Heilshorn's interests include biomaterials in regenerative medicine, engineered proteins with novel assembly properties, microfluidics and photolithography of proteins, and synthesis of materials to influence stem cell differentiation. Current projects include tissue engineering for spinal cord and blood vessel regeneration, designing injectable materials for use in stem cell therapies, and the design of microfluidic devices to study the directed migration of cells (i.e., chemotaxis).
Associate Professor, Materials Science and Engineering
Associate Professor (By courtesy), Chemical Engineering
Associate Professor (By courtesy), Bioengineering
Member, Cardiovascular Institute
Member, Child Health Research Institute
Affiliate, Precourt Institute for Energy
Faculty Fellow, Stanford ChEM-H
Member, Stanford Neurosciences Institute
Honors & Awards
New Innovator Award, National Institutes of Health (2009)
CAREER Award, National Science Foundation (2009)
New Investigator Award, Petroleum Research Fund, American Chemical Society (2009)
PhD, Caltech, Chemical Engineering (2004)
MS, Caltech, Chemical Engineering (2000)
BS, Georgia Tech, Chemical Engineering (1998)
Current Research and Scholarly Interests
- Bioengineering Materials to Heal the Body
MATSCI 81N (Aut)
- Biomaterials in Regenerative Medicine
BIOE 361, MATSCI 381 (Spr)
- Introduction to Materials Science, Biomaterials Emphasis
ENGR 50M (Win)
Independent Studies (16)
- Bioengineering Problems and Experimental Investigation
BIOE 191 (Aut, Win, Spr, Sum)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Win, Spr)
- Directed Study
BIOE 391 (Aut, Win, Spr, Sum)
- Graduate Independent Study
MATSCI 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Master's Research
MATSCI 200 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Win, Spr)
- Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr)
- Out-of-Department Graduate Research
BIO 300X (Aut)
- Participation in Materials Science Teaching
MATSCI 400 (Win, Spr)
- Ph.D. Research
MATSCI 300 (Aut, Win, Spr, Sum)
- Practical Training
MATSCI 299 (Aut, Win, Spr, Sum)
- Undergraduate Independent Study
MATSCI 100 (Aut, Win, Spr, Sum)
- Undergraduate Research
MATSCI 150 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr, Sum)
- Bioengineering Problems and Experimental Investigation
Prior Year Courses
- Bioengineering Materials to Heal the Body
MATSCI 81N (Aut)
- Biomaterials in Regenerative Medicine
BIOE 361, MATSCI 381 (Spr)
- Introduction to Materials Science, Biomaterials Emphasis
ENGR 50M (Win)
- Introductory Science of Materials
OSPPARIS 50M (Spr)
- Organic and Biological Materials
MATSCI 190, MATSCI 210 (Spr)
- Biomaterials in Regenerative Medicine
BIOE 361, MATSCI 381 (Aut)
- Introduction to Materials Science, Biomaterials Emphasis
ENGR 50M (Win)
- Materials Science Colloquium
MATSCI 230 (Win)
- Organic and Biological Materials
MATSCI 190, MATSCI 210 (Spr)
- Bioengineering Materials to Heal the Body
Graduate and Fellowship Programs
Microfluidic Investigation of BDNF-Enhanced Neural Stem Cell Chemotaxis in CXCL12 Gradients
2013; 9 (4): 585-595
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
Building stem cell niches from the molecule up through engineered peptide materials
2012; 519 (2): 138-146
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
2012; 18 (7-8): 806-815
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
Essential Regulation of CNS Angiogenesis by the Orphan G Protein-Coupled Receptor GPR124
2010; 330 (6006): 985-989
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 Web of Science ID 000284118000049
View details for PubMedID 21071672
Two-component protein-engineered physical hydrogels for cell encapsulation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (52): 22067-22072
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, three-dimensional pattern formation within enzyme-responsive hydrogels Advanced Materials 2009; 21 (41): 4148-4152
- Independent tuning of multiple biomaterial properties using protein engineering Soft Matter 2009; 5: 114-124
Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering
2015; 27 (25): 3717-3736
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 Web of Science ID 000357335900001
View details for PubMedID 25989348
Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding
NEURAL REGENERATION RESEARCH
2015; 10 (4): 514-517
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 2015; 25 (9): 1344-1351
Protein-engineered hydrogel encapsulation for 3-d culture of murine cochlea.
Otology & neurotology
2015; 36 (3): 531-538
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
2015; 11 (6): 722-730
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.
2015; 11: 48-57
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
Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis
2015; 7 (5): 569-579
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
- Multi-Site Functionalization of Protein Scaffolds for Bimetallic Nanoparticle Templating ADVANCED FUNCTIONAL MATERIALS 2014; 24 (48): 7737-7744
Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors.
Journal of controlled release
2014; 191: 71-81
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 2014; 15 (9): 3421-3428
Avidity-controlled delivery of angiogenic peptides from injectable molecular-recognition hydrogels.
Tissue engineering. Part A
2014; 20 (15-16): 2102-2114
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
Rheology and simulation of 2-dimensional clathrin protein network assembly.
2014; 10 (33): 6219-6227
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
2014; 14 (12): 2047-2056
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
Designing ECM-mimetic materials using protein engineering
2014; 10 (4): 1751-1760
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
Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids.
