| Dental implant prosthesis is the first choice for the treatment of tooth loss,and bone bonding 1s the key factor for successfiil implantation.Poor osseointegration caused by ageing osteoporosis has become a serious problem for dental implants in tihe elderly.Mesenchymal stem cells are the basis of tissue regeneration,but the functions of different tissue sources are different.We have successfully obtained BMMSCs from the jaw in the early stage,and confinned that the alveolar bone osteogenesis ability of BMMSCs in the jaw is significantly stronger than that of BMMSCs in the iliac bone marrow,and Wnt signaling pathway plays a significant role in the process of osteogenesis.However,the molecular mechanism and regulation of Wnt signaling pathway in JBMMSCs osteogenic differentiation and bone regeneration ability in aging microenvironment are still unclear.Therefore,this study aims to explore the changes of biological behavior of JBMMSCs derived from natural aging osteoporosis,and to elucidate the regulatory mechanism and regulation of Wnt signaling pathway in JBMMSCs osteogenic differentiation under natural aging osteoporosis.Objective:1.To establish an aging osteoporosis rat model.2.Isolate and extract jaw bone marrow mesenchymal stem cells from natural aging rats.3.To investigate the changes in the biological function of JBMMSCs under senile osteoporosis.4.To explore the regulation of classical Wnt signaling pathway on osteogenic differentiation of JBMMSCs in senile osteoporosis.Method:1.Micro-CT scans were performed on 2-month-old rats and 20-month-old rats(20 males each).After the scans were completed,tissue reconstruction and data analysis were carried out to quantitatively analyze the trabecular parameters of femoral metaphysis and jaw bone marrow cavity,such as bone mineral density,number of trabeculae and trabecular segregation.Deternine whether the model is successful.2.The femur and jaw of young and old rats were sectioned and HE stained to observe the histopathological changes.3.JBMMSCs were cultured by adherence method,and the surface molecules of stem cells were identified by flow cytometry.4.CCK8 method was used to detect the cell proliferation of young and aging JBMMSCs;flow cytometry was used to detect the cell cycle and apoptosis of young and aging JBMMSCs;alkaline phosphatase staihing,alizarin red staining and oil red O staining were used to detect the osteogenic and adipogenic differentiation of young and aging JBMMSCs.5.The expression of Wnt classical pathway related molecules and osteogenesis related genes and proteins(Wnt3a,Wnt4,beta-catenin,GSK-3beta,TCF4,LEF1,RUNX-2,COL-I,OCN,BSP,etc.)in JBMMSCs under young and senile conditions were detected by Real-Time PCR and Western blot.Result:1.Bone mineral density and trabecular number of jaw and femur in aging group were significantly lower than those in young group,while tOabecular spacing was significantly larger than that in young group.There were sig1ificant differences in parameters between the two groups(P<0.05).The results of HE section staining showed that the bone marrov cavity of jaw and femur in aging group was significantly adipogenic compared with that in young group.2.JBMMSCs were successfully isolated from the jaws of 2-month-old rats and 20-month-old rats.The morphology of JBMMSCs in the aging group was flatter than that in the younger group,and the positive expression of CD29,CD90 and negative expression of hematopoietic stem cell surface markers CD34 and CD45 were consistent with the characteristics of mesenchymal stem cells.3.The results of clone formation experiment showed that the number of clone heaps formed in the young group was 909.33(+21.20),the clone formation rate was 18.19(+0.42%),the number of clone heaps formed in the aging group was 299.33(+41.18),and the clone formation rate was 5.99(+0.83%).The clone formation ability of JBMMSCs in the aging group was weaker than that in the young group,with significant difference(P<0.05).4.The results of CCK8 experiment showed that the OD values of the two groups were significantly different from 3 to 7 days after culture at each time point(P<0.05).5.The results of flow cytometry showed that the percentage of GOG1 phase cells in the aging group was higher than that in the young group,while the proportion of S phase cells was lower than that in the young group,which indicated that the proliferation of JBMMSCs in the aging group was decreased;the apoptotic level in the aging group was higher than that in the young group,and the difference was significant(P<0.05).6.The results of ALP staining and alizarin red staining showed that the blue precipitation and mineralized nodules in the aging group were lower than those in the young group.O1l red O staining showed that the number of lipid droplets in the aging group was significantly higher than that in the young group.7.Real-time quantitative PCR results showed that the expression levels of osteogenesis-related genes RUNX-2,OCN,COL-1 and BSP in the aged group were lower than those in the young group 7 days after osteogenic induction.8.Western blot further confirmed that after osteogenesis induction,the expression of RUNX2 and OCN in both groups was significantly higher than that in the control group,but after osteogenesis induction,the expression of RUNX2 and OCN in the aging group was lower than that in the young group.9.The results of PCR showed that the expression levels of Wnt signaling pathway related molecules,such as beta-catenin,Wnt3a and TCF-4,were sig1ificantly lower in the aging group than those in the younger group,while the expression levels of Wnt49 GSK3? and LEF-1 did not change significantly.10.Western blot results showed that the expression levels of Wnt3a protenin,beta-catenin protein,TCF-4 protein,the related molecules of Wnt signaling pathway,was significantly lower in the aging group than in the younger group.Conclusion:1.Obvious osteoporosis symptoms were observed in 20-month-old rats,which were mainly manifested in the decrease of bone mineral density of femur and jaw,the decrease of bone trabecular strength,the adipogenesis of bone marrow cavity,and the successful establishment of aging osteoporosis rat model.2.Under the condition of senile osteoporosis,the biological function of JBMMSCs has changed significantly,which is mainly manifested in the decrease of self-renewal ability,proliferation and osteogenic of JBMMSCs.3.Under the condition of senile osteoporosis,the expression of Wnt3a,?-catenin,TCF-4,the related molecules of Wnt classical signaling pathway,decreased,which may be an important reason for the biological behavior changes of JBMMSCs. |
PDF Full Text Request