The Development Of A Bioengineered Conjunctival Epithelial Equivalent Using An Ultra–thin Polycaprolactone Membrane Substrate

Abstract

Abstract: : Purpose: To evaluate the use of an ultra–thin polycaprolactone (PCL) membrane as a substrate for the development of a bioengineered conjunctival tissue–equivalent. Methods: Ultra–thin PCL membranes 5µm in thickness were prepared by solvent casting and biaxial stretching, and analyzed by atomic force microscopy (AFM). Rabbit conjunctival epithelial cells were cultivated on sodium hydroxide (NaOH)–treated PCL membranes and untreated PCL membranes using serum–free media. Bromodeoxyuridine (BrdU) ELISA proliferation assays of the conjunctival cells were performed. The morphology and growth characteristics of the cells were monitored, and immunostaining with pancytokeratin (AE1/AE3), keratin 3 (AE5) and keratin 4 was performed. Results: AFM showed that biaxially stretched PCL membranes consisted of polymer molecules that appeared as closely packed fibrils, oriented mostly in a uniaxial direction. The membranes were highly flexible, easy to handle, and had a high strength–to–mass ratio. PCL was found to support the attachment and proliferation of conjunctival epithelial cells, forming confluent epithelial sheets on the membranes. Treatment of membranes with NaOH resulted in greater cell attachment and proliferation compared to untreated membranes. The mean BrdU absorbance of conjunctival epithelial cells cultivated on NaOH–treated PCL and untreated PCL, was 0.63±0.08 and 0.50±0.06, respectively. The cells expressed keratin 4, and not the cornea–associated keratin 3, which was consistent with the in vivo conjunctival phenotype. Conclusions:A biaxially stretched ultra–thin PCL membrane was shown to support the proliferation of conjunctival epithelial cells. This may potentially be used as a scaffold matrix in tissue–engineered conjunctival equivalents.

Keywords: conjunctiva • cornea: epithelium