Experimental study of changes of TNF-α in rabbits with steroid-induced avascular necrosis of femoral head

Objective: To explore the effect of tumor necrosis factor-α(TNF-α)on the occurrence of steroid-induced avascular necrosis of femoral head(SANFH). Methods: Twenty-four rabbits were firstly divided into void group (n=12) and model group (n=12) at random. Prednisone Acetate was injected to the model group(0.32 mg·kg^(-1)·d^(-1)). In the 6 th and 8 th week, two rabbits of the two groups were killed respectively to observe whether the model was successful. The level of TNF-α in serum of the residual rabbits of the two groups was examined in Radioimmunoassay method. Results: The level of TNF-α in model group is significantly higher than that in void group(P<0.001) under the premise of the model of SANFH success by histological observation. Conclusion: The rise of level of TNF-α may be one of the most important factors in the occurrence of SANFH.

Gubi Tongxiao granules regulate vasculization and apoptosis to intervene steroid-induced femoral head necrosis in rabbits

Objective:To investigate the effect of Gubi Tongxiao granules on vascular formation and cell apoptosis in the process of glucocorticoid-induced femoral head necrosis.Methods:Thirty experimental New Zealand rabbits were randomly divided into groups.Except the control group,the animal model of steroid-induced femoral head necrosis was established by lipopolysaccharide combined with glucocorticoids.After successful modeling,the experimental group was given Gubi Tongxiao granules intragastric treatment,the model and control group were given the same amount of normal saline intragastric treatment,once a day,for 8 weeks,the experimental animals were sacrificed,the bilateral femoral head of each group was taken out for hematoxylin-eosin staining,and protein CD34,CYR61 and VEGF were immunohistochemical staining localization.TUNEL staining was used to observe the apoptosis of femoral head cells.The expression levels of apoptosis proteins Bax and Bcl-2 were detected by WB.The expression of VEGF eNOs Bax and Bcl-2 genes was analyzed by RT-qPCR.Results:Gubi Tongxiao granules reduced the number of hollow bone lacunae and apoptotic positive cells(P<0.01),and up-regulated the expressions of CD34,CYR61 and VEGF in femoral head tissue.Compared with control group,the expression of pro-apoptotic protein Bax increased and the expression of anti-apoptotic protein Bcl-2,VEGF and eNOs mRAN decreased significantly in the model group(P<0.01),the opposite results were obtained in the experimental group after Gubi Tongxiao granule intervention(P<0.05).Conclusion:Gubi Tongxiao granules can improve the expression of angiogenic genes and regulate apoptosis-related proteins in the bone tissue of rabbit model with steroid-induced necrosis of femoral head,promote angiogenic differentiation and inhibit apoptosis,and thus achieve the effect of treating steroid-induced necrosis of femoral head.

Effect of Yang-Warming and Kidney-Tonifying Prescription on Expression of Osteogenic and Angiogenic Factors and H-Type Vascular Markers in Steroid-Induced Avascular Necrosis of Femoral Head in Rabbits

Objective: To investigate the effect of Yang-warming and Kidney-tonifying Prescription (YKP) on the treatment of steroid-induced avascular necrosis of the femoral head (SANFH) in rabbits. And to further explore whether its therapeutic mechanism is related to the expression of HIF-1α and VEGF (angiogenic factors), BMP2 and Osterix (osteogenic factor), CD31 (type H vascular marker) and MMP13 (bone destruction-related factor). Methods: Twenty-seven healthy male New Zealand white rabbits were divided into a normal group, model group, traditional Chinese medcine (TCM) group (clinical equivalent dose group of YKP), miR-130a inhibitor group and TCM + inhibitor group. The SANFH model was established by combining horse serum with methylprednisolone. After the model is successfully established, TCM group was given 6.44 g/kg·d YKP by gavage, and the miR-130a gene inhibitor group was intraperitoneally injected with 25 mg/kg miR-130a inhibitor, locked nucleic acid (LNA)-anti-miR-130a. TCM + inhibitor group was treated with YKP intragastrically and miR-130a inhibitor intraperitoneally. The rabbits in the normal group and the model group were intragastrically administered with normal saline 10 ml/d. Once a day for 4 weeks. The avascular necrosis was detected by HE staining. The contents of HIF-1α, VEGF, BMP2 and Osterix in rabbit tissues were detected by qRT-PCR kit, and the expression of CD31 and MMP13 was detected by immunofluorescence staining. Results: In the normal group, the surface of the cartilage layer of the femoral head was smooth, the bone trabeculae were intact and densely arranged, the cells of each layer were neatly arranged, the morphology of the bone cells, the chondrocytes and the adipocytes were normal. In the model group, cartilage surfaces of the femoral head showed exfoliative cracks. The bone trabecular structure was loose and incomplete, chondrocytes, osteoblasts and bone marrow cells were significantly reduced, and the number of empty bone traps was significantly increased. In the TCM-treated group, more chondrocytes, thicker cartilage layer, and more regular bone trabeculae were detected as compared to model rabbits. In contrast, the cartilage layer was thinner, the destruction and fracture of bone trabeculae was more serious, chondrocytes and osteocytes were decreased as compared to model group. The expression of HIF-1α, VEGF, BMP2, and Osterix in the model group decreased significantly as compared to the normal group (P Conclusion: YKP can regulate the expression of angiogenic-related factors (VEGF and HIF-α), osteogenic-related factors (BMP2 and Osterix), and H-type vascular marker CD31, resulting in increased expressions of VEGF, HIF-α, BMP2, and Osterix, which promote intra-femoral head revascularization. Meanwhile, YKP decreased the expression of bone-destruction-related factor MMP13, thus enhancing the ability of bone tissue to repair itself. Regulation of these molecules’ expression may be one of the mechanisms of YKP in the treatment of hormonal femoral head necrosis.

