Aucubin Promotes Osteogenic Differentiation and Facilitates Bone Formation through the lncRNA-H19 Driven Wnt/ß-Catenin Signaling Regulatory Axis.

Objectives: Traditional Chinese medicine Cortex Eucommiae has been used to treat bone fracture for hundreds of years, which exerts a significant improvement in fracture healing. Aucubin, a derivative isolated from Cortex Eucommiae, has been demonstrated to possess anti-inflammatory, immunoregulatory, and antioxidative potential. In the present study, our aim was to explore its function in bone regeneration and elucidate the underlying mechanism. Materials and Methods: The effects of Aucubin on osteoblast and osteoclast were examined in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) and RAW 264.7 cells, respectively. Moreover, the lncRNA H19 and Wnt/ß-catenin signaling were detected by qPCR examination, western blotting, and luciferase activity assays. Using the femur fracture mice model, the in vivo effect of Aucubin on bone formation was monitored by X-ray, micro-CT, histomorphometry, and immunohistochemistry staining. Results: In the present study, Aucubin was found to significantly promote osteogenic differentiation in vitro and stimulated bone formation in vivo. Regarding to the underlying mechanism, H19 was found to be obviously upregulated by Aucubin in MSCs and thus induced the activation of Wnt/ß-catenin signaling. Moreover, H19 knockdown partially reversed the Aucubin-induced osteogenic differentiation and successfully suppressed the activation of Wnt/ß-catenin signaling. We therefore suggested that Aucubin induced the activation of Wnt/ß-catenin signaling through promoting H19 expression. Conclusion: Our results demonstrated that Aucubin promoted osteogenesis in vitro and facilitated fracture healing in vivo through the H19-Wnt/ß-catenin regulatory axis.

Baicalin induces ferroptosis in osteosarcomas through a novel Nrf2/xCT/GPX4 regulatory axis.

BACKGROUND: Osteosarcomas (OS) is a kind of malignant bone tumor which occurs primarily in children and adolescents, and the clinical therapeutics remain disappointing. As a new programmed cell death, ferroptosis is characterized by iron dependent and intracellular oxidative accumulation, which provides a potential alternative intervene for the OS treatment. Baicalin, a major bioactive flavone derived from traditional Chinese medicine Scutellaria baicalensis, has been proved to have anti-tumor properties in OS. Whether ferroptosis participated in the baicalin mediated anti-OS activity is an interesting project. PURPOSE: To explore the pro-ferroptosis effect and mechanisms of baicalin in OS. METHODS/STUDY DESIGN: Pro-ferroptosis effect of baicalin on cell death, cell proliferation, iron accumulation, lipid peroxidation production was determined in MG63 and 143B cells. The levels of glutathione (GSH), oxidized (GSSG) glutathione and malondialdehyde (MDA) were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Glutathione peroxidase 4 (GPX4) and xCT were detected by western blot in baicalin-mediated ferroptosis regulation. In vivo, a xenograft mice model was adopted to explore the anticancer effect of baicalin. RESULTS: In the present study, it was found that baicalin significantly suppress tumor cell growth in vitro and in vivo. By promoting the Fe accumulation, ROS formation, MDA production and suppressing the ratio of GSH/GSSG, baicalin was found to trigger ferroptosis in OS and ferroptosis inhibitor ferrostatin-1 (Fer-1) successfully reversed these suppressive effects, indicating that ferroptosis participated in the baicalin mediated anti-OS activity. Mechanistically, baicalin physically interacted with Nrf2, a critical regulator of ferroptosis, and influenced its stability via inducing ubiquitin degradation, which suppressed the Nrf2 downstream targets GPX4 and xCT expression, and led to stimulating ferroptosis. CONCLUSIONS: Our findings for the first time indicated that baicalin exerted anti-OS activity through a novel Nrf2/xCT/GPX4-dependent ferroptosis regulatory axis, which hopefully provides a promising candidate for OS treatment.

Corrigendum to "Gallic acid mediates tumor-suppressive effects on osteosarcoma through the H19-Wnt/ß-catenin regulatory axis" [J. Orthop. Translat. 39 (2023) 34-42].

[This corrects the article DOI: 10.1016/j.jot.2022.12.003.].

LncRNA-NEF suppressed oxaliplatin resistance and epithelial-mesenchymal transition in colorectal cancer through epigenetically inactivating MEK/ERK signaling.

