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Wear particles from orthopedic implants cause aseptic loosening, the leading cause of implant revisions. The particles are phagocytosed by macrophages leading to activation of the nod-like receptor protein 3 (NLRP3) inflammasome and release of interleukin-1β (IL-1β) which then contributes to osteoclast differentiation and implant loosening. The mechanism of inflammasome activation by orthopedic particles is undetermined but other particles cause the cytosolic accumulation of the lysosomal cathepsin-family proteases which can activate the NLRP3 inflammasome. Here, we demonstrate that lysosome membrane disruption causes cathepsin release into the cytoplasm that drives both inflammasome activation and cell death but that these processes occur independently. Using wild-type and genetically-manipulated immortalized murine bone marrow derived macrophages and pharmacologic inhibitors, we found that NLRP3 and gasdermin D are required for particle-induced IL-1β release but not for particle-induced cell death. In contrast, phagocytosis and lysosomal cathepsin release are critical for both IL-1β release and cell death. Collectively, our findings identify the pan-cathepsin inhibitor Ca-074Me and the NLRP3 inflammasome inhibitor MCC950 as therapeutic interventions worth exploring in aseptic loosening of orthopedic implants. We also found that particle-induced activation of the NLRP3 inflammasome in pre-primed macrophages and cell death are not dependent on pathogen-associated molecular patterns adherent to the wear particles despite such pathogen-associated molecular patterns being critical for all other previously studied wear particle responses, including priming of the NLRP3 inflammasome.

Osteosarcoma (OS) is the most common primary bone cancer and ranks amongst the leading causes of cancer mortality in young adults. Jun activation domain-binding protein 1 (JAB1) is overexpressed in many cancers and has recently emerged as a novel target for cancer treatment. However, the role of JAB1 in osteosarcoma was virtually unknown. In this study, we demonstrate that JAB1-knockdown in malignant osteosarcoma cell lines significantly reduced their oncogenic properties, including proliferation, colony formation, and motility. We also performed RNA-sequencing analysis in JAB1-knockdown OS cells and identified 4110 genes that are significantly differentially expressed. This demonstrated for the first time that JAB1 regulates a large and specific transcriptome in cancer. We also found that JAB1 is overexpressed in human OS and correlates with a poor prognosis. Moreover, we generated a novel mouse model that overexpresses Jab1 specifically in osteoblasts upon a TP53 heterozygous sensitizing background. Interestingly, by 13 months of age, a significant proportion of these mice spontaneously developed conventional OS. Finally, we demonstrate that a novel, highly specific small molecule inhibitor of JAB1, CSN5i-3, reduces osteosarcoma cell viability, and has specific effects on the ubiquitin-proteasome system in OS. Thus, we show for the first time that the overexpression of JAB1 in vivo can result in accelerated spontaneous tumor formation in a p53-dependent manner. In summary, JAB1 might be a unique target for the treatment of osteosarcoma and other cancers.

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

Epigenetic deregulation is an emerging hallmark of cancer that enables tumor cells to escape surveillance by tumor suppressors and ultimately progress. The structure of the epigenome consists of covalent modifications of chromatin components, including acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs). Targeting these enzymes with inhibitors to restore epigenetic homeostasis has been explored for many cancers. Osteosarcoma, an aggressive bone malignancy that primarily affects children and young adults, is notable for widespread genetic and epigenetic instability. This may explain why therapy directed at unique molecular pathways has failed to substantially improve outcomes in osteosarcoma over the past four decades. In this review, we discuss the potential of targeting the cancer epigenome, with a focus on histone deacetylase inhibitors (HDACi) for osteosarcoma. We additionally highlight the safety and tolerance of HDACi, combination chemotherapy with HDACi, and the ongoing challenges in the development of these agents.

The primary conclusions of our 2014 contribution to this series were as follows: Multiple receptor tyrosine kinases (RTKs) likely contribute to aggressive phenotypes in osteosarcoma and, therefore, inhibition of multiple RTKs is likely necessary for successful clinical outcomes. Inhibition of multiple RTKs may also be useful to overcome resistance to inhibitors of individual RTKs as well as resistance to conventional chemotherapies. Different combinations of RTKs are likely important in individual patients. AXL, EPHB2, FGFR2, IGF1R, and RET were identified as promising therapeutic targets by our in vitro phosphoproteomic/siRNA screen of 42 RTKs in the highly metastatic LM7 and 143B human osteosarcoma cell lines. This chapter is intended to provide an update on these topics as well as the large number of osteosarcoma clinical studies of inhibitors of multiple tyrosine kinases (multi-TKIs) that were recently published.

Purpose The classic fracture model, described by Bonnarens and Einhorn in 1984, enlists a blunt guillotine to generate a closed fracture in a pre-stabilized rodent femur. However, in less experienced hands, this technique yields considerable variability in fracture pattern and requires highly-specialized equipment. This study describes a reproducible and low-cost model of mouse fracture healing using an open femoral osteotomy. Methods Femur fractures were produced in skeletally mature male and female mice using an open femoral osteotomy after intramedullary stabilization. Mice were recovered for up to 28 days prior to analysis with microradiographs, histomorphometry, a novel μCT methodology, and biomechanical torsion testing at weekly intervals. Results Eight mice were excluded due to complications (8/193, 4.1%), including unacceptable fracture pattern (2/193, 1.0%). Microradiographs showed progression of the fracture site to mineralized callus by 14 days and remodelling 28 days after surgery. Histomorphometry from 14 to 28 days revealed decreased cartilage area and maintained bone area. μCT analysis demonstrated a reduction in mineral surface from 14 to 28 days, stable mineral volume, decreased strut number, and increased strut thickness. Torsion testing at 21 days showed that fractured femurs had 61% of the ultimate torque, 63% of the stiffness, and similar twist to failure when compared to unfractured contralateral femurs. Conclusions The fracture model described herein, an open femoral osteotomy, demonstrated healing comparable to that reported using closed techniques. This simple model could be used in future research with improved reliability and reduced costs compared to the current options., Highlights • This study characterized a simple and reproducible model of fracture healing in mice using an open femoral osteotomy. • Analysis by x-ray, histomorphometry, µCT, and biomechanical testing demonstrated healing comparable to current models. • This simple model could be used to increase investigation into fracture healing, delayed union, and non-union.