2014; 6 (2): 127-142
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
2014; 15 (2): 445-455
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
2014; 6 (4): 438-449
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
- Dual-stage growth factor release within 3D protein-engineered hydrogel niches promotes adipogenesis BIOMATERIALS SCIENCE 2014; 2 (11): 1627-1639
- One-pot synthesis of elastin-like polypeptide hydrogels with grafted VEGF-mimetic peptides BIOMATERIALS SCIENCE 2014; 2 (5): 757-765
Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth
2013; 9 (3): 5590-5599
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
2013; 2 (3): 428-432
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
- Sequence-Specific Crosslinking of Electrospun, Elastin-Like Protein Preserves Bioactivity and Native-Like Mechanics ADVANCED HEALTHCARE MATERIALS 2013; 2 (1): 114-118
- Chemotaxis of human induced pluripotent stem cell-derived endothelial cells AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH 2013; 5 (5): 510-U96
- Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density BIOMATERIALS SCIENCE 2013; 1 (10): 1082-1090
- Engineered clathrin nanoreactors provide tunable control over gold nanoparticle synthesis and clustering JOURNAL OF MATERIALS CHEMISTRY B 2013; 1 (48): 6662-6669
- Microfluidic devices for quantifying the role of soluble gradients in early angiogenesis Mechanical and Chemical Signaling in Angiogenesis 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 2013; 10 (1): 1082-1090
- Dynamic remodelling of disordered protein aggregates is an alternative pathway to achieve robust self-assembly of nanostructures SOFT MATTER 2013; 9 (38): 9137-9145
Chemotaxis of human induced pluripotent stem cell-derived endothelial cells.
American journal of translational research
2013; 5 (5): 510-520
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 2013; 9 (29): 6781-6785
Complex chemoattractive and chemorepellent Kit signals revealed by direct imaging of murine mast cells in microfluidic gradient chambers
2013; 5 (8): 1076-1085
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
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
2012; 13 (12): 3912-3916
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
- Protein-Engineered Biomaterials to Generate Human Skeletal Muscle Mimics ADVANCED HEALTHCARE MATERIALS 2012; 1 (6): 785-789
Multifunctional Materials through Modular Protein Engineering
2012; 24 (29): 3923-3940
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
Mechanisms of Vascular Endothelial Growth Factor-Induced Pathfinding by Endothelial Sprouts in Biomaterials
TISSUE ENGINEERING PART A
2012; 18 (3-4): 320-330
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
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
2012; 109 (2): 466-471
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 Web of Science ID 000298950200030
View details for PubMedID 22190486
View details for PubMedCentralID PMC3258636
Hydrogel crosslinking density regulates temporal contractility of human embryonic stem cell-derived cardiomyocytes in 3D cultures
2012; 8 (39): 10141-10148
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
- Hydrogels from Protein Engineering Biomimetic Approaches for Biomaterials Development edited by Mano, J. F. Mannheim, Germany, Wiley-VCH Verlag.. 2012: 1
- Engineered Protein Biomaterials. Biomedical Engineering Handbook 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 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
2012; 22 (37): 19429-19437
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
Template Engineering Through Epitope Recognition: A Modular, Biomimetic Strategy for Inorganic Nanomaterial Synthesis
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2011; 133 (45): 18202-18207
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
2011; 12 (10): 3406-3411
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 Web of Science ID 000295602600006
View details for PubMedID 21861461
View details for PubMedCentralID PMC3253016
Protein-engineered biomaterials: Nanoscale mimics of the extracellular matrix
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
2011; 1810 (3): 339-349
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
- Vacuum soft lithography to direct neuronal polarization SOFT MATTER 2011; 7 (2): 343-347
- Protein-Engineered Biomaterials: Synthesis and Characterization. Comprehensive Biomaterials. edited by Ducheyne, P., Healy, K., Hutmacher, D. W. Oxford, UK, Elsevier Science.. 2011: 1
High Speed Water Sterilization Using One-Dimensional Nanostructures
2010; 10 (9): 3628-3632
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
2010; 16 (3): 285-293
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
2010; 327 (5965): 547-552
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
2010; 10 (22): 3061-3068
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
- Protein Engineered Biomaterials. Protein Engineering. 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
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
2010; 27 (1): 1-19
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
- Dynamic, 3D-Pattern Formation Within Enzyme-Responsive Hydrogels ADVANCED MATERIALS 2009; 21 (41): 4148-?
Gradient lithography of engineered proteins to fabricate 2D and 3D cell culture micro environments
2009; 11 (5): 1127-1134
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
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
ELSEVIER SCIENCE BV. 2009: 1452–58
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
Designer Protein-Based Scaffolds for Neural Tissue Engineering
Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society
IEEE. 2009: 2101–2102
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 2009; 5 (1): 114-124
Design and adsorption of modular engineered proteins to prepare customized, neuron-compatible coatings.
Frontiers in neuroengineering
2009; 2: 9-?
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
Endothelial cell polarization and chemotaxis in a microfluidic device
LAB ON A CHIP
2008; 8 (8): 1292-1299
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 Web of Science ID 000258572400009
View details for PubMedID 18651071
LKB1/STRAD promotes axon initiation during neuronal polarization
2007; 129 (3): 565-577
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 2007; 129 (16): 4874-?
Cell-binding domain context affects cell behavior on engineered proteins
2005; 6 (1): 318-323
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
2004; 5 (2): 497-504
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
2003; 24 (23): 4245-4252
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 (=100dynes/cm(2)). Finally, the levels of thrombogenic markers (tissue plasminogen activator and plasminogen activator inhibitor-1) secreted by HUVEC monolayers on aECM 1 were found to be similar to those secreted by HUVEC monolayers cultured on fibronectin. These characteristics, along with the physical strength and elasticity of crosslinked films prepared from these materials, make aECM proteins promising candidates for application in small-diameter vascular grafts.
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. 2000