Nintedanib ameliorates osteoarthritis in mice by inhibiting synovial inflammation and fibrosis caused by M1 polarization of synovial macrophages via the MAPK/PI3K-AKT pathway.

Synovial inflammation and fibrosis are important pathological changes associated with osteoarthritis (OA). Herein, we investigated if nintedanib, a drug specific for pulmonary fibrosis, plays a positive role in osteoarthritic synovial inflammation and fibrosis. We assessed the effect of nintedanib on osteoarthritic synovial inflammation and fibrosis in a mouse model of OA created by destabilization of the medial meniscus and a macrophage M1 polarization model created by stimulating RAW264.7 cells with lipopolysaccharide. Histological staining showed that daily gavage administration of nintedanib significantly alleviated articular cartilage degeneration, reduced the OARSI score, upregulated matrix metalloproteinase-13 and downregulated collagen II expression, and significantly reduced the synovial score and synovial fibrosis in a mouse OA model. In addition, immunofluorescence staining showed that nintedanib significantly decreased the number of M1 macrophages in the synovium of a mouse model of OA. In vitro results showed that nintedanib downregulated the phosphorylation levels of ERK, JNK, p38, PI3K, and AKT while inhibiting the expression of macrophage M1 polarization marker proteins (CD86, CD80, and iNOS). In conclusion, this study suggests that nintedanib is a potential candidate for OA treatment. The mechanisms of action of nintedanib include the inhibition of M1 polarization in OA synovial macrophages via the MAPK/PI3K-AKT pathway, inhibition of synovial inflammation and fibrosis, and reduction of articular cartilage degeneration.

Biomechanical comparison of the femoral neck system versus InterTan nail and three cannulated screws for unstable Pauwels type III femoral neck fracture.

BACKGROUND: There are a variety of internal fixation methods for unstable femoral neck fractures (FNFs), but the best method is still unclear. Femoral neck system (FNS) is a dynamic angular stabilization system with cross screws, and is a new internal fixation implant designed for minimally invasive fixation of FNFs. In this study, we conducted a biomechanical comparison of FNS, InterTan nail and three cannulated screws for the treatment of Pauwels III FNFs and investigate the biomechanical properties of FNS. METHODS: A total of 18 left artificial femurs were selected and randomly divide into Group A (fixation with FNS), Group B (fixation with InterTan nail) and Group C (fixation with three cannulated screws), with 6 specimens in each group. After creating Pauwels type III FNF models, the specimens in each were tested with non-destructive quasi-static tests, including torsion, A-P bending and axial compression tests. The average slope of the linear load-deformation curve obtained from quasi-static tests defines the initial torsional stiffness, A-P bending stiffness, and axial compression stiffness. After cyclic loading test was applied, the overall deformation of models and local deformation of implant holes in each group were assessed. The overall deformation was estimated as the displacement recorded by the software of the mechanical testing apparatus. Local deformation was defined as interfragmental displacement. Data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test using the SPSS software (version 24.0, IBM, New York, NY, USA). Correlation analysis was performed using Pearson's correlation analysis. RESULTS: Group B exhibited significantly higher axial stiffness and A-P bending stiffness than the other two groups (P < 0.01), while Group A had significantly higher axial stiffness and A-P bending stiffness than Group C (P < 0.01). Groups A and B exhibited significantly higher torsional stiffness than Group C (P < 0.01), no statistical significance was observed between Groups A and B (P > 0.05). Group B exhibited significantly lower overall and local deformations than the other two groups (P < 0.01), while Group A had significantly lower overall and local deformations than Group C (P < 0.01). Correlation analysis revealed positive correlation between axial stiffness and A-P bending stiffness (r = 0.925, P < 0.01), torsional stiffness (r = 0.727, P < 0.01), between torsional stiffness and A-P bending stiffness; negative correlation between overall, local deformations and axial stiffness (r = - 0.889, - 0.901, respectively, both P < 0.01), and positive correlation between the two deformations (r = - 0.978, P < 0.01). CONCLUSION: For fixation of unstable FNFs, InterTan nail showed the highest axial stiffness and A-P bending stiffness, followed by FNS, and then three cannulated screws. Torsional stiffness of FNS was comparable to that of the InterTan nail. FNS, as a novel minimally invasive implant, can create good mechanical environment for the healing of unstable FNFs. Clinical studies are needed to confirm the potential advantages of FNS observed in this biomechanical study.

