| Since nearly ten years, it has been showed that periodontal ligament cell (PDLC) is a kind of polyclonal cells. It has been believed that PDLC consists of three kinds of subgroups--osteoblasts, cementoblasts and fibroblasts, in which they have different function and quality. The three kinds of cells respectively come into being alveolar bone, cementum and periodontal ligament fibers. And osteoblasts and cementoblasts belong to osteogenetic subgroup because of their special function of forming mineralized tissue. None but proliferation of PDLC and formation of relevant tissue can induce the really regeneration of periodontal mineralized tissue and periodontal ligament fibers which embed into mineralized tissue. Meanwhile, it has been domenstrated that there are some progenitor cells in PDLC and the osteogenetic differentiation of these progenitors is the base to form new attachment of periodontal ligament. So the osteogenetic differentiation of PDLC is the key part inguided tissue regeneration(GTR). But now the perfect materials that can induce the osteogenetic differentiation of PDLC are lack.The objective of this study is to fabricate nano-hydroxyapatite powders, observe and analyze the microstructure of these powers and primarily investigate effects of nanophase powders of hydroxyapatite on proliferation and osteogenetic differentiation of PDLC.This study includes three sections as following:Part one: Analysis of the microstructure of nanophase hydroxyapatiteObjective: To analyze the microstructure and ingredient of experiment materials with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy disperse X-ray analyzer (EDAX) and primarily culture and identify the human periodontal ligament cells.Materials and Methods: Nano-hydroxyapatite powders were fabricated with sol-gel method. They were used aqueous solution of calcium nitrate tetrahydrate [Ca(NO3)2.4H2O] and alcohol solution of trimethyl phosphate((CH3O)3PO) as precursors. A small amount of citric acid (about 5%) used as a chelating reagent was added in aqueous solution of calcium nitrate tetrahydrate. The solutions were mixed at room temperature with Ca/P mole ratio of 5:3 and were kept at pH7.5 by ammonia hydroxide. After 48 h reaction, the gel thus obtained was dried at 190°C for 2 h, calcined at 600°C for 1 h and ground into fine powders. The physical and chemical characters of these nanophase hydroxyapatite powders were observed through scanning electron microscopy (SEM),transmission electron microscopy (TEM) and energy disperse X-ray analyzer (EDAX).Results: In SEM, the average dimension of nanophase hydroxyapatite powders was less than lum magnified to 7000 times. The result showed that the addition of citric acid as a chelating reagent during the sol-gel process could prevent the agglomeration of hydroxyapatite powders and separate the powers well proportioned. In TEM, a lot of nanophase particles were observed with dimension of average less than 50 nm.Conclusion: The addition of chelating reagent makes the sol be the stable gel, in which the produced hydroxyapatite gel particles through the hydrolysis and condensation reactions are separated well-proportioned and are difficult to aggregate and grow. Finally the well-proportioned dispersed nanophase particles come into being.Part two: The effects of proliferation and morphological analysis of PDLC incubated with nanophase hydroxyapatiteObjective: To observation the effects of proliferation and morphological changes of PDLC incubated with nanophase hydroxyapatite with optical microscope, scanning electron microscopy, transmission electron microscopy and energy disperse X-ray analyzer.?The primary culture and identification of human periodontal ligament cells and incubation and identification of human periodontal ligament cellMaterials and Methods: Human periodontal ligament fibroblast-like cells was obtained through different enzyme digestion method. Healthy permanent teeth, which just had been surgically extracted, were transited into DMEM and were cleaned. After periodontal ligament was scraped from mid-third of the root and cut to pieces, the sample was moved into a sterilized centrifuge tube with 0.25% collagenase and stirred at 37°C for 50-60 min to separate the tissue. Then the sample was digested with 0.125% trypsin as far as a majority of cells had been dissociated. After the cells suspension was centrifugated, supernatant fluid was seeded in culture flasks with enough medium in humidified 95% oxygen, 5% CO2 atmosphere at 37°C.Results: Primary passage PDLC began to attach after 24h and mostly cells attached after 48h. Rounded suspension cells turned to be shuttled or stellate after attachment. First medium exchange removed un-adherent cells and tissue pieces. A few adherent cells that were flat and multangular were likely to be epithelioid cells. From the third day, shuttled and stellate cells showed active proliferation and quick growth speed, but the growth speed of multangular cells was slow. Confluent monolayer cells were obtained after six days, which were a great number of shuttled cells. Epithelioid cells were mostly removed after the second passage.?The effect of nanophase hydroxyapatite on the proliferation activity of PDLCMaterials and Methods: The fourth passage periodontal ligament cells were digested by 0.25% trypsin. 