Wear debris-induced osteolysis is one of the major reasons for subsequent aseptic loosening after cementless hip arthroplasty. Increasing evidence suggests that receptor activator of nuclear factor kappa-B (NF-kappa B) ligand-mediated osteoclastogenesis and osteolysis are responsible for wear debris-induced aseptic loosening. In the present study, we explored the effect of amentoflavone (AMF) on inhibiting osteoclast generation and wear debris-induced osteolysis in vitro and in vivo. Twenty-four male C57BL/J6 mice were randomly divided into four groups: a sham group and groups with titanium wear debris treatment followed by intraperitoneal injection of various concentrations of AMF (0, 20, and 40mg/kg/day). Themicro computed tomography scanning and histological analysis were performed. Bone marrow-derived macrophages were cultured to investigate the effect of AMF on osteoclast generation and function. The results showed that AMF suppressed osteoclastogenesis, F-actin ring formation, and bone absorption without cytotoxicity. AMF prevented titanium wear debris- induced osteolysis in mice. AMF suppressed the relative proteins of NF-kappa B and mitogen-activated protein kinase (MAPKs) signaling pathways. Thus, the present study suggests that AMF derived from plants could inhibit osteoclastogenesis and titanium wear debris-induced osteolysis via suppressing NF-kappa B and MAPKs signaling pathways.

Aseptic periprosthetic osteolysis is a complication induced by wear particles after hip joint replacement, which shortens the service life of prostheses. Although many methods have been applied to prevent this complication, the results were not always successful. In the present study, we explored the prophylactic effect of notoginsenoside R1 (NG-R1) on a Ti particle-induced mouse calvarial osteolysis model and the inhibitory effect on RANKL-mediated osteoclastogenesis and osteoclast bone resorption. Our results indicated that intraperitoneal injection of NG-R1(10 or 20mg/kg/day), a constituent of notoginseng, alleviates Ti particle-induced mouse calvarial osteolysis after twelve days post-treatment. In vitro, we observed that NG-R1 inhibits RANKL-mediated mitogen-activated protein kinases (MAPKs), including the phosphorylation levels of JNK1/2, P38, ERK1/2 and NF-kappa B (NF-κB) signalling pathways, and suppresses RANKL-mediated osteoclastogenesis and osteoclast bone resorption. These results suggest that NG-R1 may be a promising therapeutic agent for preventing wear particle-induced periprosthetic osteolysis.

Surgery-induced epidural fibrosis after laminectomy often results in poor clinical outcomes. Fibroblasts proliferation is considered to be one of the major causes of epidural fibrosis formation. Previously, there was no research about the effect of Homoharringtonine(HHT) on inhibiting fibroblast proliferation and reducing epidural fibrosis. Here, we performed in vitro and in vivo experiments to explore the effect of HHT on inhibiting fibroblast proliferation, inducing fibroblast apoptosis and preventing epidural fibrosis formation. In vitro, the effect of HHT on inhibiting fibroblasts was detected by CCK-8 assay. Besides, the effect of HHT on causing fibroblast apoptosis was investigated via Western blots, flow cytometry and TUNEL assay. Results suggested that HHT could inhibit fibroblasts proliferation and induce apoptosis. And the marker proteins of endoplasmic reticulum (ER) stress were also changed positively. In vivo, histological macroscopic assessment, hydroxyproline content analysis and histological staining were used to detect the effect of HHT on reducing epidural fibrosis. The results showed that HHT had positive suppressive effects on epidural fibrosis following laminectomy in rats. TUNEL assay in epidural tissue suggested that HHT could obviously induce fibroblasts apoptosis. Immunohistochemistry staining showed that the expression of two important ER stress markers(78-kDa glucoseregulated protein and C/EBP homologous protein) were also increased. In conclusion, this research showed that HHT could reduce epidural fibrosis formation after laminectomy, and the potential mechanism might through inhibiting fibroblasts proliferation and inducing fibroblasts apoptosis via ER stress signaling pathway. It might provide a novel agent for reducing epidural fibrosis after laminectomy surgery.