Osteoclasts secrete leukemia inhibitory factor to promote abnormal bone remodeling of subchondral bone in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common chronic degenerative joint disease. At present, there is no effective treatment to check the progression of osteoarthritis. Osteochondral units are considered to be one of the most important structures affecting the occurrence and development of osteoarthritis. Osteoclasts mediate an increase in abnormal bone remodeling in subchondral bone in the early stage of osteoarthritis. Here, alendronate (ALN) that inhibit osteoclasts was used to study the regulatory effect of osteoclast-derived leukemia inhibitory factor (LIF) on early abnormal bone remodeling. METHODS: This study involved 10-week-old wild-type female C57BL/6 mice and female SOST knockout (KO) mice that were divided into the sham, vehicle, ALN, and SOST KO groups. RESULTS: The expression of LIF was found to decrease by inhibiting osteoclasts, and the histological OA score suggested that the degeneration of articular cartilage was attenuated. Additionally, micro-CT showed that osteoclasts inhibited in the early stage of OA could maintain the microstructure of the subchondral bone. The parameters of bone volume fraction (BV/TV), subchondral bone plate thickness (SBP.Th), and trabecular separation (Tb.Sp) of the treated group were better than those of the vehicle group. CONCLUSIONS: These results suggested that downregulating the expression of sclerostin in osteocytes by secreting LIF from osteoclasts, activate the Wnt/ß-catenin signaling pathway, and promote abnormal bone remodeling in OA. Therefore, clastokine LIF might be a potential molecular target to promote abnormal bone remodeling in early OA.

Extensive cytokine analysis in synovial fluid of osteoarthritis patients.

OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by articular cartilage loss and afflicts many people worldwide. However, diagnostic methods and treatment options remain limited and are often characterized by low sensitivity and low efficacy. The focus of the present study was to identify proteomic biomarkers in synovial fluid to improve diagnosis and therapy of OA patients. METHODS: Antibody array technology was utilized for protein expression profiling of synovial fluid from 24 OA patients and 24 healthy persons. RESULTS: Compared with healthy persons, twenty proteins showed lower expression levels in OA patients, while thirty proteins had higher levels. Among these differential proteins, GITRL, CEACAM-1, FSH, EG-VEGF, FGF-4, PIGF, Cystatin EM and NT-4 were found for the first time to be differentially expressed in OA. Bioinformatics analysis showed that most of these differential proteins were involved leukocytes events, and some differentially expressed proteins including IL-18, CXCL1, CTLA4, MIP-3b, CD40, MMP-1, THBS1, CCL11, PAI-1, BAFF, aggrecan, angiogenin and follistatin were located in central positions of the protein-protein interaction (PPI) network. CONCLUSION: We speculate that leukocyte proliferation and migration to the joint may be an important pathogenesis of OA, which needs a further validation. The central proteins of the PPI network may play a more pivotal role in OA. The newly identified differentially expressed proteins may be novel biomarkers for OA diagnosis and targets for OA therapy.

[Association of single nucleotide polymorphisms (SNPs) in leptin receptor gene with knee osteoarthritis in the Ningxia Hui population].

To investigate the association between primary knee osteoarthritis (OA) and single nucleotide polymorphism (SNP) (A668G) of leptin receptor gene (LEPR) in the Ningxia Hui population. A case-control association study has been adopted in this thesis. The polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis were performed to investigate the SNP of A668G site within LEPR from 148 patients with knee OA and 155 controls (asymptomatic and radiographically negative) with matched age and gender among Ningxia Hui population. In addition, genotypes of LEPR were verified by direct sequence analysis on PCR products. The result indicates that allele and genotype frequencies (P=0.024 and 0.008, respectively) in LEPR SNP A668G were significantly different in the knee OA patients group and control group, and in the knee OA patients group, the serum levels of leptin decreased significantly (P<0.001) and the serum levels of soluble leptin receptor increased significantly (P<0.001) compared with control group. Therefore, LEPR SNP A668G is associated with susceptibility to knee OA, which would be used as the genetic marker in predicting the risk of knee OA and would be one of the candidate genes in early prevention and control.

