored Dynamically Associating Polymers. ACS central science Cai, P. C., Su, B., Zou, L., Webber, M. J., Heilshorn, S. C., Spakowitz, A. J. 2022; 8 (9): 1318-1327

  Abstract

  Dynamically associating polymers have long been of interest due to their highly tunable viscoelastic behavior. Many applications leverage this tunability to create materials that have specific rheological properties, but designing such materials is an arduous, iterative process. Current models for dynamically associating polymers are phenomenological, assuming a structure for the relationship between association kinetics and network relaxation. We present the Brachiation model, a molecular-level theory of a polymer network with dynamic associations that is rooted in experimentally controllable design parameters, replacing the iterative experimental process with a predictive model for how experimental modifications to the polymer will impact rheological behavior. We synthesize hyaluronic acid chains modified with supramolecular host-guest motifs to serve as a prototypical dynamic network exhibiting tunable physical properties through control of polymer concentration and association rates. We use dynamic light scattering microrheology to measure the linear viscoelasticity of these polymers across six decades in frequency and fit our theory parameters to the measured data. The parameters are then altered by a magnitude corresponding to changes made to the experimental parameters and used to obtain new rheological predictions that match the experimental results well, demonstrating the ability for this theory to inform the design process of dynamically associating polymeric materials.

  View details for DOI 10.1021/acscentsci.2c00432

  View details for PubMedID 36188349

  View details for PubMedCentralID PMC9523779

• Rheological Characterization and Theoretical Modeling Establish Molecular Design Rules for Tailored Dynamically Associating Polymers ACS CENTRAL SCIENCE Cai, P. C., Su, B., Zou, L., Webber, M. J., Heilshorn, S. C., Spakowitz, A. J. 2022

  View details for DOI 10.1021/acscentsci.2c00432

  View details for Web of Science ID 000858451600001

• Engineering A Matrix To Improve Reproducibility Of Intestinal Organoids Martin-Villa, B. C., Klett, K. C., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2022: 39-40

  View details for Web of Science ID 000848131900042

• Biomimetic Hydrogels With Multi-component Cell Adhesive Ligands For Endothelial Cell Function Shayan, M., Suhar, R., Heilshorn, S., Huang, N. MARY ANN LIEBERT, INC. 2022: 36-37

  View details for Web of Science ID 000848131900039

• Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections. JCI insight Kratochvil, M. J., Kaber, G., Demirdjian, S., Cai, P. C., Burgener, E. B., Nagy, N., Barlow, G. L., Popescu, M., Nicolls, M. R., Ozawa, M. G., Regula, D. P., Pacheco-Navarro, A. E., Yang, S., de Jesus Perez, V. A., Karmouty-Quintana, H., Peters, A. M., Zhao, B., Buja, M. L., Johnson, P. Y., Vernon, R. B., Wight, T. N., Stanford COVID-19 Biobank Study Group, Milla, C. E., Rogers, A. J., Spakowitz, A. J., Heilshorn, S. C., Bollyky, P. L. 2022; 7 (12)

  Abstract

  Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

  View details for DOI 10.1172/jci.insight.152629

  View details for PubMedID 35730564

• Biocompatibility of photoactivated collagen-riboflavin hydrogels for corneal regeneration Arboleda, A., Cunha, G., Manche, A., Seo, Y., Logan, C., Heilshorn, S. C., Myung, D. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022

  View details for Web of Science ID 000844401300106

• Collagen hydrogels covalently crosslinked by bioorthogonal click chemistry resist cell-induced contraction while preserving encapsulated corneal stromal cell phenotype Brunel, L. G., Hull, S. M., Johansson, P. K., Myung, D., Heilshorn, S. C. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022

  View details for Web of Science ID 000844401300100

• Engineered assistive materials for 3D bioprinting: support baths and sacrificial inks. Biofabrication Brunel, L. G., Hull, S. M., Heilshorn, S. 2022

  Abstract

  3D bioprinting is a promising technique for spatially patterning cells and materials into constructs that mimic native tissues and organs. However, a tradeoff exists between printability and biological function, where weak materials are typically more suited for 3D cell culture but exhibit poor shape fidelity when printed in air. Recently, a new class of assistive materials has emerged to overcome this limitation and enable fabrication of more complex, biologically relevant geometries, even when using soft materials as bioinks. These materials include support baths, which bioinks are printed into, and sacrificial inks, which are printed themselves and then later removed. Support baths are commonly yield-stress materials that provide physical confinement during the printing process to improve resolution and shape fidelity. Sacrificial inks have primarily been used to create void spaces and pattern perfusable networks, they but can also be combined directly with the bioink to change its mechanical properties for improved printability or increased porosity. Here, we outline the advantages of using such assistive materials in 3D bioprinting, define their material property requirements, and offer case study examples of how these materials are used in practice. Finally, we discuss the remaining challenges and future opportunities in the development of assistive materials that will propel the bioprinting field forward toward creating full-scale, biomimetic tissues and organs.

  View details for DOI 10.1088/1758-5090/ac6bbe

  View details for PubMedID 35487196

• Hyaluronan and elastin-like protein (HELP) gels significantly improve microsphere retention in the myocardium. Biomaterials science Suhar, R. A., Doulames, V. M., Liu, Y., Hefferon, M. E., Figueroa, O. 3., Buabbas, H., Heilshorn, S. C. 2022

  Abstract

  Heart disease is the leading cause of death globally, and delivery of therapeutic cargo (e.g., particles loaded with proteins, drugs, or genes and cells) through direct injection into the myocardium is a promising clinical intervention. However, retention of deliverables to the contracting myocardium is low, with as much as 60-90% of payload being lost within 24 hr. Commercially-available injectable hydrogels, including Matrigel, have been hypothesized to increase payload retention but have not yielded significant improvements in quantified analyses. Here, we assess a recombinant hydrogel composed of chemically modified hyaluronan and elastin-like protein (HELP) as an alternative injectable carrier to increase cargo retention. HELP is crosslinked using dynamic covalent bonds, and tuning the hyaluronan chemistry significantly alters hydrogel mechanical properties including stiffness, stress relaxation rate, and ease of injectability through a needle or catheter. These materials can be injected even after complete crosslinking, extending the time window for surgical delivery. We show that HELP gels significantly improve in vivo retention of microsphere cargo compared to Matrigel, both 1 day and 7 days post-injection directly into the rat myocardium. These data suggest that HELP gels may assist with the clinical translation of therapeutic cargo designed for delivery into the contracting myocardium by preventing acute cargo loss.

  View details for DOI 10.1039/d1bm01890f

  View details for PubMedID 35411353

• Tuning Polymer Hydrophilicity to Regulate Gel Mechanics and Encapsulated Cell Morphology. Advanced healthcare materials Navarro, R. S., Huang, M. S., Roth, J. G., Hubka, K. M., Long, C. M., Enejder, A., Heilshorn, S. C. 2022: e2200011

  Abstract

  Mechanically tunable hydrogels are attractive platforms for three-dimensional cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, we present an alternative strategy based upon tuning the hydrophilicity of an elastin-like protein (ELP). ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. We hypothesize that increasing this transition temperature through bioconjugation with azide-containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide-modified ELPs are crosslinked into hydrogels with bicyclononyne-modified hyaluronic acid (HA-BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100-1000Pa). Human mesenchymal stromal cells, human umbilical vein endothelial cells, and human neural progenitor cells are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, we demonstrate the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/adhm.202200011

  View details for PubMedID 35373510

• Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections. medRxiv : the preprint server for health sciences Kratochvil, M. J., Kaber, G., Demirdjian, S., Cai, P. C., Burgener, E. B., Nagy, N., Barlow, G. L., Popescu, M., Nicolls, M. R., Ozawa, M. G., Regula, D. P., Pacheco-Navarro, A. E., Yang, S., de Jesus Perez, V. A., Karmouty-Quintana, H., Peters, A. M., Zhao, B., Buja, M. L., Johnson, P. Y., Vernon, R. B., Wight, T. N., Stanford COVID-19 Biobank Study Group, Milla, C. E., Rogers, A. J., Spakowitz, A. J., Heilshorn, S. C., Bollyky, P. L. 2022

  Abstract

  Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factora"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

  View details for DOI 10.1101/2022.03.28.22272848

  View details for PubMedID 35411348

• ENGINEERED BIOMIMETIC HYDROGELS WITH COMBINATORIAL CELL ADHESIVE LIGANDS FOR ANGIOGENESIS Ngan Huang, Shayan, M., Suhar, R., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2022: S583

  View details for Web of Science ID 000821187303168

• ADAPTABLE HYDROGELS FOR ORGANOID CULTURE Heilshorn, S. MARY ANN LIEBERT, INC. 2022: S280

  View details for Web of Science ID 000821187301198

• SPATIALLY NANOPATTERNED SCAFFOLDS PROMOTE THE SURVIVAL OF INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS IN THE ISCHEMIC LIMB Huang, N., Yang, G., Alcazar, C., Hu, C., Zaitseva, T., Paukshto, M. MARY ANN LIEBERT, INC. 2022: S583

  View details for Web of Science ID 000821187303167

• Bioprinted microvasculature: progressing from structure to function. Biofabrication Seymour, A. J., Westerfield, A. D., Cornelius, V. C., Skylar-Scott, M. A., Heilshorn, S. 1800

  Abstract

  Three-dimensional (3D) bioprinting seeks to unlock the rapid generation of complex tissue constructs, but long-standing challenges with efficient in vitro microvascularization must be solved before this can become a reality. Microvasculature is particularly challenging to biofabricate due to the presence of a hollow lumen, a hierarchically branched network topology, and a complex signaling milieu. All of these characteristics are required for proper microvascular - and, thus, tissue - function. While several techniques have been developed to address distinct portions of this microvascularization challenge, no single approach is capable of simultaneously recreating all three microvascular characteristics. In this review, we present a three-part framework that proposes integration of existing techniques to generate mature microvascular constructs. First, extrusion-based 3D bioprinting creates a mesoscale foundation of hollow, endothelialized channels. Second, biochemical and biophysical cues induce endothelial sprouting to create a capillary-mimetic network. Third, the construct is conditioned to enhance network maturity. Across all three of these stages, we highlight the potential for extrusion-based bioprinting to become a central technique for engineering hierarchical microvasculature. We envision that the successful biofabrication of functionally engineered microvasculature will address a critical need in tissue engineering, and propel further advances in regenerative medicine and ex vivo human tissue modeling.

  View details for DOI 10.1088/1758-5090/ac4fb5

  View details for PubMedID 35086069

• A human multi-lineage hepatic organoid model for liver fibrosis. Nature communications Guan, Y., Enejder, A., Wang, M., Fang, Z., Cui, L., Chen, S., Wang, J., Tan, Y., Wu, M., Chen, X., Johansson, P. K., Osman, I., Kunimoto, K., Russo, P., Heilshorn, S. C., Peltz, G. 2021; 12 (1): 6138

  Abstract

  To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFbeta pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

  View details for DOI 10.1038/s41467-021-26410-9

  View details for PubMedID 34686668

• 3D Bioprinting of Cell-Laden Hydrogels for Improved Biological Functionality. Advanced materials (Deerfield Beach, Fla.) Hull, S. M., Brunel, L. G., Heilshorn, S. C. 2021: e2103691

  Abstract

  The encapsulation of cells within gel-phase materials to form bioinks offers distinct advantages for next-generation 3D bioprinting. 3D bioprinting has emerged as a promising tool for patterning cells, but the technology remains limited in its ability to produce biofunctional, tissue-like constructs due to a dearth of materials suitable for bioinks. While early demonstrations commonly used viscous polymers optimized for printability, these materials often lacked cell compatibility and biological functionality. In response, advanced materials that exist in the gel phase during the entire printing process are being developed, since hydrogels are uniquely positioned to both protect cells during extrusion and provide biological signals to embedded cells as the construct matures during culture. Here, an overview of the design considerations for gel-phase materials as bioinks is presented, with a focus on their mechanical, biochemical, and dynamic gel properties. Current challenges and opportunities that arise due to the fact that bioprinted constructs are active, living hydrogels composed of both acellular and cellular components are also evaluated. Engineering hydrogels with consideration of cells as an intrinsic component of the printed bioink will enable control over the evolution of the living construct after printing to achieve greater biofunctionality.

  View details for DOI 10.1002/adma.202103691

  View details for PubMedID 34672027

• Elastin-like Proteins to Support Peripheral Nerve Regeneration in Guidance Conduits. ACS biomaterials science & engineering Suhar, R. A., Marquardt, L. M., Song, S., Buabbas, H., Doulames, V. M., Johansson, P. K., Klett, K. C., Dewi, R. E., Enejder, A. M., Plant, G. W., George, P. M., Heilshorn, S. C. 2021; 7 (9): 4209-4220

  Abstract

  Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.

  View details for DOI 10.1021/acsbiomaterials.0c01053

  View details for PubMedID 34510904

• Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling. The Journal of clinical investigation Qian, J., LeSavage, B. L., Hubka, K. M., Ma, C., Natarajan, S., Eggold, J. T., Xiao, Y., Fuh, K. C., Krishnan, V., Enejder, A., Heilshorn, S. C., Dorigo, O., Rankin, E. B. 2021; 131 (16)

  Abstract

  Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-beta signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.

  View details for DOI 10.1172/JCI146186

  View details for PubMedID 34396988

• Next-generation cancer organoids. Nature materials LeSavage, B. L., Suhar, R. A., Broguiere, N., Lutolf, M. P., Heilshorn, S. C. 2021

  Abstract

  Organotypic models of patient-specific tumours are revolutionizing our understanding of cancer heterogeneity and its implications for personalized medicine. These advancements are, in part, attributed to the ability of organoid models to stably preserve genetic, proteomic, morphological and pharmacotypic features of the parent tumour in vitro, while also offering unprecedented genomic and environmental manipulation. Despite recent innovations in organoid protocols, current techniques for cancer organoid culture are inherently uncontrolled and irreproducible, owing to several non-standardized facets including cancer tissue sources and subsequent processing, medium formulations, and animal-derived three-dimensional matrices. Given the potential for cancer organoids to accurately recapitulate the intra- and intertumoral biological heterogeneity associated with patient-specific cancers, eliminating the undesirable technical variability accompanying cancer organoid culture is necessary to establish reproducible platforms that accelerate translatable insights into patient care. Here we describe the current challenges and recent multidisciplinary advancements and opportunities for standardizing next-generation cancer organoid systems.

  View details for DOI 10.1038/s41563-021-01057-5

  View details for PubMedID 34385685

• Advancing models of neural development with biomaterials. Nature reviews. Neuroscience Roth, J. G., Huang, M. S., Li, T. L., Feig, V. R., Jiang, Y., Cui, B., Greely, H. T., Bao, Z., Pasca, S. P., Heilshorn, S. C. 2021

  Abstract

  Human pluripotent stem cells have emerged as a promising in vitro model system for studying the brain. Two-dimensional and three-dimensional cell culture paradigms have provided valuable insights into the pathogenesis of neuropsychiatric disorders, but they remain limited in their capacity to model certain features of human neural development. Specifically, current models do not efficiently incorporate extracellular matrix-derived biochemical and biophysical cues, facilitate multicellular spatio-temporal patterning, or achieve advanced functional maturation. Engineered biomaterials have the capacity to create increasingly biomimetic neural microenvironments, yet further refinement is needed before these approaches are widely implemented. This Review therefore highlights how continued progression and increased integration of engineered biomaterials may be well poised to address intractable challenges in recapitulating human neural development.

  View details for DOI 10.1038/s41583-021-00496-y

  View details for PubMedID 34376834

• 3D Printing of Microgel Scaffolds with Tunable Void Fraction to Promote Cell Infiltration. Advanced healthcare materials Seymour, A. J., Shin, S., Heilshorn, S. C. 2021: e2100644

  Abstract

  Granular, microgel-based materials have garnered interest as promising tissue engineering scaffolds due to their inherent porosity, which can promote cell infiltration. Adapting these materials for 3D bioprinting, while maintaining sufficient void space to enable cell migration, can be challenging, since the rheological properties that determine printability are strongly influenced by microgel packing and void fraction. In this work, a strategy is proposed to decouple printability and void fraction by blending UV-crosslinkable gelatin methacryloyl (GelMA) microgels with sacrificial gelatin microgels to form composite inks. It is observed that inks with an apparent viscosity greater than 100 Pa s (corresponding to microgel concentrations ≥5 wt%) have rheological properties that enable extrusion-based printing of multilayered structures in air. By altering the ratio of GelMA to sacrificial gelatin microgels, while holding total concentration constant at 6 wt%, a family of GelMA:gelatin microgel inks is created that allows for tuning of void fraction from 0.20 to 0.57. Furthermore, human umbilical vein endothelial cells (HUVEC) seeded onto printed constructs are observed to migrate into granular inks in a void fraction-dependent manner. Thus, the family of microgel inks holds promise for use in 3D printing and tissue engineering applications that rely upon cell infiltration.

  View details for DOI 10.1002/adhm.202100644

  View details for PubMedID 34342179

• Bio-orthogonally Crosslinked Matrix Therapies for Corneal Defect Repair Myung, D., Chen, F., Fernandes-Cunha, G., Le, P., Hull, S., Heilshorn, S. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2021

  View details for Web of Science ID 000690761100026

• Physical Properties of COVID-19 Acute Respiratory Distress Syndrome (ARDS) Sputum Pacheco-Navarro, A., Kratochvil, M. J., Kaber, G., Roque, J., Blish, C., Yang, S., Nadeau, K. C., Heilshorn, S. C., Milla, C. E., Rogers, A., Bollyky, P. AMER THORACIC SOC. 2021

  View details for Web of Science ID 000685468900294

• Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Hunt, D. R., Klett, K. C., Mascharak, S., Wang, H., Gong, D., Lou, J., Li, X., Cai, P. C., Suhar, R. A., Co, J. Y., LeSavage, B. L., Foster, A. A., Guan, Y., Amieva, M. R., Peltz, G., Xia, Y., Kuo, C. J., Heilshorn, S. C. 2021; 8 (10): 2004705

  Abstract

  Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

  View details for DOI 10.1002/advs.202004705

  View details for PubMedID 34026461

  View details for PubMedCentralID PMC8132048

• Reconstructing the heart using iPSCs: Engineering strategies and applications. Journal of molecular and cellular cardiology Cho, S., Lee, C., Skylar-Scott, M. A., Heilshorn, S. C., Wu, J. C. 2021

  Abstract

  Induced pluripotent stem cells (iPSCs) have emerged as a key component of cardiac tissue engineering, enabling studies of cardiovascular disease mechanisms, drug responses, and developmental processes in human 3D tissue models assembled from isogenic cells. Since the very first engineered heart tissues were introduced more than two decades ago, a wide array of iPSC-derived cardiac spheroids, organoids, and heart-on-a-chip models have been developed incorporating the latest available technologies and materials. In this review, we will first outline the fundamental biological building blocks required to form a functional unit of cardiac muscle, including iPSC-derived cells differentiated by soluble factors (e.g., small molecules), extracellular matrix scaffolds, and exogenous biophysical maturation cues. We will then summarize the different fabrication approaches and strategies employed to reconstruct the heart in vitro at varying scales and geometries. Finally, we will discuss how these platforms, with continued improvements in scalability and tissue maturity, can contribute to both basic cardiovascular research and clinical applications in the future.

  View details for DOI 10.1016/j.yjmcc.2021.04.006

  View details for PubMedID 33895197

• Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids ADVANCED SCIENCE Hunt, D. R., Klett, K. C., Mascharak, S., Wang, H. Y., Gong, D., Lou, J., Li, X., Cai, P. C., Suhar, R. A., Co, J. Y., LeSavage, B. L., Foster, A. A., Guan, Y., Amieva, M. R., Peltz, G., Xia, Y., Kuo, C. J., Heilshorn, S. C. 2021

  View details for DOI 10.1002/advs.202004705

  View details for Web of Science ID 000627768200001

• 3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks. Advanced functional materials Hull, S. M., Lindsay, C. D., Brunel, L. G., Shiwarski, D. J., Tashman, J. W., Roth, J. G., Myung, D., Feinberg, A. W., Heilshorn, S. C. 2021; 31 (7)

  Abstract

  Three-dimensional (3D) bioprinting is a promising technology to produce tissue-like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, we developed a versatile, bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers. We term this family of materials UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) were each used as backbone polymers to create inks with storage moduli spanning 200 to 10,000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix-adherent human corneal mesenchymal stromal cells and non-matrix-adherent human induced pluripotent stem cell-derived neural progenitor spheroids were printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting.

