Study The Effect On Proliferation And Cell Cycle Of Human Periodontal Ligament Cells By Nano-hydroxyapatite Suspension

Objectives1,Discussed the effect on periodontal and cell cycle of human ligament cells by hydroxyapatite suspension in the initial,so as to probe the application of nano-hydroxyapatite used in the treatment of disease.2,In preparation for the future research on apoptosis of associated fibroblasts by the drug which was loaded with nano and mesoporous materials.Method1,Using tissue culture method to culture, identificate and passage human periodontal ligament fibroblasts, the 4th-6th fibroblasts were used for the experiment.2,Prepared hydroxyapatite in the laboratory,used sodium carboxymethyl cellulose, sodium hexametaphosphate as dispersant. Compared the dispersion between the two dispersants,and compared the cell growth by cell culturing, choosed a suitable dispersant for the next researth.3,Used the better dispersant which was choosed from the two above to disperse nano-hydroxyapatite.Prepared nano-hydroxyapatite suspension in 1%concentration.then diluted the concentration to 0.5%,0.25%,0.1%, 0.05% and 0.025%. Set some controls just as the blank, sigle hydroxyap-atite group in 0.1% concentration,sodium carboxymethyl cellulose in 0.1% concentration, mesoporous materials in 0.005% concentration and calcium hydroxide in 0.01% concentration.Then cultured the human periodontal ligament fibroblasts for 24 hours,48 huors and 72 huors in mdium with the groups above.And measured the proliferation of cells in each group with MTT to evaluate the proliferation effect of human periodontal ligament fibroblasts.4^ Selected the suspension in 0.5% concentration as a new group,with the control groups together,human periodontal ligament fibroblasts were went on to culture for 48 huors.Then detected cell cycle distribution by flow cytometry to analysis the impacts of each group on culturing. Result1,Successfully cultured human periodontal ligament fibroblasts which identified by anti-vimentin and anti-keratin staining:anti-vimentin-positi-ve,and anti-keratin-negative.2,Prepared nano-hydroxyapatite by chemical precipitation in laboratory, by testing, the diameter of nano-hydroxyapatite was from 40-60nm,which was required to this experiment. By comparing the sodium hexameta-phosphate and sodium carboxymethyl cellulose on the dispersion of nano-hydroxyapatite, sodium hexametaphosphate could be drawn to better in dispersing hydroxyapatite.However,it led to a large number of dead cells when nano-hydroxyapatite suspension which dispersed by sodium hexametaphosphate that was added in medium to culture human periodontal ligament fibroblasts.But the cells grow well in another group in which nano-hydroxyapatite was dispersed by sodium carboxymethyl cellulose. So sodium carboxymethyl cellulose was choosen to went on reseach as the dispersant we wanted to disperse the nano-hydroxyapatite suspension.3,Cultured cells in different concentrations and multiple group which set for the control,then used MTT to detect proliferation of cells.The effect of proliferation was found significantly higher than other groups, when nano-hydroxyapatite was dispersed with sodium carboxymethyl cellulose in 0.5% concentration. At the same concentration(0.1%), the effect of cell proliferation was better in nano-hydroxyapatite suspension group than single nano-hydroxyapatite group.4,The result by flow cytometry showed that it would reduce the number of cells in G0/G1 phase,when cell cultured in the group which nano-hydr-oxyapatite was dispersed in sodium carboxymethyl cellulose.At the same time, the number of cells in S phase would increase. It could be infered that human periodontal ligament fibroblasts would transite G0/G1 to S phase with the effect of nano-hydroxyapatite. Conclusion1,Dispersed in sodium carboxymethyl cellulose, nano-hydroxyapatite suspension had a good nano activity in its suspension,and it significantly promoted the proliferation of human periodontal ligament fibroblasts. Within the isotonic concentration, the higher the concentration was, the deeper the effection would be on the cell proliferation,and it led an obvious effect at the first 24 hours.2,Dispersed in sodium carboxymethyl cellulose, nano-hydroxyapatite suspension acting on human periodontal ligament fibroblasts can shorten the G0/G1 phase time in human periodontal ligament fibroblasts' mitosis cycle, and made relatively large number of cells transite to S phase,for this it accelerated proliferation rate of human periodontal ligament fibroblasts3,Through this study,It further affirmed that nano-hydroxyapatite can be used for reconstruction of periodontal soft tissue,and it provide a reference for nano-hydroxyapatite using in the treatment of periodontal disease.