ß2-Adrenergic receptor expression in subchondral bone of patients with varus knee osteoarthritis.

An important causative factor in osteoarthritis (OA) is the abnormal mechanical stress-induced bone remodeling of the subchondral bone. ß2-adrenergic receptor (Adrb2) plays a major role in mechanical stresses that induce bone remodeling. The medial tibial plateau (MTP) and lateral tibial plateau (LTP) of patients with varus Knee osteoarthritis (KO) bear different mechanical stresses. The present study aimed to investigate the expression of Adrb2 in medial tibial plateau subchondral bone (MTPSB) and lateral tibial plateau subchondral bone (LTPSB) in patients with varus KO. A total of 30 tibial plateau samples from patients undergoing total knee arthroplasty for varus KO and MTPSB and LTPSB were studied. Statistical analysis was performed using paired sample t-tests. Safranin O-Fast Green staining and Micro-computed tomography showed significant differences in the bone structure between MTPSB and LTPSB. Tartrate-resistant acid phosphatase (TRAP)-positive cell density in MTPSB was higher than that in LTPSB. Immunohistochemistry, reverse transcription-quantitative polymerase chain reaction, and Western blot analysis revealed that compared to LTPSB, the levels of Adrb2, tyrosine hydroxylase (TH), and osteocalcin increased significantly in MTPSB. Double-labeling immunofluorescence showed Adrb2 was present in the majority of TRAP-positive multinuclear cells of the MTPSB. The expression of Adrb2 and TH was significantly higher in MTPSB than in LTPSB, confirming the involvement of these molecules in the development of OA.

Metformin reduces chondrocyte pyroptosis in an osteoarthritis mouse model by inhibiting NLRP3 inflammasome activation.

Osteoarthritis (OA) is an age-related degenerative disease, and its incidence is increasing with the ageing of the population. Metformin, as the first-line medication for the treatment of diabetes, has received increasing attention for its role in OA. The purpose of the present study was to confirm the therapeutic effect of metformin in a mouse model of OA and to determine the mechanism underlying the resultant delay in OA progression. The right knees of 8-week-old C57BL/6 male mice were subjected to destabilization of the medial meniscus (DMM). Metformin (200 mg/kg) was then administered daily for 4 or 8 weeks. Safranin O-fast green staining, H&E staining and micro-CT were used to analyse the structure and morphological changes. Immunohistochemical staining was used to detect type II collagen (Col II), matrix metalloproteinase 13 (MMP-13), NOD-like receptor protein 3 (NLRP3), caspase-1, gasdermin D (GSDMD) and IL-1ß protein expression. Reverse transcription-quantitative PCR was used to detect the mRNA expression of NLRP3, caspase-1, GSDMD and IL-1ß. Histomorphological staining showed that metformin delayed the progression of OA in the DMM model. With respect to cartilage, metformin decreased the Osteoarthritis Research Society International score, increased the thickness of hyaline cartilage and decreased the thickness of calcified cartilage. Regarding the mechanism, in cartilage, metformin increased the expression of Col II and decreased the expression of MMP-13, NLRP3, caspase-1, GSDMD and IL-1ß. In addition, in subchondral bone, metformin inhibited osteophyte formation, increased the bone volume fraction (%) and the bone mineral density (g/cm3), decreased the trabecular separation (mm) in early stage of osteoarthritis (4 weeks) but the opposite in an advanced stage of osteoarthritis (8 weeks). Overall, metformin inhibited the activation of NLRP3 inflammasome, decreased cartilage degradation, reversed subchondral bone remodelling and inhibited chondrocyte pyroptosis.

Metformin attenuates osteoclast-mediated abnormal subchondral bone remodeling and alleviates osteoarthritis via AMPK/NF-κB/ERK signaling pathway.

