A Study Of The Culture Of Oral Mucosal Epithelial Cells In Vitro And The Preparation Of A Temperature-sensitive Scaffold Material

As the substitution of cornea, tissue engineering is applied for the repair on many aspects of injuries successfully, which is sufficient with good physiological activity and no postoperative rejection. Among them, the construction of corneal epithelium plays a critical role in the treatment of limbal stem cell deficiency. Both scaffold material and seed cell are of utmost importance. It is valuable to explore how to construct tissue engineering cornea with good physiological activity and normal physical function. Scaffold material provides the place for cell proliferation, guides the cell growth and decides the tissue morphology. With the development of tissue engineering, synthetic intelligent polymer materials are widely applied in the biomedical field. Poly N-Isopropylacrylamide (PNIPAAm) is a fast temperature-sensitive intelligent polymer, which has a rapid and reversible structural change in accordance with minimal stimulation outside. It facilitates to separate the epithelium and the material, solving the problem of postoperative biodegradation of materials and forming the carrier-free scaffold material. Oral mucosa is easy taking and sufficient, which is full of stem cells in the epithelial layer with strong amplified ability in vitro. Therefore, it is a hotspot to construct corneal epithelium, taking advantage of oral mucosa as the seed cell.The research is of two parts as follows:the first is prepare the temperature-sensitive scaffold material by graft copolymerization of PNIPAAm onto Polystyrene (PS), insuring its temperature-sensitive property; the second is to to take advantage of epithelial cells of rabbit oral mucosa to culture in vitro, commanding the amplification rule. It may lay the foundation for further using oral mucosal epithelial cells as the seed cell and temperature-sensitive materials as the scaffold material to repair corneal epithelial defect. It may provide evidence of new clinical treatments of the corneal stem cell deficiency. Part I: A study of the preparation of a temperature-sensitive scaffold materialPurpose:Preparing the temperature-sensitive polymer membrane and understanding its chemical properties might lay the foundation for further application of "no carrier" scaffold material for cell cultivation.Methods:PNIPAAm was spread onto commercial cell culture inserts and subjected to irradiation to prepare PS-g-NIPAAm polymer membrane. The grafting ratio was measured by weighing the change of the polymer membrane. The chemical composition was evaluated by the infrared spectrometer. The property of hydrophobicity and hydrophilicity was analyzed by the contact angle measurement. The lower critical solution temperature was measured by the differential thermal analyzer. The kinetic curve of de-swelling and re-swelling was obtained by measuring the swelling ratio at different temperatures (25～45℃), ensuring the swelling property of the polymer membrane. The ultrastructure of polymer membrane was observed by electron microscopy.Results:The grafting ratio of the PS-g-NIPAAm polymer membrane was 39.2%. It was proved that PNIPAAm had been grafted on the polystyrene by the fact that the overlapping peak of amino and carbonyl were observed at about 1650cm-1 on the infrared spectrum. The contact angle was 46.9°, suggesting the hydrophilicity of PS-g-NIPAAm. The LCST was about 35℃. Membranes swelled below the LCST to the extend that the swelling ratio was 10 times more than that of the dry state, but rapidly de-swelled above the LSCT and became almost the same as that of the dry state. Though electron microscope, NIPAAm has a uniform distribution on the PS membrane.Conclusions:PS-g-NIPAAm polymer membrane which has a good temperature sensitivity and swelling properties provides the evidence for the application of the "no carrier" scaffold material. Part II:A study of the culture of oral mucosal epithelial cells in vitroPurpose:To investigate the optimal procedure for the culture and amplification of rabbit oral mucosal epithelial cells to lay the foundation for further using oral mucosal epithelial cells as the seed cell to repair corneal epithelial defect.Methods:OMEC from New Zealand rabbit were primarily cultured with dispase and trypsin-EDTA digestion, and medium was changed periodically. The biological characteristics of the cells were observed through phase microscope and electron microscope. The growth curve and cloning efficiency was used to measure basic law, proliferative ability.Results:Oral mucosal epithelial cells well-cultured were oval with large nucleus and normal karyokinesis phase, connecting like flagstones. By transmission electron microscope, epithelial cells had oval nucleus and normal organelles, especial numerous reticular or fasciculate tonofibrils in the cytoplasm. There were many microvillis on the cellular surface. After cells inoculation,1～4 days was latent phase, 6-11 days was logarithmic phase and stagnate phase was from 13 days. The cloning efficiency was 0.75% at the inoculating density of 200 cells/cm2.Conclusions:Oral mucosal epithelial cells could be cultured in vitro, keeping a good ability of proliferation in a certain time and laying the foundation for the reconstruction of the ocular surface by tissue engineering oral mucosal epithelium.