Decrease in particle‐induced osteolysis in ovariectomized mice

Postmenopausal osteoporosis is a common disorder that results from increased osteoclastic activity caused by estrogen deficiency. Whether postmenopausal bone remodeling can alter the response to particulate debris is unknown. The purpose of this study was to evaluate the bone response to polyethylene particles in an ovariectomized murine model. Polyethylene particles were implanted onto the calvaria of seven control mice and seven ovariectomized (OVX) mice, as compared with calvaria from sham‐operated and OVX mice. Calvaria were harvested after 14 days. Skulls were analyzed with a high‐resolution micro‐CT and by histomorphometry after staining with Stevenel blue and picrofuschine, and for tartrate‐specific alkaline phosphatase. As assessed by micro‐CT, particle implantation induced a significant decrease in bone thickness in control mice, while bone thickness remained stable in OVX mice. In particle‐implanted animals, the osteoclast number was 2.84 ± 0.3 in control mice and 1.74 ± 0.22 in OVX mice. Mean bone loss was ?12% ± 1.9% in control mice and ?4.7% ± 1.7% in OVX animals. The reduction of osteolytic response suggests that ovariectomy may have a protective role against particle‐induced bone resorption. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:178–183, 2010     

Antisense oligonucleotide targeting TNF‐α can suppress co‐cr‐mo particle‐induced osteolysis

The most common cause of implant failure in joint replacement is aseptic loosening due to particle‐induced osteolysis. TNF‐α has been shown to be one of the key factors in the process of osteoclastogenesis. Anti‐TNF agents are useful in the treatment of joint inflammation related to osteolysis. This study investigated the effect of a single subcutaneous dose of an antisense oligonucleotide (ASO) on particle‐induced osteolysis. We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured by the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase (TRAP) staining assay and were quantified by a TRAP quantification kit. Results show that bone resorption is 0.347 ± 0.09 mm² in mice with particle implantation, and decreased to 0.123 ± 0.05 mm² and 0.052 ± 0.02 mm² after ASO treatment with low and high doses, respectively. The number of osteoclasts in animal calvaria treated with ASO is reduced compared with that of untreated animals, and the quantification results indicate that about 90% of osteoclastogenesis is suppressed by the ASO. In addition, the osteoclastogenesis can be reestablished by the addition of TNF‐α. In conclusion, we demonstrate that the antisense oligonucleotide targeting to TNF‐α can suppress osteolysis induced by metal particles in a murine calvaria model. This new finding may be of value in the search for novel therapeutic methods for implant loosening. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1114–1120, 2008     

Expression of XBP1s in fibroblasts is critical for TiAl6V4 particle‐induced RANKL expression and osteolysis

Wear particle‐induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the expression of Receptor activation of nuclear factor (NF)‐kB (RANKL) by fibroblasts in periprosthetic membrane played a crucial role in wear particle‐induced osteolysis. However, the underlying mechanism of RANKL expression remains largely unknown. In the present study, we investigated the effect of TiAl₆V₄ particle (TiPs)‐induced XBP1s (spliced form of X‐box binding protein 1) on RANKL expression and osteoclastogenesis both in vitro and in vivo. The levels of XBP1s in peri‐implant membrane, animal models, and TiPs‐stimulated fibroblasts were determined by western blots. To assess the effect of XBP1s on RANKL expression, fibroblasts were treated with both a small interfering RNA (siRNA) and an inhibitor of XBP1 prior to exposure to TiPs. The effect of XBP1s on osteoclasts formation was determined by tartrate‐resistant acid phosphatase (TRAP) staining in vitro osteoclastogenesis assay and in animal models. The resorption of bone was assessed by micro‐computed tomography (micro‐CT) with three‐dimensional reconstruction. Our results demonstrated that XBP1s was activated in periprosthetic membrane, mouse calvaria models, and TiPs‐stimulated human synovial fibroblasts. Further, inhibition of XBP1s decreased the expression of RANKL and osteoclasts formation in vitro. In mouse calvaria models, both of the osteoclastogenesis and osteolysis were inhibited XBP1s inhibitor. Our results suggested that XBP1s mediated TiPs‐induced of RANKL expression in fibroblasts, and down regulating XBP1s may represent a potential therapy for wear particle‐induced osteolysis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:752–759, 2017.