This study explored the mechanism by which metformin (Met) inhibits osteoclast activation and determined its effects on osteoarthritis (OA) mice. Bone marrow-derived macrophages were isolated. Osteoclastogenesis was detected using tartrate-resistant acid phosphatase (TRAP) staining. Cell proliferation was evaluated using CCK-8, F-actin rings were detected by immunofluorescence staining, and bone resorption was detected using bone slices. Nuclear factor kappa-B (NF-κB) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) were detected using luciferase assays, and the adenosine monophosphate-activated protein kinase (AMPK), NF-κB, and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blotting. Finally, expression of genes involved in osteoclastogenesis was measured using quantitative polymerase chain reaction. A knee OA mouse model was established by destabilization of the medial meniscus (DMM). Male C57BL/6J mice were assigned to sham-operated, DMM+vehicle, and DMM+Met groups. Met (100 mg/kg/d) or vehicle was administered from the first day postoperative until sacrifice. At 4- and 8-week post OA induction, micro-computed tomography was performed to analyze microstructural changes in the subchondral bone, hematoxylin and eosin staining and Safranin-O/Fast Green staining were performed to evaluate the degenerated cartilage, TRAP-stained osteoclasts were enumerated, and receptor activator of nuclear factor κB ligand (RANKL), AMPK, and NF-κB were detected using immunohistochemistry. BMM proliferation was not affected by Met treatment below 2 mM. Met inhibited osteoclast formation and bone resorption in a dose-dependent manner in vitro. Met suppressed RANKL-induced activation of p-AMPK, NF-κB, phosphorylated extracellular regulated protein kinases (p-ERK) and up-regulation of genes involved in osteoclastogenesis. Met reversed decreases in BV/TV, Tb.Th, Tb.N, and CD, and an increase in Tb.Sp at 4 weeks postoperatively. The number of osteoclasts and OARSI score were decreased by Met without effect on body weight or blood glucose levels. Met inhibited RANKL, p-AMPK, and NF-κB expression in early OA. The mechanism by which Met inhibits osteoclast activation may be associated with AMPK/NF-κB/ERK signaling pathway, indicating a novel strategy for OA treatment.

The effect of strontium ranelate on titanium particle-induced periprosthetic osteolysis regulated by WNT/ß-catenin signaling in vivo and in vitro.

Aseptic loosening following periprosthetic osteolysis is the primary complication that limits the lifetime of total joint arthroplasty (TJA). The wear particles trigger a chronic inflammation response in the periprosthetic tissue and turn over the bone balance to bone resorption. The present study aimed to investigate the possible effect and mechanism of strontium ranelate (SR), a clinically safe drug for osteoporosis, on particle-induced periprosthetic osteolysis. Thirty-six female C57BL/6j mice underwent tibial Ti-nail implantation to establish an animal model of aseptic loosening. After 12 weeks, micro-CT results showed that strontium ranelate could inhibit periprosthetic bone resorption. In vitro, Ti particles were used to stimulate RAW264.7 cell line to collect conditioned medium, and co-culture MC3T3-E1 cell line with conditioned medium to establish a cell model of aseptic loosening. The results of alkaline phosphatase (ALP) detection, immunofluorescence, and flow cytometry demonstrated that strontium ranelate could regulate the expression of OPG/RANKL, promote differentiation and mineralization, and inhibit apoptosis in osteoblasts. Moreover, we revealed that SR's exerted its therapeutic effect by down-regulating sclerostin, thereby activating the Wnt/ß-catenin signal pathway. Therefore, this research suggests that strontium ranelate could be a potential drug for the prevention and treatment of particle-induced aseptic loosening post-TJA.

Hyperglycemia aggravates spinal cord injury through endoplasmic reticulum stress mediated neuronal apoptosis, gliosis and activation.

BACKGROUND: Hyperglycemia has been shown to influence prognostic outcome of spinal cord injury (SCI). However, the corresponding mechanism is not very clear. AIM: This study is expected to explore the role of endoplasmic reticulum (ER) stress in hyperglycemia aggravated SCI. METHODS: Hyperglycemia was established in rats by intraperitoneal (i.p.) injection of streptozotocin. SCI was performed at the T10 of spinal cord through weight dropping. ER stress was suppressed by oral gavage of 4-PBA. ER stress, histological change of the injured spinal cords, neuronal apoptosis, demyelination, glial proliferation, inflammatory factor production, blood-spinal cord barrier (BSCB) permeability, TJ (Occludin, Claudin5) and AJ (ß-catenin, P120) protein degradation, and locomotor recovery were determined using western blotting, immunohistochemistry, HE staining, Evan's Blue assay, BBB scores and inclined plane test, respectively. In vitro, rat spinal cord neurons cells (RSCNCs) and cerebral microvascular endothelial cells (RCMECs) were stimulated with high glucose (HG) and/or thapsigargin (TG). The effects of HG and/or TG on RSCNCs apoptosis, and AJ and TJ expression by RCMECs were evaluated with flow cytometry and western blotting, respectively. RESULTS: Hyperglycemic rats exhibited enhanced ER stress, increased neuronal apoptosis, aggravated demyelination, increased glial proliferation and inflammatory factors secretion, more serious BSCB disruption and disturbed locomotor recovery. ER stress inhibition alleviated hyperglycemia induced adverse effect on neuronal apoptosis and BSCB permeability, whereas showed little influence on glial activation and inflammation. CONCLUSION: ER stress was aggravated in hyperglycemic rats after SCI, and subsequently promoted neuronal apoptosis and BSCB disruption in rats.

