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A combination of enzymatic digestions with chondroitinases in conjunction with anion-exchange HPLC was used to determine the sequences of octa- and decasaccharides. The findings of sequencing showed that each fraction is a mixture of a major component together with one to three minor components, reflecting the heterogeneity of the parent polysaccharide.

Abstract Background Autologous chondrocyte transplantation is a promising technique for treatment of cartilage defects. Three dimensional chondrocyte cultures on a scaffold are widely used to retain the chondrogenic phenotype. Using a biodegradable gelatin scaffold is one option for the cell delivery system, but molecular and histological studies of the method have not yet been done. Methods We evaluated the chondrogenic property of the primary human chondrocyte on a gelatin scaffold as compared to a collagen scaffold over a period of 21 days. We examined the production of glycosaminoglycan by quantitative and histological analysis. Gene expression of cartilage-associated molecules was assessed by quantitative RT-PCR. Results The gelatin scaffold showed the ability to promote chondrocyte expansion, chondrogenic phenotype retention at molecular and mRNA levels. Conclusions This scaffold is thus suitable for use as an in vitro model for chondrocyte 3D culture.

Abstract Background Osteoporosis is a worldwide health problem predominantly affecting post-menopausal women. Therapies aimed at increasing bone mass in osteoporetic patients lag behind comparable investigation of therapeutic strategies focusing on the bone resorption process. Sesamin, a major lignan compound found in Sesamun indicum Linn., has a variety of pharmacological effects, though its activity on bone cell function is unclear. Herein we examine the effect of this lignan on osteoblast differentiation and function. Method Cell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 h of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 h of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stained with Alizarin Red S. MAPK signaling pathway activation was observed by using western blotting. Results Sesamin promoted the gene expression of COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19. Conclusions The data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the p38 and ERK MAPK signaling pathway and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts. Therefore, sesamin may be a promising phytochemical that could be developed for supplementation of osteoporotic therapy.

Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells, but does not affect normal cells or human leukemic cells, such as MOLT-4 and U937 cells, which are relatively resistant to TRAIL. Three flavonoids extracted from the rhizome of K. parviflora were 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF) and 3,5,7,3',4'-pentamethoxyflavone (PMF), and synthetic flavonoids including 5-methoxyflavone (5-MF) and 2'-methoxyflavone (2"-MF) were chosen for testing in this study. The aims of this study were to examine whether the treatment of TRAIL-resistant leukemia MOLT-4 and U937 cells, with methoxyflavone derivatives could enhance the apoptotic response and to identify the mechanism involved. Methods The cytotoxic effect of methoxyflavone (MF) derivatives in MOLT-4, U937 and peripheral blood mononuclear cells (PBMCs) was analyzed by the MTT assay. The induction of apoptosis and the reduction of mitochondrial transmembrane potential (ΔΨm) after staining with annexin V FITC and propidium iodide (PI), and 3,3'-dihexyloxacarbocyanine iodide (DiOC6), respectively, were performed using flow cytometry. ROS production was determined by staining with 2',7'-dichlorofluorescin diacetate and processed with a flow cytometer. DR4, DR5, cFLIP, Mcl-1, BAX and Bid expression were demonstrated by immunoblotting. Caspase-8 and -3 activities were determined by using IETD-AFC and DEVD-AFC substrates and the fluorescence intensity was measured. Results All methoxyflavone derivatives were cytotoxic to MOLT-4, U937 cells and PBMCs, except DMF, TMF and PMF were not toxic to PBMCs. All MF derivatives induced human leukemic MOLT-4 cell apoptosis, but not in U937 cells. Percentage of MOLT-4 cells with (ΔΨm) was increased when treated with DMF, TMF, PMF, 5-MF and 2'-MF in the presence of TRAIL. 5-MF and 2'-MF enhanced TRAIL-induced apoptosis through the up-regulation of both DRs and the down-regulation of cFLIP and Mcl-1. Bid was cleaved and BAX was up-regulated, followed by the activation of caspase-8 and -3. Oxidative stress was also increased. 2'-MF gave the same result compared with 5-MF but with a less effect. Conclusion Methoxyflavone derivatives enhanced TRAIL-induced apoptosis in human leukemic MOLT-4 cells through the death receptors and mitochondrial pathways.

