Corneal Wound Healing Effects of Solubilized Porcine Cornea Extracellular-Matrix

Abstract

Purpose : Extracellular matrix (ECM) has a vital role in mediating proliferation, differentiation and migration of corneal epithelial cells during homeostasis and wound healing conditions. Natural biomaterials rich in ECM components can be used therapeutically to facilitate corneal wound healing. We investigated the biochemical profile and corneal wound healing effects of decellularized and solubilized porcine cornea-ECM (PC-ECM) as a potential source for producing therapeutic ECMs comparable to human amniotic membrane (HAM)-ECM.

Methods : Porcine eyeballs and HAMs were obtained freshly from certified slaughterhouse and elective caesarean sections, respectively. HAM and PC tissues were decellularized by several freeze-thaw cycles followed by treatment with nucleases. Then, the tissues were lyophilized, cryo-milled and solubilized (20 mg/ml) in 0.01M HCl containing 1 mg/ml pepsin in 25°C with gentle shaking for 72h, and neutralized using NaOH and 10X PBS. The amount of sulfated glycosaminoglycans (sGAGs), collagen and DNA were measured using sGAG, hydroxyproline and DNA quantification assays. The proliferation of primary corneal epithelial cells (pCECs) on plates coated with solubilized-ECMs were evaluated using a Cell Counting Kit. The healing effects of fabricated ECMs were investigated in 2-mm corneal wound healing model.

Results : The amount of DNA (µg/mg dried ECM) were significantly lower in decellularized ECMs compared to non-decellularized samples (3.7±2.4 vs 6.7±3.3 and 2.7±2.6 vs 0.9±0.9 in HAM and PC-ECM, respectively, P<0.05). sGAGs contents were more in PC-ECM (0.17±0.01 mg/mg dried-ECM) than HAM-ECM (0.06±0.01 mg/mg dried ECM, p<0.05). Total collagen content was 0.75±0.07 mg/mg dried ECM in PC-ECM and 0.79±0.08 mg/mg dried ECM in HAM-ECM (P>0.05). The proliferation of pCECs was significantly faster on both PC- and HAM-ECM coatings compared to no-coating (ODs, 0.5±0.1, 0.55±0.07, and 0.41±0.05, respectively, P<0.05). Both PC-ECM and HAM-ECM led to significantly faster closure of 2-mm corneal wounds compared to controls (88.8±2.2%, 90.7±0.3%, and 58.6±6.8%, respectively, P<0.05).

Conclusions : Solubilized PC-ECM promoted corneal epithelial wound healing. Easy processing, low cost and low chance of disease transmission as well as high rate of reproducibility, make the PC-ECM a promising natural bio-active biomaterial/hydrogel for corneal tissue engineering applications.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.