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Osteoarthritis (OA) is an inflammatory and degenerative joint disease with severe effects on individuals, society, and the economy that affects millions of elderly people around the world. To date, there are no effective treatments for OA; however, there are some treatments that slow or prevent its progression. Polyfunctional nanosystems have many advantages, such as controlled release, targeted therapy and high loading rate, and have been widely used in OA treatment. Previous mechanistic studies have revealed that inflammation and ROS are interrelated, and a large number of studies have demonstrated that ROS play an important role in different types of OA development. In this review article, we summarize third-generation ROS-sensitive nanomaterials that scavenge excessive ROS from chondrocytes and osteoclasts in vivo. We only focus on polymer-based nanoparticles (NPs) and do not review the effects of drug-loaded or heavy metal NPs. Mounting evidence suggests that polyfunctional nanosystems will be a promising therapeutic strategy in OA therapy due to their unique characteristics of being sensitive to changes in the internal environment.

Osteoarthritis (OA) is a degenerative joint disease and is the main cause of chronic pain and functional disability in adults. Articular cartilage is a hydrated soft tissue that is composed of normally quiescent chondrocytes at a low density, a dense network of collagen fibrils with a pore size of 60-200 nm, and aggrecan proteoglycans with high-density negative charge. Although certain drugs, nucleic acids, and proteins have the potential to slow the progression of OA and restore the joints, these treatments have not been clinically applied owing to the lack of an effective delivery system capable of breaking through the cartilage barrier. Recently, the development of nanotechnology for delivery systems renders new ideas and treatment methods viable in overcoming the limited penetration. In this review, we focus on current research on such applications of nanotechnology, including exosomes, protein-based cationic nanocarriers, cationic liposomes/solid lipid nanoparticles, amino acid-based nanocarriers, polyamide derivatives-based nanocarriers, manganese dioxide, and carbon nanotubes. Exosomes are the smallest known nanoscale extracellular vesicles, and they can quickly deliver nucleic acids or proteins to the required depth. Through electrostatic interactions, nanocarriers with appropriate balance in cationic property and particle size have a strong ability to penetrate cartilage. Although substantial preclinical evidence has been obtained, further optimization is necessary for clinical transformation. STATEMENT OF SIGNIFICANCE: The dense cartilage matrix with high-negative charge was associated with reduced therapeutic effect in osteoarthritis patients with deep pathological changes. However, a systematic review in nanodevices for deep cartilage penetration is still lacking. Current approaches to assure penetration of nanosystems into the depth of cartilage were reviewed, including nanoscale extracellular vesicles from different cell lines and nanocarriers with appropriate balance in cationic property and size particle. Moreover, nanodevices entering clinical trials and further optimization were also discussed, providing important guiding significance to future research.

To compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects.The endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/β-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting.Endogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, β-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower.The superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/β-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.

The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein-protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.

Background Novel biomarkers for malignant glioma are urgently needed due to the poor prognosis of this cancer. Numerous discoveries have demonstrated that microRNAs (miRNAs) participate in the occurrence and progression of glioma. The objective of this study is to explore new biomarkers related to glioma prognosis by bioinformatic analysis.Methods The miRNA, mRNA expression profiles and relevant clinical information were extracted from TCGA database. Differently expressed miRNAs (DEMs) and differently expressed mRNAs/genes (DEGs) were screened, and a risk score comprising four miRNAs was constructed. We evaluated and tested the model accuracy through the ROC curve and a survival status analysis. Four-miRNA nomography were performed to quantitatively and visually evaluate the model. The potential biological functions of those network genes were performed through KEGG pathways and Gene Ontology (GO) terms analysis. Furthermore, survival analysis and the clinical information was used to verify whether the hub genes are associated with prognosis. The CGGA and GEPIA database were used to verified the accuracy of these analysis. To investigate the potential functions of those hub genes in glioma, we performed GSEA and GSVA.Results The results of univariate Cox regression showed that twenty miRNAs were significantly associated with overall survival of glioma. Multivariate Cox regression analyses were performed to screen four miRNAs (hsa-miR-744-5p, hsa-miR-1249-3p, hsa-miR-3677-3p and hsa-miR-10b-3p). The estimated area under the receiver operating characteristic (ROC) curve (AUC) were 0.902, 0.896 and 0.907 for groups train, test and entire, respectively, indicating that the model had a significant ability to predict survival in glioma patients. Furthermore, univariate and multivariate Cox regression were performed using relevant clinical factors associated with glioma, showed that the model could act as an independent prognostic factor. CGGA and GEPIA database were used to verified the accuracy of these hub genes. GSEA and GSVA revealed that the lower expressions of these hub genes were most closely associated with vascularization and tumor proliferation.Conclusions In conclusion, the four-miRNA risk score model and these hub genes may represent a novel prognostic marker to predict the survival of patients with glioma.

