miR-126 mitigates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting the ERK1/2 and Bcl-2 pathways.

Human bone marrow mesenchymal stem cells (hBMMSCs) are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The objective of the present study is to assess microRNA-126 (miR-126) and examine its effects on the osteogenic differentiation of hBMMSCs. In this study, we investigate the role of miR-126 in the progression of osteogenic differentiation (OD) as well as the apoptosis and inflammation of hBMMSCs during OD induction. OD is induced in hBMMSCs, and matrix mineralization along with other OD-associated markers are evaluated by Alizarin Red S (AR) staining and quantitative PCR (qPCR). Gain- and loss-of-function studies are performed to demonstrate the role of miR-126 in the OD of hBMMSCs. Flow cytometry and qPCR-based cytokine expression studies are performed to investigate the effect of miR-126 on the apoptosis and inflammation of hBMMSCs. The results indicate that miR-126 expression is downregulated during the OD of hBMMSCs. Gain- and loss-of function assays reveal that miR-126 upregulation inhibits the differentiation of hBMMSCs into osteoblasts, whereas the downregulation of miR-126 promotes hBMMSC differentiation, as assessed by the determination of osteogenic genes and alkaline phosphatase activity. Furthermore, the miR-126 level is positively correlated with the production of inflammatory cytokines and apoptotic cell death. Additionally, our results suggest that miR-126 negatively regulates not only B-cell lymphoma 2 (Bcl-2) expression but also the phosphorylation of extracellular signal­regulated protein kinase (ERK) 1/2. Moreover, restoring ERK1/2 activity and upregulating Bcl-2 expression counteract the miR-126-mediated suppression of OD in hBMMSCs by promoting inflammation and apoptosis, respectively. Overall, our findings suggest a novel molecular mechanism relevant to the differentiation of hBMMSCs into osteoblasts, which can potentially facilitate bone formation by counteracting miR-126-mediated suppression of ERK1/2 activity and Bcl-2 expression.

Avascular necrosis after femoral neck fracture in children and adolescents: poor prognosis and risk factors.

INTRODUCTION: Avascular necrosis (AVN) after femoral neck fracture (FNF) is a rare and severe paediatric condition, but only few studies described its prognosis and risk factors. The present study aimed to evaluate the outcomes and independent factors for poor prognosis of AVN after FNF in children and adolescents. METHOD: This retrospective study included children and adolescents with AVN after FNF who received conservative treatment (CT group) or non-vascularized bone grafting (NVBG group) between 2000 and 2018. The primary outcomes were the risk of hip arthritis (Tönnis grade) and hip deformity risk (Stulberg classification). All patients were followed for at least two years to assess AVN progression. RESULTS: Study included 81 patients. In the CT group, 23/43 patients (53.4%) developed hip arthritis, and 24/43 patients (55.8%) showed hip deformity. In the NVBG group, 23/38 patients (60.5%) developed hip arthritis, and 34/38 patients (89.5%) had a hip deformity. The multivariable analysis indicated that NVBG surgery had no significant effect on the outcomes. Post-treatment femoral head collapse (P = 0.05, OR = 3.80, 95% CI = 1.01-14.29) and post-treatment hip subluxation (P = 0.01, OR = 2.85, 95% CI = 2.31-129.56) were independent risk factors for severe hip arthritis. Post-treatment femoral head collapse (P < 0.01, OR = 7.64, 95% CI = 3.23-18.04) and pre-treatment hip subluxation (P = 0.02, OR = 7.33, 95% CI = 1.44-37.41) were independent risk factors for severe hip deformity. CONCLUSION: Neither CT nor NVBG have demonstrated superiority regarding long-term outcomes in patients with AVN after FNF. Upon the disease progression to severe collapse with subluxation and severe arthritis, further hip preservation attempts could be futile.

Cajaninstilbene acid inhibits osteoporosis through suppressing osteoclast formation and RANKL-induced signaling pathways.

Osteoporosis is a form of osteolytic disease caused by an imbalance in bone homeostasis, with reductions in osteoblast bone formation, and augmented osteoclast formation and resorption resulting in reduced bone mass. Cajaninstilbene acid (CSA) is a natural compound derived from pigeon pea leaves. CSA possesses beneficial properties as an anti-inflammatory, antibacterial, antihepatitis, and anticancer agent; however, its potential to modulate bone homeostasis and osteoporosis has not been studied. We observed that CSA has the ability to suppress RANKL-mediated osteoclastogenesis, osteoclast marker gene expression, and bone resorption in a dose-dependent manner. Mechanistically, it was revealed that CSA attenuates RANKL-activated NF-κB and nuclear factor of activated T-cell pathways and inhibited phosphorylation of key signaling mediators c-Fos, V-ATPase-d2, and ERK. Moreover, in osteoclasts, CSA blocked RANKL-induced ROS activity as well as calcium oscillations. We further evaluated the therapeutic effect of CSA in a preclinical mouse model and showed that in vivo treatment of ovariectomized C57BL/6 mice with CSA protects the mice from osteoporotic bone loss. Thus, this study demonstrates that osteolytic bone diseases can potentially be treated by CSA.

