Relative influence on cell behaviors of osteoblasts seeded onto demineralized bone matrix with diverse particle size.

Demineralized bone matrix (DBM) is a natural and collagen-based allogeneic bone graft material with good bioactivity and biocompatibility. However, there are conflicting reports on the efficiency of this product owning to the relevant factors, especially, the particle size of DBM. In the current study, osteoblasts were seeded onto DBM with diverse particle sizes of 0.6-1.0 mm, 2.2-2.6 mm, 3.8-4.2 mm and 5.4-5.8 mm, and then the cell adhesion, proliferation, differentiation and apoptosis were analyzed to evaluate the effects of particle sizes on bioactivity and biocompatibility of DBM. It could be identified that particle size had a significant influence on the surface roughness and the collagen structure of DBM. In-vitro cytological assays had confirmed that DBM with all particle sizes had good cytocompatibility, which was beneficial for cell survival. What's more, DBM with a smaller particle size had a better biological activity and could promote cell proliferation and differentiation. We hope that researchers could choose to apply DBM with particle size of 0.6-1.0 mm for further intensive study, from which researchers will acquire more comparable and reliable conclusions for different study purposes.

Silencing of CRT relieves Ang II-Induced injury of HUVECs with insulin resistance.

In this study, we investigated the effects of Angiotensin II (Ang II) on insulin-resistant endothelial cells. High glucose and insulin at series of concentrations were used to induce IR in Human Umbilical Vein Endothelial Cells (HUVECs). Successful IR induction was confirmed according to glucose consumption and glycogen content levels. Cell morphology was observed under a microscope. Expression levels of Ang II and Calreticulin (CRT) were measured by ELISA, qRT-PCR and Western blot as appropriate. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. HUVECs with IR were exposed to Ang II at series of concentrations, and then the cell viability, apoptosis and CRT were detected. Rescue assays were performed by transfection of siCRT or overexpression of CRT with or without Ang II stimulation into the HUVECs with IR. Expressions of cell apoptosis-related proteins Bcl-2 and Bax were measured by qRT-PCR and Western blot. Glucose (33.3 mmol/L) and insulin (4 µmol/L) induced significantly strong IR to the HUVECs, with a pathological appearance. Levels of Agn II and CRT were both up-regulated by IR. Cell viability of HUVECs was slightly reduced after IR induction for 2 h, and cell apoptosis rate was increased. In addition, Ang II (10-7 mol/l) suppressed cell viability and glucose uptake, promoted cell apoptosis and increased CRT, and these effects could be weakened by silencing CRT. Thus, we preliminarily proved that Ang II up-regulates CRT, and CRT knockdown can relieve Ang II-induced injury of HUVECs with IR.

TIPE2 Mediates the Suppressive Effects of Shikonin on MMP13 in Osteosarcoma Cells.

BACKGROUND/AIMS: Osteosarcoma (OS) is a primary malignant bone tumor in humans, and is notorious mainly for its distal metastases. We have recently shown that Shikonin, an effective constituent extracted from Chinese medicinal herb, inhibits OS cell invasion through suppression of matrix metalloproteinase 13 (MMP13). However, the underlying mechanisms remain unknown. METHODS: Here, we studied the levels of tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) in OS cells upon Shikonin treatment. TIPE2 levels were adapted in OS cell lines through transfection with plasmids carrying transgene or short-hairpin interference RNA (shRNA), and the effects of TIPE2 adaptation on MMP13 and cell invasiveness were evaluated by RT-qPCR, Western blot, ELISA and transwell cell migration assay, respectively. TIPE2 levels in OS specimens from patients were examined and correlated with cancer metastases and patient survival. RESULTS: We found that Shikonin dose-dependently decreased MMP13 levels, and increased TIPE2 levels in two OS cell lines, U2OS and SaOS-2. Overexpression of TIPE2 in U2OS significantly suppressed MMP13 levels and cell invasiveness. Depletion of TIPE2 in SaOS-2 cells significantly increased MMP13 levels and cell invasiveness. Moreover, TIPE2 levels in OS specimens were significantly decreased, compared to adjacent non-cancer bone tissue. Lower TIPE2 levels correlated with higher incidence of metastases and worse 5-year survival. CONCLUSION: TIPE2 mediates the suppressive effects of Shikonin on MMP13 in osteosarcoma cells, and TIPE2 may be a novel therapeutic target for OS.

Shikonin inhibits invasiveness of osteosarcoma through MMP13 suppression.

