Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

Blood Trihalomethane Concentrations and Osteoarthritis among U.S. Population Aged over 50 Years.

Exposure to trihalomethanes (THMs) has been associated with inflammation and oxidative stress, which are implicated in osteoarthritis. However, the association of THM exposure with osteoarthritis is unknown. Therefore, we pooled seven independent National Health and Nutrition Examination Survey cycles (1999-2012) among participants aged over 50 years who had quantified blood concentrations of chloroform (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and bromoform (TBM). Among 4,077 adults aged over 50 years, 781 (21.3%) reported osteoarthritis. Logistic regression models showed increased odds of osteoarthritis across the categories of blood BDCM, DBCM, and brominated THM (Br-THM, which was the sum of BDCM, DBCM, and TBM) concentrations [odds ratios = 1.46 (95% CI 1.09-1.94), 1.53 (95% CI 1.15-2.04), and 1.35 (95% CI 0.97-1.88), respectively], comparing highest versus lowest exposure categories (quartiles or tertiles). Additionally, we found positive dose-response relationships between blood BDCM, DBCM, and Br-THM concentrations and serum markers of oxidative stress (i.e., gamma-glutamyltransferase). In summary, blood Br-THM concentrations were associated with elevated serum levels of gamma-glutamyltransferase as well as an increased risk of osteoarthritis among U.S. adults aged over 50 years. However, more prospective population studies are needed to verify these findings and explore the underlying mechanisms.

Quercetin prevents osteoarthritis progression possibly via regulation of local and systemic inflammatory cascades.

Due to the lack of effective treatments, osteoarthritis (OA) remains a challenge for clinicians. Quercetin, a bioflavonoid, has shown potent anti-inflammatory effects. However, its effect on preventing OA progression and the underlying mechanisms are still unclear. In this study, Sprague-Dawley male rats were divided into five groups: control group, OA group (monosodium iodoacetate intra-articular injection), and three quercetin-treated groups. Quercetin-treated groups were treated with intragastric quercetin once a day for 28 days. Gross observation and histopathological analysis showed cartilage degradation and matrix loss in the OA group. High-dose quercetin-group joints showed failure in OA progression. High-dose quercetin inhibited the OA-induced expression of MMP-3, MMP-13, ADAMTS4, and ADAMTS5 and promoted the OA-reduced expression of aggrecan and collagen II. Levels of most inflammatory cytokines and growth factors tested in synovial fluid and serum were upregulated in the OA group and these increases were reversed by high-dose quercetin. Similarly, subchondral trabecular bone was degraded in the OA group and this effect was reversed in the high-dose quercetin group. Our findings indicate that quercetin has a protective effect against OA development and progression possibly via maintaining the inflammatory cascade homeostasis. Therefore, quercetin could be a potential therapeutic agent to prevent OA progression in risk groups.

Quercetin modulates the gut microbiota as well as the metabolome in a rat model of osteoarthritis.

Although the mechanism of osteoarthritis (OA) has been widely studied and the use of quercetin for OA therapy is well documented, the relevant characteristics of the microbiome and metabolism remain unclear. This study reports changes in the gut microbiota and metabolism during quercetin therapy for OA in a rat model and provides an integrative analysis of the biomechanism. In this study, the rats were categorized into 3 different groups: the OA model, quercetin treatment, and control groups. The OA rats was conducted using a monoiodoacetate (MIA) injection protocol. The rats in the quercetin group received daily intragastric administration of quercetin from day 1 to day 28. Stool samples were collected, and DNA was extracted. We used an integrated approach that combined the sequencing of whole 16S rRNA, short-chain fatty acid (SCFA) measurements and metabolomics analysis by mass spectrometry (MS) to characterize the functional impact of quercetin on the gut microbiota and metabolism in a rat model of OA. The use of quercetin partially abrogated intestinal flora disorder and reversed fecal metabolite abnormalities. Compared with the control rats, the OA rats showed differences at both the class level (Clostridia, Bacteroidia, and Bacilli) and the genus level (Lactobacillus and unidentified Ruminococcaceae). Acetic acid, propionic acid and 24 metabolites were significantly altered among the three groups. However, the changes were significantly abrogated in quercetin-treated OA rats. Consequently, this study provided important evidence regarding perturbations of the gut microbiome and the function of these changes in a potential new mechanism of quercetin treatment.