Changes of bone mineral in steroid-induced osteonecrosis in rabbits

To investigate the significance of bone mineral changes in the pathogenic process of steroid-induced osteonecrosis in rabbits. Methods： Seventy adult female Japanese White rabbits were randomly divided into five groups Animals in steroid group received an intramuscular injection of methylprednisolone acetate （4 mg/kg） once a week, and killed in four different time after first treatment. Animals in control group received an intramuscular injection with the same volume of normal saline, while 150 kU penicillin was injected twice a week for preventing infection in all groups. The contents of serum calcium （Ca） and phosphorus （P） were determined and the bone mineral densities of the femoral head and neck were measured at the 2nd, 4th, 8th and 10th weeks after first treatment. Meanwhile the general condition such as weight, spirit and hair of rabbits was observed. Results： Compared with the control group, the weight of rabbits in steroid group lost since the first week, and progressively aggravated; the bone mineral density of the femoral head and neck was lowered since the 2nd and 4th week respectively （P〈0.05, P〈0.01）, and lowered more quickly in femoral neck than in femoral head. In steroid group, the bone mineral density of the femoral neck at the 10th week was significantly lower than that at the 8th week （P〈0.05） Compared with the control group, the serum levels of Ca and P in steroid group were lowered since the 2nd week （P〈0.05, P〈0.01）. In steroid group, the serum Ca level at the 10th week was lower than that at the 8th week, and the serum P level waslowered more quickly than Ca level was. Conclusion： Osteonecrosis appears in rabbits shortly after corticoids are first administered, and osteonecrosis in rabbits is chronologically associated with the onset of hypocalcaemia, hypophosphatemia and lowered bone mineral density. Steroid-induced osteonecrosis may be relevant to bone mineral loss and density lowering of the femoral head and neck.

Titanium Alloy Cage Implantation for the Treatment of Ischemic Necrosis of Femoral Head in Dogs

To study the effect of titanium alloy cage on the treatment of the ischemic necrosis of femoral head in dog, the model of the ischemic necrosis of femoral head was made with the liquid nitrogen in 15 hybrid adult dogs. The titanium alloy cage made of a hollow cylinder was driven into the subchondral bone of necrotic femoral head via central channel. The dogs were divided into 3 groups, each group was sacrificed 3, 6, 12 weeks after the operation respectively. No collapse of femoral head was observed after the operation. The position of the cages was good on radiograph. Microscopically, the cancellous bone of necrotic femoral head rebuilt gradually and grew into cage. After 12 weeks of creeping substitution, the cancellous bone filled up the hollow cavity and holes of the cages. It is concluded that the titanium alloy cage can provide structural support for the subchondral bone and prevent collapse and can be used for the treatment of the ischemic necrosis of femoral head.

Arthroscopy Assisted Lesion Clearance and Bone Graft, Titanium Rod Support Treatment of Early Stage Osteonecrosis of the Femoral Head of the Postoperative Survival Rate Analysis

Objective: To assess the curative effect of the stage II femoral head necrosis treated by arthroscopy assisted lesion clearance, bone graft and titanium rod support. Methods: All the patients (including 58 patients 74 hips) were diagnosed as stage II femoral head necrosis according to the ARCO staging system during 2003-2013. In these patients, 15 hips were stage IIA, 34 hips were stage IIB and 25 hips were stage IIC. Located by C-arm and assisted by arthroscopy, minimally-invasive percutaneous pulp core decompression and lesion clearance within the femoral head were accurately performed, and then, the OAM composite of autologous bone marrow was implanted and the femoral head was supported using the titanium rod. Follow-up including the pain score, the Harris hip score and X-ray observation for disease progression were achieved at 6, 12, 24 and 36 months postoperatively, Kaplan-Meier survival curve was used for the survival analysis. Result: The VAS score and the Harris score after operation were better THRAn THRAt of before the surgery, the difference had statistical significance (p Conclusion: Arthroscopy assisted lesion clearance, bone graft and titanium rod support to treat the stage II osteonecrosis of the femoral head are effective and can prevent the femoral head from collapsing. But for stage IIC patients who had a history of the use of hormone, this surgery should be chosen carefully because the outcome is always very poor.

HuoguⅠformula prevents steroid-induced osteonecrosis in rats by down-regulating PPARγ expression and activating Wnt/LRP5/β-catenin signaling

OBJECTIVE： To investigate the effects of Huogu I formula on regulation of lipid metabolism in ste- roid-induced osteonecrosis of the femoral head （SONFH） rats and verify our hypothesis that Huogu I formula regulates lipid metabolism by down-regulating peroxisome proliferator-activated receptor gamma （PPARy） expression and activating Wnt signaling pathways. METHODS： Eighty-five rats were divided into four groups： control, model, Huogu 15 g/kg and Huogu 30 g/kg. Six weeks later, animals were anaesthe- tized, femora were dissected for histopathologicalexamination of the osteonecrotic changes and re- pair processes, micro computed tomography （Mi- cro-CT）-based micro-angiography was performed to assess vascularization. Serum lipid levels were detected by haematological examination. The ex- pressions of PPARy, Wnt3a, low density lipoprotein receptor-related protein 5 （LRP5） and 13-catenin were evaluated by immunohistochemistry, Western blot and quantitative real-time polymerase chain reaction analyses. RESULTS： The incidence of osteonecrosis, ratio of empty lacuna, adipose tissue area and adipocyte perimeter in the bone marrow were dramatically lower in the Huogu ~ formula treatment groups. By micro-CT quantification, Huogu ~ formula treat- ment dose-dependently increased vessel volume, vessel surface, percentage of vessel volume and vessel thickness of the femoral heads of SONFH rats. Levels of serum lipid in Huogu 15 g/kg and Huogu 30 g/kg groups reduced significantly. HuoguⅠformula treatment could suppress the ex- pression of PPARy and increase the expressions of Wnt3a, LRP5 and 13-catenin at both protein and mRNA levels. CONCLUSION： The results of our present study highlight the lipid-lowering potential of Huogu Ⅰ formula, and provide further evidence of the in- volvement of the PPARy inhibition and Wnt/LRPS/ 13-catenin signaling activation in the effects of Huogu Ⅰ formula.

