[Treatment of Gustilo-Anderson â ¢B type injury of distal femur complicated with bone defect by membrane induction technique].

Objective: To evaluate the effectiveness of membrane induction technique in the treatment of Gustilo-Anderson ⅢB type injury of distal femur complicated with bone defect. Methods: The clinical data of 20 patients with Gustilo-Anderson ⅢB type injury of distal femur complicated with bone defects admitted between January 2019 and December 2020 were retrospectively analyzed, including 15 males and 5 females, with an average age of 35 years (range, 19-70 years). Causes of injuries included 15 cases of traffic accidents and 5 cases of falling from height. Bone defect located at metaphyseal in 11 cases and at proximal metaphyseal in 9 cases. The time from injury to primary first-stage surgery was 4-28 hours, with an average of 11 hours. After primary radical debridement, the length of bone defect was 3-12 cm, with an average of 6 cm. Antibiotic-containing bone cement was implanted in the bone defect site to induce membrane formation. At 34-56 days (mean, 45 days) after the first-stage surgery, bone grafting was performed in the induced membrane for the repair and reconstruction of bone defects; 16 patients received a combination of autogenous cancellous and allogeneic bone grafts and 4 patients received cancellous bone grafts. The bone graft healing time after the second-stage surgery was recorded; the visual analogue scale (VAS) score and Lysholm score were compared before the second-stage bone graft and at last follow-up to evaluate the pain and functional improvement of the affected limb; and the knee joint range of motion at last follow-up was recorded. Results: None of the patients had a second revision after the first-stage surgery, 1 patient recieved flap transfer and the flap survived well after operation. All patients were followed up 12-36 months after the second-stage surgery, with an average of 23 months. All patients achieved bone union, and the bone union time was 7-10 months (mean, 8.4 months). No bone nonunion or donor site related complications occurred. The Lysholm score and VAS score at last follow-up were 85.6±4.1 and 1.7±0.8, respectively, and they were significantly improved when compared with those before the second-stage bone defect repair (42.7±4.6 and 7.1±0.8, respectively) ( t=37.410, P<0.001; t=21.962, P<0.001). Knee flexion range of motion was 60°-120°, with an average of 95°; the limit of elongation was 0°-10°, with an average of 5°. Conclusion: For Gustilo-Anderson ⅢB type injury of distal femur complicated with bone defect, induction membrane technique can effectively control infection, promote bone healing of the defect site, and effectively restore the function of lower limbs with satisfactory effectiveness.

[Flap combined with induced membrane technique in treatment of post-traumatic tibial osteomyelitis with soft tissue defect].

Objective: To investigate the effectiveness of flap combined with induced membrane technique in treatment of post-traumatic tibial osteomyelitis with soft tissue defect. Methods: A clinical data of 33 patients with post-traumatic tibial osteomyelitis with soft tissue defect who met the selection criteria between August 2015 and October 2018 was retrospectively analyzed. There were 21 males and 12 females. The age ranged from 19 to 70 years, with an average of 39 years. The osteomyelitis located in the upper 1/3 of tibia in 8 cases, in the middle 1/3 of tibia in 14 cases, and in the lower 1/3 of tibia in 11 cases. According to Cierny-Mader classification standard, 19 cases of osteomyelitis were type Ⅲ and 14 cases were type Ⅳ. The duration of osteomyelitis ranged from 2 months to 20 years (median, 3 months). In the first-stage operation, after radical debridement, the length of bone defect was 1.5-12.5 cm (mean, 5.0 cm) and the size of soft tissue defects ranged from 5 cm×4 cm to 15 cm×12 cm. Bone cement containing antibiotics was implanted into the bone defect and the personalized flap was used to repair the wound. After the wound healed at 6-8 weeks and the infection was controlled, bone grafting was performed to repair bone defects in the second-stage operation. Results: The flaps survived completely after the first-stage operation in 29 cases. Partial necrosis of the flap occurred in 4 cases and healed after surgical dressing change. All the incisions healed by first intention after the second-stage operation. All patients were followed up 24-32 months (mean, 28 months). All the bone grafts healed after operation, and the radiographic healing time was 3-9 months (mean, 5 months). The clinical healing time was 4-14 months (mean, 8 months). There was no recurrence of osteomyelitis during follow-up. At last follow-up, according to Johner-Wruhs evaluation criteria, the limb function was excellent in 27 cases, good in 4 cases, and fair in 2 cases, with an excellent and good rate of 93.9%. Conclusion: For the post-traumatic tibial osteomyelitis with soft tissue defect, the flap combined with induced membrane technique is a reliable and effective method and can effectively restore the function of lower limbs with satisfactory effectiveness.

