Effects Of Strontium Ranelate On Fracture Healing In Bilateral Ovariectomized Type 2 Diabetic Mellitus Rats

Part one Research on fracture healing in type 2 diabetic mellitus ratsObjective:An animal model of type 2 diabetes mellitus(T2DM)was established by feeding SD rats with high-fat diet and intraperitoneal injection of low-dose streptozotocin(STZ),and on the basis of this,an animal model of right femoral shaft closed fracture was established.Based on this model,the effect of T2 DM on the fracture healing at 2 and 3 weeks after fracture was studied.Methods:1.Thirty-two female healthy SD rats,2 months old,were randomly assigned to 2 groups(n = 16):control fracture group(CF),T2 DM fracture group(DF).2.The experimental animals were subjected to X-ray examination of the right femur at 2 and 3 weeks post-fracture.The animals were sacrificed and the materials were obtained.Various biochemical indicators in the serum were detected.Specimens were stained with SafraninO-Fast Green,and the ratio of bone,cartilage and fibrous callus to total callus area was calculated by histomorphometry.Immunohistochemical staining of VEGF and α-SMA was performed to analyze the expression of VEGF and vascular density in the callus.Results:1.After establishment of animal model of T2 DM,blood glucose levels were markedly higher in the DF group than the CF group during the experiment.Compared with CF group,the T-CHO and TG levels in serum showed markedly higher in the DF group at 2 and 3 weeks post-fracture(P < 0.05).2.At 2 and 3 weeks post-fracture,the markedly higher radiographic healing score was observed in the CF group compared with the DF group(P < 0.05).3.Analysing the results of SafraninO-Fast Green staining,at 2 weeks post-fracture,the DF group displayed markedly lower area ratios of bony and cartilaginous callus than the CF group(P < 0.05),but the markedly higher area ratio of fibrous callus was found in the DF group compared with the CF group(P < 0.05).At 3 weeks post-fracture,the DF group showed markedly lower area ratio of bony callus than the CF group(P < 0.05),and the markedly higher area ratio of fibrous callus was still found in the DF group compared with the CF group(P < 0.05),while no remarkable difference was observed between the two groups for the area ratio of cartilaginous callus.4.At 2 weeks post-fracture,the DF group displayed markedly lower expression of VEGF and blood vessel density than the CF group(P < 0.05).At 3 weeks post-fracture,no remarkable difference was evident between the two groups for VEGF.The DF group displayed markedly higher blood vessel density in callus compared with the CF group(P < 0.05).Part two Research on fracture healing in type 2 diabetic mellitus rats with bilateral ovariectomyObjective:Simultaneously with the establishment of animal models of T2 DM,the bilateral ovaries of rats were excised for osteoporosis,and on the basis of this,an animal model of the right femoral shaft closed fracture was created to study its effect on the fracture healing.Methods:1.Forty-eight female healthy SD rats,2 months old,were randomly assigned to 3 groups(n=16):control fracture group(CF),T2DM+fracture group(DF),T2DM+Osteoporosis+fracture group(DOF).2.The experimental animals were subjected to X-ray examination of the right femur at 2 and 3 weeks post-fracture.The animals were sacrificed and the materials were obtained.After micro-CT examination,the bone tissue was fully decalcified.The specimens were stained with Masson,and the ratio of bone and cartilage callus to total callus area was calculated by histomorph-ometry.And the expression of Col II and Caspase-3 in callus was analyzed by immunohistochemistry.Results:1.After establishment of animal model of T2 DM,blood glucose levels were markedly higher in the DF and DOF group compared with CF group.2.At 2 weeks post-fracture,both the DF and DOF groups displayed markedly smaller area of callus measured by X-ray than the CF group(P < 0.05),while the DOF group showed markedly smaller area of callus than the DF group(P < 0.05).At 3 weeks post-fracture,no remarkable differences were observed between those three groups for area of callus.3.Analysing the results of Micro-CT,at 2 weeks post-fracture,the DOF group displayed markedly lower Tb.N than the CF and DF groups(P < 0.05),while no remarkable difference was evident between the CF and DF groups.The DF and DOF group displayed markedly lower Tb.Th than the CF group(P < 0.05),while no remarkable difference was evident between the DF and DOF groups.The DOF group displayed markedly higher Tb.Sp than the CF and DF groups(P < 0.05),while no remarkable difference was evident between the CF and DF groups.At 3 weeks post-fracture,the DF and DOF groups displayed markedly lower Tb.N than the