BACKGROUND: Orthopaedic wear particles activate the NLRP3 inflammasome to produce active interleukin 1β (IL1β). However, the NLRP3 inflammasome must be primed before it can be activated, and it is unknown whether wear particles induce priming. Toll-like receptors (TLRs) are thought to mediate particle bioactivity. It remains controversial whether pathogen-associated molecular patterns (PAMPs) and/or alarmins are responsible for TLR activation by wear particles. QUESTIONS/PURPOSES: (1) Does priming of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? (2) Does priming of the NLRP3 inflammasome by wear particles depend on TLRs and TIRAP/Mal? (3) Does priming of the NLRP3 inflammasome by wear particles depend on cognate TLRs? (4) Does activation of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? METHODS: Immortalized murine macrophages were stimulated by as-received titanium particles with adherent bacterial debris, endotoxin-free titanium particles, or titanium particles with adherent ultrapure lipopolysaccharide. To study priming, NLRP3 and IL1β mRNA and IL1β protein levels were assessed in wild-type, TLR4(-/-), TLR2(-/-), and TIRAP/Mal(-/-) macrophages. To study activation, IL1β protein secretion was assessed in wild-type macrophages preprimed with ultrapure lipopolysaccharide. RESULTS: Compared with titanium particles with adherent bacterial debris, endotoxin-free titanium particles induced 86% less NLRP3 mRNA (0.05 ± 0.03 versus 0.35 ± 0.01 NLRP3/GAPDH, p < 0.001) and 91% less IL1β mRNA (0.02 ± 0.01 versus 0.22 ± 0.03 IL1β/GAPDH, p < 0.001). ProIL1β protein level was robustly increased in wild-type macrophages stimulated by particles with adherent PAMPs but was not detectably produced in macrophages stimulated by endotoxin-free particles. Adherence of ultrapure lipopolysaccharide to endotoxin-free particles reconstituted stimulation of NLRP3 and IL1β mRNA. Particles with adherent bacterial debris induced 79% less NLRP3 mRNA (0.09 ± 0.004 versus 0.43 ± 0.13 NLRP3/GAPDH, p < 0.001) and 40% less IL1β mRNA (0.09 ± 0.04 versus 0.15 ± 0.03 IL1β/GAPDH, p = 0.005) in TLR4(-/-) macrophages than in wild-type. Similarly, those particles induced 49% less NLRP3 mRNA (0.22 ± 0.10 versus 0.43 ± 0.13 NLRP3/GAPDH, p = 0.004) and 47% less IL1β mRNA (0.08 ± 0.02 versus 0.15 ± 0.03 IL1β/GAPDH, p = 0.012) in TIRAP/Mal(-/-) macrophages than in wild-type. Particles with adherent ultrapure lipopolysaccharide induced 96% less NLRP3 mRNA (0.012 ± 0.001 versus 0.27 ± 0.05 NLRP3/GAPDH, p = 0.003) and 91% less IL1β mRNA (0.03 ± 0.01 versus 0.34 ± 0.07 IL1β/GAPDH, p < 0.001) expression in TLR4(-/-) macrophages than in wild-type. In contrast, those particles did not induce less NLRP3 and IL1β mRNA in TLR2(-/-) macrophages. IL1β protein secretion was equivalently induced by particles with adherent bacterial debris or by endotoxin-free particles in a time-dependent manner in wild-type macrophages. For example, particles with adherent bacterial debris induced 99% ± 2% of maximal IL1β secretion after 12 hours, whereas endotoxin-free particles induced 92% ± 11% (p > 0.5). CONCLUSIONS: This cell culture study showed that adherent PAMPs are required for priming of the NLRP3 inflammasome by wear particles and this process is dependent on their cognate TLRs and TIRAP/Mal. In contrast, activation of the NLRP3 inflammasome by titanium particles is not dependent on adherent PAMPs. Animal and implant retrieval studies are needed to determine whether wear particles have similar effects on the NLRP3 inflammasome in vivo. CLINICAL RELEVANCE: Our findings, together with recent findings that aseptic loosening associates with polymorphisms in the TIRAP/Mal locus, support that adherent PAMPs may contribute to aseptic loosening in patients undergoing arthroplasty.

The primary conclusions of our 2014 contribution to this series were as follows: Multiple receptor tyrosine kinases (RTKs) likely contribute to aggressive phenotypes in osteosarcoma and, therefore, inhibition of multiple RTKs is likely necessary for successful clinical outcomes. Inhibition of multiple RTKs may also be useful to overcome resistance to inhibitors of individual RTKs as well as resistance to conventional chemotherapies. Different combinations of RTKs are likely important in individual patients. AXL, EPHB2, FGFR2, IGF1R, and RET were identified as promising therapeutic targets by our in vitro phosphoproteomic/siRNA screen of 42 RTKs in the highly metastatic LM7 and 143B human osteosarcoma cell lines. This chapter is intended to provide an update on these topics as well as the large number of osteosarcoma clinical studies of inhibitors of multiple tyrosine kinases (multi-TKIs) that were recently published.

Epigenetic deregulation is an emerging hallmark of cancer that enables tumor cells to escape surveillance by tumor suppressors and ultimately progress. The structure of the epigenome consists of covalent modifications of chromatin components, including acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs). Targeting these enzymes with inhibitors to restore epigenetic homeostasis has been explored for many cancers. Osteosarcoma, an aggressive bone malignancy that primarily affects children and young adults, is notable for widespread genetic and epigenetic instability. This may explain why therapy directed at unique molecular pathways has failed to substantially improve outcomes in osteosarcoma over the past four decades. In this review, we discuss the potential of targeting the cancer epigenome, with a focus on histone deacetylase inhibitors (HDACi) for osteosarcoma. We additionally highlight the safety and tolerance of HDACi, combination chemotherapy with HDACi, and the ongoing challenges in the development of these agents.

Jab1, also known as Csn5/Cops5, is a key subunit of the COP9 Signalosome, a highly conserved macromolecular complex. We previously reported that the conditional knockout of Jab1 in mouse limb buds and chondrocytes results in severely shortened limbs and neonatal lethal chondrodysplasia, respectively. In this study, we further investigated the specific role of Jab1 in osteoblast differentiation and postnatal bone growth by characterizing a novel mouse model, the Osx-cre; Jab1(flox/flox) conditional knockout (Jab1 cKO) mouse, in which Jab1 is deleted in osteoblast precursor cells. Jab1 cKO mutant mice appeared normal at birth, but developed progressive dwarfism. Inevitably, all mutant mice died prior to weaning age. The histological and micro-computed tomography analysis of mutant long bones revealed severely altered bone microarchitecture, with a significant reduction in trabecular thickness. Moreover, Jab1 cKO mouse tibiae had a drastic decrease in mineralization near the epiphyseal growth plates, and Jab1 cKO mice also developed spontaneous fractures near the tibiofibular junction. Additionally, our cell culture studies demonstrated that Jab1 deletion in osteoblast precursors led to decreased mineralization and a reduced response to TGFβ and BMP signaling. Moreover, an unbiased reporter screen also identified decreased TGFβ activity in Jab1-knockdown osteoblasts. Thus, Jab1 is necessary for proper osteoblast differentiation and postnatal bone growth, likely in part through its positive regulation of the TGFβ and BMP signaling pathways in osteoblast progenitor cells.

