Steroid-induced Osteonecrosis Research Articles

MIR31HG polymorphisms are related to steroid-induced osteonecrosis of femoral head among Chinese Han population

BackgroundsMIR31 host gene (MIR31HG) polymorphisms play important roles in the occurrence of osteonecrosis. However, the association of MIR31HG polymorphisms with the risk of steroid-induced osteonecrosis of the femoral head (SONFH) remains unclear. In this study, we aimed to investigate the correlation between MIR31HG polymorphisms and SONFH susceptibility in the Chinese Han population.MethodsA total of 708 volunteers were recruited to detect the effect of seven single nucleotide polymorphisms (SNPs) in the MIR31HG gene on SONFH risk in the Chinese Han population. Genotyping of MIR31HG polymorphisms was performed using the Agena MassARRAY platform. The odds ratio (OR) and 95% confidence interval (95% CI) were used to evaluate the correlation between MIR31HG polymorphisms and SONFH risk using logistic regression model.ResultsAccording to the results of genetic model, rs10965059 in MIR31HG was significantly correlated with the susceptibility to SONFH (OR = 0.56, p = 0.002). Interestingly, the stratified analysis showed that rs10965059 was associated with the reduced risk of SONFH in subjects aged > 40 years (OR = 0.30, p < 0.001) and male populations (OR = 0.35, p < 0 .001). Moreover, rs10965059 was associated with the reduced risk of bilateral SONFH (OR = 0.50, p = 0.002). Finally, multi-factor dimension reduction (MDR) results showed that the combination of rs1332184, rs72703442, rs2025327, rs55683539, rs2181559, rs10965059 and rs10965064 was the best model for predicting SONFH occurrence (p < 0.0001).ConclusionThe study indicated that rs10965059 could be involved in SONFH occurrence in the Chinese Han population, which might provide clues for investigating the role of MIR31HG in the pathogenesis of SONFH.

Effects of Extracellular Vesicles from Osteogenic Differentiated Human BMSCs on Osteogenic and Adipogenic Differentiation Capacity of Naïve Human BMSCs.

Osteoporosis, or steroid-induced osteonecrosis of the hip, is accompanied by increased bone marrow adipogenesis. Such a disorder of adipogenic/osteogenic differentiation, affecting bone-marrow-derived mesenchymal stem cells (BMSCs), contributes to bone loss during aging. Here, we investigated the effects of extracellular vesicles (EVs) isolated from human (h)BMSCs during different stages of osteogenic differentiation on the osteogenic and adipogenic differentiation capacity of naïve (undifferentiated) hBMSCs. We observed that all EV groups increased viability and proliferation capacity and suppressed the apoptosis of naïve hBMSCs. In particular, EVs derived from hBMSCs at late-stage osteogenic differentiation promoted the osteogenic potential of naïve hBMSCs more effectively than EVs derived from naïve hBMSCs (naïve EVs), as indicated by the increased gene expression of COL1A1 and OPN. In contrast, the adipogenic differentiation capacity of naïve hBMSCs was inhibited by treatment with EVs from osteogenic differentiated hBMSCs. Proteomic analysis revealed that osteogenic EVs and naïve EVs contained distinct protein profiles, with pro-osteogenic and anti-adipogenic proteins encapsulated in osteogenic EVs. We speculate that osteogenic EVs could serve as an intercellular communication system between bone- and bone-marrow adipose tissue, for transporting osteogenic factors and thus favoring pro-osteogenic processes. Our data may support the theory of an endocrine circuit with the skeleton functioning as a ductless gland.

