| Periodontal diseases that lead to the destruction of periodontal tissues-including periodontal ligament(PDL), cementum, and bone are a major cause of tooth loss in adults and are a substantial public-health burden worldwide.To regenerate periodontal tissue and reserve tooth is always a highlight for many researchers,many new approaches have been developed for treating periodontal defects, including guided tissue regeneration, growth factors, and enamel matrix proteins, and tissue engineering offers a new option to supplement existing treatment regimen for periodontal tissue regeneration.Tissue engineering is a newly emerging biomedical technique that involves the artificial manipulation of cells to promote tissue and organ regeneration.This can be seen as the third medical therapy in regenerative medicine following the two major clinical therapies,organ transplantation and reconstructive surgery in the 21st century.The object of tissue engineering is to regenerate natural tissues from living cells to replace defective or lost tissues and organs . Tissue or organ regeneration using tissue engineering methods will require integration of three key elements:inductive morphogenetic signal,responding progenitor/stem cells and the extracellular matrix scaffold. Moreover, to induce morphogenesis of periodontum, these elements must be combined to facilitate a developmental cascade of pattern formation. compared with other parts of the body, the oralcavity offers distinct advantages to the tissue engineering, such as ease of observation and accessibility.In recent years,many adult stem cells have been identified and successfully isolated from various tissues including bone marrow, muscle, neurtal tissue, skin ,dental pulp and so on. One discovery suggests that PDL contains stem cells that have the potential to generate cementum/PDL-like tissue in vivo. It has been that human periodontal ligament stem cell (PDLSCs) could repair periodontal destroy of nude mice. PDLSCs are similar to other adult stem cells so far have been identified and they have great potential of self-replication and can diffentiate into functional mature cell types or trans-differentiate into other cell types. Though successful isolation of human PDLSCs has been reported, investigations on dog PDLSCs and studies on tissue engineering of periodontal-like structures by cultured dog PDLSCs have not yet been documented. As one of the most frequently used animal models in periodontal disease scitientific research, dog also play an important role in periodontal disease because it's less costly and high homogeneity in comparison with human beings. In this study, dog PDLSCs clones were isolated from cultured periodontal ligament cells(PDLCs) and the isolated clones were characterized through series of biological examinations, and were implanted to artificial furcation defects combined with ceramic bovine bone (CBB) to regenerate dog allograf periodontal defects.Dental follicle, which has been implicated as the origion of alvelar bone, cementum and periodontal ligament, is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. With the advance of study of periodontal tissue engineer, some scientists have attemped to get periodontum using dental follicle cells as seeded cells. In this study, human dental follicle cells(DFCs) were isolated and charaterized , and were implanted into scoffalds to select periodontal tissue engineering scoffalds.We isolated dog PDLSCs from adult dog teeth and human DFCs from tooth germ tissue at late development, and select suitable scoffalds for periodontal tissue engineering, and PDLSCs was induced to differentiate into cementoblasts/osteoblasts for periodontal tissue engineering. Subsequently, the cultured dog PDLSCs were seeded onto different kinds of scaffolds and cell/scaffold constructs which were cultured in vitro. Through observing and comparing cell growth and proliferation on different scaffolds, we chose CBB combined with collaguous and lyophilization collagen membrane as scaffolds for periodontal tissue engineering. Furthermore, the cell-scaffold constructs were transplanted into nude mices.after 8 wks, cementum and periodontal ligament-like structures were generated in implants.Also cell-scaffold constructs were transplanted into dog artificial furcation defects, after 8 wks , cementum , periodontal ligament and alveolar bone structures were generated in implants. Finally, in vivo culture model of dog PDLSCs were established with CBB combined with hydrogel and lyophilization collagen membrane. As a result, cement-periodontal ligament- like structures were bioengineered successfully in both nude mices and dogs. This study is a trial one on periodontal tissue regenetation for the first time by combining the tissue engineer and adult stem cell biology, which will lay a reliable experiomental foundation for clinical periodontal tissue regeneration. Following experiments were involved in this study: l.Isolation and identification of dog periodontal ligament stem cellsIn this study, single cell clones generated from dog periodontal ligament tissue were culture-expanded and induced in vitro and in vivo to differentiate into various cell types. Cells originated from one of these clones were found to have a long protrude just like an osteoblast after induction by mineralizatin solution. At the same time, Immunocytochemistry assay was used to identify the source of adult PDLSCs, The cells expressed STRO-land CD 146 by immunofluorescence stain, which is the marker of mesenchymal stem cells.AlsoVimentin,osteoblast-like marker alkaline phosphatase and Collagen- I were expressed weakly , Cells didn't expressed CK. And these cells were clonegenic,highly proliferative cells and capable of differentiating into osteoblasts/cementoblasts,asipocytes and neuronal-like cells.The evidence suggests that the cells cultured from adult dog periodontal ligament. 