Hypoxia-Inducible Factor-1α (HIF-1α) as a Factor to Predict the Prognosis of Spinal Chordoma.

STUDY DESIGN: Retrospective study. OBJECTIVE: In this study, the authors explore the potential relationship between hypoxia inducible factor-1α (HIF-1α) and the prognosis of patients with spinal chordoma. SUMMARY OF BACKGROUND DATA: Currently, prognostic factors related to the clinical course in the setting of spinal chordoma are poorly understood. Although the close relationship between HIF-1α and tumor angiogenesis, metastasis, and recurrence have been widely reported, it has not been investigated in the context of spinal chordoma. MATERIALS AND METHODS: In this study, 32 samples of chordoma patients were compared with 14 nucleus pulposus tissues as controls. The specific expression of HIF-1α was detected by immunohistochemistry. Continuous disease-free survival (CDFS) was defined as the interval from tumor resection to confirmation of the first local recurrence or distant metastasis. Overall survival (OS) was defined as the interval from the date of surgery to death related to any cause. The relationship between HIF-1α expression and the clinicopathologic characteristics of patients with chordoma was analyzed using the Pearson χ 2 test. Multivariate Cox analysis was used to evaluate whether HIF-1α expression was associated with the prognosis of patients after controlling for confounders. RESULTS: HIF-1α was mainly expressed in the cytoplasm or nucleus in all of the chordoma samples, which showed significantly higher than that in the normal nucleus pulposus tissue ( P =0.004). Multivariate Cox regression analyses showed that high HIF-1α expression and location of HIF-1α expression were significantly associated with poor CDFS (hazard ratio (HR)=3.374; P =0.021) and OS (HR=4.511; P =0.012). In addition, we further found that high HIF-1α expression both in the cytoplasm and nucleus indicated a stronger prognostic factor for poor CDFS (HR=3.885; P =0.011) and OS (HR=4.014; P =0.011) in spinal chordoma patients. CONCLUSION: High HIF-1α expression may become a potential new biological indicator to predict a poor prognosis in patients with spinal chordoma. HIF-1α may also represent a novel therapeutic target for the treatment of spinal chordoma.

Study on performance of a relay-assisted UWOC system based on adaptive optics.

To improve the performance of underwater wireless optical communication (UWOC) systems, we propose a relay-assisted UWOC system model based on adaptive optics (AO). The closed expressions of the scintillation index, composite channel probability density function, and outage probability of the Gaussian beam before and after AO compensation are derived using the extended Rytov theory and Meijer G-function. The performance variation of an UWOC system with different parameters is analyzed by simulation. The results show that AO correction can compensate for the distorted wavefront and significantly reduce the intensity fluctuation at the receiving end. The proposed system can efficiently alleviate channel fading, improving the outage probability performance of the UWOC system.

Zinc oxide nanoparticles inhibit osteosarcoma metastasis by downregulating ß-catenin via HIF-1α/BNIP3/LC3B-mediated mitophagy pathway.

Osteosarcoma (OS) therapy faces many challenges, especially the poor survival rate once metastasis occurs. Therefore, it is crucial to explore new OS treatment strategies that can efficiently inhibit OS metastasis. Bioactive nanoparticles such as zinc oxide nanoparticles (ZnO NPs) can efficiently inhibit OS growth, however, the effect and mechanisms of them on tumor metastasis are still not clear. In this study, we firstly prepared well-dispersed ZnO NPs and proved that ZnO NPs can inhibit OS metastasis-related malignant behaviors including migration, invasion, and epithelial-mesenchymal transition (EMT). RNA-Seqs found that differentially expressed genes (DEGs) in ZnO NP-treated OS cells were enriched in wingless/integrated (Wnt) and hypoxia-inducible factor-1 (HIF-1) signaling pathway. We further proved that Zn2+ released from ZnO NPs induced downregulation of ß-catenin expression via HIF-1α/BNIP3/LC3B-mediated mitophagy pathway. ZnO NPs combined with ICG-001, a ß-catenin inhibitor, showed a synergistic inhibitory effect on OS lung metastasis and a longer survival time. In addition, tissue microarray (TMA) of OS patients also detected much higher ß-catenin expression which indicated the role of ß-catenin in OS development. In summary, our current study not only proved that ZnO NPs can inhibit OS metastasis by degrading ß-catenin in HIF-1α/BNIP3/LC3B-mediated mitophagy pathway, but also provided a far-reaching potential of ZnO NPs in clinical OS treatment with metastasis.

