| Implantology is the most effective way to repair missing teeth in current clinical application.Diseases such as inflammation,tumour and trauma may lead to insufficient bone mass and narrow the indications for implant repair.Although autologous bone transplantation can solve this problem to some extent,opening the second operation area will result in limited clinical application.Therefore,it is urgent for researchers to find a better way of repairing bone defects on the implant site.The emergence of tissue engineering in recent years has provided a new approach to bone defect repair.Tissue engineering has been applied to different fields mainly by forming complexes of seed cells and biomaterial scaffolds to implant defect sites to fo rm new tissues.Finding the optimal seed cells is an important step for the application of bone defect in tissue engineering.Adipose-derived stem cells(ADSCs)have attracted great attention for their advantages,such as rich sources,easy access,easy clinical transformation and potential of osteoblast differentiation,which has become an ideal choice of adult stem cells in bone tissue engineer.Since ADSCs have the potential of adipogenic differentiation,it is necessary for researchers to find the factors that can promote their osteogenic differentiation and inhibit their adipogenic differentiation at the same time.As a glucagon-like peptide-1(GLP-1)receptor agonist,Exendin-4 is the main component of the exenatide and is clinically used in the treatment of type 2 diabetes.It has been proven that Exendin-4 can prevent bone loss and accelerate fracture repair by inducing osteogenic differentiation of BMSCs.However,there is no report at home and abroad whether Exendin-4 can promote ADSCs to differentiate into osteoblasts via Wnt/β-catenin signaling pathway.Our research systematically focuses on the effects and molecular regulation mechanism of Exendin-4 on regulating the osteogenic differentiation of ADSCs both in vitro and in vivo and aims at establishing the theoretical basis of the combination of Exendin-4 and ADSCs on the bone defect repair.This research is divided into the following four parts.Part Ⅰ Isolation and identification of ADSCs and diaphragm construction of ADSCs BackgroundADSCs have been widely used for their abundant sources,easy access,and their potential of osteogenic differentiation.However,ADSCs alone can not meet the clinical and basic research application.Therefore,it is of great application value to construct highly purified ADSCs and their diaphragm for the following experimental research in vitro and in vivo.Methods ADSCs were extracted by enzyme digestion for the adipose tissue of C57BL/6J mice.The proliferation of ADSCs was observed by CCK-8 assays.The expression of surface markers(CD29,CD34,CD35 and Sca-1)was detected by flow cytometry.The osteogenic and adipogenic differentiation ability of ADSCs were identified by Alizarin Red S staining and Oil Red O staining.In addition,diaphragm of ADSCs was constructed using induction solution,and the film formation was observed by HE staining and transmission electron microscopy.Results Morphological findings showed that the density of ADSCs was uniform and the morphology was good.The results of flow cytometry showed that the positive markers of ADSCs surface antibodies(CD29 and Sca-1)were highly expressed.Alizarin Red S staining and Oil Red O staining showed obvious calcified nodules and lipid droplets.HE staining showed that the content of cells in the diaphragm of ADSCs was high and the cellular connection was good.The results of transmission electron microscopy presented abundant extracellular matrix of ADSCs and clearly visible organelles.Conclusion The purified stem cells were exactly ADSCs,which have the great potential to be differentiated into osteoblasts and adipocytes.The cells in the diaphragm of ADSCs were in good function and could be used for our follow-up research.Part Ⅱ Effects of Exendin-4 on biological behaviour and function of ADSCsBackground As a common drug of type 2 diabetes mellitus,Exendin-4 has showed promoting effects on osteogenic differentiation of BMSCs.However,the effects of Exendin-4 on the biological behaviour and function of ADSCs is unknown.In addition,it is still a challenge to reserchers whether Exendin-4 can promote the differentiation of ADSCs into osteoblasts.Methods Exendin-4 and Ex9-39(GLP-1 receptor antagonist)were used to intervene extracted ADSCs.The effects of Exendin-4 on cellular proliferation,apoptosis,and migration of ADSCs were detected by CCK-8 assays,flow cytometry and Transwell test,respectively.The effects of Exendin-4 on the osteogenic differentiation and adipogenic differentiation of ADSCs were evaluated by Alizarin Red S staining and Oil Red O staining,respectively.The effects of Exendin-4 on the expression level of genes and protein related to bone formation in ADSCs were detected by qRT-PCR and Western blotting assays.Results Exendin-4 intervention had no significant effects on the proliferation and apoptosis of ADSCs.The results of Transwell test showed that Exendin-4 significantly enhanced the migration of ADSCs.The results of Alizarin Red S staining and Oil Red O staining showed that after Exendin-4 intervention the calcification areas in ADSCs were significantly increased while the number of lipid droplets was significantly decreased.Conversely,the calcification area was significantly reduced and the number of lipid