  View details for DOI 10.1002/adfm.202007983

  View details for PubMedID 33613150

  View details for PubMedCentralID PMC7888563

• Microrheology reveals simultaneous cell-mediated matrix stiffening and fluidization that underlie breast cancer invasion. Science advances Krajina, B. A., LeSavage, B. L., Roth, J. G., Zhu, A. W., Cai, P. C., Spakowitz, A. J., Heilshorn, S. C. 2021; 7 (8)

  Abstract

  Living tissues embody a unique class of hybrid materials in which active and thermal forces are inextricably linked. Mechanical characterization of tissues demands descriptors that respect this hybrid nature. In this work, we develop a microrheology-based force spectrum analysis (FSA) technique to dissect the active and passive fluctuations of the extracellular matrix (ECM) in three-dimensional (3D) cell culture models. In two different stromal models and a 3D breast cancer spheroid model, our FSA reveals emergent hybrid dynamics that involve both high-frequency stress stiffening and low-frequency fluidization of the ECM. We show that this is a general consequence of nonlinear coupling between active forces and the frequency-dependent viscoelasticity of stress-stiffening networks. In 3D breast cancer spheroids, this dual active stiffening and fluidization is tightly connected with invasion. Our results suggest a mechanism whereby breast cancer cells reconcile the seemingly contradictory requirements for both tension and malleability in the ECM during invasion.

  View details for DOI 10.1126/sciadv.abe1969

  View details for PubMedID 33597244

• Dynamic light scattering microrheology for soft and living materials. Soft matter Cai, P. C., Krajina, B. A., Kratochvil, M. J., Zou, L., Zhu, A., Burgener, E. B., Bollyky, P. L., Milla, C. E., Webber, M. J., Spakowitz, A. J., Heilshorn, S. C. 2021

  Abstract

  We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. This microrheology technique only requires a small sample volume of 12 muL to measure up to six decades in time of rheological behavior. We demonstrate the use of dynamic light scattering microrheology (DLSmuR) on a variety of soft materials, including dilute polymer solutions, covalently-crosslinked polymer gels, and active, biological fluids. In this work, we detail the procedure for applying the technique to new materials and discuss the critical considerations for implementing the technique, including a custom analysis script for analyzing data output. We focus on the advantages of applying DLSmuR to biologically relevant materials: breast cancer cells encapsulated in a collagen gel and cystic fibrosis sputum. DLSmuR is an easy, efficient, and economical rheological technique that can guide the design of new polymeric materials and facilitate the understanding of the underlying physics governing behavior of naturally derived materials.

  View details for DOI 10.1039/d0sm01597k

  View details for PubMedID 33427280

• Defined matrices bring IBD to 3D. Nature materials LeSavage, B. L., Heilshorn, S. C. 2021; 20 (2): 124–25

  View details for DOI 10.1038/s41563-020-00904-1

  View details for PubMedID 33504987

• Transforming Growth Factor Induced Protein Promotes NF-Kappa-B Mediated Angiogenesis During Postnatal Lung Development. American journal of respiratory cell and molecular biology Liu, M., Iosef, C., Rao, S., Domingo-Gonzalez, R., Fu, S., Snider, P., Conway, S. J., Umbach, G. S., Heilshorn, S. C., Dewi, R. E., Dahl, M. J., Null, D. M., Albertine, K. H., Alvira, C. M. 2020

  Abstract

  Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that nuclear factor kappa-B (NFkappaB) promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NFkappaB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate pro-angiogenic NFkappaB signaling in the developing pulmonary vasculature. Proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis (2D-DIGE). NFkappaB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PEC) and TGFBI-regulated genes identified using RNA-sequencing. Alveolarization and pulmonary angiogenesis was assessed in WT and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PEC. Proteomic analysis identified transforming growth factor beta-induced protein (TGFBI) as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NFkappaB via alphavbeta3 integrins. RNA-sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PEC. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally-regulated protein that promotes NFkappaB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

  View details for DOI 10.1165/rcmb.2020-0153OC

  View details for PubMedID 33264084

• 3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks ADVANCED FUNCTIONAL MATERIALS Hull, S. M., Lindsay, C. D., Brunel, L. G., Shiwarski, D. J., Tashman, J. W., Roth, J. G., Myung, D., Feinberg, A. W., Heilshorn, S. C. 2020

  View details for DOI 10.1002/adfm.202007983

  View details for Web of Science ID 000590660600001

• Neural Progenitor Cells Alter Chromatin Organization and Neurotrophin Expression in Response to 3D Matrix Degradability. Advanced healthcare materials Madl, C. M., LeSavage, B. L., Khariton, M., Heilshorn, S. C. 2020: e2000754

  Abstract

  Neural progenitor cells (NPCs) are promising therapeutic candidates for nervous system regeneration. Significant efforts focus on developing hydrogel-based approaches to facilitate the clinical translation of NPCs, from scalable platforms for stem cell production to injectable carriers for cell transplantation. However, fundamental questions surrounding NPC-hydrogel interactions remain unanswered. While matrix degradability is known to regulate the stemness and differentiation capacity of NPCs, how degradability impacts NPC epigenetic regulation and secretory phenotype remains unknown. To address this question, NPCs encapsulated in recombinant protein hydrogels with tunable degradability are assayed for changes in chromatin organization and neurotrophin expression. In high degradability gels, NPCs maintain expression of stem cell factors, proliferate, and have large nuclei with elevated levels of the stemness-associated activating histone mark H3K4me3. In contrast, NPCs in low degradability gels exhibit more compact, rounded nuclei with peripherally localized heterochromatin, are non-proliferative yet non-senescent, and maintain expression of neurotrophic factors with potential therapeutic relevance. This work suggests that tuning matrix degradability may be useful to direct NPCs toward either a more-proliferative, stem-like phenotype for cell replacement therapies, or a more quiescent-like, pro-secretory phenotype for soluble factor-mediated therapies.

  View details for DOI 10.1002/adhm.202000754

  View details for PubMedID 32743903

• Bioengineered, In Situ-Crosslinked Collagen Gels for Suture-Free Stromal Defect Reconstruction of the Cornea Myung, D., Djalilian, A. R., Heilshorn, S., Chen, F., Le, P., Hull, S., Fernandes-Cunha, G., Na, K. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020

  View details for Web of Science ID 000554528301263

• Designer, injectable gels to prevent transplanted Schwann cell loss during spinal cord injury therapy. Science advances Marquardt, L. M., Doulames, V. M., Wang, A. T., Dubbin, K., Suhar, R. A., Kratochvil, M. J., Medress, Z. A., Plant, G. W., Heilshorn, S. C. 2020; 6 (14): eaaz1039

  Abstract

  Transplantation of patient-derived Schwann cells is a promising regenerative medicine therapy for spinal cord injuries; however, therapeutic efficacy is compromised by inefficient cell delivery. We present a materials-based strategy that addresses three common causes of transplanted cell death: (i) membrane damage during injection, (ii) cell leakage from the injection site, and (iii) apoptosis due to loss of endogenous matrix. Using protein engineering and peptide-based assembly, we designed injectable hydrogels with modular cell-adhesive and mechanical properties. In a cervical contusion model, our hydrogel matrix resulted in a greater than 700% improvement in successful Schwann cell transplantation. The combination therapy of cells and gel significantly improved the spatial distribution of transplanted cells within the endogenous tissue. A reduction in cystic cavitation and neuronal loss were also observed with substantial increases in forelimb strength and coordination. Using an injectable hydrogel matrix, therefore, can markedly improve the outcomes of cellular transplantation therapies.

  View details for DOI 10.1126/sciadv.aaz1039

  View details for PubMedID 32270042

• Materials for blood brain barrier modeling in vitro. Materials science & engineering. R, Reports : a review journal Ferro, M. P., Heilshorn, S. C., Owens, R. M. 2020; 140

  Abstract

  Brain homeostasis relies on the selective permeability property of the blood brain barrier (BBB). The BBB is formed by a continuous endothelium that regulates exchange between the blood stream and the brain. This physiological barrier also creates a challenge for the treatment of neurological diseases as it prevents most blood circulating drugs from entering into the brain. In vitro cell models aim to reproduce BBB functionality and predict the passage of active compounds through the barrier. In such systems, brain microvascular endothelial cells (BMECs) are cultured in contact with various biomaterial substrates. However, BMEC interactions with these biomaterials and their impact on BBB functions are poorly described in the literature. Here we review the most common materials used to culture BMECs and discuss their potential impact on BBB integrity in vitro. We investigate the biophysical properties of these biomaterials including stiffness, porosity and material degradability. We highlight a range of synthetic and natural materials and present three categories of cell culture dimensions: cell monolayers covering non-degradable materials (2D), cell monolayers covering degradable materials (2.5D) and vascularized systems developing into degradable materials (3D).

  View details for DOI 10.1016/j.mser.2019.100522

  View details for PubMedID 33551572

  View details for PubMedCentralID PMC7864217

• Materials for blood brain barrier modeling in vitro MATERIALS SCIENCE & ENGINEERING R-REPORTS Ferro, M. P., Heilshorn, S. C., Owens, R. M. 2020; 140

  View details for DOI 10.1016/j.mser.2019.100522

  View details for Web of Science ID 000528779100001

• Engineering the Microenvironment for Heart Muscle Cell Mechanobiology Castillo, E. A., Lane, K., Chirikian, O., Feinstein, S., Blair, C., Schroer, A., Pardon, G., Grancharova, T., Gunawardane, R., Heilshorn, S., Pruitt, B. L. CELL PRESS. 2020: 154A

  View details for Web of Science ID 000513023201022

• Bio-orthogonally crosslinked hyaluronate-collagen hydrogel for suture-free corneal defect repair. Biomaterials Chen, F. n., Le, P. n., Fernandes-Cunha, G. M., Heilshorn, S. C., Myung, D. n. 2020; 255: 120176

  Abstract

  Biomaterials that mimic corneal stroma could decrease the need for donor corneal tissue and could decrease the prevalence of complications associated with corneal transplantation, including infection and rejection. We developed a bio-orthogonally crosslinked hyaluronate-collagen hydrogel which can fill corneal defects in situ without the need for any sutures, initiators, or catalysts. We studied the effects of biorthogonal crosslinking on the light transmittance of the hydrogel, which was greater than 97% water. The transmittance of the optimized hydrogel in the visible light range was over 94%. We also investigated the mechanical properties, refractive index, morphology, biocompatibility, and corneal re-epithelialization capacity of the hyaluronate-collagen hydrogel. Our in vitro, in vivo, and ex vivo results demonstrated that this bio-orthogonally crosslinked hyaluronate-collagen hydrogel has excellent potential as a biomaterial for cornea repair and regeneration.

  View details for DOI 10.1016/j.biomaterials.2020.120176

  View details for PubMedID 32559566

• Bioprinting Cell- and Spheroid-Laden Protein-Engineered Hydrogels as Tissue-on-Chip Platforms. Frontiers in bioengineering and biotechnology Duarte Campos, D. F., Lindsay, C. D., Roth, J. G., LeSavage, B. L., Seymour, A. J., Krajina, B. A., Ribeiro, R. n., Costa, P. F., Blaeser, A. n., Heilshorn, S. C. 2020; 8: 374

  Abstract

  Human tissues, both in health and disease, are exquisitely organized into complex three-dimensional architectures that inform tissue function. In biomedical research, specifically in drug discovery and personalized medicine, novel human-based three-dimensional (3D) models are needed to provide information with higher predictive value compared to state-of-the-art two-dimensional (2D) preclinical models. However, current in vitro models remain inadequate to recapitulate the complex and heterogenous architectures that underlie biology. Therefore, it would be beneficial to develop novel models that could capture both the 3D heterogeneity of tissue (e.g., through 3D bioprinting) and integrate vascularization that is necessary for tissue viability (e.g., through culture in tissue-on-chips). In this proof-of-concept study, we use elastin-like protein (ELP) engineered hydrogels as bioinks for constructing such tissue models, which can be directly dispensed onto endothelialized on-chip platforms. We show that this bioprinting process is compatible with both single cell suspensions of neural progenitor cells (NPCs) and spheroid aggregates of breast cancer cells. After bioprinting, both cell types remain viable in incubation for up to 14 days. These results demonstrate a first step toward combining ELP engineered hydrogels with 3D bioprinting technologies and on-chip platforms comprising vascular-like channels for establishing functional tissue models.

  View details for DOI 10.3389/fbioe.2020.00374

  View details for PubMedID 32411691

  View details for PubMedCentralID PMC7198818

• Weekly injection of IL-2 using an injectable hydrogel reduces autoimmune diabetes incidence in NOD mice. Diabetologia Nagy, N. n., Kaber, G. n., Kratochvil, M. J., Kuipers, H. F., Ruppert, S. M., Yadava, K. n., Yang, J. n., Heilshorn, S. C., Long, S. A., Pugliese, A. n., Bollyky, P. L. 2020

  Abstract

  IL-2 injections are a promising therapy for autoimmune type 1 diabetes but the short half-life of this cytokine in vivo limits effective tissue exposure and necessitates frequent injections. Here we have investigated whether an injectable hydrogel could be used to promote prolonged IL-2 release in vivo.Capitalising on the IL-2-binding capabilities of heparin, an injectable hydrogel incorporating clinical-grade heparin, collagen and hyaluronan polymers was used to deliver IL-2. The IL-2-release kinetics and in vivo stability of this material were examined. The ability of soluble IL-2 vs hydrogel-mediated IL-2 injections to prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes were compared.We observed in vitro that the hydrogel released IL-2 over a 12-day time frame and that injected hydrogel likewise persisted 12 days in vivo. Notably, heparin binding potentiates the activity of IL-2 and enhances IL-2- and TGFβ-mediated expansion of forkhead box P3-positive regulatory T cells (FOXP3+ Tregs). Finally, weekly administration of IL-2-containing hydrogel partially prevented autoimmune diabetes while injections of soluble IL-2 did not.Hydrogel delivery may reduce the number of injections required in IL-2 treatment protocols for autoimmune diabetes. Graphical abstract.

  View details for DOI 10.1007/s00125-020-05314-1

  View details for PubMedID 33125521

• THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING Natarajan, S., Foreman, K., Soriano, M., Shehade, H., Fregoso, D., Eggold, J., Rosen, N. S., Heilshorn, S., Krieg, A. J., Krishnan, V., Dorigo, O., Sinha, S., Fuh, K. C., Rankin, E. B. AMER ASSOC CANCER RESEARCH. 2019: 168

  View details for Web of Science ID 000497337700104

• Rapid Diels-Alder Cross-linking of Cell Encapsulating Hydrogels CHEMISTRY OF MATERIALS Madl, C. M., Heilshorn, S. C. 2019; 31 (19): 8035–43

  View details for DOI 10.1021/acs.chemmater.9b02485

  View details for Web of Science ID 000489678800024

• Rapid Diels-Alder Cross-linking of Cell Encapsulating Hydrogels. Chemistry of materials : a publication of the American Chemical Society Madl, C. M., Heilshorn, S. C. 2019; 31 (19): 8035-8043

  Abstract

  Recent efforts in the design of hydrogel biomaterials have focused on better mimicking the native cellular microenvironment to direct cell fate. To simultaneously control multiple material parameters, several orthogonal chemistries may be needed. However, present strategies to prepare cell-encapsulating hydrogels make use of relatively few chemical reactions. To expand this chemical toolkit, we report the preparation of hydrogels based on a Diels-Alder reaction between fulvenes and maleimides with markedly improved gelation kinetics and hydrolytic stability. Fulvene-maleimide gels cross-link up to 10-times faster than other commonly used DA reaction pairs and remain stable for months under physiological conditions. Furthermore, fulvene-maleimide gels presenting relevant biochemical cues, such as cell-adhesive ligands and proteolytic degradability, support the culture of human mesenchymal stromal cells. Finally, this rapid DA reaction was combined with an orthogonal click reaction to demonstrate how the use of selective chemistries can provide new avenues to incorporate multiple functionalities in hydrogel materials.

  View details for DOI 10.1021/acs.chemmater.9b02485

  View details for PubMedID 32410775

  View details for PubMedCentralID PMC7224313

• Bioprinting of stem cell expansion lattices ACTA BIOMATERIALIA Lindsay, C. D., Roth, J. G., LeSavage, B. L., Heilshorn, S. C. 2019; 95: 225–35

  View details for DOI 10.1016/j.actbio.2019.05.014

  View details for Web of Science ID 000484878900016

• Engineered materials for organoid systems. Nature reviews. Materials Kratochvil, M. J., Seymour, A. J., Li, T. L., Paşca, S. P., Kuo, C. J., Heilshorn, S. C. 2019; 4 (9): 606-622

  Abstract

  Organoids are 3D cell culture systems that mimic some of the structural and functional characteristics of an organ. Organoid cultures provide the opportunity to study organ-level biology in models that mimic human physiology more closely than 2D cell culture systems or non-primate animal models. Many organoid cultures rely on decellularized extracellular matrices as scaffolds, which are often poorly chemically defined and allow only limited tunability and reproducibility. By contrast, the biochemical and biophysical properties of engineered matrices can be tuned and optimized to support the development and maturation of organoid cultures. In this Review, we highlight how key cell-matrix interactions guiding stem-cell decisions can inform the design of biomaterials for the reproducible generation and control of organoid cultures. We survey natural, synthetic and protein-engineered hydrogels for their applicability to different organoid systems and discuss biochemical and mechanical material properties relevant for organoid formation. Finally, dynamic and cell-responsive material systems are investigated for their future use in organoid research.

  View details for DOI 10.1038/s41578-019-0129-9

  View details for PubMedID 33552558

  View details for PubMedCentralID PMC7864216

• Engineered materials for organoid systems NATURE REVIEWS MATERIALS Kratochvil, M. J., Seymour, A. J., Li, T. L., Pasca, S. P., Kuo, C. J., Heilshorn, S. C. 2019; 4 (9): 606–22

  View details for DOI 10.1038/s41578-019-0129-9

  View details for Web of Science ID 000484683300006

• Adaptable protein-engineered hydrogels for organoid culture Heilshorn, S. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525061504374

• Characterization of bioorthogonally crosslinked collagen gels with encapsulated corneal stromal stem cells Hull, S., Fernandes-Cunha, G., Putra, I., Eslani, M., Djalilian, A. R., Heilshorn, S., Myung, D. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019

  View details for Web of Science ID 000488800701286

• Smart Bioinks as de novo Building Blocks to Bioengineer Living Tissues. Gels (Basel, Switzerland) Blaeser, A., Heilshorn, S. C., Duarte Campos, D. F. 2019; 5 (2)

  Abstract

  In vitro tissues and 3D in vitro models have come of age [...].

  View details for DOI 10.3390/gels5020029

  View details for PubMedID 31121889

• Collagen Remodeling in the Hypoxic Tumor-Mesothelial Niche Promotes Ovarian Cancer Metastasis CANCER RESEARCH Natarajan, S., Foreman, K. M., Soriano, M., Rossen, N. S., Shehade, H., Fregoso, D. R., Eggold, J. T., Krishnan, V., Dorigo, O., Krieg, A. J., Heilshorn, S. C., Sinha, S., Fuh, K. C., Rankin, E. B. 2019; 79 (9): 2271–84

  View details for DOI 10.1158/0008-5472.CAN-18-2616

  View details for Web of Science ID 000466765900019

• Biomimetic polymers as custom bioinks for 3D printing Heilshorn, S. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000478861204868

• Matrix Remodeling Enhances the Differentiation Capacity of Neural Progenitor Cells in 3D Hydrogels ADVANCED SCIENCE Madl, C. M., LeSavage, B. L., Dewi, R. E., Lampe, K. J., Heilshorn, S. C. 2019; 6 (4): 1801716

  Abstract

  Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material-based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell-mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC-secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter-responsive neurons. Matrix remodeling modulates expression of the transcriptional co-activator Yes-associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin-dependent manner. Thus, cell-remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.

  View details for PubMedID 30828535

• Adefinition of bioinks and their distinction from biomaterial inks BIOFABRICATION Groll, J., Burdick, J. A., Cho, D., Derby, B., Gelinsky, M., Heilshorn, S. C., Juengst, T., Malda, J., Mironov, V. A., Nakayama, K., Ovsianikov, A., Sun, W., Takeuchi, S., Yoo, J. J., Woodfield, T. F. 2019; 11 (1): 013001

  Abstract

  Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384-402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

  View details for DOI 10.1088/1758-5090/aaec52

  View details for Web of Science ID 000451083800001

  View details for PubMedID 30468151

• Antibiofilm elastin-like polypeptide coatings: functionality, stability, and selectivity ACTA BIOMATERIALIA Atefyekta, S., Pihl, M., Lindsay, C., Heilshorn, S. C., Andersson, M. 2019; 83: 245-256

  View details for DOI 10.1016/j.actbio.2018.10.039

  View details for Web of Science ID 000455068900021

• Shear Thinning Hydrogel-based 3D Tissue Modelling BIOFABRICATION AND 3D TISSUE MODELING Lindsay, C. D., Heilshorn, S. C., Cho, D. W. 2019; 3: 94–118

  View details for Web of Science ID 000565917000006

• Bioprinting of stem cell expansion lattices. Acta biomaterialia Lindsay, C. D., Roth, J. G., LeSavage, B. L., Heilshorn, S. C. 2019

  Abstract

  Stem cells have great potential in regenerative medicine, with neural progenitor cells (NPCs) being developed as a therapy for many central nervous system diseases and injuries. However, one limitation to the clinical translation of stem cells is the resource-intensive, two-dimensional culture protocols required for biomanufacturing a clinically-relevant number of cells. This challenge can be overcome in an easy-to-produce and cost-effective 3D platform by bioprinting NPCs in a layered lattice structure. Here we demonstrate that alginate biopolymers are an ideal bioink for expansion lattices and do not require chemical modifications for effective NPC expansion. Alginate bioinks that are lightly crosslinked prior to printing can shield printed NPCs from potential mechanical damage caused by printing. NPCs within alginate expansion lattices remain in a stem-like state while undergoing a 2.5-fold expansion. Importantly, we demonstrate the ability to efficiently remove NPCs from printed lattices for future down-stream use as a cell-based therapy. These results demonstrate that 3D bioprinting of alginate expansion lattices is a viable and economical platform for NPC expansion that could be translated to clinical applications.