To study role of TLR4/NF-κB pathway for early change of synovial membrane in knee osteoarthritis rats.Eighteen male SD rats weighted (200±20) g were randomly divided into 2 groups, namely control and model group, and 9 in each group. Knee OA model group was established by using modified Hulth method in model group. Control group was not treated. Synovial tissue and serum was extracted at 4 and 21 d after operation. Expression of CD14, TLR4, IL-1β, TNF-α, ADAMTS-4, MMP-13 were detected by real-time PCR respectively. NF-κB p65 protein was detected by Western-blot; serum concentrations of haluronic acid (HA), N-propeptide of type III procollagen(PIIINP) was detected by Elisa.Expression of CD14, ADAMTS-4, and NF-κB p65 in model group were higher than that of control group at 4 and 21 days after operation, while expression of TLR4, IL-1β, TNF-α and MMP-13 were higher than that of control group at 21 days after operation(NF-κB pathway could mediate occurrence of KOA by early activating and triggeringg synovial increasingly secreting inflammatory secretion CD14, TLR4, IL-1β, TNF-α, ADAMTS-4, MMP-13, PIIINP and HA.

Objective. The aim of this study is to systematically evaluate the evidence whether traditional Chinese herbal patches (TCHPs) for osteoarthritis (OA) are effective and safe and analyze their medication patterns.Methods. A systematic literature search was performed using all the possible Medical Subject Headings (MeSH) and keywords from January 1979 to July 2013. Both randomized controlled trials (RCTs) and observational studies were included. Estimated effects were analyzed using mean difference (MD) or relative risk (RR) with 95% confidence intervals (CI) and meta-analysis.Results. 86 kinds of TCHPs were identified. RCTs and controlled clinical trials (CCTs) which were mostly of low quality favored TCHPs for local pain and dysfunction relief. TCHPs, compared with diclofenac ointment, had significant effects on global effectiveness rate (RR = 0.50; 95% CI (0.29, 0.87)). Components of formulae were mainly based on the compounds “Xiao Huo Luo Dan” (Minor collateral-freeing pill) and “Du Huo Ji Sheng Tang” (Angelicae PubescentisandLoranthidecoction). Ten kinds of adverse events (AEs), mainly consisting of itching and/or local skin rashes, were identified after 3-4 weeks of follow-up.Conclusions. TCHPs have certain evidence in improving global effectiveness rate for OA; however, more rigorous studies are warranted to support their use.

OBJECTIVE: To investigate the effects of osthole on chondrocyte proliferation in vitro. METHODS: Primary rat chondrocytes were isolated from the femoral head of newborn rats using collagenase digestion and cultured in Dulbecco's modified Eagle's medium. The proliferation of primary chondrocytes was assessed in second-passage cultures using cell counting kit-8 and the growth curve was drawn. Type II procollagen gene (Col2a1) expression in chondrocytes was also identified using cell immunofluorescence assay. The second-passage chondrocytes were divided into five groups, including control group and osthole groups at 6.25, 12.5, 25 and 50 μmol/L. The growth property of rat chondrocytes was observed after 24, 48 and 72 h of culture with osthole at corresponding dose. Both protein and mRNA expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 was measured by Western blot and polymerase chain reaction methods. RESULTS: The second-passage chondrocytes were viable and showed Col2a1 expression in the cytoplasm. The proliferation of rat chondrocytes was inhibited by osthole in a dose-dependent manner. Meanwhile, there were significant decreases in both protein and mRNA expression of PCNA and cyclin D1 in the osthole groups compared with the control group. CONCLUSION: Osthole exhibits inhibitory effect on proliferation of rat chondrocytes by down-regulating PCNA and cyclin D1 expression.