Osteosarcoma (OS) is the most common primary malignant bone tumor, notorious for its metastasis. We have recently shown that shikonin, an effective constituent extracted from Chinese medicinal herb, induces necroptosis in OS cells. Nevertheless, the effects of low-dose shikonin on the invasiveness of OS cells are unknown. Here, we showed that shikonin dose-dependently decreased OS cell invasiveness in both scratch wound healing assay and transwell cell migration assay. Moreover, the direct target of shikonin on cell invasiveness was found to be matrix metalloproteinase (MMP)-13. Further, the inhibitory effects of shikonin on cell invasiveness were completely abolished in MMP13-overexpressing OS cells. Together, these data suggest that shikonin may suppress OS invasiveness through MMP13 suppression. Thus, our data highlight a previous unappreciated role for shikonin in suppressing OS cell metastasis.

Anxiety and adverse coronary artery disease outcomes in Chinese patients.

OBJECTIVES: To investigate the impact of anxiety on the prognosis of coronary artery disease (CAD) in patients of Chinese Han ethnicity and to explore the correlation between anxiety and the severity of coronary atherosclerosis. METHODS: Between June 2007 and May 2009, 1007 hospitalized patients with CAD diagnosed by coronary angiography were recruited. The anxiety symptoms were investigated within 2 days after coronary angiography or percutaneous coronary intervention, using the Zung Self-Rating Anxiety Scale. The severity of coronary atherosclerosis was assessed using the modified Gensini score. One and a half years after discharge, the participants were evaluated at the outpatient clinic or by telephone. The primary outcome was the composite of all-cause death and nonfatal myocardial infarction (MI). RESULTS: A total of 917 (91.1%) patients were evaluated during a mean follow-up period of 17 months. Thirty-five deaths (26 of cardiovascular and 9 of other causes) and 5 nonfatal MIs occurred. The Zung Self-Rating Anxiety Scale score assessed during hospitalization was significantly and independently associated with the Gensini score (ß = 1.35, p < .001). Adjusted Cox proportional hazards models showed that anxiety symptoms predicted all-cause death and nonfatal MI (adjusted hazard ratio = 2.43, 95% confidence interval = 1.24-4.75, p = .009). CONCLUSIONS: Anxiety was independently associated with the severity of coronary atherosclerosis and predicted worse outcome in patients with CAD of Chinese Han ethnicity.

The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis.

BACKGROUND: Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers. METHODS: MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo. RESULTS: The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001). CONCLUSIONS: Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Syringaresinol attenuates osteoarthritis via regulating the NF-κB pathway.

Osteoarthritis (OA) is a now regarded as a worldwide whole joint disease with synovial inflammation, cartilage degeneration, and subchondral sclerosis. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used drugs for OA treatment which only relieve the symptoms and restrain the progression of OA. However, various severe adverse effects often occur in patients with long-term NSAIDs use, which heavily burdens the healthcare system and impacts the quality of life. Therefore, it is much imperative to identify alternative drugs with increased efficacy. Syringaresinol (Syr), a naturally occurring phytochemical which belonging to the lignan group of polyphenols, shows anti-tumor and anti-oxidant activities, which to benefit human health. Studies has shown Syr can regulate the inflammatory response by modulating the secretion and expression level of cytokines IL-6, IL-8, and tumor necrosis factor (TNF)-α. it also shows the inhibitory effect on NF-κB pathway in mouse cells. In the present study, we aimed to demonstrate the anti-inflammatory effects of Syr in OA. In vitro Syr treatment in IL-1ß-activated mouse chondrocytes significantly restrained the expression of NO, PGE2, IL-6, TNF-α, INOS, COX-2 and MMP-13. Moreover, it considerably ameliorated the degradation of aggrecan and collagen II. Furthermore, the phosphorylation of the NF-kB signaling pathway was significantly suppressed by Syr. Moreover, in vivo, the cartilage degeneration was attenuated and the increased Osteoarthritis Research Society International (OARSI) scores were reversed in the DMM + Syr group, comprared to those in the DMM group. In sum, our study demonstrated that Syr can attenuate the inflammation in vitro and further verified its effect on OA in vivo. Thus, Syr might be a potent therapeautic alternative for OA treatment.

LncRNA RBM5-AS1 Promotes Osteosarcoma Cell Proliferation, Migration, and Invasion.