MUTYH and ORAI1 polymorphisms are associated with susceptibility to osteoarthritis in the Chinese Han population.

Background This study analyzed the associations between single nucleotide polymorphisms (SNPs) in the mutY homolog gene ( MUTYH) and the calcium release-activated calcium channel gene ( ORAI1) with susceptibility to osteoarthritis in the Chinese Han population. Methods A total of 350 patients diagnosed with osteoarthritis from October 2013 to May 2016 were selected as the study group, together with 350 age- and gender-matched healthy controls. MUTYH SNP rs3219463 and ORAI1 SNPs rs712853, rs12313273, rs6486795, rs12320939, and rs7135617 were analyzed by Sanger sequencing. Serum MUTYH levels were measured by enzyme-linked immunosorbent assay. The relationship between SNPs in MUTYH and ORAI1 and osteoarthritis susceptibility was analyzed and compared with the level of serum MUTYH in the osteoarthritis and control groups. Results MUTYH rs3219463 G allele carriers (GG or GA genotypes) and ORAI1 rs7135617 T allele carriers had a higher risk of osteoarthritis than patients with other genotypes. The level of serum MUTYH in the study group was significantly higher than in the control group (22.05 ± 19.14 ng/mL vs. 14.15 ± 13.54 ng/mL). Conclusions MUTYH and ORAI1 SNPs are associated with osteoarthritis susceptibility in the Chinese Han population.

Strontium ranelate inhibits wear particle-induced aseptic loosening in mice.

The imbalance between bone formation and osteolysis plays a key role in the pathogenesis of aseptic loosening. Strontium ranelate (SR) can promote bone formation and inhibit osteolysis. The aim of this study was to explore the role and mechanism of SR in aseptic loosening induced by wear particles. Twenty wild-type (WT) female C57BL/6j mice and 20 sclerostin-/- female C57BL/6j mice were used in this study. Mice were randomly divided into four groups: WT control group, WT SR group, knockout (KO) control group, and KO SR group. We found that SR enhanced the secretion of osteocalcin (0.72±0.007 in WT control group, 0.98±0.010 in WT SR group, P=0.000), Runx2 (0.34±0.005 in WT control group, 0.47±0.010 in WT SR group, P=0.000), ß-catenin (1.04±0.05 in WT control group, 1.22±0.02 in WT SR group, P=0.000), and osteoprotegerin (OPG) (0.59±0.03 in WT control group, 0.90±0.02 in WT SR group, P=0.000). SR significantly decreased the level of receptor activator for nuclear factor-κB ligand (RANKL) (1.78±0.08 in WT control group, 1.37±0.06 in WT SR group, P=0.000) and improved the protein ratio of OPG/RANKL, but these effects were not observed in sclerostin-/- mice. Our findings demonstrated that SR enhanced bone formation and inhibited bone resorption in a wear particle-mediated osteolysis model in wild-type mice, and this effect relied mainly on the down-regulation of sclerostin levels to ameliorate the inhibition of the canonical Wnt pathway.

Strontium ranelate reduces the progression of titanium particle-induced osteolysis by increasing the ratio of osteoprotegerin to receptor activator of nuclear factor-κB ligand in vivo.