  View details for PubMedID 31096043

• Antibiofilm elastin-like polypeptide coatings: functionality, stability, and selectivity. Acta biomaterialia Atefyekta, S., Pihl, M., Lindsay, C., Heilshorn, S. C., Andersson, M. 2019; 83: 245–56

  Abstract

  Antimicrobial peptides (AMPs) are currently receiving interest as an alternative to conventional antibiotics to treat biomaterial-associated infection. However, the inherent instability of such peptides often limits their efficacy in intended clinical applications. Covalent immobilization of AMPs to surfaces is one strategy to increase the long-term stability and minimize the toxicity. In this work, an antimicrobial peptide, RRPRPRPRPWWWW-NH2 (RRP9W4N), was used to modify elastin-like polypeptide (ELP) surface coatings containing cell-adhesive peptide domains (RGD) using covalent chemistry. The AMP retained its antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa when covalently bonded to ELP surfaces. Simultaneously, the AMP functionalization had insignificant effect on the viability, function, and differentiation of human osteosarcoma MG63 cells and human mesenchymal stem cells (hMSCs). Furthermore, stability of the immobilized AMP in human blood serum was investigated, and the results suggested that the AMP preserved its antibacterial activity up to 24 h. Combined, the results show that covalently attached AMPs onto RGD-containing ELP are an excellent candidate as an antimicrobial coating for medical devices. STATEMENT OF SIGNIFICANCE: Biomaterial associated infection, caused by adherent biofilm, is usually difficult to treat. There is a high demand for new materials and treatments to decrease the infection rates, especially with increasing threats concerning resistant bacteria. Formation of biofilms on medical devices lowers the bacteria susceptibility towards traditional antibiotics and also circumvent our immune system often resulting in revisional surgery and extensive use of antibiotics. One promising strategy is to develop surfaces having low bacterial attractiveness or bacterial killing properties, but still retaining the main function of the device. In this study, we have developed an implant coating that demonstrates a high antimicrobial effect and at the same time showing no negative affect on human cells.

  View details for PubMedID 30541700

• An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium. ACS nano Yang, H. n., Qin, X. n., Wang, H. n., Zhao, X. n., Liu, Y. n., Wo, H. T., Liu, C. n., Nishiga, M. n., Chen, H. n., Ge, J. n., Sayed, N. n., Abilez, O. J., Ding, D. n., Heilshorn, S. C., Li, K. n. 2019

  Abstract

  A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

  View details for DOI 10.1021/acsnano.9b03343

  View details for PubMedID 31149806

• Interrogating extracellular matrix remodeling by breast cancer spheroids using dynamic light scattering microrheology. Krajina, B. A., Zhu, A., Spakowitz, A. J., Heilshorn, S. C. AMER SOC CELL BIOLOGY. 2018

  View details for Web of Science ID 000505772703181

• Engineering Regenerative Thymic Tissues to Restore Long-Term T Cell Lymphopoiesis Gai, H., Gras-Pena, R., Verma, Y., Fateh, V., Ikeda, K., Dejene, B., Min, D., Wang, J., Swigut, T., Weinberg, K. I., Hollander, G. A., Heilshorn, S., Roncarolo, M., Sebastiano, V., Weinacht, K. G. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-117499

  View details for Web of Science ID 000454842805362

• Tuning Bulk Hydrogel Degradation by Simultaneous Control of Proteolytic Cleavage Kinetics and Hydrogel Network Architecture. ACS macro letters Madl, C. M., Katz, L. M., Heilshorn, S. C. 2018; 7 (11): 1302-1307

  Abstract

  Degradation of three-dimensional hydrogels is known to regulate many cellular behaviors. Accordingly, several elegant approaches have been used to render hydrogels degradable by cell-secreted proteases. However, existing hydrogel systems are limited in their ability to simultaneously and quantitatively tune two aspects of hydrogel degradability: cleavage rate (the rate at which individual chemical bonds are cleaved) and degraded hydrogel architecture (the network structure during degradation). Using standard peptide engineering approaches, we alter the proteolytic kinetics of the polymer cleavage rate to tune gel degradation time from less than 12 h to greater than 9 days. Independently, we vary the cross-linker functionality to achieve network architectures that initially have identical molecular weight between cross-links but upon degradation are designed to release between 5% and 100% of the polymer. Confirming the biological relevance of both parameters, formation of vascular-like structures by endothelial cells is regulated both by bond cleavage rate and by degraded hydrogel architecture. This strategy to fine-tune different aspects of hydrogel degradability has applications in cell culture, regenerative medicine, and drug delivery.

  View details for DOI 10.1021/acsmacrolett.8b00664

  View details for PubMedID 32523799

  View details for PubMedCentralID PMC7286611

• Tuning Bulk Hydrogel Degradation by Simultaneous Control of Proteolytic Cleavage Kinetics and Hydrogel Network Architecture ACS MACRO LETTERS Madl, C. M., Katz, L. M., Heilshorn, S. C. 2018; 7 (11): 1302–7

  View details for DOI 10.1021/acsmacrolett.8b00664

  View details for Web of Science ID 000451246000005

• Active DNA Olympic Hydrogels Driven by Topoisomerase Activity. Physical review letters Krajina, B. A., Zhu, A., Heilshorn, S. C., Spakowitz, A. J. 2018; 121 (14): 148001

  Abstract

  Biological systems are equipped with a diverse repertoire of proteins that regulate DNA topology with precision that is beyond the reach of conventional polymer chemistry. Here, we harness the unique properties of topoisomerases to synthesize Olympic hydrogels formed by topologically interlinked DNA rings. Using dynamic light scattering microrheology to probe the viscoelasticity of DNA topological networks, we show that topoisomerase II enables the facile preparation of active, adenosine triphosphate-driven Olympic hydrogels that can be switched between liquid and solid states on demand. Our results provide a versatile system for engineering switchable topological materials that may be broadly leveraged to model the impact of topological constraints and active dynamics in the physics of chromosomes and other polymeric materials.

  View details for DOI 10.1103/PhysRevLett.121.148001

  View details for PubMedID 30339454

• Active DNA Olympic Hydrogels Driven by Topoisomerase Activity PHYSICAL REVIEW LETTERS Krajina, B. A., Zhu, A., Heilshorn, S. C., Spakowitz, A. J. 2018; 121 (14)

  View details for DOI 10.1103/PhysRevLett.121.148001

  View details for Web of Science ID 000446138000014

• Engineered stem cell mimics to enhance stroke recovery BIOMATERIALS George, P. M., Oh, B., Dewi, R., Hua, T., Cai, L., Levinson, A., Liang, X., Krajina, B. A., Bliss, T. M., Heilshorn, S. C., Steinberg, G. K. 2018; 178: 63–72

  View details for DOI 10.1016/j.biomaterials.2018.06.010

  View details for Web of Science ID 000440959000006

• Polymers at the Interface with Biology BIOMACROMOLECULES Deming, T. J., Klok, H., Armes, S. P., Becker, M. L., Champion, J. A., Chen, E., Heilshorn, S. C., van Hest, J. M., Irvine, D. J., Johnson, J. A., Kiessling, L. L., Maynard, H. D., de la Cruz, M., Sullivan, M. O., Tirrell, M. V., Anseth, K. S., Lecommandoux, S., Percec, S., Zhong, Z., Albertsson, A. 2018; 19 (8): 3151–62

  View details for PubMedID 30099879

• Tunable Control of Hydrogel Microstructure by Kinetic Competition between Self-Assembly and Crosslinking of Elastin-like Proteins ACS APPLIED MATERIALS & INTERFACES Wang, H., Paul, A., Duong Nguyen, Enejder, A., Heilshorn, S. C. 2018; 10 (26): 21808–15

  Abstract

  The fabrication of three dimensional "bead-string" microstructured hydrogels is rationally achieved by controlling the relative timing of chemical crosslinking and physical self-assembly processes of an engineered protein. To demonstrate this strategy, an elastin-like protein (ELP) amino acid sequence was selected to enable site-specific chemical crosslinking and thermoresponsive physical self-assembly. This method allows the tuning of material microstructures without altering the ELP amino acid sequence but simply through controlling the chemical crosslinking extent before the thermally induced, physical coacervation of ELP. A loosely crosslinked network enables ELP to have greater chain mobility, resulting in phase segregation into larger beads. By contrast, a network with higher crosslinking density has restricted ELP chain mobility, resulting in more localized self-assembly into smaller beads. As a proof of concept application for this facile assembly process, we demonstrate one-pot, simultaneous, dual encapsulation of hydrophilic and hydrophobic model drugs within the microstructured hydrogel and differential release rates of the two drugs from the material.

  View details for PubMedID 29869869

• Investigating the interplay between substrate stiffness and ligand chemistry in directing mesenchymal stem cell differentiation within 3D macro-porous substrates. Biomaterials Haugh, M. G., Vaughan, T. J., Madl, C. M., Raftery, R. M., McNamara, L. M., O'Brien, F. J., Heilshorn, S. C. 2018; 171: 23–33

  Abstract

  Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior.

  View details for PubMedID 29677521

• Investigating the interplay between substrate stiffness and ligand chemistry in directing mesenchymal stem cell differentiation within 3D macro-porous substrates BIOMATERIALS Haugh, M. G., Vaughan, T. J., Madl, C. M., Raftery, R. M., McNamara, L. M., O'Brien, F. J., Heilshorn, S. C. 2018; 171: 23-33

  View details for DOI 10.1016/j.biomaterials.2018.04.026

  View details for Web of Science ID 000433643000003

• Effects of engineered cellular microenvironments on the secretome of human mesenchymal stem cells Hull, S., Fernandes-Cunha, G., Lee, H., Heilshorn, S., Myung, D. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018

  View details for Web of Science ID 000442912506282

• Mechanical properties of collagen gels crosslinked by copper-free click chemistry and their effects on encapsulated keratocytes Lee, H., Fernandes-Cunha, G., Heilshorn, S., Myung, D. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018

  View details for Web of Science ID 000442912506258

• Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche. Annual review of biomedical engineering Madl, C. M., Heilshorn, S. C. 2018; 20: 21–47

  Abstract

  Stem cells are a powerful resource for many applications including regenerative medicine, patient-specific disease modeling, and toxicology screening. However, eliciting the desired behavior from stem cells, such as expansion in a naive state or differentiation into a particular mature lineage, remains challenging. Drawing inspiration from the native stem cell niche, hydrogel platforms have been developed to regulate stem cell fate by controlling microenvironmental parameters including matrix mechanics, degradability, cell-adhesive ligand presentation, local microstructure, and cell-cell interactions. We survey techniques for modulating hydrogel properties and review the effects of microenvironmental parameters on maintaining stemness and controlling differentiation for a variety of stem cell types. Looking forward, we envision future hydrogel designs spanning a spectrum of complexity, ranging from simple, fully defined materials for industrial expansion of stem cells to complex, biomimetic systems for organotypic cell culture models.

  View details for PubMedID 29220201

• Review: Bioengineering strategies to probe T cell mechanobiology. APL bioengineering de la Zerda, A., Kratochvil, M. J., Suhar, N. A., Heilshorn, S. C. 2018; 2 (2): 021501

  Abstract

  T cells play a major role in adaptive immune response, and T cell dysfunction can lead to the progression of several diseases that are often associated with changes in the mechanical properties of tissues. However, the concept that mechanical forces play a vital role in T cell activation and signaling is relatively new. The endogenous T cell microenvironment is highly complex and dynamic, involving multiple, simultaneous cell-cell and cell-matrix interactions. This native complexity has made it a challenge to isolate the effects of mechanical stimuli on T cell activation. In response, researchers have begun developing engineered platforms that recapitulate key aspects of the native microenvironment to dissect these complex interactions in order to gain a better understanding of T cell mechanotransduction. In this review, we first describe some of the unique characteristics of T cells and the mounting research that has shown they are mechanosensitive. We then detail the specific bioengineering strategies that have been used to date to measure and perturb the mechanical forces at play during T cell activation. In addition, we look at engineering strategies that have been used successfully in mechanotransduction studies for other cell types and describe adaptations that may make them suitable for use with T cells. These engineering strategies can be classified as 2D, so-called 2.5D, or 3D culture systems. In the future, findings from this emerging field will lead to an optimization of culture environments for T cell expansion and the development of new T cell immunotherapies for cancer and other immune diseases.

  View details for DOI 10.1063/1.5006599

  View details for PubMedID 31069295

  View details for PubMedCentralID PMC6324202

• Review: Bioengineering strategies to probe T cell mechanobiology APL BIOENGINEERING de la Zerda, A., Kratochvil, M. J., Suhar, N. A., Heilshorn, S. C. 2018; 2 (2)

  View details for DOI 10.1063/1.5006599

  View details for Web of Science ID 000455057800002

• Dynamic Hyaluronan Hydrogels with Temporally Modulated High Injectability and Stability Using a Biocompatible Catalyst ADVANCED MATERIALS Lou, J., Liu, F., Lindsay, C. D., Chaudhuri, O., Heilshorn, S. C., Xia, Y. 2018; 30 (22)

  View details for DOI 10.1002/adma.201705215

  View details for Web of Science ID 000434034100001

• Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D. Journal of visualized experiments : JoVE LeSavage, B. L., Suhar, N. A., Madl, C. M., Heilshorn, S. C. 2018

  Abstract

  Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

  View details for PubMedID 29863669

• Bioengineering strategies to accelerate stem cell therapeutics NATURE Madl, C. M., Heilshorn, S. C., Blau, H. M. 2018; 557 (7705): 335–42

  Abstract

  Although only a few stem cell-based therapies are currently available to patients, stem cells hold tremendous regenerative potential, and several exciting clinical applications are on the horizon. Biomaterials with tuneable mechanical and biochemical properties can preserve stem cell function in culture, enhance survival of transplanted cells and guide tissue regeneration. Rapid progress with three-dimensional hydrogel culture platforms provides the opportunity to grow patient-specific organoids, and has led to the discovery of drugs that stimulate endogenous tissue-specific stem cells and enabled screens for drugs to treat disease. Therefore, bioengineering technologies are poised to overcome current bottlenecks and revolutionize the field of regenerative medicine.

  View details for PubMedID 29769665

• Dynamic Hyaluronan Hydrogels with Temporally Modulated High Injectability and Stability Using a Biocompatible Catalyst. Advanced materials (Deerfield Beach, Fla.) Lou, J., Liu, F., Lindsay, C. D., Chaudhuri, O., Heilshorn, S. C., Xia, Y. 2018; 30 (22): e1705215

  Abstract

  Injectable and biocompatible hydrogels have become increasingly important for cell transplantation to provide mechanical protection of cells during injection and a stable scaffold for cell adhesion post-injection. Injectable hydrogels need to be easily pushed through a syringe needle and quickly recover to a gel state, thus generally requiring noncovalent or dynamic cross-linking. However, a dilemma exists in the design of dynamic hydrogels: hydrogels with fast exchange of cross-links are easier to eject using less force, but lack long-term stability; in contrast, slow exchange of cross-links improves stability, but compromises injectability and thus the ability to protect cells under flow. A new concept to resolve this dilemma using a biocompatible catalyst to modulate the dynamic properties of hydrogels at different time points of application to have both high injectability and high stability is presented. Hyaluronic acid based hydrogels are formed through dynamic covalent hydrazone cross-linking in the presence of a biocompatible benzimidazole-based catalyst. The catalyst accelerates the formation and exchange of hydrazone bonds, enhancing injectability, but rapidly diffuses away from the hydrogel after injection to retard the exchange and improve the long-term stability for cell culture.

  View details for PubMedID 29682801

• Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D JOVE-JOURNAL OF VISUALIZED EXPERIMENTS LeSavage, B. L., Suhar, N. A., Madl, C. M., Heilshorn, S. C. 2018

  View details for DOI 10.3791/57739

  View details for Web of Science ID 000444372200136

• Protein engineering of multi-functional biomaterials for regenerative medicine Heilshorn, S. AMER CHEMICAL SOC. 2018

  View details for Web of Science ID 000435539905870

• Bioorthogonal Strategies for Engineering Extracellular Matrices ADVANCED FUNCTIONAL MATERIALS Madl, C. M., Heilshorn, S. C. 2018; 28 (11)

  View details for DOI 10.1002/adfm.201706046

  View details for Web of Science ID 000427110100021

• Bioorthogonal Strategies for Engineering Extracellular Matrices. Advanced functional materials Madl, C. M., Heilshorn, S. C. 2018; 28 (11)

  Abstract

  Hydrogels are commonly used as engineered extracellular matrix (ECM) mimics in applications ranging from tissue engineering to in vitro disease models. Ideal mechanisms used to crosslink ECM-mimicking hydrogels do not interfere with the biology of the system. However, most common hydrogel crosslinking chemistries exhibit some form of cross-reactivity. The field of bio-orthogonal chemistry has arisen to address the need for highly specific and robust reactions in biological contexts. Accordingly, bio-orthogonal crosslinking strategies have been incorporated into hydrogel design, allowing for gentle and efficient encapsulation of cells in various hydrogel materials. Furthermore, the selective nature of bio-orthogonal chemistries can permit dynamic modification of hydrogel materials in the presence of live cells and other biomolecules to alter matrix mechanical properties and biochemistry on demand. In this review, we provide an overview of bio-orthogonal strategies used to prepare cell-encapsulating hydrogels and highlight the potential applications of bio-orthogonal chemistries in the design of dynamic engineered ECMs.

  View details for DOI 10.1002/adfm.201706046

  View details for PubMedID 31558890

  View details for PubMedCentralID PMC6761700

• Protein-engineered hydrogels enhance the survival of induced pluripotent stem cell-derived endothelial cells for treatment of peripheral arterial disease BIOMATERIALS SCIENCE Foster, A. A., Dewi, R. E., Cai, L., Hou, L., Strassberg, Z., Alcazar, C. A., Heilshorn, S. C., Huang, N. F. 2018; 6 (3): 614–22

  Abstract

  A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

  View details for PubMedID 29406542

  View details for PubMedCentralID PMC5829050

• Interrogating Cell-Mediated Remodeling of the Extracellular Matrix by Dynamic Light Scattering Microrheology Krajina, B. A., Zhu, A., Heilshorn, S. C., Spakowitz, A. J. CELL PRESS. 2018: 371A–372A

  View details for Web of Science ID 000430450000347

• Engineered stem cell mimics to enhance stroke recovery. Biomaterials George, P. M., Oh, B. n., Dewi, R. n., Hua, T. n., Cai, L. n., Levinson, A. n., Liang, X. n., Krajina, B. A., Bliss, T. M., Heilshorn, S. C., Steinberg, G. K. 2018; 178: 63–72

  Abstract

  Currently, no medical therapies exist to augment stroke recovery. Stem cells are an intriguing treatment option being evaluated, but cell-based therapies have several challenges including developing a stable cell product with long term reproducibility. Since much of the improvement observed from cellular therapeutics is believed to result from trophic factors the stem cells release over time, biomaterials are well-positioned to deliver these important molecules in a similar fashion. Here we show that essential trophic factors secreted from stem cells can be effectively released from a multi-component hydrogel system into the post-stroke environment. Using our polymeric system to deliver VEGF-A and MMP-9, we improved recovery after stroke to an equivalent degree as observed with traditional stem cell treatment in a rodent model. While VEGF-A and MMP-9 have many unique mechanisms of action, connective tissue growth factor (CTGF) interacts with both VEGF-A and MMP-9. With our hydrogel system as well as with stem cell delivery, the CTGF pathway is shown to be downregulated with improved stroke recovery.

