FoxO3 Regulates Mouse Bone Mesenchymal Stem Cell Fate and Bone-Fat Balance During Skeletal Aging.

Age-related osteoporosis is characterized by an imbalance between osteogenic and adipogenic differentiation in bone mesenchymal stem cells (BMSCs). Forkhead box O 3 (FoxO3) transcription factor is involved in lifespan and cell differentiation. In this study, we explore whether FoxO3 regulates age-related bone loss and marrow fat accumulation. The expression levels of FoxO3 in BMSCs during aging were detected in vivo and in vitro. To explore the role of FoxO3 in osteogenic and adipogenic differentiation, primary BMSCs were isolated from young and aged mice. FoxO3 expression was modulated by adenoviral vector transfection. The role of FoxO3 in bone-fat balance was evaluated by alizarin red S staining, oil red O staining, quantitative reverse transcription-polymerase chain reaction, Western blot, and histological analysis. Age-related bone loss and fat deposit are associated with downregulation of FoxO3. Overexpression of FoxO3 alleviated age-related bone loss and marrow fat accumulation in aged mice. Mechanistically, FoxO3 reduced adipogenesis and enhanced osteogenesis of BMSCs via downregulation of PPAR-γ and Notch signaling, respectively. In conclusion, FoxO3 is an essential factor controlling the fate of BMSCs and is a potential target for the prevention of age-related osteoporosis.

A Bionic Thermosensitive Sustainable Delivery System for Reversing the Progression of Osteoarthritis by Remodeling the Joint Homeostasis.

Although the pathogenesis of osteoarthritis (OA) is unclear, inflammatory cytokines are related to its occurrence. However, few studies focused on the therapeutic strategies of regulating joint homeostasis by simultaneously remodeling the anti-inflammatory and immunomodulatory microenvironments. Fibroblast growth factor 18 (FGF18) is the only disease-modifying OA drug (DMOAD) with a potent ability and high efficiency in maintaining the phenotype of chondrocytes within cell culture models. However, its potential role in the immune microenvironment remains unknown. Besides, information on an optimal carrier, whose interface and chondral-biomimetic microenvironment mimic the native articular tissue, is still lacking, which substantially limits the clinical efficacy of FGF18. Herein, to simulate the cartilage matrix, chondroitin sulfate (ChS)-based nanoparticles (NPs) are integrated into poly(D, L-lactide)-poly(ethylene glycol)-poly(D, L-lactide) (PLEL) hydrogels to develop a bionic thermosensitive sustainable delivery system. Electrostatically self-assembled ChS and ε-poly-l-lysine (EPL) NPs are prepared for the bioencapsulation of FGF18. This bionic delivery system suppressed the inflammatory response in interleukin-1ß (IL-1ß)-mediated chondrocytes, promoted macrophage M2 polarization, and inhibited M1 polarization, thereby ameliorating cartilage degeneration and synovitis in OA. Thus, the ChS-based hydrogel system offers a potential strategy to regulate the chondrocyte-macrophage crosstalk, thus re-establishing the anti-inflammatory and immunomodulatory microenvironment for OA therapy.

Oroxin B Attenuates Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Formation and Activity.

BACKGROUND: Osteoclasts are the major players in bone resorption and have always been studied in the prevention and treatment of osteoporosis. Previous studies have confirmed that a variety of flavonoids inhibit osteoporosis and improve bone health mainly through inhibiting osteoclastogenesis. Oroxin B (OB) is a flavonoid compound extracted from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent, exerts potent antitumor and anti-inflammation effect, but its effect on osteoclastogensis remains unknown. METHODS: We comprehensively evaluated the effect of OB on the formation and function of osteoclasts and the underling mechanism by bone marrow-derived macrophage in vitro. In vivo, we used mice ovariectomized model to verify the protective effect of OB. RESULTS: OB was found to inhibit osteoclast formation and bone resorption function in vitro, in a dose-dependent manner and the increased osteoclastic-related genes induced by RANKL (NFATc1, c-fos, cathepsin K, RANK, MMP9 and TRAP) were also attenuated following OB treatment. Mechanistical investigation showed OB abrogated the increased phosphorylation level of MAPK and NF-κB pathway, and diminished the expression of the vital transcription factors for osteoclastogenesis. OB also prevented ovariectomy (OVX)-induced bone loss by inhibiting osteoclast formation and activity in mice. CONCLUSION: Our study demonstrated that OB may act as an anti-osteoporosis agent by inhibiting osteoclast maturation and attenuating bone resorption.

