Use of Polymeric Scaffolds as Extracellular Matrix Substitutes for Conjunctival Epithelial Repair

Abstract

Purpose: The aim of this study is to investigate the use of artificial scaffolds to replace the native extracellular matrix (ECM) and use for the repair of wounded conjunctiva. We hypothesize that a polymeric scaffold, that approximates the mechanical properties of the native conjunctiva, will induce optimum growth and cellular viability of conjunctival goblet and stratified squamous epithelial cells.

Methods: Using electrospinning techniques, we fabricated porous polymeric scaffolds made of poly(caprolactone) (PCL), poly(vinyl alcohol) (PVA) and crosslinked poly(acrylic acid) (PAA). Polyethylene terephthalate (PET) scaffolds in tissue culture inserts were purchased and used for comparison. Explants from cadaveric human conjunctival tissue were placed on each scaffold and cultured in 10% FBS supplemented RPMI-1640 medium for 12 to 15 days. After fixation and DAPI staining, outgrowth area relative to explant size was calculated. A live-dead assay was performed, and the percentage of viable cells was calculated after confocal laser scanning microscopy. Scaffolds with cultured cells were processed for immunocytochemistry (ICC) and the expression of markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were visualized by confocal laser scanning microscopy.

Results: The percentage of viable cells was 89.9±5.6% (n=5) for PAA, 3.6±2.2% (n=5) for PCL, 0% for PVA (n=3) and 92.7%±5.4% for PET (n=3). The difference between PAA and PET was non-significant (p=0.938), whereas both PCL and PVA was significantly different from PET (p<0.001). Fold growth was 4.8±3.4 for PAA (n=3), 2.3±0.3 for PCL (n=4), 0 for PVA (n=3) and 123.8±45.0 for PET (n=2). The difference was significantly different for all 3 electrospun scaffolds compared to PET (p=0.002 for PAA, p=0.001 for both PCL and PVA). On PCL ICC showed occasional islands of CK7 positive cells among many DAPI positive cells of presumed epithelial origin. Occasional CK4 positive cells on PCL did not have typical epithelial morphology. ICC was not performed for PVA, as cells did not grow on these membranes.

Conclusions: PAA can be used as a substrate for conjunctival cell culture, and its polyanionic nature at physiological pH may play an important role. PCL supports high densities of cultured conjunctival goblet cells. These materials have potential for use in conjunctival transplantation. PVA did not support cell growth.

Keywords: 474 conjunctiva • 607 nanotechnology