Bevacizumab attenuates osteosarcoma angiogenesis by suppressing MIAT encapsulated by serum-derived extracellular vesicles and facilitating miR-613-mediated GPR158 inhibition.

Targeting angiogenesis has been considered a promising treatment for a large number of malignancies, including osteosarcoma. Bevacizumab (Bev) is an anti-vascular endothelial growth factor being used for this purpose. We herein investigate the therapeutic potential of Bev in angiogenesis during osteosarcoma and the related mechanisms. Bioinformatics were performed for identification of osteosarcoma-related microarray dataset to collect related lncRNA and miRNA, with MIAT and miR-613 obtained. The predicted binding site between miR-613 and GPR158 3'UTR region was further confirmed by luciferase assay. Then, their effects combined with treatment with Bev on osteosarcoma cells were explored by the gain- and loss-of-function. After extraction from osteosarcoma patients' serum (serum-EVs) and identification, EVs were co-cultured with osteosarcoma cells, the biological behaviors of which were detected by CCK-8 assay and microtubule formation in vitro. A mouse tumor xenograft model was used to determine the effect of Bev on tumor angiogenesis in vivo. Bev inhibited osteosarcoma cell proliferation and angiogenesis in vivo and in vitro. Besides, serum-EVs could transfer MIAT (EV-MIAT) into osteosarcoma cells, where it is competitively bound to miR-613 to elevate GPR158, thus promoting osteosarcoma cell proliferation and angiogenesis. Furthermore, Bev arrested osteosarcoma cell proliferation and angiogenesis by inhibiting EV-MIAT and inducing miR-613-mediated GPR158 inhibition. In conclusion, the Bev-mediated MIAT/miR-613/GPR158 regulatory feedback revealed a new molecular mechanism in the pathogenesis of osteosarcoma angiogenesis.

Evaluation of platelet-rich plasma and fibrin matrix to assist in healing and repair of rotator cuff injuries: a systematic review and meta-analysis.

OBJECTIVE: To perform a meta-analysis examining the effectiveness of platelet-rich plasma and platelet-rich fibrin matrix for improving healing of rotator cuff injuries. Data sources/design: A meta-analysis of eligible studies was performed after searching Medline, Cochrane, and EMBASE on 14 December 2015. SETTING: University hospital. PARTICIPANTS: Patients with rotator cuff injuries. Review methods/intervention: Databases were searched using the keywords "PRP or platelet-rich plasma," "PRFM or platelet-rich fibrin matrix," "rotator cuff," and "platelet-rich" for studies comparing outcomes of patients with rotator cuff injuries that did and did not receive a platelet-rich product. MAIN MEASURES: The primary outcome was a functional score change from pre- to post-treatment (Scorepost-Scorepre). The secondary outcome was a visual analogue scale (VAS) pain score change from pre- to post-treatment (VASpost-VASpre). RESULTS: A total of 11 studies were included in the meta-analysis. The total number of patients that received platelet-rich plasma or platelet-rich fibrin matrix was 320 and the number of control patients was 318. The standard difference in means of the functional scores was similar between patients administered platelet-rich plasma/fibrin matrix and patients in the control group (standard difference in means for functional scores = 0.029; 95% confidence interval (CI): -0.132 to 0.190; p = 0.725). The standard difference in means was similar between patients administered platelet-rich plasma and the controls (standard difference in means = 0.142; 95% CI: -0.080 to 0.364; p = 0.209). CONCLUSION: The results of this meta-analysis do not support the use of platelet-rich plasma/platelet-rich fibrin matrix in patients with rotator cuff injuries.

A novel mechanism of mTORC1-mediated serine/glycine metabolism in osteosarcoma development.

Osteosarcoma is the major malignant primary bone cancer in children and adolescents, which is highly aggressive with frequent acquisition of chemoresistance phenotypes. Although much progress has been made, mechanisms of osteosarcoma rapid growth and chemoresistance are still not well elucidated. Generally, alternated metabolic characterization has been proposed to be a hallmark of cancer, yet it is lack of a systematic characterization of cancer metabolic networks. In the present study, we aim to characterize osteosarcoma metabolism and key regulators to reveal mechanisms of how osteosarcoma grows and resists apoptosis under stress conditions. The results demonstrate that mTORC1 pathway is hyperactivated in clinical osteosarcoma samples. However, inhibition of mTORC1 may not be enough to induce significant death of osteosarcoma cells. Results of GC-TOFMS suggested that inhibition of mTORC1 reduce one-carbon amino acids, serine and glycine, in osteosarcoma cells. Moreover, mTORC1 regulates serine/glycine de novo synthesis via modulating glycolysis and serine/glycine synthesis gene expressions. Further, mTORC1/serine/glycine metabolic axis promotes osteosarcoma proliferation and antioxidant ability to environmental stress, which finally leads to cell survival. Our results identify a novel mechanism of mTORC1-mediated serine/glycine metabolism as a significant protective system in osteosarcoma cells.