Accumulation of LDL/ox-LDL in the necrotic region participates in osteonecrosis of the femoral head: a pathological and in vitro study.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a common but intractable disease that appears to involve lipid metabolic disorders. Although numerous studies have demonstrated that high blood levels of low-density lipoprotein (LDL) are closely associated with ONFH, there is limited evidence to explain the pathological role of LDL. Pathological and in vitro studies were performed to investigate the role of disordered metabolism of LDL and oxidized LDL (ox-LDL) in the femoral head in the pathology of ONFH. METHODS: Nineteen femoral head specimens from patients with ONFH were obtained for immunohistochemistry analysis. Murine long-bone osteocyte Y4 cells were used to study the effects of LDL/ox-LDL on cell viability, apoptosis, and metabolism process of LDL/ox-LDL in osteocytes in normoxic and hypoxic environments. RESULTS: In the pathological specimens, marked accumulation of LDL/ox-LDL was observed in osteocytes/lacunae of necrotic regions compared with healthy regions. In vitro studies showed that ox-LDL, rather than LDL, reduced the viability and enhanced apoptosis of osteocytes. Pathological sections indicated that the accumulation of ox-LDL was significantly associated with impaired blood supply. Exposure to a hypoxic environment appeared to be a key factor leading to LDL/ox-LDL accumulation by enhancing internalisation and oxidation of LDL in osteocytes. CONCLUSIONS: The accumulation of LDL/ox-LDL in the necrotic region may contribute to the pathology of ONFH. These findings could provide new insights into the prevention and treatment of ONFH.

Pinocembrin alleviates glucocorticoid-induced apoptosis by activating autophagy via suppressing the PI3K/Akt/mTOR pathway in osteocytes.

Glucocorticoids are widely used in clinical practice, but are associated with potentially severe side effects like glucocorticoid-induced osteoporosis (GIOP) and glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH). Glucocorticoid-induced osteocyte apoptosis plays critical roles in the pathological processes of both GIOP and GA-ONFH. Pinocembrin is a natural flavonoid that may exert protective effects on osteocytes. The present study investigated the effects of pinocembrin on glucocorticoid-induced apoptosis of murine long bone osteocyte Y4 (MLO-Y4) cells and sought to elucidate the underlying molecular mechanism. We found that pinocembrin attenuated glucocorticoid-induced cell viability injury and apoptosis of MLO-Y4 cells. Moreover, pinocembrin increased Beclin-1 and LC3B-II level, but decreased p62 expression, suggesting that pinocembrin activates autophagy in glucocorticoid-treated MLO-Y4 cells. The protective effects of pinocembrin on glucocorticoid-induced apoptosis of MLO-Y4 cells were mimicked by a known stimulator of autophagy but prevented by a known inhibitor of autophagy. Pinocembrin also suppressed the PI3K/Akt/mTOR signaling pathway, which regulates cell autophagy, in glucocorticoid-treated MLO-Y4 cells. In conclusion, the results indicate that pinocembrin alleviates glucocorticoid-induced osteocyte apoptosis by activating autophagy via suppressing the PI3K/Akt/mTOR pathway. Pinocembrin may represent a potential natural agent for preventing and treating GIOP and GA-ONFH.

Serum Biomarkers Related to Glucocorticoid-Induced Osteonecrosis of the Femoral Head: A Prospective Nested Case-Control Study.