Extrusion Printed Silk Fibroin Scaffolds with Post-mineralized Calcium Phosphate as a Bone Structural Material.

Artificial bone materials are of high demand due to the frequent occurrence of bone damage from trauma, disease, and ageing. Three-dimensional (3D) printing can tailor-make structures and implants based on biomaterial inks, rendering personalized bone medicine possible. Herein, we extrusion-printed 3D silk fibroin (SF) scaffolds using mixed inks from SF and sodium alginate (SA), and post-mineralized various calcium phosphates to make hybrid SF scaffolds. The effects of printing conditions and mineralization conditions on the mechanical properties of SF scaffolds were investigated. The SF scaffolds from ~10 wt% SF ink exhibited a compressive modulus of 240 kPa, which was elevated to ~1600 kPa after mineralization, showing a significant reinforcement effect. Importantly, the mineralized SF 3D scaffolds exhibited excellent MC3T3-E1 cell viability and promoted osteogenesis. The work demonstrates a convenient strategy to fabricate SF-based hybrid 3D scaffolds with bone-mimetic components and desirable mechanical properties for bone tissue engineering.

VPS34 regulates dynamin to determine the endocytosis of mitochondria-targeted zinc oxide nanoparticles in human osteosarcoma cells.

In our previous study, zinc oxide nanoparticles (ZnO NPs) presented satisfying therapeutic effects with cancer cell selectivity in osteosarcoma cells and, thus, have been considered as a potential nanomedicine for human osteosarcoma treatment. However, the poorly investigated internalization process, including their endocytic pathway into tumor cells and intracellular fate, limits the clinical application. Here, we further clarified these aspects. First, ZnO NPs were rapidly internalized by osteosarcoma cells and accumulated in mitochondria, before being entrapped into lysosomes. Second, dynasore (a dynamin inhibitor) was demonstrated to be the most effective in blocking ZnO NP uptake and rescuing ZnO NP-induced osteosarcoma cell autophagic death and apoptosis. Third, we confirmed the key role of dynamin 2 in ZnO NP endocytosis and subsequent autophagic cell death in vitro and in vivo. Furthermore, we proved that VPS34 transferred from cell cytoplasm to cell membrane to interact with dynamin under ZnO NP treatment. Altogether, combined with our previous study, the current research further revealed that ZnO NPs entered human osteosarcoma cells through the VPS34/dynamin 2-dependent endocytic pathway, directly targeting and damaging the mitochondria before being entrapped into the lysosomes, thereby initiating mitophagy-Zn2+-reactive oxygen species-mitophagy axis mediated cell apoptosis.

HIF-1α-Mediated Mitophagy Determines ZnO Nanoparticle-Induced Human Osteosarcoma Cell Death both In Vitro and In Vivo.

Although ZnO nanoparticles (NPs) can kill human osteosarcoma cells, the underlying upstream regulatory mechanisms remain unclear. Since hypoxia inducible factor-1α (HIF-1α) regulates the tumor microenvironment, here we explored the interplay between HIF-1α regulation and mitophagy in ZnO NP-induced osteosarcoma inhibition both in vivo and in vitro. We found that ZnO NPs upregulated HIF-1α protein levels when they killed four common human osteosarcoma cell lines. This finding was consistent with our observations that additional HIF-1α upregulation by a hypoxia inducer CoCl2 or under a 1% hypoxia environment enhanced NP-induced cell death, but concurrent HIF-1α suppression by a hypoxia inhibitor YC-1 or HIF-1α siRNA inhibited NP-induced cell death. We discovered an interplay between HIF-1α and the autophagy-Zn2+-reactive oxygen species (ROS)-autophagy cycle axis and revealed that NP-induced cancer cell killing followed a HIF-1α-BNIP3-LC3B-mediated mitophagy pathway. We confirmed that NP-upregulated HIF-1α protein expression was attributed to prolyl hydroxylase inhibition by both ROS and Zn2+. In addition, the in vivo assay confirmed the therapeutic effectiveness and safety of ZnO NPs on a nude mice osteosarcoma model. Collectively, our findings clarified the upstream regulatory mechanism of autophagy induced by the NPs and further demonstrated their antitumor ability in vivo. This work provides new targets and strategies for enhancing NP-based osteosarcoma treatment.