Lipid metabolism analysis for peripheral blood in patients with alcohol-induced and steroid-induced osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH), also known as vascular necrosis of the femoral head, is combined with lipid metabolism disorders in most patients. This study aims to explore the lipid metabolism profiles in different subtypes of ONFH. The subjects were divided into an alcohol-induced osteonecrosis of the femoral head (AONFH) group, a steroid-induced osteonecrosis of the femoral head (SONFH) group, and a normal control (NC) group (n=16, 29, and 32, respectively). Ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) was used to detect the lipidomics analysis in the peripheral blood samples of subjects and identify the underlying biomarkers. The samples were preprocessed, the partial least squares discriminant analysis (PLS-DA) was adopted, and the variable importance for the projection (VIP) values were calculated to measure the expression pattern of each lipid metabolite and observe the influence and explanatory power of the expression pattern of each lipid metabolite on the classification and discrimination between the different groups. The lipid metabolites with fold change (FC)>2, P<0.05 and VIP>1 in the different groups were screened as differential lipids. Among them, the differential lipids co-existing in the AONFH group and the SONFH group were regarded as common differential lipids for ONFH, and the differential lipids that exist separately were regarded as specific differential lipids in the AONFH group or the SONFH group. Binary logistic regression was used to evaluate the diagnostic value of differential lipid metabolites on the basis of the receiver operator characteristic (ROC) curve analysis. Based on the disease stage information, the correlation between the differential lipids and the disease stage was analyzed in the AONFH group and the SONFH group. In this study, 1 358 lipid metabolites were detected in each plasma sample. Compared with the NC group, there were significant difference in the expression patterns of lipid metabolism profiles in the AONFH group and the SONFH group. A total of 62 and 64 differential lipid metabolites were screened in the AONFH and SONFH patients (FC>2, P<0.05, VIP>1) respectively, and these differential lipids were mainly up-regulated in the disease samples. Nine differential lipid metabolites were further identified, which were shared by the AONFH group and the SONFH group; the area under the curve (AUC) in 6 kinds of lipid components was greater than 0.7, including 1-myristoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, hypoxanthin, serotonin, PE (19:0/22:5), PE (19:0/22:5), and cholest-5-en-3-yl beta-D-glucopyranosiduronic acid. Fifty-three specific differential lipid metabolites were identified in the AONFH group, and 55 specific differential lipid metabolites were identified in the SONFH group. The AUC in 6 kinds of lipid components was greater than 0.9, including 1D-myo-Inositol 1,2-cyclic phosphate, L-pyroglutamic acid, DL-carnitine, 8-amino-7-oxononanoic acid, Clobetasol, and presqualene diphosphate. In the AONFH group, there were 9 differential lipid metabolites related to the disease stages, including LPG 18:1, serotonin, PC (22:4e/23:0), PC (19:2/18:5), hypoxanthin, PE (18:1/20:3), LPE 18:1, 1-stearoyl-2-arachidonoyl-sn-glycerol, and PE (16:0/18:1); with AONFH disease progresses from I/II stages to III/IV stages, the relative content of these 9 differential lipid metabolites was increased. In the SONFH group, 8 differential lipid metabolites were found to be related to the stage of the disease, including TM6076000, 4-(1,1-dimethylpropyl)phenol, D-617, asarone, phenylac-gln-OH, creatine, leu-pro, and 8-amino-7-oxononanoic acid; and with the SONFH progressed from stage I/II to stage III/IV, the content of these 8 differential lipid metabolites were gradually increased. This study analyzes the characteristics of the plasma lipid metabolism profile in the AONFH and SONFH patients, and which identifies the differential lipid metabolites related to disease diagnosis and evaluation. These results provide evidence for exploring lipid metabolism alterations and the mining of novel lipid biomarkers for the ONFH.

Huogu injection alleviates SONFH by regulating adipogenic differentiation of BMSCs via targeting the miR-34c-5p/MDM4 pathway

Although the precise pathogenesis of steroid-induced osteonecrosis of femoral head (SONFH) is not yet fully understood, evidence shows miRNAs-mediated posttranscription control directs the adipogenesis of bone marrow mesenchymal stem cells (BMSCs) and plays a pivotal role in the SONFH processes. Huogu injection formulated according to traditional Chinese medicine (TCM) theory has been used to treat SONFH by intra-articular injection. In this study, we asked whether the therapeutic effects of Huogu injection might depend on the inhibition of adipogenic differentiation of BMSCs, and if so, the pathway might be a therapeutic target to promote bone repair. Consequently, miR-34c-5p was upregulated in the dexamethasone (DEX)-treated BMSCs and might participate in the adipogenesis of BMSCs. TargetScan database and the luciferase reporter assay showed miR-34c-5p targeted on the MDM4 and negatively regulated its expression. Huogu injection in vitro inhibited the adipogenesis in the DEX-treated BMSCs by inhibiting the expression levels of PPARγ and C/EBPα, as well as reducing miR-34c-5p to prevent the degradation of MDM4. Moreover, miR-34c-5p mimic or MDM4 knockdown using shRNA neutralized the anti-adipogenesis of Huogu injection in BMSCs. In vivo, the results of X-ray imaging confirmed that Huogu injection alleviated the bone loss in rat SONFH. Consistent with results in vitro, Huogu injection reduced the lipid accumulation, removed the suppression of MDM4 by downregulating the expression of miR-34c-5p, and inhibited the expression of C/EBPα and PPARγ in bone tissues. When the lentivirus encoding miR-34c-5p was conducted by intra-articular injection, the overexpression of miR-34c-5p antagonized the therapeutic effects of Huogu injection. Our results underline the critical importance of the miR-34c-5p/MDM4 pathway in regulating the adipogenic outcome of BMSCs, suggesting the miR-34c-5p as a potentially effective therapeutic target in SONFH. These results further reinforce the potential of Huogu injection as an alternative approach in SONFH.