The CtIP-CtBP1/2-HDAC1-AP1 transcriptional complex is required for the transrepression of DNA damage modulators in the pathogenesis of osteosarcoma.

Most tumors, including osteosarcomas, have deficiencies in DNA damage repair. However, the regulatory mechanisms underlying dysregulation of DNA damage repair genes are still being investigated. In this study, we reveal that C-terminal binding protein (CtBP) interacting protein (CtIP) couples with three transcriptional regulators, CtBP1/2 heterodimer, histone deacetylase 1 (HDAC1), and two subunits of the activating protein 1 (AP1) transcription factor to assemble a transcriptional complex. This complex specifically controls the expression of four genes involved in DNA damage and repair processes: MutL homolog 1 (MLH1), MutS Homolog 3 (MSH3), breast cancer type 1 (BRCA1), and cyclin dependent kinase inhibitor 1A (CDKN1A). Chromatin immunoprecipitation (ChIP) assay results revealed that the CtIP-CtBP1/2-HDAC1-AP1 complex regulated these four genes by binding to their promoters through the TGAT/CTCA consensus sequence. The depletion of CtIP, CtBP1/2, and HDAC1 increased the expression levels of MLH1, MSH3, BRCA1, and CDKN1A and inhibited in vitro and in vivo osteosarcoma cell growth. Overexpression of MLH1, MSH3, BRCA1, or CDKN1A in osteosarcoma cells can reduce cell viability, colony formation, cell migration, and tumor growth. Our findings suggest that the CtIP-CtBP1/2-HDAC1-AP1 complex is required for mediation of DNA damage processes for the pathogenesis of osteosarcoma.

Orai1 downregulation causes proliferation reduction and cell cycle arrest via inactivation of the Ras-NF-κB signaling pathway in osteoblasts.

BACKGROUND: The purpose of this study was to determine the role of Orai1 in the regulation of the proliferation and cell cycle of osteoblasts. METHODS: The expression of Orai1 was inhibited by Orai1 small interfering RNA (siRNA) in MC3T3-E1 cells. Following Orai1 downregulation, cell proliferation and cell cycle were examined. Furthermore, the expression of cyclin D1, cyclin E, CDK4, and CDK6 was analyzed. The activity of the Ras-NF-κB signaling pathway was investigated to identify the role of Orai1 in the regulation of osteoblast proliferation. RESULTS: Orai1 was successfully downregulated in MC3T3-E1 cells by the Orai1 siRNA transfection (p < 0.05). We found that MC3T3-E1 cell proliferation was decreased, and the cell cycle was arrested by Orai1 downregulation (p < 0.05). Additionally, the expression of cyclin D1 was decreased by Orai1 downregulation (p < 0.05), as was the activity of the Ras-NF-κB signaling pathway (p < 0.05). Orai1 siRNA did not further reduce cell proliferation, the proportion of cells in the S phase, and cyclin D1 expression after chemical blockage of the Ras signaling pathway in MC3T3-E1 cells (p > 0.05). CONCLUSIONS: The results reveal that Orai1 downregulation may reduce cyclin D1 expression by inactivating the Ras-NF-κB signaling pathway thus blocking osteoblast proliferation and cell cycle.

MYC inhibition reprograms tumor immune microenvironment by recruiting T lymphocytes and activating the CD40/CD40L system in osteosarcoma.

