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

Mechanism of bone repair in the cystic lesion tissues of steroid-induced osteonecrosis of the femoral head and the effects of Huoxue Tongluo Jiaonang(活血通络胶囊)medicated serum on the osteogenic differentiation of hypertrophic chondrocytes:an experimental study

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。

Objective:To analyze the mechanism of bone repair in cystic lesion tissues of steroid-induced osteonecrosis of the femoral head(SONFH),and to observe the effects of Huoxue Tongluo Jiaonang(活血通络胶囊,HXTLJN)medicated serum on the osteogenic differentiation of hypertrophic chondrocytes(HCs).Methods:①Analyzing the mechanism of bone repair in SONFH-associated cystic lesion tissues.Six patients who underwent total hip arthroplasty for SONFH were enrolled in the study,and their necrotic femur heads were collected after the surgery.Among the 6 femur heads,3 ones were used for single-cell RNA sequencing(scRNA-seq)on cystic lesion tissues to analyze the cell categories,chondrocyte subsets,and biological processes involving in HCs in cystic lesion tissues by bioinformatics methods;while,the other 3 ones were used for pathological examination on the cystic lesion tissues to observe the distributions and characteristics of chondrocytes and HCs in cystic lesion tissues by using hematoxylin-eosin(HE)staining,Alcian blue staining,and safranin O-fast green(SO-FG)staining,respectively,and detect the expression of hypertrophic chondrocyte marker protein including collagen typeⅩα1(Col10α1)and matrix metalloproteinase(MMP)13,as well as the osteogenic differentiation-related protein including Runt-related transcription factor 2(RUNX2)and osteopontin(OPN)in the cystic lesion tissues by using immunohistochemical staining.②Analyzing the effects of HXTLJN medicated serum on the osteogenic differentiation of HCs.The rats were intervened by intragastric administration with HXTLJN(HXTLJN powders were dissolved into the distilled water)for making HXTLJN medicated serum.The knee articular cartilages were harvested from the sacrificed mice,and were digested and separated for chondrocytes,which were then cultured in DMEM/F-12.Furthermore,the chondrocytes were inoculated into complete medium containing different concentrations of HXTLJN medicated serum for determining the optimal intervention concentration of HXTLJN medicated serum.The HCs were induced from the first-generation mouse chondrocytes in the logarithmic phase,after successful induction,they were divided into osteogenic induction group,5%HXTLJN medicated serum intervention group,and control group.The HCs in osteogenic induction group were cultured with osteogenesis inducing medium(OIM),the ones in 5%HXTLJN medicated serum intervention group with OIM containing 5%HXTLJN medicated serum,while the ones in control group with hypertrophy-inducing medium.After 7-day culture,the morphological characteristics of the HCs were observed by using alkaline phosphatase(ALP)staining and alizarin red(AR)staining,respectively;and the relative mRNA expression levels of collagen Iα1(Col1α1),ALP,RUNX2 and OPN,as well as the relative protein expression levels of ALP,RUNX2 and OPN were detected by using real-time quantitative PCR(RT-qPCR)and Western blotting,respectively.Results:①The results of analysis on SONFH-associated cystic lesion tissues by scRNA-seq.The scRNA-seq results of 30224 cells were acquired.The results of cell categories indicated that the cystic lesions harbored eight categories of cells,including T cells,endothelial cells,fibroblasts,chondrocytes,macrophages,monocytes,osteoclasts,and mast cells.The results of chondrocyte subset analysis showed that the chondrocytes included five subsets,namely homeostatic chondrocytes,fibroblast chondrocytes,inflammatory chondrocytes,hypertrophic chondrocytes,and precursor hypertrophic chondrocytes.The results of enrichment analysis on the differentially expressed genes(DEGs)showed that the hypertrophic chondrocytes mainly participated in the biological processes such as positive regulation of cell migration,cell differentiation,extracellular matrix formation,ossification,cell migration,and osteoblast differentiation.②The results of examination on histopathological changes in SONFH-associated cystic lesion tissues.The cartilage cells and HCs were found at the edge of cystic lesion tissues,with some cartilage tissue presented a transition towards to bone trabeculae in morphology.Moreover,the Col10α1,MMP13,RUNX2,and OPN were highly expressed at the edge of cystic lesion tissues.③The results of analysis on the effects of HXTLJN medicated serum in interventing osteogenic differentiation of HCs.The screening results showed the optimal intervention concentration of HXTLJN medicated serum was 5%.The ALP and ARS staining results showed that the distinct calcified nodules were found in osteogenic induction group,which,however,failed to be found in control group,and they were further increased in 5%HXTLJN medicated serum intervention group compared to osteogenic induction group.Additionally,the relative mRNA expression levels of ALP,RUNX2,OPN and Col1α1,as well as the relative protein expressions levels of ALP,RUNX2 and OPN were higher in osteogenic induction group compared to control group(P=0.000,P=0.000,P=0.001,P=0.000;P=0.000,P=0.000,P=0.001),and were highest in 5%HXTLJN medicated serum intervention group(P=0.000,P=0.000,P=0.002,P=0.001;P=0.000,P=0.000,P=0.001).Conclusion:A repair mechanism of hypertrophic chondrocytes differentiating into osteoblasts is presented in the cystic lesion tissues of SONFH,which can be promoted by the HXTLJN medicated serum in vitro.