     

Promotion of bone formation by naringin in a titanium particle‐induced diabetic murine calvarial osteolysis model

Diabetic patients have an increased risk of prosthesis failure requiring revision surgery. Furthermore, skeletal defects are observed in conjunction with type 1 diabetes. Using a titanium particle‐induced calvarial osteolysis model in diabetic mice, we investigated the effect of diabetes on the osteolytic process and the role of naringin in its prevention. Three groups each of nondiabetic or diabetic mice were treated with vehicle only, with particles only, or with particles then naringin for 10 days. Alteration of bone indices near the midline suture were then analyzed by microcomputed tomography scanning and histology. Serum levels of osteocalcin (OCN) and cross‐linked N‐telopeptide of type I collagen (NTx) were measured by enzyme‐linked immunosorbent assay. The decreases in new bone formation (p < 0.05), calvaria thickness (p < 0.05), bone volume (p < 0.05), midline suture area (p < 0.05), and OCN concentration (p < 0.05) found in diabetic mice were normalized with naringin treatment. Diabetic state promoted particle‐induced osteolysis. Naringin, an osteoanabolic agent, improved bone indices apparently by stimulating bone formation. Therefore, naringin may be beneficial in preventing and treating debris‐mediated periprosthetic osteolysis after total joint replacement, especially in diabetics. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:451–456, 2010     

Tumor necrosis factor α suppresses the mesenchymal stem cell osteogenesis promoter miR‐21 in estrogen deficiency–induced osteoporosis

Inflammatory cytokines, especially tumor necrosis factor α (TNF‐α), have been shown to inhibit osteogenic differentiation of mesenchymal stem cells (MSCs) and bone formation in estrogen deficiency–induced osteoporosis, but the mechanism responsible remains poorly understood. MicroRNAs (miRNAs) have been shown to regulate MSC differentiation. Here, we identified a novel mechanism whereby TNF‐α, suppressing the functional axis of a key miRNA (miR‐21) contributes to estrogen deficiency–induced osteoporosis. In this study, we screened differentially expressed miRNAs in MSCs derived from estrogen deficiency‐induced osteoporosis and found miR‐21 was significantly downregulated. miR‐21 was suppressed by TNF‐α during the osteogenesis of MSCs. Furthermore, miR‐21 was confirmed to promote the osteoblast differentiation of MSCs by repressing Spry1, which can negatively regulate the osteogenic differentiation of MSCs. Upregulating miR‐21 partially rescued TNF‐α–impaired osteogenesis of MSCs. Blocking TNF‐α ameliorated the inflammatory environment and significantly enhanced bone formation with increased miR‐21 expression and suppressed Spry1 expression in ovariectomized (OVX) mice. Our results revealed a novel function for miR‐21 and suggested that suppressed miR‐21 may contribute to impaired bone formation by elevated TNF‐α in estrogen deficiency–induced osteoporosis. This study may indicate a molecular basis for novel therapeutic strategies against osteoporosis and other inflammatory bone diseases. © 2013 American Society for Bone and Mineral Research.     

