| Periodontal disease is the disease of periodontal supporting tissue includingalveolar bone, cementum and periodontal ligament. Periodontal disease seriouslyharms human teeth and general health. Nowadays, research of periodontologyhas become the main field of oral disease. Periodontology has made greatprogress in recent years, however the conventional periodontal therapy methodcan not entirely regenerate functional periodontal tissue. Tissue engineeringtechnology applied to the periodontal treatment can compensate the limitationof the routine therapy.The reconstruction and repair of the tissues and organs depend on thebiological characteristics of stem cells and the direct and indirect intervention ofthe surrounding environment. The superior character between stem cell andmicro-environment is still argued. But the common sense has reached that thethe imitate cooperation between the two factors mediates the colonization,migration, differetiation and proliferation of the seeding cells. Various signalingmolecules widespreaded in the host implant bed can meditated these progress.At the same time, the seed cells will act on the Host microenvirement, and even control the systemic condition to achieve the tissue regeneration. Therefore, theselect of types of the suitable seed cells is not related to the biologicalactivity of seed cells itself, but also influenced the quality of Hostmicroenvironment. Improvement of host micrenvironemnt is more favorable forthe survival, differentiation and activity of the stem cells.hPDLSC and BMMSC have multi differentiation potential ability.They candifferentiated into fibroblasts, osteoblasts, cementoblasts, neuroblast etc.PDLSC and BMMSC as seed cells can regenerate relatively completeperiodontal ligament/cementum like tissue in animal ectopic transplantation.But periodontal regeneration ability by these two stem cells is inconsistent,which are influenced by the model of cell transplantation and inducingenvironment. Recently, the application of two kinds of stem cells for tissueengineering has emerged in basic research and clinical applications. So, couldthe two stem cells with different functions be co-operatively used in theperiodontal engineering to regenerate totally functional alveolus, periodontalligament and cementum?Three parts were designed and operated in this research. The main resultswere listed as follows:Part.1the fabrication and characteristics of thehPDLSCs/BMMSCs cell sheetObjective: To isolate and culture the hPDLSCs and BMMSCs, and fabricatethe cell sheet. The cell sheets co-cultured withe TDM and HA-TCP wereexamined to show the biological activity. Methods: The flow cytometry weretested to show the cell phenotype and cell cycle of the hPDLSCs and BMMSCs.And the osteogenic and adipogenic induction were carried. MTT and coloneformation rate were examined to evaluate their proliferative capacity.Histological and immunohistochemical observation of the structure of cellsheets were observed and compared. Result: hPDLSCs and BMMSCs showed the colony growth tendency, and the positive expression of specific surfacemarkers of mesenchymal stem cells. The osteogenic and adipogenic induction ofthe two stem cells was positive. The hPDLSCs/BMMSCs cell sheets showed thedeposition of rich extracellular matrix, closely-arranged cell, the obviouspolarity. And both of cell sheets were observed to show the strongly positiveexpression of structural proteins of ECM such as Col-, OCN and Laminin, weakexpression of col-â…¢. SEMresults showed cell morphology in the cell sheet notobviously changed and the multi-layered tendency of cell sheet, the obvious cellpolarity. The extensive and dense deposition of extracellular matrixmineralization was observed in the BMMSC group. Conclusion: The isolatedhPDLSCs and BMMSCs possessed the characteristics of mesenchymal stem cell.The cell sheet was rich in extracellular matrix and can mediate cell attachmentand colonization on scaffolds.Part2. PDLSCs/BMMSCs cell sheets with scaffoldsin vivo ectopic transplantation.Objective: We transplanted the hPDLSCs/BMMSCs cell sheets co-culturedwith the TDM and HA-TCP in different micro-environment to evaluate theeffect of micro-environment on the cell differentiation. Method: To co-culturetwo types of cell sheets with TDM and HA-TCP scaffold and observe themicrostructure change dynamicly by SEM and HE. The hPDLSC, hBMMSCand hPDLSC./hBMMSC cell sheets were transplanted into the subcutaneous andskull defection in order to evaluate the tissue regeneration activity. Result: Thetexture of the double cell sheet was more dense with the richer mineralapposition on the surface and more closely-connected cells. The subcutaneoustransplantation of three kinds of cell sheets without scaffold support onlyobserved to regenerate rich fibrous bundles and not deposit the mineralizedsubstances. The subcutaneous transplantation of three kinds of cell sheet withTDM support could regenerate abundant mineralized deposition and fibrous bundles similar to ligament/cementums, among which PDLSC/BMMSC groupshowed more obvious regenerative results. The subcutaneous transplantation ofthree kinds of cell sheet with HA-TCP also formed periodontalligament/cementum like structure, but the fiber and mineral deposition wereweak compared with TDM group, BMMSC group showed the worst results. Theresult of skull defection transplantation showed insignificant inflammation.inflamatory cells and obvious new bone tissue formation at the edge of defection.Conclusion: hPDLSC/hBMMSC can integate the two kinds of stem cellbiological characteristics in the sheets to promote stem cell differentiation toperiodontal regeneration direction in a suitable micro-environment.Part three The application of the cPDLSCs/BMMSCs Cell sheetsin canine periodontal regeneration experimental research.Objective: to observe the activity of cell sheet using canine PDLSCs, BMMSCsand PDLSCs/BMMSCs in acute defection model. Method: To isolated andculture canine PDLSCs and BMMSCs and fabricate the PDLSCs/BMMSCs cellsheet. The acute periodontal defection model was established and the cell sheetswere transplanted in the defect and evaluated by histologic and radiologicexamination. Result: Canine PDLSC and BMMSC also possessed mesenchymalstem cell characteristics. Comparing with PDLSCs cell sheet and BMMSC cellsheet, the histological and radiologic results of PDLSCs/BMMSCs cell sheetgroup showed stronger bone repair and regeneration. Conclusion:PDLSCs/BMMSCs cell sheet can utilize the specialized differentiatedcharacteristics of two different stem cells to regenerate alveolar bone,periodontal ligament and cementum-like tissue in complex periodontalmicroenvironment. |
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