Fabricating Natural Tunable Bioactive Biomaterials for Corneal Repair and Regeneration

Fabricating thermoresponsive hydrogels from decellularized tissues is a trending and promising approach to develop novel biomaterials for tissue engineering and therapeutic purposes. There are differences in the characteristics of the produced hydrogels related to the source tissue as well as the decellularization and solubilization protocols employed. Detailed characterization of the hydrogels will support the efforts to optimize their application as biomaterials for tissue engineering and therapeutics. Here (Chapter II), we describe an optimized method for fabricating an in-situ thermoresponsive hydrogel from decellularized porcine cornea extracellular matrix (COMatrix), and provide a detailed characterization of its structure, thermoresponsive rheological behavior (heat-induced Sol-Gel transition), as well as exploring its protein composition using proteomics. COMatrix forms a transparent gel (10 minutes time to gelation) after in-situ curing with heat, characterized by alteration in light absorbance and rheological indexes. The rheological characterization of heat-formed COMatrix gel shows similar behavior to common biomaterials utilized in tissue engineering. The fibrillar structure of COMatrix gel was observed by scanning electron microscopy showing that the density of fibers attenuates in lower concentrations. Mass-spectrometry-based proteomic analysis revealed that COMatrix hydrogel is rich in proteins with known regenerative properties such as lumican, keratocan and laminins in addition to structural collagen proteins. COMatrix hydrogel is a naturally-driven biomaterial with favorable biomechanical properties and protein content with potential application as a therapeutic biomaterial in ocular regeneration and tissue engineering. Decellularized tissue-specific extracellular matrix (ECM), as representative of the native tissue, can be processed into hydrogel for cell delivery, tissue engineering and regenerative purposes. We have already shown that a thermoresponsive hydrogel derived from decellularized porcine COrnea Matrix (COMatrix) has promising potentials for corneal epithelial therapeutic applications. However, properties of the hydrogel can be greatly affected by decellularization methods. In this study (Chapter III), we compared the effects of two decellularization protocols, detergent-based (De) and freeze-thaw (FT) on the characteristics of fabricated thermoresponsive and light-curable (LC) COMatrix hydrogels. Both decellularization protocols retained essential ECM components such as collagens and sulfated glycosaminoglycans while significantly reducing the DNA content. Both derived hydrogels (FT-COMatrix and De-COMatrix) showed similar fibrillar structures determined by scanning electron microscopy. Furthermore, the α-Gal epitope content was significantly reduced as a result of both decellularization protocols; and, α-galactosidase treatment, removed the remaining α-Gal epitopes. The thermoresponsive De-COMatrix and FT-COMatrix have similar thermal gelation kinetics analyzed with turbidimetric assay. Moreover, the rheological characterizations revealed significantly higher shear moduli (elastic, G’ and viscous, G”) of thermoresponsive FT-COMatrix versus De-COMatrix. Both thermoresponsive and light-curable hydrogels resulting from FT decellularization have slightly higher light transmission than that of De decellularization. Although the light-curable COMatrices have significantly higher shear moduli (after curing with green light) than thermoresponsive COMatrices, the remarkable difference between those of FT-LC-COMatrix and De-LC-COMatrix is preserved after functionalization to produce light-curable COMatrices. The G’ of FT-LC-COMatrix reached to 18393±1718 Pa, which is ideal for corneal tissue engineering. Lastly, the obtained products from both decellularization methods showed excellent cytocompatibility while seeded by human corneal mesenchymal stem cells (hcMSCs). FT-LC-COMatrix was the only fabricated hydrogel that showed no significant shrinkage against the contracting capability of hcMSCs. The results of this study have shown that there is a significant difference between De and FT decellularization protocols regarding the biomechanical properties of hydrogels derived from porcine corneal ECM, which FT decellularization produces superior products. Bioactive substrates can be used therapeutically to enhance wound healing. Here (Chapter IV), we evaluated the effect of an in-situ thermoresponsive hydrogel from decellularized porcine cornea ECM, COMatrix (COrnea Matrix), for application as an ocular surface bandage for corneal epithelial defects. COMatrix hydrogel was fabricated from decellularized porcine corneas. The effects of COMatrix hydrogel on attachment and proliferation of human corneal epithelial cells (HCECs) were evaluated in vitro. The effect of COMatrix on the expressions of the inflammatory genes, IL-1β, TNF-α, and IL-6 was assessed by RT-PCR. The in-situ application and also repairing effects of COMatrix hydrogel as an ocular bandage was studied in a murine model of corneal epithelial wound. The eyes were examined by optical coherence tomography (OCT) and slit-lamp microscopy in vivo and by histology and immunofluorescence post-mortem. In vitro, COMatrix hydrogel significantly enhanced the attachment and proliferation of HCECs relative to control. HCECs exposed to COMatrix had less induced expression of TNF-α (P<0.05). In vivo, COMatrix formed a uniform hydrogel that adhered to the murine ocular surface after in-situ curing. Corneal epithelial wound closure was significantly accelerated by COMatrix hydrogel compared to control (P<0.01). There was a significant increase in the expression of proliferation marker Ki-67 in wounded corneal epithelium by COMatrix hydrogel compared to control. COMatrix hydrogel is a naturally derived bioactive material with potential application as an ocular surface bandage to enhance epithelial wound healing. There is a growing demand in the clinical practice for novel approaches for in-situ rapid repair of corneal perforations and diseased corneal stroma. Here (Chapter V), we are introducing a novel tunable material derived from decellularized porcine cornea extracellular matrix to address the clinical demand. Light-curable COrnea Matrix (LC-COMatrix) is a ready-to-apply material without the requirement of pre-application preparations (such as warming or mixing) and is representative of the natural cornea composition. It has proper consistency and cohesion before cross-linking that limits spreading to undesired areas following administration. The LC-COMatrix has remarkable adhesion strength to human corneas and can repair large human corneal macro-perforations with tissue-loss. Moreover, LC-COMatrix can seal large corneal perforations (1 mm) as well as replacing the lost partial-thickness corneal stroma in rabbit models. In general, LC-COMatrix is a promising natural bio-adhesive ready-to-apply (single syringe) biomaterial which is representative of native corneal composition and has many potential applications in corneal and ocular surgeries.