Lucia Brunel

All Publications

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• Conducting polymer-based granular hydrogels for injectable 3D cell scaffolds. Advanced materials technologies Feig, V. R., Santhanam, S., McConnell, K. W., Liu, K., Azadian, M., Brunel, L. G., Huang, Z., Tran, H., George, P. M., Bao, Z. 2021; 6 (6)

  Abstract

  Injectable 3D cell scaffolds possessing both electrical conductivity and native tissue-level softness would provide a platform to leverage electric fields to manipulate stem cell behavior. Granular hydrogels, which combine jamming-induced elasticity with repeatable injectability, are versatile materials to easily encapsulate cells to form injectable 3D niches. In this work, we demonstrate that electrically conductive granular hydrogels can be fabricated via a simple method involving fragmentation of a bulk hydrogel made from the conducting polymer PEDOT:PSS. These granular conductors exhibit excellent shear-thinning and self-healing behavior, as well as record-high electrical conductivity for an injectable 3D scaffold material (~10 S m-1). Their granular microstructure also enables them to easily encapsulate induced pluripotent stem cell (iPSC)-derived neural progenitor cells, which were viable for at least 5 days within the injectable gel matrices. Finally, we demonstrate gel biocompatibility with minimal observed inflammatory response when injected into a rodent brain.

  View details for DOI 10.1002/admt.202100162

  View details for PubMedID 34179344

  View details for PubMedCentralID PMC8225239

• 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

• Generation of a three-dimensional collagen scaffold-based model of the human endometrium INTERFACE FOCUS Abbas, Y., Brunel, L. G., Hollinshead, M. S., Fernando, R. C., Gardner, L., Duncan, I., Moffett, A., Best, S., Turco, M. Y., Burton, G. J., Cameron, R. E. 2020; 10 (2): 20190079

  Abstract

  The endometrium is the secretory lining of the uterus that undergoes dynamic changes throughout the menstrual cycle in preparation for implantation and a pregnancy. Recently, endometrial organoids (EO) were established to study the glandular epithelium. We have built upon this advance and developed a multi-cellular model containing both endometrial stromal and epithelial cells. We use porous collagen scaffolds produced with controlled lyophilization to direct cellular organization, integrating organoids with primary isolates of stromal cells. The internal pore structure of the scaffold was optimized for stromal cell culture in a systematic study, finding an optimal average pore size of 101 µm. EO seeded organize to form a luminal-like epithelial layer, on the surface of the scaffold. The cells polarize with their apical surface carrying microvilli and cilia that face the pore cavities and their basal surface attaching to the scaffold with the formation of extracellular matrix proteins. Both cell types are hormone responsive on the scaffold, with hormone stimulation resulting in epithelial differentiation and stromal decidualization.

  View details for DOI 10.1098/rsfs.2019.0079

  View details for Web of Science ID 000515395400007

  View details for PubMedID 32194932

  View details for PubMedCentralID PMC7061944

• Self-Assembling Amphiphilic Block Copolymer from Renewable delta-Decalactone and delta-Dodecalactone JOURNAL OF POLYMER SCIENCE PART A-POLYMER CHEMISTRY Ferrari, R., Agostini, A., Brunel, L., Morosi, L., Moscatelli, D. 2017; 55 (22): 3788-3797

  View details for DOI 10.1002/pola.28764

  View details for Web of Science ID 000412299600013

• RAFT Macro-Surfmers and Their Use in the ab Initio RAFT Emulsion Polymerization To Decouple Nanoparticle Size and Polymer Molecular Weight MACROMOLECULES Palmiero, U., Agostini, A., Gatti, S., Sponchioni, M., Valenti, V., Brunel, L., Moscatelli, D. 2016; 49 (22): 8387-8396

  View details for DOI 10.1021/acs.macromol.6b01827

  View details for Web of Science ID 000388913500002