Prognostic significance of miRNA‐1 (miR‐1) expression in patients with chordoma

Reliable prognostic biomarkers for chordoma have not yet been established. Recent studies revealed that expression of miRNA‐1 (miR‐1) is frequently downregulated in several cancer types including chordoma. The goal of this follow‐up study is to investigate the expression of miR‐1 as a prognostic biomarker and further confirm the functional role of miR‐1 in chordoma cell growth and proliferation. We determined the relative expression levels of miR‐1 and Met in chordoma tissue samples and correlated those to clinical variables. The results showed that miR‐1 was downregulated in 93.7% of chordoma tissues and expression was inversely correlated with Met expression. miR‐1 expression levels also correlated with clinical prognosis. To characterize and confirm the functional role of miR‐1 in the growth and proliferation of chordoma cells, miR‐1 precursors were stably transfected into chordoma cell lines UCH‐1 and CH‐22. Cell Proliferation Assay and MTT were used to evaluate cell growth and proliferation. Restoring expression of miR‐1 precursor decreased cell growth and proliferation in UCH‐1 and CH‐22 cells. These results indicate that suppressed miR‐1 expression in chordoma may in part be a driver for tumor growth, and that miR‐1 has potential to serve as prognostic biomarker and therapeutic target for chordoma patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:695–701, 2014.     

A long non‐coding RNA,GAS5, plays a critical role in the regulation of miR‐21 during osteoarthritis

Growth Arrest‐Specific 5 (GAS5) is known to negatively regulate cell survival and is aberrantly expressed in several cancers. The influence of GAS5 on osteoarthritis (OA) has not been determined. To address this, articular chondrocytes were isolated from relatively normal (Non‐OA) and clear OA regions (OA) of cartilage in total knee replacement (TKR) patients and biopsied normal cartilage. We found that GAS5 was up‐regulated in OA chondrocytes compared with Non‐OA and normal chondrocytes. The over‐expression of GAS5 increased the expression levels of several MMPs, such as MMP‐2, MMP‐3, MMP‐9, MMP‐13, and ADAMTS‐4; stimulated apoptosis; and suppressed autophagic responses. Furthermore, we subsequently identified miR‐21 as a regulator of GAS5 during OA pathogenesis. The expression level of miR‐21 was significantly reduced in OA patients, and the ectopic expression of GAS5 is capable of suppressing miR‐21 induction. Consistent with GAS5 experiments, the introduction of miR‐21 stimulated the apoptosis of chondrocytes and inhibited the expression levels of autophagic complexes, including LC‐3B. In vivo, we found that the introduction of miR‐21 into the cartilage of OA mice significantly stimulated cartilage destruction. Together, these results show that GAS5 contributes to the pathogenesis of OA by acting as a negative regulator of miR‐21 and thereby regulating cell survival. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1628–1635, 2014.     

Local delivery of mutant CCL2 protein‐reduced orthopaedic implant wear particle‐induced osteolysis and inflammation in vivo

Total joint replacement (TJR) has been widely used as a standard treatment for late‐stage arthritis. One challenge for long‐term efficacy of TJR is the generation of ultra‐high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle‐induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle‐induced osteolysis and the effects of 7ND treatment were evaluated using micro‐CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle‐induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle‐induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:58–64, 2016.     

Regulation of miR‐200c and miR‐141 by Methylation in Prostate Cancer

BACKGROUND

In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation of miRNAs is a contributing factor to their altered expression in cancer. In this study, we investigate whether expression of miR‐200c and miR‐141 in PCa is related to the DNA methylation status of their promoter.

METHODS

PCR analysis of miR‐200c and miR‐141, and CpG methylation analysis of their common promoter, was performed in PCa cell‐lines and in archived prostate biopsy specimens. The biological significance of miR‐200c and miR‐141 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets DNMT3A and TET1/TET3 was investigated. The effect on promoter methylation status in cells treated with demethylating agents was also examined.