A major cause of oxaliplatin chemoresistance in colorectal cancer (CRC) is acquired epithelial-mesenchymal transition (EMT) in cancer cells, making the cancer cells easy to metastasis and recurrence. LncRNA Neighboring Enhancer of FOXA2 (lncRNA-NEF) has been characterized as a tumor suppressor to mediate cancer metastasis in multiple cancer types. However, whether it mediated the drug resistance remains unknown. In the present study, an oxaliplatin-resistant CRC cell line (SW620R) was established and lncRNA-NEF was obviously down-regulated in this resistant cell line. The further loss and gain-of-function studies demonstrated that this lncRNA suppressed oxaliplatin resistance as well as EMT programme in vitro and inhibited metastasis in vivo. Mechanistically, lncRNA-NEF epigenetically promoted the expression of DOK1 (Downstream of Tyrosine kinase 1), a negative regulator of MEK/ERK signaling, by disrupting DNA methyltransferases (DNMTs)-mediated DNA methylation. DOK1, in turn, induced the inactivation of MEK/ERK signaling, forming the lncRNA-NEF/DOK1/MEK/ERK regulatory axis to mediate oxaliplatin resistance in CRC. Collectively, our work reveals the critical function of lncRNA-NEF in mediating the oxaliplatin chemotherapy resistance in CRC, and provides a promising therapeutic strategy for CRC patients with oxaliplatin resistance.

Gallic acid mediates tumor-suppressive effects on osteosarcoma through the H19-Wnt/ß-catenin regulatory axis.

Background: Osteosarcoma (OS) is the most common primary malignancy in bone tissues, and effective therapeutics remain absent in clinical practice. Traditional Chinese medicines (TCM) have been used for thousands of years, which provide great insights into OS management. Gallic acid (GA) is a natural phenolic acid enriched in various foods and herbs. Several pharmacological activities of GA such as anti-oxidation and anti-inflammation have been well-established. However, its biological function in OS remains not fully understood. Methods: The potential anti-cancer properties of GA were evaluated in 143 â€‹B, U2OS and MG63 â€‹cells. Its effects on cell growth, cell cycle, apoptosis and migration were examined in these OS cells. The lncRNA H19 and Wnt/ß-catenin signaling were detected by qPCR, luciferase activity and Western blotting assays. The in vivo effect of GA on tumor growth was investigated using an orthotopic mouse model. Results: In the present study, GA was found to suppress the tumor growth in vitro via inducing cell cycle arrest and apoptosis in OS cells, and inhibit the invasion and metastasis as well. Using the orthotopic animal model, GA was also found to suppress tumorigenesis in vivo. Long noncoding RNA (lncRNA) H19 was demonstrated to be down-regulated by GA, and thus disrupted the canonical Wnt/ß-catenin signaling in OS cells. Furthermore, the ectopic expression of H19 rescued the GA-induced suppressive effects on tumor growth and metastasis, and partially reversed the inactivation of Wnt/ß-catenin signaling. Conclusions: Taken together, our results indicated that GA inhibited tumor growth through an H19-mediated Wnt/ß-catenin signaling regulatory axis in OS cells. The translational potential of this article: The information gained from this study provides a novel underlying mechanism of GA mediated anti-OS activity, suggesting that GA may be a promising drug candidate for OS patients.

MLK3 silence suppressed osteogenic differentiation and delayed bone formation via influencing the bone metabolism and disturbing MAPK signaling.

Background: Mixed lineage kinase 3 (MLK3) is a member of a serine/threonine MAP3K family, and it has been demonstrated to play critical roles in various biological activities and disease progression. Previous studies showed that impaired skeletal mineralization and spontaneous tooth fracture in the MLK3-deficient mice, suggesting MLK3 actively participated in the bone formation. However, the detailed function and underlying mechanisms remain obscure. Methods: The MLK3 knockout (KO) mouse was applied in the present study, and multi-omics were performed to compare the metabolites and gene expression between wild type (WT) and KO mice. The bone fracture model was successfully established, and the healing process was evaluated by X-ray, micro-CT examination, histomorphometry and immunohistochemistry (IHC) staining. On the other hand, the effects of MLK3 on osteogenic differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red S (ARS) staining and qRT-PCR examination. Finally, the downstream signaling pathways were screened out by RNA-sequencing (RNA-seq) and then validated by Western blotting. Results: In the present study, imbalanced bone metabolism was observed in these MLK3 KO mice, suggesting MLK3 may participate in bone development. Moreover, MLK3 -/- mice displayed abnormal bone tissues, impaired bone quality, and delayed fracture healing. Further investigation showed that the inhibition of MLK3 attenuated osteoblast differentiation in vitro. According to the RNA-seq data, MAPK signaling was screened out to be a downstream pathway, and its subfamily members extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal protein kinase (JNK) were subjected to Western blotting examination. The results revealed that although no differences in their expression were observed between MSCs derived from WT and KO mice, their phosphorylated protein levels were all suppressed in MLK3 -/- MSCs. Conclusion: In conclusion, our results demonstrated that loss of MLK3 suppressed osteoblast differentiation and delayed bone formation via influencing metabolism and disturbing MAPK signaling. The translational potential of this article: The findings based on the current study demonstrated that MLK3 promoted osteogenesis, stimulated new bone formation and facilitated fracture healing, suggesting that MLK3 may serve as a potential therapeutic target for bone regeneration. MLK3 activator therefore may be developed as a therapeutic strategy for bone fracture.