The efficacy of immune checkpoint blockade (ICB) therapy depends on sufficient infiltration and activation of primed tumor-specific cytotoxic T lymphocytes (CTLs) in the tumor microenvironment. However, many tumor types, including osteosarcoma, mainly display immune-desert or immune-excluded phenotypes, which are characterized by a lack of tumor-infiltrating lymphocytes and a poor response to ICB monotherapy. Thus, novel therapeutic strategies are urgently needed to surmount these obstacles. In this study, we found that the expression of the c-Myc oncogene is negatively correlated with the T cell infiltration rate in osteosarcoma. Pharmacological inhibition of c-Myc with JQ-1 significantly reduced tumor burden and improved overall survival in an immunocompetent syngeneic murine model of osteosarcoma (K7M2). A mechanistic study revealed that JQ-1 administration dramatically reprogrammed the tumor immune microenvironment (TIME) within K7M2 tumors. On the one hand, JQ-1 can promote T cell trafficking into tumors by increasing the expression and secretion of T cell-recruiting chemokines. On the other hand, JQ-1 is capable of facilitating crosstalk between antigen-presenting dendritic cells and T cells through the CD40/CD40L costimulatory pathway, leading to activation of tumor-specific CTLs. Combined treatment with anti-PD-1 antibody and JQ-1 resulted in more pronounced tumor regression than either monotherapy, showing an obvious synergistic effect. These findings uncover for the first time that c-Myc inhibition can promote T cell infiltration and activation in osteosarcoma in multiple ways, delivering a one-two punch for modulating TIME. The present work also provides the basis for establishing c-Myc inhibitor and ICB coadministration as a novel therapeutic regimen for patients with osteosarcoma.

Targeting the CtBP1-FOXM1 transcriptional complex with small molecules to overcome MDR1-mediated chemoresistance in osteosarcoma cancer stem cells.

Chemoresistance is a major barrier for the chemotherapy of osteosarcoma. The induction of multidrug resistance protein 1 (MDR1), an ATP-dependent transporter, can efflux anti-cancer drugs, thereby decreasing chemosensitivity. However, an actual involvement of MDR1 in the chemoresistance of osteosarcoma cells has not been established. We obtained two cisplatin (CDDP)-resistant osteosarcoma cancer stem cell (CSC) lines using sphere formation medium supplemented with CDDP. These two CDDP-resistant CSC cell lines showed substantial cell proliferation, colony formation, cell invasion, and in vivo tumor growth in the presence of CDDP. Microarray analysis revealed that three genes, MDR1, FOXM1 (forkhead box M1), and CtBP1 (C-Terminal binding protein 1), showed significant overexpression in both cell lines. Mechanistically, CtBP1 assembled with FOXM1 to form a transcriptional complex, which docked onto the MDR1 promoter to activate MDR1 expression. Knockdown or inhibition of the CtBP1-FOXM1 components with specific small molecules, including NSM00158 and NSC95397 for CtBP1 and RCM1 for FOXM1, significantly repressed MDR1 expression. Administration of these three small molecules also significantly inhibited tumor growth in mouse tumor xenograft model. The MDR1-mediated chemoresistance could be reversed by NSM00158 and RCM1. Collectively, our data revealed that the CtBP1-FOXM1 complex activated MDR1 expression and that targeting this complex with their specific inhibitors could reverse MDR1-mediated chemoresistance both in vitro and in vivo. Our results indicate a new therapeutic strategy for overcoming chemoresistance during osteosarcoma treatment.

Orai1 Promotes Osteosarcoma Metastasis by Activating the Ras-Rac1-WAVE2 Signaling Pathway.

