Osthole stimulates bone formation,drives vascularization and retards adipogenesis to alleviate alcohol‐induced osteonecrosis of the femoral head

Characteristic pathological changes in osteonecrosis of the femoral head (ONFH) include reduced osteogenic differentiation of bone mesenchymal stem cells (BMSCs), impaired osseous circulation and increased intramedullary adipocytes deposition. Osthole is a bioactive derivative from coumarin with a wide range of pharmacotherapeutic effects. The aim of this study was to unveil the potential protective role of osthole in alcohol‐induced ONFH. In vitro, ethanol (50 mmol/L) remarkably decreased the proliferation and osteogenic differentiation of BMSCs and impaired the proliferation and tube formation capacity of human umbilical vein endothelial cell (HUVECs), whereas it substantially promoted the adipogenic differentiation of BMSCs. However, osthole could reverse the effects of ethanol on osteogenesis via modulating Wnt/β‐catenin pathway, stimulate vasculogenesis and counteract adipogenesis. In vivo, the protective role of osthole was confirmed in the well‐constructed rat model of ethanol‐induced ONFH, demonstrated by a cascade of radiographical and pathological investigations including micro‐CT scanning, haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling, immunohistochemical staining and fluorochrome labelling. Taken together, for the first time, osthole was demonstrated to rescue the ethanol‐induced ONFH via promoting bone formation, driving vascularization and retarding adipogenesis.     

Circulating microRNA signature of steroid‐induced osteonecrosis of the femoral head

Objectives

Steroid‐induced osteonecrosis of the femoral head (ONFH) is a common orthopaedic disease of which early detection remains clinically challenging. Accumulating evidences indicated that circulating microRNAs (miRNAs) plays vital roles in the development of several bone diseases. However, the association between circulating miRNAs and steroid‐induced ONFH remains elusive.

Materials and methods

miRNA microarray was performed to identify the differentially abundant miRNAs in the serums of systemic lupus erythematosus (SLE) patients with steroid‐induced ONFH as compared with SLE control and healthy control group. We predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Results

Our data indicated that there were 11 differentially abundant miRNAs (2 upregulated and 9 downregulated) between SLE‐ONFH group and healthy control group and 42 differentially abundant miRNAs (14 upregulated and 28 downregulated) between SLE‐ONFH group and SLE control group. We also predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Conclusions

These findings corroborated the idea that circulating miRNAs play significant roles in the development of ONFH and may serve as diagnostic markers and therapeutic targets.     

Icariin promotes angiogenesis in glucocorticoid‐induced osteonecrosis of femoral heads: In vitro and in vivo studies

The injury and dysfunction of the femoral head microvascular endothelial cells are associated with the pathogenesis of glucocorticoid‐induced osteonecrosis of the femoral head (ONFH). Reports indicate that icariin (ICA) can enhance vascular roles and also inhibit endothelial cell dysfunction. However, it still remains unclear whether ICA can promote angiogenesis in glucocorticoid‐induced ONFH. In this study, we investigate this hypothesis through in vitro and in vivo experiments. Results showed that 0.1 mg/mL hydrocortisone significantly suppressed bone microvascular endothelial cells (BMECs) proliferation while ICA at 10^?5 mol/L reversed this inhibition. ICA significantly promoted BMECs migration, tube formation, the angiogenesis‐related cytokines expression and the activation of Akt. Furthermore, ICA enhanced Bcl‐2 expression but diminished Bax expression. According to in vivo results, rats with ICA treatment exhibited a lower ratio of empty lacunae, higher volume of blood vessels and more CD31‐positive cells. This study revealed that ICA promotes angiogenesis of BMECs in vitro and improves femoral head blood vessel volume of rats treated with glucocorticoid, suggesting the efficacy of ICA in the prevention of glucocorticoid‐induced ONFH.     

Glucocorticoids decreased Cx43 expression in osteonecrosis of femoral head: The effect on proliferation and osteogenic differentiation of rat BMSCs

Glucocorticoid (GC)-induced osteonecrosis of the femoral head (GC-ONFH) is considered as one of the most serious side effects of long-term or over-dose steroid therapy. However, the underlying cause mechanisms are still not fully investigated. We firstly established a rat model of GC-ONFH and injected lipopolysaccharide (LPS) and methylprednisolone (MPS). We found that the expressions of Cx43, Runx2, ALP and COLⅠ were more decreased than the normal group. Secondly, the isolated rat bone marrow stem cells (BMSCs) were treated with dexamethasone (Dex) in vitro, and the expressions of Cx43, Runx2, ALP and COLⅠ were decreased significantly. Moreover, the results of immunofluorescence staining, alizarin red staining, EdU assay and CCK8 showed that the osteogenic differentiation and the proliferation capacity of BMSCs were decreased after induced by Dex. A plasmid of lentivirus-mediated Cx43 (Lv-Cx43) gene overexpression was established to investigate the function of Cx43 in BMSCs under the Dex treatment. Findings demonstrated that the proliferation and osteogenic differentiation abilities were enhanced after Lv-Cx43 transfected to BMSCs, and these beneficial effects of Lv-Cx43 were significantly blocked when PD988059 (an inhibitor of ERK1/2) was used. In conclusion, the overexpression of Cx43 could promote the proliferation and osteogenic differentiation of BMSCs via activating the ERK1/2 signalling pathway, which provide a basic evidence for further study on the detailed function of Cx43 in GC-ONFH.     