RESULTS

miR‐200c and miR‐141 are both highly elevated in LNCaP, 22RV1, and DU145 cells, but significantly reduced in PC3 cells. This correlates inversely with the methylation status of the miR‐200c/miR‐141 promoter, which is unmethylated in LNCaP, 22RV1, and DU145 cells, but hypermethylated in PC3. In PC3 cells, miR‐200c and miR‐141 expression is subsequently elevated by treatment with the demethylating drug decitabine (5‐aza‐2′deoxycytidine) and by knockdown of DNA methyltransferase 1 (DNMT1), suggesting their expression is regulated by methylation. Expression of miR‐200c and miR‐141 in prostate biopsy tissue was inversely correlated with methylation in promoter CpG sites closest to the miR‐200c/miR‐141 loci. In vitro, over‐expression of miR‐200c in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis. Expression of the genes DNMT3A and TET1/TET3 were down‐regulated by miR‐200c and miR‐141 respectively. Finally, treatment with the soy isoflavone genistein caused demethylation of the promoter CpG sites closest to the miR‐200c/miR‐141 loci resulting in increased miR‐200c expression.

CONCLUSIONS

Our findings provide evidence that miR‐200c and miR‐141 are under epigenetic regulation in PCa cells. We propose that profiling their expression and methylation status may have potential as a novel biomarker or focus of therapeutic intervention in the diagnosis and prognosis of PCa. Prostate 76:1146–1159, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.     

The balance between endotoxin accumulation and clearance during particle-induced osteolysis in murine calvaria.

Bacterial endotoxin may contribute to aseptic loosening of orthopedic implants even in the absence of clinical or microbiological evidence of infection. One potential source of endotoxin during aseptic loosening is systemically circulating endotoxin, derived from intestinal flora, minor infections, or dental procedures, that may bind to wear particles. The current study demonstrates that systemically derived endotoxin accumulates when 'endotoxin-free' titanium and polyethylene particles are implanted on murine calvaria. Time-course experiments and experiments using germ-free mice rule out the possibility that the observed endotoxin accumulation may be due to bacterial contamination. In contrast, endotoxin is cleared from titanium particles that originally carry high amounts of adherent endotoxin. The mechanism of endotoxin clearance is not dependent on induction of a respiratory burst. Taken together, these results indicate that a balance between endotoxin accumulation and endotoxin clearance controls the steady-state level of endotoxin surrounding orthopedic wear particles implanted on murine calvaria. This balance may regulate the rate of osteolysis in the murine calvaria model as well as in patients with aseptic loosening.     

Blockade of JNK and NFAT pathways attenuates orthopedic particle‐stimulated osteoclastogenesis of human osteoclast precursors and murine calvarial osteolysis

Particles released from orthopedic implants attract immune host defense cells to the bone‐implant interface and contribute to development of inflammation. The inflammatory microenvironment supports recruitment and differentiation of osteoclasts, the primary culprit of osteolysis. Therefore, understanding the complex signals that contribute to osteoclastogenesis and osteolysis is a sensible approach to design strategies to inhibit bone loss. The signaling cascades that coordinate osteoclastogenesis have been widely investigated. These include MAP kinases, Akt/PI3K pathway, NF‐κB signal transduction pathway, and NFAT pathway. We have recently reported that polymethylmethacrylate (PMMA) particles activate the NFAT pathway in murine osteoclast precursors and that NFAT inhibitors dose‐dependently block PMMA‐induced osteoclastogenesis. In the current study, we examined the role of JNK and NFATc1 in mice in response to PMMA particles using murine calvaria model. We show that locally administered MAPK/JNK inhibitor SP600125 and calcineurin/NFAT inhibitor cyclosporine‐A effectively blocked PMMA‐induced osteolysis in murine calvaria. To buttress the clinical relevance of JNK/NFATc1‐based regulation of PMMA‐induced osteoclastogenesis, we evaluated the effect of PMMA using human macrophages. We demonstrate that SP600125 and cyclosporine‐A abolished particle‐induced osteoclastogenesis in human osteoclast progenitors retrieved from patients undergoing total hip replacement. Thus JNK and NFATc1 appear to act as significant mediators of orthopedic particle‐induced osteolysis in humans. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:67–72, 2012     

Particle‐induced osteolysis is not accompanied by systemic remodeling but is reflected by systemic bone biomarkers