Background: Proinflammatory signaling by toll-like receptors (TLRs) likely contributes to biologic responses to wear particles causing aseptic loosening. We recently reported associations with aseptic loosening in patients with polymorphisms in the locus encoding an adapter protein specific for TLR-2 and TLR-4 known as toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal). To directly examine the contribution of TIRAP/Mal, we tested the hypothesis that TIRAP/Mal deficiency reduces the activity of wear particles. Signaling by TLR-2 and TLR-4 through TIRAP/Mal can be activated by bacterial pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide or endogenous alarmins. To distinguish between those possibilities, we tested the hypothesis that the effects of TIRAP/Mal depend on the adherence of bacterial PAMPs to the particles. Methods: In vitro mRNA levels and secretion of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were measured after incubating wild-type and TIRAP/Mal−/− macrophages in the presence or absence of titanium particles with adherent bacterial debris, so-called endotoxin-free particles, or particles with adherent lipopolysaccharide. In vivo osteolysis was measured after implanting titanium particles on the calvaria of wild-type and TIRAP/Mal−/− mice. Results: TIRAP/Mal deficiency significantly inhibited the activity of titanium particles with adherent bacterial debris to stimulate in vivo osteolysis and in vitro cytokine mRNAs and secretion. Those effects are dependent on adherent PAMPs because removal of >99% of the adherent bacterial debris from the particles significantly reduced their activity and the remaining activity was not dependent on TIRAP/Mal. Moreover, adherence of highly purified lipopolysaccharide to the endotoxin-free particles reconstituted the activity and the dependence on TIRAP/Mal. Conclusions: TIRAP/Mal deficiency reduces inflammatory responses and osteolysis induced by particles with adherent PAMPs. Clinical Relevance: Our results, coupled with the genetic associations between aseptic loosening and polymorphisms within the TIRAP/Mal locus, support TLR signaling through TIRAP/Mal as one of the factors that enhances the activity of wear particles and further support the hypothesis that bacterial PAMPs likely contribute to aseptic loosening in a subset of patients.

The most important factor contributing to short-term and long-term success of cementless total joint arthroplasties is osseointegration. Osseointegration leads to a direct structural and functional connection between living bone and the surface of an implant. Surface contaminants may remain on orthopaedic implants after sterilization procedures and impair osseointegration. For example, specific lots of hip replacement Sulzer Inter-OP(TM) acetabular shells that were associated with impaired osseointegration and early failure rates were found to be contaminated with both bacterial debris and machine oil residues. However, the effect of machine oil on implant integration is unknown. Therefore, the goal of this study was to determine if machine oil inhibits the osseointegration of orthopaedic implants. To test this hypothesis in vivo we used our murine model of osseointegration where titanium alloy implants are implanted into a unicortical pilot hole in the mid-diaphysis of the femur. We found that machine oil inhibited bone-to-implant contact and biomechanical pullout measures. Machine oil on titanium alloy discs inhibited early stages of MC3T3-E1 osteogenesis in vitro such as attachment and spreading. Inhibition of osteoblast attachment and spreading occurred in both areas with and without detectable oil. Osteoblast growth was in turn inhibited on discs with machine oil due to both a decrease in proliferation and an increase in cell death. Later stages of osteogenic differentiation and mineralization on titanium alloy discs were also inhibited. Thus, machine oil can inhibit osseointegration through cell autonomous effects on osteoblast cells. These results support routine testing by manufacturers of machine oil residues on orthopaedic implants.

Chondrocytes in the epiphyseal cartilage undergo terminal differentiation prior to their removal through apoptosis. To examine the role of ERK1 and ERK2 in chondrocyte terminal differentiation, we generated Osterix (Osx)-Cre; ERK1–/–; ERK2flox/flox mice (conditional knockout Osx [cKOosx]), in which ERK1 and ERK2 were deleted in hypertrophic chondrocytes. These cKOosx mice were grossly normal in size at birth, but by 3 weeks of age exhibited shorter long bones. Histological analysis in these mice revealed that the zone of hypertrophic chondrocytes in the growth plate was markedly expanded. In situ hybridization and quantitative real-time PCR analyses demonstrated that Matrix metalloproteinase-13 (Mmp13) and Osteopontin expression was significantly decreased, indicating impaired chondrocyte terminal differentiation. Moreover, Egr1 and Egr2, transcription factors whose expression is restricted to the last layers of hypertrophic chondrocytes in wild-type mice, were also strongly downregulated in these cKOosx mice. In transient transfection experiments in the RCS rat chondrosarcoma cell line, the expression of Egr1, Egr2, or a constitutively active mutant of MEK1 increased the activity of an Osteopontin promoter, whereas the MEK1-induced activation of the Osteopontin promoter was inhibited by the coexpression of Nab2, an Egr1 and Egr2 co-repressor. These results suggest that MEK1-ERK signaling activates the Osteopontin promoter in part through Egr1 and Egr2. Finally, our histological analysis of cKOosx mice demonstrated enchondroma-like lesions in the bone marrow that are reminiscent of human metachondromatosis, a skeletal disorder caused by mutations in PTPN11. Our observations suggest that the development of enchondromas in metachondromatosis may be caused by reduced extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK) signaling. © 2014 American Society for Bone and Mineral Research.

BACKGROUND Approximately 80% of patients with osteosarcoma harbor subclinical pulmonary micrometastases at diagnosis. Conventional chemotherapy includes methotrexate, doxorubicin, and cisplatin (MAP); however, this regimen and thus overall survival (60%-70%) have remained largely unchanged for 30 years. It therefore is necessary to identify novel therapeutics targeting the metastatic progression of osteosarcoma. QUESTIONS/PURPOSES This laboratory study explored application of osteosarcoma spheroids (sarcospheres) for drug screening with the following purposes: (1) to characterize sarcosphere size; (2) to establish accurate measurement of sarcosphere growth; (3) to confirm sarcosphere uniformity; and (4) to apply the platform to evaluate MAP chemotherapy. METHODS Sarcospheres were first characterized to establish accurate measurement of sarcosphere growth and uniform production. The refined platform then was applied to evaluate MAP chemotherapy to validate its use in drug screening. Sarcospheres were generated from highly metastatic human cell lines (143B, MG-63.3, and LM7) by centrifugation to form three-dimensional aggregates modeling micrometastases. Sarcospheres were matured for 24 hours and then incubated with or without drug from Days 0 to 2. Size was assessed by diameter and volume using brightfield microscopy. Growth was measured by volume and resazurin reduction in viable cells. Sarcosphere uniformity was assessed by diameter and resazurin reduction at Day 0 and the Z' factor, a measure of assay suitability for high-throughput screening, was calculated at Day 2. Sarcospheres were treated with individual MAP agents (0 to 1000 μmol/L) to determine concentrations at which 50% of growth from Days 0 to 2 was inhibited (GIC50). Cell lines resistant to MAP in sarcospheres were treated in monolayer for comparison. RESULTS Sarcosphere diameter and growth from Days 0 to 2 were quantitatively dependent on the number of cells seeded and the cell line used. Accurate measurement of growth occurred after resazurin incubation for 6 hours, without EDTA-mediated permeabilization, and was correlated with the number of cells seeded and sarcosphere volume for 143B (Spearman's r: 0.98; p < 0.001), MG-63.3 (0.99; p < 0.001), and LM7 (0.98; p < 0.001). Sarcospheres met established criteria for screening applications as mean Z' factors were greater than 0.5 for all cell lines. Response to MAP therapy was cell line-dependent, because MG-63.3 and LM7 sarcospheres exhibited greater than 2000-fold resistance to methotrexate (GIC50 = 88 ± 36 μmol/L and 174 ± 16 μmol/L, respectively) compared with the 143B cell line (GIC50 = 0.04 ± 0.01 μmol/L; p < 0.001 for MG-63.3 and LM7). MG-63.3 monolayers were more sensitive to methotrexate (GIC50 = 0.01 ± 0.01 μmol/L; p < 0.001) than MG-63.3 sarcospheres, whereas LM7 monolayers remained chemoresistent (GIC50 not reached). CONCLUSIONS This study developed and validated a drug screening platform for progression of osteosarcoma micrometastases. It also highlights heterogeneity among osteosarcoma cell lines. These findings appear to reflect known patient-to-patient heterogeneity and underscore the importance of evaluating multiple tumor models when testing drugs for the treatment of osteosarcoma. CLINICAL RELEVANCE The described approach is a promising starting point for drug screening in osteosarcoma because it is tailored to evaluate micrometastatic disease. A reliable and rapid method to identify novel therapeutics is critical to improve stagnant outcomes for patients with osteosarcoma.

