Betty Cai

All Publications

• In situ UNIversal Orthogonal Network (UNION) bioink deposition for direct delivery of corneal stromal stem cells to corneal wounds. Bioactive materials Brunel, L. G., Cai, B., Hull, S. M., Han, U., Wungcharoen, T., Fernandes-Cunha, G. M., Seo, Y. A., Johansson, P. K., Heilshorn, S. C., Myung, D. 2025; 48: 414-430

  Abstract

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

  View details for DOI 10.1016/j.bioactmat.2025.02.009

  View details for PubMedID 40083774

• One-step bioprinting of endothelialized, self-supporting arterial and venous networks. Biofabrication Cai, B., Kilian, D., Ghorbani, S., Roth, J., Seymour, A. J., Brunel, L. G., Ramos Mejia, D., Rios, R. J., Szabo, I. M., Iranzo, S. C., Perez, A., Rao, R. R., Shin, S., Heilshorn, S. 2025

  Abstract

  Advances in biofabrication have enabled the generation of freeform perfusable networks mimicking vasculature. However, key challenges remain in the effective endothelialization of these complex, vascular-like networks, including cell uniformity, seeding efficiency, and the ability to pattern multiple cell types. To overcome these challenges, we present an integrated fabrication and endothelialization strategy to directly generate branched, endothelial cell-lined networks using a diffusion-based, embedded 3D bioprinting process. In this strategy, a gelatin microparticle sacrificial ink delivering both cells and crosslinkers is extruded into a crosslinkable gel precursor support bath. A self-supporting, perfusable structure is formed by diffusion-induced crosslinking, after which the sacrificial ink is melted to allow cell release and adhesion to the printed lumen. This approach produces a uniform cell lining throughout networks with complex branching geometries, which are challenging to uniformly and efficiently endothelialize using conventional perfusion-based approaches. Furthermore, the biofabrication process enables high cell viability (>90%) and the formation of a confluent endothelial layer providing vascular-mimetic barrier function and shear stress response. Leveraging this strategy, we demonstrate for the first time the patterning of multiple endothelial cell types, including arterial and venous cells, within a single arterial-venous-like network. Altogether, this strategy enables the fabrication of multi-cellular engineered vasculature with enhanced geometric complexity and phenotypic heterogeneity.

  View details for DOI 10.1088/1758-5090/adab26

  View details for PubMedID 39819775

• Interpenetrating networks of fibrillar and amorphous collagen promote cell spreading and hydrogel stability. Acta biomaterialia Brunel, L. G., Long, C. M., Christakopoulos, F., Cai, B., Johansson, P. K., Singhal, D., Enejder, A., Myung, D., Heilshorn, S. C. 2025

  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.

  View details for DOI 10.1016/j.actbio.2025.01.009

  View details for PubMedID 39798635

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

  Abstract

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

  View details for DOI 10.1101/2024.09.19.613997

  View details for PubMedID 39386574

• Achieving optical transparency in live animals with absorbing molecules. Science (New York, N.Y.) Ou, Z., Duh, Y. S., Rommelfanger, N. J., Keck, C. H., Jiang, S., Brinson, K., Zhao, S., Schmidt, E. L., Wu, X., Yang, F., Cai, B., Cui, H., Qi, W., Wu, S., Tantry, A., Roth, R., Ding, J., Chen, X., Kaltschmidt, J. A., Brongersma, M. L., Hong, G. 2024; 385 (6713): eadm6869

  Abstract

  Optical imaging plays a central role in biology and medicine but is hindered by light scattering in live tissue. We report the counterintuitive observation that strongly absorbing molecules can achieve optical transparency in live animals. We explored the physics behind this observation and found that when strongly absorbing molecules dissolve in water, they can modify the refractive index of the aqueous medium through the Kramers-Kronig relations to match that of high-index tissue components such as lipids. We have demonstrated that our straightforward approach can reversibly render a live mouse body transparent to allow visualization of a wide range of deep-seated structures and activities. This work suggests that the search for high-performance optical clearing agents should focus on strongly absorbing molecules.

  View details for DOI 10.1126/science.adm6869

  View details for PubMedID 39236186

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

  View details for Web of Science ID 001319566000288

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

  Abstract

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

  View details for DOI 10.1002/advs.202306470

  View details for PubMedID 38145962

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

  Abstract

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

  View details for DOI 10.1002/adhm.202303325

  View details for PubMedID 38134346

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

  Abstract

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

  View details for DOI 10.1002/adfm.202307435

  View details for PubMedID 38646474

  View details for PubMedCentralID PMC11031202

• 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

• 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

• Enhancing Metabolome Coverage in Data-Dependent LC-MS/MS Analysis through an Integrated Feature Extraction Strategy ANALYTICAL CHEMISTRY Hu, Y., Cai, B., Huan, T. 2019; 91 (22): 14433-14441

  Abstract

  In untargeted metabolomics, conventional data preprocessing software (e.g., XCMS, MZmine 2, MS-DIAL) are used extensively due to their high efficiency in metabolic feature extraction. However, these programs present limitations in recognizing low-abundance metabolic features, thus hindering complete metabolome coverage from the analysis. In this work, we explored the possibility of enhancing the metabolome coverage of data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) results by rescuing metabolic features that are missed by conventional software. To achieve this goal, we first categorized the metabolic features into four confidence levels based on their chromatographic peak shapes and the presence of corresponding MS/MS spectra. We then assessed the false positives and quantitative accuracy of the metabolic features that contain MS/MS spectra but are not recognized by conventional software. Our results indicate that these missed features contain valid and important metabolic information and should be integrated into the conventional metabolomics results. Thus, we developed a data-preprocessing pipeline to extract low-abundance metabolic features and integrate them with the results from conventional programs. This integrated feature extraction strategy was tested on a set of fecal metabolomic data retrieved from mice who have undergone normal diet vs high-fat diet treatments. In our test data set, the integrated feature extraction approach increased the number of significant features being extracted by 24.4% and identified five additional metabolites bearing critical biological meanings. Our results show that this integrated feature extraction strategy remarkably improves the metabolome coverage beyond that of conventional data preprocessing, therefore facilitating the confirmation of metabolites of interest and accomplishment of a higher success rate in de novo metabolite identification.

  View details for DOI 10.1021/acs.analchem.9b02980

  View details for Web of Science ID 000498280100038

  View details for PubMedID 31626534