Biofunctionalization of gelatin microcarrier with oxidized hyaluronic acid for corneal keratocyte cultivation

Development of microcarriers as alternative architecture models to traditional three-dimensional porous scaffolds can potentially eliminate the concern of extensive geographic necrosis during keratocyte cultivation. For the first time, this work was performed to evaluate the in vitro expansion of rabbit corneal keratocytes (RCKs) on the gelatin microcarrier (GMC) modified with hyaluronic acid (HA). In order to confirm the successful synthesis, the oxidized HA (oHA) samples following treatment of HA with sodium periodate were investigated by chemical characterization, including Fourier transform infrared and nuclear magnetic resonance spectroscopy. Biofunctionalization of GMC with oHA was then achieved by the formation of aldimine linkage between free amino groups in protein and aldehyde groups in oxidized polysaccharide. The grafting yield of oHA onto GMC determined by alcian blue staining assay was 26.9 ± 0.1%. Tissue culture polystyrene (TCPS) plates and the microcarriers from either the GMC or GMC-oHA group were used for suspension culture of RCKs. Results of WST-1 assays, scanning electron microscopy, extracellular matrix production assays, and quantitative real-time reverse transcription polymerase chain reaction and Western blot analysis showed that among all the groups studied, the GMC-oHA samples could most effectively support a large scale growth of RCKs while enhancing their total collagen and glycosaminoglycan contents (i.e., indicative of biosynthetic capacity) and keratocan and ALDH1 gene and protein expressions (i.e., indicative of cell phenotype and function). It is concluded that the aforementioned keratocyte behaviors are found to be strongly dependent on the type of culture substrate (i.e., TCPS plate versus biopolymer microcarrier) and on the biomaterial functionalization (i.e., modification of gelatin with oHA).