  View details for PubMedID 29909038

• Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche ANNUAL REVIEW OF BIOMEDICAL ENGINEERING, VOL 20 Madl, C. M., Heilshorn, S. C., Yamush, M. L. 2018; 20: 21-47

  View details for DOI 10.1146/annurev-bioeng-062117-120954

  View details for Web of Science ID 000434421400002

• Biotemplated synthesis of inorganic materials: An emerging paradigm for nanomaterial synthesis inspired by nature PROGRESS IN MATERIALS SCIENCE Krajina, B. A., Proctor, A. C., Schoen, A. P., Spakowitz, A. J., Heilshorn, S. C. 2018; 91: 1–23

  View details for DOI 10.1016/j.pmatsci.2017.08.001

  View details for Web of Science ID 000415780300001

• Dynamic Light Scattering Microrheology Reveals Multiscale Viscoelasticity of Polymer Gels and Precious Biological Materials ACS CENTRAL SCIENCE Krajina, B. A., Tropini, C., Zhu, A., DiGiacomo, P., Sonnenburg, J. L., Heilshorn, S. C., Spakowitz, A. J. 2017; 3 (12): 1294–1303

  Abstract

  The development of experimental techniques capable of probing the viscoelasticity of soft materials over a broad range of time scales is essential to uncovering the physics that governs their behavior. In this work, we develop a microrheology technique that requires only 12 μL of sample and is capable of resolving dynamic behavior ranging in time scales from 10-6 to 10 s. Our approach, based on dynamic light scattering in the single-scattering limit, enables the study of polymer gels and other soft materials over a vastly larger hierarchy of time scales than macrorheology measurements. Our technique captures the viscoelastic modulus of polymer hydrogels with a broad range of stiffnesses from 10 to 104 Pa. We harness these capabilities to capture hierarchical molecular relaxations in DNA and to study the rheology of precious biological materials that are impractical for macrorheology measurements, including decellularized extracellular matrices and intestinal mucus. The use of a commercially available benchtop setup that is already available to a variety of soft matter researchers renders microrheology measurements accessible to a broader range of users than existing techniques, with the potential to reveal the physics that underlies complex polymer hydrogels and biological materials.

  View details for PubMedID 29296670

  View details for PubMedCentralID PMC5746858

• Adaptable hydrogels with secondary reinforcement for regenerative medicine Heilshorn, S., Wang, H. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000429556704279

• Recombinant biomaterials for treatment of spinal cord injuries Dubbin, K., Marquardt, L., Plant, G., Heilshorn, S. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000429556703370

• Peptide-crosslinking of biomaterials for 3D bio-printing Heilshorn, S., Dubbin, K. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000429556703119

• Polypeptide scaffolds as engineered neural stem cell niches Madl, C., Heilshorn, S. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000429556703312

• Covalently adaptable elastin-like protein - hyaluronic acid (ELP - HA) hybrid hydrogels with secondary thermoresponsive crosslinking for injectable stem cell delivery. Advanced functional materials Wang, H., Zhu, D., Paul, A., Cai, L., Enejder, A., Yang, F., Heilshorn, S. C. 2017; 27 (28)

  Abstract

  Shear-thinning, self-healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. We present an injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive engineered proteins. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine-modified elastin-like protein (ELP) and aldehyde-modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by Coherent anti-Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a 10-fold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks post injection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP-HA hydrogels for injectable stem cell transplantation and tissue regeneration.

  View details for DOI 10.1002/adfm.201605609

  View details for PubMedID 33041740

  View details for PubMedCentralID PMC7546546

• Covalently Adaptable Elastin-Like Protein-Hyaluronic Acid (ELP-HA) Hybrid Hydrogels with Secondary Thermoresponsive Crosslinking for Injectable Stem Cell Delivery ADVANCED FUNCTIONAL MATERIALS Wang, H., Zhu, D., Paul, A., Cai, L., Enejder, A., Yang, F., Heilshorn, S. C. 2017; 27 (28)

  View details for DOI 10.1002/adfm.201605609

  View details for Web of Science ID 000406183100001

• A novel protein-engineered hepatocyte growth factor analog released via a shear-thinning injectable hydrogel enhances post-infarction ventricular function. Biotechnology and bioengineering Steele, A. N., Cai, L., Truong, V. N., Edwards, B. B., Goldstone, A. B., Eskandari, A., Mitchell, A. C., Marquardt, L. M., Foster, A. A., Cochran, J. R., Heilshorn, S. C., Woo, Y. J. 2017

  Abstract

  In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 μg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 μg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;9999: 1-11. © 2017 Wiley Periodicals, Inc.

  View details for DOI 10.1002/bit.26345

  View details for PubMedID 28574594

• Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates BIOMATERIALS Dimarco, R. L., Hunt, D. R., Dewi, R. E., Heilshorn, S. C. 2017; 129: 152-162

  Abstract

  The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by pharmaceutical companies without major changes to their current testing protocols.

  View details for DOI 10.1016/j.biomaterials.2017.03.023

  View details for Web of Science ID 000399256500011

  View details for PubMedID 28342321

• Novel approaches to anchoring therapeutic factors to corneal stroma to promote wound healing. Myung, D., Djalilian, A. R., Heilshorn, S., Goldberg, J. L., Kreymerman, A., Kumar, A., Madl, C., Eslani, M., Shen, X., Putra, I., Fernandes-Cunha, G., Koh, W., Lee, H. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2017

  View details for Web of Science ID 000432170300117

• Dynamic Rheology for the Prediction of Surgical Outcomes in Autologous Fat Grafting. Plastic and reconstructive surgery Luan, A., Zielins, E. R., Wearda, T., Atashroo, D. A., Blackshear, C. P., Raphel, J., Brett, E. A., Flacco, J., Alyono, M. C., Momeni, A., Heilshorn, S., Longaker, M. T., Wan, D. C. 2017

  Abstract

  Due to the abundance and biocompatibility of fat, lipotransfer has become an attractive method for treating soft tissue deficits. However, it is limited by unpredictable graft survival and retention. Currently, little is known about the viscoelastic properties of fat after various injection methods. Here, we assess the effects of cannula diameter, length, and shape on the viscoelastic properties, structure, and retention of fat.Human lipoaspirate was harvested using suction-assisted liposuction and prepared for grafting. A syringe pump was used to inject fat at a controlled flow rate through cannulas of varying gauge, length, and shape. Processed samples were tested in triplicate on an oscillatory rheometer to measure their viscoelastic properties. Fat grafts from each group were placed into the scalps of immunocompromised mice. After 8 weeks, graft retention was measured using micro-CT and grafts were explanted for histological analysis.Lipoaspirate injected through narrower, longer, and bent cannulas exhibited more shear thinning with diminished quality. The storage modulus (G') of fat processed with 18-gauge cannulas was significantly lower than when processed with 14-gauge or larger cannulas, which also corresponded with inferior in vivo histological structure. Similarly, the longer cannula group had a significantly lower G' than the shorter cannula, and was associated with decreased graft retention.Discrete modifications in the methods used for fat placement can have a significant impact on immediate graft integrity, and ultimately on graft survival and quality. Respecting these biomechanical influences during the placement phase of lipotransfer may allow surgeons to optimize outcomes.

  View details for DOI 10.1097/PRS.0000000000003578

  View details for PubMedID 28574947

• Protein-Nanoparticle Hydrogels That Self-assemble in Response to Peptide-Based Molecular Recognition. ACS biomaterials science & engineering Parisi-Amon, A., Lo, D. D., Montoro, D. T., Dewi, R. E., Longaker, M. T., Heilshorn, S. C. 2017; 3 (5): 750-756

  Abstract

  Recently, supramolecular hydrogels assembled through nonspecific interactions between polymers and nanoparticles (termed PNP systems) were reported to have rapid shear-thinning and self-healing properties amenable for cell-delivery applications in regenerative medicine. Here, we introduce protein engineering concepts into the design of a new family of PNP hydrogels to enable direct control over the polymer-nanoparticle interactions using peptide-based molecular recognition motifs. Specifically, we have designed a bifunctional peptide that induces supramolecular hydrogel assembly between hydroxy apatite nanoparticles and an engineered, recombinant protein. We demonstrate that this supramolecular assembly critically requires molecular recognition, as no assembly is observed in the presence of control peptides with a scrambled amino acid sequence. Titration of the bifunctional peptide enables direct control over the number of physical cross-links within the system and hence the resulting hydrogel mechanical properties. As with previous PNP systems, these materials are rapidly shear-thinning and self-healing. As proof-of-concept, we demonstrate that these materials are suitable for therapeutic cell delivery applications in a preclinical murine calvarial defect model.

  View details for DOI 10.1021/acsbiomaterials.6b00286

  View details for PubMedID 33440497

• Protein-Nanoparticle Hydrogels That Self-assemble in Response to Peptide-Based Molecular Recognition ACS BIOMATERIALS SCIENCE & ENGINEERING Parisi-Amon, A., Lo, D. D., Montoro, D. T., Dew, R. E., Longaker, M. T., Heilshorn, S. C. 2017; 3 (5): 750-756

  View details for DOI 10.1021/acsbiomaterials.6b00286

  View details for Web of Science ID 000401053800009

• Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration. Biomaterials Zhu, D., Wang, H., Trinh, P., Heilshorn, S. C., Yang, F. 2017

  Abstract

  Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair.

  View details for DOI 10.1016/j.biomaterials.2017.02.010

  View details for PubMedID 28268018

• Tyrosine-Selective Functionalization for Bio-Orthogonal Cross-Linking of Engineered Protein Hydrogels. Bioconjugate chemistry Madl, C. M., Heilshorn, S. C. 2017

  Abstract

  Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.

  View details for DOI 10.1021/acs.bioconjchem.6b00720

  View details for PubMedID 28151642

• YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography BIOMATERIALS Mascharak, S., Benitez, P. L., Proctor, A. C., Madl, C. M., Hu, K. H., Dewi, R. E., Butte, M. J., Heilshorn, S. C. 2017; 115: 155-166

  Abstract

  Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.

  View details for DOI 10.1016/j.biomaterials.2016.11.019

  View details for Web of Science ID 000390642100014

  View details for PubMedID 27889666

• Hyaluronan content governs tissue stiffness in pancreatic islet inflammation. The Journal of biological chemistry Nagy, N. n., de la Zerda, A. n., Kaber, G. n., Johnson, P. Y., Hu, K. H., Kratochvil, M. J., Yadava, K. n., Zhao, W. n., Cui, Y. n., Navarro, G. n., Annes, J. P., Wight, T. N., Heilshorn, S. C., Bollyky, P. L., Butte, M. J. 2017

  Abstract

  We have identified a novel role for hyaluronan (HA), an extracellular matrix (ECM) polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes (T1D) of mice and humans is preceded by intra-islet accumulation of HA, a highly hygroscopic polymer. Using the DORmO double transgenic (DO11.10 x RIPmOVA) mouse model of T1D, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared to controls. Conversely, treatment with 4-methylumbelliferone (4-MU), a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

  View details for PubMedID 29183997

• Photoacoustic Imaging of Embryonic Stem Cell-Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles Advanced Functional Materials Qin, X., Chen, H., Yang, H., Wu, H., Zhao, X., Wang, H., Chour, T., Neofytou, E., Ding, D., Daldrup-Link, H., Heilshorn, S. C., Li, K., Wu, J. C. 2017

  View details for DOI 10.1002/adfm.201704939

• The Diverse Roles of Hydrogel Mechanics in Injectable Stem Cell Transplantation. Current opinion in chemical engineering Foster, A. A., Marquardt, L. M., Heilshorn, S. C. 2017; 15: 15–23

  Abstract

  Stem cell delivery by local injection has tremendous potential as a regenerative therapy but has seen limited clinical success. Several mechanical challenges hinder therapeutic efficacy throughout all stages of cell transplantation, including mechanical forces during injection and loss of mechanical support post-injection. Recent studies have begun exploring the use of biomaterials, in particular hydrogels, to enhance stem cell transplantation by addressing the often-conflicting mechanical requirements associated with each stage of the transplantation process. This review explores recent biomaterial approaches to improve the therapeutic efficacy of stem cells delivered through local injection, with a focus on strategies that specifically address the mechanical challenges that result in cell death and/or limit therapeutic function throughout the stages of transplantation.

  View details for PubMedID 29085771

  View details for PubMedCentralID PMC5659597

• Immobilization of growth factors to collagen surfaces using visible light. Biomacromolecules Fernandes Cunha, G. M., Lee, H. J., Kumar, A. n., Kreymerman, A. n., Heilshorn, S. C., Myung, D. n. 2017

  Abstract

  In the treatment of traumatic injuries, burns, and ulcers of the eye, inadequate epithelial tissue healing remains a major challenge. Wound healing is a complex process involving the temporal and spatial interplay between cells and their extracellular milieu. It can be impaired by a variety of causes including infection, poor circulation, loss of critical cells and/or proteins, and a deficiency in normal neural signaling (e.g. neurotrophic ulcers). Ocular anatomy is particularly vulnerable to lasting morbidity from delayed healing, whether it be scarring or perforation of the cornea, destruction of the conjunctival mucous membrane, or cicatricial changes to the eyelids and surrounding skin. Therefore, there is a major clinical need for new modalities for controlling and accelerating wound healing, particularly in the eye. Collagen matrices have long been explored as scaffolds to support cell growth as both two-dimensional coatings and substrates, as well as three-dimensional matrices. Meanwhile, the immobilization of growth factors to various substrates has also been extensively studied as a way to promote enhanced cellular adhesion and proliferation. Herein we present a new strategy for photochemically immobilizing growth factors to collagen using riboflavin as a photosensitizer and exposure to visible light (~458 nm). epidermal growth factor (EGF) was successfully bound to collagen-coated surfaces as well as directly to endogenous collagen from porcine corneas. The initial concentration of riboflavin and EGF, as well as the blue light exposure time, were keys to the successful binding of growth factor to these surfaces. The photocrosslinking reaction increased EGF residence time on collagen surfaces over seven days. EGF activity was maintained after the photocrosslinking reaction with a short duration of pulsed blue light exposure time. Bound EGF accelerated in vitro corneal epithelial cell proliferation and migration and maintained normal cell phenotype. Additionally, the treated surfaces were cytocompatible, and the photocrosslinking reaction was proven to be safe, preserving nearly 100% cell viability. These results suggest that this general approach is safe and versatile may be used for targeting and immobilizing bioactive factors onto collagen matrices in a variety of applications, including in the presence of live, seeded cells or in vivo onto endogenous extracellular matrix collagen.

  View details for PubMedID 28799757

• Quantitative criteria to benchmark new and existing bio-inks for cell compatibility. Biofabrication Dubbin, K. n., Tabet, A. n., Heilshorn, S. C. 2017; 9 (4): 044102

  Abstract

  Recent advancements in 3D bioprinting have led to the fabrication of more complex, more precise, and larger printed tissue constructs. As the field continues to advance, it is critical to develop quantitative benchmarks to compare different bio-inks for key cell-biomaterial interactions, including (1) cell sedimentation within the ink cartridge, (2) cell viability during extrusion, and (3) cell viability after ink curing. Here we develop three simple protocols for quantitative analysis of bio-ink performance. These methods are used to benchmark the performance of two commonly used bio-inks, poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), against three formulations of a novel bio-ink, Recombinant-protein Alginate Platform for Injectable Dual-crosslinked ink (RAPID ink). RAPID inks undergo peptide-self-assembly to form weak, shear-thinning gels in the ink cartridge and undergo electrostatic crosslinking with divalent cations during curing. In the one hour cell sedimentation assay, GelMA, the RAPID inks, and PEGDA with xanthan gum prevented appreciable cell sedimentation, while PEGDA alone or PEGDA with alginate experienced significant cell settling. To quantify cell viability during printing, 3T3 fibroblasts were printed at a constant flow rate of 75 μl min-1and immediately tested for cell membrane integrity. Less than 10% of cells were damaged using the PEGDA and GelMA bio-inks, while less than 4% of cells were damaged using the RAPID inks. Finally, to evaluate cell viability after curing, cells were exposed to ink-specific curing conditions for five minutes and tested for membrane integrity. After exposure to light with photoinitiator at ambient conditions, over 50% of cells near the edges of printed PEGDA and GelMA droplets were damaged. In contrast, fewer than 20% of cells found near the edges of RAPID inks were damaged after a 5 min exposure to curing in a 10 mM CaCl2solution. As new bio-inks continue to be developed, these protocols offer a convenient means to quantitatively benchmark their performance against existing inks.

  View details for DOI 10.1088/1758-5090/aa869f

  View details for PubMedID 28812982

  View details for PubMedCentralID PMC5811195

• Micro- and nano-patterned elastin-like polypeptide hydrogels for stem cell culture. Soft matter Paul, A. n., Stührenberg, M. n., Chen, S. n., Rhee, D. n., Lee, W. K., Odom, T. W., Heilshorn, S. C., Enejder, A. n. 2017; 13 (34): 5665–75

  Abstract

  We show that submicron-sized patterns can be imprinted into soft, recombinant-engineered protein hydrogels (here elastin-like proteins, ELP) by transferring wavy patterns from polydimethylsiloxane (PDMS) molds. The high-precision topographical tunability of the relatively stiff PDMS is translated to a bio-responsive, soft material, enabling topographical cell response studies at elastic moduli matching those of tissues. Aligned and unaligned wavy patterns with mold periodicities of 0.24-4.54 μm were imprinted and characterized by coherent anti-Stokes Raman scattering and atomic force microscopy. The pattern was successfully transferred down to 0.37 μm periodicity (width in ELP: 250 ± 50 nm, height: 70 ± 40 nm). The limit was set by inherent protein assemblies (diameter: 124-180 nm) that formed due to lower critical solution temperature behavior of the ELP during molding. The width/height of the ELP ridges depended on the degree of hydration; from complete dehydration to full hydration, ELP ridge width ranged from 79 ± 9% to 150 ± 40% of the mold width. The surface of the ridged ELP featured densely packed protein aggregates that were larger in size than those observed in bulk/flat ELP. Adipose-derived stem cells (ADSCs) oriented along hydrated aligned patterns with periodicities ≥0.60 μm (height ≥170 ± 100 nm), while random orientation was observed for smaller distances/amplitudes, as well as flat and unaligned wavy ELP surfaces. Hence, micro-molding of ELP is a promising approach to create tissue-mimicking, hierarchical architectures composed of tunable micron-sized structures with nano-sized protein aggregates, which opens the way for orthogonal screening of cell responses to topography and cell-adhesion ligands at relevant elastic moduli.

  View details for PubMedID 28737182

• Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling. Nature materials Madl, C. M., LeSavage, B. L., Dewi, R. E., Dinh, C. B., Stowers, R. S., Khariton, M. n., Lampe, K. J., Nguyen, D. n., Chaudhuri, O. n., Enejder, A. n., Heilshorn, S. C. 2017; 16 (12): 1233–42

  Abstract

  Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

  View details for PubMedID 29115291

• Regulating Stem Cell Secretome Using Injectable Hydrogels with In Situ Network Formation. Advanced healthcare materials Cai, L., Dewi, R. E., Goldstone, A. B., Cohen, J. E., Steele, A. N., Woo, Y. J., Heilshorn, S. C. 2016

  Abstract

  A family of shear-thinning hydrogels for injectable encapsulation and long-term delivery (SHIELD) has been designed and synthesized with controlled in situ stiffening properties to regulate the stem cell secretome. The authors demonstrate that SHIELD with an intermediate stiffness (200-400 Pa) could significantly promote the angiogenic potential of human adipose-derived stem cells.

  View details for DOI 10.1002/adhm.201600497

  View details for PubMedID 27709809

• Dual-Stage Crosslinking of a Gel-Phase Bioink Improves Cell Viability and Homogeneity for 3D Bioprinting. Advanced healthcare materials Dubbin, K., Hori, Y., Lewis, K. K., Heilshorn, S. C. 2016; 5 (19): 2488-2492

  Abstract

  Current bioinks for cell-based 3D bioprinting are not suitable for technology scale-up due to the challenges of cell sedimentation, cell membrane damage, and cell dehydration. A novel bioink hydrogel is presented with dual-stage crosslinking specifically designed to overcome these three major hurdles. This bioink enables the direct patterning of highly viable, multicell type constructs with long-term spatial fidelity.

  View details for DOI 10.1002/adhm.201600636

  View details for PubMedID 27581767

• Probing the Metabolomics of Stem Cell Differentiation with Biomaterials CHEM Madl, C. M., Heilshorn, S. C. 2016; 1 (2): 192-+

  View details for DOI 10.1016/j.chempr.2016.07.016

  View details for Web of Science ID 000387029500010

• Integrating concepts of material mechanics, ligand chemistry, dimensionality and degradation to control differentiation of mesenchymal stem cells CURRENT OPINION IN SOLID STATE & MATERIALS SCIENCE Haugh, M. G., Heilshorn, S. C. 2016; 20 (4): 171-179

  Abstract

  The role of substrate mechanics in guiding mesenchymal stem cell (MSC) fate has been the focus of much research over the last decade. More recently, the complex interplay between substrate mechanics and other material properties such as ligand chemistry and substrate degradability to regulate MSC differentiation has begun to be elucidated. Additionally, there are several changes in the presentation of these material properties as the dimensionality is altered from two- to three-dimensional substrates, which may fundamentally alter our understanding of substrate-induced mechanotransduction processes. In this review, an overview of recent findings that highlight the material properties that are important in guiding MSC fate decisions is presented, with a focus on underlining gaps in our existing knowledge and proposing potential directions for future research.