Particle‐induced osteolysis is caused by an imbalance in bone resorption and formation, often leading to loss of implant fixation. Bone remodeling biomarkers may be useful for identification of osteolysis and studying pathogenesis, but interpretation of biomarker data could be confounded if local osteolysis engenders systemic bone remodeling. Our goal was to determine if remote bone remodeling contributes to biomarker levels. Serum concentrations of eight biomarkers and bone remodeling rates at local (femur), contiguous (tibia), and remote (humerus and lumbar vertebra) sites were evaluated in a rat model of particle‐induced osteolysis. Serum CTX‐1, cathepsin K, PINP, and OPG were elevated and osteocalcin was suppressed in the osteolytic group, but RANKL, TRAP 5b, and sclerostin were not affected at the termination of the study at 12 weeks. The one marker tested longitudinally (CTX‐1) was elevated by 3 weeks. We found increased bone resorption and decreased bone formation locally, subtle differences in contiguous sites, but no differences remotely at 12 weeks. Thus, the skeletal response to local particle challenge was not systemic, implying that the observed differences in serum biomarker levels reflect differences in local remodeling. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:967–973, 2014.     

MicroRNA‐1 (miR‐1) inhibits chordoma cell migration and invasion by targeting slug

Recent studies have revealed that expression of miRNA‐1 (miR‐1) is frequently down‐regulated in several cancer types including chordoma. Identifying and validating novel targets of miR‐1 is useful for understanding the roles of miR‐1 in chordoma. We aimed to further investigate the functions of miR‐1 in chordoma. Specifically, we assessed whether restoration of miR‐1 affects cell migration and invasion in chordoma, and focused on the miR‐1 potential target Slug gene. Migratory and invasive activities were assessed by wound healing and Matrigel invasion assays, respectively. Cell proliferation was determined by MTT assay. Slug expression was evaluated by Western blot, immunofluorescence, and immunohistochemistry. Restoration of miR‐1 expression suppressed the migratory and invasive activities of chordoma cells. Transfection of miR‐1 inhibited cell proliferation both time‐ and dose‐dependently in chordoma. MiR‐1 transfected cells showed inhibited Slug expression. Slug was over‐expressed in chordoma cell lines and advanced chordoma tissues. In conclusion, we have shown that miR‐1 directly targets the Slug gene in chordoma. Restoration of miR‐1 suppressed not only proliferation, but also migratory and invasive activities, and reduced the Slug expression in chordoma cells. These results collectively indicate that miR‐1/Slug pathway is a potential therapeutic target because of its crucial roles in chordoma cell growth and migration. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1075–1082, 2014.     

MicroRNA‐21 (miR‐21) expression in hypothermic machine perfusate may be predictive of early outcomes in kidney transplantation

Hypothermic machine perfusion is effective in improving outcome following kidney transplantation. Molecular analyses of hypothermic machine perfusate (HMP) have the potential to identify biomarkers of organ viability prior to transplantation, offering significant advantages to the transplant surgeon, and leading to a potential increase in the organ donor pool. MicroRNAs are emerging as important biomarkers in the context of kidney injury and transplantation. Recent data demonstrate increased microRNA‐21 (miR‐21) expression in the kidney following acute kidney injury. This study investigated the potential of miR‐21 detected in HMP to act as a sentinel for early kidney transplant outcomes. MiR‐21 was found to be readily detectable in HMP by RT‐qPCR. Eleven ECD kidneys were maintained on a hypothermic machine perfusion system for a median 627 (range 117–1027) minutes, and evaluation of flow and resistance characteristics suggested stability on the machine from 60 min post‐perfusion. MiR‐21 quantification at 60 min post‐perfusion correlated with eGFR at 6 and 12 months post‐transplantation. These data suggest that miR‐21 expression in HMP may be predictive of early outcomes following kidney transplantation. In the era of ECD kidneys, a reliable measure of organ quality is urgently needed, and this study suggests miR‐21 may be such a marker.