The Cellular Biocompatibility Of Chitosan With NIH3T3 Cells Transfected HBMP2 Gene

Periodontitis is a common disease which could lead to tooth loss inadults. To recover construction and function of periodontium by means ofrebuilding new periodontal ligament, cementum and alveolar bone is theideal goal. However, almost all of the method can not attach the goal.Tissue engineering is a subject based on cytobiology, developmentalbiology and biomaterials, which creates new tissue to replace destroyedtissue with development technique. Up to date, a lot of methods onperiodontal tissue engineering have been developed and improved for thedisease in all of world.Three elements tissue engineering,including seed cells havingability of proliferation and differentiation, extracellular matrix (ECM),growth factors. How to combining seed cells with growth factors, thenseed them onto ECM to generate new organs, is a urgent problem to besolved. Bone morphogenetic protein (BMP) has been commonly used forinducing bone regeneration in periodontium. If we transfect BMP gene toseed cells before we seed the cells onto substitution material ofECM,which is called scaffold, the cells will secrete BMP locally toacquire regeneration of periodontium.Scaffold supplies environment for regeneration of organs.Nowadays, people are interested in searching nature scaffolds. Extracted from shells of crustacean, chitosan possesses weak cationoid electrolyte,which is the merely linear chain polysaccharide in nature. Chitosancontains N-acetyl polyamine which is the major elements in ECM.Chitosan can be easily degraded by lysozyme, as well as possessesbroad-spectrum antibiosis, excellent modeling properties. Chitosanpossesses suitable mechanical strengenth and homogenicity to becomescaffold after it becomes membrane. To develop organs by combiningcells transfected gene with chitosan scaffold has rarely been reported.We seeded the NIH3T3 cells transfected hBMP2 gene onto chitosanmembrane,then tested the compatibility by MTT, ALP, etc, in order toinvestigate significance in periodontal tissue engineering.PartⅠProperties and preparation of chitosan membraneObjective: To prepare chitosan membrane for cell culture as well as testwater absorbing capacity and degradation rate in vitro.Method: Chitosan membrane was fabricated by casting method. Chitosanpowder was dissolved in acetic acid (1.5%, v/v) to make 2% chitosansolution. It was poured onto round glass cover slip, whose diameter wasequal to the aperture of 24-well plastic culture plates, then was dried. Thewater absorbing capacity and degradation rate in vitro of 2% chitosanmembrane were all tested.Result: The surface of chitosan membrane was even and adhered to glasscover slip firm. The margin did not wrap. The mean water absorbing capacity of 2% chitosan membrane was 106.5%. 2% chitosan membranewas degraded more slowly with PBS than with lysozyme. The weightlose rate at 60th day was only 17.8%. However, with lysozyme, theweight lose rate was 8.8% at 10th day, 30% at 30th day, 50% at 40th day,almost dissolved at 60th day.Conclusion: It can be concluded that chitosan possesses excellent watercapacity after it becomes membrane. Chitosan membrane can bedegraded more rapidly with lysozyme in this test.PartⅡGrowth,proliferation activity of expression human bonemorphogenetic protein2 fibroblasts on chitosan membraneObjective: The behavior of expression human bone morphogeneticprotein2 fibroblasts including growth, proliferation was analyzed.Method: First, the growth curve of cells was draw to investigate thegrowth rule, then cells were digested and seeded, which were divided intochitosan membrane group (in which the cells were cultured on chitosanmembrane) and control group (in which cells were cultured on glasscover slip without chitosan).After 6 hours, the initial adherence rate wastested. On the second/fourth/sixth day, the cells were observed underinverted phase contrast microscope and scanning electric microscope.Proliferation of transfected cells was evaluated by MTT assay.Results: The proliferation of NIH3T3 cells decreased as transfected byBMP2 gene.After 6 hours, the initial adherence on the chitosan membrane was better than control groups. The cells could adhere to chitosanmembrane well and keep cellular morphology, and proliferation indexaccelerated on the membrane compared with control group (P＜0.05).Conclusion: Chitosan membrane can promote growth, adherence andproliferation of the cells transfected gene.PartⅢOsteogenic differentiation of expression human bonemorphogenetic protein2 fibroblasts on chitosan membraneObjective: The osteogenic differentiation of expression human bonemorphogenetic protein2 fibroblasts on chitosan membrane wasanalyzed.Method: The cells were digested and seeded, which were divided intochitosan membrane group (in which the cells were cultured on chitosanmembrane) and control group (in which cells were cultured on glasscover slip without chitosan membrane). On the second/fourth/sixth day,cells of each group were digested. 2×10~4/4×10~4/6×10~4 cells wereevaluated by alkaline phosphatase (ALP) assay on different days.Results: There was no significant difference between two groups.Conclusion: It can be concluded that chitosan membrane does not inhibitosteogenic differentiation of cells to keep the differentiation of cells.