Early diagnosis and prevention of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH) continues to be a challenging problem for clinicians and researchers. However, the role of circulating biomarkers for GC-induced ONFH, which may reveal individual susceptibility and facilitate earlier diagnosis, remains to be determined. The aim of this study was to identify potential biomarkers that may predict early GC-induced ONFH. A total of 123 patients scheduled for initial systemic GC therapy were enrolled in this prospective nested case-control study. The serum concentrations of 13 potential biomarkers were measured in seven patients with GC-induced ONFH, diagnosed instantly after short-term use of GCs and in 20 controls who did not develop osteonecrosis despite similar GC therapy. Biomarkers were measured both before and after taking GCs to identify any differences in marker levels and the changes during GC therapy between two groups. Type I collagen cross-linked C-telopeptide (ß-CTX; p = 0.000) was significantly lower, high-density lipoprotein cholesterol (p = 0.092) and apolipoprotein (apo)-B/apo-A1 (p = 0.085) tended to be lower and higher, respectively, before GC treatment in osteonecrosis group. After GC therapy, ß-CTX (p = 0.014) was significantly lower and amino terminal telopeptide of procollagen type I (PINP; p = 0.068) tended to be lower in the osteonecrosis group. As secondary outcomes, we observed remarkable changes in nine potential biomarkers following short-term GC therapy in both groups. In conclusion, we found that ß-CTX, could potentially be used to predict GC-induced ONFH before GC therapy. Lower ß-CTX and PINP are promising biomarkers for the early diagnosis of GC-induced ONFH. These findings need to be confirmed in large-scale prospective studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2348-2357, 2019.

Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways.

Wear particles liberated from the surface of prostheses are considered to be main reason for osteoclast bone resorption and that extensive osteoclastogenesis leads to peri-implant osteolysis and subsequent prosthetic loosening. The aim of this study was to assess the effect of rifampin on osteoclastogenesis and titanium (Ti) particle-induced osteolysis. The Ti particle-induced osteolysis mouse calvarial model and bone marrow-derived macrophages (BMMs) were used. Rifampin, at dose of 10 or 50 mg/kg/day, was respectively given intraperitoneally for 14 days in vivo. The calvariae were removed and processed for Further histological analysis. In vitro, osteoclasts were generated from mouse BMMs with receptor activator of nuclear factor-κB ligand (RANKL) and the macrophage colony stimulating factor. Rifampin at different concentrations was added to the medium. The cell viability, tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity and resorption on bone slices were analysis. Osteoclast-specific genes and RANKL-induced MAPKs signaling were tested for further study of the mechanism. Rifampin inhibited Ti-induced osteolysis and osteoclastogenesis in vivo. In vitro data indicated that rifampin suppressed osteoclast differentiation and bone resorption in a dose-dependent manner. Moreover, rifampin significantly reduced the expression of osteoclast-specific markers, including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear factor of activated T cells (NFAT) c1. Further investigation revealed that rifampin inhibited osteoclast formation by specifically abrogating RANKL-induced p38 and NF-κB signaling. Rifampin had significant potential for the treatment of particle-induced peri-implant osteolysis and other diseases caused by excessive osteoclast formation and function.

Clinical outcomes of osteonecrosis of the femoral head after autologous bone marrow stem cell implantation: a meta-analysis of seven case-control studies.

The purpose of this study was to evaluate the clinical outcomes of osteonecrosis of the femoral head after autologous bone marrow stem cell implantation. We searched the PubMed, Embase and Web of Science databases and included all case-control trials that reported on the clinical outcomes of osteonecrosis progression, incidence of total hip arthroplasty and improvement in Harris hip scores. Overall, seven case-control trials were included. Compared with the controls, patients treated with the bone marrow stem cells implantation treatment showed improved clinical outcomes with delayed osteonecrosis progression (odds ratio = 0.17, 95% CI: 0.09 - 0.32; p <0.001), a lower total hip arthroplasty incidence (odds ratio = 0.30, 95% CI: 0.12 - 0.72; p <0.01) and increased Harris hip scores (mean difference = 4.76, 95% CI: 1.24 - 8.28; p<0.01). The heterogeneity, publication bias, and sensitivity analyses showed no statistical difference significant differences between studies. Thus, our study suggests that autologous bone marrow stem cells implantation has a good therapeutic effect on osteonecrosis of the femoral, resulting in beneficial clinical outcomes. However, trials with larger sample sizes are needed to confirm these findings.

