A Hyaluronan Hydrogel Scaffold for Culture of Human Oral Mucosal Epithelial Cells in Limbal Stem-Cell Therapy

Hyaluronan (HA), a major component of the extracellular matrix, plays a key role in cell proliferation, growth, survival, polarization and differentiation. We investigated the optimization of a HA hydrogel scaffold for culture of human oral mucosal epithelial cells (OMECs) for potential application in limbal stem cell therapy. The effect of the optimized scaffold on OMEC cell sheet morphology, cell metabolic activity and expression of genes associated with stemness, adherence and cell damage was studied. The results indicate that HA hydrogels crosslinked with polyethylene glycol diacrylate (PEGDA) failed to support OMEC attachment and growth. However, HA hydrogel scaffolds dried for three days and coated with 1 mg/mL collagen IV produced a full OMEC sheet. Cell morphology was comparable to control after three weeks culture, maintaining 76% metabolic activity. Of apoptosis-related genes, the pro-apoptotic markers CASP3 and BAX2 were upregulated and downregulated, respectively, compared to control whereas the anti-apoptotic marker BCL2 was downregulated. The expression level of stemness genes Delta Np63 alpha and ABCG2 was significantly higher than control. Genes associated with improved scar-less wound healing (integrin-alpha V) and protection of the ocular surface (cadherin-1) had similar to 3-fold increased expression. These data suggest that our optimized HA-hydrogel scaffold could enhance culture of OMEC cell sheets for use in ocular reconstruction.