10%FBS 1640 was immediately added to end digestion, after a majority of cells had been rounded. Afterthe cells suspension was centrifugated (lOOOrps) for lOmin, supernatant fluid was removed and cells were resuspended by 10%FBS 1640. Cells were seeded at a concentration of lxlO4 cells/well for 1 ml in 24 well plates. After 24 h, nanophase hydroxyapatite powder (nano-HA), dense hydroxyapatite powder (dense-HA) and only medium as control were added into wells respectively. On the 2, 3, 4 day, the proliferation activity of PDLC was detected with MTT. The OD450 value was determined with ELIS A analyzer.Results: MTT showed that on the second day, the proliferation of nano-HA group was significant. On the third days, the proliferation of nano-HA group was quickened; on the fourth day, the cell numbers of nano-HA group were significantly more than others two groups. But there were not significant difference between the dense-HA group and the control. The result also showed that peak proliferation appeared at day 4 as well as the dense-HA and control group.? Morphological analysis of PDLC incubated with nanophase hydroxyapatiteMaterials and Methods: The morphological changes of cells were examined by upside-down microscope, scanning electron microscopy, transmission electron microscopy and energy disperse X-ray analyzer after had been cultured with materials for 5d.Results: In upside-down microscope, cells grew fine and had long cytoplasmic extensions after incubated three days with nanophase hydroxyapatite while cells incubated with dense-HA grew slowly. In SEM, after added nanoparticles of hydroxyapatite, isolated cellsexpressed satellite morphology, displaying numerous long cytoplasmic extensions and marked nucleoli. In nanoHA group, observation of these cells was showed as many particles-like materials sticked on the surface of cells and some were phagocytotic process in cells. The energy disperses X-ray analysis of granules showed that they were hydroxyapatite. The results of TEM showed that there was an abundant of conglobulations in cells cultured with nano-HA, which were 5nm in diameter and gathered in the PDLC cultured with the nanometer HA. The electronic density of the globe-like granules was obviously higher than the cytoplasm and organelle of cells. Cells swallowing granules were showed as abundant mitochondria and marked rough endoplasmic reticulum as well as nucleolus. The energy disperses X-ray analysis of granules showed that the content of these particles is consistent with that of hydroxyapatite. Besides, the shape of PDLC swallowing abundant hydroxyapatite was normal.Conclusion: There is important effect of nanophase hydroxyapatite on the proliferation activity of PDLC. PDLC can swallow nanophase hydroxyapatite into cytoplasm in good growth condition. It indicated that nanophase hydroxyapatite had better biological compatibility and is a new kind of absorbable biomaterial.Part three: The effect of nanophase hydroxyapatite on the osteogenetic differentiation of PDLCObjective: To investigate the effect of nanophase hydroxyapatite on the proliferation and the osteogenetic differentiation of PDLC. Toevaluate the meaning of nanophase hydroxyapatite in the differentiation function of periodontal ligament progenitor cells.? The effect of nanophase hydroxyapatite on the alkaline phosphatase (ALP) specific activity of PDLCMaterials and Methods: On the 5, 8 d of culture, cells were rinsed with PBS free Ca2+ and Mg2+ twice. Cells were scraped and collected into cell fracture solution, and frozen (-20 °C) three times to disrupt cell membranes. Thawed cell suspensions were sonicated for 5 minutes and were centrifugated for 15min. Alkaline phosphatase activity was measured on the supernatant according to the instruction of reagent package. The absorbance at 520nm was read on a plate reader.Results: There were significant differences between nano-HA group, dense-HA group and control group on the 5th and 8th day of culture. It is showed that cells of nano-HA group express the tendency of osteogenetic differentiation.?The effects of nanophase hydroxyapatite on PDLC with ALP immunohistochemical assay and flow cytometry analysisMaterials and Methods: After cells had been incubated with three kinds of materials as above in humidified 95% oxygen, 5% CO2 atmosphere at 37°C for 5 d, the cells that attached on sterile glass slice were fixed with 90% alcohol for 2h. Then cells sample were stained with ALP immunohistochemical assay. The residual cells were digested with 0.25% trypsin, centrifugated and resuspended. At last the suspension cells were analyzed with ALP positive flow cytometry.Results: A majority of nano-HA group and dense-HA group cellssample showed positive ALP immunohistochemical stain. But the ALP positive stain of nano-HA group cells sample was denser than dense-HA group. In FCM, distribution of ALP positive cells cultured with nanoparticles were significantly greater than others groups. The number of positive cells on nano-HA group was the highest one and the control was the lowest.Conclusion: The nano-hydroxyapatite, as a calcium phosphate biomaterial, has ability to promotes the activity of osteogenetic differentiation for periodontal ligament cells compared with dense-HABased on these findings, we can get the conclusion that nanohydroxyapatite appears significant effects on proliferation and differentition of periodontal ligament cells compared with dense hydroxyapatite. Besides, the particles of nanophase hydroxyapatite can be swallowed by PDLC and it, from our experiments, is a available biomaterial in guided periodontal tissue regeneration.
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