The Value of the Frog Lateral View Radiograph for Detecting Collapse of Femur Head Necrosis: A Retrospective Study of 1001 Cases.

Aims: The collapse in femur head necrosis is generally detected by CT or MRI which are not primary routine examination at every follow-up in developing countries. The purpose of this study was to verify the reliability of the frog lateral view radiograph in detecting the collapse of femoral head. Methods: We retrospectively included 1001 hips of 620 patients with femur head necrosis. The anteroposterior view and frog lateral view of X-ray standard radiographs, CT and MRI of patients were collected and simultaneously evaluated by three orthopedists to evaluate the condition of collapse according to the unified standard. The inter-observer reliability of each view of X-ray for detecting the collapse were analyzed through the weighted Cohen's kappa index. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of each evaluation method were also calculated. Results: A moderate or substantial reliability was indicated in the evaluation of frog lateral view radiograph, whereas the anteroposterior view only showed fair or poor reliability. Using the CT or MRI results of collapse as the gold standard, the frog lateral view indicated higher sensitivity and accuracy than the anteroposterior view (sensitivity: 82.8 vs. 64.9%; accuracy: 87.1 vs. 73.9%). The combination of the anteroposterior view and frog lateral view indicated higher reliability than individual views. Conclusion: The frog lateral view radiograph has higher sensitivity and accuracy than anteroposterior view. It is a complementary method to AP view for detecting the collapse in femur head necrosis during the follow-up, which has moderate or substantial inter-observer reliability.

SOCS1, the feedback regulator of STAT1/3, inhibits the osteogenic differentiation of rat bone marrow mesenchymal stem cells.

Our study showed that Signal transducer and activator of transcription (STAT)1 and STAT3 phosphorylation was firstly upregulated in the early stage of osteogenic differentiation (OD), and quickly eliminated in hours. Following with phosphorylation of STAT1/3, its downstream feedback regulator Suppressor of cytokine signaling 1 (SOCS1) protein also underwent a quick elevation. Further activation and deactivation of STAT1/3, by administrated with Colivelin and Nifuroxazide in Bone mesenchymal stem cells (BMSCs), increased and decreased SOCS1 expression, inhibited and promoted OD of BMSCs, respectively, as evidenced by Alizarin staining, alkaline phosphatase (ALP) activity, and determination of Run-related transcription factor 2 (RUNX2), Osteocalcin (OCN), ALP, and Bone sialoprotein (BSP). In addition, administration of Colivelin and Nifuroxazide caused and blocked inflammation and apoptosis of BMSCs. To further elucidate the role of STAT1/3-SOCS1 regulatory loop on OD of BMSCs, we overexpressed or silenced SOCS1 in BMSCs during OD. WB data showed that overexpression of SOCS1 repressed STAT1/3 phosphorylation, and knockdown of SOCS1 increased the phosphorylated STAT1/3. Further mechanism study showed that OD of BMSCs was elevated or reduced by SOCS1 overexpression or knockdown, respectively. The findings presenting indicated that the STAT1/3-SOCS1 axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.

MiRNA-320a-5p contributes to the homeostasis of osteogenesis and adipogenesis in bone marrow mesenchymal stem cell.

