Bone Regeneration Of Peri-implant Defects Using Cell Sheets Of Adipose-derived Mesenchymal Stem Cells And Platelet-rich Fibrin Membrane:an Experimental Study In The Canine

Background:With the development of the times,implant restoration is a proven and effective therapy for the replacement of missing teeth.Unfortunately,bone defects around the implant as a result of traumatic injury,periodontal disease,typically bring a daunting prospect and a questionable prognosis for the implant.Currently,the most commonly used methods of treatment are autologous bone,allograft,xenogeneic bone and artificial bone transplantation,but no matter which method there are some drawbacks are difficult to solve.Recently,bone tissue engineering has been considered the most promising and potential method for repairing bone defect,which could replace autologous bone,allogenic bone,and xenograft bone by constructing a cell-scaffold complex in vitro.As seed cells,compared with bone marrow mesenchymal stem cells(BMSCs),adipose-derived stem cells(ASCs)are more easily obtained,more abundant,and have less donor-associated morbidity.What's more,ASCs also have multipotent and angiogenic ability.In addition,the cell sheet technique,which provides a high density of transplanted cells containing extracellular matrix(ECM) with intact cell–cell junctions,is a novel approach in tissue engineering.At the same time,platelet-rich fibrin(PRF)obtained from whole blood after high-speed centrifugation is characterized by three-dimensional network structure,rich in platelet and growth factors,which can be used as a source of growth factorss in tissue engineering.Purpose:The objective of this study was to explore the effect of PRF membrane on the proliferation and osteogenic differentiation of ASCs and to provide a clinical basis for the clinical application of PRF in the repair of bone defects.To explore the effects of a tissue engineering bone consisting of adipose-derived stem cells(ASCs)sheet and autologous Platelet-rich fibrin(PRF)on the new bone formation and osseointegration around dental implants.Methods:1.Isolate and culture canine ASCs,and characterization of ASCs by osteogenic,adipogenic and chondrogenic differentiation.At the same time,we build adipose-derived stem cells into cell sheet,H-E staining and electron microscopy were performed to assess cell sheet.2.Whole blood was collected from healthy beagle's forelimb vein to prepare PRF,and H-E staining and electron microscopy were used to observe structures of PRF.Cell proliferation and osteogenic potential of ASCs treated with autologous PRF were evaluated by CCK-8 assay,alkaline phosphatase(ALP)staining,and real-time quantitative PCR.3.In vivo: The third and fourth premolar teeth of bilateral mandibular were extracted.A three wall bone defect(4 × 4 × 3 mm)around each immediate implant was generated in the mandible and randomly treated with one of the following four groups: A,ASC sheets and PRF;B,ASC sheets only;C,PRF only;D,nothing was used.Micro-CT,biomechanical test,fluorescent bone labeling and histological assessments were carried out to evaluate bone regeneration capacity.4.Statistical analysis: All results were statistically analyzed with SPSS 17.0 software(SPSS,Chicago,USA).Independent-samples T test was used for comparison between two groups.One-way analysis of variance(ANOVA)and the LSD post hoc test were used to evaluate differences between each group,and p < 0.05 was considered statistically.Results:1.The ASCs were induced to osteogenic/adipogenic/chondrogenic differentiation in vitro.ASC sheets were constructed successfully.H-E staining revealed that cell sheet was composed of 2-4layers of cells and SEM images showed that cells were embedded in extracellular matrix(ECM).2.PRF was like a milky white fibrin clot,and the bottom connected with the red corpuscles.H-E staining and SEM showed that PRF membrane consisted of numerous arranged densely red-stained fibrin bundles,many red cells and nuclear blue-stained leukocytes were distributed in fibrin bundles.The results of CCK-8 assay indicated that the PRF group had a significantly increased proliferative capacity from 4th day.After osteogenic induction,the expression of alkaline phosphatase and osteoblast gene were significantly increased in ASCs co-cultured with PRF(P <0.05).3.Peri-implant bone defect model was successfully created.Postoperative results showed that only a small amount of new bone was formed in the control group,and new bone was found in ASC sheets/PRF group,ASC sheets group and PRF group,especially in ASC sheets/PRF group.The results of Micro-CT showed that the repairing rate of bone defect(BV/TV%)were 60.90 ± 4.83% and 80.85 ± 3.93% in ASC sheets/PRF group,49.22 ± 6.38% and 68.43 ± 2.70% in ASC sheets group at,46.92 ± 6.70% and 57.48 ± 1.47% in PRF group,32.89 ± 2.22% and 39.00 ± 9.57% in the control group at 4 and 8 weeks respectively.In addition,there were significant differences between ASC sheets/PRF group and other three groups.Histomorphological analysis showed that both BF and BIC in the ASC sheets/PRF group increased significantly.The MAR and biomechanical test results showed that the ASC sheets/PRF group significantly increased at 8 weeks(P <0.05).Conclusion:1.PRF can promote the proliferation and osteogenic differentiation of ASCs in vitro.2.The results suggest that ASC sheets combined with autologous PRF may be a promising tissue-engineering strategy for bone formation and osseointegration in immediate implantation.