BACKGROUND The purpose of this study was to investigate whether Orai1 plays a role in the metastasis of osteosarcoma. MATERIAL AND METHODS The expression of Orai1 was silenced by small interfering RNAs against Orai1 (Orai1 siRNA) in osteosarcoma MG-63 cells. Various experiments were carried out to detect the changes in migration, invasion, and adhesion ability of these osteosarcoma cells. Furthermore, the activity of Rac1, Wave2, and Ras was detected using Western blot analysis. Moreover, the Rac1 and Ras inhibitors were used to confirm whether the Ras-Rac1-WAVE2 signaling pathway was involved in osteosarcoma metastasis promoted by Orai1. RESULTS We found that the migration, invasion, and adhesion ability of MG-63 cells were significantly reduced after silencing Orai1 expression (p<0.05). Moreover, the activity of the Rac1-WAVE2 signaling pathway was significantly inhibited after silencing of Orai1 expression (p<0.05). After the Rac1 inhibitor was added, Orai1 siRNA could not further inhibit migration, invasion, and adhesion of the osteosarcoma cells. Further experiments showed that Ras activity was significantly inhibited after silencing Orai1 expression (p<0.05). Moreover, Orai1 siRNA did not further inhibit the activity of the Rac1-WAVE2 signaling pathway nor did it further inhibit the migration, invasion, and adhesion ability of osteosarcoma cells following the addition of Ras inhibitors. CONCLUSIONS Orai1 activates the Ras-Rac1-WAVE2 signaling pathway to promote metastasis of osteosarcoma. Abnormal expression or function of Orai1 may be an important cause of osteosarcoma metastasis.

Downregulation of Bach1 protects osteoblasts against hydrogen peroxide-induced oxidative damage in vitro by enhancing the activation of Nrf2/ARE signaling.

Oxidative-stress-induced osteoblast dysfunction plays an important role in the development and progression of osteoporosis. BTB and CNC homology 1 (Bach1) has been suggested as a critical regulator of oxidative stress; however, whether Bach1 plays a role in regulating oxidative-stress-induced osteoblast dysfunction remains unknown. Thus, we investigated the potential role and mechanism of Bach1 in regulating oxidative-stress-induced osteoblast dysfunction. Osteoblasts were treated with hydrogen peroxide (H2O2) to mimic a pathological environment for osteoporosis in vitro. H2O2 exposure induced Bach1 expression in osteoblasts. Functional experiments demonstrated that Bach1 silencing improved cell viability and reduced cell apoptosis and reactive oxygen species (ROS) production in H2O2-treated cells, while Bach1 overexpression produced the opposite effects. Notably, Bach1 inhibition upregulated alkaline phosphatase activity and osteoblast mineralization. Mechanism research revealed that Bach1 inhibition increased the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling and upregulated heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 mRNA expression. The Bach1 inhibition-mediated protective effect was partially reversed by silencing Nrf2 in H2O2-exposed osteoblasts. Taken together, these results demonstrate that Bach1 inhibition alleviates oxidative-stress-induced osteoblast apoptosis and dysfunction by enhancing Nrf2/ARE signaling activation, findings that suggest a critical role for the Bach1/Nrf2/ARE regulation axis in osteoporosis progression. Our study suggests that Bach1 may serve as a potential therapeutic target for treating osteoporosis.

MicroRNA-499a-5p inhibits osteosarcoma cell proliferation and differentiation by targeting protein phosphatase 1D through protein kinase B/glycogen synthase kinase 3ß signaling.