Abstract Background Zearalenone (ZEA) is a phytoestrogen from Fusarium species. The aims of the study was to identify mode of human leukemic cell death induced by ZEA and the mechanisms involved. Methods Cell cytotoxicity of ZEA on human leukemic HL-60, U937 and peripheral blood mononuclear cells (PBMCs) was performed by using 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Reactive oxygen species production, cell cycle analysis and mitochondrial transmembrane potential reduction was determined by employing 2',7'-dichlorofluorescein diacetate, propidium iodide and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. Caspase-3 and -8 activities were detected by using fluorogenic Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) and Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (IETD-AMC) substrates, respectively. Protein expression of cytochrome c, Bax, Bcl-2 and Bcl-xL was performed by Western blot. The expression of proteins was assessed by two-dimensional polyacrylamide gel-electrophoresis (PAGE) coupled with LC-MS2 analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) approach. Results ZEA was cytotoxic to U937 > HL-60 > PBMCs and caused subdiploid peaks and G1 arrest in both cell lines. Apoptosis of human leukemic HL-60 and U937 cell apoptosis induced by ZEA was via an activation of mitochondrial release of cytochrome c through mitochondrial transmembrane potential reduction, activation of caspase-3 and -8, production of reactive oxygen species and induction of endoplasmic reticulum stress. Bax was up regulated in a time-dependent manner and there was down regulation of Bcl-xL expression. Two-dimensional PAGE coupled with LC-MS2 analysis showed that ZEA treatment of HL-60 cells produced differences in the levels of 22 membrane proteins such as apoptosis inducing factor and the ER stress proteins including endoplasmic reticulum protein 29 (ERp29), 78 kDa glucose-regulated protein, heat shock protein 90 and calreticulin, whereas only ERp29 mRNA transcript increased. Conclusion ZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.

Abstract Background Glucosamine (GlcN) is a well-recognized candidate for treatment of osteoarthritis. However, it is currently used in derivative forms, such as glucosamine-hydrochloride (GlcN-HCl) or glucosamine sulfate (GlcN-S). However, the molecular mode of action remains unclear. In this study, we compared the effects of Glucose (Glc), Glucuronic acid (GlcA), Glucosamine hydrochloride (GlcN-HCl) and Glucosamine sulfate (GlcN-S) on cartilage degradation. Methods Porcine cartilage explants were co-cultured with recombinant human IL-1β and each tested substance for 3 days. HA, s-GAG and MMP-2 releases to media were measured using ELISA, dye-binding assay and gelatin zymography, respectively. Similar studies were performed in a human articular chondrocytes (HAC) monolayer culture, where cells were co-treated with IL-1β and each reagent for 24 hours. Subsequently, cells were harvested and gene expression measured using RT-PCR. All experiments were carried out in triplicate. Student's t-tests were used for statistical analysis. Results In cartilage explants treated with IL-1β, GlcN-S had the highest chondroprotective activity of all four chemicals as shown by the inhibition of HA, s-GAG and MMP-2 released from cartilage. The anabolic (aggrecan core protein; AGG, SOX9) and catabolic (MMP-3, -13) genes in HACs treated with IL-1β and with/without chemicals were studied using RT-PCR. It was found that, GlcN-HCl and GlcN-S could reduce the expression of both MMP-3 and -13 genes. The IL-1β induced-MMP-13 gene expression was decreased maximally by GlcN-S, while the reduction of induced-MMP-3 gene expression was greatest with GlcN-HCl. Glc and GlcA reversed the effect of IL-1β on the expression of AGG and SOX9, but other substances had no effect. Conclusion This study shows that glucosamine derivatives can alter anabolic and catabolic processes in HACs induced by IL-1β. GlcN-S and GluN-HCl decreased induced MMP-3 and -13 expressions, while Glc and GlcA increased reduced-AGG and SOX9 expression. The chondroprotective study using porcine cartilage explant showed that GlcN-S had the strongest effect.