Clinical outcomes of osteonecrosis of the femoral head after autologous bone marrow stem cell implantation: a meta-analysis of seven case-control studies

The purpose of this study was to evaluate the clinical outcomes of osteonecrosis of the femoral head after autologous bone marrow stem cell implantation. We searched the PubMed, Embase and Web of Science databases and included all case-control trials that reported on the clinical outcomes of osteonecrosis progression, incidence of total hip arthroplasty and improvement in Harris hip scores. Overall, seven case-control trials were included. Compared with the controls, patients treated with the bone marrow stem cells implantation treatment showed improved clinical outcomes with delayed osteonecrosis progression (odds ratio = 0.17, 95% CI: 0.09 - 0.32; p <0.001), a lower total hip arthroplasty incidence (odds ratio = 0.30, 95% CI: 0.12 - 0.72; p <0.01) and increased Harris hip scores (mean difference = 4.76, 95% CI: 1.24 - 8.28; p<0.01). The heterogeneity, publication bias, and sensitivity analyses showed no statistical difference significant differences between studies. Thus, our study suggests that autologous bone marrow stem cells implantation has a good therapeutic effect on osteonecrosis of the femoral, resulting in beneficial clinical outcomes. However, trials with larger sample sizes are needed to confirm these findings.

Involvement of MicroRNA-210 Demethylation in Steroid-associated Osteonecrosis of the Femoral Head.

Angiogenesis is an important event in steroid-associated osteonecrosis of the femoral head (SONFH). Here we performed miRNA microarray with SONFH tissues (ONs) and the adjacent normal tissues (NLs) to select the angiogenic miRNA. The results showed that miR-210 was differentially expressed in SONFH versus normal tissues. Unexpectedly, its specific transcription factor, hypoxia-inducible factor-1α, was shown of no significant changes in ONs compared with NLs. Further Bisulfite sequencing revealed that miR-210 is embedded in a CpG island and miR-210 gene has 2 CpG sites with lower methylation percentage in ONs compared with NLs. Additionally, ONs with lower miR-210 gene methylation exhibited higher miR-210 expression. Next, we found that the endothelial cells treated with demethylating agents could significantly increase the expression of miR-210, along with promoted cell viability and differentiation. Some angiogenic genes (VEGF, bFGF, TNF-α and PCNA) were up-regulated as well. In addition, the supernatant of the cells after demethylation treatment displayed an enhanced ability of recruiting new microvessels in vivo. Taken together, our study not only provides novel insights into the regulation of angiogenesis in this disease, but also reveals a therapeutic opportunity for treatment of SONFH patients with demethylating agents.

Predictive value of single photon emission computerized tomography and computerized tomography in osteonecrosis after femoral neck fracture: a prospective study.