股骨颈骨折空心钉内固定后股骨近端骨质疏松的有限元分析

背景:股骨颈骨折空心钉内固定术后由于患肢短期内常常不能负重,且高刚度的内植物对骨折断端存在应力遮挡效应,易导致患肢出现骨质疏松,股骨近端生物力学分布发生变化,术后股骨头坏死发病率较高,目前关于股骨颈骨折术后股骨近端骨质疏松对股骨近端及空心钉生物力学影响的研究较少。目的:通过有限元分析探讨股骨颈骨折术后发生骨质疏松对空心钉内固定治疗的生物力学影响,探究生物力学因素在股骨头坏死进程中的作用。方法:获取1例股骨颈骨折患者股骨CT扫描数据,利用Mimics 19.0、3-Matic、UG 11.0、Hypermesh 14.0、Abaqus软件建立空心钉治疗股骨颈骨折的股骨近端模型,利用Abaqus软件分析1种术后股骨近端无骨质疏松、3种术后股骨近端骨质疏松的有限元模型,测量分析4种模型不同部件的应力、接触压力、位移峰值及云图,对比分析股骨头内部应力变化及分布情况。结果与结论:股骨头及下前空心钉的应力、接触压力随骨质疏松程度变化较大,4种模型的位移峰值随着骨质疏松程度加重而缓慢增长。通过单因素方差分析,结果显示骨质疏松程度对不同部件的应力、接触压力、位移峰值无显著性影响,股骨头内部应力分布随骨质疏松发生相应变化,股骨近端的生物力学环境变化对股骨头坏死有着重要影响。

基于“久病入络”理论探讨“从虚而始,因虚致瘀,瘀痹骨络”的激素性股骨头坏死核心病机观

激素性股骨头坏死(Steroid-induced osteonecrosis of femoral head, SONFH)是骨科常见疾病,随着激素应用率提升其发病率也逐年增加。随着“久病入络”理论的研究不断深入,特此提出“从虚而始,因虚致瘀,瘀痹骨络”为激素性股骨头坏死发病病机,强调药邪是SONFH的致病因素,肝脾肾三脏亏虚是SONFH发病基础,脏腑亏虚,功能减退,血行涩滞导致SONFH进展,瘀血痹阻骨络是SONFH的病机关键,以期建立“从虚而始,因虚致瘀,瘀痹骨络”的特色辨治体系,为中医药治疗SONFH提供理论基础。

基于3D黑骨序列测量Legg-Calvé-Perthes病股骨头突出指数并评估其Herring分型

目的观察基于3D黑骨序列测量Legg-Calvé-Perthes病(LCPD)股骨头突出指数并评估其Herring分型的价值。方法回顾性分析38例单侧LCPD患儿髋关节MRI,分别比较冠状位3D黑骨序列类X线图及冠状位脂肪抑制T2WI与髋关节正位X线片所测患/健侧股骨头突出指数;以髋关节X线片为金标准,绘制受试者工作特征曲线,计算曲线下面积(AUC),分析3D黑骨序列及脂肪抑制T2WI评估LCPD Herring分型的效能,并以DeLong检验进行比较。结果基于X线片及3D黑骨序列类X线图测得LCPD健侧股骨头突出指数分别为(11.31±6.16)%及(11.47±5.83)%,差异无统计学意义(P>0.05);患侧股骨头突出指数分别为(22.45±9.21)%及(22.82±7.92)%,差异无统计学意义(P>0.05)且均显著高于健侧(P均<0.001)。基于脂肪抑制T2WI测得LCPD患侧及健侧股骨头突出指数分别为(28.01±2.01)%及(27.25±1.92)%,差异无统计意义(P>0.05)且分别高于基于X线片所获患侧与健侧股骨头突出指数(P均<0.001)。3D黑骨序列及脂肪抑制T2WI评估LCPD Herring分型的AUC为0.750~0.938及0.625~0.705。3D黑骨序列评估LCPD Herring B型的AUC高于脂肪抑制T2WI(P<0.05),二者评估LCPD其他Herring分型的AUC差异均无统计学意义(P均>0.05)。结论3D黑骨序列可用于测量LCPD股骨头突出指数并评估其Herring分型;以之联合脂肪抑制T2WI可在一定程度上取代髋关节X线检查。

三七活骨丸联合体外冲击波对气滞血瘀型早期股骨头缺血性坏死患者的影响

目的 观察三七活骨丸联合体外冲击波(ESW)对气滞血瘀型早期股骨头缺血性坏死的影响。方法 选取2021年4月至2022年10月黑龙江中医药大学附属第二医院收诊的72例气滞血瘀型早期股骨头缺血性坏死患者,采用随机数字表法将其分为对照组和观察组,各36例。对照组采用ESW治疗,观察组在对照组的基础上口服三七活骨丸,两组均治疗4周。观察治疗前及治疗2、4周后两组视觉模拟评分法(VAS)评分、髋关节Harris评分、中医证候评分,并进行疗效评价。比较两组治疗前后血清白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)、骨保护素(OPG)水平。结果 对照组有4例患者在治疗期间服用其他药物,故剔除。观察组临床疗效优于对照组(P<0.05)。两组治疗2、4周后VAS、中医证候评分均低于治疗前,髋关节Harris评分高于治疗前,两组治疗4周后VAS、中医证候评分均低于治疗2周后,髋关节Harris评分高于治疗2周后(P<0.05)。治疗2、4周后,观察组髋关节Harris评分高于同期对照组,中医证候评分低于同期对照组(P<0.05)。治疗4周后,观察组VAS评分低于同期对照组(P<0.05)。治疗4周后,两组血清IL-1、IL-6、TNF-α水平低于治疗前,血清OPG水平高于治疗前,且观察组血清IL-1、IL-6、TNF-α水平低于对照组,血清OPG水平高于对照组(P<0.05)。结论 三七活骨丸联合ESW对于改善气滞血瘀型早期股骨头缺血性坏死患者的症状疗效良好,适合临床应用。

