| T2DM is no longer a contraindication for implantation with the development of dental implant technology, but the failure rate in T2 DM is still much higher than normal patients. In clinical work, unheal of the tooth extraction area is very common in T2 DM patients. Even though the conventional bone substitute materials is transplanted to the bone defect area, the outcome is still limited. Therefore, it is significant to research better bone substitute materials that suitable for T2 DM patients. ASCs has brought revolutionary change in bone tissue regeneration and has become an ideal stem cells source for tissue engineering. Therefore, we recommend that the ASCs based biomaterial may also promote bone formation in T2 DM condition. Prior to evaluation of biomaterial, the standard CSD model is crucial. Unfortunately, present studies are mainly concentrated on normal animal CSD model and the exact value under T2 DM remains unknown. Therefore, this experiment will be divided into two parts. First of all, a serial of calvarial defects wereprepared on both T2 DM and normal rats to estimate the CSD value under T2 DM condition. Secondly, chitosan sponge combined with ASCs cell sheet was transplanted into the T2 DM rats’ calvarial CSD area to observe the bone healing behavior.PART ONE: The experimental research of rat calvarial CSD in T2DMObjective: To explore the calvarial critical size defect(CSD) in type 2 diabetes mellitus(T2DM) rat model. Methods: SPF level male Sprague-Dawley rats weight 300-320 g were used to induce the T2 DM model by high fat and high sugar diet with low dose streptozotocin(STZ) injected intraperitoneally. After the model was made successfully, both T2 DM and normal rats were randomly divided into 4 groups with 3 animals each. The 2, 3, 4 and 5 mm diameter defects were fabricated on the central calvarias throughout calvarial whole thickness outside the endocranium. General observation, X-ray examination and histological study were performed 8 weeks post operation. Results: In T2 DM group, only the 2 mm diameter defects were able to heal completely, the X-ray were resistance, and histological examination showed good new bone formation; the 3, 4, 5 mm diameter defects unhealed, the X-ray transmission and new formed bone is insufficient. In the control group, the 2, 3,4 mm diameter defects healed completely, the X-ray were resistance, and histological examination showed good new bone formation; the 5 mm diameter defects were unhealed, the X-ray were transmission and new formed bone is insufficient. Conclusion: T2 DM rats calvarial bone defect healing worse than normal rats significantly, and the calvarial CSD of T2 DM rat model can be defined as 3 mm.PART TWO: The experimental research of T2 DM rat calvarial CSD healing by using chitosan sponge loaded with ASCs sheetObjective: To observe the ability of chitosan sponge loaded ASCs sheet in enhancing wound healing of calvarial CSD in rat T2 DM model. Methods: ASCs was isolated and culture in vitro. The osteogenic differentiation was confirmed by BCIP/NBT, Sirius Red and Alizarin Red staining while the adipogenic differentiation was observed by Oil Red staining. Chitosan sponge was prepared by freeze drying. The ASCs loading efficiency of chitosan sponge was observed by DAPI staining and SEM observation. The collagen secretion of ASCs sheet was checked by Sirius Red staining. The chitosan sponge loadedwith ASCs sheet was transplanted into the calvarial CSD of T2 DM rat, while the defect of control group left unfilled. Each group was operated in 5 rats. Animals were sacrificed after 8 weeks of healing and the bone formation was evaluated via general observation, Micro-CT scanning and histological staining. Results: ASCs has good abilities of both osteogenic and adipogenic differentiation. ASCs can be loaded by chitosan sponge efficiently. The healing capability of ASCs sheet was significantly better than the control group. Conclusion: Chitosan sponge loaded with ASCs sheet can promote the healing of calvarial CSD in rat T2 DM model. |
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