Objective Human chondrocytes and annulus fibrosus cells of intervertebral disc (IVD) express osteoprotegerin (OPG), but the effect of OPG on the pathogenesis of IVD degeneration remains unknown. Here we assessed the phenotype change of IVD in OPG−/− mice. Methods The IVDs from 12-, 20-, and 28-week-old OPG−/− mice and WT controls were subjected to histologic analyses including TRAP staining for osteoclasts, immunostaining for OPG and type I collagen protein expression, and TUNEL staining for apoptosis. The IVD tissues were also subjected to real time RT-PCR for mRNA expression of genes for osteoblast-osterix, ALP, and osteocalcin; for osteoclasts-trap, rank, mmp9 and cathepsin K, and for chondrocytes-aggrecan, mmp13 and Col10. Results OPG protein expresses at the cells of endplate cartilage and annulus fibrosis in IVDs of WT mice. Compared to WT mice, OPG−/− mice developed aging related cartilage loss and bony tissue appearance at the endplate. Stating from 20 weeks of age, IVDs from OPG−/− mice expressed significantly increased mmp13 and Col10 levels, which is associated with increased osteoblast number and elevated expression of osteoblast marker genes. Furthermore, TRAP+ osteoclasts were presented in the endplate cartilage of OPG−/− mice. These osteoclasts localized adjacently to and erosion into the cartilage. Increased expression of RANK, mmp9 and cathepsin k was detected in OPG−/− IVDs. Conclusions OPG at IVD plays an important role for maintaining the integrity of endplate cartilage during aging by preventing endplate cartilage from osteoclast-mediated resorption.

Study design Several senescence biomarkers were observed to investigate cell senescence in degenerative intervertebral lumbar discs of foreleg-amputated rats. Objective To determine if cell senescence is accelerated in degenerative intervertebral lumbar disc cells in an upright-rat model. Summary of background data Cellular senescence was accelerated in human and sand rat degenerative intervertebral disc (IVD) cells. Repeated use of upright posture by rats contributed to degenerative disc changes. No convincing evidence of cell senescence was observed in the lumbar disc of the foreleg amputated rat. Methods The forelimbs of 20 rats were amputated at 1 month of age such that they maintained an upright stance; rats were housed in custom-made cages. Nonamputated rats, also 1 month of age, were kept in regular cages and served as a control group. The lumbar IVDs were harvested from rats in 2 groups, at 5 or 9 months following amputation. Senescence-associated-β-galactosidase-positive staining was used to detect cell senescence. p16INK4a and p27KIP were assessed by immunohistochemistry analysis. Total RNA isolated from these samples was used to measure the gene expression of p16INK4a, RB, cyclin D1, CDK4, PTEN, p27KIP, p19ARF, p21, TERT, and RAGE by real-time polymerase chain reaction assay. Results The highest levels of SA-β-GAL activity were detected in 9-month amputated rats. Quantitative immunohistochemical analysis showed that there were highest rates of p16INK4a and p27KIP protein expression in the cartilage endplate and anulus fibrosus of 9-month amputated rats. The mRNA levels of p16INK4a, RB, PTEN, p27KIP, p19ARF, and RAGE were upregulated. The increased cyclin D1 mRNA level was statistically significant only at the ninth month following amputation; CDK4 and TERT mRNA levels were downregulated to a similar extent at both points compared with nonamputated controls. mRNA expression of p21 was significantly downregulated. Conclusion Accelerated cell senescence was associated with forelimb amputation that causes abnormal loading in rat lumbar IVDs.

Objective: The aim of this study was to characterize the mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) mobilization, and bone turnover in osteoporotic fracture healing in ovariectomized mice. Methods: In total, 112 female C57/BL mice were divided into two groups. The first group was sham-operated (SO), and the other group was ovariectomized (OVX). After three weeks, the right femora of the mice were fractured under anesthesia and internally fixed with steel pin. Peripheral blood and bone marrow were was collected for flow cytometry analysis, at 0 hours (h), 12 h, 24 h, 72 h and 168 h after fracture. MSCs and EPCs levels were assessed using cell surface antigens in different combinations (CD44+ CD34-CD45-, and CD34+ KDR+CD45-) by flow cytometry. At 0, 14, 28 and 42 days after fracture, sera were assayed for circulating levels of procollagen type I-N-terminal propeptide (P1NP) and C-terminal telopeptide of type I-collagen (CTX) by ELISA. Femurs were harvested at 2 weeks and 6 weeks after fracture for X-ray radiography, micro-computed tomography (micro-CT) and histology. Results: Our results showed that bone marrow and peripheral blood MSCs numbers of the OVX mice were significantly lower than the SO mice, at 12 h, 24 h and 72 h after fracture. In addition, circulating P1NP and CTX levels of the OVX mice were significantly higher than the SO mice, at 2 and 4 weeks. Conclusion: Results of the present study revealed disorders of bone marrow MSCs mobilization and bone turnover may partially account for the delay of osteoporotic fracture healing.