CF group(P < 0.05),while the DOF group showed markedly lower Tb.N than the DF group(P < 0.05).The DF and DOF groups displayed markedly lower Tb.Th than the CF group(P < 0.05),while no remarkable difference was evident between the DF and DOF groups.The DF and DOF groups displayed markedly higher Tb.Sp than the CF group(P < 0.05),and the DOF group showed markedly higher Tb.Sp than the DF group(P < 0.05).4.Analysing the results of masson’s staining,at 2 weeks post-fracture,the DF and DOF groups displayed markedly smaller area ratio of bony and cartilaginous callus than the CF group(P < 0.05),and the DOF group showed lower area ratio of cartilaginous callus than the DF group(P < 0.05),but no remarkable difference was observed between the DF and DOF groups for the area ratio of bony callus.At 3 weeks post-fracture,the DF and DOF groups displayed markedly smaller area ratio of bony callus than the CF group(P < 0.05),while the DOF group showed markedly smaller area ratio of bony callus than the DF group(P < 0.05),but no remarkable difference was observed between those three groups for the area ratio of cartilaginous callus.5.At 2 weeks post-fracture,the DF and DOF groups displayed markedly lower expression of Col Ⅱ than the CF group(P < 0.05),while the DOF group showed markedly lower expression of Col Ⅱ than the DF group(P < 0.05).At 3 weeks post-fracture,the DOF group displayed markedly lower expression of Col Ⅱ than the CF and DF groups(P < 0.05),but no remarkable difference was evident between the CF and DF groups.At 2 weeks post-fracture,the DF and DOF groups displayed markedly higher expression of Caspase-3 than the CF group(P < 0.05),and the DOF group showed markedly greater expression of Caspase-3 than the DF group(P < 0.05).At 3 weeks post-fracture,the DF and DOF groups displayed markedly greater expression of Caspase-3 than the CF group(P < 0.05),while no remarkable difference was evident between the DOF and DF groups.Part three Effects of strontium ranelate combined with insulin on fracture healing in bilateral ovariectomized type 2 diabetic mellitus ratsObjective:Based on the rat model of T2 DM,bilateral ovariectomy and closed femoral fracture,the effects of insulin alone or combined with strontium ranelate on fracture healing were studied.Methods:1.Eighty female healthy SD rats,2 months old,were randomly assigned to 5 groups(n=16):control fracture group(CF),T2DM+fracture group(DF),T2DM+Osteoporosis+fracture group(DOF),T2DM+Osteoporosis+Fracture+ Insulin group(DOFI),T2DM+Osteoporosis+Fracture+Insulin+Strontium ranelate group(DOFIS).2.The experimental animals were subjected to X-ray examination of the right femur at 2 and 3 weeks post-fracture.The animals were sacrificed and the materials were obtained.After micro-CT examination,the bone tissue was fully decalcified.The specimens were stained with SafraninO-Fast Green,and the ratio of bony and cartilaginous callus to total callus area was calculated by histomorphometry.The number of osteoclasts per callus area was analyzed by TRAP staining.And the expression of OCN and Col Ⅰ in callus was analyzed by immunohistochemistry.Results:1.After establishment of animal model of T2 DM,blood glucose levels were markedly higher in the DF,DOF,DOFI and DOFIS groups compared with CF group.After insulin intervention,blood glucose levels of the DOFI and DOFIS groups tended to be normal.2.At 2 weeks post-fracture,the markedly lower radiographic healing scores were observed in the DF and DOF groups compared with the CF group(P < 0.05),while no remarkable difference was observed between the DF and DOF groups,or between the DOF,DOFI and DOFIS groups.At 3 weeks post-fracture,the CF,DF,DOFI and DOFIS groups displayed markedly higher radiographic healing scores than the DOF group(P < 0.05),and the CF group showed higher radiographic healing scores than the DF group(P < 0.05),but no remarkable difference was observed between the DOFI and DOFIS groups.3.Analysing the results of Micro-CT,at 2 weeks post-fracture,the DF and DOF groups displayed markedly lower BMD,BV,TV and BV/TV than the CF group(P < 0.05),and the DOF group showed markedly lower BV,TV and BV/TV than the DF group(P < 0.05),but no remarkable difference was evident between the DF and DOF group for BMD.At 3 weeks post-fracture,the DF and DOF groups displayed markedly lower BMD,BV and BV/TV than the CF group(P < 0.05),and the DOF group showed markedly lower BMD,BV and BV/TV than the DF group(P < 0.05),but no remarkable difference was evident between those three groups for TV.At 2 weeks post-fracture,the DOFI and DOFIS groups displayed markedly higher BV,TV and BV/TV than the DOF group(P < 0.05),but no remarkable difference was evident between the DOFI and DOFIS groups for those three parameters,and