  View details for DOI 10.1016/j.cossms.2016.04.001

  View details for Web of Science ID 000382408500001

  View details for PubMedCentralID PMC5404745

• Integrating Concepts of Material Mechanics, Ligand Chemistry, Dimensionality and Degradation to Control Differentiation of Mesenchymal Stem Cells. Current opinion in solid state & materials science Haugh, M. G., Heilshorn, S. C. 2016; 20 (4): 171-179

  Abstract

  The role of substrate mechanics in guiding mesenchymal stem cell (MSC) fate has been the focus of much research over the last decade. More recently, the complex interplay between substrate mechanics and other material properties such as ligand chemistry and substrate degradability to regulate MSC differentiation has begun to be elucidated. Additionally, there are several changes in the presentation of these material properties as the dimensionality is altered from two- to three-dimensional substrates, which may fundamentally alter our understanding of substrate-induced mechanotransduction processes. In this review, an overview of recent findings that highlight the material properties that are important in guiding MSC fate decisions is presented, with a focus on underlining gaps in our existing knowledge and proposing potential directions for future research.

  View details for DOI 10.1016/j.cossms.2016.04.001

  View details for PubMedID 28458610

  View details for PubMedCentralID PMC5404745

• An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy. Nature biotechnology Quarta, M., Brett, J. O., DiMarco, R., de Morree, A., Boutet, S. C., Chacon, R., Gibbons, M. C., Garcia, V. A., Su, J., Shrager, J. B., Heilshorn, S., Rando, T. A. 2016; 34 (7): 752-759

  Abstract

  A promising therapeutic strategy for diverse genetic disorders involves transplantation of autologous stem cells that have been genetically corrected ex vivo. A major challenge in such approaches is a loss of stem cell potency during culture. Here we describe an artificial niche for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state. Using a machine learning method, we identified a molecular signature of quiescence and used it to screen for factors that could maintain mouse MuSC quiescence, thus defining a quiescence medium (QM). We also engineered muscle fibers that mimic the native myofiber of the MuSC niche. Mouse MuSCs maintained in QM on engineered fibers showed enhanced potential for engraftment, tissue regeneration and self-renewal after transplantation in mice. An artificial niche adapted to human cells similarly extended the quiescence of human MuSCs in vitro and enhanced their potency in vivo. Our approach for maintaining quiescence may be applicable to stem cells isolated from other tissues.

  View details for DOI 10.1038/nbt.3576

  View details for PubMedID 27240197

• Bio-Orthogonally Crosslinked, Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation ADVANCED FUNCTIONAL MATERIALS Madl, C. M., Katz, L. M., Heilshorn, S. C. 2016; 26 (21): 3612-3620

  Abstract

  Covalently-crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross-react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio-orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin-like proteins (ELPs) are developed that cross-link via strain-promoted azide-alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC-crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC-ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin-derived arginine-glycine-aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.

  View details for DOI 10.1002/adfm.201505329

  View details for Web of Science ID 000377597400006

  View details for PubMedCentralID PMC5019573

• Bio-Orthogonally Crosslinked, Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation. Advanced functional materials Madl, C. M., Katz, L. M., Heilshorn, S. C. 2016; 26 (21): 3612-3620

  Abstract

  Covalently-crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross-react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio-orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin-like proteins (ELPs) are developed that cross-link via strain-promoted azide-alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC-crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC-ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin-derived arginine-glycine-aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.

  View details for DOI 10.1002/adfm.201505329

  View details for PubMedID 27642274

  View details for PubMedCentralID PMC5019573

• Multifunctional coatings to simultaneously promote osseointegration and prevent infection of orthopaedic implants. Biomaterials Raphel, J., Holodniy, M., Goodman, S. B., Heilshorn, S. C. 2016; 84: 301-314

  Abstract

  The two leading causes of failure for joint arthroplasty prostheses are aseptic loosening and periprosthetic joint infection. With the number of primary and revision joint replacement surgeries on the rise, strategies to mitigate these failure modes have become increasingly important. Much of the recent work in this field has focused on the design of coatings either to prevent infection while ignoring bone mineralization or vice versa, to promote osseointegration while ignoring microbial susceptibility. However, both coating functions are required to achieve long-term success of the implant; therefore, these two modalities must be evaluated in parallel during the development of new orthopaedic coating strategies. In this review, we discuss recent progress and future directions for the design of multifunctional orthopaedic coatings that can inhibit microbial cells while still promoting osseointegration.

  View details for DOI 10.1016/j.biomaterials.2016.01.016

  View details for PubMedID 26851394

• A Comparative Study of Collagen Matrix Density Effect on Endothelial Sprout Formation Using Experimental and Computational Approaches ANNALS OF BIOMEDICAL ENGINEERING Shamloo, A., Mohammadaliha, N., Heilshorn, S. C., Bauer, A. L. 2016; 44 (4): 929-941

  Abstract

  A thorough understanding of determining factors in angiogenesis is a necessary step to control the development of new blood vessels. Extracellular matrix density is known to have a significant influence on cellular behaviors and consequently can regulate vessel formation. The utilization of experimental platforms in combination with numerical models can be a powerful method to explore the mechanisms of new capillary sprout formation. In this study, using an integrative method, the interplay between the matrix density and angiogenesis was investigated. Owing the fact that the extracellular matrix density is a global parameter that can affect other parameters such as pore size, stiffness, cell-matrix adhesion and cross-linking, deeper understanding of the most important biomechanical or biochemical properties of the ECM causing changes in sprout morphogenesis is crucial. Here, we implemented both computational and experimental methods to analyze the mechanisms responsible for the influence of ECM density on the sprout formation that is difficult to be investigated comprehensively using each of these single methods. For this purpose, we first utilized an innovative approach to quantify the correspondence of the simulated collagen fibril density to the collagen density in the experimental part. Comparing the results of the experimental study and computational model led to some considerable achievements. First, we verified the results of the computational model using the experimental results. Then, we reported parameters such as the ratio of proliferating cells to migrating cells that was difficult to obtain from experimental study. Finally, this integrative system led to gain an understanding of the possible mechanisms responsible for the effect of ECM density on angiogenesis. The results showed that stable and long sprouts were observed at an intermediate collagen matrix density of 1.2 and 1.9 mg/ml due to a balance between the number of migrating and proliferating cells. As a result of weaker connections between the cells and matrix, a lower collagen matrix density (0.7 mg/ml) led to unstable and broken sprouts. However, higher matrix density (2.7 mg/ml) suppressed sprout formation due to the high level of matrix entanglement, which inhibited cell migration. This study also showed that extracellular matrix density can influence sprout branching. Our experimental results support this finding.

  View details for DOI 10.1007/s10439-015-1416-2

  View details for Web of Science ID 000373741800009

  View details for PubMedID 26271521

• Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants. Biomaterials Raphel, J., Karlsson, J., Galli, S., Wennerberg, A., Lindsay, C., Haugh, M. G., Pajarinen, J., Goodman, S. B., Jimbo, R., Andersson, M., Heilshorn, S. C. 2016; 83: 269-282

  Abstract

  Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.

  View details for DOI 10.1016/j.biomaterials.2015.12.030

  View details for PubMedID 26790146

• Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials INTEGRATIVE BIOLOGY Benitez, P. L., Mascharak, S., Proctor, A. C., Heilshorn, S. C. 2016; 8 (1): 50-61

  Abstract

  While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell-matrix interface may serve as design principles when developing future generations of implantable biomaterials.

  View details for DOI 10.1039/c5ib00258c

  View details for Web of Science ID 000368348900006

  View details for PubMedCentralID PMC4771524

• Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials. Integrative biology : quantitative biosciences from nano to macro Benitez, P. L., Mascharak, S., Proctor, A. C., Heilshorn, S. C. 2016; 8 (1): 50-61

  Abstract

  While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell-matrix interface may serve as design principles when developing future generations of implantable biomaterials.

  View details for DOI 10.1039/c5ib00258c

  View details for PubMedID 26692238

  View details for PubMedCentralID PMC4771524

• Design of Injectable Materials to Improve Stem Cell Transplantation. Current stem cell reports Marquardt, L. M., Heilshorn, S. C. 2016; 2 (3): 207–20

  Abstract

  Stem cell-based therapies are steadily gaining traction for regenerative medicine approaches to treating disease and injury throughout the body. While a significant body of work has shown success in preclinical studies, results often fail to translate in clinical settings. One potential cause is the massive transplanted cell death that occurs post injection, preventing functional integration with host tissue. Therefore, current research is focusing on developing injectable hydrogel materials to protect cells during delivery and to stimulate endogenous regeneration through interactions of transplanted cells and host tissue. This review explores the design of targeted injectable hydrogel systems for improving the therapeutic potential of stem cells across a variety of tissue engineering applications with a focus on hydrogel materials that have progressed to the stage of preclinical testing.

  View details for PubMedID 28868235

  View details for PubMedCentralID PMC5576562

• Injectable, Covalently Adaptable Hydrogels with Secondary Thermoresponsive Reinforcement for Cartilage Engineering Wang, H., Zhu, D., Yang, F., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2015: S253

  View details for Web of Science ID 000360205202081

• Injectable Hydrogels with Double Network Formation to Promote Angiogenesis Cai, L., Heilshorn, S. MARY ANN LIEBERT, INC. 2015: S131

  View details for Web of Science ID 000360205201113

• Hybrid Elastin-like Polypeptide-Polyethylene Glycol Hydrogels for 3D Cell Culture with Tunable Matrix Stiffness and Cell Ligand Density Wang, H., Cai, L., Paul, A., Enejder, A., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2015: S308–S309

  View details for Web of Science ID 000360205202298

• Injectable Hydrogels to Deliver and Engraft Schwann Cells in Spinal Cord Lesions Dubbin, K., Cai, L., Madl, C., Marquardt, L., Plant, G., Heilshorn, S. MARY ANN LIEBERT, INC. 2015: S81

  View details for Web of Science ID 000360205200319

• Design of Self-assembling Bio-inks for Cell-based 3d Printing Dubbin, K., Hori, Y., Pimienta, R., Heilshorn, S. MARY ANN LIEBERT, INC. 2015: S41-S42

  View details for Web of Science ID 000360205200158

• Matrix Remodeling Regulates Neural Progenitor Cell Phenotype in Engineered Elastin-Like Protein Hydrogels Madl, C. M., Lampe, K. J., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2015: S150

  View details for Web of Science ID 000360205201187

• An Experimental and Computational Investigation of 3D Matrix Mechanics in Directing Stem Cell Lineage Haugh, M. G., Vaughan, T. J., McNamara, L. M., O'Brien, F. J., Heilshorn, S. C. MARY ANN LIEBERT, INC. 2015: S300

  View details for Web of Science ID 000360205202264

• Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering ADVANCED MATERIALS Wang, H., Heilshorn, S. C. 2015; 27 (25): 3717-3736

  Abstract

  Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-formation of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, an overview of adaptable-hydrogel design considerations and linkage selections is presented, with a focus on various cell-compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering.

  View details for DOI 10.1002/adma.201501558

  View details for PubMedID 25989348

• Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding NEURAL REGENERATION RESEARCH Madl, C. M., Heilshorn, S. C. 2015; 10 (4): 514-517

  Abstract

  Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding; however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration.

  View details for DOI 10.4103/1673-5374.155426

  View details for Web of Science ID 000354156200002

  View details for PubMedID 26170800

  View details for PubMedCentralID PMC4424732

• Injectable Hydrogels with In Situ Double Network Formation Enhance Retention of Transplanted Stem Cells ADVANCED FUNCTIONAL MATERIALS Cai, L., Dewi, R. E., Heilshorn, S. C. 2015; 25 (9): 1344-1351

  View details for DOI 10.1002/adfm.201403631

  View details for Web of Science ID 000350541700003

  View details for PubMedCentralID PMC4529129

• Injectable Hydrogels with In Situ Double Network Formation Enhance Retention of Transplanted Stem Cells. Advanced functional materials Cai, L., Dewi, R. E., Heilshorn, S. C. 2015; 25 (9): 1344-1351

  View details for DOI 10.1002/adfm.201403631

  View details for PubMedID 26273242

  View details for PubMedCentralID PMC4529129

• Protein-engineered hydrogel encapsulation for 3-d culture of murine cochlea. Otology & neurotology Chang, D. T., Chai, R., DiMarco, R., Heilshorn, S. C., Cheng, A. G. 2015; 36 (3): 531-538

  Abstract

  Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture.Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose.A protein-engineered ELP hydrogel was used to encapsulate organ of Corti isolated from neonatal mice. Cultured cochleae were immunostained for markers of hair cells and supporting cells. Organ of Corti hair cell and supporting cell density and organ dimensions were compared between the ELP and nonencapsulated systems. These culture systems were then compared with noncultured cochlea.After 3 days in vitro, vital dye uptake and immunostaining for sensory and nonsensory cells show that encapsulated cochlea contain viable cells with an organized architecture. In comparison with nonencapsulated cultured cochlea, ELP-encapsulated cochleae exhibit higher densities of hair cells and supporting cells and taller and narrower organ of Corti dimensions that more closely resemble those of noncultured cochleae. However, we found compromised cell viability when the culture period extended beyond 3 days.We conclude that the ELP hydrogel can help preserve the 3-D architecture of neonatal cochlea in short-term culture, which may be applicable to in vitro study of the physiology and pathophysiology of the inner ear.

  View details for DOI 10.1097/MAO.0000000000000518

  View details for PubMedID 25111520

• Microfluidic Gradients Reveal Enhanced Neurite Outgrowth but Impaired Guidance within 3D Matrices with High Integrin Ligand Densities SMALL Romano, N. H., Lampe, K. J., Xu, H., Ferreira, M. M., Heilshorn, S. C. 2015; 11 (6): 722-730

  Abstract

  The density of integrin-binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell-adhesive ligands on neurite chemoattraction is not well understood. A platform is presented here that combines gradient-generating microfluidic devices with 3D protein-engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia-derived spheroids. Spheroids are encapsulated in elastin-like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade-off between neurite extension and neurite guidance is reminiscent of the well-known trade-off between adhesive forces at the leading and trailing edges of a migrating cell, implying that a similar matrix-mediated balance of forces regulates neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels.

  View details for DOI 10.1002/smll.201401574

  View details for Web of Science ID 000349977500010

  View details for PubMedID 25315156

• Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth. Acta biomaterialia Romano, N. H., Madl, C. M., Heilshorn, S. C. 2015; 11: 48-57

  Abstract

  The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell-matrix and Schwann cell-neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell-matrix and cell-cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell-matrix interactions by presenting integrin-binding RGD (Arg-Gly-Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell-neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell-neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell-matrix and cell-cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.

  View details for DOI 10.1016/j.actbio.2014.10.008

  View details for PubMedID 25308870

  View details for PubMedCentralID PMC4528982

• Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids BIOMATERIALS SCIENCE Dimarco, R. L., Dewi, R. E., Bernal, G., Kuoc, C., Heilshorn, S. C. 2015; 3 (10): 1376-1385

  Abstract

  Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  View details for DOI 10.1039/c5bm00108k

  View details for Web of Science ID 000361194900004

  View details for PubMedID 26371971

• Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis INTEGRATIVE BIOLOGY Ferreira, M. M., Dewi, R. E., Heilshorn, S. C. 2015; 7 (5): 569-579

  Abstract

  Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (10(0)-10(3) minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 10(2) minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm h(-1)) than either fibronectin (10.6 μm h(-1)) or collagen (7.6 μm h(-1)) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm h(-1) across all ECMs tested. We observed relatively mild chemoattraction (∼67% of myoblast population) in response to bFGF gradients of 3.2 ng mL(-1) mm(-1) regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.

  View details for DOI 10.1039/c5ib00060b

  View details for Web of Science ID 000354362000008

  View details for PubMedID 25909157

  View details for PubMedCentralID PMC4528978

• Multi-Site Functionalization of Protein Scaffolds for Bimetallic Nanoparticle Templating ADVANCED FUNCTIONAL MATERIALS Huggins, K. N., Schoen, A. P., Arunagirinathan, M. A., Heilshorn, S. C. 2014; 24 (48): 7737-7744

  View details for DOI 10.1002/adfm.201402049

  View details for Web of Science ID 000346498900020

• Dual-stage growth factor release within 3D protein-engineered hydrogel niches promotes adipogenesis. Biomaterials science Greenwood-Goodwin, M., Teasley, E. S., Heilshorn, S. C. 2014; 2 (11): 1627-1639

  Abstract

  Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a Mixing-Induced Two-Component Hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes.

  View details for DOI 10.1039/C4BM00142G

  View details for PubMedID 25309741

  View details for PubMedCentralID PMC4188404

• Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors. Journal of controlled release Mulyasasmita, W., Cai, L., Dewi, R. E., Jha, A., Ullmann, S. D., Luong, R. H., Huang, N. F., Heilshorn, S. C. 2014; 191: 71-81

  Abstract

  To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein-polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol-gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C-P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.

  View details for DOI 10.1016/j.jconrel.2014.05.015

  View details for PubMedID 24848744

• Hybrid elastin-like polypeptide-polyethylene glycol (ELP-PEG) hydrogels with improved transparency and independent control of matrix mechanics and cell ligand density. Biomacromolecules Wang, H., Cai, L., Paul, A., Enejder, A., Heilshorn, S. C. 2014; 15 (9): 3421-3428

  Abstract

  Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP's lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell-matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.

  View details for DOI 10.1021/bm500969d

  View details for PubMedID 25111283

• Hybrid Elastin-like Polypeptide-Polyethylene Glycol (ELP-PEG) Hydrogels with Improved Transparency and Independent Control of Matrix Mechanics and Cell Ligand Density BIOMACROMOLECULES Wang, H., Cai, L., Paul, A., Enejder, A., Heilshorn, S. C. 2014; 15 (9): 3421-3428

  View details for DOI 10.1021/bm500969d

  View details for Web of Science ID 000341409800024

• The Optimal Fat Graft: Evaluating the Biomechanical Effects of Diameter, Length, and Flow Rate during Fat Placement Atashroo, D., Wearda, T., Raphel, J., Paik, K., Zielins, E. R., Walmsley, G. G., Tevlin, R., Wan, D. C., Heilshorn, S., Longaker, M. T. ELSEVIER SCIENCE INC. 2014: S90

  View details for DOI 10.1016/j.jamcollsurg.2014.07.215

  View details for Web of Science ID 000342420900186

• Injectable hydrogels with in situ double network formation for cell transplantation Cai, L., Heilshorn, S. AMER CHEMICAL SOC. 2014

  View details for Web of Science ID 000349167404541

• Self-assembled protein cages as nanoreactors for inorganic nanomaterials synthesis Heilshorn, S. AMER CHEMICAL SOC. 2014

  View details for Web of Science ID 000349165105623

• Avidity-controlled delivery of angiogenic peptides from injectable molecular-recognition hydrogels. Tissue engineering. Part A Mulyasasmita, W., Cai, L., Hori, Y., Heilshorn, S. C. 2014; 20 (15-16): 2102-2114

  Abstract

  Peptide mimics of growth factors represent an emerging class of therapeutic drugs due to high biological specificity and relative ease of synthesis. However, maintaining efficacious therapeutic dosage at the therapy site has proven challenging owing to poor intestinal permeability and short circulating half-lives in the blood stream. In this work, we present the affinity immobilization and controlled release of QK, a vascular endothelial growth factor (VEGF) mimetic peptide, from an injectable mixing-induced two-component hydrogel (MITCH). The MITCH system is crosslinked by reversible interactions between WW domains and complementary proline-rich peptide modules. Fusion of the QK peptide to either one or two units of the proline-rich sequence creates bifunctional peptide conjugates capable of specific binding to MITCH while preserving their angiogenic bioactivity. Presenting two repeats of the proline-rich sequence increases the binding enthalpy 2.5 times due to avidity effects. Mixing of the drug conjugates with MITCH components results in drug encapsulation and extended release at rates consistent with the affinity immobilization strength. Human umbilical vein endothelial cells (HUVECs) treated with the soluble drug conjugates exhibit morphogenetic events of VEGF receptor 2 signal transduction followed by cell migration and organization into networks characteristic of early angiogenesis. In a three-dimensional model where HUVECs were cultured as spheroids in a matrix of collagen and fibronectin, injection of drug-releasing MITCH resulted in significantly more cell outgrowth than drugs injected in saline. This ability to sustain local drug availability is ideal for therapeutic angiogenesis applications, where spatiotemporal control over drug distribution is a key requirement for clinical success.