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.

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.

Baicalein mediates the anti-tumor activity in Osteosarcoma through lncRNA-NEF driven Wnt/ß-catenin signaling regulatory axis.

Background: Osteosarcoma (OS) is a common type of malignant bone tumor in adolescents with high risk of metastasis. However, the clinical management still remains unsatisfactory. Traditional Chinese medicine (TCM) has been widely considered as an alternative treatment, and their extracts have proved to possess great potential for drug discovery. Baicalein (BA), the active pharmaceutical ingredient of rhizoma coptidis, was proved to have anti-tumor properties in OS, but the mechanism remains poorly understood. Methods: The potential anti-cancer effects on cell growth, cell cycle, apoptosis and migration were examined in OS cells. Moreover, the lncRNA-Neighboring Enhancer of FOXA2 (lncRNA-NEF) and Wnt/ß-catenin signaling were detected by qPCR and Western blotting assays. The in vivo effect of GA on tumor growth was investigated using a xenograft mice model. Results: In the present study, BA was found to significantly suppress tumor growth in vitro and in vivo. And it was also found to inhibit the invasion and metastasis as well. As for the mechanism investigation, lncRNA-NEF was obviously upregulated by BA in OS cells, and thus induced the inactivation of Wnt/ß-catenin signaling. Moreover, lncRNA-NEF knockdown partially reversed the BA-induced anti-cancer activities; and successfully compensated the suppressive effect on Wnt/ß-catenin signaling. We therefore suggested that BA induced the inactivation of Wnt/ß-catenin signaling through promoting lncRNA-NEF expression. Conclusions: In conclude, our results demonstrated that BA suppressed tumor growth and metastasis in vitro and in vivo through an lncRNA-NEF driven Wnt/ß-catenin regulatory axis, in which lncRNA-NEF was upregulated by BA, and thus induced the inactivation of Wnt/ß-catenin signaling. The Translational potential of this article: The findings derived from this study validates the anti-cancer activity of BA in OS and provides a novel underlying mechanism, which suggest that BA may be a potential candidate to develop the effective drug for OS patients.

Diagnostic value of necrotic lesion boundary in bone collapse of femoral head osteonecrosis.

PURPOSE: Our research developed a novel approach to quantitatively evaluate the boundary of necrotic lesions in osteonecrosis of the femoral head (ONFH) and to explore its diagnostic value in predicting bone collapse of the femoral head. METHODS: A retrospective cross-sectional study was conducted in our institution, and 146 hips (121 cases) identified as ONFH were recruited. The anterior and lateral boundaries of each enrolled subject were measured in standard anteroposterior (AP) view and frog-leg (FL) view of plain radiographic images, the intact rate of which was then calculated and presented as the anteroposterior view intact ratio (APIR) and frog-leg view intact ratio (FLIR), respectively. Univariate and multivariate logistic regression analyses were performed to identify risk factors for collapse. A receiver operating characteristic (ROC) curve analysis was performed to determine the sensitivity, specificity and cutoff value of the APIR and FLIR. A Kaplan-Meier (K-M) analysis was applied to calculate the survival rate of the femoral head, and bone collapse of the femoral head was regarded as the endpoint. RESULTS: Femoral head collapse was observed in 61 hips during the follow-up period. Patients with or without femoral head collapse were categorized into the collapse group and non-collapse group, respectively. The mean follow-up time was 3.7 years (2-9) for the collapse group and 7.7 years (5-20) for the non-collapse group. Univariate and multivariate logistic regression analysis and ROC analysis showed that APIR (< 25.61%) and FLIR (< 24.43%) were significantly associated with femoral head collapse. The K-M survival curves indicated that the overall survival rate of APIR (≥ 25.61%) was 94.8% at 7.5 years and 76.6% at 10 years, while that of FLIR (≥ 24.43%) was 87.3% at 7.5 years and ten years. CONCLUSION: The present study demonstrates that APIR and FLIR are of high diagnostic value in the early and middle stages of ONFH. APIR and FLIR can be used to predict the occurrence of femoral head collapse in patients with JIC classification types B and C1. The measurement of these two parameters in plain radiography images may contribute to the selection of a proper hip preservation strategy.