Identification of potential miRNA biomarkers for traumatic osteonecrosis of femoral head

Traumatic osteonecrosis of femoral head (TONFH) is a common orthopedic disease caused by physical injury in hip. However, the unclear pathogenesis mechanism of TONFH and lacking of simple noninvasive early diagnosis method cause the necessity of hip replacement for most patients with TONFH. In this study, we aimed to identify circulating microRNAs (miRNAs) by integrated bioinformatics analyses as potential biomarker of TONFH. mRNA expression profiles were downloaded from the Gene Expression Omnibus database. Then we combined two miRNA screen methods: Weighted gene co-expression network analysis and fold change based differentially expressed miRNAs analysis. As a result, we identified 14 key miRNAs as potential biomarkers for TONFH. Besides, 302 target genes of these miRNAs were obtained and the miRNA–mRNA interaction network was constructed. Furthermore, the results of Kyoto Encyclopedia of Gene and Genome pathway analysis, Gene Ontology function analysis, protein–protein interaction (PPI) network analysis and PPI network module analysis showed close correlation between these 14 key miRNAs and TONFH. Then we established receiver operating characteristic curves and identified 6-miRNA signature with highly diagnosis value including miR-93-5p (area under the curve [AUC] = 0.93), miR-1324 (AUC = 0.92), miR-4666a-3p (AUC = 0.92), miR-5011-3p (AUC = 0.92), and miR-320a (AUC = 0.89), miR-185-5p (AUC = 0.89). Finally, the results of quantitative real-time polymerase chain reaction confirmed the significantly higher expression of miR-93-5p and miR-320a in the serum of patients with ONFH. These circulating miRNAs could serve as candidate early diagnosis markers and potential treatment targets of TONFH.     

骨源性细胞因子在激素性股骨头坏死发生发展中的研究进展

股骨头坏死是骨外科常见的难治性疾病,其机制仍有待研究.目前为止,医源性糖皮质激素是非创伤性股骨头坏死的主要原因.激素的长期使用可导致股骨头骨细胞凋亡、血液循环障碍所致缺血缺氧,最终导致股骨头塌陷.激素性股骨头坏死的发生发展与骨组织中细胞直接接触和其间接分泌的细胞因子调控相关.本文综述了骨组织中成骨细胞分泌的核因子κB受...     

The role of biomechanical forces and MALAT1/miR-329-5p/PRIP signalling on glucocorticoid-induced osteonecrosis of the femoral head

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common orthopaedic disease. GIONFH primarily manifests clinically as hip pain in the early stages, followed by the collapse of the femoral head, narrowing of the hip joint space and damage to the acetabulum, resulting in severely impaired mobility. However, the pathogenesis of GIONFH is not clearly understood. Recently, biomechanical forces and non-coding RNAs have been suggested to play important roles in the pathogenesis of GIONFH. This study aimed to evaluate the role of biomechanical forced and non-coding RNAs in GIONFH. We utilized an in vivo, rat model of GIONFH and used MRI, μCT, GIONFH-TST (tail suspension test), GIONFH-treadmill, haematoxylin and eosin staining, qRT-PCR and Western blot analysis to analyse the roles of biomechanical forces and non-coding RNAs in GIONFH. We used RAW264.7 cells and MC3T3E1 cells to verify the role of MALAT1/miR-329-5p/PRIP signalling using a dual luciferase reporter assay, qRT-PCR and Western blot analysis. The results demonstrated that MALAT1 and PRIP were up-regulated in the femoral head tissues of GIONFH rats, RAW264.7 cells, and MC3T3E1 cells exposed to dexamethasone (Dex). Knockdown of MALAT1 decreased PRIP expression in rats and cultured cells and rescued glucocorticoid-induced osteonecrosis of femoral head in rats. The dual luciferase reporter gene assay revealed a targeting relationship for MALAT1/miR-329-5p and miR-329-5p/PRIP in MC3T3E1 and RAW264.7 cells. In conclusion, MALAT1 played a vital role in the pathogenesis of GIONFH by binding to (‘sponging’) miR-329-5p to up-regulate PRIP. Also, biomechanical forces aggravated the pathogenesis of GIONFH through MALAT1/miR-329-5p/PRIP signalling.     

Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3‐E1 osteoblast‐like cells

Osteoblasts in culture can differentiate into mature mineralizing osteoblasts when stimulated with osteogenic agents. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) may benefit bone health. The aim of this study was to investigate whether arachidonic acid (AA) and docosahexaenoic acid (DHA) affect osteogenesis in osteoblasts and the transdifferentiation into adipocytes. Results from this study show that long‐term exposure to AA inhibited alkaline phosphatase (ALP) activity in these cells, which might be prostaglandin E₂ (PGE₂)‐mediated. DHA exposure also inhibited ALP activity which was evident after both short‐ and long‐term exposures. The mechanism whereby DHA inhibits ALP activity is not clear and needs to be investigated. Although long‐term exposure to PUFAs inhibited ALP activity, the mineralizing properties of these cells were not compromised. Furthermore, PUFA exposure did not induce adipocyte‐like features in these cells as evidenced by the lack of cytoplasmic triacylglycerol accummulation. More research is required to elucidate the cellular mechanisms of action of PUFAs on bone. Copyright © 2008 John Wiley & Sons, Ltd.     

Microarray analysis of long-noncoding RNAs and mRNA expression profiles in human steroid-induced avascular necrosis of the femoral head

This study performed the first microarray analysis of long-noncoding RNA (lncRNA) and mRNA expression profiles in human steroid-induced avascular necrosis of the femoral head (SAVNFH). Expression levels of lncRNAs and mRNAs in three human SAVNFH samples and three human femoral head fracture samples (controls) were detected using third-generation lncRNA microarrays (KangChen Biotech, Shanghai, China). The fold change, false discovery rate, and P value were utilized to filter genes with significant differential expression in the SAVNFH samples compared with the control samples. In total, there were 1179 upregulated and 3214 downregulated lncRNAs (P2. zerofold, P < 0.05). Meanwhile, 1092 upregulated and 565 downregulated mRNAs were found in the SAVNFH samples compared with the control samples. Then, quantitative real-time polymerase chain reaction was used to confirm the previous microarray results using 8 and 20 selected dysregulated lncRNAs and mRNAs, respectively, and the results generally confirmed the microarray findings. Finally, we used Gene Ontology (GO) and pathway analysis to investigate the functions of the altered mRNAs and their associated GO terms and biological pathways. The Immune system process term (GO:0002376) was the most significantly upregulated GO term, and the Regulation of blood coagulation term (GO:0030193) was the most significantly downregulated GO term in the biological process category for the SAVNFH samples. “Hematopoietic cell lineage - Homo sapiens (human) (Pathway ID: hsa04640)” and “Complement and coagulation cascades - Homo sapiens (human) (Pathway ID: hsa04610)” were the most significantly up- and downregulated pathways in the SAVNFH samples compared with the controls. In conclusion, the differential expression of lncRNAs and mRNAs may be correlated with the pathogenesis of SAVNFH, and these significantly dysregulated lncRNAs and mRNAs may function through networks or participate in several specific biological processes. Further research is needed to understand their exact functions and mechanisms in SAVNFH.     

TNF‐α regulates the early development of avascular necrosis of the femoral head by mediating osteoblast autophagy and apoptosis via the p38 MAPK/NF‐κB signaling pathway

Previous studies have shown that the tumor necrosis factor‐α (TNF‐α) levels in serum and bone tissues formed in avascular necrosis of femoral head (ANFH) patients were higher than those of normal individuals, indicating TNF‐α might play a role in the pathogenesis of ANFH. However, the underlying mechanisms remain unclear. Hematoxylin and eosin staining was performed to show the pathological changes of ANFH bone tissues. TNF‐α expression in normal and ANFH tissues was examined by quantitative real‐time polymerase chain reaction and western blot analyses. Osteoblast autophagy and apoptosis, as well as signaling pathways activation, were measured by their corresponding marker proteins. Osteoblast proliferation, autophagy, and apoptosis were evaluated using cell counting kit‐8, transmission electron microscopy, and flow cytometry. The structures of bone tissues of ANFH were obviously damaged. TNF‐α expression was significantly upregulated in ANFH bone tissues compared to normal tissues. Autophagy and apoptosis were remarkably promoted, and p38 mitogen‐activated protein kinase (MAPK)/nuclear factor‐κB (NF‐κB) signaling pathways were markedly activated in ANFH. Suppression of the p38 MAPK/NF‐κB pathway significantly attenuated the TNF‐α‐induced autophagy, however, enhanced the TNF‐α‐induced apoptosis in osteoblasts. Increased TNF‐α in ANFH regulated osteoblast autophagy and apoptosis by p38 MAPK/NF‐κB signaling pathways, blocking the pathway by inhibitors exacerbated TNF‐α‐induced apoptosis through impairing autophagy flux.     

