Repair of osteonecrosis of the femoral head : 3D printed Cervi cornus Colla deproteinized bone scaffolds.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a common joint disease and a major cause of morbidity. OBJECTIVE: In this study Cervi cornus Colla (CCC) deproteinized bone scaffolds were designed and three dimensional (3D)-printed for the repair of ONFH in rats. MATERIAL AND METHODS: The CCC-deproteinized bone scaffolds were 3D-printed using polycaprolactone mixed with the CCC-deproteinized bone powder. The scaffolds were viewed under a scanning electron microscope and subjected to compression analysis. Osteoblasts were isolated from rats and coated onto the scaffolds. Cell proliferation assays were performed with the MTT (3­[4,5-dimethylthiazole­2]-2,5-diphenyltetrazolium bromide) kit from Promega. An ONFH was induced in rats and a CCC-deproteinized bone scaffold was implanted into the necrotic femoral head. General observations, X­ray imaging, and pathological examination of the femoral head were performed to evaluate the treatment of ONFH in the rats. RESULTS: The scaffolds were porous with a mean pore diameter of 315.70 ± 41.52 nm and a porosity of 72.86 ± 5.45% and exhibited favorable mechanical properties and degradation. In vitro assays showed that osteoblasts accumulated in the pores and adhered to the scaffolds. The CCC-deproteinized bone scaffolds enhanced the proliferation of osteoblasts. The in vivo experiments revealed that the general observation score of rats in the CCC-scaffold implanted group was significantly higher than that in the control group. The X­ray images showed significant alleviation of ONFH in the CCC-deproteinized bone scaffold implanted rats. The femoral heads of rats in the treatment group showed less destruction or ossification of cartilage cells, few bone cement lines, very little necrosis or irregularities on the cartilage surface and only a small amount of inflammatory cell infiltration in the medullary cavity. CONCLUSION: These results suggest that CCC-deproteinized bone scaffold implants facilitated the repair of ONFH in rats. This research provides a new therapeutic approach for the repair of early and mid-term ONFH.

EFFECT OF COLLA CORNUS CERVI COMBINED WITH LV-MEDIATED BMP7 TRANSFECTED BMSCs ON ANFH IN RATS.

In the present study, we investigated the combined effect of Colla Comus Cervi (CCC) and BMP7-overexpressing bone marrow-derived mesenchymal stem cells (BMSCs) on osteogenic induction and the treatment of avascular necrosis of the femoral head (ANFH). BMSCs were isolated from rats. BMP7-overexpressing BMSCs were generated by lentiviral-mediated gene transduction. Cell proliferation, alkaline phosphatase (ALP) activity, osteogenesis related gene expression, osteocalcin levels, and calcified nodules were quantified and compared between four groups: untreated controls, BMSCs cultured with CCC complex medium, BMP7-overexpressing BMSCs, and BMP7-overexpressing BMSCs cultured with CCC complex medium (CCC+BMP7). CCC+BMP7 BMSCs showed higher proliferation rate. ALP activity and osteaocalcin content were significantly increased in CCC+BMP7 BMSCs. The osteogenesis related genes, COLI, and integrin-α2, -α5, and -ß1, were expressed significantly higher in CCC+BMP7 BMSCs. The number of calcified nodules in the CCC+BMP7 group was significantly higher than that in other groups. For in vivo assays, ANFH was induced in rats, and BMSCs were injected into the femoral head of the lower left extremity. In rats with induced ANFH, general observation scores of the CCC+BMP7 injected group were significantly higher than the model group. X-ray and microscopic observations revealed that ANFH was significantly improved and femoral head cells gradually recovered in rats treated with CCC+BMP7 BMSCs. Our results suggest that CCC+BMP7 significantly promote the proliferation and osteogenic differentiation of BMSCs in vitm. CCC+BMP7 BMSCs promote the ability of repairing ANFH in rats, providing a new therapeutic paradigm for the treatment of ANFH.

Role and interrelationship of Gα protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves.

Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m(-2) UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including Gα protein, hydrogen peroxide (H2O2), and nitric oxide (NO). UV-B triggered a significant increase in H2O2 or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H2O2 and NO generation are regulated by GPA1, the Gα-subunit of heterotrimeric G proteins. UV-B-dependent H2O2 and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, exogenously applied H2O2 or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cGα AtrbohD/AtrbohF and cGα nia1 constructs exhibited a similar response to AtrbohD/AtrbohF and Nia1, respectively. Finally, we demonstrated that Gα activation of NO production depends on H2O2. The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H2O2 accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H2O2 production and subsequent Nia1-dependent NO accumulation.

No evidence of genetic causality between diabetes and osteonecrosis: a bidirectional two-sample Mendelian randomization analysis.

OBJECTIVE: This study aimed to examine whether diabetes mellitus is causally associated with osteonecrosis. METHOD: Using publicly accessible genome-wide association study statistics, a bidirectional two-sample Mendelian randomization analysis was carried out. In order to determine whether diabetes has a causal effect on osteonecrosis and whether osteonecrosis has a causal effect on diabetes, we extracted six date on diabetes in Europeans from IEU OpenGWAS and GWAS Catalogue and osteonecrosis in Europeans from FinnGen. We then evaluated the data using inverse variance weighting, MR-Egger regression, weighted median, weighted mode, and simple mode. The results' stability and dependability were then evaluated using sensitivity analysis and heterogeneity analysis. Finally, meta-analysis is used to further confirm if there is a relationship between diabetes and osteonecrosis. RESULTS: When diabetes was used as an exposure factor, MR-Egger regression showed that directional fold product was unlikely to bias the results. Cochran's Q test showed only minor heterogeneity in a few data sets. Multidirectional tests Egger-intercept, MR-PRESSO and funnel plots for most data did not show multidirectional and asymmetry at the gene level. Most of the IVW results showed no causal relationship between diabetes mellitus and osteonecrosis. The results of meta-analysis of IVW methods further confirmed the absence of a causal relationship. Inverse MR analysis also showed no causal relationship between osteonecrosis and diabetes. CONCLUSION: Results of bidirectional MR analysis show no evidence of causal relationship between diabetes and osteonecrosis.

The Separation of Antler Polypeptide and Its Effects on the Proliferation and Osteogenetic Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Colla Cornus Cervi (CCC) has been used as a traditional Chinese medicine in the treatment of osteoporosis and osteonecrosis of the femoral head. However, the bioavailability of CCC is seriously limited owing to its large molecular weight and complex ingredients. In the present study, antler polypeptide was separated from CCC, and the effects of antler polypeptide on rat bone marrow mesenchymal stem cells (BMSCs) were investigated. METHODS: Antler polypeptide was separated from Colla Cornus Cervi by ultrafiltration into different samples according to the molecular weight. The total peptide content of these samples was determined by the biuret method. The content of antler polypeptide in different samples was quantified by high-performance liquid chromatography (HPLC). The effects of antler polypeptide at different concentrations on the proliferation, cell cycle, alkaline phosphatase activity, and BMP7 expression of BMSCs were investigated. RESULTS: Antler polypeptide was separated by ultrafiltration into different samples: A (molecular weight <800 Da), B (molecular weight 800-1500 Da), and C (molecular weight >1500 Da). The total peptide contents of A, B, and C were 0.602 mg/mL, 8.976 mg/mL, and 38.88 mg/mL. Antler polypeptide B eluted at 14.279∼15.351 min showed that the content of antler polypeptide was significantly higher than that of A and C with a peak area of 933.80927. The BMSCs proliferation rate (84.66%) of polypeptide B was the highest at the concentration of 1.578 × 10-2 g/mL. Antler polypeptide B significantly promoted the proliferation of BMSCs with a proliferation index of 38.68%, which was significantly higher than that of the other groups. Antler polypeptide B significantly enhanced the activity of alkaline phosphatase in BMSCs compared to that of the blank group (P < 0.001). Antler polypeptide B increased the BMP7 protein expression in BMSCs. CONCLUSIONS: Results suggested that antler polypeptide may promote the proliferation and osteogenic differentiation of BMSCs. Our study lays an experimental foundation for the further development and application of antler polypeptide in medicine.