[Predictive effect of femoral neck strength composite indexes on femoral head collapse in non-traumatic osteonecrosis of femoral head].

OBJECTIVE: To explore the predictive effect of the femoral neck strength composite indexes on femoral head collapse in non-traumatic osteonecrosis of the femoral head (ONFH) compared with bone turnover marker. METHODS: The non-traumatic ONFH patients who were admitted and received non-surgical treatment between January 2010 and December 2016 as the research object. And 96 cases (139 hips) met the selection criteria and were included in the study. There were 54 males (79 hips) and 42 females (60 hips), with an average age of 40.2 years (range, 22-60 years). According to whether the femoral head collapsed during follow-up, the patients were divided into collapsed group and non-collapsed group. The femoral neck width, hip axis length, height, body weight, and bone mineral density of femoral neck were measured. The femoral neck strength composite indexes, including the compressive strength index (CSI), bending strength index (BSI), and impact strength index (ISI), were calculated. The bone turnover marker, including the total typeⅠcollagen amino terminal elongation peptide (t-P1NP), ß-crosslaps (ß-CTx), alkaline phosphatase (ALP), 25 hydroxyvitamin D [25(OH)D], and N-terminal osteocalcin (N-MID), were measured. The age, gender, height, body weight, body mass index (BMI), bone mineral density of femoral neck, etiology, Japanese Osteonecrosis Investigation Committee (JIC) classification, femoral neck strength composite indexes, and bone turnover marker were compared between the two groups, and the influencing factors of the occurrence of femoral head collapse were initially screened. Then the significant variables in the femoral neck strength composite indexes and bone turnover marker were used for logistic regression analysis to screen risk factors; and the receiver operating characteristic (ROC) curve was used to determine the significant variables' impact on non-traumatic ONFH. RESULTS: All patients were followed up 3.2 years on average (range, 2-4 years). During follow-up, 46 cases (64 hips) had femoral head collapse (collapsed group), and the remaining 50 cases (75 hips) did not experience femoral head collapse (non-collapsed group). Univariate analysis showed that the difference in JIC classification between the two groups was significant ( Z=-7.090, P=0.000); however, the differences in age, gender, height, body weight, BMI, bone mineral density of femoral neck, and etiology were not significant ( P>0.05). In the femoral neck strength composite indexes, the CSI, BSI, and ISI of the collapsed group were significantly lower than those of the non-collapsed group ( P<0.05); in the bone turnover marker, the t-P1NP and ß-CTx of the collapsed group were significantly lower than those of the non-collapsed group ( P<0.05); there was no significant difference in N-MID, 25(OH)D or ALP between groups ( P>0.05). Multivariate analysis showed that the CSI, ISI, and t-P1NP were risk factors for femoral collapse in patients with non-traumatic ONFH ( P<0.05). ROC curve analysis showed that the cut-off points of CSI, BSI, ISI, t-P1NP, and ß-CTx were 6.172, 2.435, 0.465, 57.193, and 0.503, respectively, and the area under the ROC curve (AUC) were 0.753, 0.642, 0.903, 0.626, and 0.599, respectively. CONCLUSION: The femoral neck strength composite indexes can predict the femoral head collapse in non-traumatic ONFH better than the bone turnover marker. ISI of 0.465 is a potential cut-off point below which future collapse of early non-traumatic ONFH can be predicted.

Osteoporosis is associated with varus deformity in postmenopausal women with knee osteoarthritis: a cross-sectional study.

