Glucocorticoid-induced Osteonecrosis Of Femoral Head Research Articles

SOST/Sclerostin impairs the osteogenesis and angiogesis in glucocorticoid-associated osteonecrosis of femoral head.

Glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH) is a progressive bone disorder which frequently results in femoral head collapse and hip joint dysfunction. Sclerostin (SOST) is principally secreted by osteocytes in bone and plays an important role in bone homeostasis and homeostasis of skeletal integrity. Our previous study reported that short-term use of glucocorticoid increased serum sclerostin levels. Here this study is aimed to identify whether sclerostin played an essential role in the occurrence and development of GA-ONFH. Glucocorticoid-induced osteonecrosis of femoral head (ARCO stage II) samples were collected and sclerostin staining was conducted. Osteocyte cell line Ocy454, MC3T3-E1 and endothelial cells was used. MC3T3-E1 or endothelial cells were co-cultured with Ocy454 or SOST-silencing Ocy454 in presence of dexamethasone to mimic the crosstalk of various cells in the bone niche. GA-ONFH rat model and SOST knockout model was built to better understand the phenomenon in vivo. Sclerostin was highly concentrated in osteonecrosis patient sample in the necrotic area. Co-culture with osteocytes aggravated the inhibition of dexamethasone on MC3T3-E1 and endothelial cells. Sclerostin derived from osteocytes impaired osteogenesis and angiogenesis via inhibiting the Wnt pathway. In GA-ONFH rat model, SOST knockout ameliorated the incidence of osteonecrosis and improved bone metabolism compared with the wild type group through histological, immunohistochemical and bone metabolic analyses. Sclerostin contribute to pathologic process of GA-ONFH by impairing osteogenesis and angiogenesis.

β-catenin inhibition disrupts the homeostasis of osteogenic/adipogenic differentiation leading to the development of glucocorticoid-induced osteonecrosis of the femoral head.

Glucocorticoid-induced osteonecrosis of the femoral head (GONFH) is a common refractory joint disease characterized by bone damage and the collapse of femoral head structure. However, the exact pathological mechanisms of GONFH remain unknown. Here, we observed abnormal osteogenesis and adipogenesis associated with decreased β-catenin in the necrotic femoral head of GONFH patients. In vivo and in vitro studies further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation of bone marrow mesenchymal cells (BMSCs) by inhibiting β-catenin signaling in glucocorticoid-induced GONFH rats. Col2+ lineage largely contributes to BMSCs and was found an osteogenic commitment in the femoral head through 9 mo of lineage trace. Specific deletion of β-catenin gene (Ctnnb1) in Col2+ cells shifted their commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice. Overall, we uncover that β-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of GONFH and identify an ideal genetic-modified mouse model of GONFH.

Exosomes from bone marrow mesenchymal stem cells ameliorate glucocorticoid-induced osteonecrosis of femoral head by transferring microRNA-210 into bone microvascular endothelial cells

ObjectivesBone microvascular endothelial cells (BMECs) played an important role in the pathogenesis of glucocorticoid-induced osteonecrosis of femoral head (GCS-ONFH), and exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) may provide an effective treatment. This study aimed to evaluate the effects of BMSC-Exos and internal microRNA-210-3p (miRNA-210) on GCS-ONFH in an in vitro hydrocortisone-induced BMECs injury model and an in vivo rat GCS-ONFH model.MethodsBMECs, BMSCs and BMSC-Exos were isolated and validated. BMECs after the treatment of hydrocortisone were cocultured with different concentrations of BMSC-Exos, then proliferation, migration, apoptosis and angiogenesis of BMECs were evaluated by CCK-8, Annexin V-FITC/PI, cell scratch and tube formation assays. BMSCs were transfected with miRNA-210 mimics and miRNA-210 inhibitors, then BMSC-ExosmiRNA−210 mimic and BMSC-ExosmiRNA−210 inhibitor secreted from such cells were collected. The differences between BMSC-Exos, BMSC-ExosmiRNA−210 mimic and BMSC-ExosmiRNA−210 inhibitor in protecting BMECs against GCS treatment were analyzed by methods mentioned above. Intramuscular injections of methylprednisolone were performed on Sprague–Dawley rats to establish an animal model of GCS-ONFH, then tail intravenous injections of BMSC-Exos, BMSC-ExosmiRNA−210 mimic or BMSC-ExosmiRNA−210 inhibitor were conducted after methylprednisolone injection. Histological and immunofluorescence staining and micro-CT were performed to evaluate the effects of BMSC-Exos and internal miRNA-210 on the in vivo GCS-ONFH model.ResultsDifferent concentrations of BMSC-Exos, especially high concentration of BMSC-Exos, could enhance the proliferation, migration and angiogenesis ability and reduce the apoptosis rates of BMECs treated with GCS. Compared with BMSC-Exos, BMSC-ExosmiRNA−210 mimic could further enhance the proliferation, migration and angiogenesis ability and reduce the apoptosis rates of BMECs, while BMECs in the GCS + BMSC-ExosmiRNA−210 inhibitor group showed reduced proliferation, migration and angiogenesis ability and higher apoptosis rates. In the rat GCS-ONFH model, BMSC-Exos, especially BMSC-ExosmiRNA−210 mimic, could increase microvascular density and enhance bone remodeling of femoral heads.ConclusionsBMSC-Exos containing miRNA-210 could serve as potential therapeutics for protecting BMECs and ameliorating the progression of GCS-ONFH.

