| BackgroundOsteoporosis and its related fragility fractures have become the main health burden of China’s elderly population.The osteogenic ability of elderly patients with osteoporosis is weak,and the healing cycle of fragility fracture is long,which leads to many osteoporosis related complications.Most patients with osteoporosis only use anti-osteoporosis drugs for the first time when they suffer from fragility fractures.In addition,the effect of anti-osteoporosis drugs on fragility fracture healing is not completely clear,and there are some disadvantages,such as long treatment cycle,low patient compliance,high cost and so on.Therefore,how to effectively treat osteoporosis and accelerate the healing of osteoporotic fractures is an urgent clinical difficulty to be solved.It is very important to correctly deal with osteoporosis and fragility fracture and explore the action mechanism of anti-osteoporosis drugs,especially how to affect bone metabolism and fracture healing.In recent years,the role of sclerotin secreted by osteocyte in bone remodeling has attracted more and more attention.Sclerotin is an important regulator of Wnt/β-catenin pathway,which can competitively inhibit the binding of Wnt protein to related receptors and block the downstream osteogenesis process.By inhibiting the release of sclerotin,sclerotin monoclonal antibody can reduce the antagonism of sclerotin to Wnt pathway,which may be a new way to treat osteoporosis and fragility fracture.However,the effect of Scl-Ab on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells has not been clearly reported.In addition,the effects of Scl-Ab on callus remodeling and fracture healing of osteoporotic fractures need to be further studied.ObjectiveThis study intended to explore the effects of Scl-Ab on the proliferation and osteogenic differentiation of BMSCs at the cellular level.At the same time,we established animal models of osteoporosis and fragility fracture to study the effects of Scl-Ab on bone metabolism and bone healing of fragility fracture.Method(1)Isolation,culture and identification of rabbit BMSCs.Rabbit BMSCs were obtained by gradient density centrifugation combined with adherence method.BMSCs were identified by morphological observation,detection of cell surface antigen markers,induction of osteogenic,adipogenic and chondrogenic differentiation and related gene expression detection.(2)The effect of Scl-Ab on the proliferation and osteogenic differentiation of BMSCs was verified at the cellular level.Different drug concentrations of Scl-Ab were co-cultured with BMSCs,and the cell growth curve was drawn to detect the effect of Scl-Ab on the proliferation of BMSCs.On the 3rd,7th and 14 th days after Scl-Ab intervention,the expressions of osteogenesis-related genes and proteins were detected.(3)The effect of Scl-Ab on bone metabolism in osteoporotic state was verified at the animal level.An animal model of osteoporosis in New Zealand white rabbits was established by bilateral ovariectomy(OVX).The successfully modeled rabbits were randomly divided into 3 groups with 10 rabbits in each group,namely the Scl-Ab-OVX group,the PBS-OVX group and the OVX control group.Scl-Ab drug intervention for 8 weeks,bone metabolism indexes,bone mineral density and Micro CT bone microstructure parameters were detected to verify the therapeutic effect of Scl-Ab on osteoporosis.(4)The effect of Scl-Ab on the healing of osteoporotic fractures was verified at the animal level.A rabbit model of osteoporotic femoral fracture was established,and the successful rabbits were randomly divided into 3 groups with 10 rabbits in each group,namely the Scl-Ab-OVX group,the PBS-OVX group and the OVX control group.After 8 weeks of Scl-Ab drug intervention,the femur was taken out.Micro CT bone microstructure parameters at the fracture end,callus immunohistochemical analysis at the fracture end,Masson staining,ALP and TRAP staining,and femoral three-point bending mechanical property test were performed to evaluate the new bone.formation,callus remodeling,and fracture healing strength.Results(1)The experiment successfully obtained rabbit BMSCs with adherent,consistent morphology,stable proliferation and good condition.The expression rates of BMSCs cell surface antigen markers CD90,CD73,and CD105 detected by flow cytometry were 94.970%,98.394%,and 98.557%,respectively,which were judged to be positive;while the expression rates of CD34 and CD45 were only 4.976% and5.389%,which were judged to be negative expression.BMSCs were induced to differentiate into osteogenic,adipogenic and chondrogenic lineages.With time,the expression levels of Runx2,PPARγ and Sox9 genes increased,indicating that BMSCs had the ability to differentiate into osteogenic,adipogenic and chondrogenic cells.Therefore,the cells obtained in this experiment were identified as rabbit BMSCs.(2)Scl-Ab was co-cultured with BMSCs,and the cell growth curve was drawn by CCK-8 method.The results showed that compared with the control group,Scl-Ab had no significant effect on the proliferation of BMSCs(P>0.05).The positive rates of alkaline phosphatase and calcium nodule staining in the Scl-Ab group were higher than those in the control group,and the difference was statistically significant(P<0.05).RT-PCR results showed that compared with the control group,the expressions of osteogenesis-related genes Col1,Runx2 and OPN were up-regulated in the Scl-Ab group(P<0.05).The expressions of osteogenesis-related proteins Col1 and osteocalcin were higher in the Scl-Ab group than in the control group,and the difference was statistically significant(P<0.05).(3)Six months after bilateral oophorectomy,the bone mineral density of New Zealand white rabbits decreased significantly by 35.8%,indicating that the osteoporosis model was successfully established.After 8 weeks of Scl-Ab drug intervention,compared with the PBS-OVX group and the OVX group,the serum ALP of the rabbits was significantly increased and the level of TRACP5 b was significantly decreased(P<0.05).The results of Micro CT test showed that the bone microstructure parameters of the Scl-Ab group were better than those of the PBS-OVX group and the OVX group,and the difference was statistically significant(P<0.05).There was no significant difference in the biological data between the PBS-OVX group and the OVX group(P>0.05).(4)The experiment successfully established a rabbit osteoporotic femoral fracture model.After 8 weeks of Scl-Ab drug intervention,the results of Micro CT showed that there was more new bone formation at the fracture end in the Scl-Ab group,and Ct.Ar,Ct.Th,Tb.N and Ct.Wi were higher than those in the PBS-OVX group and In the OVX group,the difference was statistically significant(P<0.05).The results of immunohistochemistry showed that more callus formed at the fracture end of the Scl-Ab group,the callus was more mature,and the mineralization rate of the callus at the fracture end was faster(P<0.05).The maximum load,elastic modulus,maximum stress and energy absorption of the femur in the Ab group were significantly higher than those in the PBS-OVX group and the OVX group,and the differences were statistically significant(P<0.05).There was no significant difference in the biological and biomechanical data between the PBS-OVX group and the OVX group(P>0.05).ConclusionScl-Ab can promote the osteogenic differentiation of BMSCs,but has no significant effect on the proliferation of BMSCs.Scl-Ab can improve bone metabolism and bone microstructure in the pathological state of osteoporosis by promoting osteogenesis and inhibiting bone resorption.Scl-Ab can promote callus growth and callus mineralization and maturation,enhance the mechanical properties of new bone,and ultimately promote the early healing of osteoporotic fractures. |
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