自体外周血干细胞移植联合髓芯减压治疗早中期股骨头缺血性坏死的临床研究

目的对比研究单纯股骨头髓芯减压与联合自体外周血干细胞移植治疗早中期股骨头缺血性坏死（ANFH）的临床疗效。方法 2004年10月至2012年3月期间就诊于福建省龙岩市第一医院,经影像学检查及术后病理检查确诊为ANFH的患者90例。A组采用单纯髓芯减压共34例,男22例,女12例,年龄22~62岁（中位年龄41.5岁）。B组采用髓芯减压联合自体外周血干细胞移植（用rhG-CSF皮下注射4 d进行外周血干细胞动员,第5天分离外周造血干细胞混悬液。在髓芯减压基础上沿减压隧道注入自体外周血干细胞10~15 ml）56例,男35例,女21例,年龄21~62岁（中位年龄39岁）。按世界骨髓循环研究学会（ARCO）国际骨坏死标准：A组：Ⅰ期7髋,Ⅱ期26髋,Ⅲ期17髋;B组：Ⅰ期10髋,Ⅱ期50髋,Ⅲ期29髋。术后及随访期间进行Harris评分和疼痛视觉模拟（VAS）评分,对X线片、CT及MRI检查结果进行评估。两组Harris评分和VAS评分比较用成组t检验。结果 A组34例,B组49例随访13~89个月,平均38.5个月。术后3、6、12个月与术前的Harris评分比较有明显提高,VAS评分有明显的下降;术前、术后3个月两时间点的A、B组Harris评分比较差异无统计学意义（t=0.342、0.628,P=0.781、0.516）;术后6、12个月的B组评分优于A组（t=2.991、7.753,P=0.009、0.001）。术后12个月A、B两组T1相低信号区所占股骨头体积的比例分别为（17.24±3.71）﹪和（12.11±3.14）﹪,差异有统计学意义（t=4.360,P=0.001）。结论髓芯减压联合自体外周血干细胞移植治疗早中期ANFH,可显著减轻关节疼痛,改善股骨头血液供应,明显恢复关节功能,加速病灶区骨组织修复,有效防止股骨头进一步塌陷。     

Mesenchymal stem cells‐derived and siRNAs‐encapsulated exosomes inhibit osteonecrosis of the femoral head

Osteonecrosis of the femoral head (ONFH) is a progressive, obstinate and disabling disease. At present, the treatment of ONFH is still a global medical problem. We aim to explore the role of bone mesenchymal stem cells (BMSCs)‐derived and siRNAs‐encapsulated exosomes (siRNAs‐encapsulated BMSCexos) in ONFH. We first isolated BMSCexos and screened siRNAs of 6 ONFH‐related genes for siRNAs‐encapsulated BMSCexo. The expression of these 6 ONFH‐related genes in dexamethasone (DXM)‐treated MC3T3‐E1 cell, cell model of ONFH, was detected by RT‐qPCR and Western blot analysis. And then, we performed CCK‐8 assay, angiogenesis assay and HE staining analysis to test the promotion role of the siRNAs‐encapsulated BMSCexo for angiogenesis during ONFH repair. The results suggest that the obtained particles were BMSCexos. The screened effective siRNAs could effectively knock down their expression in VECs. Moreover, siRNAs‐encapsulated BMSCexo could effectively knock down the expression of these genes in VECs. In addition, siRNAs‐encapsulated BMSCexo promote angiogenesis during ONFH repair. In conclusion, we found siRNAs‐encapsulated BMSCexos could promote ONFH repair by angiogenesis, and indicated exosome as the new siRNA carrier is of great significance to improve the efficiency of RNAi.     

股骨头缺血坏死相关影像学误诊分析

目的:分析与股骨头缺血坏死相关临床与影像资料,以期提高影像科医生对该病的认识水平,提高股骨头缺血坏死的诊断正确率,防止与其相关的髋关节疾病的误诊。方法:回顾性分析26例误诊为股骨头缺血坏死的髋关节疾患,归纳分类疾病的种类,找出原因。结果:类风湿性髋关节炎、强直性脊柱炎髋关节炎和痛风性髋关节炎、先天性髋臼发育不良、股骨颈干角发育异常、髋关节骨关节病等疾病易与股骨头缺血坏死相混淆,引起误诊。结论:病史采集不详细、思路狭窄,对类风湿、强直性脊柱炎及痛风性关节炎所致髋关节病变认识不足或不了解、股骨头缺血坏死病理过程不清楚、临床误导等因素与股骨头缺血坏死的误诊有关。