The effect of endostatin on angiogenesis and osteogenesis of steroid-induced osteonecrosis of the femoral head in a rabbit model.

This study aimed to investigate whether endostatin, a crucial anti-angiogenic factor, plays a negative role in angiogenesis and osteogenesis and aggravates the progression of osteonecrosis of the femoral head induced by steroid use in a rabbit model. 66 New Zealand white rabbits were randomly divided into four groups: glucocorticoid model (GC) group (GC group, n = 18), glucocorticoid model and endostatin group (GC;ES group, n = 18), ES group (ES group, n = 18), and blank control group (CON group, n = 12). In the GC group, 10 μg/ kg lipopolysaccharide (LPS) was intravenously injected into the ear margin, and 24h after LPS injection, 20 mg/kg GC methylprednisolone (MPS) was injected into the gluteus muscle three times, each time at an interval of 24h. The animals of the GC;ES group were given as same treatment as the GC group, except for the addition of ES. MPS was not used in the ES group and CON group. ES group was only given ES, while the CON group was only given the same amount of normal saline. All animals successfully established models of femoral head necrosis, and then the difference among the Immunohistochemistry, Quantitative polymerase chain reaction (qPCR) analysis, Enzyme-linked immunosorbent assay, Biomechanical test, etracyclline-calcein double labeling, and Van Gieson staining indices were compared among the four groups. The combination of MPS and LPS was successful in establishing the femoral head necrosis model in New Zealand white rabbits. The incidence of osteonecrosis after MPS and LPS intervention was 70% (7/10), while that plus ES was 100% (10/10). At the same time, after MPS and LPS intervention, while the empty bone lacuna rate of the femoral head was significantly increased, the number of osteo- blasts was decreased. Also, the expressions of CD31 positive cells, Runx2, Osterix, COL1A1, and VEGF mRNA in the femoral head were decreased, and the levels of osteogenesis-related protein b-ALP, OCN, and angiogenic factor VEGF in the femoral head were decreased. The percentage of the trabecular bone area (%Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), labeled perimeter percent (%L.Pm), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the femoral head after MPs and LPS intervention detected by tetracycline calcein double labeling and Van Gieson staining decreased significantly, except trabecular separation (Tb.Sp) increased significantly. The compressive strength (CS), elastic modulus (EM), and strain energy (SE) of the femoral head examed by biomechanical measurement decreased significantly. All the above changes were more obvious after adding ES intervention. ES mRNA in the femoral head was undifferentiated and increased in the GC, ES, and GC;ES group compared with group CON. This study has revealed that ES can inhibit angiogenesis and osteogenesis in the femoral head and aggravate the occurrence and development of femoral head necrosis. Thus, antiangiogenic factors may play an important role in the pathogenesis of ONFH.

Circ_0058792 regulates osteogenic differentiation through miR-181a-5p/Smad7 axis in steroid-induced osteonecrosis of the femoral head

ABSTRACT Osteonecrosis of the femoral head (ONFH) caused by steroids is a severe orthopedic disorder resulting from the use of high-dose steroid drugs, characterized by structural changes in the bone, joint dysfunction, and femoral head collapse. CircRNAs and miRNAs have increasingly been suggested to play pivotal roles in osteogenic differentiation and osteogenesis. Significant upregulation of circ_0058792 was observed in patients with steroid-induced ONFH. Bioinformatic analysis showed that circ_0058792 might act as a sponge for miR-181a-5p. This study further investigated the mechanisms underlying the role of circ_0058792 and miR-181a-5p in osteogenic differentiation in methylprednisolone-induced ONFH rats and MC3T3-E1 cells. The results showed a notable decrease in the serum of miR-181a-5p in methylprednisolone-induced ONFH rats. Silencing of circ_0058792 using siRNAs and overexpression of miR-181a-5p significantly increased alkaline phosphatase activity and matrix mineralization capacity. Additionally, markers for osteogenic differentiation were significantly upregulated in miR-181a-5p-transfected cells. However, overexpression of circ_0058792 and the addition of the miR-181a-5p inhibitor reversed this increase. Smad7 was identified to be miR-181a-5p’s direct target and circ_0058792 was confirmed to be miR-181a-5p’s competing endogenous RNA (ceRNA). Upregulation of miR-181a-5p promotes phosphorylation of Smad2 and Smad3. Furthermore, circ_0058792 and miR-181a-5p had opposing effects on Smad7 expression. Collectively, these findings indicate that circ_0058792 regulates osteogenic differentiation by sponging miR-181a-5p via the TGF-β/Smad7 pathway. These findings elucidated the functions of circ_0058792 and miR-181a-5p in the regulation of steroid-induced ONFH. Our findings also indicated that circ_0058792 and miR-181a-5p are possible diagnostic markers and therapeutic targets for treating steroid-induced ONFH.

