Treated Dentin Matrix Particles Combined With Dental Follicle Cell-sheet Stimulated Periodontal Wound Healing/Regeneration

Objectives:Tissue engineering has shown an increasing development to provide suitable strategies for regeneration of damaged tissue or organ.The association of biomaterials,stem cells,growth and differentiation factors formed to be the three essential elements for tissue repair and organ regeneration.In perspective,regeneration of lost periodontal tissues remained an elusive objective,requiring synchronous formation of all periodontal tissues via cementogenesis,osteogenesis and formation of a periodontal ligament,generating a similar form and function found in the intact,native periodontal attachment.Therefore,it becomes the key point to choose exact elements to achieve tissue remolding.The development biology might provide rational strategy and guideline for tissue engineering reconstruction as the regeneration process itself is a recurrence of tissue development.The present study tried to establish a novel method for tissue reconstruction from development point,which was performed on a periodontal defect model.While tooth roots are forming,the supporting tissues of tooth also develop,which are formed from the dental follicle.As the root sheath fragments,ecotomesenchymal cells of the dental follicle penetrate between the epithelial fenestrations and the newly formed root dentin,all of which become the vital components of periodontal tissue micro-environment.Meanwhile,the development and features of root dentin make it to possess similar physicochemical properties and structure with bone.Thus,this study intend to reconstruct periodontal tissue complex by combination of dental follicle with dentin matrix.The search of feasible seeded cells and corresponding scaffold or other cell loading method would exert significant effect to improve periodontium regeneration.Methods and materials:1.Dental follicle cells from fresh tissue and dental follicle fragments were stored in liquid nitrogen for 3 months.After thawing,the isolation,morphology,proliferation,cell cycle,colony-forming-unit ability,stemness-related marker expression,apoptosis,and multi-lineage differentiation potential of C-cdf were tested compared with cDFCs.2.Dental follicle cell were cultured in the cell sheet induction medium to create cell sheet.To examine the microscopic structure of DFCSs were evaluated via hematoxylin and eosin(HE)staining and immunofluorescence.The expression of cementogenesis,osteogenesis and periodontal ligament related genes was detected by RT-PCR.3.Fabrication of dentin matrix particles using grinding method and then undergo gradient demineralization,the physicochemical and biological properties of TDMP were detected by laser particle analyzer,SEM,XRD,XPS,FI-IR,ELISA,et.al.The biological effect of TDM on bone mesenchymal stem cells were analyzed by CCK8,RT-PCR,WB,et.al.Furthermore,bone inductive potentiality of TDMP was investigated by establishment of critical size cranial defects in SD rats.4.The effect of TDMP extracts on DFCSs were assessed by RT-PCR and WB.5.One wall intrabony defect model on Beagle were used to test the capacity to repair periodontium defects of combination of DFCSs with TDMP.Results:1.Autologous dental follicle cells were successfully obtained from cryoperserved dental follicle.C-cdf expressed mesenchymal stem cells marker,had good proliferation rate,showed similar levels of mRNA for sternness-and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to cDFCs.However,compared with cDFC,C-cdf s mRNA level of ALP,OSX,PLAP-1 Periostin were decreased(P<0.05).2.After 2 week's culture,DFCs can form cell sheets by secreting extracellular matrix(ECM)proteins.Both DFCs and DFCSs expressed PLAP-1,Periostin,Scleraxis,cp-23,CAP,Runx2,ALP,OSX,indicating the abilities of periodontal differentiation.2 weeks after culturing DFCs with cell sheet induction medium,mRNA level of PLAP-1,Periostin,Scleraxis were increased significantly(P<0.05).3.The fundamental element of TDM particles were hydroxyapatite with low crystallinity,large amount of organic molecules like CO32-,and Mg,Na,Cu except of abundant Ca,P.The TDMP could constant release factors such as TGF-?,VEGF,BMP-2,PDGF-BB,and the extracted liquid of which could induce MSCs to proliferation and osteogenic differentiation.Meanwhile,it was showed that TDMP possess the abilities to repair bone defects with good biocompatibility and osteoconduction,which was no obvious difference with HA/?-TCP.4.When DFCSs were induced by TDMP,mRNA and protein level of fibronectin,collagen I,osteopontin,PLAP-1,Periostin,collagen ? were increased or maintained,while ALP,OSX,Runx2 were decreased(P<0.05).5.Eight weeks after the transplantation,periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the DFCSs,TDMP+ DFCSs,HA/?-TCP+DFCSs groups than the other groups.The amount of alveolar bone regeneration was more when TDMP or HA/?-TCP used.Periodontal regeneration was significantly observed with both newly formed cementum,well-oriented PDL fibers and the amount of alveolar bone regeneration in the TDMP+DFCSs,HA/?-TCP+DFCSs groups.Conclusions:Cryopreservation of human dental follicle fragment could be used as a resource of mesenchymal stem cells.DFCSs could be an ideal targeted cells and matrix for regeneration of periodontal tissue complex.It was proved that TDMP possess good biocompatibility and the capacity to repair bone defects.Furthermore,TDMP could induce DFCSs to periodontium differentiation.In vivo results indicate that DFCSs combined with TDMP scaffold serve as a promising tool for periodontal regeneration.