A number of studies have attempted to elucidate the association between mircoRNAs (miRNAs/miRs) and cancer-associated processes. The aim of the present study was to determine how miR-499a-5p intervenes in human osteosarcoma cell proliferation and differentiation. The cancerous tissues and adjacent non-cancerous tissues of 62 patients with osteosarcoma (OS) were collected. miRNA microarray analysis revealed that 29 miRNAs were upregulated while 26 were downregulated, among which miR-499a-5p expression was the most decreased. Western blot analysis and reverse transcription-quantitative polymerase chain reaction demonstrated that the mRNA and protein expression of miR-499a-5p was lower, while that of protein phosphatase 1D (PPM1D) was higher in OS tissues compared with expression levels in normal tissues. Furthermore, miR-499a-5p expression was markedly decreased in the metastatic tumors and in those at stage III+IV compared with the non-metastatic tumors and those at stage I, respectively. In addition, following transfection of the human OS MG-63 cell line with an miR-499a-5p mimic, the expression of miR-499a-5p was elevated while the protein and mRNA expression of PPM1D was decreased. When combining these findings with the information obtained from the Targetscan predictive software, it was confirmed that PPM1D was targeted by miR-499a-5p. In MG-63 cells transfected with an miR-499a-5p mimic, PPM1D-associated downstream proteins phosphorylated protein kinase B (p-Akt) and phosphorylated glycogen synthase kinase 3ß (p-GSK-3ß) were significantly downregulated compared with the negative control (NC) group, while the expression of p-Akt and p-GSK-3ß were significantly elevated in the tumor tissues compared with the adjacent non-tumor tissues. Simultaneously, the growth and proliferation activity of MG-63 cells were notably reduced when transfected with the miR-499a-5p mimic, compared with the NC group. Therefore, it may be concluded that miR-499a-5p suppresses OS cell proliferation and differentiation by targeting PPM1D through modulation of Akt/GSK-3ß signaling.

Blockade of Interleukin 6 by Rat Anti-mouse Interleukin 6 Receptor Antibody Promotes Fracture Healing.

Level of interleukin 6 (IL-6) is associated with fracture healing. This study was performed to explore the effect of IL-6 blockade on fracture healing. Clinical serum levels of IL-6 and tumor necrosis factor-α (TNF-α) were evaluated by enzyme-linked immunosorbent assay (ELISA). For animal experiments, the IL-6 levels after fracture and treatment with rat anti-mouse IL-6 receptor antibody (MR16-1) were assessed. Then, mice were assigned into four or seven groups: control group, fracture group, isotype IgG group, and MR16-1 groups. Serum levels of IL-6 and TNF-α, relative flexural rigidity, and mRNA levels of osteoblast-specific genes were respectively assayed by ELISA, three-point bending test, and quantitative reverse transcription PCR (qRT-PCR). Serum levels of IL-6 and TNF-α after fracture in humans and mice were increased. The increase in IL-6 and TNF-α levels in murine serum was attenuated by MR16-1 treatment. The three-point bending test showed the relative flexural rigidity of the femur was decreased after fracture, whereas the decrease was alleviated by MR16-1 treatment. The qRT-PCR results demonstrated mRNA levels of osteoblast-specific genes were upregulated after fracture and then further upregulated by MR16-1 treatment in a dose-dependent manner. Collectively, the serum level of IL-6 was elevated after fracture both in clinical and murine samples. IL-6 blockade by MR16-1 promoted fracture healing, which might be associated with changes in expression of osteoblast-specific genes.

Ginsenoside Rb2 inhibits osteoclast differentiation through nuclear factor-kappaB and signal transducer and activator of transcription protein 3 signaling pathway.

Ginsenoside-Rb2 (Rb2) is a 20(S)-protopanaxadiol glycoside extracted from ginseng possessing various bioactivities which has drawn considerable interest regarding the area of bone metabolism. However, the effect of Rb2 on osteoclast differentiation remains unknown. In this study, we aimed to investigate the potential role of Rb2 in regulating osteoclast differentiation and the underlying molecular mechanisms. Osteoclast differentiation was induced by receptor activator nuclear factor-kappaB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in mouse RAW 264.7 cells. The results showed that Rb2 dose-dependently inhibited the formation of the tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP expression. Furthermore, Rb2 promoted osteoprotegerin expression and bone resorption. The expression of osteoclast marker genes including nuclear factor of activated T cells c1 (NFATc1), c-Fos, OSCAR, and cathepsin K were also markedly inhibited by Rb2 treatment. Moreover, Rb2 significantly inhibited the RANKL-induced NF-κB activation. In addition, Rb2 also markedly suppressed the activation of signal transducer and activator of transcription protein 3 (STAT3) signaling pathway. Interestingly, the knockdown of STAT3 significantly strengthened the inhibitory effect of Rb2 on osteoclast differentiation. Taken together, our study suggests that Rb2 inhibits osteoclast differentiation associated with blocking NF-κB and STAT3 signaling pathways.

