Influences of IL-6R antibody on PMMA bone cement-mediated expression of OPG and RANKL in synovial fibroblasts.

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 µg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 µg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.

Inhibitory effects of SRT1720 on the apoptosis of rabbit chondrocytes by activating SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

SRT1720, a new discovered drug, was reported to activate silent information regulator 1 (SIRT1) and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside (SNP) (2.5 mmol/L) and divided into five groups: 1, 5, 10, and 20 µmol/L SRT1720 groups and blank control group (0 µmol/L SRT1720). MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) were detected by Western blotting and the expression levels of SIRT1, type II collagen, and aggrecan mRNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the mRNA expression levels of type II collagen and aggrecan increased ({ptP}<0.05), and the expression levels of p53, NF-κB and bax decreased (P<0.05). It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1+/+ control group (group A, n=6); SIRT1+/+ osteoarthritis group (group B, n=6); SIRT1-/- control group (group C, n=6); SIRT1-/- osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-ß-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1-/- osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1+/+ osteoarthritis group and SIRT1-/- control group, SIRT1 protein expression was not obviously changed in the SIRT1-/- osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

Arthroscopic treatment of bony loose bodies in the subacromial space.

INTRODUCTION: Multiple bony loose bodies in the subacromial space caused form cartilage or bone cells and continue to grow. PRESENTATION OF CASE: A 58-year-old man with two-year history of swelling and pain of the right shoulder. He had no history of tuberculosis and rheumatoid arthritis. Magnetic resonance (MR) images showed some bony loose bodies in the subacromial space. The removal of loose bodies and bursa debridement were performed arthroscopically. Histological diagnosis of them was synovitis with fibrous bodies. DISCUSSION: Extra-articular loose bodies is extremely rare, especially in the subacromial space, which maybe originated in the proliferative synovial bursa. Most authors recommend open removal to relive the pain, but there were choice to apply arthroscopy to remove them. CONCLUSION: The mechanism of formation of bony loose bodies is not clear, may be associated with synovial cartilage metaplasia. Arthroscopic removal of loose bodies and bursa debridement is a good option for treatment of the loose body in the subacromial space, which can receive good function.

Steroid-associated hip joint collapse in bipedal emus.

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients.

Resveratrol inhibits interleukin 1ß-mediated inducible nitric oxide synthase expression in articular chondrocytes by activating SIRT1 and thereby suppressing nuclear factor-κB activity.

In chondrocytes, resveratrol, a natural SIRT1 activator, exerts an anti-inflammatory response via inhibition of nuclear factor kappaB (NF-κB). Given that SIRT1 inhibits the transactivation potential of NF-κB by deacetylating acetylated lysines in p65, the NF-κB subunit, we investigated the effects of resveratrol-activated SIRT1 on articular chondrocytes. We found that when chondrocytes were stimulated with interleukin 1ß (IL-1ß), the time- and dose-dependent expression of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production was suppressed by resveratrol. Resveratrol-activated SIRT1 mediated this suppression. SIRT1 suppressed not only the nuclear translocation of NF-κB but also the acetylation of p65. Furthermore, acetylated Lys310 in p65, which must be present for transactivation activity, was the immediate downstream target of SIRT1. Therefore, SIRT1 protects against the inflammatory response induced by IL-1ß in articular chondrocytes. Resveratrol, as an activator of SIRT1, merits consideration as a therapeutic agent in the treatment and prevention of osteoarthritis.

Hydrogen sulfide protects MC3T3-E1 osteoblastic cells against H2O2-induced oxidative damage-implications for the treatment of osteoporosis.