Identification of CD20, ECM, and ITGA as Biomarkers for Osteosarcoma by Integrating Transcriptome Analysis.

BACKGROUND Osteosarcoma is the most frequent primary bone cancer derived from primitive mesenchymal cells. The aim of this study was to explore the molecular mechanism of the development and progression of osteosarcoma. MATERIAL AND METHODS The gene expression profiles of osteosarcoma from 17 specimens (3 normal and 14 osteosarcoma) were downloaded from the GEO database. The differentially expressed genes were identified by use of the Limma package. DAVID and Enrichment Map were used to perform GO and KEGG pathways enrichment analysis and to integrate enrichment results of differentially expressed genes (DEGs). Protein-protein interaction network was constructed and analyzed to screen out the potential regulatory proteins using the STRING online tools. RESULTS A total of 417 DEGs were screened, including 215 up-regulated and 202 down-regulated ones, accounting for 51.56% and 48.4%, respectively. In GO term, a total of 12 up-regulated expression genes were enriched in Cellular Component. The up-regulated DEGs were enriched in 6 KEGG pathways while the down-regulated expression genes were enriched in 2 KEGG pathways. The constructed PPI network was aggregated with 1006 PPI relationships and 238 nodes, accounting for 57.07% of DEGs. We found that CD20, MCM, and CCNB1 (down-regulated) in cell cycle and ECM, ITGA, RTKin (up-regulated) in focal adhesion had important roles in the progression of osteosarcoma. CONCLUSIONS The identified DEGs and their enriched pathways provide references for the exploration of the molecular mechanism of the development and progression of osteosarcoma. Moreover, the key genes (CD20, ECM, and ITGA) may be useful in treatment and diagnosis of osteosarcoma.

Allogeneic hand transplantation and rehabilitation of hand function: a 10-year follow-up study.

The purpose of this study is to present the long-term outcomes of allogenic hand transplantations performed at our centre. Between January 2001 and October 2002, five allogeneic limb transplantations were performed in three patients (two bilateral forearm and one left hand transplantation). Donors and recipients were matched for blood types (ABO/Rh) and had at least two human leukocyte antigen (HLA) matches. A comprehensive rehabilitation plan integrating preoperative, intraoperative and postoperative management was developed for each patient. After 10 years, all transplantations were performed successfully without complications. As of 2014, all grafts were viable. The transplanted hands showed palmate morphology, perceived superficial pain and tactile sensations, and the static two-point discrimination ranged from 2·5 to 4·0 mm. Chronic rejection at 4 years after surgery reduced hand function in case 2. Grip strength ranged from 3 kg (case 2) to 16-18 kg (case 1) to 41-43 kg for case 3. Lifting strength ranged from 3 kg (case 2) to 21-23 kg (case 1) to 47-51 kg for case 3. They lead a completely independent life. In summary, hand function following allogeneic limb transplantation allows the ability to perform tasks of daily living.

Apoptosis induced by sonodynamic therapy in human osteosarcoma cells in vitro.

The aim of the present study was to investigate the potential effect of hematoporphyrin monomethyl ether-sonodynamic therapy (HMME-SDT) on MG-63 osteosarcoma cells. The HMME concentration was kept constant at 20 µg/ml and the MG-63 osteosarcoma cell line was exposed to ultrasound with an intensity of 1.0 W/cm2 for 30 sec. Cell cytotoxicity was quantified using an MTT assay 6 h after HMME-SDT. The intracellular localization of HMME was imaged using inverted confocal laser scanning microscopy. Apoptosis was investigated using flow cytometry with Annexin V-fluorescein isothiocyanate and propidium iodine staining. The cytotoxicity of HMME-mediated sonodynamic action on MG-63 cells was significantly higher than that of other treatments, including ultrasound alone, HMME alone and sham treatment. Flow cytometry demonstrated that HMME­SDT action markedly enhanced the apoptotic rate of MG-63 cells. The mechanisms of apoptosis were analyzed by measuring the protein expression of poly ADP-ribose polymerase (PARP), cleaved PARP, procaspase-3, cleaved caspase-3 and cleaved caspase-9. The data demonstrated that HMME-SDT action markedly induced the apoptosis of MG-63 cells.

Inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats.