The present study aimed to investigate the effects of strontium ranelate (SR), an anti­osteoporotic drug, on osteolysis in an experimental mouse model of aseptic loosening. A total of 45 female C57BL/6J mice each received implantation of one titanium alloy pin into the tibia, followed by intraarticular injection of titanium particles. One week following surgery, mice were randomly divided into three groups: Control group (no additional treatment), SR625 group (treated with SR at a dose of 625 mg/kg/day), and SR1800 group (treated with SR at a dose of 1,800 mg/kg/day). SR was administered via oral gavage once every day for 12 weeks. Micro­computed tomography scanning and hematoxylin/eosin staining were used to assess osteolysis around the prosthesis. Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction analysis were used to measure the expression of receptor activator of nuclear factor­κB ligand (RANKL) and osteoprotegerin (OPG). Compared with the control, the SR625 and SR1800 groups exhibited a significantly increased pulling force of the titanium alloy pin. Bone volume and the bone surface/volume ratio in the periprosthetic tissue were significantly increased in the SR­treated groups. Significant differences were observed between the SR1800 group and control group with respect to trabecular thickness and trabecular number. Mechanistically, SR downregulated the expression of RANKL and upregulated the expression of OPG in the periprosthetic tissue. In addition, SR was observed to inhibit wear particle­associated osteolysis in a dose­dependent manner. In conclusion, the present data illustrated that SR inhibited titanium particle­induced osteolysis in vivo.

Therapeutic efficacy of cyclosporin A against spinal cord injury in rats with hyperglycemia.

The present study aimed to explore the therapeutic effects of cyclosporin A (CsA) on spinal cord injury (SCI) in rats with hyperglycemia and to identify a novel potential method to treat SCI in the presence of hyperglycemia. Female Sprague­Dawley (SD) rats were randomly allocated into four groups: Sham, SCI, SCI+hyperglycemia and SCI+hyperglycemia+CsA groups. Streptozotocin­induced hyperglycemic SD rats and a weight­drop contusion SCI model were established. The Basso, Beattie, Bresnahan scale and inclined plane test were used to evaluate the neurological function of the rats. Flow cytometric assay was performed to detect the apoptotic rates of cells in the spinal cord. ELISA and western blot analysis were performed to determine the levels of interleukin (IL)­10, tumor necrosis factor (TNF)­α, cyclophilin­D (Cyp­D) and apoptosis­inducing factor (AIF). The results demonstrated that CsA significantly improved the neurological function of the SCI rats with hyperglycemia. CsA markedly reduced the number of apoptotic cells exaggerated by hyperglycemia in the spinal cord of the SCI rats. CsA significantly decreased the expression levels of IL­10, TNF­α, Cyp­D and AIF in the spinal cord of the SCI rats. Overall, the present study revealed a significant role of CsA in the treatment of SCI in the presence of hyperglycemia by inhibiting the apoptosis of spinal cord cells.

Effects of strontium ranelate on wear particle­induced aseptic loosening in female ovariectomized mice.

Aseptic loosening and menopause­induced osteoporosis are caused by an imbalance between bone formation and osteolysis. With an aging population, the probability of simultaneous occurrence of such conditions in an elderly individual is increasing. Strontium ranelate (SR) is an anti­osteoporosis drug that promotes bone formation and inhibits osteolysis. The present study compared the effects of SR with those of the traditional anti­osteoporosis drug alendronate (ALN) using an ovariectomized mouse model of osteolysis. The degree of firmness of the prosthesis and the surrounding tissue was examined, a micro­CT scan of the prosthesis and the surrounding tissue was performed, and the levels of inflammatory and osteogenic and osteoclast factors were examined. It was observed that treatment with SR and ALN improved the bond between the prosthesis and the surrounding bone tissue by reducing the degree of osteolysis, thus improving the quality of bone around the prosthesis. SR increased the secretion of osteocalcin, runt­related transcription factor 2 and osteoprotegerin (OPG). It additionally decreased the expression of the receptor activator of nuclear factor­κB ligand (RANKL) and consequently increased the protein ratio OPG/RANKL, whereas ALN exhibited the opposite effect. Furthermore, SR and ALN suppressed tumor necrosis factor­α and interleukin­1ß production, with SR exerting a more marked effect. The present results demonstrate that SR and ALN may stimulate bone formation and inhibit bone resorption in the ovariectomized mouse model of wear particle­mediated osteolysis, with SR demonstrating better effects compared with ALN.

Particle-induced SIRT1 downregulation promotes osteoclastogenesis and osteolysis through ER stress regulation.