Differential Diagnosis of Rare Diseases Involving Bilateral Lower Extremities with Similar 99mTc-MDP Bone Scan Patterns: Analysis of a Case Series.

Here, we reported a panel of rare diseases involving bilateral lower extremities with similar imaging patterns on Tc-MDP bone scans. Glucose-6-phosphate dehydrogenase deficiency (G-6PD deficiency), Gaucher disease (GD), steroid-induced osteonecrosis, progressive diaphyseal dysplasia (PDD), Erdheim–Chester disease (ECD) and Langerhans cell sarcoma (LCS) were included and imaging characteristics were analyzed. The rare properties of these diseases and mimicking features on Tc-MDP bone scans rendered differential diagnosis difficult but necessary. We believe that the rarely known imaging features of the reported diseases will undoubtedly help nuclear medicine physicians make differential diagnoses in clinical practice.

Knowledge structure and emerging trends on osteonecrosis of the femoral head: a bibliometric and visualized study

BackgroundOsteonecrosis of the femoral head (ONFH) is a common disabling disease with considerable social and economic impacts. Although extensive studies related to ONFH have been conducted in recent years, a specific bibliometric analysis on this topic has not yet been performed. Our study attempted to summarize the comprehensive knowledge map, development landscape, and future directions of ONFH research with the bibliometric approach.MethodsAll publications concerning ONFH published from 2001 to 2020 were identified from Web of Science Core Collection. Key bibliometric indicators were calculated and evaluated using CiteSpace, VOSviewer, and the online bibliometric analysis platform.ResultsA total of 2594 publications were included. Our analysis revealed a significant exponential growth trend in the annual number of publications over the past 20 years (R2 = 0.9663). China, the USA, and Japan were the major contributors both from the quality and quantity points of view. Correlation analysis indicated that there was a high positive correlation between the number of publications and gross domestic product (r = 0.774), and a moderate positive correlation between publications and demographic factor (r = 0.673). All keywords were categorized into four clusters including Cluster 1 (etiology and risk factors study); Cluster 2 (basic research and stem cell therapy); cluster 3 (hip-preserving study); and Cluster 4 (hip replacement study). Stem cell therapy-related research has been recognized as an important research hotspot in this field. Several topics including exosomes, autophagy, biomarkers, osteogenic differentiation, microRNAs, steroid-induced osteonecrosis, mesenchymal stem cells, double-blind, early-stage osteonecrosis, and asymptomatic osteonecrosis were considered as research focuses in the near future.ConclusionOver the past two decades, increasing attention has been paid to global ONFH-related research. Our bibliometric findings provide valuable information for researchers to understand the basic knowledge structure, identify the current research hotspots, potential collaborators, and future research frontiers in this field.

Hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis.

BackgroundSteroid‐induced osteonecrosis of femoral head (SONFH) is a serious complication of glucocorticoid overused. Recent evidence has demonstrated that circRNAs exert key pathophysiological roles in a variety of disease processes. However, the role of circRNA in SONFH remains largely unknown. The current study sought to evaluate how hsa_circ_0058122 affects SONFH in dexamethasone (DEX) treated human umbilical vein endothelial cells (HUVECs) model.MethodsRT‐PCR was used to demonstrate the hsa_circ_0058122 expression level in Dex‐treated HUVECs cells. The effects of hsa_circ_0058122 on HUVECs apoptosis were evaluated via overexpression plasmid and siRNA. Using dual‐luciferase and fluorescence in situ hybridization assays, we demonstrated that hsa_circ_0058122 binds to miR‐7974 thereby facilitating HUVECs apoptosis. Bioinformatics analysis and western blot were performed to confirm target genes of hsa‐miR‐7974.ResultsIn our previous work, we revealed the top 20 elevated circRNAs in SONFH patients were hsa_circ_0010027, hsa_circ_0058115, hsa_circ_0010026, hsa_circ_0058839, hsa_circ_0056886, hsa_circ_0056885, hsa_circ_0058146, hsa_circ_0058105, hsa_circ_0058112, hsa_circ_0058143, hsa_circ_0058102, hsa_circ_0058090, hsa_circ_0075353, hsa_circ_0058126, hsa_circ_0058130, hsa_circ_0058140, hsa_circ_0058122, hsa_circ_0058123, hsa_circ_0058103, and hsa_circ_0058121. Among these, hsa_circ_0058122 was finally selected for further investigation. We found hsa_circ_0058122 expression was markedly elevated in Dex‐treated HUVECs cells, and the Dex‐mediated HUVEC apoptosis was impaired in hsa_circ_0058122‐silenced cells and increased in hsa_circ_0058122‐overexpressing cells. hsa_circ_0058122 competitively binds to hsa‐miR‐7974, which in turn interacts with insulin‐like growth factor binding protein 5 (IGFBP5).Conclusionshsa_circ_0058122/miR‐7974/IGFBP5 was proposed to be a key regulatory pathway for SONFH. DEX treatment upregulated hsa_circ_0058122 expression in HUVECs, which sponged miR‐7974, thereby increasing IGFBP5 expression, the hsa_circ_0058122/miR‐7974/IGFBP5 axis contributed to the Dex‐mediated apoptosis. These findings may identify novel targets for SONFH molecular therapy.

