Abstract
Purpose :
Due to the dearth of graft tissue available globally for patients with corneal blindness, improved corneal regeneration will require bioengineered strategies. Collagen hydrogels have demonstrated potential as matrices for corneal mesenchymal stromal cells (cMSCs) but commonly suffer from opacity and cell-induced contraction, affecting the optical properties of the material. We have developed a transparent, bioorthogonally-crosslinked collagen hydrogel with enhanced stability against cMSC-induced contraction while maintaining cell phenotype for improved regenerative potential.
Methods :
Covalently-crosslinked collagen hydrogels were formed by strain-promoted azide-alkyne cycloaddition (SPAAC), a bioorthogonal click chemistry. The hydrogel fibrillar microstructure and corresponding transparency were characterized with second harmonic generation microscopy and UV/Vis spectroscopy, respectively. Human cMSCs were encapsulated within SPAAC-crosslinked or non-chemically crosslinked control collagen hydrogels, and the contraction was monitored over time. In addition, cMSC viability, differentiation markers, and secretome within the hydrogels were analyzed using a Live/Dead assay, immunocytochemistry, and a Luminex immunoassay, respectively.
Results :
We demonstrated the improved transparency of the SPAAC-crosslinked collagen across the visible light range compared to non-chemically crosslinked collagen (e.g., 98% vs. 52% transmittance at 500 nm), which resulted from the disruption of collagen fibril formation. The SPAAC-crosslinked collagen resisted deformation from encapsulated human cMSCs over 72 h: the non-chemically crosslinked collagen hydrogels contracted to 20% of their initial diameters, while SPAAC-crosslinked collagen did not detectably contract. In addition, the cMSCs in the SPAAC-crosslinked collagen maintained their characteristic phenotype, with high viability (> 90%), expression of the keratocyte differentiation marker aldehyde dehydrogenase 3A1, and secretion of cytokines similar to cMSCs within the non-chemically crosslinked collagen.
Conclusions :
Our results indicate that human cMSCs may be successfully encapsulated within SPAAC-crosslinked collagen to create transparent, bioengineered hydrogels that resist cell-induced contraction while simultaneously preserving the corneal cell phenotype.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.