PURPOSE: Osteosarcoma (Os) is the most frequent malignant tumor of the bone in the pediatric age group, and accumulating evidences show that lncRNAs play a key role in the development of Os. Thus, we investigated the role of RBM5-AS1 and its molecular mechanism. METHODS: The expression of RBM5-AS1 in Os tissues and cell lines was detected by real-time polymerase chain reaction (QPCR). The effect of RBM5-AS1 on the proliferation of Os cells was detected using CCK8 assays and flow cytometry. The effect of RBM5-AS1 on the migration and invasion of Os cells was detected by transwell assays. And we performed QPCR and western blotting assays to investigate the relationship between RBM5-AS1 and RBM5. Finally, western blotting assays were performed to explore the mechanism of RBM5. RESULTS: LncRNA RBM5-AS1 was overexpressed in the Os tissues and cell lines. And lncRNA RBM5-AS1 promoted Os cell proliferation, migration, and invasion in vitro and tumor growth in vivo. LncRNA RBM5-AS1 targets RBM5 in Os cells. CONCLUSION: To sum up, the results showed that lncRNA RBM5-AS1 promotes cell proliferation, migration, and invasion in Os.

[Psychological status in 1083 hospitalized patients with coronary artery disease].

OBJECTIVE: To explore the prevalence and associated factors of anxiety and depression symptoms in hospitalized Chinese patients with coronary artery disease (CAD). METHODS: From June 2007 to May 2009, 1083 hospitalized patients with confirmed coronary artery disease were recruited in this study. The ZUNG Self-rating Anxiety Scale (SAS) and the ZUNG Self-rating Depression Scale (SDS) were used for the psychological assessment. Economic status, living condition and the environment of both living and working places were evaluated by epidemiological questionnaires. RESULTS: The prevalence of pure anxiety, pure depression symptoms and the combination of anxiety and depression symptoms were 7.9%, 28.3% and 14.3% respectively. Incidence of anxiety and depression symptoms was significantly higher in female patients compared with in male patients (P = 0.003, 0.012 respectively) and in aged patients than in middle-aged patients (P = 0.001). The elderly, less than 9 years of education and poor sleep quality increased the risk of anxiety symptom with ORs of 1.63 (95%CI: 1.21 - 2.21), 1.54 (95%CI: 1.15 - 2.07) and 1.62 (95%CI: 1.34 - 1.96), respectively, while workplace noise, history of chronic disease and poor sleep quality increased the risk of depression symptom with ORs of 1.52 (95%CI: 1.18 - 1.98), 1.36 (95%CI: 1.06 - 1.75) and 1.27 (95%CI: 1.08 - 1.50), respectively. Female (OR = 1.91, 95%CI: 1.22-2.98), aged patient (OR = 1.84, 95%CI: 1.23 - 2.76), workplace noise (OR = 1.61, 95%CI: 1.07 - 2.42), history of chronic disease (OR = 1.84, 95%CI: 1.24 - 2.71) and poor sleep quality (OR = 1.73, 95%CI: 1.35 - 2.21) were significantly correlated with the combined incidence of anxiety and depression symptoms. CONCLUSION: Around half of the Chinese hospitalized CAD patients were complicated with various degrees of anxiety and/or depression symptoms. Female and aged patients were at higher risk for anxiety and depression symptoms. Sleep quality, workplace noise, years of education and history of chronic disease were independent risk factors for anxiety or depression symptoms.

Clinical Study of 3D Imaging and 3D Printing Technique for Patient-Specific Instrumentation in Total Knee Arthroplasty.

Patient-specific instrumentation (PSI) was designed to improve the accuracy of preoperative planning and postoperative prosthesis positioning in total knee arthroplasty (TKA). However, better understanding needs to be achieved due to the subtle nature of the PSI systems. In this study, 3D printing technique based on the image data of computed tomography (CT) has been utilized for optimal controlling of the surgical parameters. Two groups of TKA cases have been randomly selected as PSI group and control group with no significant difference of age and sex (p > 0.05). The PSI group is treated with 3D printed cutting guides whereas the control group is treated with conventional instrumentation (CI). By evaluating the proximal osteotomy amount, distal osteotomy amount, valgus angle, external rotation angle, and tibial posterior slope angle of patients, it can be found that the preoperative quantitative assessment and intraoperative changes can be controlled with PSI whereas CI is relied on experience. In terms of postoperative parameters, such as hip-knee-ankle (HKA), frontal femoral component (FFC), frontal tibial component (FTC), and lateral tibial component (LTC) angles, there is a significant improvement in achieving the desired implant position (p < 0.05). Assigned from the morphology of patients' knees, the PSI represents the convergence of congruent designs with current personalized treatment tools. The PSI can achieve less extremity alignment and greater accuracy of prosthesis implantation compared against control method, which indicates potential for optimal HKA, FFC, and FTC angles.