目的：建立大鼠心肌缺血再灌注损伤模型和氧化应激损伤细胞模型，研究抗肌萎缩蛋白Dp71在损伤心肌及细胞中的表达，探讨其拮抗心肌细胞凋亡的作用及其分子机制。方法：阻断SD大鼠冠状动脉左前降支血流30 min后恢复血流复制心肌缺血再灌注损伤模型，观察心肌形态学、LDH变化及心肌细胞凋亡；检测再灌注不同时间心脏Dp71蛋白和mRNA表达。建立H9c2细胞氧化应激损伤模型，检测H2 O2刺激后细胞中Dp71蛋白和mRNA的表达。 H9c2细胞转染Dp71过表达质粒，流式细胞术检测Dp71高表达对H2 O2诱导H9c2细胞凋亡率的影响。 Western blot检测过表达Dp71对细胞中lamin B1和Bcl－2蛋白表达的影响；对H2 O2刺激下lamin B1和Bcl2蛋白表达改变的影响。结果：与假手术组相比，再灌注损伤组HE染色心肌出现明显形态改变，LDH明显增加， IR组心肌细胞凋亡数显著增加。 IR后各时点Dp71 mRNA和蛋白表达水平均增加（ P＜0．05）。0．2 mmol／L H2 O2刺激细胞16 h Dp71蛋白及mRNA表达明显升高（ P＜0．05）。 H9c2细胞中转染Dp71过表达质粒可抑制H2 O2所诱导的细胞凋亡；H9c2细胞中过表达Dp71引起lamin B1和Bcl2表达增高，高表达的Dp71可以抑制过氧化氢刺激引起的lamin B1和Bcl2表达下降。结论：Dp71 mRNA和蛋白表达在大鼠心肌缺血再灌注损伤模型和H2 O2诱导H9c2氧化应激损伤模型中明显升高。 H9c2细胞过表达Dp71通过提高Bcl2和lamin B1表达而抑制H2 O2诱导的细胞凋亡。

Lumbar laminectomy is one of the most common treatments for lumbar disc herniation and other lumbar disorders with serious complications, such as failed back surgery syndrome, mainly caused by epidural fibrosis (EF). The developing fibrosis causes radicular pain after the laminectomy or discectomy. Methotrexate (MTX) is a folic acid antagonist that has shown anti-proliferative effects in previous studies. The aim of our experiment is to study whether MTX has positive effects on the outcome of the laminectomy in rats. Our finding first demonstrated the beneficial effect of topical application of MTX in laminectomy models. As the results of a macroscopic scoring system, hydroxyproline content analysis, histological evaluation, the number of fibroblasts and immunohistochemistry showed that MTX suppressed the EF compared with the control group, and the inhibiting effect was in a dose-dependent manner. Furthermore, we hypothesized that the endoplasmic reticulum (ER) stress mediated the suppression effect of the EF. To verify this point of view, fibroblast cells cultured from epidural scar tissues of rats were used. CCK-8 assay, Western blot (for apoptotic genes, such as cleaved PARP) and annexin V-FITC/PI double-labelling showed that MTX could induce cell apoptosis. The expression of CHOP and GRP78 and the activation of ER stress-associated genes strongly suggested that ER stress mediated the apoptotic signalling pathway; immunohistochemistry of GRP78 and CHOP further verified this. Our findings indicate that topical application of MTX could indeed reduce EF, and the application of MTX could induce apoptosis through ER stress in rats.

BACKGROUND:The pathogenesis of knee intraarticular adhesion is yet unknown. Excessive proliferation of fibroblasts is considered to cause knee intraarticular adhesion. OBJECTIVE:To study the preventive effects of methotrexate on knee intraarticular adhesion through fibroblast apoptosis induced by endoplasmic reticulum stress. METHODS:The viability of the cultured fibroblasts treated with methotrexate(10-5-10-9mol/L)or PBSwas determined after 24 hours. Fibroblast apoptosis was detected by Hoechst33342 staining. Endoplasmic reticulum stress-and apoptosis-related proteins, including cleaved-PARP, CHOP, Bax and Bcl-2, were determined by western blotassay. Eighteen healthy male New Zealand white rabbits were used to establish the knee intraarticular adhesion models, and equaly randomized into three groups, and received topical application of 2 or 1 g/L methotrexate, or normal saline (control). The preventive effects of methotrexate on knee intraarticular adhesion and CHOP expression in scar tissue were observed. RESULTS AND CONCLUSION:Methotrexate inhibited the proliferation and viability of fibroblasts in a dose-dependent manner. The number of apoptotic fibroblasts was significantly increased compared with control group. Protein expression of cleaved-PARP, CHOP, and bax was increased, while protein expression of bcl-2 was decreased with time. The animal experiment showed that preventive effects of 2 g/L methotrexate on knee intraarticular adhesion were superior to 1 g/L methotrexate treatment. CHOP expression in the scar tissue in the methotrexate groups was higher than the control group and that was higher in high-dose methotrexate group. Our results suggest that methotrexate prevents knee intraarticular adhesionviaendoplasmic reticulum stress-induced fibroblast apoptosis.