droplets was also significantly increased when Ex9-39 was applied.The results of m RNA and protein quantification showed that Exendin-4 significantly up-regulated the expression of osteogenic-related genes and proteins(e.g.Runx-2,ALP,COL-1,osterix and OPG)but significantly down-regulated the expression levels of adipogenic related genes and proteins(e.g.PPAR-γ,ADPN and PLIN).However,the expression of osteogenic-related genes and proteins were significantly decreased while the expression of adipogenic-related genes and proteins were significantly decreased after Ex9-39 treatment.Conclusion Exendin-4 can significantly promote the differentiation of ADSCs into osteoblasts and meanwhile greatly inhibit the differentiation of ADSCs into adipocytes through the enhanment of ADSCs migration ability.Part Ⅲ Effects of Exendin-4 combined with ADSCs on bone defect repairBackground The technology of cell diaphragm has provided a scientific and feasible method to improve the implanted cellular viability in clinical application.However,it has not been systematically reported whether the combination of Exendin-4 with ADSCs diagram has a positive effect on bone defect repair by inducing the osteogenic differentiation of ADSCs in the complex internal environment and immune system.Methods The mice model with femur defect was established and was injected the Exendin-4.The new bone formation and the repair of bone injury were observed and analyzed by MicroCT scanning,HE staining and immunohistochemical staining.The bone mineralization deposition rate(MAR)and bone formation rate(BFR/BS)were detected by fluorescence double labeling.The expression of genes and proteins related to bone formation was quantitatively analyzed by qRT-PCR and Western blotting assays.Results The results of Micro-CT scanning showed that the trabeculae mass,BV/TV and Tb.N in the damaged femur implanted with ADSCs diaphragm were significantly increased compared with the injured group,and the trabeculae mass at the injured site was further increased after Exendin-4 injection.The results of HE staining,immumohistochemical staining and fluorescence double labeling assays showed that both ADSCs diaphragm implantion and the combination ADSCs of with Exendin-4 could significantly promoted the bone formation of trabecular,inhibit the adipogenic differentiation and increase the MAR and BFR/BS of trabecular bone.The results of m RNA and protein quantification showed that the expression levels of osteogenicrelated genes and proteins(Runx-2,ALP,osterix,COL-1 and OPG)was increased after ADSCs diaphragm implantion.The expression of higher gene and protein related to bone formation was demonstrated after the combination of ADSCs diaphragm implantion and Exendin-4 intervene.Conclusion ADSCs diaphragm combined with Exendin-4 can increase the trabeculae mass and significantly improve the deterioration of trabecular microstructure caused by bone defect.Besides,the combination of ADSCs diaphragm and Exendin-4 can promote the differentiation of ADSCs into osteoblasts by activating the expression of osteogenic related factors,which plays an important role in bone injury repair.Part Ⅳ Mechanism of Exendin-4 on promoting osteogenic differentiation of ADSCs via classical Wnt signaling pathwayBackground It has been reported that Wnt/β-catenin signaling pathway is closely related to skeletal diseases.However,whether Exendin-4 can regulate the differentiation of ADSCs into osteoblasts to promote bone formation through Wnt/β-catenin signaling pathway has not been reported at home and abroad.Methods The siRNA technology was used to make Wnt3 a in ADSCs into targeted silence and then Exendin-4 intervention was applied.Immunofluorescence and Western blotting assays were used to detect the protein expression in Wnt/β-catenin signaling pathway.CCK-8 assays,flow cytometry,Alizarin Red S staining,qRT-PCR and Western blotting assays were used to detect the cellular viability,apoptosis,mineralization,RNA and protein expression related to bone formation in ADSCs after Wnt3 a was silenced.Results Exendin-4 activated the protein levels of Wnt3 a,GSK-3β and β-catenin in ADSCs.After Wnt3 a was silenced,the cellular proliferation of ADSCs was significantly decreased while the apoptosis level was significantly increased with no significant effects of Exendin-4 intervention on the cell viability of ADSCs.The results of Alizarin Red S staining showed that the promoting effects of Exendin-4 on osteogenic differentiation of ADSCs was greatly inhibited after Wnt3 a was silenced.Forthermore,there was no significant difference in the expression of osteogenic-related genes and proteins in ADSCs with or without Exendin-4 intervention after the gene expression of Wnt3 a was silenced.Conclusion Exendin-4 can activate classical Wnt/β-catenin signaling pathway in ADSCs and regulate osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.In conclusion,Exendin-4 can improve the behavioral function of ADSCs,promote the differentiation of ADSCs into osteoblasts,enhance the deterioration of trabecular microstructure caused by bone defect and facilitate bone repair.Our research has revealed the mechasim of the effects of Exendin-4 on promoting the osteoblastic differentiation of ADSCs via Wnt/β-catenin signaling pathway,which provides a scientific experiment evidence for the clinical application of Exendin-4 on bone injury. |
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