To date, no study has investigated the antiresorptive property of lunasin. Hence, the present study aimed to assess the ability of lunasin to inhibit the osteoclast formation using RAW 264.7 cells. We hypothesized that lunasin is able to inhibit osteoclast formation.In the present study, the murine monocytic cell line RAW 264.7 was induced to differentiate into mature osteoclasts in the presence of recombinant receptor activator of nuclear factor kappa-B ligand. Tartrate-resistant acid phosphatase, a marker of osteoclasts, was used to identify osteoclasts. Cell lines were divided into different groups and exposed to different concentrations of 50 μmol/L, 75 μmol/L, and 100 μmol/L active and inactive lunasin. The control group was RAW 264.7 cells with receptor activator of nuclear factor kappa-B ligand. Tartrate-resistant acid phosphatase-positive cells of 3 or more nuclei, indicative of mature osteoclasts, were counted by 3 observers. The mean number of the data collected was analyzed using 1-way analysis of variance and the multiple comparison post hoc Bonferroni correction.There was a significant difference in the reduction of osteoclast formation in all the active lunasin groups (P .001) compared with the control group and the inactive lunasin group (P .001).Considering the suppressive effect of lunasin on osteoclastogenesis, the use of lunasin as a potential antiresorptive agent can be evaluated in future studies.

Wear particle-induced inflammatory bone loss (osteolysis) is the leading cause of total hip arthroplasty (THA) failure. Individual susceptibility to osteolysis is modulated by genetic variation. In this 2-stage case-control association study we examined whether variation within candidate genes in inflammatory and bone turnover signaling pathways associates with susceptibility to osteolysis and time to prosthesis failure. We examined two cohorts, comprising 758 (347 male) Caucasian subjects who had undergone THA with a metal on polyethylene bearing couple; 315 of whom had developed osteolysis. Key genes within inflammatory, bone resorption, and bone formation pathways were screened for common variants by pairwise-SNP tagging using a 2-stage association analysis approach. In the discovery cohort four SNPs within RANK, and one each within KREMEN2, OPG, SFRP1, and TIRAP (p < 0.05) were associated with osteolysis susceptibility. Two SNPs within LRP6, and one each within LRP5, NOD2, SOST, SQSTM1, TIRAP, and TRAM associated with time to implant failure (p < 0.05). Meta-analysis of the two cohorts identified four SNPs within RANK, and one each within KREMEN2, OPG, SFRP1, and TIRAP associated with osteolysis susceptibility (p < 0.05). Genetic variation within inflammatory signaling and bone turnover pathways may play a role in susceptibility to osteolysis.

Overwhelming evidence supports the concept that wear particles are the primary initiator of aseptic loosening of orthopaedic implants. It is likely, however, that other factors modulate the biologic response to wear particles. This review focuses on three potential other factors: genetic susceptibility, Toll-like receptors (TLRs), and bacterial pathogen-associated molecular patterns (PAMPs). WHERE ARE WE NOW?: Considerable evidence is emerging that both genetic susceptibility and TLR activation are important factors that modulate the biologic response to wear particles, but it remains controversial whether bacterial PAMPs also do so. WHERE DO WE NEED TO GO?: Detailed understanding of the roles of these other factors may lead to identification of novel therapeutic targets for patients with aseptic loosening. HOW DO WE GET THERE?: Highest priority should be given to polymorphism replication studies with large numbers of patients and studies to replicate the reported correlation between bacterial biofilms and the severity of aseptic loosening.

The protein kinase inhibitor (Pki) gene family inactivates nuclear protein kinase A (PKA) and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in murine embryonic fibroblasts (MEFs), murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown also simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of leukemia inhibitory factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. Stem Cells 2013;31:2789–2799

Osseointegration is the process by which an orthopaedic implant makes direct bone-to-implant contact and is crucial for the long-term function of the implant. Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopaedic implants after sterilization and impair osseointegration. For example, specific lots of implants that were associated with impaired osseointegration and high failure rates were discovered to have contaminants including bacterial debris. Therefore, the goals of this study were to determine if bacterial debris exists on sterile orthopaedic implants and if adherent bacterial debris inhibits the osseointegration of orthopaedic implants. We found that debris containing lipopolysaccharide (LPS) from Gram-negative bacteria exists on both sterile craniofacial implants and wrist implants. Levels of bacterial debris vary not only between different lots of implants but within an individual lot. Using our murine model of osseointegration, we found that ultrapure LPS adherent to the implants inhibited bone-to-implant contact and biomechanical pullout measures. Analysis of osseointegration in knock-out mice demonstrated that adherent LPS inhibited osseointegration by signaling through its primary receptor, Toll-like receptor 4, and not by signaling through Toll-like receptor 2. Ultrapure LPS adherent to titanium alloy discs had no detectable effect on early stages of MC3T3-E1 osteogenesis in vitro such as attachment, spreading or growth. However, later stages of osteogenic differentiation and mineralization were inhibited by adherent LPS. Thus, LPS may inhibit osseointegration in part through cell autonomous effects on osteoblasts. These results highlight bacterial debris as a type of surface contaminant that can impair the osseointegration of orthopaedic implants.

Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopaedic implants after sterilization procedures and affect osseointegration. The goals of this study were to develop a murine model of osseointegration in order to determine whether removing surface contaminants enhances osseointegration. To develop the murine model, titanium alloy implants were implanted into a unicortical pilot hole in the mid-diaphysis of the femur and osseointegration was measured over a five week time course. Histology, backscatter scanning electron microscopy and x-ray energy dispersive spectroscopy showed areas of bone in intimate physical contact with the implant, confirming osseointegration. Histomorphometric quantification of bone-to-implant contact and peri-implant bone and biomechanical pullout quantification of ultimate force, stiffness and work to failure increased significantly over time, also demonstrating successful osseointegration. We also found that a rigorous cleaning procedure significantly enhances bone-to-implant contact and biomechanical pullout measures by two-fold compared with implants that were autoclaved, as recommended by the manufacturer. The most likely interpretation of these results is that surface contaminants inhibit osseointegration. The results of this study justify the need for the development of better detection and removal techniques for contaminants on orthopaedic implants and other medical devices.

