Increased circulating microRNA-21 level as a potential indicator for predicting a higher risk of incident fragility fractures.

CONTEXT: As a common disease in the elderly, osteoporosis clearly increases the risk of fractures, leading to higher mortality, but the current markers to estimate the risk of fractures are limited. MicroRNA-21 (miR-21) may play an important role in osteoporosis, but the link of this biomarker with fractures was undetermined. OBJECTIVES: We aimed to investigate the association between miR-21 levels and the presence of fragility fractures. METHODS: A total of 200 patients were recruited and miR-21 was collected from baseline serum. The correlation between miR-21 and the fracture risk assessment tool (FRAX) score was analyzed. The incidence of fragility fractures was presented by Kaplan-Meier analysis, and Cox regression analysis was utilized to evaluate risk factors. The diagnostic value of miR-21 was conducted by the area under curve (AUC). RESULTS: The FRAX score was significantly associated with miR-21 level (p<0.001). According to the 50th percentile of miR-21 content in the overall distribution, the cumulative incidence of fragility fractures was significantly higher in patients with higher miR-21 levels than those with lower levels (75.4, 95 % CI: 69.0-81.8 vs. 59.2, 95 % CI: 42.1-76.3, p<0.001). The results of the Cox regression analysis showed that the miR-21 level was an independent risk factor linked to the incidence of fracture (p=0.005). The optimal cut-off value of the miR-21 was 6.08, and the AUC for predicting fracture was 0.718 (95 % CI, 0.645-0.790). CONCLUSIONS: This study showed that miR-21 has optimal diagnostic performance in the discrimination of fragility fracture, and the circulating miR-21 level in predicting the risk of fragility fracture may have a certain value.

PINK1 regulates apoptosis of osteosarcoma as the target gene of cisplatin.

BACKGROUND: Osteosarcoma is a common primary bone malignancy prevalent among adolescents and young adults. PTEN-induced kinase 1 (PINK1) regulates Parkinson's disease, but its role in cancers is unknown. OBJECTIVE: This study was designed to analyze the mechanism by which PINK1 affects osteosarcoma using bioinformatics and cell experiments. MATERIALS AND METHODS: The gene expression profiles were downloaded from the TARGET database. Several online databases were used to analyze the expression and protein‒protein interaction networks. CCK-8 cell viability assays and cisplatin treatment were used to assess cell activity with or without cisplatin treatment. Acridine orange/ethidium bromide (AO/EB) fluorescence staining was used to calculate the percentage of apoptotic cells. RESULTS: Through bioinformatics analysis, we found that high expression of PINK1 was associated with poor prognosis in patients with osteosarcoma, and PINK1 inhibited apoptosis and promoted proliferation pathways. Next, we found that both PINK1 mRNA and protein levels were upregulated in osteosarcoma tissues. Additionally, we found that PTEN was reduced, while FOXO3a was markedly increased in osteosarcoma, suggesting that FOXO3a and not PTEN induced the overexpression of PINK1. CCK-8 and clonogenic assays showed that the knockdown of PINK1 decreased the growth of U2OS osteosarcoma cells. Ki67 immunofluorescence staining revealed that reduced cell proliferation in U2OS cells resulted in the depletion of PINK1. In addition, our AO/EB staining results indicated that the knockdown of PINK1 resulted in an increase in apoptotic cells and increased the levels of cleaved caspase-3. Furthermore, our experiments revealed that cisplatin promotes OS cell apoptosis by downregulating PINK1. CONCLUSION: Collectively, our findings demonstrate that PINK1 is crucially involved in osteosarcoma and suggests that it can promote the apoptosis of OS cells as the downstream target gene of cisplatin.