BACKGROUND: Varus deformity of the knee is a common pathological characteristic in knee osteoarthritis (KOA), and not enough attention has been given to the relationship between knee varus deformity and the state of systemic bone mass. The purpose of this study was to evaluate the potential relationship between bone mineral density (BMD) and varus deformity in postmenopausal women with KOA. METHODS: A total of 202 postmenopausal women with KOA(KL grade ≥ 2)in our department from January 2018 to June 2020 were reviewed in this cross-sectional study. The hip-knee-ankle angle of the lower extremity (HKA), medial distal femoral angle (MDFA), medial proximal tibial angle (MPTA), and the angle of the joint line (JLCA) were measured in all patients. According to the HKA Angle, these participants were divided into the varus deformity group (HKA < 175.3°) and the normal limb alignment group (175.3°≤ HKA ≤ 180.3°). The BMD of the lumbar (L1-L4), left femoral neck, and left hip were measured by dual-energy X-ray absorptiometry in all patients. The difference in BMD between the knee varus deformity group and the normal limb alignment group was compared, and the relationship between the different angles of limb alignment and the BMD values at different sites was evaluated. RESULTS: There were 144 cases (71.3 %) in the varus deformity group and 58 cases (28.7 %) in the normal limb alignment group. BMD at different joint sites within the knee varus deformity group was lower than of the normal limb alignment group, and the prevalence of osteoporosis was higher. After adjusting for confounding factors such as age, BMI, pain duration, and affected side, binary logistic regression showed that osteoporosis was an independent risk factor for varus deformity of KOA, and multiple linear regression showed that the BMD of spine, femoral neck, and hip was significantly associated with varus deformity of KOA. Pearson correlation analysis showed that BMD of the lumbar spine (L1-L4), left femoral neck and left hip joint were positively correlated with the HKA, but negatively correlated with JLCA. MPTA was positively correlated with the left femoral neck and left hip joint BMD, but not correlated with lumbar bone density. Furthermore, in the normal limb alignment group, the HKA was only negatively correlated with JLCA, but not significantly correlated with MDFA and MPTA. In the varus deformity group, the HKA was not only negatively correlated with JLCA but also positively correlated with MDFA and MPTA. CONCLUSIONS: Osteoporosis should be a major risk factor for varus deformity in postmenopausal women with KOA. The progression of varus deformity of the knee should be concerned in postmenopausal women who simultaneously has KOA and osteoporosis.

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.

Preliminary proposal: a classification system for reconstruction with autologous femoral head after periacetabular tumors resection.

BACKGROUND: Although researchers have adopted various methods for the resection and reconstruction of periacetabular tumors, the total incidence rate of complications remains high. Aiming for preserving the acetabulum and reducing the risk of complications, we applied a surgery method using tumor-free autologous femoral head to reconstruct the defective acetabulum after resection of periacetabular tumors followed by performing a conventional total hip arthroplasty (THA). Moreover, we proposed a preliminary classification system for these surgery methods. METHODS: We retrospectively reviewed 6 patients treated with acetabulum reconstruction combined with autologous femoral head following peri-acetabulum resection between April 2010 and May 2018. All patients were diagnosed as periacetabular tumors including chondrosarcoma (n = 5) and chondroblastoma (n = 1). Clinical data such as age, diagnosis, complications, local recurrence or metastasis, and function (Musculoskeletal Tumor Society 1993, MSTS93) were documented. The average time of follow-up was 62.5 months (range, 17 to 106 months). RESULTS: A total of 5 patients survive with average MSTS93 score of 27.8 points (range, 26-30). One patient, suffering from multiple bone metastasis prior treatment, ended up dying. One who had received radiotherapy before surgery had poor incision healing. Further, a classification system was preliminary proposed in 2 patients involving the pubis (type A) and 4 patients involving ischium (type B). CONCLUSIONS: Based on the results, we preliminary proposed a classification system for reconstruction with autologous femoral head after periacetabular low malignant tumors resection. The clinical results suggested that surgery methods involving pubis (type A) and ischium (Type B) are safe and feasible. However, further researches should be conducted to verify our classification system.

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.

Finite element modeling of proximal femur with quantifiable weight-bearing area in standing position.