Aseptic loosening of orthopedic implants is initiated by macrophage activation by wear particles derived from the implant.1 The activated macrophages produce pro-inflammatory cytokines that induce osteoclast differentiation and local osteolysis.1-3 This production of pro-inflammatory cytokines is due to activation in the macrophages of a number of intracellular signaling pathways associated with inflammation, including the ERK1/2 and NF-κB pathways.4-10 We have recently shown that the phosphoinositol-3-kinase (PI3K)/Akt pathway is another signal transduction pathway that mediates the in vitro activation of macrophages by orthopedic wear particles.11 In that study, we showed that wear particles activate the PI3K/Akt pathway and that inhibitors of either PI3K or Akt reduce production of TNFα induced by the wear particles.11 The PI3K/Akt pathway has diverse effects on cellular processes, including proliferation, survival, differentiation, and migration and is especially important in both innate and adaptive immunity.12 PI3K consists of heterodimers of catalytic and regulatory subunits. The γ isoform of the catalytic subunit is primarily responsible for PI3K activity during inflammation.13-15 We therefore hypothesized that PI3Kγ mediates particle-induced osteolysis. This hypothesis was of interest since pharmacological agents that selectively inhibit PI3Kγ are being developed for inflammatory conditions such as rheumatoid arthritis15-17 and lupus.16 Moreover, PI3Kγ−/− mice are substantially protected from both rheumatoid arthritis and lupus.17,18 Moreover, osteoclast differentiation and survival are reduced in PI3Kγ−/− mice leading to an increase in bone mass.19 The primary goal of this project was therefore to determine whether PI3Kγ−/− mice are also protected from particle-induced osteolysis. We also determined whether repair of particle-induced osteolysis is altered in the PI3Kγ−/− mice.

Adherent pathogen-associated molecular patterns (PAMPs) act through toll-like receptor 2 (TLR2) and TLR4 to increase the biological activity of orthopedic wear particles in cell culture and animal models of implant loosening. This study tested whether this is dependent on TLR association with lipid rafts as reported for the response to soluble TLR ligands. For this purpose, RAW264.7 murine macrophages were activated by exposure to titanium particles with adherent PAMPs, soluble lipopolysaccharide (LPS), soluble lipotecichoic acid (LTA), or heat-killed bacteria that had been extensively washed to remove soluble PAMPs. Lipid rafts were isolated by two independent methods and the location of TLR4 and TLR2 was analyzed by Western blotting. The cognate TLRs associated with lipid rafts when the macrophages were activated with soluble LPS and LTA but not after stimulation with either titanium particles with adherent PAMPs or heat-killed bacteria. The lipid raft disruptor, methyl-β-cyclodextrin, dose-dependently inhibited TNF-α release in response to LPS but had no affect on TNF-α release in response to titanium particles with adherent PAMPs. We conclude, therefore, that titanium particles with adherent PAMPs and heat-killed bacteria activate TLR2 and TLR4 in macrophages without inducing either TLR to associate with lipid rafts. These results have important implications for the mechanisms of orthopedic implant loosening as well the mechanisms for TLR activation in other inflammatory situations.

Background: Bacterial biofilms play a major role in chronic orthopaedic infections. Recently, farnesol (an antifungal agent) has been shown to express antimicrobial activities against Staphylococcus aureus and Streptococcus mutans . However, the effects of farnesol on the formation of bacterial biofilms on orthopaedic biomaterials and its effects on osteoblasts have not been investigated, to our knowledge, and are therefore the focus of this study. Methods: Biofilms of Staphylococcus aureus (Seattle 1945GFPuvr) were grown on titanium alloy discs. The effects of soluble farnesol on biofilm formation with or without gentamicin were examined with fluorescence microscopy and in quantitative cultures. The effect of farnesol coated on titanium alloy discs was also investigated, as was the effect of the agent on MC3T3-E1 pre-osteoblastic cells cultured on titanium alloy discs. Results: Soluble farnesol at a 30-mM concentration reduced the number of viable bacteria 104-fold and completely inhibited biofilm formation. Low concentrations of soluble farnesol (0.03 to 3 mM) did not inhibit biofilm formation and did not potentiate the effect of a submaximal concentration of gentamicin. Dried farnesol on titanium alloy discs reduced the number of viable bacteria fiftyfold. The effect of farnesol on bacterial biofilm formation lasted for at least three days. Soluble farnesol added after the biofilm had already formed also reduced the final number of viable bacteria, by fifty-six-fold. Soluble farnesol (3-mM and 30-mM concentrations) inhibited spreading of the MC3T3-E1 cells. Conclusions: In vitro, a high concentration of farnesol (30 mM) shows antimicrobial properties against bacterial biofilms; however, it also has a negative effect on pre-osteoblasts. Farnesol can also express antimicrobial activity when predried on titanium discs and when added to preformed biofilms. Clinical Relevance: As musculoskeletal infections remain a complicated problem, development of novel interventions to enhance prevention or treatment is necessary. Our study provides a basic knowledge of the effectiveness of farnesol against Staphylococcus aureus biofilms on titanium alloy surfaces. Although farnesol may have a clinical role, additional investigations, both in vitro and in vivo, are needed.

Bone loss that causes aseptic loosening of orthopedic implants is initiated by pro-inflammatory cytokines produced by macrophages in response to implant-derived wear particles. MAPK and NF-kappaB signaling pathways are activated by the particles; however, it is not clear which of the signaling pathways are important for the initial response to the wear particles and which are only involved at later steps in the process, such as osteoclast differentiation. Here, we show that the ERK1/2, p38, JNK, and NF-kappaB pathways are rapidly activated by the wear particles but that only the ERK1/2 and NF-kappaB pathways are required for the initial response to the wear particles, which include increases in TNFalpha promoter activity, TNFalpha mRNA expression, and secretion of TNFalpha protein. Moreover, ERK1/2 activation by wear particles is also required for increased expression of the transcription factor Egr-1 as well as Egr-1's ability to bind to and activate the TNFalpha promoter. These results, together with our previous studies of the PI3K/Akt pathway, demonstrate that wear particles coordinately activate multiple signaling pathways and multiple transcription factors to stimulate production of pro-inflammatory cytokines, such as TNFalpha. The current study also demonstrates that the signaling pathways are activated to a much greater extent by wear particles with adherent endotoxin than by "endotoxin-free" wear particles. These results, together with those demonstrating the requirement for ERK1/2/Egr-1 and NF-kappaB, show that activation of these signaling pathways is responsible for the ability of adherent endotoxin to potentiate cytokine production, osteoclast differentiation, and bone loss induced by wear particles.

Understanding the complex cellular and tissue mechanisms and interactions resulting in periprosthetic osteolysis requires a number of experimental approaches, each of which has its own set of advantages and limitations. In vitro models allow for the isolation of individual cell populations and have furthered our understanding of particle-cell interactions; however, they are limited because they do not mimic the complex tissue environment in which multiple cell interactions occur. In vivo animal models investigate the tissue interactions associated with periprosthetic osteolysis, but the choice of species and whether the implant system is subjected to mechanical load or to unloaded conditions are critical in assessing whether these models can be extrapolated to the clinical condition. Rigid analysis of retrieved tissue from clinical cases of osteolysis offers a different approach to studying the biologic process of osteolysis, but it is limited in that the tissue analyzed represents the end-stage of this process and, thus, may not reflect this process adequately.