In vitro and in vivo Repair Effects of the NCF-Col-NHA Aerogel Scaffold Loaded With SOST Monoclonal Antibody and SDF-1 in Steroid-Induced Osteonecrosis.

In the current study, we synthesized nanocellulose (NCF)-collagen (Col)-nano hydroxyapatite (NHA) organic-inorganic hybrid aerogels loaded with stromal cell derived factor-1 (SDF-1) and sclerostin monoclonal antibody (SOST McAb) and investigated their ability to repair steroid-induced osteonecrosis. Rabbit bone marrow mesenchymal stem cells (BMSCs) and human vascular endothelial cells (HUVECs) were used for the in vitro study. A rabbit steroid-induced osteonecrosis model was used for the in vivo study. The best elastic modulus reached 12.95 ± 4.77 MPa with a mean compressive property of 0.4067 ± 0.084 MPa for the scaffold containing 100% mass fraction. The average pore diameter of the aerogel was 75 ± 18 µm with a porosity of more than 90% (96.4 ± 1.6%). The aerogel-loaded SDF-1 and SOST were released at 40–50% from the material within the initial 3 h and maintained a stable release for more than 21 days. The in vitro study showed osteogenesis and vascularization capabilities of the scaffold. The in vivo study showed that rabbits received implantation of the scaffold with SOST McAb and SDF-1 showed the best osteogenesis of the osteonecrosis zone in the femoral head. Imaging examination revealed that most of the necrotic area of the femoral head was repaired. These results suggest that this hybrid aerogel scaffold could be used for future steroid-induced osteonecrosis repair.

CircHGF suppressed cell proliferation and osteogenic differentiation of BMSCs in ONFH via inhibiting miR-25-3p binding to SMAD7

Steroid-induced osteonecrosis of the femoral head (ONFH) is a common and devastating bone disorder, which often results in progressive collapse of the femoral head and subsequent osteoarthritis. The proliferation ability and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) play critical roles in maintaining the structural and functional integrity of the femoral head to prevent ONFH. Until now, little has been known about the underlying mechanism of BMSCs differentiation disorder during ONFH progression. Circular RNAs (circRNAs) are considered to be vital non-coding RNAs functionally involved in various human diseases. However, whether and how circRNA regulates the proliferation and osteogenic differentiation of BMSCs in ONFH remain unclear. In this study, we analyzed the circRNA expression profile of five samples of BMSCs in ONFH and five samples of control by using circRNA microarray assays. We identified 182 differentially expressed circRNAs, among which 108 circRNAs were upregulated. We further investigated the effects of a significantly upregulated circRNA, circHGF, on the proliferation and osteogenic differentiation of BMSCs in vitro. Results showed that circHGF suppressed the proliferation and osteogenic differentiation of BMSCs in ONFH by targeting miR-25-3p/SMAD7 axis. Our findings provided a potential diagnostic and therapeutic strategy for ONFH.

LncRNA FGD5-AS1 Regulates Bone Marrow Stem Cell Proliferation and Apoptosis by Affecting miR-296-5p/STAT3 Axis in Steroid-Induced Osteonecrosis of the Femoral Head.