Genetic landscape and ligand-dependent activation of sonic hedgehog-Gli1 signaling in chordomas: a novel therapeutic target.

Chordoma, a rare neoplasm derived from intraosseous notochordal remnants, is unresponsive to conventional chemotherapy and radiotherapy. Sonic Hedgehog (Shh) is a crucial fetal notochord-secreted morphogen that directs notochordal development. The aim of this study was to determine the functional roles and therapeutic potential of Shh-Gli1 signaling in chordomas. Tissue samples and clinical profiles were collected from 42 patients with chordoma. The chordoma cell lines U-CH1 and MUG-Chor1 were used for functional experiments. Shh-Gli1 signaling pathway genetic alterations were screened, and the functions of the identified novel variants were analyzed using in silico analyses, real-time quantitative PCR, and minigene assays. Ligand-dependent Shh-Gli1 signaling activation was assessed using single- and dual-label immunostaining, western blot analysis, and a Shh-responsive Gli-luciferase reporter assay. The small-molecule inhibitor vismodegib was used to target Shh-Gli1 signaling in vitro and in vivo. Overall, 44 genetic alterations were identified, including four novel variants (c.67_69dupCTG in SMO, c.-6_-4dupGGC and c.3306 + 83_3306 + 84insG in PTCH1, and c.183-67_183-66delinsA in SUFU). Shh, PTCH1, SMO, SUFU, and Gli1 were extensively expressed in chordomas, and higher Gli1 expression correlated with poorer prognosis. A luciferase reporter assay and dual-label immunostaining indicated the occurrence of juxtacrine ligand-dependent Shh-Gli1 signaling activation. Vismodegib significantly inhibited cell proliferation and induced apoptosis and G1/S cell cycle arrest. In vivo investigation demonstrated that vismodegib effectively inhibited chordoma xenograft growth. This current preclinical evidence elucidates the therapeutic potential of Shh-Gli1 signaling pathway targeting for chordoma treatment. Vismodegib may be a promising targeted agent, and further clinical trials are warranted.

IL17RC affects the predisposition to thoracic ossification of the posterior longitudinal ligament.

BACKGROUND: Thoracic ossification of the posterior longitudinal ligament (T-OPLL) can cause thoracic spinal stenosis, which results in intractable myelopathy and radiculopathy. The etiology of T-OPLL is unknown and the condition is difficult to treat surgically. Whole-genome sequencing identified a genetic variant at rs199772854 of the interleukin 17 receptor C (IL17RC) gene as a potentially pathogenic locus associated with T-OPLL. We aimed to determine whether the rs199772854A site mutation causes abnormal expression of the IL17RC in Han Chinese patients with T-OPLL and predict the possible pathogenic mechanisms of T-OPLL. Analyses were performed to determine whether IL17RC is involved in the pathogenicity of T-OPLL. METHODS: Peripheral blood and OPLL tissue were collected from a total of 72 patients with T-OPLL disease (36 patients carrying the rs199772854A site mutation in IL17RC and 36 wild-type patients). The expression of IL17RC was analyzed by enzyme-linked immunosorbent assay, reverse transcription-quantitative polymerase chain reaction, immunohistochemistry, and Western blotting. RESULTS: rs199772854A mutation resulted in markedly increased IL17RC gene expression levels in peripheral blood samples and the OPLL tissue obtained following clinical surgery (P < 0.05). CONCLUSIONS: The results suggest that the rs199772854A site mutation of IL17RC can significantly increase the expression of IL17RC. The IL17RC gene rs199772854A site polymorphism is a potential pathogenic mutation in T-OPLL disease, which may be associated with the occurrence of T-OPLL.