Activation of cannabinoid receptor 2 alleviates glucocorticoid-induced osteonecrosis of femoral head with osteogenesis and maintenance of blood supply

In glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH), downregulated osteogenic ability and damaged blood supply are two key pathogenic mechanisms. Studies suggested that cannabinoid receptor 2 (CB2) is expressed in bone tissue and it plays a positive role in osteogenesis. However, whether CB2 could enhance bone formation and blood supply in GC-induced ONFH remains unknown. In this study, we focused on the effect of CB2 in GC-induced ONFH and possible mechanisms in vitro and in vivo. By using GC-induced ONFH rat model, rat-bone mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) to address the interaction of CB2 in vitro and in vivo, we evaluate the osteogenic and angiogenic effect variation and possible mechanisms. Micro-CT, histological staining, angiography, calcein labeling, Alizarin red staining (ARS), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) staining, TUNEL staining, migration assay, scratch assay, and tube formation were applied in this study. Our results showed that selective activation of CB2 alleviates GC-induced ONFH. The activation of CB2 strengthened the osteogenic activity of BMSCs under the influence of GCs by promotion of GSK-3β/β-catenin signaling pathway. Furthermore, CB2 promoted HUVECs migration and tube-forming capacities. Our findings indicated that CB2 may serve as a rational new treatment strategy against GC-induced ONFH by osteogenesis activation and maintenance of blood supply.

Protective Effect of Human Umbilical Cord Mesenchymal Stem Cells in Glucocorticoid-induced Osteonecrosis of Femoral Head.

To evaluate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on preventing rats from glucocorticoid-induced osteonecrosis of femoral head (GCONFH) in the early stage in vivo and to investigate the possible mechanism of hUC-MSCs in regulating the balance of osteogenesis and adipogenesis. All rats were randomly divided into 3 groups: control group (C group), model group (M group), and intervention group (I group). The model of GC-ONFH was developed by a sequential administration of lipopolysaccharide and methylprednisolone. The rats in the I group were treated with caudal vein injection of hUC-MSCs. Six weeks later, the blood samples were obtained to measure the activity of alkaline phosphatase (ALP) and the content of triglyceride (TG) in serum, and the femoral heads were harvested and observed by hematoxylin-eosin staining, Micro-CT, Western blot and real-time quantitative polymerase chain reaction. After intervention of hUC-MSCs, the necrosis rate of femoral head decreased from 83% (10/12) to 33% (4/12), the rate of empty bone lacuna was significantly decreased, the activity of ALP increased significantly, the content of TG decreased significantly, the bone density increased obviously, the expression of RUNX2 and Col I increased significantly and the expression of PPARγ decreased significantly. These results revealed that caudal vein injection of hUC-MSCs can effectively reduce the incidence of GC-ONFH in rats by increasing ALP activity and reducing TG content in serum, increasing bone mineral density, promoting the expression of RUNX2 and Col I, and inhibiting the expression of PPARγ.

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.