活血通络法对激素性股骨头坏死囊性变患者血清骨吸收/成血管/成骨蛋白调节作用的研究

背景激素性股骨头坏死(SIONFH)中囊性变具有“双刃剑”作用,活血通络法对早期激素性股骨头坏死疗效佳,但对囊性变骨吸收/成血管/成骨蛋白调节作用尚未明确。目的探讨活血通络法对激素性股骨头坏死囊性变患者血清骨吸收/成血管/成骨蛋白作用的影响。方法纳入2019年1月—2021年1月在广州中医药大学髋关节研究中心收治的60例SIONFH患者为研究对象,按随机数字表法分成两组,对照组和治疗组各30例。另选取同时期在医院体检正常且无激素使用史30例志愿者作为正常组。治疗组给予活血通络胶囊(2 g/次,3次/d)及碳酸钙(600 mg/次,1次/d)治疗,对照组给予等量碳酸钙治疗,两组患者疗程均为12个月,同时患肢加以限制性负重。采集空腹血清,采用酶联免疫吸附试验检测血清核因子κB受体活化因子配体(RANKL)、血小板衍生生长因子-BB(PDGF-BB)、血管内皮生长因子A(VEGFA)、骨保护素(OPG)、钙黏蛋白相关蛋白(CTNNB1)的表达水平,以门诊复诊方式分别于患者出院后第6、12个月进行2次随访,以股骨头塌陷定义为终点事件。以髋关节疼痛视觉模拟评分(VAS)、髋关节功能(Harris)评分和坏死面积评分进行疗效评估。结果3组研究对象基线RANKL、PDGF-BB、OPG、CTNNB1水平比较,差异均有统计学意义(P<0.05),组间比较结果显示,对照组、治疗组RANKL、PDGF-BB均高于正常组,OPG、CTNNB1均低于正常组(P<0.05)。重复测量方差分析结果示,时间和组别对RANKL、PDGF-BB、VEGFA、OPG、CTNNB1水平存在交互作用(P<0.05),时间和组别对RANKL、PDGF-BB、VEGFA、OPG、CTNNB1水平主效应显著(P<0.05)。其中第6个月治疗组RANKL水平高于对照组,第12个月治疗组PDGF-BB水平高于对照组,第6、12个月VEGFA、OPG、CTNNB1水平高于对照组(P<0.05)。治疗后第12个月治疗组髋关节VAS评分、坏死面积评分低于对照组,髋关节Harris评分高于对照组(P<0.05)。组内比较结果显示,治疗后第12个月治疗组髋关节VAS评分、坏死面积评分低于治疗前,髋关节Harris评分高于治疗前(P<0.05),对照组髋关节VAS评分、髋关节Harris评分高于治疗前(P<0.05)。结论活血通络法能上调激素性股骨头坏死患者RANKL、PDGF-BB、VEGFA、OPG和CTNNB1蛋白表达水平,有效促进SIONFH患者骨修复及改善临床症状,推测该疗法通过“骨吸收/成血管/成骨”三元修复网络促进囊性变骨修复作用。

补肾壮骨方治疗非创伤性股骨头坏死肝肾亏虚证的功效内涵解析及临床验证

目的:探讨补肾壮骨方治疗非创伤性股骨头坏死(non-traumatic osteonecrosis of the femoral head,NONFH)肝肾亏虚证的功效内涵。方法:①网络药理学研究。检索TCMIP v2.0、ETCM2.0、HERB数据库,收集补肾壮骨方的靶标基因,从项目组前期研究中获取NONFH肝肾亏虚证相关差异表达基因,以NONFH肝肾亏虚证典型症状为关键词检索NONFH肝肾亏虚证典型症状相关基因。将上述基因合并,构建补肾壮骨方治疗NONFH肝肾亏虚证蛋白质互作网络,从中筛选关键靶标基因,并对其进行KEGG信号通路富集分析,结合文献对相关信号通路进行药理作用分析。以靶标基因与信号通路、信号通路与药理作用的映射关系为基础,结合文献报道,构建补肾壮骨方治疗NONFH肝肾亏虚证“组方中药-关键靶标-信号通路-功效-药理作用-临床症状”关联网络。②临床研究。选择77例(120髋)NONFH肝肾亏虚证患者,采用口服补肾壮骨方治疗6个月。分别采用股骨头坏死临床疗效评价标准和北京中医药大学X线评价系统进行临床疗效和影像学疗效评价。结果:①网络药理学研究结果。从构建的补肾壮骨方治疗NONFH肝肾亏虚证蛋白质互作网络中共筛选出556个关键靶标基因,这些基因共参与39条与NONFH肝肾亏虚证有关的信号通路,主要涉及纠正骨代谢紊乱、纠正脂代谢紊乱、矫正免疫-炎症失衡、改善血液循环受阻4种药理作用。纠正骨代谢紊乱涉及靶标基因183个,纠正脂代谢紊乱涉及靶标基因176个,矫正免疫-炎症失衡涉及靶标基因171个,改善血液循环受阻涉及靶标基因125个。构建的“组方中药-关键靶标-信号通路-功效-药理作用-临床症状”关联网络直观显示了补肾壮骨方治疗NONFH肝肾亏虚证的功效内涵。②临床研究结果。临床疗效评价结果显示,治疗6个月后患者的髋关节疼痛强度评分、髋关节屈曲功能评分、行走距离评分及临床疗效总分均较治疗前降低[(5.86±1.96)分,(2.26±1.84)分,t=25.672,P=0.000;(5.76±1.62)分,(3.46±1.68)分,t=38.411,P=0.000;(6.31±1.41)分,(0.78±0.74)分,t=46.771,P=0.000;(5.92±1.18)分,(2.32±1.06)分,t=48.263,P=0.000];临床疗效优88髋、良28髋、可2髋、差2髋。影像学疗效评价结果显示,治疗后3年影像学疗效优17髋、良25髋、可67髋、差11髋;股骨头形态稳定109髋、进展11髋,坏死灶改善67髋、稳定34髋、进展19髋,骨关节炎稳定100髋、进展20髋。结论:补肾壮骨方治疗NONFH肝肾亏虚证的机制为纠正骨代谢和脂代谢紊乱、免疫-炎症失衡及血液循环受阻,其中纠正骨代谢紊乱可能是最主要的机制。