Oleandrin sensitizes human osteosarcoma cells to cisplatin by preventing degradation of the copper transporter 1.

A major problem in osteosarcoma treatment is cisplatin resistance. We have reported the anti-osteosarcoma effect of oleandrin; however, whether oleandrin sensitizes osteosarcoma to cisplatin is unknown. We investigated the chemosensitization of oleandrin and potential mechanisms in osteosarcoma cells U-2OS, SaOS-2, and MG-63. The median-effect analysis demonstrated that cisplatin + oleandrin exerted synergistic (U-2OS and MG-63) or additive effects (SaOS-2), which were consistent with the changes of the intracellular accumulation of platinum (Pt) and Pt-DNA adducts. Immunohistochemistry staining showed that the expression level of the mature form CTR1, the major influx transporter of cisplatin, was low in osteosarcoma tissue. However, oleandrin with or without cisplatin significantly increased the expression and membrane localization of the mature CTR1. Furthermore, CTR1 knockdown reversed the synergistic effect and decreased cisplatin uptake. The mRNA microarray analysis suggested that oleandrin downregulated the expression of proteasome-related genes, which was verified by the proteasome activity assay. Besides, the proteasome inhibitor MG132 upregulated the expression of the mature CTR1 in U-2OS and MG-63 cells. Overall, we conclude that oleandrin sensitizes osteosarcoma cells to cisplatin in synergistic or additive manners. The synergy results from the enhanced cisplatin uptake via oleandrin-mediated inhibition of proteasome activity and subsequent blockage of the mature CTR1 degradation.

Melatonin benefits to the growth of human annulus fibrosus cells through inhibiting miR-106a-5p/ATG7 signaling pathway.

BACKGROUND: Disc degeneration (DD) is one of the common diseases worldwide, which deeply influences normal life and leads to excruciating pain. However, an effective treatment for DD is still not identified. METHOD: The present study systemically examined the effect of melatonin on annulus fibrosus (AF) cells of patients with DD. RESULTS: Melatonin had the effect of promoting proliferation, inducing autophagy, and suppressing apoptosis on AF cells of patients with DD. Moreover, melatonin contributed to the translation and transcription of autophagy-related protein ATG7 and inhibited the function of miR-106a-5p in AF cells. In addition, the results suggested that miR-106a-5p mediated the expression of ATG7 by directly binding to its 3'UTR in AF cells. CONCLUSION: This research not only gained a deep insight of melatonin mode of action, but also indicated its potential target signaling pathway in AF cells.

Potential role of the IL17RC gene in the thoracic ossification of the posterior longitudinal ligament.

The thoracic ossification of the posterior longitudinal ligament (T­OPLL) can cause thoracic spinal stenosis, which results in intractable myelopathy and radiculopathy. Our previous whole­genome sequencing study first reported rs199772854 in the interleukin 17 receptor C (IL17RC) gene as a potentially pathogenic loci for T­OPLL. The aim of the present study was to examine the effects of the IL17RC gene rs199772854A site mutation on osteogenesis by establishing a model of osteogenic differentiation. IL17RC gene mutation site and wild­type site mouse embryonic osteoblast (3T3­E1) models were constructed in order to induce the differentiation of the cells into osteoblasts. Whether the mutation site causes the abnormal expression of the IL17RC gene and osteogenic markers was analyzed by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. The IL17RC gene rs199772854A site mutation was demonstrated to play a biological role through the overexpression of its own gene, and also to significantly increase the expression levels of osteogenic markers. Furthermore, the mutation upregulated the expression of the key proteins, tumor necrosis factor receptor (TNFR)­associated factor 6 (TRAF6) and nuclear factor (NF)­κB, in the interleukin (IL)­17 signaling axis. On the whole, the findings of this study suggest that the IL17RC gene rs199772854A loci mutation propels mouse embryonic osteoblasts towards osteogenic differentiation and may play an important role in the pathogenesis of T­OPLL. The IL17RC gene may promote osteogenesis through the IL­17 signaling pathway and may thus be involved in the process of ectopic osteogenesis in T­OPLL.