  View details for DOI 10.1089/ten.tea.2013.0357

  View details for PubMedID 24490588

  View details for PubMedCentralID PMC4137330

• Rheology and simulation of 2-dimensional clathrin protein network assembly. Soft matter VanDersarl, J. J., Mehraeen, S., Schoen, A. P., Heilshorn, S. C., Spakowitz, A. J., Melosh, N. A. 2014; 10 (33): 6219-6227

  Abstract

  Clathrin is a three-legged protein complex that assembles into lattice structures on the cell membrane and transforms into fullerene-like cages during endocytosis. This dynamic structural flexibility makes clathrin an attractive building block for guided assembly. The assembly dynamics and the mechanical properties of clathrin protein lattices are studied using rheological measurements and theoretical modelling in an effort to better understand two dynamic processes: protein adsorption to the interface and assembly into a network. We find that percolation models for protein network formation are insufficient to describe clathrin network formation, but with Monte Carlo simulations we can describe the dynamics of network formation very well. Insights from this work can be used to design new bio-inspired nano-assembly systems.

  View details for DOI 10.1039/c4sm00025k

  View details for PubMedID 25012232

• Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator. Lab on a chip Xu, H., Ferreira, M. M., Heilshorn, S. C. 2014; 14 (12): 2047-2056

  Abstract

  A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron-friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 h exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.

  View details for DOI 10.1039/c4lc00162a

  View details for PubMedID 24781157

• One-pot Synthesis of Elastin-like Polypeptide Hydrogels with Grafted VEGF-Mimetic Peptides. Biomaterials science Cai, L., Dinh, C. B., Heilshorn, S. C. 2014; 2 (5): 757-765

  Abstract

  Immobilization of growth factors to polymeric matrices has been a common strategy in the design of tissue engineering scaffolds to promote tissue regeneration, which requires complex cell signaling events with the surrounding matrix. However, the use of large protein growth factors in polymeric scaffolds is often plagued by immunogenicity, short in vivo half-lives, and reduced bioactivity. To address these concerns, we develop a single-step, cell-compatible strategy to tether small, growth-factor-mimetic peptides into a protein-engineered hydrogel with tunable biomaterial properties. Specifically, we covalently immobilize the QK peptide, an angiogenic peptide mimicking the receptor-binding region of vascular endothelial growth factor (VEGF), within tunable elastin-like polypeptide (ELP) hydrogels that include a cell-adhesive RGD sequence. Using a cell-compatible, amine-reactive crosslinker, we conducted a one-pot synthesis to simultaneously encapsulate cells while precisely controlling the QK grafting density (10 nM - 100 μM) in the ELP hydrogels without altering other material properties. Fluorescence analysis of fluor-labeled QK peptides demonstrated that the conjugation efficiency to ELP hydrogels was >75% and that covalent immobilization effectively eliminates all QK diffusion. Compared with pristine ELP hydrogels, human umbilical vein endothelial cell (HUVEC) proliferation was significantly enhanced on ELP hydrogels immobilized with 10 nM or 1 μM QK. Moreover, upon encapsulation within tethered QK-ELP hydrogels, HUVEC spheroids maintained near 100% viability and demonstrated significantly more three-dimensional outgrowth compared to those supplemented with soluble QK peptide at the same concentration. These results encourage the further development of protein-engineered scaffolds decorated with growth-factor-mimetic peptides to provide long-term biological signals using this versatile, single-step synthesis.

  View details for DOI 10.1039/C3BM60293A

  View details for PubMedID 24729868

  View details for PubMedCentralID PMC3979545

• Designing ECM-mimetic materials using protein engineering ACTA BIOMATERIALIA Cai, L., Heilshorn, S. C. 2014; 10 (4): 1751-1760

  Abstract

  The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsive properties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy to create ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds. This modular design strategy involves combining multiple protein domains with different functionalities into a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunability of the individual domain functions. These eECMs often enable decoupled control over multiple material properties for fundamental studies of cell-matrix interactions. In addition, since the eECMs are frequently composed entirely of bioresorbable amino acids, these matrices have immense clinical potential for a variety of regenerative medicine applications. This brief review demonstrates how fundamental knowledge gained from structure-function studies of native proteins can be exploited in the design of novel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existed for over 20years, the community is only now beginning to fully explore the diversity of functional peptide modules that can be incorporated into these materials. We have chosen to highlight recent examples that either (i) demonstrate exemplary use as matrices with cell-instructive and cell-responsive properties or (ii) demonstrate outstanding creativity in terms of novel molecular-level design and macro-level functionality.

  View details for DOI 10.1016/j.actbio.2013.12.028

  View details for Web of Science ID 000334137700025

  View details for PubMedID 24365704

  View details for PubMedCentralID PMC3973542

• Injectable materials for co-delivery of stem cells and nanoparticles Heilshorn, S. C. AMER CHEMICAL SOC. 2014

  View details for Web of Science ID 000348457605196

• Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids. Integrative biology Dimarco, R. L., Su, J., Yan, K. S., Dewi, R., Kuo, C. J., Heilshorn, S. C. 2014; 6 (2): 127-142

  Abstract

  Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport. The culture microenvironment was further tailored through variation of collagen type-I matrix density, addition of exogenous R-spondin1, and specification of culture geometry. "Air-liquid interface" cultures resulted in significantly higher numbers of contractile cultures relative to traditional submerged cultures. These interface cultures were confirmed to have enhanced and more symmetric oxygen transport relative to traditional submerged cultures. While oxygen availability was found to impact in vitro contraction rate and the orientation of contractile movement, it was not a key factor in enabling contractility. For all conditions tested, reproducible contractile behavior only occurred within a consistent and narrow range of collagen type-I matrix densities with porosities of approximately 20% and storage moduli near 30 Pa. This suggests that matrix density acts as a "permissive switch" that enables contractions to occur. Similarly, contractions were only observed in cultures with diameters less than 15.5 mm that had relatively large interfacial surface area between the compliant matrix and the rigid culture dish. Taken together, these data suggest that spatial geometry and mechanics of the microenvironment, which includes both the encapsulating matrix as well as the surrounding culture device, may be key determinants of intestinal organoid functionality. As peristaltic contractility is a crucial requirement for normal digestive tract function, this achievement of reproducible organoid contraction marks a pivotal advancement towards engineering physiologically functional replacement tissue constructs.

  View details for DOI 10.1039/c3ib40188j

  View details for PubMedID 24343706

• Presentation of BMP-2 Mimicking Peptides in 3D Hydrogels Directs Cell Fate Commitment in Osteoblasts and Mesenchymal Stem Cells BIOMACROMOLECULES Madl, C. M., Mehta, M., Duda, G. N., Heilshorn, S. C., Mooney, D. J. 2014; 15 (2): 445-455

  Abstract

  Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.

  View details for DOI 10.1021/bm401726u

  View details for Web of Science ID 000331342200001

  View details for PubMedID 24400664

  View details for PubMedCentralID PMC3930060

• A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration INTEGRATIVE BIOLOGY McLaughlin, L. M., Xu, H., Carden, S. E., Fisher, S., Reyes, M., Heilshorn, S. C., Monack, D. M. 2014; 6 (4): 438-449

  Abstract

  Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration.

  View details for DOI 10.1039/c3ib40177d

  View details for Web of Science ID 000333331800007

  View details for PubMedID 24599496

• Recombinant Protein Hydrogels for Cell Injection and Transplantation HYDROGELS IN CELL-BASED THERAPIES Benitez, P. L., Heilshorn, S. C., Connon, C. J., Hamley, I. W. 2014; 2: 48-72

  View details for Web of Science ID 000387103100003

• Biological biomaterials structure, function, property design across the molecular-nano-micro-macro scales. Acta biomaterialia Barker, T. H., Heilshorn, S. C. 2014; 10 (4): 1487

  View details for PubMedID 24530557

• Dual-stage growth factor release within 3D protein-engineered hydrogel niches promotes adipogenesis BIOMATERIALS SCIENCE Greenwood-Goodwin, M., Teasley, E. S., Heilshorn, S. C. 2014; 2 (11): 1627-1639

  Abstract

  Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a Mixing-Induced Two-Component Hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes.

  View details for DOI 10.1039/c4bm00142g

  View details for Web of Science ID 000343034200008

  View details for PubMedCentralID PMC4188404

• One-pot synthesis of elastin-like polypeptide hydrogels with grafted VEGF-mimetic peptides BIOMATERIALS SCIENCE Cai, L., Dinh, C. B., Heilshorn, S. C. 2014; 2 (5): 757-765

  Abstract

  Immobilization of growth factors to polymeric matrices has been a common strategy in the design of tissue engineering scaffolds to promote tissue regeneration, which requires complex cell signaling events with the surrounding matrix. However, the use of large protein growth factors in polymeric scaffolds is often plagued by immunogenicity, short in vivo half-lives, and reduced bioactivity. To address these concerns, we develop a single-step, cell-compatible strategy to tether small, growth-factor-mimetic peptides into a protein-engineered hydrogel with tunable biomaterial properties. Specifically, we covalently immobilize the QK peptide, an angiogenic peptide mimicking the receptor-binding region of vascular endothelial growth factor (VEGF), within tunable elastin-like polypeptide (ELP) hydrogels that include a cell-adhesive RGD sequence. Using a cell-compatible, amine-reactive crosslinker, we conducted a one-pot synthesis to simultaneously encapsulate cells while precisely controlling the QK grafting density (10 nM - 100 μM) in the ELP hydrogels without altering other material properties. Fluorescence analysis of fluor-labeled QK peptides demonstrated that the conjugation efficiency to ELP hydrogels was >75% and that covalent immobilization effectively eliminates all QK diffusion. Compared with pristine ELP hydrogels, human umbilical vein endothelial cell (HUVEC) proliferation was significantly enhanced on ELP hydrogels immobilized with 10 nM or 1 μM QK. Moreover, upon encapsulation within tethered QK-ELP hydrogels, HUVEC spheroids maintained near 100% viability and demonstrated significantly more three-dimensional outgrowth compared to those supplemented with soluble QK peptide at the same concentration. These results encourage the further development of protein-engineered scaffolds decorated with growth-factor-mimetic peptides to provide long-term biological signals using this versatile, single-step synthesis.

  View details for DOI 10.1039/c3bm60293a

  View details for Web of Science ID 000333579600017

  View details for PubMedCentralID PMC3979545

• Engineered clathrin nanoreactors provide tunable control over gold nanoparticle synthesis and clustering. Journal of materials chemistry. B Schoen, A. P., Huggins, K. N., Heilshorn, S. C. 2013; 1 (48): 6662-6669

  Abstract

  The use of biomolecules to direct nanomaterial synthesis has been an area of growing interest due to the complexity of structures that can be achieved in naturally occurring systems. We previously reported the functionalization of self-assembled clathrin protein cages to enable synthesis of nanoparticles from a range of inorganic materials. Here, we investigate the ability of this engineered biomolecule complex to act as a tunable nanoreactor for the formation of different arrangements of gold nanoparticles in three dimensions. We find that self-assembled clathrin cages functionalized with engineered bi-functional peptides induce formation of gold nanoparticles to generate solutions of either dispersed or clustered gold nanoparticles on demand. The 3D arrangement of nanoparticles is dependent on the concentration of the engineered peptide, which fulfills multiple roles in the synthesis process including stabilization of the nanoparticle surface and localization of the nanoparticles within the self-assembled clathrin cage. We propose and evaluate a mechanism that allows us to predict the peptide concentration at which the nanoreactor behavior switches. This work provides insight into peptide-based surfactants and the potential for incorporating them into strategies for tuning biological mineralization processes in mild solution conditions to generate complex structures.

  View details for DOI 10.1039/c3tb21145b

  View details for PubMedID 32261275

• Cellular fate in 3D elastin-like scaffolds is regulated by stiffness and integrin ligand density Heilshorn, S. C. AMER CHEMICAL SOC. 2013

  View details for Web of Science ID 000323851303720

• Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth ACTA BIOMATERIALIA Lampe, K. J., Antaris, A. L., Heilshorn, S. C. 2013; 9 (3): 5590-5599

  Abstract

  The design of bioactive materials allows tailored studies probing cell-biomaterial interactions, however, relatively few studies have examined the effects of ligand density and material stiffness on neurite growth in three-dimensions. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within three-dimensional (3-D) materials. To promote neurite out-growth and better understand the effects of common biomaterial design parameters on neuronal cultures we here focused on the cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins these 3-D ELP hydrogels enabled decoupled investigations into the effects of biomechanics and biochemistry on neurite out-growth from dorsal root ganglia. Increasing the cell-adhesive RGD ligand density from 0 to 1.9×10(7)ligands μm(-3) led to a significant increase in the rate, length, and density of neurite out-growth, as quantified by a high throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite out-growth was observed in materials with the higher ligand density at all time points up to 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest out-growth, with some neurites extending over 1800μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite out-growth within defined and tunable 3-D microenvironments these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron-biomaterial interactions.

  View details for DOI 10.1016/j.actbio.2012.10.033

  View details for Web of Science ID 000315536000019

  View details for PubMedID 23128159

• Protein-Engineered Injectable Hydrogel to Improve Retention of Transplanted Adipose-Derived Stem Cells ADVANCED HEALTHCARE MATERIALS Parisi-Amon, A., Mulyasasmita, W., Chung, C., Heilshorn, S. C. 2013; 2 (3): 428-432

  Abstract

  Improved retention of transplanted stem cells is achieved through minimally invasive delivery in MITCH, a mixing-induced two-component hydrogel that was engineered to possess shear-thinning and self-healing thixotropic properties. MITCH, an ideal injectable cell-delivery vehicle, supports 3D stem-cell culture, resulting in high cell viability and physiologically relevant cell morphology.

  View details for DOI 10.1002/adhm.201200293

  View details for Web of Science ID 000315899900004

  View details for PubMedID 23184882

• Microfluidic Investigation of BDNF-Enhanced Neural Stem Cell Chemotaxis in CXCL12 Gradients SMALL Xu, H., Heilshorn, S. C. 2013; 9 (4): 585-595

  Abstract

  In vivo studies have suggested that gradients of CXCL12 (aka stromal cell-derived factor 1α) may be critical for neural stem cell (NSC) migration during brain development and neural tissue regeneration. However, traditional in vitro chemotaxis tools are limited by unstable concentration gradients and the inability to decouple cell migration directionality and speed. These limitations have restricted the reproducible and quantitative analysis of neuronal migration, which is required for mechanism-based studies. Using a microfluidic gradient generator, nestin and Sox-2 positive human embryonic NSC chemotaxis is quantified within a linear and stable CXCL12 gradient. While untreated NSCs are not able to chemotax within CXCL12 gradients, pre-treatment of the cells with brain-derived neurotrophic factor (BDNF) results in significant chemotactic, directional migration. BDNF pre-treatment has no effect on cell migration speed, which averages about 1 μm min(-1). Quantitative analysis determines that CXCL12 concentrations above 9.0 nM are above the minimum activation threshold, while concentrations below 14.7 nM are below the saturation threshold. Interestingly, although inhibitor studies with AMD 3100 revealed that CXCL12 chemotaxis requires receptor CXCR4 activation, BDNF pre-treatment is found to have no profound effects on the mRNA levels or surface presentation of CXCR4 or the putative CXCR7 scavenger receptor. The microfluidic study of NSC migration within stable chemokine concentration profiles provides quantitative analysis as well as new insight into the migratory mechanism underlying BDNF-induced chemotaxis towards CXCL12.

  View details for DOI 10.1002/smll.201202208

  View details for Web of Science ID 000315103300013

  View details for PubMedID 23109183

• Sequence-Specific Crosslinking of Electrospun, Elastin-Like Protein Preserves Bioactivity and Native-Like Mechanics ADVANCED HEALTHCARE MATERIALS Benitez, P. L., Sweet, J. A., Fink, H., Chennazhi, K. P., Nair, S. V., Enejder, A., Heilshorn, S. C. 2013; 2 (1): 114-118

  View details for DOI 10.1002/adhm.201200115

  View details for Web of Science ID 000315121900009

  View details for PubMedID 23184558

  View details for PubMedCentralID PMC3641778

• Protein-Engineered Hydrogels BIOMATERIALS SURFACE SCIENCE Raphel, J., Parisi-Amon, A., Heilshorn, S. C., Taubert, A., Mano, J. F., RodriguezCabello, J. C. 2013: 207-237

  View details for Web of Science ID 000337029600008

• Chemotaxis of human induced pluripotent stem cell-derived endothelial cells AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH Huang, N. F., Dewi, R. E., Okogbaa, J., Lee, J. C., Jalilrufaihah, A., Heilshorn, S. C., Cooke, J. P. 2013; 5 (5): 510-U96

  Abstract

  This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1α (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.

  View details for Web of Science ID 000323539100004

  View details for PubMedID 23977410

  View details for PubMedCentralID PMC3745438

• Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density BIOMATERIALS SCIENCE Chung, C., Pruitt, B. L., Heilshorn, S. C. 2013; 1 (10): 1082-1090

  Abstract

  Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.

  View details for DOI 10.1039/c3bm60139k

  View details for Web of Science ID 000330137700009

  View details for PubMedCentralID PMC3987919

• Engineered clathrin nanoreactors provide tunable control over gold nanoparticle synthesis and clustering JOURNAL OF MATERIALS CHEMISTRY B Schoen, A. P., Huggins, K. N., Heilshorn, S. C. 2013; 1 (48): 6662-6669

  Abstract

  The use of biomolecules to direct nanomaterial synthesis has been an area of growing interest due to the complexity of structures that can be achieved in naturally occurring systems. We previously reported the functionalization of self-assembled clathrin protein cages to enable synthesis of nanoparticles from a range of inorganic materials. Here, we investigate the ability of this engineered biomolecule complex to act as a tunable nanoreactor for the formation of different arrangements of gold nanoparticles in three dimensions. We find that self-assembled clathrin cages functionalized with engineered bi-functional peptides induce formation of gold nanoparticles to generate solutions of either dispersed or clustered gold nanoparticles on demand. The 3D arrangement of nanoparticles is dependent on the concentration of the engineered peptide, which fulfills multiple roles in the synthesis process including stabilization of the nanoparticle surface and localization of the nanoparticles within the self-assembled clathrin cage. We propose and evaluate a mechanism that allows us to predict the peptide concentration at which the nanoreactor behavior switches. This work provides insight into peptide-based surfactants and the potential for incorporating them into strategies for tuning biological mineralization processes in mild solution conditions to generate complex structures.

  View details for DOI 10.1039/c3tb21145b

  View details for Web of Science ID 000327499100010

• Microfluidic devices for quantifying the role of soluble gradients in early angiogenesis Mechanical and Chemical Signaling in Angiogenesis Benitez, P., Heilshorn, S. C. edited by Reinhart-King, C. A. Heidelberg, Germany, Springer.. 2013: 1
• Spontaneous cardiomyocyte differentiation of mouse and embryoid bodies regulated by hydrogel crosslink density. Biomaterials Science Chung, C., Pruitt, B. L., Heilshorn, S. C. 2013; 10 (1): 1082-1090
• Dynamic remodelling of disordered protein aggregates is an alternative pathway to achieve robust self-assembly of nanostructures SOFT MATTER Schoen, A. P., Cordella, N., Mehraeen, S., Arunagirinathan, M. A., Spakowitz, A. J., Heilshorn, S. C. 2013; 9 (38): 9137-9145

  View details for DOI 10.1039/c3sm50830g

  View details for Web of Science ID 000324423700012

• Chemotaxis of human induced pluripotent stem cell-derived endothelial cells. American journal of translational research Huang, N. F., Dewi, R. E., Okogbaa, J., Lee, J. C., Jalilrufaihah, A., Heilshorn, S. C., Cooke, J. P. 2013; 5 (5): 510-520

  Abstract

  This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1α (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.

  View details for PubMedID 23977410

• Tuning colloidal association with specific peptide interactions SOFT MATTER Schoen, A. P., Hommersom, B., Heilshorn, S. C., Leunissen, M. E. 2013; 9 (29): 6781-6785

  View details for DOI 10.1039/c3sm50230a

  View details for Web of Science ID 000321273000023

• Complex chemoattractive and chemorepellent Kit signals revealed by direct imaging of murine mast cells in microfluidic gradient chambers INTEGRATIVE BIOLOGY Shamloo, A., Manchandia, M., Ferreira, M., Mani, M., Nguyen, C., Jahn, T., Weinberg, K., Heilshorn, S. 2013; 5 (8): 1076-1085

  Abstract

  Besides its cooperating effects on stem cell proliferation and survival, Kit ligand (KL) is a potent chemotactic protein. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization precludes dynamic chemotaxis studies. In response, we utilize microfluidic chambers that enable direct observation of murine bone marrow-derived mast cells (BMMC) within stable KL gradients. Using this system, individual Kit+ BMMC were quantitatively analyzed for migration speed and directionality during KL-induced chemotaxis. Our results indicated a minimum activating threshold of ∼3 ng ml(-1) for chemoattraction. Analysis of cells at KL concentrations below 3 ng ml(-1) revealed a paradoxical chemorepulsion, which has not been described previously. Unlike chemoattraction, which occurred continuously after an initial time lag, chemorepulsion occurred only during the first 90 minutes of observation. Both chemoattraction and chemorepulsion required the action of G-protein coupled receptors (GPCR), as treatment with pertussis toxin abrogated directed migration. These results differ from previous studies of GPCR-mediated chemotaxis, where chemorepulsion occurred at high ligand concentrations. These data indicate that Kit-mediated chemotaxis is more complex than previously understood, with the involvement of GPCRs in addition to the Kit receptor tyrosine kinase and the presence of both chemoattractive and chemorepellent phases.

