Differential circRNA and mRNA Expression Profiling During Osteogenic Differentiation in Rat Bone Marrow Mesenchymal Stem Cells.

BACKGROUND Circular RNAs (circRNAs) play important roles in gene expression and signaling pathways. The study aimed to identify the differential expression of circRNAs and mRNAs in the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) and to explore the biological function of circRNAs in the osteogenic differentiation of rBMSCs. MATERIAL AND METHODS High-throughput sequencing was used to detect differentially expressed circular RNA and mRNA during osteogenic differentiation of rBMSCs. The RNAs were analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment to predict their potential role in regulating rBMSC osteogenesis. MiRanda, Circatlas, and miRDB databases were used to predict target relationships between circRNA, miRNA, and mRNA.The regulatory network was constructed by Cytoscape (version 3.6.1). The RNA-Seq findings were validated by quantitative real-time PCR (qRT-PCR). RESULTS The results revealed that 29 differentially expressed circRNAs and 2453 differentially expressed mRNAs were detected during the osteogenic differentiation of rBMSCs. Many differentially expressed circRNAs were closely related to osteogenic differentiation of cells. Among them, circRNAs_1809 and Kitlg were the significantly increased circRNA and mRNA during osteogenic differentiation of rBMSCs. The ceRNA network showed that circRNA_1809 could target the Kitlg gene through miR-370-3p. CONCLUSIONS CircRNAs may play an important role in the osteogenic differentiation of rBMSCs. CircRNA_1809 may acts as a sponge for miR-370-3p and regulate the osteogenic differentiation of rBMSCs by targeting Kitlg; however, this hypothesis needs further verification. This study laid a theoretical foundation for further understanding the mechanism of circRNAs regulating osteogenic differentiation of rBMSCs.

CircularRNA CDR1as promotes osteogenic differentiation and angiogenesis related genes expression in mouse bone marrow mesenchymal stem cells / 口腔疾病防治

Objective@# To investigate the effects of over expression and low expression of antisense transcripts of circular RNA cerebellar degeneration associated protein 1 (CDR1as) in Balb/C mouse bone marrow mesenchymal stem cells (BMSCs) on factors related to osteogenesis and angiogenesis.@*Methods@#BMSCs were cultured and identified in vitro. The lentiviral (LV) vector containing the overexpressed and silenced circRNA CDR1as genes and the control lentivirus were respectively transfected into mouse BMSCs, and stable cell lines were screened. The cells were divided into the circRNACDR1as over expression group and the over expression control group, and the CircRNACDR1as low expression group and the low expression control group. The components were stained with Alizarin Red S and alkaline phosphatase after 14 and 21 days of osteoinduction; qRT-PCR was used to detect the target genes circRNA CDR1as, osteogenic differentiation markers alkaline phosphatase (ALP), runt- related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), osterix(Osx), collagen I (COL-1), and the mRNA expression levels of vascular endothelial grown factor (VEGF) and angiogenin-1 (Ang-1). @*Results@# The results of alizarin red staining and alkaline phosphatase staining showed that the extracellular matrix calcium precipitation and ALP staining area of the over expression experimental group was greater than its control group, and those of the low expression experimental group was less than its control group. As the number of days of osteogenic induction increased, the calcium precipitation and ALP staining in each group also increased. RT-PCR results showed that the mRNA expression levels of circRNA CDR1as, ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in the over expression experimental group BMSCs were significantly increased (P<0.001). In the low expression experimental group, the mRNA expression levels of circRNA CDR1as, ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in BMSCs were significantly reduced (P<0.001). @*Conclusion@# Over expression of the circRNA CDR1as gene promotes the osteogenic differentiation and angiogenesis of BMSCs. Low expression of the circRNA CDR1as gene inhibits the osteogenic differentiation and angiogenesis of BMSCs.