Osteoporosis is a bone disease that leads to an increased risk of fracture. Oxidative damage is an important contributor to the morphological and functional changes in the development of osteoporosis. We found in this study that hydrogen sulfide (H2S), a novel endogenous gaseous mediator, protected MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative injury. NaHS, an H2S donor, increased cell viability and reduced cell apoptosis caused by H2O2. NaHS also stimulated osteoblast proliferation by enhancing both transcription and activity of alkaline phosphatase in MC3T3-E1 osteoblastic cells. Moreover, treatment with NaHS stimulated the transcriptional level of osteocalcin, the main bone matrix protein, and the protein expression of collagen, a major constituent of bone tissue. The above effects were mediated by the antioxidant effect of H2S. NaHS reversed the reduced superoxide dismutase activity, decreased reactive oxygen species production, and suppressed NADPH oxidase activity in H2O2-treated osteoblasts. In addition, NaHS treatment also produced anti-inflammatory effects via inhibition of the production of nitric oxide and TNF-α, suggesting an anti-inflammatory effect of H2S. Cell viability and Western blotting analysis demonstrated that the protective effects of H2S were mediated by p38 and ERK1/2 MAPKs. In conclusion, H2S protects osteoblastic cells against oxidative stress-induced cell injury and suppression of proliferation and differentiation via a MAPK (p38 and ERK1/2)-dependent mechanism. Our findings suggest that H2S may have a potentially therapeutic value for osteoporosis.

[Construction of eukaryotic expression vectors of basic fibroblast growth factor and transfection in rabbit bone marrow stromal cells].

OBJECTIVE: To investigate approach and possibility of transferring basic fibroblast growth factor (bFGF) gene into rabbit bone marrow stromal cells (BMSCs). METHODS: The eukaryotic expression vectors harboring bFGF cDNA were constructed and transfected into rabbit BMSCs mediated by liposome. The transcription and expression of bFGF gene in the transfected BMSCs were detected by means of morphological observation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and RT-PCR. The changes in the biological characteristics of the transfected MSCs were also observed. RESULTS: Stable overexpression of bFGF protein was detected in the transfected BMSCs, which showed differentiation towards chondrocyte lineage. CONCLUSION: Stable expression of bFGF gene in transfected BMSCs can induce cell differentiation into chondrocyte lineage.

[Biocompatibility of polylactic-co-glycolic acid for culturing bFGF gene-transfected bone marrow stromal cells and application of the cell complex for repairing rabbit cartilage defect].

OBJECTIVE: To evaluate the biocompatibility of polylactic-co-glycolic acid (PLGA) for culturing bFGF gene-transfected bone marrow stromal cells (BMSCs) and assess the feasibility of this cell complex for repairing cartilage defect in rabbits using tissue engineering method. METHODS: BMSCs transfected by bFGF gene were cultured on PLGA matrix to assess the biocompatibility of PLGA. The cell complex was then implanted into the cartilage defect in rabbits, and its effect in cartilage defect repair was evaluated by histological observation and immunohistochemical staining. RESULTS: BMSCs transfected by bFGF gene grew normally on PLGA matrix. After implantation, the complex showed good effect for cartilage defect repair in rabbits. CONCLUSION: PLGA has good biocompatibility with the transfected BMSCs, and the cell complex can be used for repairing rabbit cartilage defect and may potentially serve as a substitute of cartilage autograft.

[Role of hepatitis C virus superinfection in hepatic pathologic changes of patients with chronic hepatitis B: a retrospective short-term and long-term comparative study].

OBJECTIVE: To observe the effect of hepatitis C virus (HCV) superinfection on the short-term and long-term hepatic pathological changes in patients with chronic hepatitis B (CHB). METHODS: HCV-RNA of twice corresponding period serum samples was detected via reverse transcription polymerase chain reaction assay from 230 patients with CHB for whom liver biopsy was performed at an interval of 0.5-15 years, respectively. The hepatic pathological changes of the patients with CHB who were serum HCV-RNA positive at the beginning of observation and persistently positive between the starting and ending of observation were respectively compared with those of serum HCV-RNA negative and persistently negative patients. RESULTS: 41 patients (17.83%) were positive for serum HCV-RNA at the beginning of observation. There were significant differences in the severity of hepatic inflammatory activity grade and fibrosis stage between serum HCV-RNA positive and negative patients with CHB (P < 0.05). Twenty-nine patients were persistently positive for serum HCV-RNA in the beginning and end of observation. Compared with persistently negative patients who were 116 patients selected from the above-mentioned 230 patients and they were comparable with HCV-RNA persistently positive patients in mean follow-up time, age and sex, the long-term progression of hepatic inflammatory activity grade and fibrosis stage in persistently positive patients were more speedy (P < 0.01). CONCLUSION: HCV superinfection worsens the hepatic pathological changes of patients with CHB and speeds up its progression.