Research On Combined Regeneration Of Pulp And Periodontal Tissue On Delayed Replantation Of Avulsed Immature Tooth By Using Dental Pulp Stem Cell Aggregates From Human Exfoliated Deciduous Teeth

In most cases, the avulsed immature teeth after delayed replantation will lose pulp and periodontal tissue. In recent years, regenerative pulp and periodontium have been successfully obtained on animal model by applying tissue engineering. However, for dislocation teeth subject to delayed implantation, there is no precedent on combined regeneration of dental pulp and periodontium. Dental pulp stem cell from human exfoliated deciduous teeth deciduous teeth(SHED) is a type of junior mesenchymal stem cell, which has excellent capacity in proliferation and multipotential differentiation. On nude mice and large animal dental pulp necrosis model, regenerative pulp tissue similar to physiological structure of dental pulp tissue can be obtained by utilizing SHED aggregates. Regeneration of periodontium can be obtained by PDLSC cell sheet wrapping on the surface of replanted teeth root. However, SHED has stronger differentiation potency. Under certain environmental induction, it is capable of differentiating into osteogenic and odontogenic cells, adipogenic, neural, endothelial cells and hepatocytes. There is no report on applying SHED on periodontium regeneration at present. Through detecting SHED biological performance, the research further determined the potency of SHED applying on joint regeneration of pulp and periodontal tissue; and then apply SHED on Beagle dog dislocation tooth model, and verify the possibility of dental pulp and periodontium of joint regeneration on animal model; eventually, apply SHED on clinical record of delayed implantation after dislocation of immature teeth to observe the effects of SHED on complete dislocation of immature tooth in clinical cases. It is to develop new ways, explore new ideas and provide experimental basis for extensive clinical application of stem cell therapy in the future.Part one: The study on biological properties of SHED and its aggregateObject: To further clarify biological properties of SHED and its aggregate. Method: SHED were separated and cultivated to detect molecular surface markers, clone formation ability, proliferation ability, multi-directions differentiation ability; cell aggregate was prepared and through HE, MASSON staining to detect the biologicalproperties. Result: SHED can positively express mesenchymal cell marks on STRO-1, CD90 and CD146, while negatively express the source of hematopoietic system CD34. SHED does well on clone formation, proliferation and osteogenesis, adipogenic differentiation. HE staining of SHED aggregate presents abundant undifferentiated cells and extracellular matrix; MASSON staining can detect collagenous fiber in cross-linked network structure. Conclusion:SHED and its aggregate have excellent biological performance. They are ideal seed cells of dental tissue engineering.Part two: Combined Regeneration of pulp and periodontal tissue on Beagle dog avulsion model by using autologous dental pulp stem cell aggregates from deciduous teethObject:To explore the regeneration conditions of dental pulp and periodontium by using autologous dental pulp stem cell aggregates from deciduous teeth on teeth avulsion of Beagle dog model. Method: Beagle dog deciduous teeth dental pulp stem cells were separated and cultivated to produce cell aggregate; Beagle dog teeth dislocation model was established; experimental group was set up, including regeneration group, negative control group namely conventional treatment group and positive control namely normal teeth group; Beagle dog autologous deciduous teeth dental pulp stem cells aggregate was prepared and transported to avulsion root canal and the tooth root was wrapped by the aggregate in experimental group. Fill Vitapex to tooth root canal of conventional treatment group leaving tooth root untreated. And then implant the two groups of teeth into alveolar socket respectively.Take materials after certain time, take Micro-CT, and adopt HE, MASSON for tissue staining after demineralization. Result: The radiological results show that regeneration group root canal wall is smooth with periodontal ligament lacuna and bone white line of alveolar bone clear. The root canal of conventional treatment group displays Vitapex high density images. Periodontal ligament lacuna has been disappear with he root and alveolar bone being indistinguishable. HE results show that the dental pulp tissue of regeneration group are arranged in order and are distributed among collagenous fiber. Cementum surface contains cementoblast. MASSON staining result displays periodontal fiber being parallel to each other in regeneration group. Mineralized nodule is found in periapical area and absorption lacuna on root surface in conventional treatment group, the root and alveolar bone mutually be indistinguishable. Conclusion: Autologous dental pulp stem cells aggregate from deciduous teeth could realize functional regeneration of dental pulp tissue and periodontium on teeth avulsion of animal model. Part three: The prospective clinical trial: combined regeneration of pulp and periodontal tissue on delayed replantation of avulsed immature tooth by using autologous SHED aggregateObject: SHED is applied on clinical treatment of delayed replantation of avulsedimmature tooth to explore the effects of combined regeneration of dental pulp and periodontal tissue. Method: We screen the clinical patients with avulsed teeth in accordance with certain standard. On first visit, after cleaning the whole tooth root, fill Vitapex to the root canal and fix it to alveolar socket. Then we obtain deciduous teeth dental pulp and prepare SHED aggregatein the experimental group; On the subsequent visit,extract the teeth of the experimental group and clean up the root canal, then implant the SHED aggregate to root canal and wrap the root surface. At last, reimplanted to alveolar socket and fix it for one month, leaving the control group untreated. Take intraoral photographs, X film, CBCT and carry out Doppler blood flow detection, pulp electricity test, gingival pocket detection and tooth mobility testing at 6 and 12 months after the operation. Result: The color of dental crowns in experimental group presents light yellow, transparent and glossy. Doppler blood flow test results, dental pulp vitality electricity test results, gingiva pocket depth results and tooth mobility are the same with those of normal teeth. Radiographic results display thickening of root canal wall, clear periodontal ligament lacuna and lamina dura. The teeth of control group present grayish yellow without gloss. Doppler blood flow test result shows the minimum value, while the dental pulp vitality electricity test result shows the maximum value. The tooth mobility gradually diminishes until at last totally loses. The depth of gingival pocket is the same to that of normal teeth. Radiographic results show high density images in root canal. The periodontal ligament lacuna and lamina dura disappeared. Conclusion: SHED aggregate could reconstruct the dental pulp and periodontium, and restore physiological functions of avulsed immature teeth in clinic.