Effect of cell sheet combined with 3D printing an antler powder/silk fibroin/polyvinyl alcohol scaffold on the repair of mandibular defects in sheep / 口腔疾病防治

Objective @#To explore the feasibility of antler powder/silk fibroin/polyvinyl alcohol scaffolds as tissue engineering bone scaffolds and the relationship between their degradation performance and the healing speed of bone defects.@*Methods @# Antler powder/silk fibroin/polyvinyl alcohol scaffolds and nano hydroxyapatite/silk fibroin/polyvinyl alcohol scaffolds were prepared by 3D printing. The whole bone marrow culture method was used to prepare blood cell sheets of Altay big tail sheep’s iliac bone marrow. With observation times of 1, 2 and 3 months, the mandibular defects of 4 sheep were established. The experimental group was coated with antler powder/silk fibroin/polyvinyl alcohol scaffolds. The control group was coated with nanohydroxyapatite/silk fibroin/polyvinyl alcohol scaffolds. The negative control group was coated with gel-free sponges. According to the self-control method of the bilateral mandible defect area, scaffolds wrapped with cell membranes or gel sponges wrapped with cell membranes were implanted. At the ends of the first, second and third months after implantation, the experimental animals were killed, cone beam CT was performed, and paraffin sections were taken for HE staining to evaluate the effect of different scaffold materials on bone regeneration and scaffold degradation.@* Results@# Scanning electron microscopy showed that both groups had regular pores and good continuity, and there was no difference in pore size and porosity between the two groups (P > 0.05). The results of CBCT imaging showed that in 3 months after operation, the experimental group had significantly better repair effects on bone defects than the control group, and the degradation rate matched the bone repair rate. The bone mineral density in the center of the defect was higher than that of the control group, which was close to that of normal bone tissue. The central bone mineral density of the experimental group at each time point was higher than those of the control group and the negative control group, and the difference was statistically significant (P < 0.05). HE staining results showed that the bone cells in the experimental group were more active, with more new capillaries and bone trabeculae formed, and the scaffold material absorbed more than the control group. @*Conclusion @#The antler powder/silk fibroin/polyvinyl alcohol scaffold can promote the repair of critical bone defects. Its degradability matches its bone tissue healing rate. It is expected to become a promising scaffold material for bone tissue engineering.

Effects of hypoxia inducible factor-1α on osteogenic differentiation and angiogenesis related factors of bone marrow mesenchymal stem cells / 口腔疾病防治

Objective@#To investigate the level of hypoxia inducible factor-1α (HIF-1α) on osteoblasts and angiogenesis-associated cytokines in bone marrow mesenchymal stem cells (BMSCs) from SD rats.@*Methods@#BMSCs were isolated and cultured and identified by flow cytometry. Plasmid vectors containing upregulated and downregulated HIF-1α gene and a control vector were constructed. The plasmids were transfected into BMSCs by Lipofectamine®LTX transfection reagent, and the cells were divided into an overexpression experimental group, an overexpression control group, a low expression experimental group and a low expression control group. All components were stained with a lizarin red 3 d and 7 d after osteogenesis induction. The mRNA expression levels of the target gene HIF-1α, osteogenic differentiation-specific markers, including Runt-related transcription factor 2 (Runx2) and angiogenic markers, including platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β), were detected by RT-PCR. Western blot was used to detect the protein expression of the target proteins HIF-1α, Runx2, and PDGF-BB.@*Results@# The CD29- and CD45-positivity rates of BMSC surface markers identified by flow cytometry were 98.2% and 4.2%, respectively. RT-PCR results showed that the mRNA expression of HIF-1α, Runx2, TGF-β and PDGF-BB was observably increased (P < 0.001). The mRNA expression levels of HIF-1α, Runx2, TGF-β and PDGF-BB in BMSCs from the low expression experimental group were significantly reduced (P < 0.001). Western blot results showed that the expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the overexpression experimental group were all increased (P < 0.001). The expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the low expression experimental group were reduced (P < 0.001). Alizarin red staining results showed that the area of calcium nodules in the low expression experimental group was smaller than that in low expression control group, the area of red calcium nodules in the over expression experimental group was larger than that in over expression control group, and with the increase of osteogenic induction time, the calcification area of each group also increased.@*Conclusion@# Upregulation and downregulation of HIF-1α can regulate the osteogenic differentiation and the expression of angiogenesis related factors of BMSCs.