Wear debris contributes to implant loosening after total joint arthroplasty, and few advances have been made in our ability to inhibit the biological response to wear particles. Bacterial endotoxins augment the effects of wear particles in vitro and in vivo. The cytokine, tumor necrosis factor-alpha (TNF-alpha), is produced by macrophages in response to bacterial endotoxins and wear particles, and it increases osteoclast activity resulting in bone resorption and implant loosening. The phosphoinositol-3-kinase (PI3K)-Akt intracellular signal transduction pathway contributes to cytokine production in response to soluble endotoxin. We investigated the role of the PI3K-Akt pathway in the production of TNF-alpha in response to wear particles with adherent endotoxin and so-called endotoxin-free wear particles.Cultured RAW264.7 murine macrophages were incubated with titanium particles with adherent endotoxin or with endotoxin-free titanium particles in the presence and absence of specific inhibitors of PI3K (LY294002) or Akt (SH-5). Akt activation was assessed with use of Western blot. TNF-alpha production was measured with use of enzyme-linked immunosorbent assay. Cytotoxicity was determined by measuring lactic dehydrogenase release.Titanium particles with adherent endotoxin increased Akt activation, whereas endotoxin-free titanium particles did not. The PI3K inhibitor reduced TNF-alpha production by 70% in response to titanium with adherent endotoxin without increasing cytotoxicity. Similarly, the Akt inhibitor reduced TNF-alpha production by 83% in response to titanium particles with adherent endotoxin without increasing cytotoxicity. High concentrations of endotoxin-free titanium particles resulted in a small delayed increase in TNF-alpha production that was completely blocked by the PI3K inhibitor.Inhibition of the PI3K-Akt pathway reduces macrophage TNF-alpha production in response to titanium particles with adherent endotoxin and endotoxin-free particles in vitro.

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.

Innate defense regulator peptide-1018 (IDR-1018) is a 12-amino acid, synthetic, immunomodulatory host defense peptide that can reduce soft tissue infections and is less likely to induce bacterial resistance than conventional antibiotics. However, IDRs have not been tested on orthopaedic infections and the immunomodulatory effects of IDR-1018 have only been characterized in response to lipopolysacharide, which is exclusively produced by Gram-negative bacteria.We sought (1) to more fully characterize the immunomodulatory effects of IDR-1018, especially in response to Staphylococcus aureus; and (2) to determine whether IDR-1018 decreases S aureus infection of orthopaedic implants in mice and thereby protects the implants from failure to osseointegrate.In vitro effects of IDR-1018 on S aureus were assessed by determining minimum inhibitory concentrations in bacterial broth without and with supplementation of physiologic ion levels. In vitro effects of IDR-1018 on macrophages were determined by measuring production of monocyte chemoattractant protein-1 (MCP-1) and proinflammatory cytokines by enzyme-linked immunosorbent assay. In vivo effects of IDR-1018 were determined in a murine model of S aureus implant infection by quantitating bacterial burden, macrophage recruitment, MCP-1, proinflammatory cytokines, and osseointegration in nine mice per group on Day 1 postimplantation and 20 mice per group on Day 15 postimplantation.IDR-1018 demonstrated antimicrobial activity by directly killing S aureus even in the presence of physiologic ion levels, increasing recruitment of macrophages to the site of infections by 40% (p = 0.036) and accelerating S aureus clearance in vivo (p = 0.008) with a 2.6-fold decrease in bacterial bioburden on Day 7 postimplantation. In vitro immunomodulatory activity of IDR-1018 included inducing production of MCP-1 in the absence of other inflammatory stimuli and to potently blunt excess production of proinflammatory cytokines and MCP-1 induced by lipopolysaccharide. Higher concentrations of IDR-1018 were required to blunt production of proinflammatory cytokines and MCP-1 in the presence S aureus. The largest in vivo immunomodulatory effect of IDR-1018 was to reduce tumor necrosis factor-α levels induced by S aureus by 60% (p = 0.006). Most importantly, IDR-1018 reduced S aureus-induced failures of osseointegration by threefold (p = 0.022) and increased osseointegration as measured by ultimate force (5.4-fold, p = 0.033) and average stiffness (4.3-fold, p = 0.049).IDR-1018 is potentially useful to reduce orthopaedic infections by directly killing bacteria and by recruiting macrophages to the infection site.These findings make IDR-1018 an attractive candidate to explore in larger animal models to ascertain whether its effects in our in vitro and mouse experiments can be replicated in more clinically relevant settings.

Expression of many genes induced by cAMP-dependent protein kinase (PKA) signaling is rapidly terminated. Although many mechanisms contribute to regulation of PKA signaling, members of the endogenous protein kinase inhibitor (PKI) family may be particularly important for terminating nuclear PKA activity and gene expression. Here we used both siRNA and antisense knockdown strategies to examine PKA signaling activated by parathyroid hormone or the β-adrenergic agonist, isoproterenol. We found that endogenous PKIγ is required for efficient termination of nuclear PKA activity, transcription factor phosphorylation, and immediate-early genes. Moreover, PKIγ is required for export of PKA catalytic subunits from the nucleus back to the cytoplasm following activation of PKA signaling because this is also inhibited by PKIγ knockdown. Leptomycin B blocks PKA nuclear export but has little or no effect on nuclear PKA activity or immediate-early gene expression. Thus, inactivation of PKA activity in the nucleus is sufficient to terminate signaling, and nuclear export is not required. These results were the first in any cell type showing that endogenous levels of PKI regulate PKA signaling.

cAMP/PKA signaling transiently stimulates mRNA expression of immediate-early genes, including IL-6 and c- fos. We confirmed that these mRNAs are transiently stimulated by parathyroid hormone (PTH) in ROS 17/2.8 osteoblastic cells. Consistent with the role for cAMP/PKA signaling in this response, PTH induces transient cAMP elevation, PKA activation, and cAMP-responsive element-binding protein (CREB) phosphorylation. Our goal was to determine whether termination of immediate-early gene expression is due to receptor desensitization or cAMP degradation. The approaches used were 1) inhibition of PTH receptor desensitization with G protein-coupled receptor kinase 2 (GRK2) antisense oligonucleotides or antisense plasmids, 2) sustained activation of adenyl cyclase with forskolin, and 3) inhibition of cAMP degradation with 3-isobutyl-1-methylxanthine. These experiments show that mechanisms downstream of receptor desensitization and cAMP degradation are primarily responsible for termination of PKA activity, CREB phosphorylation, and immediate-early gene expression. Similar conclusions were also obtained in response to PTH in a second osteoblastic cell line (MC3T3-E1) and in response to isoproterenol in NIH3T3 fibroblasts. This conclusion may therefore reflect a general mechanism for termination of immediate-early gene expression after induction by cAMP/PKA.

The major cause of orthopaedic implant loosening is thought to be accelerated osteoclastic bone resorption due to the action of cytokines produced in response to phagocytosis of implant-derived wear particles. This accelerated osteoclastic bone resorption could be due to increases in any of the following processes: recruitment of osteoclast precursors to the local microenvironment, differentiation of precursors into mature multinucleated osteoclasts. activation of mature osteoclasts, and/or survival of osteoclasts. Our studies have focused on differentiation and survival to complement work by others who have focused on recruitment of precursors and activation. Taken together, our studies and those of other investigators provide strong evidence that increased recruitment of osteoclast precursors and their subsequent differentiation play major roles in wear particle-induced osteolysis. In contrast, increased osteoclast activation and survival appear to play minor roles. These studies suggest that development of therapeutic interventions that reduce either recruitment or differentiation of osteoclast precursors would improve the performance of orthopaedic implants.