lncRNA HAND2-AS1 is downregulated in osteoarthritis and regulates IL-6 expression in chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a leading cause of disability. The incidence of OA is progressively rising due to the diminishing levels of physical activity and ever-expanding aging population. However, the mainstay for OA treatment only can improve symptoms without delay the progression of this severe disease. This study aimed to explore the biological role and clinical function of lncRNA HAND2-AS1 in OA. METHODS: Blood samples and synovial fluid were collected from OA patients and normal subjects. HAND2-AS1 expression was detected by qRT-PCR and IL-6 expression was detected by ELISA. The plasma levels of HAND2-AS1 were also detected in different ages, stages, and gender of OA patients and controls. Furthermore, the ROC curve was used to analyze whether HAND2-AS1 can distinguish OA patients from normal subjects. Also, Pearson correlation coefficient analysis was used to analyze the correlation between lncRNA HAND2-AS1 and IL-6. In addition, Western blot was used to detect the IL-6 level upon HAND2-AS1 over-expression in chondrocytes and qRT-PCR was used to detect the HAND2-AS1 level after endogenous IL-6 treatment. RESULTS: HAND2-AS1 and IL-6 were dysregulated in plasma and synovial fluid of OA patients. The expression of HAND2-AS1 in plasma of OA patients was decreased with aging and progression. Furthermore, HAND2-AS1 downregulation effectively distinguished OA patients from the healthy controls. Over-expression of HAND2-AS1 inhibited IL-6 expression in chondrocytes, while treatment with exogenous IL-6 did not affect HAND2-AS1 expression. CONCLUSION: HAND2-AS1 effectively distinguished OA patients from the healthy controls and regulates IL-6 expression in human chondrocytes. TRIAL REGISTRATION: ChiCTR, ChiCTR2000038635 . Registered 11 February 2019.

Glycyrrhizin, an HMGB1 inhibitor, Suppresses Interleukin-1ß-Induced Inflammatory Responses in Chondrocytes from Patients with Osteoarthritis.

BACKGROUND: High mobility group box 1 (HMGB1) is increased in osteoarthritis (OA) tissue and chondrocytes stimulated with interleukin-1ß (IL-1ß). Suppression of HMGB1 expression is correlated with reduced inflammatory responses induced by IL-1ß. This study aimed to investigate how inhibition of HMGB1 by glycyrrhizin might affect inflammatory responses and viability of OA patient-derived chondrocytes treated with IL-1ß. DESIGN: The amounts of HMGB1 in the cartilage tissue and synovial fluid in patients with OA were assessed by Western blot and enzyme-linked immunosorbent assay (ELISA). Chondrocytes were extracted from OA patients and maintained in culture. The impact of glycyrrhizin on IL-1ß-induced cell toxicity and inflammatory mediators and cytokines, including prostaglandin E2 (PGE2), nitric oxide (NO), proinflammatory cytokines, and metalloproteases (MMPs), were assessed by ELISA, Western blot, quantitative real-time polymerase chain reaction, and the Griess reagent assay. RESULTS: We confirmed that HMGB1 was significantly upregulated in specimens acquired from patients with OA. HMGB1 inhibition by glycyrrhizin improved cell viability of chondrocytes treated with IL-1ß. Glycyrrhizin suppressed IL-1ß-induced upregulation of HMGB1 and inflammatory mediators and cytokines, including PGE2, NO, proinflammatory cytokines, and MMPs. CONCLUSION: Our results indicate that glycyrrhizin may be a potential therapy for OA patients and these promising findings warrant further study for clinical application.

High-Throughput Metabolomics Discovers Metabolic Biomarkers and Pathways to Evaluating the Efficacy and Exploring Potential Mechanisms of Osthole Against Osteoporosis Based on UPLC/Q-TOF-MS Coupled With Multivariate Data Analysis.

Postmenopausal osteoporosis (PMOP) is the most common metabolic bone illness among the elderly especially in postmenopausal women resulting from a reduction in bone mineral density, but there is no effective drug at present. The study was aimed at evaluating efficacy of osthole against osteoporosis using high-throughput metabolomics method. The blood samples for illustrating the pathological mechanism of PMOP and exploring the efficacy of osthole treatment (ST) were collected to perform metabolites and metabolic profiles and pathways analysis using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and pattern recognition methods. In addition, backbone weight, the bone density, and some vital biochemical indexes were also detected. A total of 28 metabolites were identified as biomarkers for ovariectomized-osteoporosis model, and ST could significantly regulate 19 of them including lysine, linoleic acid, 3-hydroxybutyric acid, prostaglandin F2a, taurocholic acid, LysoPC(15:0), l-carnitine, glucose, arginine, citric acid, corticosterone, ornithine, tryptophan, arachidonic acid, Cer(d18:0/18:0), glutamine, uric acid, 8-HETE, estriol, which mainly related with 13 metabolic pathways, such as linoleic acid metabolism, starch, and sucrose metabolism, arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, citrate cycle (TCA cycle), and arginine biosynthesis. The ovariectomized model (OVX) rats display a significant decrease bone density, TGF-ß1, NO, and NOS level, and a significant increase bone weight, IL-6, TNF-α, and Ca 2+ level. These parameters in the ST rats were evidently improved as compared to the OVX group. ST effectively mitigated ovariectomy-induced osteoporosis in rats by affecting endogenous metabolite-related metabolic mechanism and showed the natural alternative with potential for the treatment of PMOP.