多参数MRI纹理分析预测直肠癌患者放射治疗后早期股骨头坏死

目的 探讨多参数MRI(mpMRI)纹理分析预测直肠癌患者放射治疗(放疗)后早期股骨头坏死(FHN)的价值。方法 回顾性分析52例接受盆腔放疗直肠癌患者放疗前2周及放疗后3个月内的盆腔MRI,其中31例未见FHN,21例出现早期FHN(Ⅰ期16例,Ⅱ期5例)。采用3D-slicer软件基于T1WI、T2WI和弥散加权成像(DWI)分割右侧股骨头,并提取其纹理参数;对比放疗前后纹理参数,将差异有统计学意义的放疗前参数纳入多因素logistic回归分析,建立模型T1WI、模型T2WI、模型DWI及模型T1WI+T2WI+DWI。根据放疗后影像学所见行股骨头坏死骨循环研究协会(ARCO)分期,以受试者工作特征(ROC)曲线分析单一纹理参数及联合模型预测直肠癌患者放疗后早期FHN的效能。结果 T1WI及T2WI纹理参数中,放疗前股骨头能量及均匀性高于放疗后;DWI纹理参数中,放疗前股骨头能量高于、而长行程高灰度强调及短行程高灰度强调低于放疗后。上述放疗前纹理参数单一预测直肠癌患者放疗后早期FHN的曲线下面积(AUC)为0.581~0.712,模型T1WI、模型T2WI、模型DWI和模型T1WI+T2WI+DWI的AUC分别为0.660、0.713、0.770和0.853。结论 mpMRI纹理分析可有效预测直肠癌患者放疗后早期FHN。

激素性股骨头坏死囊性变组织骨修复机制分析与活血通络胶囊含药血清对肥大软骨细胞成骨分化影响的实验研究

目的:分析激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)囊性变组织的骨修复机制,并观察活血通络胶囊含药血清对肥大软骨细胞成骨分化的影响。方法:(1)SONFH囊性变组织的骨修复机制分析。纳入6例采用全髋关节置换术治疗的SONFH患者,术后收集坏死股骨头6个,其中3个股骨头用于囊性变组织的单细胞转录组测序,采用生物信息学方法分析囊性变组织中的细胞分类、软骨细胞的细胞亚群、肥大软骨细胞参与的生物过程;另外3个股骨头用于囊性变组织的病理学检查,分别采用苏木素-伊红染色、阿利新蓝染色、番红O-固绿染色观察囊性变组织中软骨细胞、肥大软骨细胞的分布与特征,采用免疫组织化学染色观察囊性变组织中肥大软骨细胞标志蛋白Ⅹ型胶原α1(collagen typeⅩα1,Col10α1)、基质金属蛋白酶(matrix metalloproteinase,MMP)13和成骨分化相关蛋白Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨桥蛋白(osteopontin,OPN)的表达情况。(2)活血通络胶囊含药血清对肥大软骨细胞成骨分化影响的分析。采用活血通络胶囊(活血通络胶囊粉末溶于蒸馏水)给大鼠灌胃,制备活血通络胶囊含药血清。取小鼠膝关节软骨,消化分离后进行培养。将小鼠软骨细胞接种于含有不同浓度的活血通络胶囊含药血清的完全培养基中,筛选活血通络胶囊含药血清的最佳干预浓度。取处于对数生长期的第1代小鼠软骨细胞,行肥大软骨细胞诱导成功后,分为成骨诱导组、5%含药血清干预组和对照组,成骨诱导组更换成骨诱导培养基培养,5%含药血清干预组更换含5%活血通络胶囊含药血清的成骨诱导培养基培养,对照组继续采用肥大诱导培养基培养;培养7 d后,分别采用碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红染色,观察细胞形态特征,分别采用实时定量PCR和蛋白质印迹法检测Ⅰ型胶原蛋白α1(collagenⅠα1,Col1α1)、ALP、RUNX2、OPN的mRNA相对表达量和ALP、RUNX2、OPN的蛋白相对表达量。结果:(1)SONFH囊性变组织单细胞转录组测序分析结果。共获得30224个细胞的单细胞转录组测序结果;细胞分类结果显示,囊性变组织中有8种细胞,包括T细胞、内皮细胞、成纤维细胞、软骨细胞、巨噬细胞、单核细胞、破骨细胞和肥大细胞;软骨细胞亚群分析共鉴定出5类亚群细胞,包括稳态软骨细胞、成纤维软骨细胞、炎症软骨细胞、肥大软骨细胞、前体肥大软骨细胞;差异基因富集分析结果显示,肥大软骨细胞主要参与细胞迁移正向调节、细胞分化、细胞外基质形成、骨化、细胞迁移和成骨细胞分化等生物过程。(2)SONFH囊性变组织的病理学检查结果。囊性变组织的边缘可见软骨细胞、肥大软骨细胞,且部分软骨组织呈现向骨小梁过渡的形态;Col10α1、MMP13、RUNX2、OPN在囊性变组织的边缘高表达。(3)活血通络胶囊含药血清干预肥大软骨细胞成骨分化的分析结果。经筛选,5%为活血通络胶囊含药血清的最佳干预浓度。ALP染色和茜素红染色结果显示,对照组无明显钙化结节,成骨诱导组有明显钙化结节,5%含药血清干预组钙化结节较成骨诱导组进一步增多;成骨诱导组细胞ALP、RUNX2、OPN、Col1α1的mRNA相对表达量和ALP、RUNX2、OPN的蛋白相对表达量均高于对照组(P=0.000,P=0.000,P=0.001,P=0.000;P=0.000,P=0.000,P=0.001),5%含药血清干预组细胞ALP、RUNX2、OPN、Col1α1的mRNA相对表达量和ALP、RUNX2、OPN的蛋白相对表达量均高于成骨诱导组(P=0.000,P=0.000,P=0.002,P=0.001;P=0.000,P=0.000,P=0.001)。结论:SONFH囊性变组织中存在肥大软骨细胞向成骨细胞分化的修复机制,而活血通络胶囊含药血清能够促进体外培养的肥大软骨细胞向成骨细胞分化。