  View details for DOI 10.1039/c3ib40025e

  View details for Web of Science ID 000322076800007

  View details for PubMedID 23835699

• Engineered Protein Templates Synthesize Inorganic Nanomaterials CHEMICAL ENGINEERING PROGRESS Schoen, A. P., Schoen, D. T., Huggins, K. N., Adhimoolam, A. M., Heilshorn, S. C. 2012; 108 (12): 47-50

  View details for Web of Science ID 000312283600020

• Tetrakis(hydroxymethyl) Phosphonium Chloride as a Covalent Cross-Linking Agent for Cell Encapsulation within Protein-Based Hydrogels BIOMACROMOLECULES Chung, C., Lampe, K. J., Heilshorn, S. C. 2012; 13 (12): 3912-3916

  Abstract

  Native tissues provide cells with complex, three-dimensional (3D) environments comprised of hydrated networks of extracellular matrix proteins and sugars. By mimicking the dimensionality of native tissue while deconstructing the effects of environmental parameters, protein-based hydrogels serve as attractive, in vitro platforms to investigate cell-matrix interactions. For cell encapsulation, the process of hydrogel formation through physical or covalent cross-linking must be mild and cell compatible. While many chemical cross-linkers are commercially available for hydrogel formation, only a subset are cytocompatible; therefore, the identification of new and reliable cytocompatible cross-linkers allows for greater flexibility of hydrogel design for cell encapsulation applications. Here, we introduce tetrakis(hydroxymethyl) phosphonium chloride (THPC) as an inexpensive, amine-reactive, aqueous cross-linker for 3D cell encapsulation in protein-based hydrogels. We characterize the THPC-amine reaction by demonstrating THPC's ability to react with primary and secondary amines of various amino acids. In addition, we demonstrate the utility of THPC to tune hydrogel gelation time (6.7±0.2 to 27±1.2 min) and mechanical properties (storage moduli ∼250 Pa to ∼2200 Pa) with a recombinant elastin-like protein. Lastly, we show cytocompatibility of THPC for cell encapsulation with two cell types, embryonic stem cells and neuronal cells, where cells exhibited the ability to differentiate and grow in elastin-like protein hydrogels. The primary goal of this communication is to report the identification and utility of tetrakis(hydroxymethyl) phosphonium chloride (THPC) as an inexpensive but widely applicable cross-linker for protein-based materials.

  View details for DOI 10.1021/bm3015279

  View details for Web of Science ID 000312035000004

  View details for PubMedID 23151175

  View details for PubMedCentralID PMC3556456

• Protein-Engineered Biomaterials to Generate Human Skeletal Muscle Mimics ADVANCED HEALTHCARE MATERIALS Sengupta, D., Gilbert, P. M., Johnson, K. J., Blau, H. M., Heilshorn, S. C. 2012; 1 (6): 785-789

  View details for DOI 10.1002/adhm.201200195

  View details for Web of Science ID 000315120500014

  View details for PubMedID 23184832

  View details for PubMedCentralID PMC3508759

• Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings. Journal of materials chemistry Raphel, J., Parisi-Amon, A., Heilshorn, S. 2012; 22 (37): 19429-19437

  Abstract

  Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

  View details for DOI 10.1039/C2JM31768K

  View details for PubMedID 23015764

  View details for PubMedCentralID PMC3449156

• Elastin-like matrices regulate embryonic stem cell-derived cardiomyocyte differentiation Chung, C., Pruitt, B. L., Heilshorn, S. C. AMER CHEMICAL SOC. 2012

  View details for Web of Science ID 000324621808073

• Multifunctional Materials through Modular Protein Engineering ADVANCED MATERIALS Dimarco, R. L., Heilshorn, S. C. 2012; 24 (29): 3923-3940

  Abstract

  The diversity of potential applications for protein-engineered materials has undergone profound recent expansion through a rapid increase in the library of domains that have been utilized in these materials. Historically, protein-engineered biomaterials have been generated from a handful of peptides that were selected and exploited for their naturally evolved functionalities. In recent years, the scope of the field has drastically expanded to include peptide domains that were designed through computational modeling, identified through high-throughput screening, or repurposed from wild type domains to perform functions distinct from their primary native applications. The strategy of exploiting a diverse library of peptide domains to design modular block copolymers enables the synthesis of multifunctional protein-engineered materials with a range of customizable properties and activities. As the diversity of peptide domains utilized in modular protein engineering continues to expand, a tremendous and ever-growing combinatorial expanse of material functionalities will result.

  View details for DOI 10.1002/adma.201200051

  View details for Web of Science ID 000307048200002

  View details for PubMedID 22730248

• Building stem cell niches from the molecule up through engineered peptide materials NEUROSCIENCE LETTERS Lampe, K. J., Heilshorn, S. C. 2012; 519 (2): 138-146

  Abstract

  The native stem cell niche is a dynamic and complex microenvironment. Recapitulating this niche is a critical focus within the fields of stem cell biology, tissue engineering, and regenerative medicine and requires the development of well-defined, tunable materials. Recent biomaterial design strategies seek to create engineered matrices that interact with cells at the molecular scale and allow on-demand, cell-triggered matrix modifications. Peptide and protein engineering can accomplish these goals through the molecular-level design of bioinductive and bioresponsive materials. This brief review focuses on engineered peptide and protein materials suitable for use as in vitro neural stem cell niche mimics and in vivo central nervous system repair. A key hallmark of these materials is the immense design freedom to specify the exact amino acid sequence leading to multi-functional bulk materials with tunable properties. These advanced materials are engineered using rational design strategies to recapitulate key aspects of the native neural stem cell niche. The resulting materials often combine the advantages of biological matrices with the engineering control of synthetic polymers. Future design strategies are expected to endow these materials with multiple layers of bi-directional feedback between the cell and the matrix, which will lead to more advanced mimics of the highly dynamic neural stem cell niche.

  View details for DOI 10.1016/j.neulet.2012.01.042

  View details for Web of Science ID 000306146800009

  View details for PubMedID 22322073

• Improving Viability of Stem Cells During Syringe Needle Flow Through the Design of Hydrogel Cell Carriers TISSUE ENGINEERING PART A Aguado, B. A., Mulyasasmita, W., Su, J., Lampe, K. J., Heilshorn, S. C. 2012; 18 (7-8): 806-815

  Abstract

  Cell transplantation is a promising therapy for a myriad of debilitating diseases; however, current delivery protocols using direct injection result in poor cell viability. We demonstrate that during the actual cell injection process, mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we hypothesize that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. We use a controlled in vitro model of cell injection to demonstrate success of this acute protection strategy for a wide range of cell types including human umbilical vein endothelial cells (HUVEC), human adipose stem cells, rat mesenchymal stem cells, and mouse neural progenitor cells. Specifically, alginate hydrogels with plateau storage moduli (G') ranging from 0.33 to 58.1 Pa were studied. A compliant crosslinked alginate hydrogel (G'=29.6 Pa) yielded the highest HUVEC viability, 88.9% ± 5.0%, while Newtonian solutions (i.e., buffer only) resulted in 58.7% ± 8.1% viability. Either increasing or decreasing the hydrogel storage modulus reduced this protective effect. Further, cells within noncrosslinked alginate solutions had viabilities lower than media alone, demonstrating that the protective effects are specifically a result of mechanical gelation and not the biochemistry of alginate. Experimental and theoretical data suggest that extensional flow at the entrance of the syringe needle is the main cause of acute cell death. These results provide mechanistic insight into the role of mechanical forces during cell delivery and support the use of protective hydrogels in future clinical stem cell injection studies.

  View details for DOI 10.1089/ten.tea.2011.0391

  View details for Web of Science ID 000302137200013

  View details for PubMedID 22011213

  View details for PubMedCentralID PMC3313609

• Utilizing protein-engineered biomaterials to create human muscle tissue constructs Sengupta, D., Gilbert, P. M., Johnson, K. J., Blau, H. M., Heilshorn, S. C. AMER CHEMICAL SOC. 2012

  View details for Web of Science ID 000324475100684

• Self-assembly of Clathrin protein nanostructures Arunagirinathan, M., Gibbons, B. J., Schoen, A. P., Huggins, K. L., Heilshorn, S. C. AMER CHEMICAL SOC. 2012

  View details for Web of Science ID 000324475104134

• Molecular recognition enables biotemplating at distinct protein sites Huggins, K. N., Schoen, A. P., Arunagirinathan, M. A., Heilshorn, S. C. AMER CHEMICAL SOC. 2012

  View details for Web of Science ID 000324503203830

• Mechanisms of Vascular Endothelial Growth Factor-Induced Pathfinding by Endothelial Sprouts in Biomaterials TISSUE ENGINEERING PART A Shamloo, A., Xu, H., Heilshorn, S. 2012; 18 (3-4): 320-330

  Abstract

  A critical property of biomaterials for use in regenerative medicine applications is the ability to promote angiogenesis, the formation of new vascular networks, to support regenerating tissues. Recent studies have demonstrated that a complex interplay exists between biomechanical and biochemical regulators of endothelial cell sprouting, an early step in angiogenesis. Here, we use a microfluidic platform to study the pathfinding behaviors induced by various stable vascular endothelial growth factor (VEGF) gradients during sprouting morphogenesis within biomaterials. Quantitative, time-lapse analysis of endothelial sprouting demonstrated that the ability of VEGF to regulate sprout orientation during several stages of sprouting morphogenesis (initiation, elongation, and turning navigation) was biomaterial dependent. Identical VEGF gradients induced different types of coordinated cell movements depending on the density of the surrounding collagen/fibronectin matrix. In denser matrices, sprouts were more likely to have an initial orientation aligned parallel to the VEGF gradient. In contrast, in less dense matrices, sprouts were more likely to initially misalign with the VEGF gradient; however, these sprouts underwent significant turning and navigation to eventually reorient to be parallel to the VEGF gradient. These less dense matrices required shallower VEGF gradients and demonstrated lower activating VEGF thresholds to induce proper sprout alignment and pathfinding. These results encourage the future use of microfluidic platforms to probe fundamental aspects of matrix effects on angiogenesis, to screen biomaterials for angiogenic potential, and to design ex vivo tissues with aligned vascular networks.

  View details for DOI 10.1089/ten.tea.2011.0323

  View details for Web of Science ID 000300003300010

  View details for PubMedID 21888475

  View details for PubMedCentralID PMC3267969

• The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yan, K. S., Chia, L. A., Li, X., Ootani, A., Su, J., Lee, J. Y., Su, N., Luo, Y., Heilshorn, S. C., Amieva, M. R., Sangiorgi, E., Capecchi, M. R., Kuo, C. J. 2012; 109 (2): 466-471

  Abstract

  The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

  View details for DOI 10.1073/pnas.1118857109

  View details for PubMedID 22190486

• Hydrogel crosslinking density regulates temporal contractility of human embryonic stem cell-derived cardiomyocytes in 3D cultures SOFT MATTER Chung, C., Anderson, E., Pera, R. R., Pruitt, B. L., Heilshorn, S. C. 2012; 8 (39): 10141-10148

  Abstract

  Systematically tunable in vitro platforms are invaluable in gaining insight to stem cell-microenvironment interactions in three-dimensional cultures. Utilizing recombinant protein technology, we independently tune hydrogel properties to systematically isolate the effects of matrix crosslinking density on cardiomyocyte differentiation, maturation, and function. We show that contracting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) remain viable within four engineered elastin-like hydrogels of varying crosslinking densities with elastic moduli ranging from 0.45 to 2.4 kPa. Cardiomyocyte phenotype and function was maintained within hESC embryoid bodies for up to 2 weeks. Interestingly, increased crosslinking density was shown to transiently suspend spontaneous contractility. While encapsulated cells began spontaneous contractions at day 1 in hydrogels of the lowest crosslinking density, onset of contraction was increasingly delayed at higher crosslinking densities up to 6 days. However, once spontaneous contraction was restored, the rate of contraction was similar within all materials (71 ± 8 beats/min). Additionally, all groups successfully responded to electrical pacing at both 1 and 2 Hz. This study demonstrates that encapsulated hESC-CMs respond to 3D matrix crosslinking density within elastin-like hydrogels and stresses the importance of investigating temporal cellular responses in 3D cultures.

  View details for DOI 10.1039/c2sm26082d

  View details for Web of Science ID 000308882800024

  View details for PubMedCentralID PMC3511866

• Hydrogels from Protein Engineering Biomimetic Approaches for Biomaterials Development Greenwood-Goodwin, M., Heilshorn, S. C. edited by Mano, J. F. Mannheim, Germany, Wiley-VCH Verlag.. 2012: 1
• Engineered Protein Biomaterials. Biomedical Engineering Handbook Parisi-Amon, A., Heilshorn, S. C. edited by Bronzino, J. D., Peterson, D. R., FIsher, J. P. Boca Raton, FL, CRC Press. 2012; 4th: 1
• Protein-Engineered Hydrogels. Biomaterials Surface Science Raphel, J., Parisi-Amon, A. P., Heilshorn, S. C. edited by Taubert, A., Mano, J., Rodriquez-Cabello, J. C. Mannheim, Germany, Wiley-VCH Verlag.. 2012: 1
• Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings JOURNAL OF MATERIALS CHEMISTRY Raphel, J., Parisi-Amon, A., Heilshorn, S. C. 2012; 22 (37): 19429-19437

  Abstract

  Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

  View details for DOI 10.1039/c2jm31768k

  View details for Web of Science ID 000308099900010

  View details for PubMedCentralID PMC3449156

• Template Engineering Through Epitope Recognition: A Modular, Biomimetic Strategy for Inorganic Nanomaterial Synthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Schoen, A. P., Schoen, D. T., Huggins, K. N., Arunagirinathan, M. A., Heilshorn, S. C. 2011; 133 (45): 18202-18207

  Abstract

  Natural systems often utilize a single protein to perform multiple functions. Control over functional specificity is achieved through interactions with other proteins at well-defined epitope binding sites to form a variety of functional coassemblies. Inspired by the biological use of epitope recognition to perform diverse yet specific functions, we present a Template Engineering Through Epitope Recognition (TEThER) strategy that takes advantage of noncovalent, molecular recognition to achieve functional versatility from a single protein template. Engineered TEThER peptides span the biologic-inorganic interface and serve as molecular bridges between epitope binding sites on protein templates and selected inorganic materials in a localized, specific, and versatile manner. TEThER peptides are bifunctional sequences designed to noncovalently bind to the protein scaffold and to serve as nucleation sites for inorganic materials. Specifically, we functionalized identical clathrin protein cages through coassembly with designer TEThER peptides to achieve three diverse functions: the bioenabled synthesis of anatase titanium dioxide, cobalt oxide, and gold nanoparticles in aqueous solvents at room temperature and ambient pressure. Compared with previous demonstrations of site-specific inorganic biotemplating, the TEThER strategy relies solely on defined, noncovalent interactions without requiring any genetic or chemical modifications to the biomacromolecular template. Therefore, this general strategy represents a mix-and-match, biomimetic approach that can be broadly applied to other protein templates to achieve versatile and site-specific heteroassemblies of nanoscale biologic-inorganic complexes.

  View details for DOI 10.1021/ja204732n

  View details for Web of Science ID 000297381200043

  View details for PubMedID 21967307

• Molecular-Level Engineering of Protein Physical Hydrogels for Predictive Sol-Gel Phase Behavior BIOMACROMOLECULES Mulyasasmita, W., Lee, J. S., Heilshorn, S. C. 2011; 12 (10): 3406-3411

  Abstract

  Predictable tuning of bulk mechanics from the molecular level remains elusive in many physical hydrogel systems because of the reliance on nonspecific and nonstoichiometric chain interactions for network formation. We describe a mixing-induced two-component hydrogel (MITCH) system, in which network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with a simple polymer physics model, we manipulate the polypeptide binding interactions and demonstrate the direct ability to predict the resulting effects on network cross-linking density, sol-gel phase behavior, and gel mechanics.

  View details for DOI 10.1021/bm200959e

  View details for PubMedID 21861461

• Protein-engineered biomaterials: Nanoscale mimics of the extracellular matrix BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS Romano, N. H., Sengupta, D., Chung, C., Heilshorn, S. C. 2011; 1810 (3): 339-349

  Abstract

  Traditional materials used as in vitro cell culture substrates are rigid and flat surfaces that lack the exquisite nano- and micro-scale features of the in vivo extracellular environment. While these surfaces can be coated with harvested extracellular matrix (ECM) proteins to partially recapitulate the bio-instructive nature of the ECM, these harvested proteins often exhibit large batch-to-batch variability and can be difficult to customize for specific biological studies. In contrast, recombinant protein technology can be utilized to synthesize families of 3 dimensional protein-engineered biomaterials that are cyto-compatible, reproducible, and fully customizable.Here we describe a modular design strategy to synthesize protein-engineered biomaterials that fuse together multiple repeats of nanoscale peptide design motifs into full-length engineered ECM mimics.Due to the molecular-level precision of recombinant protein synthesis, these biomaterials can be tailored to include a variety of bio-instructional ligands at specified densities, to exhibit mechanical properties that match those of native tissue, and to include proteolytic target sites that enable cell-triggered scaffold remodeling. Furthermore, these biomaterials can be processed into forms that are injectable for minimally-invasive delivery or spatially patterned to enable the release of multiple drugs with distinct release kinetics.Given the reproducibility and flexibility of these protein-engineered biomaterials, they are ideal substrates for reductionist biological studies of cell-matrix interactions, for in vitro models of physiological processes, and for bio-instructive scaffolds in regenerative medicine therapies. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

  View details for DOI 10.1016/j.bbagen.2010.07.005

  View details for Web of Science ID 000287470900012

  View details for PubMedID 20647034

  View details for PubMedCentralID PMC3033985

• Vacuum soft lithography to direct neuronal polarization SOFT MATTER Nevill, J. T., Mo, A., Cord, B. J., Palmer, T. D., Poo, M., Lee, L. P., Heilshorn, S. C. 2011; 7 (2): 343-347

  View details for DOI 10.1039/c0sm00869a

  View details for Web of Science ID 000286110900005

• Protein-Engineered Biomaterials: Synthesis and Characterization. Comprehensive Biomaterials. Mulyasasmita, W., Heilshorn, S. C. edited by Ducheyne, P., Healy, K., Hutmacher, D. W. Oxford, UK, Elsevier Science.. 2011: 1
• Essential Regulation of CNS Angiogenesis by the Orphan G Protein-Coupled Receptor GPR124 SCIENCE Kuhnert, F., Mancuso, M. R., Shamloo, A., Wang, H., Choksi, V., Florek, M., Su, H., Fruttiger, M., Young, W. L., Heilshorn, S. C., Kuo, C. J. 2010; 330 (6006): 985-989

  Abstract

  The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.

  View details for DOI 10.1126/science.1196554

  View details for PubMedID 21071672

• High Speed Water Sterilization Using One-Dimensional Nanostructures NANO LETTERS Schoen, D. T., Schoen, A. P., Hu, L., Kim, H. S., Heilshorn, S. C., Cui, Y. 2010; 10 (9): 3628-3632

  Abstract

  The removal of bacteria and other organisms from water is an extremely important process, not only for drinking and sanitation but also industrially as biofouling is a commonplace and serious problem. We here present a textile based multiscale device for the high speed electrical sterilization of water using silver nanowires, carbon nanotubes, and cotton. This approach, which combines several materials spanning three very different length scales with simple dying based fabrication, makes a gravity fed device operating at 100000 L/(h m(2)) which can inactivate >98% of bacteria with only several seconds of total incubation time. This excellent performance is enabled by the use of an electrical mechanism rather than size exclusion, while the very high surface area of the device coupled with large electric field concentrations near the silver nanowire tips allows for effective bacterial inactivation.