To investigate the effects of serum containing Gumibao (Chinese character: see text) on the proliferation and differentiation of osteoblast induced by dexamethasone.Osteoblasts were extracted from skulls in newly born (within 24 hours) SD rats, and digested with collagenase. The first passage of cells were used for experiments. Cells were cultured in the medium containing different concentrations of dexamethasone (0, 10(-8), 10(-7), 10(-6), 10(-5) ,10(-4) mol/L). Alkaline phosphatase staining were carried out after 1 week and numbers of mineralized nodes with alizarin red staining were observed after 3 weeks. Accordingly, following the treatment of 10(-5) mol/L dexamethasone for 1 week, cells were cultured in the medium with serum containing Gumibao (Chinese character: see text). One week after Cumibao (Chinese character: see text) treatment, cells were stained with Alkaline phosphatase and collagen I and PCNA were examined by Western-blot. However, the observation of numbers of mineralized nodes with alizarin red stain required one more week.High concentration of dexamethasone could inhibit the expression of PCNA, collagen I, alkaline phosphatase and reduce the number of mineralized nodes of osteoblast, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.High concentration of dexamethasone could inhibit the proliferation and differentiation of osteoblastic cells, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

To investigate regulation function of anodonta glucan HBP-A on chondrocytes through Wnt pathway in vitro.Rat chondrocytes were cultured and differentiated induced with IL-1beta (10 ng/ml) in vitro. Chondrocytes were divided into five groups:IL-13 group,IL-1beta + IWP-2 (5 microM,Wnt pathway inhibitor) group, IL-1beta + HBP-A (0.3 mg/ml) group and IL-1beta + IWP-2 + HBP-A group. Wnt-3a, beta-catenin (24 h,48 h,72 h) and MMP-13(72 h) genes expression were detected by Rt-PCR, while beta-catenin, MMP-13, Sox-9 and coll-II (48 h) protein expression were measured by Western-blot.After induction of IL-1beta, gene expression of Wnt-3a, beta-catenin and MMP-13 were increased,so were the protein expression of beta-catenin and MMP-13. In contrast,protein expression of Sox-9 and Coll-II were declined. Following addition of HBP-A, Wnt-3a, beta-catenin and MMP-13 were shown as induction of IL-1beta, but protein expression of Sox-9 and Coll-II were upgraded. Combining HBP-A with IWP-2 led to the lowest level in Wnt-3a, beta-catenin gene and beta-catenin protein expression and highest expression of Sox-9 protein.HBP-A could not only delay the differentiation of chondrocytes through downgrading the signal expression of Wnt/beta-catenin,but also adjust the expression of Wnt-3a, beta-catenin and Sox-9 when combinated with the Wnt inhibitor.

To investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism.Ten 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, the cranium were digested by type I collagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. To identify the cells, ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100, 50, 25, 12.5, 0 micromol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and beta-catenin protein were detected through the method of Western Blot after one week.The cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 micromol/L inhibited the proliferation of osteoblast after 24 h, while the osthole with final concentrations of 50 micromol/L and 25 micromol/L displayed the inhibition effect after 48 h. The osthole of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and beta-catenin protein in a dose-dependent manner.The osthole with final concentrations of 100, 50, 25 micromol/L inhibited the proliferation of osteoblast (P0.05). The osthole with final concentrations of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast (P0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/beta-catenin signaling in osteoblast.

To compared the activity and yield rate of osteoblast obtained by different collagenase digestion methods, to find a better way to extract osteoblast for the experimental researches of osteoporosis.Ten 24-hour-old SD rats were were euthanized. The cranium of rats were removed and cuted into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, all the cranium were divided into two equal parts, and randomly divided into two groups which would be digested by type I collagenase and type II collagenase separately for two times. The rat cells of the two groups were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. The primary culture osteoblasts were counted by using a haemacytometer after digestion and 72 hours later. The second generation osteoblasts cultured 48 h were dyed by NBT/BCIP staining solution, and were detected by quantitative measurement with PNPP.The cells had irregular shapes. The results of cell counting showed that the cell number of type I group was larger than type 11 group. Alkaline phosphatase dyeing were positive. Detecting of alkaline phosphatase using the method of PNPP showed that the absorbance value in type I group were higher than type II group (P0.05).Two types of collagenase are both suitable for the in vitro culture of rat osteoblasts. The activity and yield rate of osteoblasts in type I group are higher which could provide more stable seed cells for the treatment of osteoporosis.