| Steroid-induced osteonecrosis of the femoral head(SONFH)is a disease caused by long-term or high-dose use of glucocorticoid hormones,which reduces or interrupts the blood supply to the femoral head,necrosis of the bone cells,and synchronous formation of neovascularization for repair,leading to structural deterioration of the femoral head to collapse.Disorders of local microcirculatory blood supply to the femoral head are the initial factors in the development of osteonecrosis.The study of bone blood flow is important for diagnosing and treating early femoral head necrosis.Currently,clinical imaging techniques observe the destruction of bone structure and fail to effectively detect blood flow changes in the femoral head.Objective1.A rat model of SONFH was constructed,and contrast-enhanced ultrasound(CEUS),superb microvascular imaging(SMI)and shear wave elastography(SWE)were used to detect the local microcirculatory flow and articular cartilage elasticity characteristics in rats,aiming at exploring the multimodal ultrasound evaluation of the microcirculatory perfusion value of SONFH and the characteristics related to pathological stage.2.The CEUS and SMI were applied to monitor the microcirculatory perfusion characteristics of SONFH intervened by the Chinese herbal compound Shengmai Chenggu capsule in real time,and the mechanism of angiogenesis-osteogenesis response to Huoxue Tongluo recipe Shengmai Chenggu Capsule was preliminarily analyzed.Methods(1)SD rats were randomly divided into control group,the 3rd,5th,7thweek model group.The local microcirculatory perfusion of the rat femoral head was examined by CEUS at the corresponding time points,respectively,and large specimens of the femoral head were obtained at the end of the examination to clarify the pathology.The local imaging characteristics,contrast agent perfusion and contouring patterns of osteonecrosis were observed,and ultrasound quantitative parameters were analyzed.Investigate the value of CEUS in quantitatively evaluating blood perfusion in rats with SONFH.(2)The stages of osteonecrosis in rats were determined by pathological histological examination.CEUS,SMI and SWE multimodal ultrasound techniques were applied to examine the sonographic characteristics,contrast perfusion and contouring patterns,microvascular distribution and articular cartilage elasticity characteristics of SONFH rats at different pathological stages.Peak Intensity(PI),Ascending Slope(AS),Area Under Curve(AUC),Time To Peak(TTP),Mean Transit Time(MTT),Vascular Index(Ⅵ),and Young’s modulus were analyzed,and the correlation between the parameters and the pathological stage of SONFH was evaluated.(3)A rat SONFH model was constructed and randomly divided into control group,model group and FSCC group.CEUS and SMI multimodal ultrasound techniques were used to observe the ultrasound imaging characteristics of local microcirculation perfusion of femoral head in SONFH rats with FSCC intervention,the patterns of contrast agent perfusion and clearance,the distribution of microvessels,and the characteristics of multimodal ultrasound parameters PI,AS,AUC,TTP,MTT,VI at different time quantitative analysis in each group.The expression of H-type vascular markers CD31hi and Emcnhi were examined by pathological HE staining to analyze the rate of empty bone lacuna and osteonecrosis in each group,and the expression of H-type vascular angiogenesis-osteogenesis coupling factors RUNX2 and VEGF were examined by immunohistochemistry to explore the possible mechanism of FSCC intervention in SONFH by activating blood circulation formula.Results1.Quantitative evaluation of local microcirculation perfusion by contrastenhanced ultrasound in SONFH rats(1)Pathological histology:There were no histopathological changes in the femoral head of the control rats.In the model group,the number of empty bone traps increased,the bone trabeculae were sparse and disorganized and fractured,and new bone tissue was seen locally.The rate of empty lacuna in the control group was lower than that in the model groups of 3th,5th and 7th weeks(P<0.05).(2)Sonazoid(SZ)-CEUS Results:①Two-dimensional ultrasound signs:The femoral head in the model group had a normal profile,was smooth and semicircular,without hairiness,joint cavity effusion,flattening and collapse,and there was no significant difference compared with the control group.