BACKGROUND: Sirtuin 1 (SIRT1) downregulation has been found to be induced by wear particles in aseptic prosthesis loosening (APL). Osteoclastogenesis and osteoclast activation are the main pathological factors associated with APL. However, whether SIRT1 downregulation contributes to the formation and activation of osteoclasts through the induction of endoplasmic reticulum (ER) stress is unclear. METHODS: To address this, an osteolysis mouse model was used in which animals were treated with the SIRT1 activator, resveratrol (RES), or an ER stress inhibitor, 4-PBA, for two weeks. Osteolysis, osteoclastogenesis, and morphologic alteration of calvariae were observed by toluidine blue, TRAP, and H&E staining. SIRT1 expression and ER stress were evaluated by western blot analysis. In vitro, mouse macrophage RAW 264.7 cells were treated with polyethylene (PE) particles alone or combined with either RES or 4-PBA, and SIRT1 expression and ER stress were measured using western blot assays. Osteoclast differentiation was determined through TRAP staining. Osteoclast activation was evaluated by culturing osteoclast cells on bone slices followed by toluidine blue staining. Mechanistically, osteoclastogenesis-related MAPK activation, NFATc1 and c-Fos expression, and NF-κB translocation were determined. RESULTS: Both in vivo and in vitro experimental results indicated that PE particles induced SIRT1 downregulation and enhanced ER stress. SIRT1 activator RES and ER stress inhibitor 4-PBA significantly suppressed PE particle-induced osteoclast differentiation and osteolysis. In vitro experimental results showed that 4-PBA suppressed PE particle-induced ERK1/2, p38, and JNK activation, NFATc1 and c-Fos upregulation, as well as NF-κB p65 nucleus translocation. CONCLUSIONS: PE particle-induced downregulation of SIRT1 enhances ER stress and promotes osteoclast proliferation and bone resorption through regulation of c-Fos, NFATc1, and the MAPK and NF-κB signaling pathways.

Sclerostin expression in the subchondral bone of patients with knee osteoarthritis.

The aim of this study was to assess the expression of ß-catenin, transcription factor-4 (TCF-4) and sclerostin in the subchondral bone of patients with primary knee osteoarthritis (OA). Tibial plateau specimens from patients with OA who underwent total knee arthroplasty were classified into the early stage (n=15), intermediate stage (n=13) and late stage (n=17) groups using the Mankin score. Structural parameters, including total articular cartilage (TAC), subchondral bone plate (SCP) thickness and trabecular bone volume (BV/TV), were assessed using Image-Pro Plus 6.0 analysis software. Subsequently, ß-catenin and sclerostin expression levels in subchondral bone were determined by immunohistochemistry. In addition, the mRNA and protein levels of ß-catenin, TCF-4 and sclerostin were evaluated by RT-qPCR and western blot analysis, respectively. As regards the cartilage and subchondral bone structural parameters, TAC was reduced, while SCP thickness and BV/TV were increased due to OA, with significant differences observed among the different stages (all P<0.05). The results of immunohistochemistry revealed that the ß-catenin levels in the intermediate- and late-stage samples were significantly increased, while the levels of sclerostin were markedly decreased compared with the values in the early-stage samples (all P<0.05). Compared with the intermediate-stage samples, the sclerostin levels were decreased, and SCP thickness and the ß-catenin levels were increased in the late-stage samples (all P<0.05). The results of RT-qPCR and western blot analysis revealed that the ß-catenin and TCF-4 mRNA and protein levels in the intermediate- and late-stage samples were significantly increased, while sclerostin expression was significantly decreased compared with the early-stage samples; a similar trend was observed between the intermediate- and late-stage samples (all P<0.05). Finally, the ß-catenin and TCF-4 levels positively correlated with the Mankin scores, while there was a negative correlation with sclerostin expression. Our findings demonstrate that sclerostin expression is closely associated with the degree of joint damage in patients with OA, confirming its involvement in the development of OA.

Adenovirus-mediated small interfering RNA targeting tumor necrosis factor-α inhibits titanium particle-induced osteoclastogenesis and bone resorption.