Analysis and Construction of a Competitive Endogenous RNA Regulatory Network of Baicalin-Induced Apoptosis in Human Osteosarcoma Cells.

BACKGROUND: Baicalin is an extract from the traditional Chinese herb Scutellaria baicalensis and has the potential to treat osteosarcoma (OS). However, the transcriptome-level mechanism of baicalin-mediated antitumor effects in OS has not yet been investigated. The aim of this study was to analyze the competitive endogenous RNA (ceRNA) regulatory network involved in baicalin-induced apoptosis of OS cells. METHODS: In this study, CCK-8 and flow cytometry assays were used to detect the antitumor effects of baicalin on human OS MG63 cells. Furthermore, transcriptome sequencing was employed to establish the long noncoding RNA (lncRNA), microRNA (miRNA), and mRNA profiles. RESULTS: Baicalin inhibited MG63 cell proliferation and induced apoptosis. Totals of 58 lncRNAs, 31 miRNAs, and 2136 mRNAs in the baicalin-treated MG63 cells were identified as differentially expressed RNAs compared to those in control cells. Of these, 2 lncRNAs, 3 miRNAs, and 18 mRNAs were included in the ceRNA regulatory network. The differentially expressed RNAs were confirmed by quantitative real-time PCR (qRT-PCR). CONCLUSIONS: By identifying the ceRNA network, our results provide new information about the possible molecular basis of baicalin, which has potential applications in OS treatment.

Increased EHHADH Expression Predicting Poor Survival of Osteosarcoma by Integrating Weighted Gene Coexpression Network Analysis and Experimental Validation.

Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH), a member of the 3-hydroxyacyl-CoA dehydrogenase family, were previously demonstrated to be involved in the tumorigenesis of various cancer types. This study is aimed at determining of the diagnostic and prognostic value of EHHADH in osteosarcoma (OS). The overexpression of EHHADH was found both in OS and also other sarcoma types, and according to the retrospective cohort study, the EHHADH level was related to the overall survival and disease-free survival of the OS patients. Furthermore, knockdown of EHHADH under the influence of EHHADH small interfering RNA significantly suppressed the proliferation ability of the tumor cells. Moreover, EHHADH overexpressed was found in human OS tissues. In summary, the progression of OS could be enhanced by EHHADH, which may be a potential diagnostic and prognostic biomarker for OS patients.

Investigation of normal knees kinematics in walking and running at different speeds using a portable motion analysis system.

Walking and running at different speeds are common in daily life. This study investigated 6 degrees of freedom (DOF) kinematics of normal knees of Chinese during walking and running. Forty healthy participants were investigated in 4 conditions: comfortable walking, normal walking, slow running and ordinary running. The range of motion (ROM) and peak values in 6 DOF kinematics were analysed. As the speed increased, a general increase in flexion, lateral and proximal translations occurred. Significant increases of ROM in flexion/extension, axial rotation and medial/lateral translations were observed. The ROM of adduction/abduction, anterior/posterior and proximal/distal translations were greatest during normal walking. The maximum and minimum flexion/extension, maximum internal rotation and tibial lateral translations increased with the increase of speed. The maximum and minimum tibial proximal translations in running were found being greater than walking. A phenomenon between walking and running was observed: both tibial proximal/distal and medial/lateral translations increased when changed from walking to running. Non-linear transition exists in 6 DOF kinematics during walking to running. Discoveries in this study may have potential clinical values to serve as references of normal walking and running in the management of knee injury and knee rehabilitation.

A Traditional Chinese Medicine Plant Extract Prevents Alcohol-Induced Osteopenia.