Research status of 3D⁃printed composite PVA bone tissue engineering scaffolds / 口腔疾病防治

@# Three dimensionally printed composite porous bone tissue engineering scaffolds have become a research focus. Composite polyvinyl alcohol (PVA) has good biocompatibilityand degradability, but it cannot be prepared indepen⁃dently because it cannot resist highmechanical resistance. This material shows many advantages, such as good biocom⁃patibility, degradability and mechanical properties, when compounded with other materials with good mechanical proper⁃ties and good biocompatibility. Therefore, 3D printed composite PVA scaffold material can optimize the performance of PVA scaffolds. This article reviews 3D printing bone scaffold technology, polyvinyl alcohol (PVA), and composite PVA scaffolds for in vivo and in vitro bone formation.

[Distribution of WNT10A gene rs10177996 polymorphism between Han and Uygur populations in Xinjiang area].

PURPOSE: To explore the characteristics of distribution of WNT10A gene rs10177996 polymorphism between Han and Uygur populations in Xinjiang area. METHODS: A cross-sectional survey on 154 Han individuals in Urumqi area and 134 Uygur individuals in Kashgar area was performed. Buccal epithelial cells were harvested using Cotton swab scraping, and DNA was extracted by special kit. After screening, the corresponding SNP segments of qualified samples were propagated by PCR. Dideoxy-mediated chain termination method was used for gene sequencing, and then, genotyping was conducted with corresponding software. Statistical analysis of genetic data was performed by SPSS 23.0 software package. RESULTS: Among Uygur nationality in Kashgar area, the frequencies of CC, CT, TT genetypes in rs10177996 were 8.21%, 30.60% and 61.19%, respectively. The allele frequency of C was 23.51% and T was 76.49%. Among Han nationality in Urumqi area, the frequencies on CC, CT, TT genetypes of rs10177996 were 9.74%, 43.51% and 46.75%, respectively. The allele frequency of C was 31.49% and T was 68.51%. When compared with Han nationality, the frequency of TT was significantly higher in Uygur nationality(P=0.046). When compared with European, the frequency of TT was significantly lower in Uygur nationality (P=0.05). When compared with European, the frequency of TT was significantly lower in Han nationality(P＜0.01). Compared with European, the distribution on C allele frequency was significantly higher, the distribution on T allele frequency was significantly lower in Han nationality (P=0.033). However, there was no significant difference between Han nationality in Urumqi area and Uygur nationality in Kashgar area (P＞0.05), and, between Uygur nationality in Kashgar area and European (P＞0.05). Meanwhile, there was no significant difference in gender between Uygur nationality in Kashgar area and Han nationality in Urumqi area (P＞0.05). CONCLUSIONS: The distributions of WNT10A gene rs10177996 SNP among Han nationality in Urumqi area, Uygur nationality in Kashgar area and the reported European population are obviously different.

Effect of different hydrofluoric acid etching methods on the surface and adhesion strength of two ceramic inlays / 口腔疾病防治

Objective@#o study the effect of cleaning treatment with hydrofluoric acid (HF) on the surface and bonding strength of IPS e.max and Vita Mark II ceramic inlays. @*Methods@#Fifty pieces of IPS e.max and Vita Mark II ceramic inlay specimens were made separately using CAD/CAM. After uniformly bonding surfaces using 9% HF etching, they were randomly divided into an untreated control group (group A) and the following experimental groups: neutralizing powder (B group), 37% phosphoric acid (group C), ultrasonic cleaning (group D) and neutralizing powder + 37% phosphoric acid + ultrasonic cleaning (group E). Each set of 8 specimens was bonded to Variolink N resin adhesive under standard conditions. The shear adhesive strength was measured after exposure to a constant-temperature water bath at 37 ℃ for 24 h. The location of the fracture and the type of adhesion failure were recorded. The shear adhesion and the average strength of the connection were analyzed. The remaining 2 specimens were used for scanning electron microscopy (SEM) to observe the surface morphology, including the crystal structure, pore pattern, and residue.@*Results @# The results were similar for the IPS e.max and Vita Mark II inlays. The maximum bond strength was observed in the IPS e.max ceramic inlays in group E, with an average bond strength 11.96 MPa higher than that in group A. Among the Vita Mark II porcelain inlays, the maximum bond strength was observed in group E. The average bond strength was 9.74 MPa higher than that in group A. The results of the statistical analysis were similar for the IPS e.max and Vita Mark II porcelain inlays, with significant differences in the bond strengths between groups C, D, and E and the control group (P < 0.05). There was no significant difference in the adhesive strength between groups B and A. At the same time, there was no significant difference in the bond strength between the treatment groups B, C, D, and E (P > 0.05). SEM revealed that the pores on the surface of ceramics subjected to the acid etching treatment were broadened and uniform, with less residue than observed in the control group. The effects of treatments D and E were the best. @*Conclusion@#The HF etching treatment can enhance the bonding strength of IPS e.max and Vita Mark Ⅱ ceramic inlays while leaving little residue, and the joint strength is highest when the joints are treated together.