②SZ-CEUS Results:Contrast agent perfusion characteristics:The contrast agent in the control group was uniformly perfused and faded,with a relatively uniform distribution.In the 3th,5th and 7th week model groups,the microbubbles of contrast agent were rapidly perfused and faded,and they were rapidly collected in the subchondral bone area after entering the femoral neck,and the distribution of microbubbles was not uniform.The distribution of microbubbles in the 5th week model group was significant.TIC curve trend:The TIC curve of the control group rose and fell slowly,the time width of half of the peak fall was larger,and the wave peak was rounded and blunt,showing a "slow rise and fall" trend.The TIC curve in the 3rd,7th week model group rose and fell faster,and the time width of half of the peak fall was smaller than that of the control group,with a sharper peak.The TIC curve in the 5th week model group rose and fell rapidly,and the time width of half of the peak fall was smaller,with sharp peaks and significant "rapid rise and fall" characteristics.TIC parameter results:The AUC of the 3rd week model group was higher than that of control group(P<0.05).PI and AS were higher and TTP was lower than control group,but there was no significant difference(P>0.05).The parameters of PI,AS and AUC in the 5th week model group were significantly higher than those in the control group,while TTP was significantly lower(P<0.05),indicating that the filling of microbubbles in the subchondral bone region of SONFH rats was increased and rapid.In the 5th week,the characteristics were significant.The parameters of AS and AUC in 7th week model group were significantly higher than those in control group(P<0.05).PI was higher than that of the control group,but there was no significant difference(P>0.05).TTP was significantly lower than the control group(P<0.001).③Parameter diagnostic efficacy results:PI,TTP,MTT,AS,and AUC could be used to detect local microcirculatory perfusion in SONFH(P<0.05),with good detection efficacy.(5)Sonovue(SV)-CEUS Results:Compared with the control group,the parameters of TTP,AS,MTT and AUC in the model 3rd week group had no significant difference(P>0.05).TTP and MTT in the model group were lower than those in the control group(P<0.05),and the AS and AUC were higher than those in the control group(P<0.05).In the 7th week,the MTT of the model group was lower than that of the control group(P<0.05),and the AS and AUC of the model group were higher than that of the control group(P<0.05).TTP of the model 7th week group was lower than that of control group,but there was no significant difference(P>0.05).The amount of microvesicles of contrast agent SZ into the subchondral bone region of the femoral head in the acoustic images was more than that of SV,and the amount of SZ in the surrounding muscle tissue was also relatively high.The TIC curves obtained by SZ-CEUS and SV-CEUS at different time points basically maintained the same trend.(6)Comparative results of SV-CEUS and SZ-CEUS parameters:Local microcirculatory perfusion was detected in the 5th week model group of rats,and the parameters MTT and AUC were statistically different(P<0.05),while PI,AS and TTP were not statistically different(P>0.05).The difference in local microcirculatory perfusion between SZ-CEUS and SV-CEUS detection of osteonecrosis was mainly in the time width of half of the peak decrease(MTT)and the area under the curve(AUC).2.Study on the correlation between multimodal ultrasound parameters and pathological staging of SONFH in rats(1)Pathological histology:No obvious empty bone traps were seen in the control group.the number of empty bone traps increased and bone trabeculae were sparse in the stage Ⅰgroup,and the number of empty bone traps increased and bone trabeculae were sparse and disorganized and broken in the stage Ⅱ group.The rate of empty lacuna in stage Ⅰ and Ⅱwere higher than that in control group(P<0.001).The rate of empty lacuna in stage Ⅰ was significantly lower than that in stage Ⅱ(P<0.001).(2)Kappa consistency test:There is good consistency in the pathological staging results evaluated by two physicians,kappa=0.843,P<0.001.Pathological staging results of rats:twelve in the control group,twenty-four in the stage Ⅰ group,and eighteen in the stageⅡ group.(3)cSMI test results:The number of microvessels in the subchondral bone region increased as the degree of femoral head necrosis increased.VI in stage Ⅰ and Ⅱ was higher than that in control group(P<0.05),and Ⅵ in Stage Ⅱ was higher than that in stage Ⅰ(P<0.001).(4)SWE test results:The more severe the osteonecrosis,the less elastic the cartilage,the slower the shear wave propagation.The Young’s modulus of the control group was significantly higher than that of the stage Ⅰ and Ⅱ group(P<0.001),and the Young’s modulus of the stage Ⅱ group was lower than that of the stage Ⅰ group(P<0.05).(5)CEUS test results:①Contrast agent perfusion characteristics:The contrast agent perfused and faded evenly in the control group,and the microbubbles perfused and faded quickly and distributed less evenly in the phase I group.In the phase Ⅱ group,the microbubbles of contrast agent were rapidly perfused and faded,and the microbubbles were rapidly collected in the subchondral bone area after entering the femoral neck,and the distribution of microbubbles was not uniform.②TIC curve trend:The control group had a small slope of TIC curve,a long time to peak,and a slow rise of the curve.Phase Ⅰ group had a large slope,a short time to peak and a fast rising curve.The phase Ⅱ group had a large slope,short time to peak,and a fast rising curve with significant features.