激素性股骨头坏死滑膜病变分子机制的实验研究

目的:探讨激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)滑膜病变的分子机制。方法:(1)转录组测序及生物信息学分析。收集9例接受全髋关节置换术的患者(SONFH患者3例、髋骨关节炎患者3例和股骨颈骨折患者3例)术中切除的髋关节滑膜组织,进行转录组测序和生物信息学分析,筛选SONFH滑膜病变核心基因。(2)滑膜组织检测。根据标本来源将髋关节滑膜组织分为SONFH组、髋骨关节炎组和股骨颈骨折组。观察各组髋关节滑膜组织的组织形态,采用实时定量PCR检测3组髋关节滑膜组织中SONFH滑膜病变核心基因的mRNA相对表达量,采用蛋白质印迹法和免疫组织化学染色法检测3组髋关节滑膜组织中SONFH滑膜病变核心基因的蛋白相对表达量。采用免疫荧光染色法检测SONFH滑膜病变的靶细胞。(3)细胞验证。培养大鼠滑膜成纤维细胞,构建滑膜炎细胞模型。采用实时定量PCR及蛋白质印迹法检测空白细胞(空白细胞组)和滑膜炎模型细胞(模型细胞组)中SONFH滑膜病变核心基因的表达。结果:(1)转录组测序和生物信息学分析结果。经对不同患者来源的髋关节滑膜组织进行差异基因分析,共筛选出1001个与SONFH髋关节滑膜病变相关的基因,这些基因与免疫反应和外泌体有关,其中干扰素调节因子(interferon regulatory factor,IRF)4和IRF7是SONFH髋关节滑膜病变的核心基因,两者均为参与Ⅰ型干扰素应答的关键转录因子。(2)滑膜组织形态观察结果。苏木素-伊红染色显示,股骨颈骨折组髋关节滑膜组织形态正常,无细胞增生、肥大或间质水肿;髋骨关节炎组髋关节滑膜组织细胞增生,有少量新生血管和细胞聚集;SONFH组髋关节滑膜组织细胞大量增殖和聚集,有新生血管。(3)滑膜组织中SONFH滑膜病变核心基因表达检测结果。SONFH组髋关节滑膜组织中IRF4、IRF7、干扰素-α(interferon-α,IFN-α)的mRNA相对表达量均高于股骨颈骨折组和髋骨关节炎组(P=0.000,P=0.000,P=0.000;P=0.001,P=0.000,P=0.036),髋骨关节炎组髋关节滑膜组织中IRF4、IRF7、IFN-α的mRNA相对表达量均高于股骨颈骨折组(P=0.000,P=0.000,P=0.000)。IRF4、IRF7、IFN-α蛋白在SONFH组髋关节滑膜组织中高表达,SONFH组髋关节滑膜组织中IRF4、IRF7、IFN-α的蛋白相对表达量均高于股骨颈骨折组和髋骨关节炎组(P=0.001,P=0.000,P=0.000;P=0.000,P=0.014,P=0.000),髋骨关节炎组髋关节滑膜组织中IRF4、IRF7、IFN-α的蛋白相对表达量均高于股骨颈骨折组(P=0.002,P=0.005,P=0.000)。(4)SONFH滑膜病变靶细胞检测结果。SONFH组髋关节滑膜组织中IRF7、钙黏附蛋白11(cadherin-11,CDH-11)的蛋白相对表达量均高于股骨颈骨折组和髋骨关节炎组(P=0.001,P=0.000;P=0.000,P=0.001),髋骨关节炎组髋关节滑膜组织中IRF7、CDH-11的蛋白相对表达量高于股骨颈骨折组(P=0.001,P=0.000)。滑膜成纤维细胞为SONFH滑膜病变的靶细胞。(5)细胞验证结果。模型细胞组IRF4、IRF7、IFN-α的mRNA和蛋白相对表达量均高于空白细胞组(P=0.001,P=0.002,P=0.000;P=0.001,P=0.007,P=0.000)。结论:SONFH滑膜病变与免疫炎症反应关系密切,滑膜成纤维细胞可能是SONFH滑膜病变的靶细胞,IRF4和IRF7可能是其潜在的靶点。