Wear particle-induced inflammatory bone loss (osteolysis) is the leading cause of total hip arthroplasty (THA) failure. Individual susceptibility to osteolysis is modulated by genetic variation. In this 2-stage case-control association study we examined whether variation within candidate genes in inflammatory and bone turnover signaling pathways associates with susceptibility to osteolysis and time to prosthesis failure. We examined two cohorts, comprising 758 (347 male) Caucasian subjects who had undergone THA with a metal on polyethylene bearing couple; 315 of whom had developed osteolysis. Key genes within inflammatory, bone resorption, and bone formation pathways were screened for common variants by pairwise-SNP tagging using a 2-stage association analysis approach. In the discovery cohort four SNPs within RANK, and one each within KREMEN2, OPG, SFRP1, and TIRAP (p0.05) were associated with osteolysis susceptibility. Two SNPs within LRP6, and one each within LRP5, NOD2, SOST, SQSTM1, TIRAP, and TRAM associated with time to implant failure (p0.05). Meta-analysis of the two cohorts identified four SNPs within RANK, and one each within KREMEN2, OPG, SFRP1, and TIRAP associated with osteolysis susceptibility (p0.05). Genetic variation within inflammatory signaling and bone turnover pathways may play a role in susceptibility to osteolysis.

The most important factor contributing to short-term and long-term success of cementless total joint arthroplasties is osseointegration. Osseointegration leads to a direct structural and functional connection between living bone and the surface of an implant. Surface contaminants may remain on orthopaedic implants after sterilization procedures and impair osseointegration. For example, specific lots of hip replacement Sulzer Inter-OP(TM) acetabular shells that were associated with impaired osseointegration and early failure rates were found to be contaminated with both bacterial debris and machine oil residues. However, the effect of machine oil on implant integration is unknown. Therefore, the goal of this study was to determine if machine oil inhibits the osseointegration of orthopaedic implants. To test this hypothesis in vivo we used our murine model of osseointegration where titanium alloy implants are implanted into a unicortical pilot hole in the mid-diaphysis of the femur. We found that machine oil inhibited bone-to-implant contact and biomechanical pullout measures. Machine oil on titanium alloy discs inhibited early stages of MC3T3-E1 osteogenesis in vitro such as attachment and spreading. Inhibition of osteoblast attachment and spreading occurred in both areas with and without detectable oil. Osteoblast growth was in turn inhibited on discs with machine oil due to both a decrease in proliferation and an increase in cell death. Later stages of osteogenic differentiation and mineralization on titanium alloy discs were also inhibited. Thus, machine oil can inhibit osseointegration through cell autonomous effects on osteoblast cells. These results support routine testing by manufacturers of machine oil residues on orthopaedic implants.

Despite aggressive surgical and chemotherapy protocols, survival rates for osteosarcoma patients have not improved over the last 30 years. Therefore, novel therapeutic agents are needed. Receptor tyrosine kinases have emerged as targets for the development of new cancer therapies since their activation leads to enhanced proliferation, survival, and metastasis. In fact, aberrant expression and activation of RTKs have been associated with the progression of many cancers. Studies from our lab using phosphoproteomic screening identified RTKs that are activated and thus may contribute to the signaling within metastatic human osteosarcoma cells. Functional genomic screening using siRNA was performed to distinguish which of the activated RTKs contribute to in vitro phenotypes associated with metastatic potential (motility, invasion, colony formation, and cell growth). The resulting RTK hits were then validated using independent validation experiments. From these results, we identified four RTKs (Axl, EphB2, FGFR2, and Ret) that have not been previously studied in osteosarcoma and provide targets for the development of novel therapeutics.

Aseptic loosening of orthopedic implants is thought to be caused primarily by osteoclast differentiation induced by bone resorptive cytokines produced in response to phagocytosis of implant-derived wear particles. This study examined whether adherent endotoxin on the wear particles is responsible for inducing osteoclast differentiation as well as production of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor a (TNF-alpha). Removal of adherent endotoxin almost completely inhibited the responses to titanium (Ti) particles by both murine marrow cells and human peripheral blood monocytes. In vivo experiments showed that endotoxin removal reduced particle-induced osteolysis by 50-70%. Addition of lipopolysaccharide (LPS) to the "endotoxin-free" particles restored their ability to induce cytokine production and osteoclast differentiation in vitro. Moreover, marrow cells from mice that are hyporesponsive to endotoxin because of mutation of Toll-like receptor 4 induced significantly less cytokine production and osteoclast differentiation in response to Ti particles with adherent endotoxin than did marrow cells from normoresponsive mice. This mutation also resulted in significantly less particle-induced osteolysis in vivo. Taken together, these results show that adherent endotoxin is involved in many of the biological responses induced by orthopedic wear particles and should stimulate development of new approaches designed to reduce the activity of adherent endotoxin in patients with orthopedic implants.

Background: Loosening of orthopaedic implants is mediated by cytokines that elicit bone resorption and are produced in response to phagocytosis of implant-derived wear particles. This accelerated bone resorption could be due to increased osteoclastic activity, survival, or differentiation. Although a number of in vitro studies have shown that wear particles increase osteoclastic activity, the increase was less than twofold in all cases. The objective of the current study was to test the hypothesis that wear particles stimulate bone resorption by inducing osteoclast differentiation. Methods: Conditioned media were prepared from murine marrow cells or human peripheral blood monocytes incubated in the presence or absence of titanium particles. The effects of conditioned media on osteoclast differentiation were examined with use of a recently developed assay in which osteoclast precursors are co-cultured with mesenchymal support cells. Results: The present study showed that titanium particles induced both murine marrow cells and human peripheral blood monocytes to produce factors that stimulated osteoclast differentiation. The mean increase in osteoclast differentiation was 29.3 ± 9.4-fold. The stimulation of osteoclast differentiation led to a parallel increase in bone resorption. The amount of stimulation was regulated in a dose-dependent manner by the concentration of both titanium particles and conditioned media. The stimulation of osteoclast differentiation required interactions between the cells and the particles themselves and, therefore, was not due to metal ions, soluble contaminants released from the particles, or submicrometer particles. In contrast, conditioned media from control cells incubated in the absence of titanium particles had no detectable effect on any of the examined parameters. Conclusions: The present study showed that titanium particles stimulate in vitro bone resorption primarily by inducing osteoclast differentiation. In contrast, the titanium particles had only small effects on osteoclast activity or survival. Clinical Relevance: The present study provides strong support for the hypothesis that osteoclast differentiation is an important factor in the development of aseptic loosening. The development of therapeutic interventions to reduce osteoclast differentiation may be a useful approach for improving the performance of orthopaedic implants.