BACKGROUND: After injury, axonal regeneration of the adult central nervous system (CNS) is inhibited by myelin-derived growth-suppressing proteins. These axonal growth inhibitory proteins are mediated via activation of Rho, a small GTP-binding protein. The activated form of Rho, which is bound to GTP, is the direct activator of Rho kinase (ROCK) through serial downstream effector proteins to inhibit axonal regeneration. The objective of this study was to observe the therapeutic effect of inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats. METHODS: One hundred and twenty adult female Sprague-Dawley rats were randomly divided into three groups. Laminectomies alone were conducted in 40 rats in the sham group. Laminectomies and spinal cord transections were performed in 40 rats in the control group (treated with normal saline administered intraperitoneally). Laminectomies and spinal cord transections were performed in 40 rats in the fasudil-treated group (treated with fasudil administered intraperitoneally). Neurologic recovery was evaluated before surgery and 3 days, and 1, 2, 3, and 4 weeks after surgery using the Basso-Beattie-Bresnahan (BBB) scale of hind limb movement. At the same time, the expression of RhoA mRNA was determined with RT-PCR. Histopathologic examinations and immunofluorescence staining of NF were performed 1 month after surgery. RESULTS: Compared with the control group, the BBB scores of the fasudil-treated group were significantly increased and the expression of RhoA mRNA was significantly decreased. In the fasudil-treated group, a large number of NF-positive regenerating fibers was observed; some fibers crossed the slit of the lesion. CONCLUSION: Inactivation of the Rho-ROCK signaling pathway promotes CNS axonal regeneration and neurologic recovery after spinal cord injuries in rats.

Hypoxic preconditioning increases iron transport rate in astrocytes.

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.

Improved blood compatibility of Mg-1.0Zn-1.0Ca alloy by micro-arc oxidation.

Magnesium and its alloys have been used in the recent development of lightweight, biodegradeable implant materials. However, the corrosion properties of magnesium limit its usefulness. In a previous study, a micro-arc oxidation (MAO) method was used to modify a Mg-1.0 wt % Zn-1.0 wt % Ca alloy surface, with the purpose of improving the corrosion resistance of Mg alloys. However, the blood compatibility of MAO-treated Mg alloy is unknown. Results of cytotoxicity assays with bone marrow-derived mesenchymal stem cells showed that extracts of MAO-treated alloy significantly decreased cytotoxicity compared to titanium alloy extract. Results of blood compatibility tests showed that the MAO group had a decreased hemolytic ratio (2.25%) compared to the untreated Mg alloy group (24.58%) (p < 0.001). The MAO group showed significantly shorter prothrombin and thrombin times and significantly longer activated partial thromboplastin time than the untreated Mg alloy group. Arachidonic acid- and adenosine diphosphate-induced platelet aggregations were significantly decreased by the untreated Mg alloy extract, and they were less affected by extract of MAO-treated Mg alloy. In conclusion, MAO-treated Mg-1.0 wt % Zn-1.0 wt % Ca alloy exhibits favorable blood compatibility characteristics and may be useful in the development of magnesium implant materials.

Glutathione S-transferase polymorphisms and bone tumor risk in China.

AIM: We aimed to study the potential role of GSTM1 and GSTT1 in the risk of osteosarcoma in Chinese population. METHODS: We collected 110 osteosarcomas by pathologic examination and 226 controls from the First Affiliated Hospital of Harbin Medical University during December 2008 to December 2010. Genotyping was based upon duplex polymerase-chain-reaction with the PCR-CTPP method. RESULTS: Individuals carrying null GSTM1 and GSTT1 had 1.50 and 2.07 fold risks of osteosarcoma when compared with non-null genotypes, respectively. The increased risk associated with the GSTT1 polymorphism seemed more evident among males (Null GSTT1 genotype vs. non-null genotype, adjusted OR= 2.43, 95% CI: 1.29-3.30) than females (adjusted OR =1.66, 95% CI: 1.02-2.78). The increased risk was also more evident among individuals aged 15 years or less (adjusted OR for null GSTT1 genotype vs. non-null genotype = 2.24, 95% CI: 1.20-3.24) than those aged more than 15 years (adjusted OR = 1.82, 95% CI: 1.07-2.95). CONCLUSION: Our study of the association between polymorphisms in GSTM1 and GSTTI and the risk of osteosarcoma in a Chinese population provided evidence that null GSTTI might be a useful marker of susceptibility to osteosarcoma development, especially for male sand young age individuals.

Integrated assessment of potential value of erbB-2 amplification or expression status as novel therapy target in Chinese children and adolescents with osteosarcoma.

BACKGROUND: Targeted tumor therapies have been making rapid progress in recent years, and the erbB-2 oncogene is a suitable target. There was much discussion about the level of erbB-2 in osteosarcoma. The aim of this study was to investigate the erbB-2 amplification or expression status in osteosarcoma. METHODS: Fluorescence in situ hybridization (FISH) and DNA probes for erbB-2 and centromere 17 were used to examine the erbB-2 gene amplification status in 32 osteosarcoma samples, and expression of erbB-2 was analyzed by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: None of the 32 osteosarcomas was observed by FISH to have the erbB-2 gene amplified, and no distinguishable membrane staining was seen in any case yet, nevertheless, erbB-2 overexpression was present in 6 tumor samples by RT-PCR. CONCLUSIONS: The status of erbB-2 gene amplification and membrane overexpression is rare in osteosarcomas, and might suggest that the erbB-2 target agent should not be applied to osteosarcomas as single treatment.