BACKGROUND: The positional distribution and size of the weight-bearing area of the femoral head in the standing position as well as the direct active surface of joint force can directly affect the result of finite element (FE) stress analysis. However, the division of this area was vague, imprecise, and un-individualized in most studies related to separate FE models of the femur. The purpose of this study was to quantify the positional distribution and size of the weight-bearing area of the femoral head in standing position by a set of simple methods, to realize individualized reconstruction of the proximal femur FE model. METHODS: Five adult volunteers were recruited for an X-ray and CT examination in the same simulated bipedal standing position with a specialized patented device. We extracted these image data, calculated the 2D weight-bearing area on the X-ray image, reconstructed the 3D model of the proximal femur based on CT data, and registered them to realize the 2D weight-bearing area to 3D transformation as the quantified weight-bearing surface. One of the 3D models of the proximal femur was randomly selected for finite element analysis (FEA), and we defined three different loading surfaces and compared their FEA results. RESULTS: A total of 10 weight-bearing surfaces in 5 volunteers were constructed, and they were mainly distributed on the dome and anterolateral of the femoral head with a crescent shape, in the range of 1218.63-1,871.06 mm2. The results of FEA showed that stress magnitude and distribution in proximal femur FE models among three different loading conditions had significant differences, and the loading case with the quantized weight-bearing area was more in accordance with the physical phenomenon of the hip. CONCLUSION: This study confirmed an effective FE modeling method of the proximal femur, which can quantify the weight-bearing area to define a more reasonable load surface setting without increasing the actual modeling difficulty.

Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/ß-catenin signaling pathway.

OBJECTIVES: Polydatin (PD), extracted from Polygonum cuspidatum, has shown potential therapeutic applications due to its antiosteoporotic and anti-inflammatory activities. Our previous study suggested that PD promotes the osteogenesis of human bone marrow stromal cells (hBMSCs) via the BMP2-Wnt/ß-catenin pathway. The aim of our present study was to further explore the role of PD-mediated regulation of Tafazzin (TAZ), a transcriptional coactivator with a PDZ-binding motif, in osteogenesis. MATERIALS AND METHODS: hBMSCs were isolated and treated with PD at various concentrations. Alizarin red staining and RT-qPCR were performed to identify calcium complex deposition in hBMSCs as well as the expression of specific osteoblast-related markers, respectively, in each group. Next, TAZ-silenced hBMSCs were generated by lentivirus-produced TAZ shRNA. After treatment with PD, the osteogenic abilities of the TAZ-silenced and control hBMSCs were estimated by ALP activity assay, and expression of the TAZ protein was detected by Western blot analysis and immunofluorescence staining. In vitro, an ovariectomized (OVX) mouse model was established and used to evaluate the effect of PD on bone destruction by micro-CT, immunohistochemistry, and ELISA. RESULTS: In vitro, 30 µM PD significantly improved the proliferation and calcium deposition of hBMSCs and markedly stimulated the expression of the mRNAs RUNX2, Osteopontin, DLX5, ß-catenin, TAZ, and Osteocalcin (OCN). Osteogenic differentiation induced by PD was blocked by lentivirus-mediated TAZ shRNA. Furthermore, Noggin (a regulator of bone morphogenic protein 2 (BMP2)) and DKK1 (an inhibitor of the Wnt/ß-catenin pathway) were found to inhibit the increase in TAZ expression induced by PD. In vivo, PD prevented estrogen deficiency-induced bone loss in the OVX mouse model. CONCLUSION: Taken together, our findings suggest that PD improved the osteogenic differentiation of hBMSCs and maintained the bone matrix in the OVX mouse model through the activation of TAZ, a potential target gene of the BMP2-Wnt/ß-catenin pathway.

lncRNA-TINCR Functions as a Competitive Endogenous RNA to Regulate the Migration of Mesenchymal Stem Cells by Sponging miR-761.

Mounting evidences have indicated that terminal differentiation-induced lncRNA (TINCR) contributes to various cellular processes, such as proliferation, apoptosis, autophagy, migration, invasion, and metastasis. However, the function of TINCR in regulating migration of MSCs is largely unknown. In this study, the effects of TINCR on the migration of rat MSCs from the bone marrow were studied by Transwell assays and wound healing assays. Our results suggested that TINCR positively regulated migration of rMSCs. miR-761 mimics suppressed rMSC migration, whereas miR-761 inhibitor promoted migration. Target prediction analysis tools and dual-luciferase reporter gene assay identified Wnt2 as a direct target of miR-761. miR-761 could inhibit the expression of Wnt2. Further, the investigation about the function of TINCR in miR-761-induced migration of rMSCs was completed. These results demonstrated that TINCR took part in the regulation of miR-761-induced migration in rMSCs through the regulation of Wnt2 and its Wnt2 signaling pathway. Taken together, our results demonstrate that lncRNA-TINCR functions as a competitive endogenous RNA (ceRNA) to regulate the migration of rMSCs by sponging miR-761 which modulates the role of Wnt2. These findings provide evidence that lncRNA-TINCR has a chance to serve as a potential target for enhancing MSC homing through the miR-761/Wnt2 signaling pathway.

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.