PURPOSE: Osteonecrosis of the femoral head (ONFH) is a very common complication after femoral neck fracture. The purpose of this study was to assess the femoral head vascularity after femoral neck fracture using single photon emission computerized tomography and computerized tomography (SPECT/CT), and to evaluate its value in predicting ONFH. METHODS: Between January 2008 and March 2011, 120 patients diagnosed with femoral neck fracture underwent SPECT-CT before the internal fixation. The fracture was classified according to the Garden classification. The ratios of the radionuclide uptake of the fractured femoral head to that of the contralateral femoral head (F/N) were calculated to assess the femoral head vascularity. After a minimum of two years' follow-up, magnetic resonance imaging (MRI) was used as the "gold standard" for the diagnosis of possible ONFH. RESULTS: A total of 114 patients completed the study. The SPECT/CT examination showed that the F/N ratios of Garden I, II, III and IV were 2.6, 1.8, 0.8, and 0.6, respectively. At the time of the most recent follow-up, osteonecrosis developed in two of the 27 patients who had a Garden Stage-II fracture, in eight of the 34 patients who had a Garden Stage-III fracture, and nine of the 27 patients who had a Garden Stage-VI fracture. With a cutoff of 0.55 from the receiver operating characteristic (ROC) curve, F/N ratio showed a sensitivity of 97%, a specificity of 79%, a positive predictive value of 95%, and a negative predictive value of 19%. CONCLUSION: SPECT-CT proved to be reliable and valid for predicting ONFH after femoral neck fracture.

Protective effects of total saponins of panax notoginseng on steroid-induced avascular necrosis of the femoral head in vivo and in vitro.

This research was designed to investigate the protective effects of TSPN on steroid-induced avascular necrosis of the femoral head (ANFH) and the likely mechanisms of those effects. As an in vivo study, TSPN was shown to be protective against steroid-induced ANFH due to the upregulation of VEGF-A. Furthermore, TSPN attenuated the apoptosis of osteocytes and reduced the expression of Caspase-3 relative to the model group. As an in vitro study, TSPN exerted a concentration-dependent protective effect against apoptosis in MC3T3-E1 cells. Moreover, TSPN (at a dose of 100 µg/mL) significantly reversed the dexamethasone-induced augmentation of Caspase-3 expression and activity. Therefore, our study demonstrated that TSPN had a protective effect against steroid-induced ANFH that was related to the upregulation of VEGF-A and the inhibition of apoptosis and Caspase-3 activation.

Analysis of altered microRNA expression profile in the reparative interface of the femoral head with osteonecrosis.

The reparative reaction is considered to be important during the occurrence of collapse in the femoral head with osteonecrosis (ONFH), but little is known about the long-term reparative process. The aim of this study was to determine and analyze the altered microRNA expression profile in the reparative interface of ONFH, and further validate the expression of the involved genes in the predicted pathways. Microarray analysis was performed comparing the reparative interface of patients with ONFH and normal tissue of patients with fresh femoral neck fracture (FNF) and partly validated by real-time PCR. Potential target genes of differentially expressed miRNAs were predicted by TargetScan and miRanda, and the target genes were used for further bioinformatics analysis such as Gene Ontology and Pathway assay. The filtered miRNAs and genes in the predict pathways were further examined by real-time PCR in another 6 independent ONFH patients. Among the 2578 miRNAs identified, 17 were consistently differentially expressed, 12 of which are up-regulated and 5 down-regulated. GO classification showed that the predicted target genes of these miRNAs are involved in signal transduction, cell differentiation, methylation, cell growth and apoptosis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) classification indicated that these genes play a role in angiogenesis and Wnt signaling pathways. The expression of miR-34a and miR-146a and genes in the predict pathways were significantly up-regulated. This study presented a global view of miRNA expression in the reparative interface of osteonecrosis. In addition, our data provided novel and robust information for further researches in the pathogenesis and molecular events of ONFH.

Mefloquine inhibits chondrocytic proliferation by arresting cell cycle in G2/M phase.