MicroRNA-34c promotes osteoclast differentiation through targeting LGR4.

MicroRNAs have emerged as important regulators of osteoclast differentiation in recent years. Of these, miR-34c has been reported to play an important role in bone development. However, its role and the underlying mechanism in osteoclast differentiation remains poorly understood. In this study, we aimed to investigate the precise role and molecular mechanism of miR-34c in osteoclast differentiation. We found an obvious increase in miR-34c expression during osteoclast differentiation in osteoclast precursors induced by receptor activator of nuclear factor κB (NF-κB) ligand and macrophage colony-stimulating factor in vitro. Further experiments showed that overexpression of miR-34c significantly promoted osteoclast differentiation while suppression of miR-34c showed the opposite effect. Interestingly, bioinformatics analysis and dual-luciferase reporter assays showed that miR-34c targets the 3'-untranslated region of leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4). The expression of LGR4 was regulated by miR-34c in osteoclasts. Moreover, miR-34c regulated NF-κB and glycogen synthase kinase 3-ß signaling during osteoclast differentiation. Overexpression of LGR4 partially reversed the promoting effect of miR-34c overexpression on osteoclast differentiation. Taken together, our study suggests that miR-34c contributes to osteoclast differentiation by targeting LGR4, providing novel insights into understanding the molecular mechanism underlying osteoclast differentiation.

Approach to osteomyelitis treatment with antibiotic loaded PMMA.

BACKGROUND: To reduce the incidence of osteomyelitis infection, local antibiotic impregnated delivery systems are commonly used as a promising and effective approach to deliver high antibiotic concentrations at the infection site. OBJECTIVE: The objective of this review was to provide a literature review regarding approach to osteomyelitis treatment with antibiotic loaded PMMA. STUDY DESIGN: Literature study regarding osteomyelitis treatment with antibiotic loaded carriers using key terms Antibiotic, osteomyelitis, biodegradable PMMA through published articles. Hands searching of bibliographies of identified articles were also undertaken. CONCLUSION: We concluded that Antibiotic-impregnated PMMA beads are useful options for the treatment of osteomyelitis for prolonged drug therapy.

Gastrodin: an ancient Chinese herbal medicine as a source for anti-osteoporosis agents via reducing reactive oxygen species.

Increased levels of reactive oxygen species (ROS) are a crucial pathogenic factor of osteoporosis. Gastrodin, isolated from the traditional Chinese herbal agent Gastrodia elata, is a potent antioxidant. We hypothesized that gastrodin demonstrates protective effects against osteoporosis by partially reducing reactive oxygen species in human bone marrow mesenchymal stem cells (hBMMSCs) and a macrophage cell line (RAW264.7 cells). We investigated gastrodin on osteogenic and adipogenic differentiation under oxidative stress in hBMMSCs. We also tested gastrodin on osteoclastic differentiation in RAW264.7 cells. Hydrogen peroxide (H2O2) was used to establish an oxidative cell injury model. Our results showed that gastrodin significantly promoted the proliferation of hBMMSCs, improved some osteogenic markers, reduced lipid generation and inhibited the mRNA expression of several adipogenic genes in hBMMSCs. Moreover, gastrodin reduced the number of osteoclasts, TRAP activity and the expression of osteoclast-specific genes in RAW264.7 cells. Gastrodin suppressed the production of reactive oxygen species in both hBMMSCs and RAW264.7 cells. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Our data revealed that gastrodin treatment reduced the activity of serum bone degradation markers, such as CTX-1 and TRAP. Importantly, it ameliorated the micro-architecture of trabecular bones. Gastrodin decreased osteoclast numbers in vivo by TRAP staining. To conclude, these results indicated that gastrodin shows protective effects against osteoporosis linking to a reduction in reactive oxygen species, suggesting that gastrodin may be useful in the prevention and treatment of osteoporosis.

Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway.

Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5'-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.

MicroRNA­125b suppresses the proliferation and osteogenic differentiation of human bone marrow­derived mesenchymal stem cells.

The regressive biological function of human bone marrow­derived mesenchymal stem cells (hBMSCs) is one of the key factors resulting in the decrease of bone mass in senile osteoporosis. MicroRNAs (miRs) are non­coding small RNAs involved in various gene regulation processes. Whether any miR(s) are involved in the progression of osteoporosis by regulating the biological function of hBMSCs remains to be elucidated. The present study aimed to compare the expression levels of miR­125b in hBMSCs derived from senile osteoporotic patients with that of control (normal) subjects. A significantly upregulated expression of miR­125b in osteoporotic hBMSCs was detected. To elucidate the biological function of miR­125b in senile osteoporosis, the effects of miR­125b expression on proliferation and osteogenic differentiation of hBMSCs were assessed using gain­ and loss­of­function studies. It was evident that the overexpression of a miR­125b mimic was able to suppress the proliferative and osteogenic differentiation of senile hBMSCs. In contrast, repression of the function of miR­125b by transfection of an miR­125b inhibitor promoted the proliferation and osteogenic differentiation of hBMSCs. Furthermore, the potential target gene of miR­125b, osterix (Osx), was examined. The results of the present study strongly suggested that miR­125b may regulate osteogenic differentiation of hBMSCs through the modulation of Osx expression.

Overexpression of integrin a2 promotes osteogenic differentiation of hBMSCs from senile osteoporosis through the ERK pathway.

UNLABELLED: Osteoporosis is a major health problem affecting the aging population, especially in patients 65 years of age and older. The imbalance between bone formation and bone resorption is generally accepted as the essential mechanism leading to osteoporosis. In addition to the abnormal activation of osteoclast-mediated bone resorption, the dysfunction of bone marrow stromal cells (BMSCs) in mediating bone formation has been accepted as a major contributor to the progression of senile osteoporosis. RESULTS: In our study, senile osteoporotic hBMSCs displayed a decreasing capacity for proliferation and osteoblast differentiation, which was associated with the downregulation of integrin α2. Forced ectopic integrin α2 expression using a lentivirus vector reversed the dysfunction of senile osteoporotic hBMSCs. Additionally, the overexpression of integrin α2 upregulated the levels of Runx2 and Osterix. Mechanically, Western blot analyses revealed that integrin α2 phosphorylated ERK1/2 and the inactivation of ERK by PD98059 suppressed the osteoblastic differentiation of hBMSCs, suggesting that integrin α2 promotes osteoblast proliferation through the activation of ERK1/2 MAPK. CONCLUSION: Taken together, our results show that hBMSCs obtained from senile osteoporotic patients gradually lose their capability to differentiate along the osteogenic lineage and proliferate, which might be associated with the abnormal regulation of the integrin α2/ERK/Runx2 signaling pathway undergoing senile osteoporosis.

Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells.

Oxidative stress can induce apoptosis and decrease activities of osteoblasts. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), is a potent antioxidant derived from a Chinese herb Polygonum multiflorum Thunb. To evaluate the protective effect provided by TSG to osteoblastic MC3T3-E1 cells, the cells were pretreated with TSG for 24h before being treated with 0.3mM hydrogen peroxide (H(2)O(2)) for 24 h, then some markers of osteoblast function and oxidative damage of the cells were examined. Our data demonstrated that TSG significantly (P< 0.05) increased cell survival, alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of ALP, collagen I (COL-I) and osteocalcin (OCN) in the presence of H(2)O(2). In addition, TSG decreased the production of receptor activator of nuclear factor-κB ligand (RANKL), interleukin-6 (IL-6), intracellular reactive oxygen species and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells induced by H(2)O(2). Taken together, these results demonstrated that the protective effect provided by TSG to osteoblastic MC3T3-E1 cells was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage of the cells. Our results indicated that TSG may be effective in providing protection against osteoporosis associated with oxidative stress.