The Exper Imental And Cl Inical Study On Huoxue Quyu Method Preventing Steroid-induced Osteonecrosis Of The Femoral Head Collapse

Objective1.Observe the rabbit steroid-induced avascular necrosis(SIONFH)model in the process of natural restoration bone trabecular space structure parameters and reconstruction method.2.The observation method of huoxue quyu effects on femoral head necrosis restoration approach.3.Comparison of Micro-CT in vitro and in vivo HRMRI detect change the consistency of the animal model of bone trabecular space structure.4.Compare the model around the necrotic bone tissue under the change of cytokines,analysis method of huoxue quyu osteogenesis-osteoclasts coupling control.5.Evaluation of huoxue quyu TCM clinical treatment SIONFH cases bone trabecular space structure parameters and the trabecular bone reconstruction method,analysis method of huoxue quyu mechanism to prevent collapse of the femoral head necrosis.Methods1.The first part,the evaluation method of activating blood and remove stasis of rabbit femoral head necrosis repair way:the influence of 50 healthy adult New Zealand white rabbit,randomly assigned into the necrosis group,the Chinese medicine group and each group of 20,blank group and 10.Endotoxin(LPS)methylprednisolone(MPS)in accordance with the LPSX1(10?g/kg)ear marginal vein injection combined with MPS×6(40mg/kg)muscle injection production model of the hormone osteonecrosis in rabbits.Chinese traditional medicine group use the "Golden mirror of medicine" Taohongsiwu decoction in rabbit and equivalent dose lavage treatment.In 2,4,8,12 go HE dyeing and HRMRI,Micro-CT examination,observation necrosis and repair.Spatial resolution HRMRI minimum 0.03 mm or less,Micro-CT the minimum spatial resolution of 0.035 mm or less,set the structural parameters of trabecular bone imaging,comparative HRMRI and Micro-CT in the role in the process of measuring femoral head necrosis trabecular bone reconstruction.Using Western Blot techniques testing RANK,RANKL,VEGF,and OPG,RUNX2,OPN,ABCB1,PPAR-? protein expression levels of femoral head in rabbits at the end of the experiments.2.The second part,discusses the prevention of huoxue quyu method of femoral head necrosis collapse mechanism:refer to "standard of adult femoral head necrosis diagnosis expert consensus"(2012 edition)and "the diagnosis and treatment of avascular necrosis of the expert advice of diagnostic criteria",screening since December 2011 to October 2014,the first affiliated hospital of guangzhou medical university joint bone were collecting cases,a total of 36 cases(64 hips)of hormone osteonecrosis,20 cases of male,female 16 cases,28 cases were bilateral.20?50 years old,35 years old on average.A joint pain in patients with time is 2?24 weeks,average 17 weeks.Sign the informed consent,the hospital medical ethics committee approval into the group.Every 3 months a X-ray line double hip is frog,measured records femoral head collapse time and collapse height.Line HRMRI scan every six months,measuring the trabecular bone related spatial structure parameters.Follow-up time 14?38 months,an average of 27 months.Before treatment,medication for 3 months,6 months,1 year,the medicine for two years,Harris scoring record patients respectively.Based on the patient's MRI image data,using Mimics 13.1 medical imaging control system reconstruction necrosis area,three-dimensional structure of hardening area;Using image processing software ImageJ evaluation bearing area coronal necrotic area and hardening of the area ratio of longitudinal section area of the femoral head.Through the analysis of the apparent bone trabecular space structure change,huoxue quyu method to prevent femoral head necrosis collapse mechanism.Results1.SIONFH building assessment:40 New Zealand white rabbit,traditional Chinese medicine(TCM)group(n = 20)and necrosis group(n = 20)after injection of endotoxin,the seven died within 48 h,33 only survival,the survival rate is 17.5%.Necrosis group have only 14 necrotic sign,necrosis incidence rate of 82.4%(14/17);Chinese traditional medicine group have 5 only appear necrotic sign,necrosis incidence rate of 31.3%(5/16).2.Histological observation:Femoral head section of HE staining showed that necrosis group:The cartilage surface wrinkles,trabecular bone sparse significantly compared with normal group,part of the trabecular bone fracture,bone nucleus pycnosis,empty pit of bone formation,bone marrow fat cell accumulation;Groups:Normal trabecular bone integrity and rules,the trabecular bone of the bone cells can be clearly seen,empty pit is rare,bone marrow hematopoietic cells in rich,fat cells is relatively small,form normal;Chinese traditional medicine group:The trabecular bone thinning,configuration is fair,osteoblast active.4 weeks when empty bone necrosis group pit rate was(17.24± 1.08)%,the normal group(5.33±0.96)%,have significant difference(P<0.05),Chinese medicine group at 4 weeks no difference between the histology and necrosis group,8 weeks and 12 weeks of visible osteogenesis osteoblast active detection,trabecular bone form complete earlier,empty pit rate is reduced,there is a significant difference(P<0.05).3.HRMRI scanning analysis:T1WI visible in the femoral head model group was obviously low signal;Chinese traditional medicine group within the femoral head near the epiphyseal small patch sample in low signal area,but most of the bone tissue grey value is normal.HRMRI structure parameter analysis,according to the results of model group compared with normal group,the former's ROE value(that is,necrosis and edema area