Objective: A number of miRNAs and their targets were dragged in the differentiation of bone marrow mesenchymal stem cells (BMSCs). We aimed to elaborate the underlying molecular mechanisms of miRNA-320a in the osteoblast and adipocyte differentiation. Methods: Trauma-induced osteonecrosis of the femoral head (TIONFH) and normal control samples (n = 10 for each group) were collected, followed by miRNA chip analysis to identify the differentially expressed miRNAs. H&E staining was used to observe the pathological development of TIONFH. Lentiviral vector was used for overexpression and inhibition of miRNA-320a in vitro. Quantitative real-time PCR (qPCR), Western blotting and immunohistochemistry staining were employed to determine the expression of interested genes at mRNA or protein level. Luciferase report assay was employed to determine the binding of miRNA-320a and RUNX2. Alkaline phosphatase (ALP) and Alizarin red staining were performed to observe the osteogenesis and Oil red O staining were conducted to visualize the adipogenesis. Results: Expression of miRNA-320a was up-regulated while RUNX2 expression was down-regulated in TIONFH than Normal control. Luciferase report assay confirmed that miRNA-320a directly targeted to the 3'UTR of RUNX2. miRNA-320a overexpression significantly declined the expressions of osteogenesis-related markers: RUNX2, OSTERIX, Collagen I, Osteocalcin and Osteopontin. ALP and Alizarin red staining confirmed the inhibition function of miRNA-320a in osteogenesis of BMSCs. miRNA-320a inhibition significantly decreased the expression of adipogenesis-related markers: AP2, C/EBPα, FABP4 and PPARγ. Oil Red O staining confirmed the miRNA-320a inhibition reduced adipogenesis of BMSCs. Conclusions: miRNA-320a inhibits osteoblast differentiation via targeting RUNX2 and promotes adipocyte differentiation of BMSCs.

Predicting Collapse in Osteonecrosis of the Femoral Head Using a New Method: Preserved Angles of Anterior and Lateral Femoral Head.

BACKGROUND: Femoral head collapse (FHC) is associated with a poor prognosis in osteonecrosis of the femoral head (ONFH). Preserved angles (PAs), including the lateral preserved angle (LPA), the anterior preserved angle (APA) and the combined preserved angle (CPA), can be used to quantify the extent of femoral head necrosis and predict the risk of femoral head collapse. The purpose of this retrospective cohort study was to assess the efficacy of these preserved angles in the prediction of femoral head collapse using plain radiographs. METHODS: Patients with ONFH treated conservatively between January 2010 and January 2019 were analyzed retrospectively to assess the risk of FHC. A logistic regression model was used to evaluate the independent prognostic factors associated with FHC, including age, sex, etiology, onset of symptom, The Japanese Investigation Committee classification, and PAs (LPA, APA, and CPA). RESULTS: A total of 137 patients, with 180 hips, had follow-up of at least two years and were included. During the follow-up period, FHC occurred in 89 hips (49.44%) after the initial diagnosis. Multivariable analysis indicated that CPA (odds ratio [OR] = 0.95; 95%CI = 0.93-0.97; P < 0.01) was a stronger predictor of femoral head collapse compared with the Japanese Investigation Committee classification (OR = 2.40, 95%CI = 0.92-6.25; P > 0.01). The receiver operating characteristic and survival curve analyses revealed that the predictive cutoff point for the CPA was 118.7° (sensitivity = 96.70%, specificity = 79.78%, log-rank test: P < 0.01). CONCLUSIONS: Assessment of preserved angles on plain radiographs is a simple method to quantify the extent of lateral and anterior necrosis of the femoral head. Specifically, CPA has a potential value in predicting femoral head collapse.

Long noncoding RNA XIST modulates microRNA-135/CREB1 axis to influence osteogenic differentiation of osteoblast-like cells in mice with tibial fracture healing.

Fracture healing is a complex event with the involvement of many cell systems, cytokines, as well as mRNAs. Herein, we report the interactions among long noncoding RNA X-inactive specific transcript (XIST)/microRNA-135 (miR-135)/cAMP response element-binding protein 1 (CREB1) axis during fracture healing. We observed increased expression of XIST in patients with long-term unhealed fracture by microarray analysis. Subsequently, a mouse model with tibial fracture and a cell model using osteoblast-like MC3T3-E1 cells were generated. The XIST overexpression during fracture healing decreased proliferation and differentiation of MC3T3-E1 cells, while silencing of XIST facilitated MC3T3-E1 cell growth. Furthermore, miR-135 targeted CREB1 and negatively regulated its expression. XIST acted as a sponge for miR-135, thereby upregulating CREB1 and promoting the activity of the TNF-α/RANKL pathway. Transfection of miR-135 inhibitor or CREB1 overexpression blocked the stimulating effects of XIST knockdown on MC3T3-E1 cell growth. Besides, specific inhibitors of the TNF-α/RANKL pathway reversed the repressive role of XIST in cell osteogenic differentiation. All in all, these findings suggest that XIST knockdown induces the differentiation of osteoblast-like cells via regulation of the miR-135/CREB1/TNF-α/RANKL axis. XIST, as a consequence, represents an attractive therapeutic strategy to accelerate fracture healing.

CircRNA_25487 inhibits bone repair in trauma-induced osteonecrosis of femoral head by sponging miR-134-3p through p21.

