| ObjectiveTo investigate the effect of whether Kidney Deficiency Blood Stasis and Phlegm-Blood stasis cementation Syndrome can accelerate the collapsing progress of osteonecrosis of femoral head,the Microbiomechanical property of trabeculae and Ultramorphorlogical Changes in osteocytes in various pathogenical area inside necroctic femoral head and the stress distribution in trabeculae in various pathogenical by finite element analysis.Methods1.Cases were recruited and X ray,MRI and CT-scan were collected for analysis.Patients were divided into two Chinese medicine syndrome groups,namely,Kidney Deficiency Blood Stasis and Phlegm-Blood stasis cementation.Ages,gender,the period from acquiring pain to collapses confirmed and JIC types were collected for analysis.2.Femoral head samples were harvested during operation.Micro-CT,HE staining and ratio of empty osteocytes lacuna,Immunohistology and westernblotting of OPG and RANKL,nanoindentation and acid-etched scan electronic microscope were perform to evaluate the difference between two Chinese medicine syndrome groups(Kidney Deficiency Blood Stasis and Phlegm-Blood stasis cementation),also among various pathogencial area in necrotic femoral head.3.High resolution MicroCT data were collected to rebuild a trabeculae level finite element analysis model.Combining with the mico-biomechanical properties from nanoindentaiton test,models consisted of numbers of trabeculae were acquired in Abaqus.Together with the images from scan electronic microscope,both the feature of stress distribution and the displacement in trabeculae level were analyzed.Results1.A total of 37 patients(40 hips)were recruited.There were 24 patients(26 hips)in Kidney Deficiency Blood Stasis group and 13 patiens(14 hips)in and Phlegm-Blood stasis cementation group.According to the analysis,no difference were found in gender(16/8 vs 12/1,P=0.18),sides(9/17 vs 9/5,P=0.07)and ages(34.0±10.8 vs 39.4±8.1,P=0.11).VAS score and the period from acquiring pain to collapses confirmed in Kidney Deficiency Blood Stasis Syndrome group were found higher than those in Phlegm-Blood Stasis Cementation group(3.2±0.9 vs 2.5±0.7,P=0.01,5.0±1.9 vs 11.8±4.9,P<0.01).The period from acquiring pain to collapses confirmed in JIC C2 was shorter than that in JIC Cl(5.8±2.9 vs 9.3±5.5,P=0.04),but the distribution of JIC types in Kidney Deficiency Blood Stasis Syndrome group and Phlegm-Blood Stasis Cementation group was found no difference(9/17 vs 9/5,P=0.07).2.Twenty necrotic femoral head samples were harvested(10 in Kidney Deficiency Blood Stasis Syndrome group and 10 in Phlegm-Blood Stasis Cementation Syndrome group),no differences in gender and age were found between groups.The appearance of fresh femoral head were found collapsing or flatten on the articular surfaces.Weared and disserted cartilages(expecially in weigh-bearing area)were observed in all samples from both groups.Necrotic area with soft texture and osteocystic and sclerotic area with hard texture,dense property and dark color were observed in the coronal section of the samples.Micro-CT revealed that difference in BV/TV of trabecular was found among different pathogenical area(F=88.97,P<0.01).The value of BV/TV in necrotic area was found significantly lower than that in normal area(27.5±6.1 vs 53.7±7.2,P<0.05)and that in sclerotic area was significantly higher than normal area(76.4±7.8 vs 53.7±7.2,P<0.05).The value of BV/TV in necrotic area in Deficiency Blood Stasis Syndrome group was found signicant lower than that in Phlegm-Blood Stasis Cementation Syndrome group(22.6±4.8 vs 35.3±4.2,P<0.05).Difference in Tb.N(mm)was found among different pathogenical area(F=92.27,P<0.01).The value of Tb.N(mm)in necrotic area was found significantly lower than that in sclerotic area(0.93±0.12 vs 3.45±0.42,P<0.05)and that in sclerotic area was significantly higher than normal area(3.45±0.42 vs 1.96±0.24,P<O.05).The value of Tb.N in necrotic area in Deficiency Blood Stasis Syndrome group was found signicant lower than that in Phlegm-Blood Stasis Cementation Syndrome group(F=84.96,P<0.01).Difference in Tb.Sp(μm)was found among different pathogenical area(F=92.27,P<0.01).The value of Tb.Sp in necrotic area was found significant higher than that in sclerotic area and normal area(1431.7±348.4 vs 353.7±41.2,P<0.05;1431.7±348.4 vs 531.1±38.7,P<0.05).The value of Tb.Sp in necrotic area in Deficiency Blood Stasis Syndrome group was found signicant higher than that in Phlegm-Blood Stasis Cementation Syndrome group(1586.2±290.3 vs 1277.2±344.9,P<O.05).The results of HE staining and ratio of empty osteocytes lacuna showed significant difference was found in among various pathogenical area(F=92.27,P<0.01).The ratio of empty osteocytes lacuna was found higher in necrotic area compared to sclerotic area and normal area in both Deficiency Blood Stasis Syndrome group(0.81±0.07 vs 0.26±0.06,P<0.05,0.81±0.07 vs 0.28±0.09,P<0.05)and Phlegm-Blood Stasis Cementation Syndrome group(0.84±0.05 vs 0.23±0.11,P<0.05,0.84±0.05 vs 0.18±0.07,P<0.05).The results of western blotting of OPG showed that,in Deficiency Blood Stasis Syndrome group,related expression value of OPG was found lower in necrotic area compared to sclerotic area and normal area(P<0.05).In Phlegm-Blood Stasis Cementation Syndrome group,related expression value of OPG was found higher in sclerotic area compared to necrotic area and normal area(P<0.05).The related expression value RANKL in sclerotic area in Deficiency Blood Stasis Syndrome group was found signicant higher than that in Phlegm-Blood Stasis Cementation Syndrome group(P<0.05).The results of nanoindentaion test showed that the value of elastic modulus in sclerotic area was remarkably higher than