Oleandrin synergizes with cisplatin in human osteosarcoma cells by enhancing cell apoptosis through activation of the p38 MAPK signaling pathway.

PURPOSE: Our previous studies have reported the antitumor effect of oleandrin on osteosarcoma; however, its chemosensitizing effect in osteosarcoma treatment is still unknown. Therefore, we explored the sensitizing effects of oleandrin to cisplatin in osteosarcoma and investigated the potential mechanisms. METHODS: After exposure to oleandrin and/or cisplatin, CCK-8 and colony formation assays, DAPI staining and flow cytometry were performed to detect cell proliferation and apoptosis in 143B, U-2OS and MG-63 osteosarcoma cells. The median-effect analysis was applied to evaluate the combined effect. Western blot was used to determine the expression of related proteins. Osteosarcoma xenografts and histological observations were applied to confirm the combined effect in vivo. RESULTS: Compared with cisplatin or oleandrin alone, the combined treatment significantly inhibited cell proliferation and induced cell apoptosis. The median-effect analysis indicated a synergistic cytotoxic effect. The combined treatment downregulated Bcl-2 and upregulated Bax and cleaved caspase-3, -8 and -9. And the suppression of caspases reduced cell death. Furthermore, oleandrin alone or with cisplatin, activated the p38 MAPK/Elk-1 pathway. The inhibition of the p38 MAPK pathway increased cell viability and reduced apoptosis. In vivo, the combined treatment was also verified to significantly inhibit tumor growth, induce apoptosis and activate the p38 MAPK pathway. CONCLUSIONS: The combination of oleandrin with cisplatin exerts a synergistic antitumor effect in osteosarcoma, which relates to the activation of the p38 MAPK pathway.

Cross Talk Between Autophagy and Apoptosis Contributes to ZnO Nanoparticle-Induced Human Osteosarcoma Cell Death.

Killing osteosarcoma cells by zinc oxide nanoparticles (NPs) and its underlying subcellular mechanism are never studied. Here, it is found that the NPs induce cross talk between apoptosis and autophagy, which leads to osteosarcoma cell death. Specifically, the NP uptake promotes autophagy by inducing accumulation of autophagosomes along with impairment of lysosomal functions. The autophagy further causes the uptaken NPs to release zinc ions by promoting their dissolution. These intracellular zinc ions, together with those that are originally released from the extracellular NPs and flowed into the cells, collectively target and damage mitochondria to produce reactive oxygen species (ROS). Then the ROS inhibit cell proliferation by arresting S phase and trigger apoptosis by extrinsic and intrinsic pathways, ultimately leading to cell death. More importantly, suppression of the early stage autophagy restores cell viability by abolishing apoptosis whereas blockade of the late stage autophagy inversely enhances apoptosis. In contrast, inhibition of apoptosis shows a limited ability to restore cell viability but obviously enhance autophagy. Notably, cell viability is strongly ameliorated by the combination of inhibitors for both the late stage autophagy and the apoptosis. These findings provide a mechanistic understanding of the NP-directed autophagy and apoptosis in osteosarcoma cells.

Association of IL17RC and COL6A1 genetic polymorphisms with susceptibility to ossification of the thoracic posterior longitudinal ligament in Chinese patients.