Lithium Modified Mesoporous Hydroxyapatite Nanoparticles Mediate the Polarization of M2 Macrophages to Promote Bone Defect Repair in Glucocorticoid-Induced Osteonecrosis of the Femoral Head

Glucocorticoid (GC)-induced osteonecrosis of femoral head(GI-ONFH) is a chronic inflammatory disease characterized by abnormal activation of macrophages and infiltration of necrotic sites. However, previous studies on osteonecrosis repair have neglected the immunomodulatory effect of bone, which leads to the failure of bone repair. Therefore, in this study, we produced an immunomodulatory bone repair material with lithium (Li)-modified mesoporous hydroxyapatite nanoparticles (Li-mHANP) that has the potential to enhance bone regeneration by manipulating macrophage (M) polarization to create an immunomodulatory microenvironment that promotes bone healing in GIONFH. In order to investigate whether Li incorporation into mHANP can synergistically improve bone formation and repair osteonecrosis by changing the polarization of macrophages, we evaluated Li-mHANP and macrophages in the process of GIONFH bone regeneration through in vitro and in vivo biological tests. We found that, compared with mHANP, Li-mHANP activated the JAK1/STAT6/STAT3 signaling pathway and promoted the transformation of macrophages to M2 phenotype. In addition, Li-mHANP-treated macrophages promote osteogenesis of BMSCs and endothelial cell angiogenesis in vitro. After Li-mHANP nanoparticles were implanted into the model of GIONFH, we found that Li-mHANP mediated the transformation of pro-inflammatory M1 macrophages into pro-tissue repair M2 phenotype, restored the M2/M1 ratio to normal levels, effectively alleviated inflammation, and ultimately promoted osteoblast differentiation and bone regeneration, compared with mHANP without Li modified. In conclusion, these findings suggest that Li-mHANP is an immunomodulatory material that can direct immunoregulatory bone repair related to tissue repair and regeneration and promote bone regeneration of bone defect in the condition of GIONFH. Funding Information: This study was supported by The National Natural Science Foundation of China (grant no. 81974333) and Science and Technology Program of Chengdu (grant no.2019-YF05-01188-SN). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: All animal operations were approved by the Animal Research Committee of West China Hospital of Sichuan University (Chengdu, China).

Potential roles of extracellular vesicles in osteonecrosis of femoral head: A systematic review.

Extracellular vesicles (EVs), have been utilised for the diagnosis and treatment of osteonecrosis of femoral head (ONFH). Hence, we aimed to review the available evidence on the potential roles of EVs in ONFH systematically. The PubMed, EMBASE, and Science Citation Index databases were searched exhaustively from inception to June 2020. All in vitro and in vivo studies on the use of EVs in ONFH diagnosis and treatment were included. The final analysis included 14 studies. These studies were categorised according to the cell source, target cell types, secreted product types, production method, storage, and study design. The animal model, together with the ONFH induction method, used in the in vivo studies was also considered. For in vivo studies, effects on cellular proliferation and apoptosis and bone and vascular tissues and biomarkers for ONFH diagnosis were assessed, whereas in the in vitro studies, effects on EVs internalization; proliferation, viability, and migration; osteogenic and adipogenic differentiation; apoptosis; and angiogenesis were evaluated. Both in vitro and in vivo studies showed promoting effects of EVs on proliferation, osteogenic differentiation, and angiogenesis and inhibitory effects on apoptosis and adipogenic differentiation in both glucocorticoid-induced and traumatic ONFH models. Additionally, EVs in blood showed diagnostic potential for ONFH. This systemic review provides a deeper understanding of the role of EVs, thereby facilitating the development of novel therapeutic strategies.

Administration of mircoRNA-135b-reinforced exosomes derived from MSCs ameliorates glucocorticoid-induced osteonecrosis of femoral head (ONFH) in rats.