③TIC parameter results:PI and AUC in stage Ⅰ group were higher than those in control group(P<0.05).There was no significant difference in AS and TTP(P>0.05).PI,AS and AUC in stage Ⅱ group were higher than those in control group(P<0.05),while TTP was lower than that in control group(P<0.05).AS and AUC in stage Ⅱ group were higher than those in stage Ⅰ group(P<0.05),but PI and TTP had no significant difference(P<0.05).(6)The results of correlation analysis:Multimodal ultrasound parameters PI,AS,AUC,and VI were positively correlated with pathological staging(P<0.01),and TTP,Young’s modulus,and MTT were negatively correlated with pathological staging(P<0.05).(7)Multimodal ultrasound parametric diagnostic efficacy results:PI,AS,AUC,VI,Young’s modulus could be used to evaluate the pathological staging of SONFH in rats(P<0.05),and the detection efficiency is good.The combined diagnostic efficacy of multiple parameters was superior to that of a single parameter(P<0.001).3.Study on the mechanism of angiogenesis-osteogenesis of Huoxue Tongluo recipe in treating SONFH in rats based on multimodal ultrasound(1)Pathological histology:The area of necrotic area expanded and the number of empty bone traps increased in the model group as the degree of osteonecrosis increased,while the number of necrotic foci decreased and the number of empty bone traps decreased in the FSCC group as the administration period was prolonged.(2)cSMI detection results:The number of microvessels in the local area of subchondral bone increased in the model group as the degree of femoral head necrosis increased.The number of microvessels in the local area of FSCC group increased with the prolongation of the dosing cycle.(3)CEUS test results:①Contrast agent perfusion characteristics:The contrast agent in the control group was evenly perfused and dissipated,and the microbubbles were evenly distributed.The contrast agent microbubbles in both the model group and FSCC group showed rapid perfusion and regression,with uneven distribution.The contrast agent perfusion characteristics were significant in the 5th week.②TIC curve trend:In the control group,the TIC curve showed a small slope,slow rise and fall,a large time width of half of the peak fall,a rounded peak,and an overall trend of"slow rise and fall".In the model group and FSCC group,the slope was large,fast rising and falling,the time width of half of the peak drop was small,the peak was sharp,and the trend of "fast rising and falling" was significant in the FSCC group.③TIC parameter results:In the 3rd week,the AS and AUC values of the model group were higher than those of the control group(P<0.05).The parameters of AS and AUC in the control group and model group were lower than those in the FSCC group(P<0.05).In the 5th week,PI,AS and AUC in model group were lower than those in FSCC group,while TTP was higher than that in FSCC group(P<0.05).There was no significant difference in MTT(P>0.05).The parameters of PI,AS and AUC in FSCC group were higher than those in the model group in the 7th week,while TTP was lower than that in the model group(P<0.05).There was no significant difference in MTT(P>0.05).(4)Immunofluorescence double-staining results:CD31hi and Emcnhi expression in the H-type vascular marker model group gradually diminished as osteonecrosis worsened.With increasing weeks of administration,the number of CD31hi and Emcnhi localization was significantly increased in the FSCC group compared with the model group.The Chinese medicine FSCC may promote neovascularization in the necrotic area and promote osteonecrosis repair by increasing the abundance of H-type vessels.(5)Immunohistochemical results:There were fewer brown particles and lower expression of RUNX2 and VEGF in bone tissues of rats in the model group.In the FSCC group,the brown particles were slightly increased and darker than those in the model group,and the expression levels of RUNX2 and VEGF were increased,suggesting that FSCC could increase the expression of angiogenic-osteogenic coupling factor VEGF and RUNX2,promote angiogenesis and bone regeneration in SONFH rats,and promote bone repair.Conclusion(1)CEUS could quantitatively assess local microcirculatory blood perfusion in rats with SONFH.(2)The quantitative parameters of CEUS,SMI,and SWE could be used to assess the pathological stage of SONFH in rats,which indicates the degree of femoral head necrosis in a certain sense,and the combined multimodal ultrasound improves the the predictive efficacy of pathological stage.(3)CEUS and SMI monitor the increase in the number of local microvessels after the treatment of osteonecrosis with revitalizing blood circulation formula FSCC,the filling and fast perfusion of contrast agent,the high intensity of entering into the microvessels,and the fast contouring of vessels.(4)The expression of H-type vessel was low in necrosis of femoral head in rats.FSCC could increase the number of CD31hi and Emcnhi,increase the abundance of H-type vessels,increase the expression of VEGF and RUNX2,and promote angiogenesis and bone regeneration in the necrotic area,promote the repair of osteonecrosis. |
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