BACKGROUND: In our previous whole-genome sequencing study of 30 unrelated northern Chinese Han patients, we identified six single nucleotide polymorphisms (SNPs) in the interleukin 17 receptor C (IL17RC) and collagen type VI α1 chain (COL6A1) genes that were potentially associated with thoracic ossification of the posterior longitudinal ligament (T-OPLL). To determine whether these six SNPs are associated with susceptibility to T-OPLL in the northern Chinese Han population, we performed a case-control association study to confirm specific susceptible loci in the expanded samples. METHODS: The six SNPs in the IL17RC and COL6A1 genes were analyzed in 200 northern Chinese individuals (100 patients and 100 control subjects) using the Sequenom system. RESULTS: The genotype distributions and allele frequencies of each SNP in the control and patient groups were compared. rs201153092, rs13051496, rs199772854, rs76999397, and rs189013166 showed potential pathogenic loci for T-OPLL in the northern Chinese Han population, whereas rs151158105 did not. At the genotype level, the differences in the genotype frequencies of rs201153092, rs13051496, rs199772854, rs76999397, and rs189013166 between T-OPLL cases and controls reached statistical significance. CONCLUSIONS: To the best of our knowledge, this is the first association study of susceptibility genes in Han Chinese patients with T-OPLL. The results revealed five SNPs in the IL17RC and COL6A1 genes that represented potentially pathogenic mutations in patients with T-OPLL.

Identification of susceptibility loci for thoracic ossification of the posterior longitudinal ligament by whole-genome sequencing.

Ossification of the posterior longitudinal ligament (OPLL) is a myelopathy commonly observed in the cervical spine. By contrast, thoracic OPLL (T­OPLL) is rare but more severe. Previous studies have identified several polymorphisms in osteogenic genes that are associated with the occurrence and development of cervical OPLL. However, few genetic studies have evaluated T­OPLL. The present study aimed to identify the genetic factors for OPLL by performing whole­genome sequencing (WGS) in 30 unrelated northern Chinese Han patients with T­OPLL. Using bioinformatics analyses and damaging­variant prediction algorithms, two deleterious variants [c.1534G>A(p.Gly512Ser)/collagen, type VI, α1 (COL6A1)] and [c.2275C>A(p.Leu759Ile)/inteleukin-17 receptor C (IL17RC)] were identified in seven unrelated patients. These two mutations resulted in markedly increased gene expression levels in peripheral blood samples. To the best of our knowledge, this is the first report to describe the use of WGS analysis of T­OPLL in the northern Chinese Han population. The results revealed two novel potentially pathogenic mutations in patients with T­OPLL.

iASPP overexpression is associated with clinical outcome in spinal chordoma and influences cellular proliferation, invasion, and sensitivity to cisplatin in vitro.

The oncogenetic function of inhibitory member of the apoptosis stimulating protein of p53 family (iASPP) in chordoma is unclear and remains to elucidate. The expression of iASPP in chordoma tissues and cells, its correlation to clinicopathological parameters and the effect on the patients' prognosis were evaluated. Cellular proliferation, invasion and cisplatin-response were observed after the iASPP knockdown or overexpression in vitro. Co-Immunoprecipitation assay was used to explore the interaction between iASPP and p53. The regulation of miRNA-124 on the expression and apoptotic function of iASPP was explored after transiently transfecting cells with miRNA-124 mimics or inhibitor. Results indicated that iASPP overexpressed in chordoma tissues and cells. Its overexpression was associated with tumor invasion and local recurrence, and was predictive of patients' poor prognosis. Cells with iASPP-silence showed a decreased ability of proliferation and invasion, but an increasing sensitivity to cisplatin. Besides, iASPP could combine with p53 in either endogenous or exogenous detection. Post-transcriptionally, miRNA-124 negatively regulated the expression of iASPP, which further led to the changes of apoptosis-related proteins. Thus, iASPP overexpression is associated with the clinical outcome in spinal chordoma and influences cellular proliferation, invasion, and the sensitivity to cisplatin.

Unique antitumor property of the Mg-Ca-Sr alloys with addition of Zn.

