Experimental Research Of Culturing And Characterizing HDFCs And Inducing To Orientate Differentiation By Trigger Factors

BACKGROUND:Periodontal regeneration is expected to reconstitute the lost or injured tissues to restore the architecture and function of the periodontal tissues, including cementum, periodontal ligament and alveolar bone. And the basis of periodontal regeneration is periodontal ligament cells(PDLCs), which are the main responsive cells to form the new attachment. But the sources of PDLCs are very limited in the periodontal defects, so that it is difficult to obtain the satisfactory regeneration. Tissue engineering is supposed to be able to solve the problem. Tissue engineering is the emerging field of science aimed at developing techniques for the fabrication of new tissues to replace damaged tissues. To engineer living tissues, cultured cells are coaxed to grow on bioactive degradable scaffolds that provide the physical and chemical cues to guide their differentiation and assembly into three-dimensional tissues.There are three types of cells attending to periodontal regeneration: periodontal ligament fibroblasts, osteoblasts, and cementoblasts. Ideal periodontal regeneration should have alveolar bone, periodontium, and cementum formation. So it is very particular and difficult for periodontal tissue engineering. Cementum formation playsa critical role in penodontai regeneration. Nowadays, several types of cells have been used as seeding cells in penodontai tissue engineering research, such as periodontal ligament cells, bone marrow stem cells, cementoblasts, embryonic stem cells, and gene transfection gingival fibroblasts et al.It has been thought that periodontal tissues are formed from dental follicle. Dental follicle contains subpopulations of mesenchymal progenitors that give rise to periodontal tissue consisting of cementum, periodontal ligament, and alveolar bone. When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. So it has therapic basis to use dental follicle cells as seeding cells in periodontal tissue regeneration research, following treated with inducing factors and obtain the biological features of cementoblast/osteoblast. Enamel matrix proteins which secreted by Hertwig root sheath cells pay its important actions during the period of periodontal tissue development. Many of the BMPs, which belong to the transforming growth factor (TGF)8 superfamily, have been found in high concentration in mineralized tissue and exhibit osteogenetic activity. Bone morphologic protein 2(BMP-2) have emerged as inducing factors to promote regeneration of periodontal tissues. BMP-2 is an active factor in early tooth development. It has been confirmed that EMPs and BMP-2 have the ability to affect the biological behavior of dental follicle cells and to induce them differentiate to osteoblasts/cementoblasts in vitro. Therefore, it is possible to use dental follicle cells as seeding cells in periodontal regeneration research, after induced by trigger factors such as EMPs, BMP-2 to obtain the differentiate tendency. In addition, isolating and culturing dental follicle cells and observing their characterization could not only provide some advantageous pathways for periodontal regeneration research, but also pay its critical actions for raise teeth developmental biological theory. OBJECTIVES:In order to study the biological features of dental follicle cells and to investigatethe potential mechanism that dental follicle cells differentiate into periodontal tissue, and to demonstrate the dental follicle cells can be used as seeding cells in periodontal tissue engineering, we cultured human dental follicle cells in vitro and studied their characterization. Through detecting cell proliferation, alkaline phosphatase activity, observing the cells' ability of mineralization, and proteins' synthesis, we focused on determining the ability of EMPs and BMP-2 to promote the differentiation of follicle cells along a cementoblast/osteoblast pathway. We did some prime study using HDFC combined with EMPs and BMP-2 in periodontal tissue engineering and tend to provide a new pathway for the therapy of periodontal disease. Then cells-made carrier, Collagen gel, CBHA had been used as scaffolds to cultured with HDFC, their compatibility were observed by microscopy and SEM, and then cell/scaffolds were transplanted into nude mice for 6 weeks to elucidate the auto-differentiation capacity of HDFC.METHODS:Part 1, Culture and identification of dental follicle cells.Dental follicle tissue was dissected from the root surface of human embryo tooth germs. HDFC were isolated and purified by digestion with