2.Culture and characterization of the DFCs from human dental germ in vitroTo develop a culture model for human DFCs and investigate it as seed cell for periodontal tissue engineering. Metheds: Developing mamdibular germ from human embry were subjected to trypsin in Hank's balanced salt solution. After trypsinization, the dental follicle was enucleated from the tooth germ and seprarated from the associated epithelial root sheath. Pure dental follicle tissue were cultured in vitro. Cells were obtained from human dental dental follicle by collagenase digesting and purified it combining several methods. The cultured cells were identified by its morphological and ultrastructure observation by light microscope and TEM resperatively, and combined with immunostaining for expression vimemtin, type I collagen, type III collagen, fibronectin, osteopontin and bone sialapretion.Also, RT-PCR result show the cultured cells expressed type I collagen. Result: The cultured human DFCs were typical fibroblast cells in shape, and transmission electronic microscrope revealed that the cells contain electron-dense granules, aboundant rough endoplasmic reticulum, but did not form in desmosome.Vimemtin, type I collagen,type III collagen, fibronectin,osteopontin and bone sialapretion were present in DFCs. Conclusion:We succeeded in culture human DFCs, it may be useful as seeding cell for periodontal tissue engineering. 3.Selection of scaffolds for tissue engineering of periodontum-lile structuresThe cultured human DFCs and dog PDLSCs were seeded onto collagen materials,hydrogel(HG),lyophilization collagen membrane(LCM),and ceramic bovine bone(CBB).After in vitro culture,cell growth and proliferation on different scaffolds were observed and compared with SEM and cell countingmethods.The results showed that cells adhered and proliferated well on collagen materials,CBB and collagraft,and many secretary particles could be found on the surface of cells,while on the surface of coral, cells could not extend and proliferate well,indicating dental mesenchymal cell have good biocompatibilith with collagen materials, CBB and Collagraft.But collagen materials can't provide sufficient mineralization microenvironment for cells. So,ultimately CBB and collagen materials were chosen as scaffolds for further bioengineering of periodontal tissue structures.^Experimental study on construction of tissue engineered periodontal ligament-like constructure in vitroTo investigate the feasilibility of construction of tissue engineered periodontal ligament in vitro using expanded PDLC and lyophilization collagen membrane. Methods:PDLCs were isolated by tissue explant method and expanded in vitro. PDLSCs(107/ml)of 3rd passage were collected and then seeded onto lyophilization collagen membrane to form a cell-scaffold complexes. The complexes were cultured in DMEM containing 10%FBS.The cell-scaffold complexes were observed continuously by inverted microscopy. The cultured complexes were harvested at 2 week for electromicroscope, histological analysis. Results: At 2 week, a new- periodontal ligament formation was observed within the curtured complexes. The cells were in good condition, and the formation of collagen fibers can also be observed. Conclusion: periodontal ligament like-constructure is possible to be constructed in vitro using PDLCs and collagen membrane. At this basis, the tissue engineered periodontal ligament can be used to repair periodontal defects in dog in the future.5. Estabishment of cementum-periodontal ligament—like constructure in vivo nude mice The cultured dog PDLSCs as well as DFCs were taken as seeded cells,and implanted into processed CBB combined by HG .Then cell/scaffold constructs were transplanted into nude mice hyperdermic in order to establish in vivo culture model of dog PDLSCs and DFCs.Control groupswere scaffolds without dog PDLSCs. 8wks later, implants were excised for histological anolyses. Histological analysis revealed that periodontal tissue-like structures including periodontal ligament-like tissue and cement-like complex were generated within implants of experimental groups but not of control groups. These results will provide reliable experimental evidence for tooth regeneration and its clinical application ,and operate a nocel approach for bioengineering periodontal tissues. 6.Reconstruction dog periodontum in vivo by tissue engineer methodsThe cultured dog PDLSCs were taken as seeded cells,and implanted into processed CBB combined with hydrogel and lyophilization collagen membrane. Then cell/scaffold constructs were transplanted into dog in order to establish in vivo culture model of PDLSCs. Control groups were scaffold without PDLSCs,8wks later,implants were excised for histological analyses.Histological analysis revealed that periodontal tissue structures including periodontal ligament tissue and cementum and alvolar complex were generated within implants of experimental groups but not of control grouips. Immunohistochemical analysis of implants demonstrated that dog Col I was expressed in newly-formed fibres-like tissues and dog BSP, OPN was expressed in cementum-like and alveolar tissues. These results will provide reliable experimental evidence for periodontal tissue regeneration...
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