Wear particles are phagocytosed by macrophages, resulting in cellular activation and the release of pro-inflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty (TJA) failure. During this pathological process, tumor necrosis factor (TNF)-α plays an important role in wear particle-induced osteolysis. Therefore, in this study, we used adenovirus-mediated small interfering RNA (siRNA) targeting TNF-α to suppress the TNF-α release from activated macrophages in response to titanium particles. Our results showed that recombinant adenovirus (Ad-TNF-α-siRNA) suppressed the TNF-α release from activated macrophages in response to titanium particles, and reduced titanium particle-induced osteoclastogenesis and bone resorption in the presence of receptor activator of nuclear factor-κB ligand (RANKL). In addition, the conditioned medium of macrophages challenged with titanium particles (Ti CM) stimulated osteoprogenitor RANKL expression. The conditioned medium of macrophages challenged with titanium particles and Ad-TNF-α-siRNA (Ti-Ad CM) reduced the mRNA expression in MC3T3-E1 cells compared to Ti CM. Based on these data, TNF-α strongly synergizes with RANKL to promote osteoclast differentiation. Furthermore, TNF-α promoted osteoclast differentiation by stimulating osteoprogenitor RANKL expression. Ad-TNF-α-siRNA effectively suppressed osteoclast differentiation and bone resorption following exposure to titanium particles in the presence of RANKL. In addition, recombinant adenovirus (Ad-TNF-α-siRNA) does not have a toxic effect on the murine macrophage cell line, RAW264.7. Consequently, it can be concluded that recombinant adenovirus-mediated siRNA targeting TNF-α (Ad-TNF-α-siRNA) may provide a novel therapeutic approach for the treatment of periprosthetic osteolysis.

Adenovirus-mediated expression of bone morphogenetic protein-2 activates titanium particle-induced osteoclastogenesis and this effect occurs in spite of the suppression of TNF-α expression by siRNA.

The phagocytosis of wear particles by macrophages results in the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), which play a major role in promoting osteoclast recruitment. The inhibition of TNF-α expression decreases osteoclastogenesis. In a previous study, we demonstrated that bone morphogenetic protein-2 (BMP-2) can activate wear debris-induced osteoclast recruitment in the presence of receptor activator of nuclear factor (NF)-κB ligand (RANKL); however, whether these effects are associated with pro-inflammatory cytokines remains unclear. In this study, we constructed an adenoviral vector carrying TNF-small interfering RNA (siRNA) (Ad-TNF-siRNA), as well as a vector carrying both the BMP-2 gene and TNF-α-siRNA (Ad-BMP-2-TNF-siRNA). The two adenoviral vectors significantly suppressed the expression of TNF-α; however, only treatment with Ad-TNF-siRNA significantly inhibited osteoclastogenesis. We demonstrate that the overexpression of BMP-2, despite the suppression of TNF-α expression by Ad-BMP-2-TNF-siRNA, increases the size and number of titanium (Ti) particle-induced multinuclear osteoclasts, the expression of osteoclast genes, as well as the resorption area. There were no differences observed between Ti particle-induced and Ad-BMP-2-TNF-siRNA-induced osteoclast formation. Moreover, Ad-BMP-2-TNF-siRNA directly acted upon osteoclast precursors by increasing the level of c-Fos, regulating other signaling pathways, such as p38 phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated IκB (p­IκB). Taken together, these data demonstrate that treatment with Ad-BMP-2-TNF-siRNA increases wear debris-induced osteoclast formation by activating c-Fos and that these effects are not associated with pro-inflammatory cytokines.

[Experimental study on small interfering RNA silencing expression of tumor necrosis factor alpha and inhibiting osteolysis].

OBJECTIVE: To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor alpha (TNF-alpha). METHODS: The primer of small interfering RNA (siRNA) coding sequence of silent TNF-alpha was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-a-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 x 10(9) PFU/mL), and Ad5-TNF-alpha-siRNA-CMVeGFP (1 x 10(9) PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-a was detected by Western blot at 12 weeks after operation. RESULTS: The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-alpha-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A < group D < group B < group C, P < 0.05). Western blot showed that the TNF-a expression levels were 0.235 +/- 0.022, 0.561 +/- 0.031, 0.731 +/- 0.037, and 0.329 +/- 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P < 0.05). CONCLUSION: The recombinant adenovirus for silencing TNF-alpha is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-alpha expression in the tissues around prosthesis in mice.