Background Osteonecrosis of the femoral head (ONFH) is a common hip joint disease, which is more harmful and seriously affects the lives of patients. This study aims to clarify the regulatory mechanism of lncRNA FGD5-AS1 in ONFH. Methods The expression of the protein and mRNA was detected by RT-qPCR and Western blot assay. The regulatory mechanism of lncRNA FGD5-AS1 was detected by the dual-luciferase reporter assay, CCK-8 assay, and flow cytometry assay. Results Dex can inhibit cell proliferation and differentiation and induce apoptosis in hBMSCs in a dose-dependent manner. Overexpression of lncRNA FGD5-AS1 promoted cell proliferation and restrained apoptosis in Dex-treated hBMSCs. In addition, lncRNA FGD5-AS1 acts as a sponge for miR-296-5p. Also, miR-296-5p directly targets STAT3. More importantly, miR-296-5p and STAT3 can affect the function of lncRNA FGD5-AS1 in Dex-treated hBMSCs. Conclusion lncRNA FGD5-AS1 promotes cell proliferation and inhibits apoptosis in steroid-induced ONFH through acting as a sponge for miR-296-5p and upregulation of STAT3.

Identification of potential autophagy-related genes in steroid-induced osteonecrosis of the femoral head via bioinformatics analysis and experimental verification

PurposeSteroid-induced osteonecrosis of the femoral head (SONFH) is a refractory orthopaedic hip joint disease that occurs in young- and middle-aged people. Previous experimental studies have shown that autophagy might be involved in the pathological process of SONFH, but the pathogenesis of autophagy in SONFH remains unclear. We aimed to identify and validate the key potential autophagy-related genes involved in SONFH to further illustrate the mechanism of autophagy in SONFH through bioinformatics analysis.MethodsThe GSE123568 mRNA expression profile dataset, including 10 non-SONFH (following steroid administration) samples and 30 SONFH samples, was downloaded from the Gene Expression Omnibus (GEO) database. Autophagy-related genes were obtained from the Human Autophagy Database (HADb). The autophagy-related genes involved in SONFH were screened by intersecting the GSE123568 dataset with the set of autophagy genes. The differentially expressed autophagy-related genes involved in SONFH were identified with R software. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially expressed autophagy-related genes involved in SONFH were conducted by using R software. Then, the correlations between the expression levels of the differentially expressed autophagy-related genes involved in SONFH were confirmed with R software. Moreover, the protein–protein interaction (PPI) network was analysed by using the Search Tool for the Retrieval of Interacting Genes (STRING), significant gene cluster modules were identified with the MCODE Cytoscape plugin, and hub genes among the differentially expressed autophagy-related genes involved in SONFH were screened by using the CytoHubba Cytoscape plugin. Finally, the expression levels of the hub genes of the differentially expressed autophagy-related genes involved in SONFH were validated in hip articular cartilage specimens from necrotic femur heads (NFHs) by using the GSE74089 dataset and further verification by qRT-PCR.ResultsA total of 34 differentially expressed autophagy-related genes were identified between the peripheral blood samples of SONFH patients and non-SONFH patients based on the defined criteria, including 25 upregulated genes and 9 downregulated genes. The GO and KEGG pathway enrichment analyses revealed that these 34 differentially expressed autophagy-related genes involved in SONFH were particularly enriched in death domain receptors, the FOXO signalling pathway and apoptosis. Correlation analysis revealed significant correlations among the 34 differentially expressed autophagy-related genes involved in SONFH. The PPI results demonstrated that the 34 differentially expressed autophagy-related genes interacted with each other. Ten hub genes were identified by using the MCC algorithms of CytoHubba. The GSE74089 dataset showed that TNFSF10, PTEN and CFLAR were significantly upregulated while BCL2L1 was significantly downregulated in the hip cartilage specimens, which was consistent with the GSE123568 dataset. TNFSF10, PTEN and BCL2L1 were detected with consistent expression by qRT-PCR.ConclusionsThirty-four potential autophagy-related genes involved in SONFH were identified via bioinformatics analysis. TNFSF10, PTEN and BCL2L1 might serve as potential drug targets and biomarkers because they regulate autophagy. These results expand the autophagy-related understanding of SONFH and might be useful in the diagnosis and prognosis of SONFH.

