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The aim of this study was to investigate the role of the mitogen-activated protein kinase kinase-extracellular signal-regulated kinases 1/2 (MEK-ERK1/2) signaling pathway in chondrocyte differentiation and cartilage tissue construction in vitro . Chondrocytes were stimulated with rat serum (RS) and fetal bovine serum (FBS), and chondrocyte phenotypes were investigated microscopically. Chondrocyte proliferation was analyzed using fluorescence activated cell sorting (FACS) and the CCK8 method. Protein and mRNA expressions were assessed by western blot and RT-qPCR. Constructed cartilage tissues were examined by Safranin O-Fast Green FCF staining and immunofluorescence. In contrast to FBS, RS induced rapid dedifferentiation of chondrocytes and decreased type II collagen expression and proteoglycan synthesis. ERK1/2 and type I collagen expression increased during dedifferentiation and decreased during redifferentiation. Increased MEK-ERK1/2 pathway activity resulted in chondrocyte dedifferentiation, and inhibition of ERK1/2 by the inhibitor PD0325901 reversed dedifferentiation and led to redifferentiation. These data suggest strongly that inhibition of MEK-ERK1/2 activation prevents chondrocyte dedifferentiation and fibrocartilage formation., Competing Interests: None.

Background: Knee osteoarthritis (KOA) is the most common form of arthritis, leading to pain disability in seniors and increased health care utilization. Manual therapy is one widely used physical treatment for KOA., Objective: To evaluate the effectiveness and adverse events (AEs) of manual therapy compared to other treatments for relieving pain, stiffness, and physical dysfunction in patients with KOA., Study Design: A systematic review and meta-analysis of manual therapy for KOA., Methods: We searched PubMed, EMBASE, the Cochrane Library, and Chinese databases for relevant randomized controlled trials (RCTs) of manual therapy for patients with KOA from the inception to October 2015 without language restrictions. RCTs compared manual therapy to the placebo or other interventional control with an appropriate description of randomization. Two reviewers independently conducted the search results identification, data extraction, and methodological quality assessment. The methodological quality was assessed by PEDro scale. Pooled data was expressed as standard mean difference (SMD), with 95% confident intervals (CIs) in a random effects model. The meta-analysis of manual therapy for KOA on pain, stiffness, and physical function were conducted., Results: Fourteen studies involving 841 KOA participants compared to other treatments were included. The methodological quality of most included RCTs was poor. The mean PEDro scale score was 6.6. The meta-analyses results showed that manual therapy had statistically significant effects on relieving pain (standardized mean difference, SMD = -0.61, 95% CI -0.95 to -0.28, P = 76%), stiffness (SMD = -0.58, 95% CI -0.95 to -0.21, P = 81%), improving physical function (SMD = -0.49, 95% CI -0.76 to -0.22, P = 65%), and total score (SMD = -0.56, 95% CI -0.78 to -0.35, P = 50%). But in the subgroups, manual therapy did not show significant improvements on stiffness and physical function when treatment duration was less than 4 weeks. And the long-term information for manual therapy was insufficient., Limitations: The limitations of this systematic review include the paucity of literature and inevitable heterogeneity between included studies., Conclusion: The preliminary evidence from our study suggests that manual therapy might be effective and safe for improving pain, stiffness, and physical function in KOA patients and could be treated as complementary and alternative options. However, the evidence may be limited by potential bias and poor methodological quality of included studies. High-quality RCTs with long-term follow-up are warranted to confirm our findings.Key words: Knee osteoarthritis, manual therapy, systematic review.

Objective: To investigate the effects of over expression of Mash-1 gene on the differentiation of embryonic stem cells (ESC) into neural cells in vitro., Methods: The ESC of rats (CE3 cells) were transfected with MSCV-Mash-1 (MSCV-Mash-1-CE3 group) or MSCV (MSCV-CE3 group). The expression of Mash-1 gene was detected by RT-PCR. After transfection, hanging-drop culture was used to form embryonic bodies, and then embryonic bodies were cultured with neural induction medium. The cell morphology was observed under inverted phase contrast microscopy at 7 and 21 days; the positive rates of neural stem cells marker protein (nestin) and neuron marker protein (β-tubulin III) were measured by immunofluorescence staining after cell attachment; and the gene expressions of a-fetal protein (AFP), Brachyury, fibroblast growth factor 5 (FGF-5), Oct3/4, nestin, and β-tubulin III were detected by real-time fluorescence quantitative PCR at 0, 1, 7, 14, and 21 days after culture. The CE3 cells were used as control (CE3 group)., Results: Compared with MSCV-CE3 and CE3 groups, the expression of Mash-1 gene in MSCV-Mash-1-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-1-CE3 group had axons growth and showed neural stem cell-like and neuron cell-like morphology (unipolar, bipolar, and multipolar neurons), but few cells had axons growth in MSCV-CE3 and CE3 groups. The positive rates of nestin at 7 days and β-tubulin III at 21 days in MSCV-Mash-1-CE3 group were significantly higher than those in MSCV-CE3 and CE3 groups (P < 0.05). Real-time fluorescence quatitative PCR results showed that the gene expression of Brachyury was significantly decreased after 1 day (P < 0.05), and the gene expressions of FGF-5 and nestin were significantly increased after 1 day (P < 0.05) in MSC V-Mash- 1-CE3 group when compared with CE3 and MSCV-CE3 groups; the gene expression of β-tubulin III was significantly increased after 7 days (P < 0.05). There was no significant difference in above indexes between CE3 and MSCV-CE3 groups (P > 0.05). The expressions of AFP and Oct3/4 showed no significant difference among groups at each time point (P>0.05)., Conclusion: Over expression of Mash-1 gene can promote differentiation of ESC into neural cells in vitro.