Mefloquine (MQ), an analog of chloroquine, exhibits a promising cytotoxic activity against carcinoma cell lines and for the treatment of glioblastoma patients. The present study demonstrates the effect of mefloquine on proliferation and cell cycle in chondrocytes. MTT assay and propidium iodide staining were used for the analysis of proliferation and cell cycle distribution, respectively. Western blot analysis was used to examine the expression levels of cyclin B1/cdc2, cdc25c, p21WAF1/CIP1 and p53. The results revealed that mefloquine inhibited the proliferation of chondrocytes and caused cell cycle arrests in the G2/M phase. The proliferation of chondrocytes was reduced to 27% at 40 µM concentration of mefloquine after 48 h. The population of chondrocytes in G2/M phase was found to be 15.7 and 48.4%, respectively at 10 and 40 µM concentration of mefloquine at 48 h following treatment. The expression of the cell cycle regulatory proteins including, cyclin B1/cdc2 and cdc25c was inhibited. On the other hand, mefloquine treatment promoted the expression of p21WAF1/CIP1 and p53 at 40 µM concentration after 48 h. Therefore, mefloquine inhibits proliferation and induces cell cycle arrest in chondrocytes.

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering.

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF-Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF-Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.

A "two-incision" approach for en bloc resection of periacetabular tumors with illustrations from a cadaver.

A "two-incision" approach for en bloc resection of periacetabular tumors and prosthesis reconstruction is described. The first incision begins in the middle of the iliac crest, continues over the inguinal ligament, extends to the symphysis pubis and then turns down to ischial tuberosity. The muscles attached to the iliac crest are only separated from its internal side. All the attachments of the abductor muscles to the iliac crest are left intact. The second incision runs from the sacroiliac joint to the greater trochanter and is curved in shape, providing external exposure of the sciatic notch and ischial tuberosity. Communication between the two approaches is possible inside and outside under the abductor muscles or through the sciatic notch. En bloc resection of the tumor is performed by cutting the pubic symphysis and iliac as planned preoperatively. The iliac osteotomy is performed by using a Gigli saw that has been led through the sciatic notch and under the abductor muscles. This "two-incision" approach not only provides enough exposure to perform en bloc resection of periacetabular tumors, but also protects the continuity of the abductor muscles between the pelvis and greater trochanter, thus preventing prosthesis dislocation.

Fabrication of modified dextran-gelatin in situ forming hydrogel and application in cartilage tissue engineering.

Hydrogels play a very important role in cartilage tissue engineering. Here, we oxidized dextran (Odex) and modified gelatin (Mgel) to fabricate a fast forming hydrogel without the addition of a chemical crosslinking agent. The dynamic gelling process was measured through rheological measurements. The microstructure was examined by lyophilizing to get porous scaffolds. Biological assessment was performed through CCK-8 assays by using synovium-derived mesenchymal cells (SMSCs) at 1, 3, 7 and 14 days. In vivo evaluation for application in cartilage tissue engineering was performed 8 weeks after subcutaneous injection of SMSC-loaded Odex/Mgel hydrogels combined with TGF-ß3 in the dorsa of nude mice. According to the results, a fast forming hydrogel was obtained by simply modifying dextran and gelatin. Moreover, the Odex/Mgel hydrogel exhibited good biocompatibility in cultures of SMSCs and a homogeneous distribution of live cells was achieved inside the hydrogels. After 8 weeks, newly formed cartilage was achieved in the dorsa of nude mice; no inflammatory reaction was observed and high production of GAGs was shown. The method provides a strategy for the design and fabrication of fast in situ forming hydrogels. The Odex/Mgel hydrogel could be used for the regeneration of cartilage in tissue engineering.

Stat3 upregulates leucine-rich repeat-containing g protein-coupled receptor 4 expression in osteosarcoma cells.

The activation of signal transducer and activator of transcription 3 (Stat3) signaling is the common hallmark in various human cancers including osteosarcoma. In the present study, according to PCR-based microarrays using cDNA prepared from interleukin-6 (IL-6) treated osteosarcoma cells, we found that leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) was a transcriptional target of Stat3. Overexpression of Stat3 promoted LGR4 expression, while its deficiency using small interfering RNA (siRNA) reduced LGR4 expression. Furthermore, we identified a Stat3 binding motif located at -556 to -549 bp in the LGR4 promoter that is able to interact with Stat3. Thus, our results suggest a previously unknown Stat3-LGR4 molecular network, which may control osteosarcoma development and progression.