Screening of Potential Biomarkers in the Peripheral Serum for Steroid-Induced Osteonecrosis of the Femoral Head Based on WGCNA and Machine Learning Algorithms

Background Steroid-induced osteonecrosis of the femoral head (SONFH) has produced a substantial burden of medical and social experience. However, the current diagnosis is still limited. Thus, this study is aimed at identifying potential biomarkers in the peripheral serum of patients with SONFH. Methods The expression profile data of SONFH (number: GSE123568) was acquired from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in SONFH were identified and used for weighted gene coexpression network analysis (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to investigate the biological functions. The protein-protein interaction (PPI) network and machine learning algorithms were employed to screen for potential biomarkers. Gene set enrichment analysis (GSEA), transcription factor (TF) enrichment analysis, and competing endogenous RNA (ceRNA) network were used to determine the biological functions and regulatory mechanisms of the potential biomarkers. Results A total of 562 DEGs, including 318 upregulated and 244 downregulated genes, were identified between SONFH and control samples, and 94 target genes were screened based on WGCNA. Moreover, biological function analysis suggested that target genes were mainly involved in erythrocyte differentiation, homeostasis and development, and myeloid cell homeostasis and development. Furthermore, GYPA, TMCC2, and BPGM were identified as potential biomarkers in the peripheral serum of patients with SONFH based on machine learning algorithms and showed good diagnostic values. GSEA revealed that GYPA, TMCC2, and BPGM were mainly involved in immune-related biological processes (BPs) and signaling pathways. Finally, we found that GYPA might be regulated by hsa-miR-3137 and that BPGM might be regulated by hsa-miR-340-3p. Conclusion GYPA, TMCC2, and BPGM are potential biomarkers in the peripheral serum of patients with SONFH and might affect SONFH by regulating erythrocytes and immunity.

Research Progress of Tonifying Kidney and Promoting Blood Circulation in the Treatment of Steroid-induced Necrosis of Femoral Head

Osteonecrosis of the femoral head is a common disability disease of the hip joint in China, and osteonecrosis of the femoral head caused by hormone factors is the most common, which is related to the gradual increase in the utilization rate of glucocorticoids in recent years. It is a refractory disease in orthopaedics with a poor prognosis. For this disease, the treatment of traditional Chinese medicine has certain advantages. In view of this, the author reads, analyzes and summarizes its materials by searching China Journal full-text Database and Wanfang Database. This paper reviews the mechanism and clinical research progress of tonifying kidney and activating blood circulation in the prevention and treatment of steroid-induced osteonecrosis of the femoral head, hoping to provide help for the clinical treatment of hormone-related osteonecrosis of the femoral head.

Identification of key biomarkers in steroid-induced osteonecrosis of the femoral head and their correlation with immune infiltration by bioinformatics analysis

ObjectiveThis study aimed to identify key diagnostic markers and immune infiltration of (SONFH) by bioinformatics analysis.MethodsRelated SONFH datasets were downloaded from the Gene Expression Omnibus (GEO) database. First, we identified the differentially expressed genes (DEGs) and performed the functional enrichment analysis. Then weighted correlation network analysis (WGCNA) and the MCODE plug-in in Cytoscape were used to identify the diagnostic markers of SONFH. Finally, CIBERSORT was used to analyze the immune infiltration between SONFH and healthy controls, and the correlation between infiltrating immune cells and diagnostic markers was analyzed.ResultsTYROBP, TLR2, P2RY13, TLR8, HCK, MNDA, and NCF2 may be key diagnostic markers of SONFH. Immune cell infiltration analysis revealed that Memory B cells and activated dendritic cells may be related to the SONFH process. Moreover, HCK was negatively correlated with CD8 T cells, and neutrophils were positively correlated with those key diagnostic markers.ConclusionsTYROBP, TLR2, P2RY13, TLR8, HCK, MNDA, and NCF2 may be used as diagnostic markers of SONFH, and immune-related mechanism of SONFH and the potential immunotherapy are worthy of further study.

Extracorporeal shockwave relieves endothelial injury and dysfunction in steroid-induced osteonecrosis of the femoral head via miR-135b targeting FOXO1: in vitro and in vivo studies.

Injury and dysfunction of endothelial cells (ECs) are closely related to the pathogenesis of steroid-induced osteonecrosis of the femoral head (ONFH), while MicroRNAs (miRNAs) play an essential role in the processes. Extracorporeal shockwave treatment (ESWT) has been used in the non-invasive treatment of various diseases including musculoskeletal and vascular disorders. In particular, ESWT with low energy levels showed a beneficial effect in ischemic tissues. However, there has been no comprehensive assessment of the effect of ESWT and miRNAs on steroid-induced ONFH. In the present study, we investigated the role and mechanism of ESWT and miRNAs both in vitro and in vivo. Using a steroid-induced ONFH rat model, we found that ESWT significantly enhances proliferation and angiogenesis as well as alleviates apoptosis. In two types of ECs, ESWT can promote cell proliferation and migration, enhance angiogenesis, and inhibit apoptosis. Notably, our study demonstrates that miR-135b is downregulated and modulated forkhead box protein O1 (FOXO1) in ECs treated with dexamethasone. Remarkably, both miR-135b knockdown and FOXO1 overexpression reversed the beneficial effect of ESWT on ECs. Additionally, our data suggest that ESWT activates the FOXO1-related pathway to impact proliferation, apoptosis, and angiogenesis. Taken together, this study indicates that ESWT relieves endothelial injury and dysfunction in steroid-induced ONFH via miR-135b targeting FOXO1.

