Research Of Transformation And Application For Clinical On Tooth Regeneration By Using Freeze-dried Tooth Combined With SHED Aggregates

In recent years,tooth regeneration has became a hot research area with important clinical value and research significance,while more and more tissue engineering and regenerative medicine research focus on it.Multiple teeth ectomesenchymal stem cell has been cultured and then used as seed cells in pulp-dentin and even the whole tooth tissue regeneration for experimental research.Among which are the dental pulp stem cells from human exfoliated deciduous teeth(SHED)with its strong ability of proliferation and potential to multi-directional differentiation still with easier access to become the ideal seed cells for tissue engineering and regenerative medicine,.Early experiments in our team using SHED cell aggregate successfully regenerate the dental pulp-dentin complex,and applied it to the treatment of avulsion teeth through replantation to obtained a good effect of periodontal healing with dental pulp regeneration.At the same time to find ascaffold material with well biocompatibility,non-toxic stimulation,and can promote the tooth tissue regeneration is also important for tooth regeneration.Recent years?study found that the human dentin matrix is a natural extracellular scaffolds materials,with not only a good biocompatibility,low toxicity and low immunogenicity,also the cell growth factors and protein factors within the dentin tubule can provide cells with a similar setting to natural tooth development environment.Our team hope to utilize freeze drying method dealing with teeth for the preparation of tissue engineering scaffold material,to get freeze-dried teeth with intact shape,low immunogenicity and is rich in induced factors for tissue regeneration.Research plans to prove that freeze-drying tooth is a good scaffold material and its feasibility for dental pulp and periodontal tissue regeneration through the process of preparation and the biological performance testing.Then the freeze-dried teeth are composite with SHED cells aggregate for animal experimental to observe the situation of periodontal tissue healing and pulp regeneration after treating with lyophilization and replantation.Finally conducted a prospective clinical trials,to observe the curative effect to treat avulsion young permanent teeth through delayed replantation with the application of autologous freeze-dried tooth composite with SHED cells aggregate.in order to explore a new concept and strategy for regeneration to repair the defect of tooth tissue.Finally to provide theoretical basis and technical support for the clinical application of the freeze-dried teeth and SHED cell aggregate.Experiment one: The study on biological properties of freeze-dried toothObjective: To test the mechanical properties of freeze-dried tooth and its biocompatibility with SHED,and explore the feasibility of freeze-dried tooth as scaffold materials for tooth tissue regeneration.Methods: The supernumerary teeth and the orthodontic extracted teeth with single root were collected clinically.Then the freeze-dried teeth were manufactured after the process of tooth preparation,derosination anddeproteinization,deep freezing,freeze drying,and sterilization.The surface morphology and microstructure of the freeze-dried teeth were detected under scanning electron microscope(SEM),and their mechanical properties were examined by microhardness test.The SHED co-cultured with freeze-dried teeth were observed to evaluate the cellular adhesion,growth and proliferation by SEM,Hocest staining,and MTT methods.Results: After freeze drying,the teeth roots and root canals presented the visible cleaner surface,and still remained the original microstructure.The Vickers hardness of the freeze-dried teeth had no statistical difference with normal control teeth.At the same time,SHED co-cultured with freeze-dried teeth had a better cellular adhesion and growth than the control,but there is no significant difference of the cell number.Moreover,the freeze-dried teeth had smaller influence on SHED proliferation compared with control.Conclusion: The reeze-dried teeth not only remained the original microstructure and mechanical properties,but also had the better cellular compatibility with SHED.So the freeze-dried tooth could act as a kind of scaffold material for tooth tissue regeneration.Experiment two: The study on the ectopic pulp regeneration using freeze-dried tooth combined with SHED aggregateObjective: To observe the results of tooth tiusse regeneration using SHED aggregate combined with freeze-dried tooth,to explore new methods for tooth tissue regeneration.Methods: The prepared freeze-dried tooth or normal tooth combined with SHED aggregate were respectively implanted into the back subcutaneous of 6 weeks? female nude mice.after obtained materials and demineralization,HE and immunohistochemical methods were used to observe the condition of tissue regeneration.Results: histological examination revealed regenerate tissue were full of puip root cannal and covered the root surface in freeze-dried tooth,similar to normal dental pulp tissue,which revealed the regeneration of the secondary dentine,odontoblasts,vascular and nerve tissue structure.Conclusion: The implantation of SHED aggregate combined with freeze-dried tooth into the back subcutaneous of nude mice could form dental pulp and periodontal tissue regeneration.Experiment three: The study on the pulp and periodontal regeneration using freeze-dried tooth combined with dental pulp stem cells aggregate from minipig exfoliated deciduous teethObject: To detecte the application effect of pulp and periodontal regeneration using dental pulp stem cells(SPED)aggregate from minipig exfoliated deciduous teeth combined With autologous freeze-dried teeth in the autograft teeth replantation model.Methods: The stem cells from mini-pig exfoliated deciduous teeth(SPED)were separated and cultivated,then preparated to be aggregate;The experimental animal model of teeth replantation were established,and divided to groups of freeze-dried tooth combined with autologous SPED aggregate and normal tooth combined with autologous SPED aggregate as control;6 months after replantation,the clinical examination of treated tooth were detected,then the treated tooth were removed to detect the effective of tissue regeneration using the histological,immunohistochemistry examinations.Results: the replanted freeze-dried teeth and normal teeth had no difference with the nomal permanent teeth in color and appearance,teeth bitting surface had varying degrees of wear loose without abnormal mobility degree;Mirco CT showed that all replantated teeth had no abnormal pathology change in periapical tissue or tooth root canal,and had a good condition of periodontal healing with clearly periodontal membrane space,continuously and completely alveolar bone wall;HE staining found the structure of dental pulp tissue regeneration were visible in replantation group,similar to normal dental pulp.Conclusion: autologous freeze-dried tooth combined with stem cells from mini-pig exfoliated deciduous teeth(SPED)aggregate through teeth replantation can achieve a good effect of pulp regeneration and periodontal healing,and the regenerated tissue had similar morphological struture with normal dental pulp tissue.Experiment four: The translational application research of tooth regeneration by using freeze-dried tooth combined with SHED aggregatesObjective: to explore the the feasibility of using Autologous freeze-dried tooth combined with SHED aggregate to treat avulsion teeth of delayed replantation.To provide new tissue regeneration concept and methods for the clinical treatment of avulsion teeth or even lost teeth.Methods: according to the inclusion criteria,the avulsion teeth in vitro for longer than 3 hour were undergo freeze-drying process,dental pulp stem cells exfoliated deciduous teeth in the patient were separated and cultured at the same time;The freeze-dried tooth combined with SHED aggregate were replantated into the alveolar socket,fixed for 3 months,regular visit for each month for oral,X-ray,dental pulp vitality test and periodontal examination.Results: clinical examination revealed that the replantated freeze-dried tooth had normal crowns color with no loose,positive results of dental pulp vitality test and normal gingival groove depth with Periodontal probing;radiographic results showed that periapical tissue had no shadow and pathological symptoms,athough root wall had no obvious thickening,the periodontal membrane was complete and continuous.Conclusion: using Autologous freeze-dried tooth combined with SHED aggregate to treat avulsion teeth of delayed replantation can achieve a good periodontal healing and pulp regeneration effect.