Background/objectives Maxillary tooth distal movement is a treatment option for Class II malocclusion. This prospective clinical study (split-mouth design) was aimed to compare chondroitin sulphate (CS) levels in gingival crevicular fluid (GCF), the rates of tooth movement, and patient pain and discomfort during segmental maxillary posterior tooth distal movement using either 120 or 180 g of retraction force. Materials and methods Twenty patients (6 males and 14 females; aged 18.85 ± 4.38 years) with Class II malocclusion were recruited. The force magnitudes were controlled at 120 or 180 g, randomly assigned to either the right or left five-tooth segments. Gingival crevicular fluid samples were collected with Periopaper® strips. Competitive ELISA with monoclonal antibody was used to measure CS levels in GCF. The rates of segmental maxillary posterior tooth distal movement, and the amount of pain and discomfort were evaluated. Results The median CS levels during the segmental distal movement period were significantly greater than those before the segmental distal movement period (P < 0.05). At each 1-week period during segmental distal movement, the differences between the median CS levels induced by the two different force magnitudes were not significantly different. The rates of segmental distal movement induced by the two different force magnitudes were not significantly different. The mean visual analog scale scores for pain and discomfort with 180 g of retraction force was significantly greater than that with 120 g (P < 0.05). Conclusions One hundred and twenty grams of retraction force was sufficient to cause segmental distal movement, as indicated by biochemically assessed bone remodeling activity and a similar rate of tooth movement to that caused by 180 g of retraction force; it also produced less patient pain and discomfort. Trial Registration The study has been registered as TCTR20170728001. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common form of arthritis. Obesity has been believed to be an important risk factor for OA development and the progression of not only load-bearing joints, but low-load-bearing joints as well. Increased leptin has been the focus of a link between obesity and OA. In this study, the effects of pathological (100ng/ml) or supra-pathological (10μg/ml) concentrations of leptin alone or in combination with IL1β on cartilage metabolisms were studied in porcine cartilage explant. The involved mechanisms were examined in human articular chondrocytes (HACs). Moreover, the protective effect of omega-3 polyunsaturated acids, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was also investigated. Leptin (10μg/ml) alone or in combination with IL1β could induce cartilage destruction, although lower concentrations had no effect. Leptin activated NFκB, ERK, JNK and p38 in HACs, which led to the induction of MMP3, MMP13 and ADAMTS4 secretions. The combined effect could further induce those enzymes through the additive effect on activation of NFκB and JNK. Interestingly, both EPA and DHA could inhibit cartilage damage induced by leptin plus IL1β by reducing the activation of NFκB and JNK, which led to the decrease of ADAMTS4 secretion. Altogether, only a supra-pathological concentration of leptin alone or in combination with IL1β could induce cartilage destruction, whereas a pathological one could not. This effect could be inhibited by EPA and DHA. To gain greater understanding of the link between leptin and OA, the effect of different levels of leptin on several states of OA cartilage requires further investigation. [ABSTRACT FROM AUTHOR]

Preclinical studies have demonstrated impaired osteoblast differentiation in type 2 diabetes (T2DM), which is related to skeletal accumulation of advanced glycation end products (AGEs). However, the role of AGE in osteoblast differentiation in patients with T2DM is unclear. This cross-sectional study was performed to investigate osteoblast differentiation and its association with serum pentosidine and soluble receptor of AGEs (sRAGE). Twenty-seven patients with T2DM and 15 age-matched controls were included to measure sRAGE and osteogenic differentiation in mononuclear cells derived from peripheral blood. The mononuclear cells isolated from patients with T2DM showed a significantly lower rate of osteogenic differentiation (7.4% vs 86.7%, p < 0.0001) with a lower level of ALPL, COL1A1, and BGLAP expression than those of controls by 11-, 44-, and 15-fold respectively, together with nonvisualized mineralization by alizarin red S staining. The levels of pentosidine and sRAGE were comparable in both groups. AGER expression was significantly higher in the T2DM group. BAX expression was also significantly higher in the T2DM group, and showed a strong correlation with AGER expression (r = 0.86, p < 0.0001). Fasting plasma glucose (FPG) level, AGER expression, and BAX expression showed a strong correlation with osteogenic differentiation defects on univariate analysis. However, only FPG showed a correlation with this defect in a multivariate analysis. In conclusion, patients with T2DM showed impairment of osteoblast differentiation, and FPG was an independent risk factor for this impairment. Moreover, T2DM showed a higher cellular sensitivity for activation of receptor of AGEs and higher cellular apoptosis, which may contribute to the defect in osteoblast differentiation. [ABSTRACT FROM AUTHOR]