Construction and validation of steroid-induced rabbit osteonecrosis model

Osteonecrosis is a common orthopedic disease in clinic, resulting in joint collapse if appropriate treatment is not given in time. The clinical usage of high-dose steroid is one of the common causes of osteonecrosis. In several studies, the intravenous injection of steroid with or without lipopolysaccharide is the most commonly used strategy to construct osteonecrosis animal model. However, the injection dose, frequency, and interval of steroid and validation of successful model construction lack generally accepted protocol, and the survival and model formation rates are unsatisfactory. We have optimized the construction protocol of osteonecrosis animal model based on the previously reported ones and established a mature animal model of osteonecrosis for future studies.•A rabbit model of osteonecrosis was constructed by multiple injections of high-dose methylprednisolone.•The multidisciplinary biomedical examinations demonstrated the successful construction of osteonecrosis model in the rabbit.

IL-34 Aggravates Steroid-Induced Osteonecrosis of the Femoral Head via Promoting Osteoclast Differentiation

IL-34 can promote osteoclast differentiation and activation, which may contribute to steroid-induced osteonecrosis of the femoral head (ONFH). Animal model was constructed in both BALB/c and IL-34 deficient mice to detect the relative expression of inflammation cytokines. Micro-CT was utilized to reveal the internal structure. In vitro differentiated osteoclast was induced by culturing bone marrow-derived macrophages with IL-34 conditioned medium or M-CSF. The relative expression of pro-inflammation cytokines, osteoclast marker genes, and relevant pathways molecules was detected with quantitative real-time RT-PCR, ELISA, and Western blot. Up-regulated IL-34 expression could be detected in the serum of ONFH patients and femoral heads of ONFH mice. IL-34 deficient mice showed the resistance to ONFH induction with the up-regulated trabecular number, trabecular thickness, bone value fraction, and down-regulated trabecular separation. On the other hand, inflammatory cytokines, such as TNF-α, IFN-γ, IL-6, IL-12, IL-2, and IL-17A, showed diminished expression in IL-34 deficient ONFH induced mice. IL-34 alone or works in coordination with M-CSF to promote osteoclastogenesis and activate ERK, STAT3, and non-canonical NF-κB pathways. These data demonstrate that IL-34 can promote the differentiation of osteoclast through ERK, STAT3, and non-canonical NF-κB pathways to aggravate steroid-induced ONFH, and IL-34 can be considered as a treatment target.

Bone Regeneration and Angiogenesis by Co-transplantation of Angiotensin II–Pretreated Mesenchymal Stem Cells and Endothelial Cells in Early Steroid-Induced Osteonecrosis of the Femoral Head

Mesenchymal stem cells (MSCs) have been shown to exert a positive impact on osteonecrosis of the femoral head (ONFH) in preclinical experiments and clinical trials. After the femoral head suffers avascular necrosis, the transplanted MSCs undergo a great deal of stress-induced apoptosis and senescence in this microenvironment. So, survival and differentiation of MSCs in osteonecrotic areas are especially important in ONFH. Although MSCs and endothelial cells (ECs) co-culture enhancing proliferation and osteogenic differentiation of MSCs and form more mature vasculature in vivo, it remains unknown whether the co-culture cells are able to repair ONFH. In this study, we explored the roles and mechanisms of co-transplantation of angiotensin II (Ang II)-MSCs and ECs in repairing early ONFH. In vitro, when MSCs and ECs were co-cultured in a ratio of 5:1, both types of cells managed to proliferate and induce both osteogenesis and angiogenesis. Then, we established a rabbit model of steroid-induced ONFH and co-transplantation of Ang II-MSCs and ECs through the tunnel of core decompression. Four weeks later, histological and Western blot analyses revealed that ONFH treated with Ang II-MSCs and ECs may promote ossification and revascularization by increasing the expression of collagen type I, runt-related transcription factor 2, osteocalcin, and vascular endothelial growth factor in the femoral head. Our data suggest that co-transplantation of Ang II-MSCs and ECs was able to rescue the early steroid-induced ONFH via promoting osteogenesis and angiogenesis, which may be regarded as a novel therapy for the treatment of ONFH in a clinical setting.