We aimed to identify specific circular RNAs (circRNAs) involved in bone repair of trauma-induced osteonecrosis of femoral head (TIONFH) and to explore the potential mechanism. CircRNA sequencing on the blood sample collected from patients with and without TIONFH was performed to select cirRNAs that were significantly differentially expressed, followed by qRT-PCR confirmation. Furthermore, the functions of one selected circRNA and the potential mechanisms in bone repair of TIONFH were validated based on the bone marrow mesenchymal stem cells (BMSCs) and osteoclast-like cells (OLCs) through CCK-8, flow cytometry, transwell assay, luciferase reporter assay, and western blot. A total of 234 upregulated and 148 downregulated differentially expressed circRNAs were identified, and qRT-PCR showed that circRNA_25487 was significantly upregulated in the peripheral blood of TIONFH patients. Luciferase reporter assay confirmed the binding effect between miR-134-3p and circRNA_25487. CircRNA_25487 suppression and miR-134-3p overexpression could promote cell proliferation and invasion while inhibited apoptosis of BMSCs and OLCs. miR-134-3p could target p21. CircRNA_25487 inhibited bone repair in TIONFH by sponging miR-134-3p to upregulate the expression of p21.

Inhibition of miR-93-5p promotes osteogenic differentiation in a rabbit model of trauma-induced osteonecrosis of the femoral head.

Trauma-induced osteonecrosis of the femoral head (TIONFH) is characterized by femoral head collapse accompanied by degenerative changes of the hip. We previously reported that miR-93-5p expression is abnormally high in patients with TIONFH, but the role of miR-93-5p in the TIONFH process remains unclear. Herein, we investigated the role of miR-93-5p in TIONFH in a rabbit model. Bone marrow mesenchymal stem cells (BMSCs) were used for both in vivo and in vitro experiments. A rabbit model of TIONFH was injected with BMSCs transfected with miR-93-5p inhibitor. In addition, both an miR-93-5p mimic and negative control were transfected into BMSCs. Expression of miR-93-5p was significantly increased in the model group compared with control samples. An miR-93-5p inhibitor induced the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase. Furthermore, expression of osteogenesis-related markers (BMP-2, secreted phosphoprotein 1, RUNX family transcription factor 2 and Osterix) was higher in the miR-93-5p inhibitor group, as revealed by quantitative PCR and western blotting. In addition, in vitro experimentation revealed that an miR-93-5p mimic decreased BMP-2 and TNF receptor superfamily member 11b expression, but increased receptor activator of nuclear factor-kappaB ligand expression. In summary, the miR-93-5p inhibitor could promote osteogenic differentiation by increasing BMP-2 expression during the development of TIONFH. Thus, miR-93-5p may have potential as a therapeutic target for TIONF treatment.

A novel RANKL-targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species.

BACKGROUND AND PURPOSE: Osteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti-inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms. METHODS: The docking pose of Rob and RANKL was identified by protein-ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor-κB (NF-κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL-induced ROS generation in osteoclasts was detected by H2 DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real-time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen-deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo. RESULTS: Computational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL-mediated osteoclastogenesis dose-dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL-induced mitochondrial ROS production or enhanced activities of ROS-scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL-induced mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c-Fos, and Mmp9) and reducing Ca2+ oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function. CONCLUSIONS: Rob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug.

An emerging potential therapeutic target for osteoporosis: LncRNA H19/miR-29a-3p axis.

Osteoporosis (OP) is a complex systemic disease characterized by a loss of bone density, leading to bone fragility and an increase risk of fractures of the hip, spine and wrist. The clinical therapeutic effect is still far from satisfactory. Thus, further studies are urgently needed to explore the pathogenesis of OP. In this study, our aim is to explore the underlying molecular mechanism of lncRNA H19/miR-29a-3p axis for regulating of inflammation, proliferation and apoptosis in OP. The expression of lncRNA H19 was significantly upregulated in OP samples compared with the health control. Subsequently, we found that miR-29a-3p is the target of lncRNA H19 in OP. Furthermore, the knockdown of lncRNA H19 was validated to promote the expression of pro-inflammatory mediators, repress cell proliferation and inhibit cell apoptosis in vitro. Moreover, the modulating effects of lncRNA-H19 on the expressions of pro-inflammatory mediators, cell proliferation and apoptosis in vitro were diminished after co-transfecting with miR-29a-3p inhibitor and siRNA-H19. Thus, we concluded that lncRNA H19/miR-29a-3p axis was involved in the development of OP. This study might provide a better understanding of OP development and a potential therapeutic target for OP intervention.

Optimizing indications of impacting bone allograft transplantation in osteonecrosis of the femoral head.

