Amniotic Membrane as a Substrate for Cultivating Limbal Corneal Epithelial Cells for Autologous Transplantation in Rabbits.

Purpose. To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. Methods. An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. Results. A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. Conclusion. Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier

Growth factor mRNA and protein in preserved human amniotic membrane.

Purpose. To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). Methods. RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2, -3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80°C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2) in preserved human corneas and in AM both with and without amniotic epithelium. Results. RT-PCR revealed that human AM expresses mRNA for EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2, -3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. Conclusions. Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation