The development of a serum-free derived bioengineered conjunctival epithelial equivalent using an ultrathin poly(epsilon-caprolactone) membrane substrate

PURPOSE: To evaluate the use of an ultrathin poly(epsilon-caprolactone) (PCL) membrane as a substrate for the development of a serum-free-derived conjunctival epithelial equivalent. METHODS: Ultrathin PCL membranes 6 microm in thickness were prepared by solvent casting and biaxial stretching and analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), tensile testing, and water-contact angle measurement. Rabbit conjunctival epithelial cells were cultivated on sodium hydroxide (NaOH)-treated PCL membranes and untreated PCL membranes in serum-free medium. The proliferative capacity of cultivated cells was analyzed with a bromodeoxyuridine (BrdU) ELISA proliferation assay. Conjunctival equivalents were xenografted into severe combined immune-deficient (SCID) mice. Immunostaining for tissue-specific and basement membrane-related proteins was performed. RESULTS: After biaxial stretching, the tensile strength of PCL membranes increased from 21 to 42 MPa, with a Young's modulus of 225 MPa. AFM and SEM showed that biaxially stretched PCL membranes consisted of closely packed microfibrils. PCL membranes supported the attachment and proliferation of conjunctival epithelial cells to form confluent stratified epithelial sheets. Surface modification with NaOH resulted in greater hydrophilicity and cellular proliferation than that of untreated membranes. Transplanted conjunctival equivalents underwent greater proliferation and stratification in vivo. Cultivated conjunctival cells expressed K4, K19, MUC5AC, and Ki67, whereas collagen IV and integrin beta4 were detected at the basement membrane junction. CONCLUSIONS: An ultrathin PCL membrane was shown to be biocompatible, mechanically strong enough to stand up to handling, and able to support conjunctival epithelial cell proliferation. This membrane may have potential for use as a scaffold matrix for tissue-engineered conjunctival equivalents.