活血通络胶囊对激素性股骨头坏死囊性变成血管修复的影响

目的:探讨活血通络胶囊对激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)囊性变成血管修复的影响。方法:(1)将从全髋关节置换术中收集的一部分SONFH囊性变组织制备成单细胞悬液,采用单细胞转录组学分析囊性变组织中的细胞组分。将另一部分SONFH囊性变组织制成病理切片,采用苏木素-伊红染色法观察囊性变中血管形态,并采用免疫荧光法观察囊性变中血管内皮细胞特异性蛋白的表达情况。(2)分别按照每天20 mg·kg^(-1)、40 mg·kg^(-1)、80 mg·kg^(-1)体质量以浓度1 mg·mL^(-1)、2 mg·mL^(-1)、4 mg·mL^(-1)的活血通络胶囊溶液和20 mL·kg^(-1)生理盐水给大鼠灌胃,获取低、中、高剂量活血通络胶囊含药血清及空白血清。(3)取生长良好的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),分为空白血清组和低、中、高剂量含药血清组,空白血清组细胞常规培养,低、中、高剂量含药血清组细胞分别采用低、中、高剂量活血通络胶囊含药血清干预。干预48 h后,通过细胞计数试剂盒-8法测定活血通络胶囊含药血清对HUVECs增殖能力的影响。(4)将HUVECs分为对照组、地塞米松组和活血通络组,对照组细胞常规培养,地塞米松组细胞采用地塞米松干预,活血通络组细胞采用地塞米松和中剂量活血通络胶囊含药血清干预。干预至细胞长满后进行细胞划痕实验,观察活血通络胶囊含药血清对HUVECs迁移能力的影响。(5)HUVECs分组与干预方法同(4),干预7 d后进行成血管实验,观察活血通络胶囊含药血清对HUVECs成血管能力的影响。结果:(1)SONFH囊性变组织中细胞组分分析结果。SONFH囊性变组织中存在内皮细胞及具有血管生成功能的内皮细胞亚群。(2)SONFH囊性变组织病理学观察结果。苏木素-伊红染色结果显示,SONFH囊性变组织中存在椭圆形的血管腔,血管内膜稍增厚、弹力纤维增生分层。免疫荧光染色结果显示,SONFH囊性变组织内存在血管内皮细胞特异性蛋白(CD31蛋白和血管内皮生长因子蛋白),且可见明显的管形。(3)HUVECs相对增殖率测定结果。各组HUVECs相对增殖率比较,差异有统计学意义[0%,(0.15±0.83)%,(18.70±6.10)%,(24.9±15.10)%,F=7.541,P=0.001];低剂量含药血清组HUVECs相对增殖率与空白血清组的差异无统计学意义(P=0.999),中剂量含药血清组和高剂量含药血清组HUVECs相对增殖率均高于空白血清组(P=0.046,P=0.006),中剂量含药血清组HUVECs相对增殖率与高剂量含药血清组的差异无统计学意义(P=0.782)。(4)HUVECs迁移率测算结果。地塞米松组HUVECs迁移率(10%)明显低于对照组(26%),活血通络组HUVECs迁移率(24%)明显高于地塞米松组(10%)。(5)HUVECs形成的管形分支数测算结果。每个放大40倍视野下,各组HUVECs形成的管形分支数比较,差异有统计学意义[(112.20±7.96)个,(83.40±14.02)个,(122.40±14.59)个,F=2.980,P=0.001];地塞米松组HUVECs形成的管形分支数少于对照组和活血通络组(P=0.006,P=0.001),对照组HUVECs形成的管形分支数与活血通络组的差异无统计学意义(P=0.292)。结论:活血通络胶囊可通过提高血管内皮细胞的增殖、迁移和成血管能力,促进SONFH囊性变的成血管修复。

健脾活骨方治疗非创伤性股骨头坏死的生物力学机制研究

目的:探讨健脾活骨方治疗非创伤性股骨头坏死的生物力学机制。方法:选择20例单侧非创伤性股骨头坏死患者作为研究对象。男7例,女13例;左髋8例,右髋12例;年龄21~58岁,中位数48岁;国际骨微循环研究学会分期均为Ⅱ期。所有患者均采用口服健脾活骨方和药物动脉灌注治疗,健脾活骨方每日1剂,共服用180 d;药物动脉灌注共治疗1次。分别于治疗开始前、治疗开始后3个月及治疗开始后1年进行CT扫描,建立股骨近端和坏死骨三维有限元模型,测定股骨头负重区与坏死骨的最大总形变量和最大等效应力。结果:(1)股骨头负重区最大总形变量和最大等效应力。治疗开始前、治疗开始后3个月和治疗开始后1年,股骨头负重区最大总形变量比较,差异有统计学意义[(4.101±1.341)mm,(3.296±1.203)mm,(3.356±1.022)mm,F=5.646,P=0.008]。治疗开始后3个月和治疗开始后1年股骨头负重区最大总形变量均较治疗开始前减小(P=0.036,P=0.007);治疗开始后3个月和治疗开始后1年股骨头负重区最大总形变量的差异无统计学意义(P=0.576)。治疗开始前、治疗开始后3个月和治疗开始后1年,股骨头负重区最大等效应力比较,差异无统计学意义[(1.837±0.804)MPa,(1.717±0.649)MPa,(1.586±0.593)MPa,F=1.145,P=0.332]。(2)坏死骨最大总形变量和最大等效应力。治疗开始前、治疗开始后3个月和治疗开始后1年,坏死骨最大总形变量和最大等效应力总体比较,差异有统计学意义[最大总形变量:(3.963±1.302)mm,(3.157±1.126)mm,(3.202±1.012)mm,F=5.930,P=0.007;最大等效应力:(1.246±0.445)MPa,(1.470±0.601)MPa,(1.323±0.433)MPa,F=4.224,P=0.024]。治疗开始后3个月和治疗开始后1年坏死骨最大总形变量均较治疗开始前减小(P=0.032,P=0.008);治疗开始后3个月和治疗开始后1年坏死骨最大总形变量的差异无统计学意义(P=0.517)。治疗开始后3个月坏死骨最大等效应力较治疗开始前增大(P=0.030),治疗开始后1年与治疗开始前坏死骨最大等效应力的差异无统计学意义(P=0.076);治疗开始后3个月和治疗开始后1年坏死骨最大等效应力的差异无统计学意义(P=0.202)。结论:健脾活骨方治疗非创伤性股骨头坏死的生物力学机制,可能是通过增强坏死骨的强度与刚度,使坏死骨可以提供有效支撑,减轻股骨头负重区的应力集中现象、解除坏死骨区域正常骨组织高应力状态,从而起到治疗作用。