no remarkable difference was evident between those three groups for BMD.At 3 weeks post-fracture,the DOFI and DOFIS groups displayed markedly higher BMD,BV and BV/TV than the DOF group(P < 0.05),but no remarkable difference was observed between the DOFI and DOFIS groups for those three parameters,and no remarkable difference was evident between the DOF,DOFI and DOFIS groups for TV.4.Analysing the results of SafraninO-Fast Green staining,at 2 weeks post-fracture,the DF and DOF group displayed markedly smaller area ratio of bony callus than the CF group(P < 0.05),but no remarkable difference was observed between the DF and DOF groups.There was no remarkable difference in the area ratio of bony callus between the DOF,DOFI and DOFIS groups.At 3 weeks post-fracture,the DF and DOF groups displayed markedly smaller area ratio of bony callus than the CF group(P < 0.05),while the DOF group showed smaller area ratio of bony callus than the DF group(P < 0.05).The DOFI and DOFIS groups displayed markedly higher area ratio of bony callus than the DOF group(P < 0.05),but no remarkable difference was observed between the DOFI and DOFIS groups.At 2 weeks post-fracture,the DF and DOF groups displayed markedly smaller area ratio of cartilaginous callus than the CF group(P < 0.05),while the DOF group showed smaller area ratio of cartilaginous callus than the DF group(P < 0.05).The DOFI and DOFIS groups displayed markedly larger area ratio of cartilaginous callus than the DOF group(P < 0.05),but no remarkable difference was observed between the DOFI and DOFIS groups.At 3 weeks post-fracture,there was no remarkable difference in the area ratio of cartilaginous callus between those groups.5.Analysing the results of TRAP staining,at 2 and 3 weeks post-fracture,the DF and DOF groups displayed markedly larger number of osteoclasts per callus area than the CF group(P < 0.05),and the DOF group showed markedly larger number of osteoclasts per callus area than the DF group(P < 0.05).At 2 weeks post-fracture,the number of osteoclasts per callus area was no remarkable difference between the DOF,DOFI and DOFIS groups.At 3 weeks post-fracture,the DOFI and DOFIS groups displayed markedly lower number of osteoclasts per callus area than the DF group(P < 0.05),while the DOFIS group showed markedly lower number of osteoclasts per callus area than the DOFI group(P < 0.05).6.At 2 weeks post-fracture,the DF and the DOF groups displayed markedly lower expression of OCN and Col I than the CF group(P < 0.05),while no remarkable difference was evident between the DF and DOF group.At 3 weeks post-fracture,the DF and the DOF groups displayed markedly lower expression of OCN and Col I than the CF group(P < 0.05),while the DOF group showed markedly lower expression of OCN and Col I than the DF group(P < 0.05).At 2 weeks post-fracture,the DOFIS group displayed markedly higher expression of OCN and Col I than the DOF and DOFI groups(P < 0.05),while no remarkable difference was observed between the DOF and DOFI groups.At 3 weeks post-fracture,the DOFI and DOFIS groups displayed markedly higher expression of OCN and Col I than the DOF group(P < 0.05),and the DOFIS group showed markedly higher expression of Col I than the DOFI group(P < 0.05),but no remarkable difference was observed between the DOFI and DOFIS groups for OCN.Conclusion:1.The T2 DM animal model can be successfully established by high-fat diet combined with low-dose STZ injection,and the animal model was consistent with the characteristics of human T2 DM.Analysis by X-ray,histomorphology and immunohistochemistry showed that T2 DM had a significant inhibitory effect on angiogenesis in the early stage of fracture healing,which may be an important reason for the delayed fracture healing.2.In the T2 DM animal model,bilateral ovarian resection can significantly reduce the area of the callus in the early stage of fracture healing,and the spatial microstructure of the new bone in the callus was further damaged,and the fracture healing was delayed.The possible reason for this was that it increases the apoptosis of chondrocytes in callus and inhibits the endochondral ossification process of fracture healing.3.Insulin alone treatment may promote the fracture healing process in bilateral ovariectomized T2 DM rats,and improve the bone mass and microstructure in callus.And its role in promoting bone formation and inhibiting bone resorption may be an important reason for this result.On this basis,combined strontium raniate interventions have been shown to further promote bone formation and inhibit bone resorption,but in the context of this experimental cycle and drug dose,the fracture healing process,bone mass and microstructure in callus have not significantly been improved.