bacterial collagenase. Their morphological traits were observed under phase-contrast microscopy, and their characterizations were identified by immunohistochemistry and RT-PCR method. Biomineralization assays were performed after HDFC incubated with mineralization media. Part 2, Research of inducing HDFC orientating differentiation by trigger factors.HDFC were treated with EMPs, BMP-2 alone or combined to explore whether EMPs and BMP-2 have the ability to induce HDFC into osteoblasts/ cementoblasts. MTT method and ALPase immunocytochemistry were used to examine the proliferative and ALPase activity. Immunohistochemistry and image analyze methods were used to determine the expression of mineral-associated markers and cementum attachment protein of HDFC which had been treated with EMPs and/or BMP-2.Part 3, Initial study on tissue engineering by HDFC.HDFC cultured in vitro to obtain cells-made carrier as the first scaffold. Collagen gel and CBHAwere used as the other scaffolds cultured with HDFC. The cell growth in the scaffolds was observed by scanning electronic microscope(SEM). The three kinds of scaffolds containing HDFC were transplanted into nude mice. The transplants were cut for examination by histochemical analysis after implantation for 6 weeks. RESULTS:1. Cultivating human dental follicle cells in vitro and observing their characteristics: It was high efficacious for obtaining HDFC in vitro by digested method. Cells exhibited fibroblastic morphology in culture. Vimentin was expressed in the cells, while cytokeration presented negative, so it was proved that cells derived from ectomesenchyme. The immunohistochemistry effects showed that HDFC expressed mineralized associated proteins such as COL I, COLIII, BSP, OPN, and ON. The results of RT-PCR showed that BSP, OCN, ALP, mcprl mRNA were detected, while DSPP and cbfal did not be detected. HDFC were capable of inducing mineralization by day 20, with mineral nodule formation noted by day 30 which were positive by Vonkossa staining.2. The results of HDFC treated by EMPs and BMP-2:(D 50,100, 200mg/LEMPs and 50,100, 200ug/L BMP-2 could significantly promote HDFC's proliferative activity when compared with control group. And EMPs of lOOmg/L, BMP-2 of lOOug/L was the best concerntration. The effect was observed by day 3 and maintained to day 7. When treated associated with EMPs and BMP-2, HDFCs also obtained strong proliferative activity, but had no differences compared with using them alone.(D 100,200mg/L EMPs and 25,50,100, 200ug/L BMP-2 could promote HDFC to secrete ALP when compared with control group. This effect was observed at day 3. When treated associated with EMPs and BMP-2, HDFCs also expressed strong ALPactivity, and it was stonger than using EMPs and BMP-2 alone. In the mineralization assay, HDFC were incubated in mineralizing media, supplemented with EMPs(100mg/L), BMP-2(100ug/L), and associated with EMPs and BMP-2. We observed that BMP-2 and associated group could promote HDFC to form mineralize nodule, EMPs had no effect for HDFC to form mineralize matrix.(3)100mg/L EMPs and lOOug/L BMP-2 could promote the ability of synthesis of COL I , COL1II, BSP, OPN, and ON of HDFC (P<0.01). The effect was observed by day 3 and maintained to day 7. Compared with EMPs, the effect of BMP-2 for HDFC had the significance of difference in statistics.(4) It was detected CAP positive staining in HDFC by immunohistochemistry after treated with lOOug/L BMP-2 for 7 days, and negative staining after treated with lOOmg/LEMPs either 3 or 7 days.3. Initial study on tissue engineering by HDFC: Cells-made carrier, Collagen I, CBHA had good cell compatibility, so they were all valued to further study as the scaffolds of tissue engineering. Only cells-made carrier implants formed a few mineralized matrix but stained negative for anti-cementum attachment protein monocleonal antibody. CONCLUSIONS:1. HDFC could be isolated and cultured by digested method and presented some characterization of osteoblast/cementoblast.2. EMPs and BMP-2 act as trigger factors to induce dental follicle cells differentiate toward a cementoblast/osteoblast phenotype by stimulating proliferation, enhancing ALPase activity and the expression of major phenotypic markers.3. Cells-made carrier, Collagen gel, and CBHA were all valued to further study for three-dimentional cell culture and using as scaffolds of periodontal tissue engineering.4. Human dental follicle cells couldn't auto-differentiate to cementoblast/ osteoblast phenoltype without trigger factors.5. We can use HDFC as seeding cells and combine with EMPs and BMP-2 as growth factors in periodontal tissue engineering research.