area)increases,the higher proportion of ROE/NR,Chinese traditional medicine group compared with model group,Chinese medicine group reduce the value of the ROE,ROE/with a decline in the proportion of NR,significant differences(P<0.05),suggesting the remove stasis method "can promote area repair hormone osteonecrosis.4.Micro-CT scan analysis:Each time node groups of rabbits were taken right hind leg femoral head line of Micro-CT,apparent trabecular bone necrosis group sparse in the normal group,part of the fracture.Relative bone to improve Chinese traditional medicine group,the trabecular bone has a local repair.Micro-CT testing found that necrosis group compared with normal group,the former BV,Conn.,D.,SMI,Tb.N,Tb.Th value decreased,BV/TV decline,BS/BV,SMI,Tb.Sp value increases.Compared to traditional Chinese medicine(TCM)and necrosis group,the former BV,Conn.,D.,SMI,Tb.N value increases,rising rates of BV/TV,BS/BV,Tb.Sp,Tb.Th values fell,the significant difference(P<0.05).5.SIONFH related protein detection:Western blot detection ABCB1,RUNX2,OPN,RANK,RANKL,PPAR gamma,OPG and VEGF protein expression.Group compared with normal group,the necrosis of ABCB1 protein expression significantly reduced,significant differences(P<0.05),while Chinese medicine group relative to the necrosis of ABCB1 higher protein expression,significant differences(P<0.05).Compared with normal group,the necrosis group RANK,RANKL,RUNX2 protein were increased,OPG protein is reduced,significant differences(P<0.05);And Chinese medicine group than necrosis,RUNX2,elevated OPG and RANK,RANKL expression reduced(P<0.05).Compared with the normal group,reduced protein expression of VEGF in necrosis group,significant differences(P<0.05).Relative to the necrosis group,Chinese medicine group of VEGF protein increased,significant differences(P<0.05).Each group PPAR gamma and protein expression of OPN level,there was no significant difference(P>0.05).6.Clinical curative effect evaluation:42 hips SIONFH compared with before treatment,reduce necrosis and edema zone area(42/64).At the latest follow-up of 2 years,8 hips collapsed,total collapse rate was 27.6%(8/29),subsidence time 3 months?14 months,an average of subsidence time 8.7 months.The collapse<2 mm 4 cases;2 mm to 4 mm 3 cases;higher than 4 mm in 1 case.Before treatment,medication for 3 months,6 months,1 year,the medicine 2 years Harris scores are:81.5±10.8,83.7±9.1,87.3±4,7,87.5±7.8,79.4±5.2.The medication for 6 months,1 year compared with before taking the medicine had significant difference(P<0.05).7.Correlation analysis of three-dimensional reconstruction of MRI findings and clinical symptoms:negatively correlated with necrosis of the femoral head necrosis in the necrotic lesion volume,surface area,volume ratio,bearing the necrosis area of necrosis area and Harris score(r=0.171,P<0.05;r=0.288,P<0.05;r=0.452,P<0.01;r=0.452,P<0.01).The volume of the hardened zone and the area of the bearing hardening area were positively correlated(r=-0.321,P<0.01,r=0.452,P<0.01).Conclusion1.Remove stasis method of femoral head necrosis after treatment intervention,using HRMRI and Micro-CT can detect the rabbit femoral head necrosis bone trabecular structure change is better than that of natural repair,indicated that "remove stasis method" herbal medicine to improve the role of femoral head necrosis imaging findings.From the pathological observation,pathological changes in group at the end of 12 weeks 3 differences significant statistical significance(P<0.05),suggesting the remove stasis method "can repair faster prompted the rabbit model of femoral head necrosis,and 12 weeks to repair completely.2.2 weeks' necrosis HRMRI namely appear when signs and proven by pathology necrosis occurred,at the same time not present bone trabecular Micro-CT and bone mineral density change obviously.Experimental results show that HRMRI with Micro-CT as high resolution imaging techniques are effective evaluation on "dispelling stasis method" the rabbit femoral head necrosis treated by Chinese medicine,but HRMRI to femoral head necrosis in the diagnosis performs earlier than the Micro-CT,consistent with clinical practice.3.The Micro-CT,according to Chinese traditional medicine group compared with necrosis group BV,Conn.D.,SMI,Tb.The higher the value of N,BS/BV,Tb.Sp value decreased,but Tb.Th value decreased,suggesting Chinese medicine can promote bone mineral density increased,promote to repair,but not significantly increased bone mass at the same time.4.Compared with normal group,the necrosis of RUNX2,RANK,the protein expression of RANKL,ABCB1,OPG and VEGF protein expression down;Compared with necrosis group,except RANK and RANKL expression by traditional Chinese medicine group,the rest are raised."Remove stasis method" to promote the new necrosis repair mechanism may be related to inhibit the RANK,the expression of RANKL,promote ABCB1,OPG,VEGF,RUNX2 expression.5.Traditional Chinese medicine "dispelling stasis method" can improve patients not collapse SIONFH Harris score,maintain has collapsed the hip joint function.And 3 d reconstruction of MRI manifestations and clinical symptoms of correlation analysis showed that:In the necrosis of femoral head necrosis,necrotic lesions total surface area,volume necrosis area and volume ratio,bearing necrotic area Harris score negatively correlated;Hardened zone volume,bearing hardening zone area were positively correlated with them.The enlargement of the sclerotic region can increase the balance of the micro structure in the head,increase the stability of the structure and improve the Harris score.