股骨头前、外侧保留角和联合保留角在股骨头坏死经髋关节外科脱位打压植骨支撑术适应证选择中的作用

目的:探讨股骨头前侧保留角(anterior preserved angle,APA)、外侧保留角(lateral preserved angle,LPA)和联合保留角(combined preserved angle,CPA)在股骨头坏死(osteonecrosis of the femoral head,ONFH)经髋关节外科脱位打压植骨支撑术适应证选择中的作用。方法:以2020年1月至2022年12月在广州中医药大学第三附属医院接受经髋关节外科脱位打压植骨支撑术治疗的ONFH患者为研究对象。收集患者的性别、年龄、ONFH分期和分型,以及手术前后、随访期间双侧髋关节蛙位和正位X线片等信息。在患者术前双侧髋关节蛙位和正位X线片上分别测量股骨头APA和LPA,二角之和为股骨头CPA。以行全髋关节置换术为终点事件,采用Kaplan-Meier法分析经髋关节外科脱位打压植骨支撑术后股骨头生存情况;采用受试者工作特征(receiver operating characteristic,ROC)曲线确定股骨头APA、LPA和CPA预测经髋关节外科脱位打压植骨支撑术治疗失败的最佳临界值,并根据此临界值进行二分类后采用Kaplan-Meier法分析其对股骨头生存情况的预测效果。结果:(1)一般情况。共纳入109例患者,随访时间8~47个月,中位数25个月。患者术前股骨头APA 43.71°±14.43°,LPA 53.08°±15.62°,CPA 96.79°±24.60°。随访期间,18髋改行全髋关节置换术,其中17髋发生在经髋关节外科脱位打压植骨支撑术后2年内。(2)经髋关节外科脱位打压植骨支撑术后股骨头生存情况。术后1年、2年和3年股骨头生存率分别为97.5%、86.1%和81.6%。(3)股骨头APA、LPA和CPA对经髋关节外科脱位打压植骨支撑术治疗失败的预测价值评价结果。股骨头APA、LPA和CPA预测经髋关节外科脱位打压植骨支撑术治疗失败的曲线下面积分别为0.835、0.765、0.854,敏感度分别为67.3%、75.2%、91.1%,特异度分别为88.9%、72.2%、72.2%,临界值分别为39.5°、46.5°、75.0°。(4)股骨头APA、LPA和CPA对经髋关节外科脱位打压植骨支撑术后股骨头生存情况的预测结果。与术前股骨头APA≤39.5°、LPA≤46.5°、CPA≤75.0°相比,术前股骨头APA>39.5°、LPA>46.5°、CPA>75.0°时,股骨头生存率更高。结论:股骨头APA、LPA和CPA可用于指导ONFH经髋关节外科脱位打压植骨支撑术适应证的选择,且术前股骨头APA>39.5°、LPA>46.5°、CPA>75.0°的患者有望获得更好的手术疗效。

Efficacy and safety evaluation of intravenous infusion of cervus and cucumis polypeptides for treatment of avascular necrosis of the femoral head: a randomized clinical trial

OBJECTIVE: To investigate the efficacy and safety of intravenous cervus and cucumis polypeptides for treating avascular necrosis of the femoral head(ANFH) in regard to pain and hip function in a randomized clinical trial.METHODS: A total of 96 subjects with ANFH who were recruited at the Orthopaedic Hospital Affiliated with Hebei United University and Qian Hai Femoral Head Hospital of Beijing were assigned by lottery to an intervention group(n = 48) or a control group(n = 48). All subjects underwent physical therapy and rehabilitation exercises. In addition,subjects in the intervention group were given intravenous infusions of cervus and cucumis polypeptides. Visual analogue scale(VAS), Harris hip score,and radiography or magnetic resonance imaging were applied to assess all subjects at the beginning of treatment and 3, 6, and 9 months afterward. All the subjects were followed up for 2 years.RESULTS: At the beginning of treatment, there were no statistically significant differences between the two groups in terms of the general condition of patients or the VAS and Harris hip scores(all P > 0.05). At 3, 6, and 9 months after treatment,however, the VAS score decreased and the Harris hip score increased in all patients, with the improvement of intervention group significantly greater than that of the control group(P < 0.05).The total effectiveness rates for the intervention and control groups were 89.58% and 70.83%, respectively, with the difference being statistically significant(P < 0.05). There was no statistically significant difference between the two groups in terms of the safety of the injections(P > 0.05).CONCLUSION: Intravenous infusion of cervus and cucumis polypeptides relieved pain and improved hip function of subjects with ANFH.Thus, the intravenous infusion of cervus and cucumis polypeptides was a safe, effective treatment for ANFH.