Objective: To investigate the role of human Wnt7b gene in rat chondrocyte degeneration., Methods: Wnt7b gene obtained by PCR was cloned to PCDH-GFP. 293ft cell line was transfected with PCDH-GFP and PCDH-Wnt7b, and the supernatant and transfected cells were collected. The expression level of Wnt7b in 293ft cells was detected by Western blotting. The first passage of chondrocytes were isolated from articular cartilages of newborn born (within 24 h) SD rats were cultured in the supernatants from the transfected cells (at 10- and 50-fold dilutions). The cell morphology of the rat chondrocytes was observed under inverted microscope, and the protein expressions of MMP13, MMP3 and type II collagen and mRNA expressions of A-can, ADAMTS5, Col X and Sox9 were examined by Western blotting or real-time PCR., Results: Human Wnt7b gene cloned to PCDH-GFP was expressed efficiently in 293ft cell line. Rat chndrocytes cultured for 24 h in the supernatants from PCDH-Wnt7b-transfected 293ft cells underwent changes from a polygonal to a spindle-shaped morphology. The protein expression levels of MMP13 and MMP3 increased while type II collagen decreased significantly, and the mRNA levels of A-can and Sox9 were down-regulated while Col X and ADMATS5 up-regulated in ratchondrocytes after incubation in supernatants from PCDH-Wnt7b-transfected 293ft cells., Conclusion: Human Wnt7b gene can be expressed efficiently in 293ft cell line and can induce rat chondrocyte degeneration in vitro.

Objective: To investigate the effect of inhibition and activation of MAPK-ERK1/2 pathway on the expression of osteogenic genes and proliferation of rat osteoblasts in vitro., Methods: Primarily cultured rat osteoblasts, identified by cell morphology studies and ALP staining, were exposed to 1% or 5% rat serum for 24 h or to the specific MAPK-ERK1/2 inhibitor PD0325901. The downstream molecules of MAPK-ERK1/2 pathway including p-ERK1/2 and ERK1/2, osteogenic genes such as Runx2 and Type I collagen, and proliferating cell nuclear antigen (PCNA) were detected by Western Blotting, and alkaline phosphatase activities were analyzed quantitatively., Results: Compared with 1% rat serum-treated cells, exposure of the cells to a higher concentration (5%) of rat serum caused a significantly increased phosphorylation level of p-ERK1/2 (P<0.05) and obviously enhanced expressions of the osteogenic genes (Runx2, type I collagen and ALP) and PCNA (P<0.05). Inhibition of the MAPK-ERK1/2 pathway with PD0325901 resulted in suppressed expressions of the osteogenic genes and PCNA., Conclusion: The activation of MAPK-ERK1/2 pathway promotes the expression of osteogenic genes such as Runx2, type I collagen and ALP and enhances the proliferative activity of the osteoblasts, while inhibition of this pathway suppresses the expressions of these genes and the cell proliferation, suggesting that this pathway may potentially serve as a therapeutic target for osteoporosis.

Objective: To investigate the role of bone morphogenetic protein 4 (BMP4) in culturing induced pluripotent stem cells (iPSCs) and the related signal pathways., Methods: We amplified the mature peptide of BMP4 from the placenta through RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing BMP4 were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell phenotype, cell differentiation capacity into lineages of the 3 germ layers and expression levels of pluripotency-associated genes were investigated., Results: Smad1 was phosphorylated by BMP4 from the culture medium. iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages maintained the phenotype of stem cells with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to differentiate into cell lineages of the 3 germ layers., Conclusion: BMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.