Background: Epidural fibrosis is a common complication after laminectomy. It is associated with intractable lower back pain and additional complications. To date, no study has evaluated whether the local application of rapamycin (RAPA) can inhibit fibroblast proliferation and reduce epidural scar adhesion after laminectomy. The results of the present study showed that the local application of RAPA reduces epidural fibrosis after laminectomy in rats.Methods: In this study, 32 male Sprague-Dawley rats were randomly divided into four groups (0.2 mg/ml RAPA-treated group, 0.1 mg/ml RAPA-treated group, 0.05 mg/ml RAPA-treated group and physiological saline group). Laminectomy was performed at the level of lumbar segment 1 to 2, and different concentrations of RAPA or saline were applied to the laminectomy sites for 10 min. Four weeks after laminectomy, the rats were sacrificed, and the degrees of epidural adhesion in each group were evaluated. Macroscopic assessment, analysis of hydroxyproline content, and histological analysis were used to determine the therapeutic effect of the local application of RAPA on the inhibition of fibroblast proliferation and the reduction of epidural fibrosis after laminectomy. Next, we cultured fibroblasts from epidural scar tissues of rats that had undergone laminectomy. Fibroblasts were exposed to the indicated concentrations of RAPA, and western blotting and TUNEL assays were used to assess the effects of RAPA on inhibiting fibroblasts proliferation and promoting fibroblast apoptosis.Results: The results of macroscopic assessments, analysis of hydroxyproline content, and histological analyses indicated that RAPA significantly inhibited fibroblast proliferation and reduced epidural fibrosis in the treated groups in the rat model. The western blotting results indicated that the expression levels of the pro-apoptotic proteins cleaved-PARP and Bax were up-regulated, whereas those of Bcl-2 were reduced. TUNEL assay indicated that the apoptosis rates of fibroblasts were significantly increased after exposure to the indicated concentrations of RAPA.Conclusions: The local application of RAPA reduced epidural fibrosis after laminectomy by inhibiting the proliferation of fibroblasts, stimulating their apoptosis, and decreasing collagen synthesis. This protocol may be used in new clinical treatment strategies to reduce epidural fibrosis after laminectomy.

Aseptic prosthetic loosening is a major complication after hip joint replacement. Wear particle-induced periprosthetic osteolysis plays a key role in aseptic prosthetic loosening. Attempting to modulate receptor activator of nuclear factor-κB (RANKL) mediated signaling pathways is a promising strategy to prevent aseptic prosthetic loosening. In the present study, we determined the effect of scutellarin (SCU) on titanium (Ti) particle-induced osteolysis in a mouse calvarial model and RANKL-mediated osteoclastogenesis. We determined that SCU, the major effective constituent of breviscapine isolated from a Chinese herb, has potential effects on preventing Ti particle-caused osteolysis in calvarial model of mouse. In vitro, SCU could suppress RANKL-mediated osteoclastogenesis, the function of osteoclast bone resorption, and the expression levels of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, c-Fos, NFATc1). Further investigation indicated that SCU could inhibit RANKL-mediated MAPK and NF-κB signaling pathway, including JNK1/2, p38, ERK1/2, and IκBα phosphorylation. Taken together, these results indicate that SCU could inhibit osteoclastogenesis and prevent Ti particle-induced osteolysis by suppressing RANKL-mediated MAPK and NF-κB signaling pathway. These results suggest that SCU is a promising therapeutic agent for preventing wear particle-induced periprosthetic osteolysis.

