| In recent years, periodontal ligament cells were found to be heterogeneous cell population at different stages of differentiation, containing fibroblasts,osteoblasts,cementoblasts,precursor cells and other components,which contribute to the unique corresponding compartments of the periodontium.The heterogeneity of periodontal ligament cells is closely related to periodontal tissue regeneration and repair. During onset of root, the interaction of dental follicle cells (DFCs), Hertwig's epithelial root sheath (HERS) and dental papilla cells (DPCs) gives rise to formation of root and periodontium. Apart from above, it has been well recognized that DFCs which are the periodontal progenitor cells penetrate disintegrated HERS and contact with root dentin surface prior to any cementum formation. For actual periodontium regeneration, direct contact between the conditioned or denuded root surfaces and periodontal cells seems to be a prerequisite. Clinically,it is a prerequisite for periodontal regeneration that periodontal scaling,root planing and local anti-inflammatory treatment for the teeth with periodontitis. After the smear layer are removed, the new dentin and cementum are exposed, which are supposed to be a chemo-attractant for periodontal fibroblasts.It had been demonstrated that enamel matrix proteins (EMP) could facilitate the PDL cell on the ability of attachment,stretchment and inducing the PDL cell to secrete many important growth factors and cytokines which can improve the micro-environment conditions from time to time. Its biological behavior is of more as multifunctional growth factors. Scholar approved that EMP can induce osteoblastic cell line into proliferational and differentiational stage. Combined EMP and GTR can stimulate the cementum regeneration at periodontal damnification with newly forming fibrin embedded. This kind of completely regeneration mimics the structure of original one implies that EMP made a important part during this process. Generally speaking, the most important step for periodontal regeneration is the rebuild of cementum, so the basement of the periodontal regeneration is to improve the cells can form new cementum. During the development of tooth, the inner cell of Hertwig's epithelial sheath secrets a kind of matrix protein which belongs to the families of enamel matrix protein. They may act as a guider for hydroapatite forming and also take part in the periodontal regeneration and cementum forming. Basing on this theory, EMD should be a stimulator to improve the proliferation and differentiation of PDL cells, which can benefits the process of new periodontum forming. Therefore we considers that EMD make a crucial role in differentiate HPDLP(human periodontal ligament cell population)into cementoblasts.It is very difficult to regenerate cementum in periodontal tissue regeneration. At present, we know little about the cementum. Although numerous studies in vivo and in vitro have shown that it can promote periodontal tissue regeneration, including cementum regeneration through physical, chemical treatment, it has been obscure how the HPDLP differentiate into cementoblasts. In this experiment, we want to investigate if the HPDLP can be differentiated into cementoblasts, to learn the possible mechanism of induction with EMD, to further understand the differential potential of the HPDLP, and to provide a basis for periodontal tissue regeneration. Chapter One:isolation and identification of HPDLPObjectives:To isolate and identify the HPDLP in vitro successfully. Methods:1. Isolation and culture of HPDLP:Normal premolar for orthodontic treatment were collected. HPDLP was gently separated from the surface of the root and then cultured by tissue culture.2. Identification of HPDLP:The expression of vimentin and keratin were detected in the HPDLP by immunocytochemistry.Results:1. Morphological characteristics of HPDLP:Cells swim out of the edge of the tissues after the original tissue was cultured for 3-6 days, and the cells aren't significant difference in cell morphology, and the majority are fibrous long shuttle, and a small number of the cells presented as polygonal, spindle-shaped or oval-shaped.The cells grow fast and begin to converge after about 7-14 days.2. characteristics of HPDLP:Immunohistochemistry showed that cells positive for vimentin staining, negative keratin staining. It is confirmed that the cultured cells derived from the mesoderm.Chapter 2 Observation of the HPDLP morphology and its attachment and proliferation in the cementum-clip after induced by EMDObjectives:To observe the morphology of the HPDLP induced by EMD and the effects of EMD on cell attachment and poliferation in the cementum-clip.To learn whether the HPDLP differente into cmentoblast-like, and provide the experimental basis for the periodontal tissue regeneration.Methods:1. The HPDLP were induced by EMD for 7 days, and then the cell morphology was observed with the inverted phase contrast microscope2. The extracted root were used to make cementum-chip and divided into the test group and the control group. The cementum-chip samples were placed in 24-well plates, which were either precoated with 100mg/L EMD(EMD-treated group) or left uncoated (control group). After soaking for 24h, EMD-treated cells were seeded in the test group and the untreated cells were seeded in the control group.And then the cell morphology and the quantity of the HPDLP in the cementum-clip were observated by the Scanning Electron Microscropy.3. Attachment and proliferation of HPDLP in the cementum-chip were examined with MTT method. We established two groups in this study, the induced group and uninduced group.Results:1. After induced with EMD in vitro, HPDLP were changed from spindle-shaped fibroblast-like cells into polygonal cementoblast-like cells.2. More cells were adhered to the EMD-treated surface than to the untreated group. Compared with the control group, cell adhesion and proliferation of the EMD-treated group was significantly enhanced.3. Cell morphology on cementum-chip was studied by Scanning Electron Microscropy. The cells of the control group revealed an extended shape similar to that typical of fibroblasts grown on their natural habitat. These cells were long and spindle shaped, which had lots of microvillus on the cell surface. In the EMD-treated surface, cellular body is characterized by polygon or cubic figure, less microvillus and massive extracellular matrix around the surface. The number of adherent cells treated with EMD was higher than the number of untreated group.Chapter 3 Expression of mineralize-related protein of cemeoblasts in the HPDLP after induced by EMDObjectives:To study expression of mineralize-related protein of cemeoblasts in the HPDLP induced by EMD, and to understand the possible differentiation mechanisms of HPDLP into cementoblasts.Methods: Expression of cementoblastic mineralized-related proteins were analyzed between EMD-treated group and the control group by real-time polymerase chain reaction and immunohistochemical staining, including alkaline phosphatase (ALP), typeâ… collagen (COLI),osteocalcin(OCN) and bone sialoprotein (BSP).Results:1:Immunocytochemical staining showed that expression of ALP,BSP,COLI significantly increased in the experimental group compared with the control group, however, OCN showed no significant changes.2:The results of RT-PCR showed that the relative expression levels of ALP,COLâ… in the experimental group was higher than the control group. the relative expression levels of OCN showed no significant change.Conclusions:1. After induced by EMD, attachment and proliferation were increased obviously mong the HPDLP in the cementum-chip, and HPDLP can differented into cementoblasts-like cells.2. Expression of Mineral-associated protein was significantly increased in the HPDLP induced by EMD. This may be one of the mechanisms of the HPDLP differented into cementoblasts.3. Differentiation potential of the HPDLP was increased obviously after induced by EMD. |
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