Risk factors for dislocation after revision total hip arthroplasty: A systematic review and meta-analysis.

BACKGROUND: No formal systematic review or meta-analysis was performed up to now to summarize the risk factors of dislocation after revision total hip arthroplasty(THA). AIMS: The present study aimed to quantitatively and comprehensively conclude the risk factors of dislocation after revision total hip arthroplasty. METHODS: A search was applied to CNKI, Embase, Medline, and Cochrane central database (all up to October 2016). All studies assessing the risk factors of dislocation after revision THA without language restriction were reviewed, and qualities of included studies were assessed using the Newcastle-Ottawa Scale. Data were pooled and a meta-analysis completed. RESULTS: A total of 8 studies were selected, which altogether included 4656 revision THAs. 421 of them were cases of dislocation occurred after surgery, suggesting the accumulated incidence of 9.04%. Results of meta-analyses showed that age at surgery (standardized mean difference -0.222; 95% CI -0.413-0.031), small-diameter femoral heads (≤28 mm) (OR 1.451; 95%CI 1.056-1.994), history of instability (OR 2.739; 95%CI 1.888-3.974), number of prior revisions ≥ 3 (OR, 2.226; 95% CI, 1.569-3.16) and number of prior revisions ≥ 2 (OR 1.949; 95% CI 1.349-2.817), acetabular components with elevated rim liner were less likely to develop dislocation after revision THA (OR 0.611; 95% CI 0.415-0.898). CONCLUSIONS: Related prophylaxis strategies should be implemented in patients involved with above-mentioned risk factors to prevent dislocation after revision THA.

MicroRNA-126 Overexpression Inhibits Proliferation and Invasion in Osteosarcoma Cells.

This study investigated the biological effects of microRNA-126 overexpression in human MG63 osteosarcoma cells. A recombinant plasmid expressing microRNA-126, pcDNA6.2-microRNA-126, was constructed and transfected into MG63 cells. Using real-time fluorogenic quantitative polymerase chain reaction, the microRNA-126 expression was measured in microRNA-126-MG63 group, Ctrl-MG63 group, and blank group. Cell proliferation, cell cycle distribution, cell migration, and invasion were analyzed using methyl thiazolyl tetrazolium assay, flow cytometer, wound-healing assay, and transwell assay, respectively. As expected, microRNA-126 expression was higher in microRNA-126-MG63 group than in Ctrl-MG63 group and blank group (both P < .05). After 48/72 hours of transfection, cell proliferation in microRNA-126-MG63 group was significantly reduced compared to blank group (both P < .05). Compared to blank group, cell population in G0/G1 stage was significantly higher in microRNA-126-MG63 group, accompanied by lower cell numbers in the S and G2/M phases and decreased proliferation index (all P < .05). Wound-healing assay showed a wider scratch width in microRNA-126-MG63 group and reduced cell migration than blank group (both P < .05). Cells overexpressing microRNA-126 exhibited reduced ADAM9 expression levels compared to other 2 groups (all P < .05), suggesting ADAM9 is a target of microRNA-126. Cell proliferation, migration, and invasion rates were reduced in microRNA-126 group after 48/72 hours of transfection, compared with blank group (all P < .05). Cotransfection of pcDNA6.2-microRNA-126 and pMIR-ADAM9 into MG63 cells led to higher cell proliferation, invasion, and migration rates, compared with transfection of pcDNA6.2-microRNA-126 alone (all P < .05). In summary, our data show that microRNA-126 inhibits cell proliferation, migration, and invasion in human osteosarcoma cells by targeting ADAM9.