BACKGROUND:The formation of intraarticular adhesion is a common complication after total knee arthroplasty or anterior cruciate ligament reconstruction. Previously, little research was reported regarding whether the local application of rapamycin (RAPA) could reduce intraarticular adhesion following knee surgery. In our present study, we determined the therapeutic effect of RAPA by local application on the reduction of intraarticular adhesion following knee surgery in rabbits.METHODS:In this study, we built the model of knee surgery according to a previous study. The decorticated areas of the cortical bone were exposed and covered with cotton pads soaked with different concentrations of RAPA or physiological saline for 10 min. All of the rabbits were euthanized 4 weeks after the surgery. Macroscopic evaluation of the hydroxyproline content, the histological morphological analysis and collagen density and fibroblast density were used to evaluate the effect of RAPA on reducing intraarticular adhesion.RESULTS:The results shown that RAPA could significantly inhibit the proliferation of fibroblasts and reduce collagen synthesis; in the rabbit model of knee surgery, there were weak scar tissues around the decorticated areas in the 0.2 mg/ml RAPA group; moderate scar tissues were found in the 0.1 mg/ml RAPA group. However, severe fibrous adhesions were found in the 0.05 mg/ml RAPA group and the control group. The hydroxyproline content and the fibroblast density in the 0.2 mg/ml and 0.1 mg/ml RAPA groups were significantly less than those of the control group.CONCLUSIONS:We concluded that the local application of RAPA could reduce intraarticular adhesion after knee surgery in the rabbit model; this effect was mediated by inhibition of fibroblast proliferation and collagen synthesis, which may provide a new method for reducing intraarticular adhesion after clinical knee surgery.

BACKGROUND:Osteoprotegerin and nitrogen monoxidum play a key role in the prevention and treatment of osteoporosis. However, the correlation of the two in inhibiting the proliferation and differentiation of osteoclasts remains unclear. <br> OBJECTIVE:To observe the effect of different doses of osteoprotegerin on nitric oxide production and endothelial nitric oxide synthase activity in osteoclasts. <br> METHODS:Tartrate resistant acid phophatase staining was used to test whether the induced cells are osteoclasts. Osteoclasts were divided into six groups:blank control group (no reagent);negative control group (DMEM);four osteoprotegerin groups (10, 25, 50, 75μg/L of osteoprotegerin). Annexinv-FITC kit and flow cytometry were used to test the apoptosis rate of osteoclasts. Real-time PCR was used to detect the expression of osteoclasts marker gene, TRAP mRNA and protein kinase K mRNA. Nitric oxide production and nitric oxide synthase activity were determined using the corresponding kits. Four osteoprotegerin groups were added with L-NAME, a kind of nitric oxide synthase inhibitor, to test the changes of the apoptosis rates of osteoclasts and the changes of the expression of TRAP mRNA and protein kinase K mRNA of osteoclasts. <br> RESULTS AND CONCLUSION:Osteoprotegerin inhibited the differentiation of osteoclasts and induced the apoptosis. Osteoprotegerin concentration had a positive correlation with the apoptosis rate of osteoclasts, and a negative correlation with the numbers of osteoclasts and expression of marker gene TRAP mRNA and protein kinase K mRNA in osteoclasts. Osteoprotegerin boosted the nitric oxide production and endothelial nitric oxide synthase activity, and osteoprotegerin concentration was positively correlated to the nitric oxide production and endothelial nitric oxide synthase activity. After Raw264.7 cells were in vitro cultured, osteoprotegerin and nitric oxide play a synergic role in inhibiting osteoclasts production and promoting the apoptosis. We speculate that there is osteoprotegerin/endothelial nitric oxide synthase/nitric oxide signal pathway.

OBJECTIVE To investigate the isolation and the drug resistance of Enterobacter cloacae strains from the orthopedics department between 2007 and 2011,and analyze the related factors so as to provide basis for the clinical treatment and prevention of E.cloacae infections.METHODS A total of 1218 wound secretion specimens were collected from hospitalized patients with limbs trauma from 2007 to 2011.All the specimens were sampled under the rigid aseptic operation,and the bacterial culture and drug susceptibility testing were performed in accordance with the standard procedure.RESULTS Of totally 1218 specimens collected,there were 154 specimens with the E.cloacae detected positive,the detection rate was 12.64%,and the detection rates were all higher than 10.00% in the four years except 2007.The drug resistance rates to imipenem and amikacin were less than 50.00% in 2011,and the strains were highly resistant to the commonly used penicillins and cephalosporins antibiotics.The study concluded that both the detection rate and the drug resistance rate were significantly increased during the past five years.CONCLUSION E.cloacae is the prevalent pathogen causing infections in the orthopedics department and is highly resistant to the commonly used penicillin and cephalosporin antibiotics,which is closely related to the unreasonable use of antibiotics in the hospital,thus,it is necessary for the clinicians to reasonably choose antibiotics on the basis of the drug susceptibility testing.