  View details for DOI 10.1021/nl101944e

  View details for Web of Science ID 000281498200068

  View details for PubMedID 20726518

• Protein-Engineered Biomaterials: Highly Tunable Tissue Engineering Scaffolds TISSUE ENGINEERING PART B-REVIEWS Sengupta, D., Heilshorn, S. C. 2010; 16 (3): 285-293

  Abstract

  A common goal in tissue engineering is to attain the ability to tailor specific cell-scaffold interactions and thereby gain control over cell behavior. The tunable nature of protein-engineered biomaterials enables independent tailoring of a range of biomaterial properties, creating an attractive alternative to synthetic polymeric scaffolds or harvested natural scaffolds. Protein-engineered biomaterials are comprised of modular peptide domains with various functionalities that are encoded into a DNA plasmid, transfected into an organism of choice, and expressed and purified to yield a biopolymer with exact molecular-level sequence specification. Because of the modular design strategy of protein-engineered biomaterials, these scaffolds can be easily modified to enable optimization for specific tissue engineering applications. By including multiple peptide domains with different functionalities in a single, modular biomaterial, the scaffolds can be designed to mimic the diverse properties of the natural extracellular matrix, including cell adhesion, cell signaling, elasticity, and biodegradability. Recently, the field of protein-engineered biomaterials has expanded to include functional modules that are not normally present in the extracellular matrix, thus expanding the scope and functionality of these materials. For example, these modules can include noncanonical amino acids, inorganic-binding domains, and DNA-binding sequences. The modularity, tunability, and sequence specificity of protein-engineered biomaterials make them attractive candidates for use as substrates for a variety of tissue engineering applications.

  View details for DOI 10.1089/ten.teb.2009.0591

  View details for Web of Science ID 000278640000002

  View details for PubMedID 20141386

• Local and Long-Range Reciprocal Regulation of cAMP and cGMP in Axon/Dendrite Formation SCIENCE Shelly, M., Lim, B. K., Cancedda, L., Heilshorn, S. C., Gao, H., Poo, M. 2010; 327 (5965): 547-552

  Abstract

  Cytosolic cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) often mediate antagonistic cellular actions of extracellular factors, from the regulation of ion channels to cell volume control and axon guidance. We found that localized cAMP and cGMP activities in undifferentiated neurites of cultured hippocampal neurons promote and suppress axon formation, respectively, and exert opposite effects on dendrite formation. Fluorescence resonance energy transfer imaging showed that alterations of the amount of cAMP resulted in opposite changes in the amount of cGMP, and vice versa, through the activation of specific phosphodiesterases and protein kinases. Local elevation of cAMP in one neurite resulted in cAMP reduction in all other neurites of the same neuron. Thus, local and long-range reciprocal regulation of cAMP and cGMP together ensures coordinated development of one axon and multiple dendrites.

  View details for DOI 10.1126/science.1179735

  View details for Web of Science ID 000274020500029

  View details for PubMedID 20110498

• Matrix density mediates polarization and lumen formation of endothelial sprouts in VEGF gradients LAB ON A CHIP Shamloo, A., Heilshorn, S. C. 2010; 10 (22): 3061-3068

  Abstract

  Endothelial cell (EC) sprouting morphogenesis is a critical step during angiogenesis, the formation of new blood vessels from existing conduits. Here, three-dimensional sprouting morphogenesis was examined using in vitro microfluidic devices that enabled the separate and simultaneous tuning of biomechanical and soluble biochemical stimuli. Quantitative analysis of endothelial sprout formation demonstrated that the ability of vascular endothelial growth factor (VEGF) to regulate stable sprout formation was mediated by the density of the surrounding collagen/fibronectin matrix. The coordinated migration and proliferation of multiple ECs to form stable sprouts were enhanced at intermediate matrix densities (1.2-1.9 mg ml(-1)), while lower densities resulted in uncoordinated migration (0.3-0.7 mg ml(-1)) and higher densities resulted in broad cell clusters that did not elongate (2.7 mg ml(-1)). Within the permissive range of matrix biomechanics, higher density matrices resulted in shorter, thicker, and slower-growing sprouts. The sprouts in higher density matrices also were more likely to polarize towards higher VEGF concentrations, included more cells per cross-sectional area, and demonstrated more stable lumen formation compared to sprouts in lower density matrices. These results quantitatively demonstrate that matrix density mediates VEGF-induced sprout polarization and lumen formation, potentially by regulating the balance between EC migration rate and proliferation rate.

  View details for DOI 10.1039/c005069e

  View details for Web of Science ID 000283600900006

  View details for PubMedID 20820484

• Novel Receptor-Mediated Endothelial Cell Chemotaxis Shamloo, A., Kuhnert, F., Choksi, V., Kuo, C., Heilshorn, S. CELL PRESS. 2010: 497A

  View details for DOI 10.1016/j.bpj.2009.12.2705

  View details for Web of Science ID 000208762004500

• Matrix Rigidity Mediates Growth Factor Response during 3D Endothelial Cell Sprouting Shamloo, A., Heilshorn, S. C. CELL PRESS. 2010: 730A

  View details for DOI 10.1016/j.bpj.2009.12.4000

  View details for Web of Science ID 000208762007137

• Protein Engineered Biomaterials. Protein Engineering. Wong, C. P., Heilshorn, S. C. edited by Park, S. J., Cochran, J. R. Boca Raton, FL, CRC Press. 2010: 1
• The Interplay between Biomechanical and Biochemical Factors Regulates Lumen Formation and Navigation of Endothelial Cell Sprouts 12th ASME Summer Bioengineering Conference Shamloo, A., Heilshorn, S. C. AMER SOC MECHANICAL ENGINEERS. 2010: 429–430

  View details for Web of Science ID 000290705300215

• Biomaterial Design Strategies for the Treatment of Spinal Cord Injuries JOURNAL OF NEUROTRAUMA Straley, K. S., Foo, C. W., Heilshorn, S. C. 2010; 27 (1): 1-19

  Abstract

  The highly debilitating nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Many experts agree that the greatest hope for treatment of spinal cord injuries will involve a combinatorial approach that integrates biomaterial scaffolds, cell transplantation, and molecule delivery. This manuscript presents a comprehensive review of biomaterial-scaffold design strategies currently being applied to the development of nerve guidance channels and hydrogels that more effectively stimulate spinal cord tissue regeneration. To enhance the regenerative capacity of these two scaffold types, researchers are focusing on optimizing the mechanical properties, cell-adhesivity, biodegradability, electrical activity, and topography of synthetic and natural materials, and are developing mechanisms to use these scaffolds to deliver cells and biomolecules. Developing scaffolds that address several of these key design parameters will lead to more successful therapies for the regeneration of spinal cord tissue.

  View details for DOI 10.1089/neu.2009.0948

  View details for Web of Science ID 000273983200001

  View details for PubMedID 19698073

  View details for PubMedCentralID PMC2924783

• Two-component protein-engineered physical hydrogels for cell encapsulation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Foo, C. T., Lee, J. S., Mulyasasmita, W., Parisi-Amon, A., Heilshorn, S. C. 2009; 106 (52): 22067-22072

  Abstract

  Current protocols to encapsulate cells within physical hydrogels require substantial changes in environmental conditions (pH, temperature, or ionic strength) to initiate gelation. These conditions can be detrimental to cells and are often difficult to reproduce, therefore complicating their use in clinical settings. We report the development of a two-component, molecular-recognition gelation strategy that enables cell encapsulation without environmental triggers. Instead, the two components, which contain multiple repeats of WW and proline-rich peptide domains, undergo a sol-gel phase transition upon simple mixing and hetero-assembly of the peptide domains. We term these materials mixing-induced, two-component hydrogels. Our results demonstrate use of the WW and proline-rich domains in protein-engineered materials and expand the library of peptides successfully designed into engineered proteins. Because both of these association domains are normally found intracellularly, their molecular recognition is not disrupted by the presence of additional biomolecules in the extracellular milieu, thereby enabling reproducible encapsulation of multiple cell types, including PC-12 neuronal-like cells, human umbilical vein endothelial cells, and murine adult neural stem cells. Precise variations in the molecular-level design of the two components including (i) the frequency of repeated association domains per chain and (ii) the association energy between domains enable tailoring of the hydrogel viscoelasticity to achieve plateau shear moduli ranging from approximately 9 to 50 Pa. Because of the transient physical crosslinks that form between association domains, these hydrogels are shear-thinning, injectable, and self-healing. Neural stem cells encapsulated in the hydrogels form stable three-dimensional cultures that continue to self-renew, differentiate, and sprout extended neurites.

  View details for DOI 10.1073/pnas.0904851106

  View details for Web of Science ID 000273178700008

  View details for PubMedID 20007785

  View details for PubMedCentralID PMC2791665

• Dynamic, 3D-Pattern Formation Within Enzyme-Responsive Hydrogels ADVANCED MATERIALS Straley, K. S., Heilshorn, S. C. 2009; 21 (41): 4148-?

  View details for DOI 10.1002/adma.200901865

  View details for Web of Science ID 000272110000012

• Gradient lithography of engineered proteins to fabricate 2D and 3D cell culture micro environments BIOMEDICAL MICRODEVICES Wang, S., Foo, C. W., Warrier, A., Poo, M., Heilshorn, S. C., Zhang, X. 2009; 11 (5): 1127-1134

  Abstract

  Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research.

  View details for DOI 10.1007/s10544-009-9329-1

  View details for Web of Science ID 000270679400019

  View details for PubMedID 19495986

  View details for PubMedCentralID PMC2777213

• Formation and properties of magnetic chains for 100nm nanoparticles used in separations of molecules and cells 7th International Conference on Scientific and Clinical Applications of Magnetic Carriers Wilson, R. J., Hu, W., Fu, C. W., Koh, A. L., Gaster, R. S., Earhart, C. M., Fu, A., Heilshorn, S. C., Sinclair, R., Wang, S. X. ELSEVIER SCIENCE BV. 2009: 1452–58

  Abstract

  Optical observations of 100 nm metallic magnetic nanoparticles are used to study their magnetic field induced self assembly. Chains with lengths of tens of microns are observed to form within minutes at nanoparticle concentrations of 10(10) per mL. Chain rotation and magnetophoresis are readily observed, and SEM reveals that long chains are not simple single particle filaments. Similar chains are detected for several 100 nm commercial bio-separation nanoparticles. We demonstrate the staged magnetic condensation of different types of nanoparticles into composite structures and show that magnetic chains bind to immunomagnetically labeled cells, serving as temporary handles which allow novel magnetic cell manipulations.

  View details for DOI 10.1016/j.jmmm.2009.02.066

  View details for Web of Science ID 000265278000028

  View details for PubMedCentralID PMC2757286

• Formation and properties of magnetic chains for 100 nm nanoparticles used in separations of molecules and cells. Journal of magnetism and magnetic materials Wilson, R. J., Hu, W., Fu, C. W., Koh, A. L., Gaster, R. S., Earhart, C. M., Fu, A., Heilshorn, S. C., Sinclair, R., Wang, S. X. 2009; 321 (10): 1452-1458

  Abstract

  Optical observations of 100 nm metallic magnetic nanoparticles are used to study their magnetic field induced self assembly. Chains with lengths of tens of microns are observed to form within minutes at nanoparticle concentrations of 10(10) per mL. Chain rotation and magnetophoresis are readily observed, and SEM reveals that long chains are not simple single particle filaments. Similar chains are detected for several 100 nm commercial bio-separation nanoparticles. We demonstrate the staged magnetic condensation of different types of nanoparticles into composite structures and show that magnetic chains bind to immunomagnetically labeled cells, serving as temporary handles which allow novel magnetic cell manipulations.

  View details for DOI 10.1016/j.jmmm.2009.02.066

  View details for PubMedID 20161001

  View details for PubMedCentralID PMC2757286

• Designer Protein-Based Scaffolds for Neural Tissue Engineering Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Straley, K., Heilshorn, S. C. IEEE. 2009: 2101–2102

  Abstract

  A key attribute missing from many current biomaterials is the ability to independently tune multiple biomaterial properties without simultaneously affecting other material parameters. Because cells are well known to respond to changes in the initial elastic modulus, degradation rate, and cell adhesivity of a biomaterial, it is critical to develop synthetic design strategies that allow decoupled tailoring of each individual parameter in order to systematically optimize cell-scaffold interactions. We present the development of a family of biomimetic scaffolds composed of chemically crosslinked, elastin-like proteins designed to support neural regeneration through a combination of cell adhesion and cell-induced degradation and remodeling. Through use of a modular protein-design strategy, a range of biomaterials is created that allows independent tuning over the initial elastic modulus, degradation rate, cell adhesivity, and neurite outgrowth. By combining these engineered proteins into composite structures, biomaterials are created with 3D patterns that emerge over time in response to cell-secreted enzymes. These dynamic 3D structures enable the delivery of multiple drugs with precise spatial and temporal resolution and also enable the design of biomaterials that adapt to changing scaffold needs.

  View details for Web of Science ID 000280543601259

  View details for PubMedID 19964779

• Independent tuning of multiple biomaterial properties using protein engineering SOFT MATTER Straley, K. S., Heilshorn, S. C. 2009; 5 (1): 114-124

  View details for DOI 10.1039/b808504h

  View details for Web of Science ID 000263272100015

• Independent tuning of multiple biomaterial properties using protein engineering Soft Matter Straley KS, Heilshorn SC 2009; 5: 114-124
• Dynamic, three-dimensional pattern formation within enzyme-responsive hydrogels Advanced Materials Straley KS, Heilshorn SC 2009; 21 (41): 4148-4152
• Design and adsorption of modular engineered proteins to prepare customized, neuron-compatible coatings. Frontiers in neuroengineering Straley, K. S., Heilshorn, S. C. 2009; 2: 9-?

  Abstract

  Neural prosthetic implants are currently being developed for the treatment and study of both peripheral and central nervous system disorders. Effective integration of these devices upon implantation is a critical hurdle to achieving function. As a result, much attention has been directed towards the development of biocompatible coatings that prolong their in vivo lifespan. In this work, we present a novel approach to fabricate such coatings, which specifically involves the use of surface-adsorbed, nanoscale-designed protein polymers to prepare reproducible, customized surfaces. A nanoscale modular design strategy was employed to synthesize six engineered, recombinant proteins intended to mimic aspects of the extracellular matrix proteins fibronectin, laminin, and elastin as well as the cell-cell adhesive protein neural cell adhesion molecule. Physical adsorption isotherms were experimentally determined for these engineered proteins, allowing for direct calculation of the available ligand density present on coated surfaces. As confirmation that ligand density in these engineered systems impacts neuronal cell behavior, we demonstrate that increasing the density of fibronectin-derived RGD ligands on coated surfaces while maintaining uniform protein surface coverage results in enhanced neurite extension of PC-12 cells. Therefore, this engineered protein adsorption approach allows for the facile preparation of tunable, quantifiable, and reproducible surfaces for in vitro studies of cell-ligand interactions and for potential application as coatings on neural implants.

  View details for DOI 10.3389/neuro.16.009.2009

  View details for PubMedID 19562090

  View details for PubMedCentralID PMC2701681

• Endothelial cell polarization and chemotaxis in a microfluidic device LAB ON A CHIP Shamloo, A., Ma, N., Poo, M., Sohn, L. L., Heilshorn, S. C. 2008; 8 (8): 1292-1299

  Abstract

  The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18-32 ng mL(-1)) when the concentration gradient was 14 ng mL(-1) mm(-1). In contrast, shallow gradients (2 ng mL(-1) mm(-1)) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.

  View details for DOI 10.1039/b719788h

  View details for PubMedID 18651071

• LKB1/STRAD promotes axon initiation during neuronal polarization CELL Shelly, M., Cancedda, L., Heilshorn, S., Sumbre, G., Poo, M. 2007; 129 (3): 565-577

  Abstract

  Axon/dendrite differentiation is a critical step in neuronal development. In cultured hippocampal neurons, the accumulation of LKB1 and STRAD, two interacting proteins critical for establishing epithelial polarity, in an undifferentiated neurite correlates with its subsequent axon differentiation. Downregulation of either LKB1 or STRAD by siRNAs prevented axon differentiation, and overexpression of these proteins led to multiple axon formation. Furthermore, interaction of STRAD with LKB1 promoted LKB1 phosphorylation at a PKA site S431 and elevated the LKB1 level, and overexpressing LKB1 with a serine-to-alanine mutation at S431 (LKB1(S431A)) prevented axon differentiation. In developing cortical neurons in vivo, downregulation of LKB1 or overexpression of LKB1(S431A) also abolished axon formation. Finally, local exposure of the undifferentiated neurite to brain-derived neurotrophic factor or dibutyryl-cAMP promoted axon differentiation in a manner that depended on PKA-dependent LKB1 phosphorylation. Thus local LKB1/STRAD accumulation and PKA-dependent LKB1 phosphorylation represents an early signal for axon initiation.

  View details for DOI 10.1016/j.cell.2007.04.012

  View details for Web of Science ID 000246373600022

  View details for PubMedID 17482549

• Lithographic patterning of photoreactive cell-adhesive proteins JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Carrico, I. S., Maskarinec, S. A., Heilshorn, S. C., Mock, M. L., Liu, J. C., Nowatzki, P. J., Franck, C., Ravichandran, G., Tirrell, D. A. 2007; 129 (16): 4874-?

  View details for DOI 10.1021/ja070200b

  View details for Web of Science ID 000245782800009

  View details for PubMedID 17397163

• Cell-binding domain context affects cell behavior on engineered proteins BIOMACROMOLECULES Heilshorn, S. C., Liu, J. C., Tirrell, D. A. 2005; 6 (1): 318-323

  Abstract

  A family of artificial extracellular matrix proteins developed for application in small-diameter vascular grafts is used to examine the importance of cell-binding domain context on cell adhesion and spreading. The engineered protein sequences are derived from the naturally occurring extracellular matrix proteins elastin and fibronectin. While each engineered protein contains identical CS5 cell-binding domain sequences, the lysine residues that serve as cross-linking sites are either (i) within the elastin cassettes or (ii) confined to the ends of the protein. Endothelial cells adhere specifically to the CS5 sequence in both of these proteins, but cell adhesion and spreading are more robust on proteins in which the lysine residues are confined to the terminal regions of the chain. These results may be due to altered protein conformations that affect either the accessibility of the CS5 sequence or its affinity for the alpha(4)beta(1) integrin receptor on the endothelial cell surface. Amino acid choice outside the cell-binding domain can thus have a significant impact on the behavior of cells cultured on artificial extracellular matrix proteins.

  View details for DOI 10.1021/bm049627q

  View details for Web of Science ID 000226344300041

  View details for PubMedID 15638535

• Comparative cell response to artificial extracellular matrix proteins containing the RGD and CS5 cell-binding domains BIOMACROMOLECULES Liu, J. C., Heilshorn, S. C., Tirrell, D. A. 2004; 5 (2): 497-504

  Abstract

  This study addresses endothelial cell adhesion and spreading on a family of artificial extracellular matrix (aECM) proteins designed for application in small-diameter vascular grafts. The aECM proteins contain domains derived from elastin and from fibronectin. aECM 1 contains the RGD sequence from the tenth type III domain of fibronectin; aECM 3 contains the fibronectin CS5 cell-binding domain. Negative control proteins aECM 2 and 4 are scrambled versions of aECM 1 and 3, respectively. Competitive peptide inhibition studies and comparisons of positive and negative control proteins confirm that adhesion of HUVECs to aECM proteins 1 and 3 is sequence specific. When subjected to a normal detachment force of 780 pN, 3-fold more HUVECs remained adherent to aECM 1 than to aECM 3. HUVECs also spread more rapidly on aECM 1 than on aECM 3. These results (i) indicate that cellular responses to aECM proteins can be modulated through choice of cell-binding domain and (ii) recommend the RGD sequence for applications that require rapid endothelial cell spreading and matrix adhesion.

  View details for DOI 10.1021/bm034340z

  View details for Web of Science ID 000220109200032

  View details for PubMedID 15003012

• Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins BIOMATERIALS Heilshorn, S. C., DiZio, K. A., Welsh, E. R., Tirrell, D. A. 2003; 24 (23): 4245-4252

  Abstract

  This study examines the spreading and adhesion of human umbilical vein endothelial cells (HUVEC) on artificial extracellular matrix (aECM) proteins containing sequences derived from elastin and fibronectin. Three aECM variants were studied: aECM 1 contains lysine residues periodically spaced within the protein sequence and three repeats of the CS5 domain of fibronectin, aECM 2 contains periodically spaced lysines and three repeats of a scrambled CS5 sequence, and aECM 3 contains lysines at the protein termini and five CS5 repeats. Comparative cell binding and peptide inhibition assays confirm that the tetrapeptide sequence REDV is responsible for HUVEC adhesion to aECM proteins that contain the CS5 domain. Furthermore, more than 60% of adherent HUVEC were retained on aECM 1 after exposure to physiologically relevant shear stresses (

  View details for DOI 10.1016/S0142-9612(03)00294-1

  View details for Web of Science ID 000184239500017

  View details for PubMedID 12853256

• Liquid personal cleansing compositions which contain a  complex coacervate for improved sensory perception Assignee: The Procter & Gamble Company. Glenn, R. W., Sine, M. R., Evans, M. D., Carethers, M. E., Heilshorn, S. C. 2000