| Objective:The pathogenesis of osteoporosis is not clear at present.However,some studies have shown that the occurrence of osteoporosis is closely related to the decrease of blood vessels in bone.Although bone marrow mesenchymal stem cells have great potential in the treatment of osteoporosis,whether the specific mechanism of treating osteoporosis is related to promoting angiogenesis in bone still needs further verification.In this study,animal models of ovariectomized mice with osteoporosis and hind limb ischemia were established by bilateral oophorectomy and unilateral femoral artery ligation,and the effects of bone marrow mesenchymal stem cells on bone mass of ovariectomized mice were observed.To further explore the specific mechanism of its treatment of osteoporosis,provide theoretical basis for the clinical application of bone marrow mesenchymal stem cells,and also provide new ideas for the clinical treatment of osteoporosis.Methods:1.8-week-old mouse bone marrow mesenchymal stem cells were cultured by monolayer culture method,and then transplanted and passaged.They were identified by osteogenesis,adipogenesis,chondrogenesis and flow cytometry.2.Sixty healthy C57BL/6 female mice aged 8 weeks were used to establish animal models of ovariectomy and unilateral femoral artery ligation.They were randomly divided into five groups:Sham operation group(A),bilateral oophorectomy plus solvent control group(OVX+PBS treatment group),lateral oophorectomy plus cell treatment group(OVX+MSCT group),and bilateral oophorectomy plus unilateral femoral artery ligation plus solvent control group(OVX+FAL+PBS treatment)Bone marrow mesenchymal stem cells were transplanted into the established animal model 1 week and 3 weeks after bilateral oophorectomy,that is,1 day and 2 weeks after femoral artery ligation.3.At the 4th week after bilateral oophorectomy,the mice were killed,and the femurs of the mice were completely separated.Micro-CT scanning and 3D reconstruction were performed,and trabecular parameters of cortical bone and cancellous bone were analyzed to observe the changes of bone mass of ovariectomized mice,so as to evaluate the effect of MSCT on bone mass of ovariectomized mice.4.At different time points(1 day,3 days,7 days,14 days and 21 days after MSCT),the blood flow of hind limbs was monitored and the recovery of hind limb blood vessels was evaluated.At the same time,at different time points(1 day,14 days and 28 days after MSCT),the femoral intraosseous blood vessels of mice were imaged,and the angiogenesis was further observed by Micro-CT scanning.5.HE staining,Masson staining,oil red O staining and Trap staining were performed on the femur samples of mice after Micro-CT scanning,so as to detect the changes of bone tissue of ovariectomized mice and the changes of bone tissue of mice after MSCT.Results:1.The results of flow cytometry showed that the surface markers CD29,Sca-1 and CD44 were highly expressed,while the surface markers CD11b,CD45 and CD117 were poorly expressed.It conforms to the expression law of surface markers of BMSCs,and also shows good osteogenesis,adipogenesis and chondrogenesis.2.The results of Micro-CT and bone mass analysis of femur of mice in each group showed that the parameters of cancellous bone such as Tb.BT/TV,Tb.N and Tb.BMD in OVX group were significantly lower than those in Sham group(P<0.05),and the MSCT group was significantly higher than that in OVX group(P<0.05).There is no significant difference among the three groups in cortical bone parameters such as Ct.Ar,Ct.Th and Ct.BMD(P>0.05).3.The results of pathological section analysis of femur of mice in each group show that the bone mass of ovariectomized mice is lost,the collagen fibers in bone tissue are disordered,and the osteoclasts and adipose tissue in bone are increased,but for MSCT mice,the damage degree of bone tissue is obviously reduced(HE staining);Moreover,the collagen content in bone tissue decreased(Masson staining)and osteoclasts decreased significantly(Trap staining).At the same time,the fat content in bone tissue decreased obviously(oil red O staining).4.In ovariectomized mice with femoral artery ligation,bone loss and trabecular continuity were found in both femurs,but the ligation side was more serious.In both OVX group and MSCT group,the Tb.BT/TV,Tb.N and Tb.BMD of ligated side were lower than those of non-ligated side(P<0.05).Meanwhile,the bone tissue parameters in MSCT group were higher than those in OVX group.As for cortical bone parameters,Ct.Ar,Ct.Th and Ct.BMD have no obvious changes,and there is no obvious difference between MSCT group and OVX group(P>0.05).5.The results of laser speckle blood flow meter showed that after femoral artery ligation,the blood flow of hind limbs on the ligated side of mice decreased significantly compared with that on the non-ligated side.Compared with OVX group,the blood flow of hind limbs in MSCT group increased significantly(P<0.05).At the same time,the blood flow of hind limbs in OVX group also increased slowly.However,there was no significant difference in hind limb blood flow between MSCT group and PBS group(P>0.05).6.Three-dimensional reconstruction results of Micro-CT scan of intraosseous angiography showed that the number of blood vessels in the hind limbs of mice after MSCT was significantly higher than that of the control group(P<0.05).On the non-ligated side,the number of hind limb blood vessels in mice decreased slowly with time,while in MSCT group,it increased slowly,and there was a significant difference between them on the 28th day(P<0.05).Conclusions:1.MSCT can effectively prevent bone loss in ovariectomized mice.2.The loss of bone mass in ovariectomized mice is accompanied by the decrease of blood vessels in bone.Blocking the blood flow of bone tissue can aggravate the degree of bone mass loss in ovariectomized mice.3.MSCT increased the intraosseous blood flow of ovariectomized mice after femoral artery ligation,and reduced the bone loss of ovariectomized mice after femoral artery ligation.4.Bone marrow mesenchymal stem cells can reduce the degree of bone loss by increasing the number of blood vessels in bone and restoring the blood flow in bone tissue,thus achieving the purpose of protecting bone tissue. |
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