Traditional Chinese medicine (TCM) has been practiced in the treatment of bone diseases and alcoholism. Chronic excessive alcohol use results in alcohol-induced bone diseases, including osteopenia and osteoporosis, which increases fracture risk, deficient bone repair, and osteonecrosis. This preclinical study investigated the therapeutic effects of TCM herbal extracts in animal models of chronic excessive alcohol consumption-induced osteopenia. TCM herbal extracts (Jing extracts) were prepared from nine Chinese herbal medicines, a combinative herbal formula for antifatigue and immune regulation, including Astragalus, Cistanche deserticola, Dioscorea polystachya, Lycium barbarum, Epimedium, Cinnamomum cassia, Syzygium aromaticum, Angelica sinensis, and Curculigo orchioides. In this study, Balb/c male mice were orally administrated alcohol (3.2 g/kg/day) with/without TCM herbal extracts (0.125 g/kg, 0.25 g/kg, or 0.5 g/kg) by gavage. Our results showed that after 50 days of oral administration, TCM herbal extracts prevented alcohol-induced osteopenia demonstrated by µ-CT bone morphological analysis in young adults and middle-aged/old Balb/c male mice. Biochemical analysis demonstrated that chronic alcohol consumption inhibits bone formation and has a neutral impact on bone resorption, suggesting that TCM herbal extracts (Jing extracts) mitigate the alcohol-induced abnormal bone metabolism in middle-aged/old male mice. Protocatechuic acid, a natural phenolic acid in Jing extracts, mitigates in vivo alcohol-induced decline of alkaline phosphatase (ALP) gene expression in the bone marrow of Balb/c male mice and in vitro ALP activity in pre-osteoblast MC3T3-E1 cells. Our study suggests that TCM herbal extracts prevent chronic excessive alcohol consumption-induced osteopenia in male mice, implying that traditional medicinal plants have the therapeutic potential of preventing alcohol-induced bone diseases.

Tartary buckwheat extract alleviates alcohol-induced acute and chronic liver injuries through the inhibition of oxidative stress and mitochondrial cell death pathway.

Alcohol use disorder (AUD) is an enormous public health problem that poses significant social, medical, and economic burdens. Under AUD, the liver is one of the most adversely affected organs. As current therapies and protective drugs for AUD-mediated liver injury are very limited, the prevention and therapy of alcoholic liver disease are urgently needed. The present study aims to investigate the beneficial effects of tartary buckwheat extract (TBE), the important component of Maopu tartary buckwheat liquor, on both alcoholic-induced acute and chronic liver injuries. We show that the TBE administration, similar to curcumin, significantly reduces the elevated serum aspartate aminotransferase and alanine aminotransferase levels, improves liver index, alleviates the elevated contents of hepatic malondialdehye, and restores the decreased contents of hepatic glutathione both in acute and chronic liver injuries in alcohol-exposed rats. Furthermore, histopathological analyses show that a medium dose of TBE (16.70 ml/kg body weight) alleviates hepatocyte morphology changes in both acute and chronic alcohol exposure models. We also show the protective effects of TBE on the cell death rates of alcohol-exposed primary cultured hepatocytes, HepG2 hepatoma, and Huh 7 hepatoma cells. Furthermore, we demonstrate that TBE exerts hepatoprotection partly through inhibiting the mitochondrial cell death pathway by reducing cytochrome c release, caspase-9 and -3 activities, and the number of TUNEL-positive cells. These effects of TBE were accompanied by enhanced levels of Bcl-2 and Bcl-xL and autophagic cell death pathway by reducing Beclin-1 expression, as well as through promoting its anti-oxidant capacity by suppressing reactive oxygen species production. This study demonstrates, for the first time, the protective effect of TBE against alcohol-induced acute and chronic liver injury in vivo and in vitro. Given the dietary nature of tartary buckwheat, pueraria, lycium barbarum, and hawthorn, the oral intake of TBE or liquor contained TBE, e.g., Maopu Tartary buckwheat liquor, compared with pure liquor consumption alone, may have the potential to alleviate alcoholic-induced liver injuries.

CTX-II and YKL-40 in early diagnosis and treatment evaluation of osteoarthritis.