Apoptosis prediction via inhibition of AKT signaling pathway by neogrifolin.

Neogrifolin, a natural biologically active substance isolated from the edible bodies of the mushroom Albatrellus confluens, has been shown to possess several pharmacological properties. No studies were investigated against osteosarcoma cancer. Hence, in this study, we investigated the apoptosis-inducing effects and the mechanisms of neogrifolin on human osteosarcoma cells. Our results demonstrated that neogrifolin induced concentration- and time-dependent suppression of proliferation. Further, induction of apoptosis in U2OS and MG63 osteosarcoma cell lines were also observed. Neogrifolin induced the release of cytochrome c accompanied by activation of caspase-9, caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, z-VAD-fmk, a universal inhibitor of caspases, prevented caspase-3 activation and PARP cleavage and inhibited neogrifolin-induced cell growth inhibition. Furthermore, neogrifolin treatment resulted in a reduction of phosphorylated AKT level, FOXO transcription factor, and glycogen synthase kinase 3 (GSK3). Knockdown of GSK3 with siRNA inhibited the apoptotic effects of neogrifolin. On the other hand, neogrifolin treatment also down-regulated the expression of the inhibitor of apoptosis protein (IAP) in both osteosarcoma cells. Collectively, our results suggested that neogrifolin is a potential candidate for osteosarcoma.

Transglutaminase-2 is Involved in Cell Apoptosis of Osteosarcoma Cell Line U2OS Under Hypoxia Condition.

Osteosarcoma is the most common type of solid bone cancer, which is the second leading cause of cancer-related death. Hypoxia is an ordinary phenomenon in solid tumor tissues and can induce cell apoptosis but the specific molecular mechanism remains unclear. In this study, we explored the effect and the molecular mechanism of Transglutaminase 2 (TG2) on cell apoptosis in osteosarcoma U2OS cells under hypoxia. We found the enzymatic activity of TG2 is significantly increased and the expression of TG2 is remarkably up-regulated under hypoxia condition. Cell apoptotic rate is markedly increased upon knockdown of TG2 by siRNA under hypoxia. We further investigated the mechanism of cell apoptosis and found Bax protein is significantly increased after depletion of TG2 under hypoxia. Moreover, our data also show that cytochrome C (Cyt C) is significantly increased in cytoplasm and markedly decreased in mitochondria of U2OS cells after depletion of TG2 under hypoxia. Our results suggest that TG2 can inhibit tumor cell apoptosis through down-regulation of Bax and prevention of release Cyt C from mitochondria into cytoplasm.

Serum levels of 25-hydroxyvitamin D and functional outcome among postmenopausal women with hip fracture.

OBJECTIVE: The main objective of the current study was to assess the distribution and its prognostic value of serum 25-hydroxyvitamin D (25[OH] D) levels assessed at admission in Chinese postmenopausal women with hip fracture. METHODS: From January 1, 2012 to December 31, 2013, all postmenopausal women with first-ever hip fracture were recruited to participate in the study. Serum 25[OH] D levels were measured at admission. The functional evaluation at the time of discharge was performed by the Barthel Index (BI). The prognostic value of 25[OH] D to predict the functional outcome within discharge was analyzed by logistic regression analysis, after adjusting for the possible confounders. RESULTS: In our study, 261 patients were included and assessed. In the 76 patients with an unfavorable functional outcome, serum 25(OH) D levels were lower compared with those in patients with a favorable outcome [11.8(IQR, 9.9-16.1) ng/ml; 16.8(IQR, 13.6-21.4) ng/ml, respectively; P<0.0001]. In multivariate analysis, there was an increased risk of unfavorable outcome associated with serum 25(OH) D levels ≤ 20 ng/ml (OR 5.24, 95%CI: 3.11-8.15; P<0.0001) after adjusting for possible confounders. CONCLUSIONS: Our data support an association between serum 25[OH] D levels and prognosis in Chinese postmenopausal women with hip fracture.