Diya Singhal

All Publications

• Dual-orientation of collagen fibers to guide cell alignment in 3D-printed constructs. Acta biomaterialia Singhal, D., Christakopoulos, F., Brunel, L. G., Borkar, S., Doulames, V. M., Mozipo, E. A., Myung, D., Fuller, G. G., Heilshorn, S. C. 2025

  Abstract

  Natural tissue comprises fibrous proteins with complex fiber alignment patterns. Here, we develop a reproducible method to fabricate biomimetic scaffolds with patterned fiber alignment along two independent orientations. While extrusion-based approaches are commonly used to align fibrous polymers in a single orientation parallel to the direction of flow, we hypothesized that extrusion-based 3D printing could be utilized to achieve more complex patterns of fiber alignment. Specifically, we show that control of lateral spreading of a printed filament can induce fiber alignment that is either parallel or perpendicular to the flow direction. Theoretical prediction of the printing parameters that control fiber orientation was experimentally validated using a collagen biomaterial ink. The velocity ratio of the printhead movement relative to the ink extrusion rate was found to dictate collagen fiber alignment, allowing for the informed fabrication of collagen scaffolds with prescribed patterns of fiber alignment. For example, controlled variation of the ink extrusion rate during a single print resulted in scaffolds with specified regions of both parallel and perpendicular collagen fiber alignment. Human corneal mesenchymal stromal cells seeded onto the printed scaffolds adopted a spread morphology that aligned with the underlying collagen fiber patterns. This technique worked well for filaments either printed onto a printbed in air or extruded within a support bath using embedded 3D printing, enabling the fabrication of 3D structures with aligned collagen fibers. Taken together, this work demonstrates a theoretical and experimental framework to achieve the reproducible fabrication of 3D printed structures with controlled collagen fiber patterns that guide cellular alignment. Statement of Significance Natural tissues contain collagen fibers aligned in multiple directions, which are essential for guiding cell behavior; however, most existing fabrication methods can achieve only unidirectional fiber alignment. Here, we introduce an extrusion-based 3D printing strategy that enables precise control over collagen fiber orientation in both parallel and perpendicular directions. This allows multidirectional collagen fiber alignment patterned spatially within a single construct, thereby guiding corneal mesenchymal stromal cells to align multidirectionally. This approach works when printing in air or with embedded printing in a support bath. Thus, this strategy can enable the fabrication of complex 3D scaffolds that mimic the anisotropic architecture of native tissues.

  View details for DOI 10.1016/j.actbio.2025.11.013

  View details for PubMedID 41232895

• Reinforcement of Fibrillar Collagen Hydrogels with Bioorthogonal Covalent Crosslinks. Biomacromolecules Brunel, L. G., Long, C. M., Christakopoulos, F., Cai, B., de Paiva Narciso, N., Johansson, P. K., Singhal, D., Baugh, N. J., Zhang, D., Enejder, A., Myung, D., Heilshorn, S. C. 2025

  Abstract

  Bioorthogonal covalent crosslinking stabilizes collagen type I hydrogels, improving their structural integrity for tissue engineering applications with encapsulated living cells. The chemical modification required for crosslinking, however, interferes with the fibrillar nature of the collagen, leading instead to an amorphous network without fibers. We demonstrate an approach to perform bioconjugation chemistry on collagen with controlled localization such that the modified collagen retains its ability to self-assemble into a fibrillar network while also displaying functional groups for covalent crosslinking with bioorthogonal click chemistry. The collagen matrix is formed through a sequential crosslinking process, in which the modified collagen first physically assembles into fibers and then is covalently crosslinked. This approach preserves the fibrous architecture of the collagen, guiding the behavior of encapsulated human corneal mesenchymal stromal cells while also reinforcing fibers through covalent crosslinks, strengthening the stability of the cell-laden collagen hydrogel against cell-induced contraction and enzymatic degradation.

  View details for DOI 10.1021/acs.biomac.5c00398

  View details for PubMedID 40554673

• 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

• Macromolecular complex viscosity from space-filling equilibrium structure PHYSICS OF FLUIDS Chakraborty, R., Singhal, D., Kanso, M. A., Giacomin, A. J. 2022; 34 (9)

  View details for DOI 10.1063/5.0116558

  View details for Web of Science ID 000860335200004

• Complex viscosity of poly[n]catenanes including olympiadanes PHYSICS OF FLUIDS Singhal, D., Kanso, M. A., Coombs, S. J., Giacomin, A. J. 2022; 34 (3)

  View details for DOI 10.1063/5.0087283

  View details for Web of Science ID 000806402000002