AIMS: The aim of this study was to report the medium-term outcomes of impaction bone allograft and fibular grafting for osteonecrosis of the femoral head (ONFH) and to define the optimal indications. METHODS: A total of 67 patients (77 hips) with ONFH were enrolled in a single centre retrospective review. Success of the procedure was assessed using the Harris Hip Score (HHS) and rate of revision to total hip arthroplasty (THA). Risk factors were studied, including age, aetiology, duration of hip pain, as well as two classification systems (Association Research Circulation Osseous (ARCO) and Japanese Investigation Committee (JIC) systems). RESULTS: After a mean follow-up period of 8.61 years (SD 1.45), 81.3% (52/64) of enrolled cases had a good or excellent HHS at latest follow-up (declining to 76.0% (38/50) for those with more than eight years of follow-up). Overall survival was 92.1% at eight years' follow-up (95% CI 83.2% to 96.4%). A total of 12 hips (19.0%) failed (reoperation or HHS < 70 points) at final follow-up. Rate of success was adversely affected by increasing age, duration of pain, and more severe disease as measured using the ARCO and JIC classifications, but not by aetiology of the ONFH. CONCLUSION: We report favourable medium-term results of this procedure. Best outcomes can be expected in patients matching the following indications: younger than 40 years; less 12-month hip pain before surgery; femoral head collapse being less than 2 mm; and integrated lateral wall of femoral head. Cite this article: Bone Joint J 2020;102-B(7):838-844.

Pathological progress of traumatic femur head necrosis after femoral neck fracture in children and adolescents: a case series study.

The pathological progression and prognosis of traumatic femur head necrosis (TFHN) after femoral neck fracture (FNF) in children and adolescent is relatively unknown and has never been specifically characterized. As we speculated, the prognosis in such population would be poor and characterized as the high risk of femoral head collapse, hip deformity and degeneration in a short term. This retrospective case series enrolled 64 children and adolescent with TFHN who treated with observational treatment from 2000.1 to 2018.1. The primary outcomes, the progression of femoral head collapse, hip deformity (Stulberg classification) and hip degeneration (Tönnis grade), and their prognostic factors were analysed. Sixty-four patients with a mean age of 13 years (6-16 years) were included. A total of 28 hips (44%) showed unsatisfactory outcome and 25 (39%) hips collapsed progressively during a mean follow-up of 48 months (24-203 months). Finally, 38 hips (59%) experienced hip deformity, 20 of them were Class IV/V. Thirty-four hips (53%) generally progressed to osteoarthritis, 14 of them were classified as Grades II/III. The location of the lesion and the presence of subluxation were found to be related to progression of collapse; however, the presence of subluxation was the only independent risk factor of severe hip deformity and degeneration. TFHN in children and adolescent is a rapidly progressing disease with a poor prognosis characterized by a high risk of femoral head collapse progression. If the subluxation emerged, collapsed cases showed increasingly tendency towards hip deformity and degeneration.

The clinical and radiographic characteristics of avascular necrosis after pediatric femoral neck fracture: a systematic review and retrospective study of 115 patients.

BACKGROUND: Avascular necrosis (AVN) after pediatric femoral neck fracture (PFNF) showed poor prognosis, but its clinical and radiographic characteristics remained unclear. METHODS: A systematic review and a retrospective study were performed to evaluate the clinical and radiographic characteristics of patients with AVN after PFNF. RESULTS: A total of 686 patients with PFNF and 203 patients with AVN from 21 articles were analyzed. Ratliff's classification was used in 178 patients, with types I, II, and III AVN accounting for 58.4%, 25.3%, and 16.3%, respectively. Ratliff's assessment was used in 147 patients, of whom 88.4% had an unsatisfactory prognosis. In retrospective study, 115 patients with a mean age of 13.6 ± 2.0 years were included. The mean interval between AVN and PFNF was 13.7 ± 9.5 months. At the time of diagnosis, 59.1% cases were symptomatic and 65.2% progressed to collapsed stage. Fifty (43.5%), 61 (53.0%), and 4 patients (3.5%) were defined as types I, II, and III , respectively, via Ratliff's classification. Thirteen (11.3%), 40 (34.8%), and 62 patients (53.9%) showed types A/B, C1, and C2 disease, respectively, via the JIC classification. Multivariate analysis demonstrated a strong relation between collapsed stage and symptomatic cases (OR = 6.25, 95% CI = 2.39-16.36) and JIC classification (OR = 3.41, 95% CI = 1.62-7.17). CONCLUSION: AVN after PFNF showed a tendency toward extensive necrotic lesions, presumably resulting in a rapid progression of femoral head collapse. And the symptoms and the JIC classification are other two risk factors of collapse progression.