necrotic area and normal area(22.6±2.1 vs 16.7±2.1,P<0.05,22.6±2.1 vs 18.5±1.9,P<0.05)and the necrotic area was found lower than the normal area(16.7±2.1vs18.5±1.9,P<0.05).The value of elastic modulus in necrotic area in Deficiency Blood Stasis Syndrome group was found signicant lower than those in Phlegm-Blood Stasis Cementation Syndrome group(15.6±2.0 vs 17.7±1.7,P=0.02).The value of hardness in sclerotic area was remarkably higher than necrotic area and normal area(0.98±0.18 vs 0.70±0.15,P<O.05,0.98±0.18 vs 0.84±0.14,P<0.05)and the necrotic area was found lower than the normal area(0.70±0.15vs 0.84±0.14,P<0.05).The results of scan electronic microscope showed that the cell number in sclerotic area was dramaticly higher than that in necrotic area or normal area(50.8±9.3 vs 9.1 ± 4.9,P<0.05;50.8±9.3 vs 35.2±4.6,P<0.05).The osteocyte’ s number in necrotic area was significantly lower than that in normoal area(9.1±4.9 vs 35.2±4.6,P<0.05).The number of dendrite from osteocytes in sclerotic area was dramaticly higher than that in necrotic area or normal area(28.2±6.7 vs 14.4±3.4,P<0.05;24.2±2.9 vs 14.4±3.4,P<0.05).The number of dendrite from osteocytes in necrotic area was significantly lower than that in normoal area(14.4±3.4 vs 24.2±2.9,P<0.05).3.Results of finite element analysis:the peak stress in necrotic area of Deficiency Blood Stasis Syndrome group was found higher than that in sclerotic area and normal area(16.67±5.89 vs 9.51±1.66,P=0.009;16.67±5.89 vs 9.25±1.45,P<0.01).No difference was found in peak stress in model from sclerotic area and normal area(2.47±1.64 vs 1.40±0.43,P=0.51).The max displacement in necrotic area was significantly higher than those in sclerotic area and normal area(17.56±4.09 vs 2.47±1.64,P<0.01;17.56 ± 4.09 vs 1.40±0.43,P<0.01).The peak stress in necrotic area of Phlegm-Blood Stasis Cementation Syndrome group was found higher than that in sclerotic area and normal area(18.76±2.08 vs 8.79±1.71,P<0.01;18.76±2.08 vs 8.30±1.42,P<0.01).No difference was found in peak stress in model from sclerotic area and normal area(8.79±1.71 vs 8.30±1.42,P=0.69).The max displacement in necrotic area was significantly higher than those in sclerotic area and normal area(8.18±3.61 vs 1.30±0.78,P<0.01;8.18±3.61 vs 1.10 ± 0.42,P<0.01).Dramatic changes were found in the stress map,beside the observable osteoporosis changes in necrotic trabecular bone cube,great amounts of sphere-shape empty space was found in necrotic trabecular bone.The stress concentration appeared around the thin trabecular structure when stress was loaded.In the stress map in sclerotic area,trabecular bone were much thicker but in unregular shapes.Model from trabecular bone in normal area was found in good shapes with multiple connection which can significantly transfer the stress inside trabecular.Images in 1kX magnification from necrotic area in Deficiency Blood Stasis Syndrome group revealed that multiple craps were found in the connective area of trabecular indicated that the breakage of trabeculae might occur in those area.ConclusionPatients in Kidney Deficiency Blood Stasis Syndrome were found to have a short period from acquiring pain to collapses confirmed than those in Phlegm-Blood Stasis Cementation Syndrome,and JIC type C2 can significantly accelerate the process of collapsing.According to a serial of results,patients with Kidney Deficiency Blood Stasis Syndrome had a poorer quanity and quality of trabecular bone in necrotic area inside a necrotic femoral head and this results might closely related to the effect of corticosteriod,induced expression of RANKL,reduced expression of OPG and the absence of the ability of sensing mechanical stimulation.The pathogenical property might manifest as bone destruction with strengthening bone absorption.The quanity and quality of trabecular bone in necrotic area from patients with Phlegm-Blood Stasis Cementation Syndrome were improved with induced OPG expression,indicated the osteogenesis was higher in Phlegm-Blood Stasis Cementation Syndrome group.Those differences might affect the stability of necrotic femoral heads in different syndromes,causing a different process in femoral head collapsing.The quanity and quality of trabecular bone in sclerotic area were significantly evaluated.Stress distribution under healthy blood supply condition might be one of the most important reason for bone repair and reconstruction in this area.The unstability of necrotic femoral head,even the occurrence of femoral head collapsing,might result from the poor microstructure and low elastic modulus of necrotic trabecular bone.The flattened femoral head might be the consequence of accumulation of compression and breakage of those weaken trabecular bone.Trabecular bone in sclerotic area was responsed for the bone reconstruction and repair and the improvement of microstructure and mechanical property can resist the deformation of trabeculae under mechanical loading providing mechanical stability through the reconstruction process.By validating the correlation of "clinical results-microproperty of trabecular bone-finite element analysis based on trabecular bone scale",our research has successfully revealed the different micro-environment in various pathogenical area in necrotic femoral head and partially unmasked the the reason of femoral head collapsing.Also,a considerable situation alerted us that patients with large necrotic lesion in Kidney Deficiency Blood Stasis Syndrome might not have a great opportunity of joint preserving treatment because femoral head collapsing might occur in a short period since.The necrotic trabecular bone might response for this phenomenon. |
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