| Currently, dental implants technology develops rapidly in our country, a majority of partial and total agomphiasis patients preferred this repair method. The key point of implant surgery techniques is how to repair alveolar bone defects in the edentulous areas. Diabetes has negative impacts on the normal bone metabolism, making the problem even more difficult. China has the largest amount of diabetic patients in the world, and how to solve the problem of alveolar bone defects of this group is the focus of our attention. Bone tissue engineering is a commonly used technique, which is seeding suspension stem cells on biological scaffolds with or without growth factors and co-cultured for a period of time before implanting to the bone defect sites. Adipose-derived stem cells(ASCs) compared with bone marrow mesenchymal stem cells(BMSCs) can be easily harvest with less surgery pain, and have a wide variety of sources and strong proliferative capacity, thus providing ideal seed cells for bone tissue engineering. Cell sheet engineering is one of the most promising research fields in recent years which can be harvest without pancreatin digestion, can avoid the loss of cells when seeded onto biomaterial scaffolds, and can perfectly preserve extracellular matrix. Semaphorin 3A(Sema3A) is a member of the semaphorin family. The latest research shows Sema3 A can promote the osteogenic differentiation and inhibite osteoclast differentiation, in addition to the important role on neurological development and healing. Thus, this study aims to use tissue engineering bone which established by adipose-derived stem cell sheet(ASC sheet) to improve the reparing of edentulous bone defect in type 2 diabetes mellitus(T2DM) patients, and to study the role of Sema3 A on the ostogenesis differentiation of ASC sheet, and ultimately to provide a theoretical basis for clinical transformation of adipose-derived stem cell sheets.Part ?: A Comparative Study of the osteogenic capability of ASCs and ASC sheet in vitro.Objective: To construct rat ASC sheets, observed the macro and micro structure of them, and compared the osteogenic capability with ASCs in vitro. Methods: ASCs were isolated from the inguinal adipose tissue of male rats ASCs using collagenase I. The third passage ASCs were seeded on petri dishes and cultured in comlete medium with ascorbic acid. The inverted microscope, HE staining and transmission electron microscopy(SEM) were used to observe the morphology of the cultured ASC sheets; Alkaline phosphatase(ALP) staining, sirius-Red staining, real-time quantitative PCR and alizarin red staining were used to investigate the osteogenesis potential of the ASCs and ASC sheets after osteoinductive culture. Results: The thickness of rat ASC sheets are gradually increased over time, and has a certain strength; The TEM observation revealed that the ASCs cytoplasm was rich in endoplasmic reticulum, ribosomes and Golgi, shows extracellular matrix had a large number of collagen fibers. The percent of positive cells of ASC sheet stained for ALP staining, Sirius red staining and alizarin red staining was higher than that of ASCs; The expression level of ALP, OPG expression of, BMP2, OCN and RUNX2 gene in ASC sheet was higher than that of ASCs. Conclusion: The rat ASC sheet in day 7 has a large number of extracellular matrix, and the exuberant cell viability. Rat ASC sheet has stronger osteogenic ability than ASCs.Part ?: Experimental study on calvarial bone healing in T2 DM rats with the implantation of the combination of rat ASC sheet and Bio-oss? bone powderObjective: To assess the bone regeneration potential of rat ASC sheet combined with Bio-oss? bone powder in a critical T2 DM rat calvarial defect. Methods: The high sugar diet fat feeding combined with low-dose long streptozotocin(STZ) intraperitoneal injection was used to constructed T2 DM rat model. Full-thickness critical-sized defects(CSD) of 5mm diameter were created in a total of 40 T2 DM rats. Then ASC sheet, Bio-oss? bone powder or ASC sheet mixed with Bio-oss? bone powder was loaded into the defect site. In the control group, the defects were left untreated. Histological analysis and Micro-CT analysis were used to evalue the healing of defects at 4 or 8 weeks after surgery. Results: Micro-CT showed bone healing of experimental groups were better than the control group; ASC sheet only could significantly increase bone trabecular thickness; the group of compound had a better effect on bone healing than other groups. Histological staining showed: ASC sheet was better than Bio-oss? bone powder on osteogenesis, bone induction, and angiogenesis; the compound group had both the advantages of ASC sheet and Bio-oss? bone powder, which had good effect on bone healing. Conclusions: The combined use of ASC sheet with Bio-oss? bone powder did enhance the potential for bone healing of T2 DM rat.Part ?: Comparative study of rat ASC sheet/ Bio-oss? complex with ASCs / Bio-oss? complex on bone healing of T2 DM rat calvarial CSDObjective: To compare the impact of rat ASC sheet/ Bio-oss? complex with ASCs/ Bio-oss? complex on bone healing of T2 DM rat calvarial CSD. Methods: Firstly, SEM(Scanning electron microscopy, SEM) results of ASC sheet/Bio-oss? complex and ASCs/bone powder complex were compared. Then, the successfully established T2 DM rat models were randomly divided into two groups: ASC sheet/Bio-oss? complex group and ASCs/bone powder complex group depending on the different implants. After 4 weeks and 8 weeks after operation, Micro-CT and histological staining methods were used to evaluate CSD healing and new bone formation. Results: SEM result showed, only a small amount of ASCs were seen on bone powder after 4 hours co-culturing, while a large accumulation of ASCs and rich cell connections were seen on ASC sheet group. Micro-CT and histological staining results showed that ASC sheet/Bio-oss? complex group had a better healing and more new blood vessels. Conclusion: The curative effect of ASC sheet/Bio-oss? complex group on bone healing was better than ASCs/bone powder complex group, and the former method is more convenient and practical.Part ?: Experimental study on calvarial bone healing in T2 DM rats with the implantation of the combination of Sema3 A, ASC sheet and Bio-oss? bone powderObjective: To observe the effect of Sema3 A on osteogenic differentiation capacity of ASC sheet, and the effect of local injection of Sema3 A combined with ASC sheet/Bio-oss? complex on bone healing of T2 DM rat calvarial CSD. Methods: To compare the osteogenic potential of ASC sheet culturing in the osteogenic inducing medium with or without 1?g/m L Sema3 A membrane, ALP staining and alizarin red staining were made after 7 days or 28 days osteoblastic induction. Then,the successfully established T2 DM rat models were randomly divided into two groups: the Sema3 A group and control group. 1, 4, 7 days after the impantation of ASC sheet/Bio-oss? complex, 20ug/kg body weight of sema3 A was injected to the surgical site, while an equal volume of saline was injected to control group. 4 weeks and 8 weeks after operation, Micro-CT and histological staining methods were used to evaluate CSD healing and new bone formation. Results: Both Micro-CT and histological staining results showed Sema3 A group had better healing effect than the control group; but the Sema3 A group had less blood vessels while no significant difference was found in blood vessel number between the control group. Conclusion: Sema3 A could promote the osteogenic differentiation of ASC sheet, and local injection Sema3 A could promote T2 DM rat calvarial bone regeneration based on ASC sheet and Bio-oss? bone powder complex treatment. Sema3 A may lead to less neovascularization at the early time of bone healing, but over time no significantly different was found between the Sema3 A group and the control group. |
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