[The in vitro differentiation and the variant expression of protein of bone marrow stromal stem cells when treating the spinal cord injury].

OBJECTIVE: To explore the differentiation and the variant expression of protein of the bone marrow stromal stem cells (BMSCs) when the BMSCs differentiated into the neuronal cells in the analogous micro-environment of spinal cord injury. METHODS: BMSCs were isolated from bone marrow of Wistar rats and labeled with PKH26 (control group), and then were cocultured with neural cells, which were isolated from the spinal cord of the fetal rats, in the same plate well (co-culture group) or in the two-layer Petri well (two-layer group). Eight days later, the BMSCs were identified by immunofluorescence staining of NSE and GFAP respectively. The apparently changing proteins were analyzed by SELDI-TOF-MS while the BMSCs differentiated into neurons. RESULTS: Eight days after co-culturing with neural cells in the same plate well or in the two-layer Petri well, BMSCs appeared more similar with neural cells. The immunofluorescence identification showed that, NSE and GFAP of which the BMSCs of the two-layer group expressed were obviously higher than control group (P < 0.05); and these two proteins of co-culture group were also obviously higher than the other two groups (P < 0.05). Five proteins in the co-culture group changed obviously as followed: TIP39_RAT and CALC_RAT were 5.360 and 2.807 times of that in the control group; INSL6_RAT, PNOC_RAT and PCSK1_RAT were 38.0, 49.9 and 43.8 percent of those in the control group. CONCLUSIONS: BMSCs could differentiate into neural cells in vitro, and the differentiation ratio of BMSCs in the co-culture group is higher than that of the two-layer group. Five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT and PCSK1_RAT, are correlated closely to the mechanisms of which the BMSCs differentiated into neurons.

Reconstruction of large limb bone defects with a double-barrel free vascularized fibular graft.

BACKGROUND: The use of a free, vascularized fibular graft is an important technique for the reconstruction of large defects in long bones. The technique has many advantages in strong, tubular bones; a more reliable vascular anatomy with a large vascular diameter and long pedicle is used, minimizing donor-site morbidity. Due to limitations in both fibular anatomy and mechanics, they cannot effectively be used to treat large limb bone defects due to their volume and strength. METHODS: From 1990 to 2001, 16 clinical cases of large bone defects were treated using vascularized double-barrel fibular grafts. Patients were evaluated for an average of 10 months after surgery. RESULTS: All the patients achieved bony union; the average bone union took 10 months post surgery, and no stress fractures occurred. Compared with single fibular grafts, the vascularized double-barrel fibular grafts greatly facilitate bony union and are associated with fewer complications, suggesting that the vascularized double-barrel fibular graft is a valuable procedure for the correction of large bone defects in large, long bones in addition to enhancing bone intensity. CONCLUSIONS: The vascularized double-barrel fibular graft is superior to the single fibular graft in stimulating osteogenous activity and biological mechanics for the correction of very large bone defects in large, long bones. Free vascularized folded double-barrel fibular grafts can not only fill up large bone defects, but also improve the intensity margin. Therefore, this study also widens its application and enlarges the treatment targets. However, in the case of bone deformability, special attention should be paid to bone fixation and protection of donor and recipient sites.

[Study of reduction rat T-lymphocytes activation and proliferation by inhibition inducible co-stimulator gene expression].

OBJECTIVE: To observe the change of activation and proliferation ability of rat T-lymphocytes after suppress ICOS gene expression by RNA interference. METHODS: Four interference sites targeting at rat ICOS gene were designed and four pairs of oligonucleotide fragments were cloned into the pSilencer 4.1-CMV neo plasmid vectors then transfected into rat lymphocytes with cationic liposome. The expression of mRNA and protein of ICOS was detected by RT-PCR and flow cytometry. The alteration of lymphocyte proliferation ability was evaluated by mix lymphocyte reaction, and the secretion levels of IFN-gamma and IL-4 were measured by ELISA procedure. RESULTS: After transfection, the expression of mRNA and protein of ICOS in test groups were lower than that in control groups (P < 0.05). The ability of T-lymphocytes in proliferation was poor and the levels of IFN-gamma and IL-4 were reduced with ICOS gene shut down. CONCLUSIONS: RNA interference plasmid vector can suppress ICOS expression in rat T-lymphocytes significantly, and it may be useful for further study on transplantation immunity tolerance.