ABSTRACT
The development of transplantable cell sheets of functional keratocytes embedded within an aligned collagen type I matrix is a viable approach for constructing a bioequivalent of corneal stroma. Thermoresponsive materials based on poly(N-isopropylacrylamide) (PolyNIPA) have been utilized to recover carrier-free corneal cell sheets by inducing temperature changes. In this study, we employed direct-write assembly (DWA) to develop microperiodic parallel patterns of silk-PolyNIPA and gelatin-PolyNIPA. Semi-interpenetrating networks of PolyNIPA hybrids (with silk/gelatin) exhibited temperature-responsive nature and thereby have potential use in cell sheet engineering. Silk-PolyNIPA and gelatin-PolyNIPA hybrids demonstrated a hydrophobic surface at 37 °C (i.e. above their lower critical solution temperature) with a contact angle of 59°± 0.3° and 55°± 3°, respectively, whereas the surface roughness of silk-PolyNIPA was double that of gelatin-PolyNIPA. The reduction of temperature to 20 °C resulted in a decrease in the value of surface roughness and water contact angle for both hybrids. All four parallel patterned substrates guided corneal cell alignment along the direction of the patterns. Collagen type-I and aggrecan gene expression was higher when the cells were grown over the gelatin-PolyNIPA matrix after 3 weeks of culture when compared to silk-PolyNIPA. In addition, a significantly higher metabolic activity as well as enhanced vinculin expression of keratocytes on the gelatin-PolyNIPA matrix indicated the improved cytocompability compared to the silk, gelatin and silk-PolyNIPA matrices. Interestingly, the detachment of keratocytes cell sheet was achieved from the silk-PolyNIPA and gelatin-PolyNIPA planar films only within 10 min and 30 min, respectively, but the patterns could not yield intact sheet recovery. Hence, we conclude that while gelatin-PolyNIPA hybrids with parallel patterns fabricated using DWA will benefit from the application of cellular alignment, some optimization in the pattern parameters may be required for rapid sheet recovery from such substrates. Understanding the keratocytes responses to such hybrid biomaterials suggests viable options to develop a corneal stromal bioequivalent.
ABSTRACT
PURPOSE: To culture and characterize oral mucosal epithelial cells (OMEC) grown on de-epithelialized human amniotic membrane (HAM) to explore their suitability as autografts in patients with bilateral ocular surface disease (OSD) and limbal stem cell deficiency. METHODS: Oral biopsy samples were obtained from 20 patients undergoing oral reconstructive surgery, with informed consent and Institutional Ethics Committee approval. Morphologic studies, transmission electron microscopy (TEM), reverse transcriptase (RT) PCR and immunocytochemistry were used to characterize the OMEC. RESULTS: Morphologic studies and TEM revealed a confluent sheet of proliferating, stratified oral epithelial cells connected to each other by desmosomes, containing intracellular cytokeratins and abundant mucin granules. These characteristics were further corroborated and elucidated by RT-PCR and immunocytochemistry. The presence of markers of differentiated, stratified epithelial cells (cytokeratin K3, K4, K13, and connexin 43), progenitor stem cell cell markers (p63, p75, ß1-integrin/CD29, and ABCG2), and a variety of predominantly membrane-bound and a few gel-forming mucins (MUC 1, 5B, 6, 13, 15, and 16) was established. CONCLUSIONS: Cultured OMEC have the potential to act as autografts for ocular surface reconstruction in patients with bilateral ocular surface disease and can prove to be particularly beneficial to ameliorate the mucin deficiency state in dry eye associated with OSD.