This study investigated the value of C-terminal telopeptides of collagen type II (CTX-II) and YKL-40 in early diagnosis and treatment evaluation of osteoarthritis (OA). A total of 90 patients with OA diagnosed and treated in The First Affiliated Hospital, Guangzhou Medical University from March 2015 to January 2018 were selected as the study group. At the same time, 50 healthy elderly were included as the control group. The study group was divided into three subgroups including group A (29 cases, 500 mg glucosamine sulfate), group B (29 cases, 50 mg diacerein) and group C (32 cases, 500 mg glucosamine sulfate and 50 mg diacerein). Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was used to assess the severity and treatment of arthritis. Enzyme-linked immunosorbent assay was used to measure the concentration of CTX-II and YKL-40 in serum. WOMAC scores in the study A, B and C groups were significantly higher than those in the control group (P<0.001). Serum CTX-II and YKL-40 concentrations were higher in the study group than in the control group (P<0.001). Sensitivity of serum CTX-II combined with YKL-40 in the diagnosis of OA was 90% and the specificity was 78%. CTX-II and YKL-40 levels in different Kellgren Lawrence (K-L) grades were significantly different (P<0.001), and increased with the increase of K-L grade. Concentrations of serum CTX-II and YKL-40 before treatment in the study group was positively correlated with WOMAC score (P<0.001). At 3, 6 and 9 weeks after the beginning of treatment, serum concentrations of CTX-II and YKL-40 decreased significantly (P<0.001). At 3 weeks of treatment, CTX-II was positively correlated with YKL-40 concentration and WOMAC score (r=0.406, P<0.001; r=0.430, P<0.001); CTX-II was positively correlated with YKL-40 concentration and WOMAC score at 6 weeks of treatment (r=0.350, P<0.001; r=0.358, P<0.001); CTX-II was positively correlated with YKL-40 concentration and WOMAC score at 9 weeks after treatment (r=0.370, P<0.001; r=0.394, P<0.001). Combined detection of serum CTX-II and YKL-40 can improve the sensitivity of early OA diagnosis, and it has an important diagnostic value for early OA patients. Therefore, it can be used as a biological indicator for early OA diagnosis, severity assessment, and evaluation of treatment effects.

Quercetin Inhibits Cell Migration and Invasion in Human Osteosarcoma Cells.

BACKGROUND/AIMS: Osteosarcoma is a malignant tumor associated with high mortality; however, no effective therapies for the disease have been developed. Several studies have focused on elucidating the pathogenesis of osteosarcoma and have aimed to develop novel therapies for the disease. Quercetin is a vital dietary flavonoid that has been shown to have a variety of anticancer effects, as it induces cell cycle arrest, apoptosis, and differentiation and is involved in cell adhesion, metastasis and angiogenesis. Herein, we aimed to investigate the effects of quercetin on osteosarcoma migration and invasion in vitro and in vivo and to explore the molecular mechanisms underlying its effects on osteosarcoma migration and invasion. METHODS: Cell viability, cell cycle activity and cell apoptosis were measured using CCK-8 assay and flow cytometry, and cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The mRNA and protein expression levels of several proteins of interest were assessed by real-time quantitative PCR and western blotting, respectively. Moreover, a nude mouse model of human osteosarcoma lung metastasis was established to assess the anti-metastatic effects of quercetin in vivo. RESULTS: We noted no significant differences in cell cycle activity and apoptosis between HOS and MG63 cells and control cells. Treatment with quercetin significantly attenuated cell migration and invasion in HOS and MG63 cells compared with treatment with control medium. Moreover HIF-1α, VEGF, MMP2, and MMP9 mRNA and protein expression levels were significantly downregulated in HOS cells treated with quercetin compared with HOS cells treated with controls. Additionally, treatment with quercetin attenuated metastatic lung tumor formation and growth in the nude mouse model of osteosarcoma compared with treatment with controls. CONCLUSION: Our findings regarding the inhibitory effects of quercetin on cell migration and invasion suggest that quercetin may have potential as a therapy for human osteosarcoma.

Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel.