Deferoxamine enhances neovascularization and accelerates wound healing in diabetic rats via the accumulation of hypoxia-inducible factor-1α.

AIMS: Hypoxia-inducible factor (HIF)-1α plays a pivotal role during the process of wound healing. Previous studies reported that deferoxamine (DFO) could increase HIF-1α stability. This study aimed to investigate the effects of DFO on wound healing in diabetic rats and explore the underlying mechanism both in vivo and in vitro. METHODS: An excisional diabetic wound model was established and the wound healing among vehicle control, DFO and vascular endothelial growth factor (VEGF) treatment groups was evaluated by macroscopy, histology and Western blot analysis. Human umbilical vein endothelial cells (HUVECs) were treated with DFO or HIF-1α siRNA, and then endothelial tube formation, cell proliferation and migration were examined. RESULTS: DFO-treated wounds exhibited accelerated wound healing with enhanced granulation formation and increased re-epithelialization. Compared to the vehicle or VEGF treatment, DFO significantly increased neovascularization through up-regulation of HIF-1α and target genes including VEGF and stromal cell-derived factor-1α (SDF-1α). DFO failed to stimulate the expression of VEGF and SDF-1α in HUVECs depleted of HIF-1α. In addition, DFO promoted the angiogenic-associated processes of endothelial tube formation, cell proliferation and migration in HIF-1α dependent manner. CONCLUSIONS: DFO enhances neovascularization and accelerates diabetic wound healing through the accumulation of HIF-1α and the regulation of endothelial cell function.

Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis.

Despite advances in wound closure techniques and devices, there is still a critical need for new methods of enhancing the healing process to achieve optimal outcomes. Recently, stem cell therapy has emerged as a new approach to accelerate wound healing. Adipose-derived stem cells (ASCs) hold great promise for wound healing, because they are multipotential stem cells capable of differentiation into various cell lineages and secretion of angiogenic growth factors. The aim of this study was to evaluate the benefit of ASCs on wound healing and then investigate the probable mechanisms. ASCs characterized by flow cytometry were successfully isolated and cultured. An excisional wound healing model in rat was used to determine the effects of locally administered ASCs. The gross and histological results showed that ASCs significantly accelerated wound closure in normal and diabetic rat, including increased epithelialization and granulation tissue deposition. Furthermore, we applied GFP-labeled ASCs on wounds to determine whether ASCs could differentiate along multiple lineages of tissue regeneration in the specific microenvironment. Immunofluorescent analysis indicated that GFP-expressing ASCs were costained with pan-cytokeratin and CD31, respectively, indicating spontaneous site-specific differentiation into epithelial and endothelial lineages. These data suggest that ASCs not only contribute to cutaneous regeneration, but also participate in new vessels formation. Moreover, ASCs were found to secret angiogenic cytokines in vitro and in vivo, including VEGF, HGF, and FGF2, which increase neovascularization and enhance wound healing in injured tissues. In conclusion, our results demonstrate that ASC therapy could accelerate wound healing through differentiation and vasculogenesis and might represent a novel therapeutic approach in cutaneous wounds.

Successful repair of a critical-sized bone defect in the rat femur with a newly developed external fixator.

Regeneration of segmental bone defects has been a clinical challenge. Recent advances in the field of tissue engineering have developed new procedures enabling bone regeneration. Small animal models capable of supporting weight-bearing femoral defects are integral parts of orthopedic biomedical research. However, a drawback of bone healing research is the lack of stable and adaptable fixation devices for small animals. Therefore, we developed and evaluated an adjustable external fixation device in the maintenance of non-healable (ie critical-sized) segmental defects, and in the fixation of tissue-engineered bone grafts in a rat model. Male Sprague-Dawley rats (n = 24) underwent a femoral osteotomy to create a non-healing segmental defect (6 mm size), which was stabilized with the fixator. A treatment group (12 rats) received tissue-engineered bone graft implants consisting of biphasic calcium phosphate blocks seeded with bone mesenchymal stem cells, while other 12 animals received no bone graft (non-treatment group). The osteotomy gap remained unchanged in the non-treatment group over the 12-week period, indicating that the 6-mm bone defect is really non-healable in the rat femur and that the device has sufficient stability for the management of critical-sized femoral defects. At 12 weeks, the treatment group maintained the bone length throughout the study period and showed bridging of the defect, with remarkable new bone formation. In contrast, the non-treatment group showed marginal new bone formation, but no apparent healing. In conclusion, the novel device provides substantial benefits in the maintenance of critical-sized femoral defects and tissue-engineered bone grafts in a rat model.