Delivery of alginate-chitosan hydrogel promotes endogenous repair and preserves cardiac function in rats with myocardial infarction.

The regenerative potential of alginate-chitosan composite in bone and cartilage tissue has been well documented, but its potential utility in cardiac tissue engineering has remained unknown. This study sought to determine whether early intramyocardial injection of alginate-chitosan could prevent left ventricular (LV) remodeling after myocardial infarction (MI), leading to a more favorable course of tissue restoration. In a rat model of acute MI, local injection of alginate-chitosan hydrogel into the peri-infarct zone preserved scar thickness, attenuated infarct expansion, and reduced scar fibrosis after 8 weeks, concomitantly with promoting increased angiogenesis and greater recruitment of endogenous repair at the infarct zone. Furthermore, this treatment prevented cell apoptosis, induced cardiomyocyte cell cycle re-entry. The cardiac function of the control-injected animals deteriorated over the 8-week course, while that of the hydrogel-injected animals did not.In addition, the hydrogel did not exacerbate inflammation in the heart. Intramyocardial injection of alginate-chitosan hydrogel represents a useful strategy to treat MI. It demonstrated marked therapeutic efficacies on various tissue levels after extensive MI, as well as potential to induce endogenous cardiomyocyte proliferation and recruit cardiac stem cells.

Establishing an osteosarcoma associated protein-protein interaction network to explore the pathogenesis of osteosarcoma.

BACKGROUND: The aim of this study was to establish an osteosarcoma (OS) associated protein-protein interaction network and explore the pathogenesis of osteosarcoma. METHODS: The gene expression profile GSE9508 was downloaded from the Gene Expression Omnibus database, including five samples of non-malignant bone (the control), seven samples for non-metastatic patients (six of which were analyzed in duplicate), and 11 samples for metastatic patients (10 of which were analyzed in duplicate). Differentially expressed genes (DEGs) between osteosarcoma and control samples were identified by packages in R with the threshold of |logFC (fold change)| > 1 and false discovery rate < 0.05. Osprey software was used to construct the interaction network of DEGs, and genes at protein-protein interaction (PPI) nodes with high degrees were identified. The Database for Annotation, Visualization and Integrated Discovery and WebGestalt software were then used to perform functional annotation and pathway enrichment analyses for PPI networks, in which P < 0.05 was considered statistically significant. RESULTS: Compared to the control samples, the expressions of 42 and 341 genes were altered in non-metastatic OS and metastatic OS samples, respectively. A total of 15 significantly enriched functions were obtained with Gene Ontology analysis (P < 0.05). The DEGs were classified and significantly enriched in three pathways, including the tricarboxylic acid cycle, lysosome and axon guidance. Genes such as HRAS, IDH3A, ATP6ap1, ATP6V0D2, SEMA3F and SEMA3A were involved in the enriched pathways. CONCLUSIONS: The hub genes from metastatic OS samples are not only bio-markers of OS, but also help to improve therapies for OS.

Expression level of Pre B cell leukemia homeobox 2 correlates with poor prognosis of gastric adenocarcinoma and esophageal squamous cell carcinoma.

Pre B cell leukemia homeobox 2 (PBX2), a member of PBX family, acts as a co-factor of homeobox proteins to regulate proliferation and differentiation of tumor cells. Our recent study revealed prognostic significance of PBX2 expression in non-small cell lung carcinoma. The significance of PBX2 expression was examined in cases with gastric cancer (GC) and esophageal squamous cell carcinoma (ESCC), and the role of PBX2 in tumor behavior was evaluated in GC and ESCC cell lines of knocked-down PBX2 expression. Expression level of PBX2 was immunohistochemically examined in 94 patients of GC and 64 patients of ESCC. Staining intensity for PBX2 was categorized as equal to or stronger (level 1) and weaker (level 2) than that of endothelial cells. Cases with level 1 expression in more than 20% of tumor were defined as high and others low expression. Patients with low PBX2 expression showed a better prognosis than those with high expression in both GC and ESCC. Multivariate analysis revealed PBX2 expression to be an independent prognosticator for both GC and ESCC. Knocked-down expression of PBX2 in GC and ESCC cell lines resulted in decrease of in vitro colony formation and in vivo tumorigenic activities, but proliferative and invasive activities did not change. Under serum depletion, apoptotic cell proportion was higher in PBX2 knocked-down cells than in control cells. The knock-down of PBX2 reduced Bcl-2 expression. Taken together, the high expression level of PBX2 was an independent negative prognosticator for both GC and ESCC, and PBX2 might promote tumor growth through suppression of apoptosis.