The present study introduced a direct co-culture of mouse ATDC5 cells and primary porcine chondrocytes into a microcavitary hydrogel, which possessed advantages in promoting the growth of chondrocytes and retaining the phenotype. These two types of cells were encapsulated with gelatin microspheres in alginate hydrogels in either of the three ratios (3:1, 1:1, or 1:3 of ATDC5 cells to chondrocytes) and cultured in chondrogenic medium for 28 days. Simultaneously, the single encapsulation of ATDC5 cells or chondrocytes was set as a control. Cell Counting Kit-8 (CCK-8), real-time PCR, and immunohistochemistry staining were used to evaluate the effect of various ratios of co-cultured ATDC5 cells and chondrocytes on the expression of the cartilaginous phenotype. The CCK-8 data indicated that the ratio of 3:1 group had an outstanding ability of cell growth. The other results demonstrated that higher the ATDC5 ratios and longer the culture duration, greater the expression of cartilage-specific genes (including type II collagen and aggrecan) and more the synthesized cartilaginous extracellular matrix. Also, the Western blot analysis suggested that p44/42 MAP Kinase was involved in cell proliferation. However, due to the direct co-culture of the two cell types, the underlying mechanism necessitates further investigation. Overall, the co-culture system in microcavitary hydrogel improved the effect of chondrogenesis and exhibited promising strategy for cartilage tissue engineering therapies. J. Cell. Biochem. 118: 3607-3615, 2017. © 2017 Wiley Periodicals, Inc.

Differential circRNA expression profiles during the BMP2-induced osteogenic differentiation of MC3T3-E1 cells.

OBJECTIVE: Recent studies have indicated that circular RNAs (circRNAs) might play important roles in various diseases. However, little is known about the functions of circRNAs in the skeletal system, and the role of circRNAs in the mechanism by which bone morphogenetic protein 2 (BMP2) promotes bone differentiation remains unknown. Here, we performed RNA-seq to analyze differential expression of circRNA during different osteoblast differentiation stages and investigated the relevant mechanisms. MATERIALS AND METHODS: Alkaline phosphatase (ALP) staining and activity were performed to assess osteogenic differentiation in MC3T3-E1 cells. The expression of osteogenic markers in MC3T3-E1 cells and the differential expression levels of circRNAs were measured and validated by qRT-PCR. Osteogenic marker proteins were measured by western blot. RNA-seq was performed to detect differential expression of circRNAs during the osteogenic differentiation of MC3T3-E1 cells induced by BMP2. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER pathway analyses were performed to predict the functions of differentially expressed circRNAs and potentially co-expressed target genes. The microRNA (miRNA) targets of the circRNAs and circRNA-miRNA interactions were predicted by miRanda. The circRNA-miRNA co-expression network was constructed based on the correlation analysis between the differentially expressed circRNAs and miRNAs. A graph of the circRNA-miRNA network was created using Cytoscape 3.01. RESULTS: The Cell Counting Kit 8 (CCK-8) assay showed that BMP2 promoted the proliferation of osteoblasts in vitro. Both the intracellular ALP content and activity were increased in BMP2-treated MC3T3-E1 cells. In addition, the mRNA and protein levels of the osteoblastic markers ALP, Sp7 transcription factor (SP7) and runt-related transcription factor 2 (RUNX2) were substantially up-regulated. In the present study, 158 circRNAs were differentially expressed by a fold-change ≥2.0, P<0.05 and false discovery rate <0.05. Among these, 74 circRNAs were up-regulated, while 84 circRNAs were down-regulated. In addition, the expression levels of circRNA.5846, circRNA.19142 and circRNA.10042 were significantly increased in the BMP2 group. Furthermore, by analyzing the target mRNAs of miR-7067-5p using GO and PANTHER pathway analyses, circ19142 and circ5846 were found to be not only strongly associated with the biological process of the positive regulation of developmental processes but also related to the fibroblast growth factor, epidermal growth factor, platelet-derived growth factor and Wnt signaling pathways, which are involved in cell growth and differentiation. CONCLUSION: The present study identified circ19142 and circ5846 as being associated with osteoblast differentiation and BMP2 may induce osteogenic differentiation through a circ19142/circ5846-targeted miRNA-mRNA axis.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair.

OBJECTIVES: The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. METHODS: The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. KEY FINDINGS: An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. CONCLUSIONS: According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.