Exosomes were found to exert a therapeutic effect in the treatment of osteonecrosis of the femoral head (ONFH), while miR‐135b was shown to play an important role in the development of ONFH. In this study, we investigated the effects of concomitant administration of exosomes and miR‐135b on the treatment of ONFH. A rat mode of ONFH was established. TEM, Western blotting and nanoparticle analysis were used to characterize the exosomes collected from human‐induced pluripotent stem cell–derived mesenchymal stem cells (hiPS‐MSC‐Exos). Micro‐CT was used to observe the trabecular bone structure of the femoral head. Real‐time PCR, Western blot analysis, IHC assay, TUNEL assay, MTT assay and flow cytometry were performed to detect the effect of hiPS‐MSC‐Exos and miR‐135b on cell apoptosis and the expression of PDCD4/caspase‐3/OCN. Moreover, computational analysis and luciferase assay were conducted to identify the regulatory relationship between PDCD4 mRNA and miR‐135b. The hiPS‐MSC‐Exos collected in this study displayed a spheroidal morphology with sizes ranging from 20 to 100 nm and a mean concentration of 1 × 1012 particles/mL. During the treatment of ONFH, the administration of hiPS‐MSC‐Exos and miR‐135b alleviated the magnitude of bone loss. Furthermore, the treatment of MG‐63 and U‐2 cells with hiPS‐MSC‐Exos and miR‐135b could promote cell proliferation and inhibit cell apoptosis. Moreover, PDCD4 mRNA was identified as a virtual target gene of miR‐135b. HiPS‐MSC‐Exos exerted positive effects during the treatment of ONFH, and the administration of miR‐135b could reinforce the effect of hiPS‐MSC‐Exos by inhibiting the expression of PDCD4.

CircUSP45 inhibited osteogenesis in glucocorticoid-induced osteonecrosis of femoral head by sponging miR-127-5p through PTEN/AKT signal pathway: Experimental studies

Purposelarge doses of glucocorticoids (GCs) are the most common cause of glucocorticoid-induced osteonecrosis of femoral head (GIONFH). Although awareness of GIONFH among patients with GCs history has increased over recent years, several studies indicate that its mechanism remains unclear. MethodsTo evaluate the function of circUSP45 in GIONFH, femoral heads in GIONFH patients or femoral heads in fracture patients were collected. In vitro, RT-PCR, FISH, RNA pull down and Western blotting assay were used to evaluate the function of circUSP45. In addition, we also verified the effects of circUSP45 on osteogenesis using alizarin red staining. In vivo, we used HE staining and microCT analysis to evaluate the bone mass. Moreover, the mechanism of circUSP45 regulating osteogenesis through the miR-127-5p/PTEN/AKT pathway was also investigated. ResultsThe results showed that expression of circUSP45 increased in GIONFH patients. The overexpression of circUSP45 decreases osteogenic gene expression and inhibits the proliferation of BMSCs. Furthermore, circUSP45 was located mainly in the cytoplasm and directly interacted with miR-127-5p. MiR-127-5p acts with its targets PTEN to regulate the osteogenesis. MicroCT and HE staining verify the function of circUSP45 in GIONFH rat model. ConclusionCircUSP45 decreases osteogenesis in bone GIONFH by sponging miR-127-5p through PTEN/AKT signal pathway.

Prevention for glucocorticoid-induced osteonecrosis of femoral head: a long-term clinical follow-up trail

Objective: To evaluated the outcome of prevention and treatment for glucocorticoid-induced osteonecrosis of femoral head with anticoagulant and vasodilator drugs. Methods: A prospective, randomized, double-blind study was performed. From August 2003 to August 2006, 58 patients with large amounts of hormone therapy in the Zhongshan Hospital Affiliated Dalian University were enrolled and randomly assigned to the control group (placebo) or preventive group (anticoagulant and vasodilator drugs). And we prospectively analyzed the clinical outcomes of 24 patients with glucocorticoid-induced osteonecrosis of femoral head early stage (treatment group)treated by anticoagulant and vasodilator drugsat the same time. Disease incidence rate and progression were evaluated by radiography and magnetic resonance imaging (MRI), Follow-up of patients with femoral head survival curve was drawn. The Harris Hip Score and the Short Form 36 health survey were used to rate hip function and quality of life, respectively. Results: Thus, a total of 80 patients were assessed in this study, 24 cases in control group[follow up from 7.5 to 13.0(10.7±1.6)years], 22 cases in preventive group and 24 cases in treatment group. There was significant difference in theincidence rate of Osteonecrosis of femoral head, survive rate of femoral head and HHS score between the control groupand preventive group(41.7% vs 13.6%, 66.7% vs 70.8% , P<0.01). Conclusion: Anticoagulant and vasodilator drugs could effect on preventing theglucocorticoid-induced osteonecrosis of femoral head, reducing disease progression, or improving life quality.

The new concept of prevention and treatment for Glucocorticoid-induced osteonecrosis of femoral head

The new concept of prevention and treatment for Glucocorticoid-induced osteonecrosis of femoral head