In clinical practice, tumor recurrence and metastasis after orthopedic prosthesis implantation is an intensely troublesome matter. Therefore, to develop implant materials with antitumor property is extremely necessary and meaningful. Magnesium (Mg) alloys possess superb biocompatibility, mechanical property and biodegradability in orthopedic applications. However, whether they possess antitumor property had seldom been reported. In recent years, it showed that zinc (Zn) not only promote the osteogenic activity but also exhibit good antitumor property. In our present study, Zn was selected as an alloying element for the Mg-1Ca-0.5Sr alloy to develop a multifunctional material with antitumor property. We investigated the influence of the Mg-1Ca-0.5Sr-xZn (x = 0, 2, 4, 6 wt%) alloys extracts on the proliferation rate, cell apoptosis, migration and invasion of the U2OS cell line. Our results show that Zn containing Mg alloys extracts inhibit the cell proliferation by alteration the cell cycle and inducing cell apoptosis via the activation of the mitochondria pathway. The cell migration and invasion property were also suppressed by the activation of MAPK (mitogen-activated protein kinase) pathway. Our work suggests that the Mg-1Ca-0.5Sr-6Zn alloy is expected to be a promising orthopedic implant in osteosarcoma limb-salvage surgery for avoiding tumor recurrence and metastasis.

Inhibitory effect of bone morphogenetic protein-2 on the proliferation of giant cell tumor of bone stromal cells in vitro.

The inhibitory effect of bone morphogenetic protein-2 (BMP-2) on the proliferation of giant cell tumor of bone stromal cells (GCTSCs) has not been fully elucidated. Therefore, the aim of this study was to evaluate the role of recombinant human BMP-2 (rhBMP-2) in the growth of GCTSCs. The effects of exposure to different concentrations of rhBMP-2 (0, 10, 100 and 300 ng/ml) for 1, 3, 5 and 7 days on GCTSC proliferation were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, the effect of treatment with rhBMP-2 (0 or 10 ng/ml) for 48 h on the cell cycle pattern of GCTSCs was examined by flow cytometry. The apoptosis-inducing effect of rhBMP-2 (0 or 10 ng/ml) in GCTSCs was also determined by flow cytometry after 48 and 72 h. In addition, western blot assays were conducted to determine whether rhBMP-2 acts on non-Smad mitogen-activated protein kinase (MAPK) signaling pathways, namely extracellular signal-regulated kinase (ERK1/2), p38 and c-jun-N-terminal kinase (JNK) pathways. The proliferation of GCTSCs treated with rhBMP-2 (10, 100 or 300 ng/ml) for 5 or 7 days was significantly inhibited in a non dose-dependent and non-time-dependent manner (P<0.05). The treatment of GCTSCs with rhBMP-2 (10 ng/ml) for 48 h had no effect on cell cycle distribution. The apoptosis of GCTSCs induced by exposure to rhBMP-2 (10 ng/ml) for 48 or 72 h was significant (P<0.05). Expression levels of phospho-ERK1/2, phospho-p38 and phospho-JNK increased significantly when GCTSCs were treated with rhBMP-2 (10 ng/ml) for 72 h (P<0.05). The results indicate that rhBMP-2 has no stimulatory effect on GCTSC growth. However, it may lead to the apoptosis of GCTSCs by non-Smad MAPK signaling pathways.

Addition of Zn to the ternary Mg-Ca-Sr alloys significantly improves their antibacterial property.

Most of the magnesium (Mg) alloys possess excellent biocompatibility, mechanical property and biodegradability in orthopedic applications. However, these alloys may suffer from bacterial infections due to their insufficient antibacterial capability. In order to reduce the post-surgical infections, a series of biocompatible Mg-1Ca-0.5Sr-xZn (x=0, 2, 4, 6) alloys were fabricated with the addition of antibacterial Zn with variable content and evaluated in terms of their biocompatibility and antibacterial property. The in vitro corrosion study showed that Mg-1Ca-0.5Sr-6Zn alloys exhibited a higher hydrogen evolution volume after 100 h immersion and resulted in a higher pH value of the immersion solution. Our work indicated that Zn-containing Mg alloys exhibited good biocompatibility with high cell viability. The antibacterial studies reveal that the number of bacteria adhered on all of these Mg alloy samples diminished remarkably compared to the Ti-6Al-4V control group. We also found that the proliferation of the bacteria was inhibited by these Mg alloys extracts. Among the prepared alloys, Mg-1Ca-0.5Sr-6Zn alloy not only exhibited a strong antibacterial effect, but also promoted the proliferation of MC3T3-E1 osteoblasts, suggesting that it is a promising alloy with both good antibacterial property and good biocompatibility for use as an orthopedic implant.