Cytokines that stimulate bone resorption are produced by cells found in bone marrow. However, marrow cells produce multiple factors, some of which may be inhibitors of osteoclast differentiation or activity. Thus, it is not possible to predict a priori whether the mixture of factors produced by marrow cells will have a net stimulatory or inhibitory effect on bone resorption. In this study, we showed that the net effect of whole marrow is to inhibit osteoclast activity induced by parathyroid hormone. Fractionation of the marrow revealed that the inhibitory activity was in the marrow fluid. However, conditioned media obtained from marrow cell cultures also inhibited osteoclast activity. Thus, it is likely that the inhibitory factors are produced in vivo by cells residing in the marrow. These inhibitory factors may represent a physiological regulatory process that plays an important role in maintaining the balance between bone resorption and formation. Because we have previously shown that interleukin-6 is one of the cytokines that parathyroid hormone induces in osteoblastic cells to stimulate osteoclast activity, one potential mechanism by which the marrow-derived inhibitory factors might act is by preventing this production of interleukin-6. However, we found that the marrow cell-conditioned media do not inhibit the production or activity of interleukin-6. Thus, the inhibitory factors appear to block osteoclast activity through a mechanism that does not involve interleukin-6. Taken together, these results demonstrate the importance of factors that inhibit bone resorption and emphasize that the presence of cytokines that stimulate bone resorption in conditions such as osteoporosis and orthopaedic implant loosening should be interpreted with caution unless evidence exists demonstrating their functional importance.

Osteoclast differentiation assays are usually conducted in alpha minimal essential medium (alpha-MEM). We reasoned that determining which components of this media are critical for osteoclast differentiation might provide insight into the mechanisms that regulate osteoclast differentiation. This study demonstrates that ascorbic acid is the crucial component of alpha-MEM that stimulates differentiation of murine osteoclasts in cocultures with murine mesenchymal support cells. Thus, supplementation with ascorbic acid allows osteoclast differentiation to occur in basal MEM media as well as in RPMI-1640 and basal media Eagle (BME) media. The conclusion that osteoclast differentiation is stimulated by ascorbic acid was obtained whether osteoclast differentiation was induced by 1,25-dihydroxyvitamin D3 or parathyroid hormone, whether ST2 or CIMC-2 cells were used as mesenchymal support cells, and whether osteoclast precursors were obtained from spleen or bone marrow. Time course studies revealed that although ascorbic acid only modestly increases the rate at which osteoclast precursors begin to express tartrate-resistant acid phosphatase, it strongly increases the rate at which precursors fuse into mature, multinucleated cells. Moreover, ascorbic acid strongly increases the life span of both osteoclasts and their precursors. The increases in precursor formation, fusion, and life span induced by ascorbic acid are together responsible for the stimulation of osteoclast differentiation by ascorbic acid. Given the known effects of ascorbic acid on differentiation of mesenchymal cells, it may stimulate osteoclast differentiation indirectly by regulating the differentiation state of the mesenchymal cells that support osteoclast differentiation.

Despite aggressive surgical and chemotherapy protocols, survival rates for osteosarcoma patients have not improved over the last 30 years. Therefore, novel therapeutic agents are needed. Receptor tyrosine kinases have emerged as targets for the development of new cancer therapies since their activation leads to enhanced proliferation, survival, and metastasis. In fact, aberrant expression and activation of RTKs have been associated with the progression of many cancers. Studies from our lab using phosphoproteomic screening identified RTKs that are activated and thus may contribute to the signaling within metastatic human osteosarcoma cells. Functional genomic screening using siRNA was performed to distinguish which of the activated RTKs contribute to in vitro phenotypes associated with metastatic potential (motility, invasion, colony formation, and cell growth). The resulting RTK hits were then validated using independent validation experiments. From these results, we identified four RTKs (Axl, EphB2, FGFR2, and Ret) that have not been previously studied in osteosarcoma and provide targets for the development of novel therapeutics.

Bones consist of a number of cell types including osteoblasts and their precursor cells at various stages of differentiation. To analyze cellular organization within the bone, we generated Col1a1CreER-DsRed transgenic mice that express, in osteoblasts, CreER and DsRed under the control of a mouse 3.2kb Col1a1 promoter. We further crossed Col1a1CreER-DsRed mice with Prx1CreER-GFP mice that express CreER and GFP in osteochondro progenitor cells under the control of a 2.4kb Prx1 promoter. Since the 3.2kb Col1a1 promoter becomes active in osteoblasts at early stages of differentiation, and Prx1CreER-GFP-expressing periosteal cells show endogenous Col1a1 expression, we expected to find a cell population in which both the 2.4kb Prx1 promoter and the 3.2kb Col1a1 promoter are active. However, our histological and flow cytometric analyses demonstrated that these transgenes are expressed in distinct cell populations. In the periosteum of long bones, Col1a1CreER-DsRed is expressed in the innermost layer directly lining the bone surface, while Prx1CreER-GFP-expressing cells are localized immediately outside of the Col1a1CreER-DsRed-expressing osteoblasts. In the calvaria, Prx1CreER-GFP-expressing cells are also localized in the cranial suture mesenchyme. Our experiments further showed that Col1a1CreER-DsRed-expressing cells lack chondrogenic potential, while the Prx1CreER-GFP-expressing cells show both chondrogenic and osteogenic potential. Our results indicate that Col1a1CreER-DsRed-expressing cells are committed osteoblasts, while Prx1CreER-GFP-expressing cells are osteochondro progenitor cells. The Prx1CreER-GFP and Col1a1CreER-DsRed transgenes will offer novel approaches for analyzing lineage commitment and early stages of osteoblast differentiation under physiologic and pathologic conditions.

β-nitrostyrene compounds, such as 3,4-methylenedioxy-β-nitrostyrene (MNS), inhibit growth and induce apoptosis in tumor cells, but no reports have investigated their role in osteosarcoma. In this study, human osteosarcoma cell families with cell lines of varying tumorigenic and metastatic potential were utilized. Scrape motility assays, colony formation assays, and colony survival assays were performed with osteosarcoma cell lines, both in the presence and absence of MNS. Effects of MNS on human osteoblasts and airway epithelial cells were assessed in monolayer cultures. MNS decreased metastatic cell line motility by 72–76% and colony formation by 95–100%. MNS consistently disrupted preformed colonies in a time-dependent and dose-dependent manner. MNS had similar effects on human osteoblasts but little effect on airway epithelial cells. An inactive analog of MNS had no detectable effects, demonstrating specificity. MNS decreases motility and colony formation of osteosarcoma cells and disrupts preformed cell colonies, while producing little effect on pulmonary epithelial cells.

Orthopedic wear particles activate a number of intracellular signaling pathways associated with inflammation in macrophages and we have previously shown that the phosphoinositol-3-kinase (PI3K)/Akt pathway is one of the signal transduction pathways that mediates the in vitro activation of macrophages by orthopedic wear particles. Since PI3Kγ is primarily responsible for PI3K activity during inflammation, we hypothesized that PI3Kγ mediates particle-induced osteolysis in vivo. Our results do not strongly support the hypothesis that PI3Kγ regulates the overall amount of particle-induced osteolysis in the murine calvarial model. However, our results strongly support the conclusion that variability in the amount of particle-induced osteolysis between individual mice is reduced in the PI3Kγ(-/-) mice. These results suggest that PI3Kγ contributes to osteolysis to different degrees in individual mice and that the mice, and patients, that are most susceptible to osteolysis may be so, in part, due to an increased contribution from PI3Kγ.

Aseptic loosening of orthopaedic implants is induced by wear particles generated from the polymeric and metallic components of the implants. Substantial evidence suggests that activation of Toll-like receptors (TLRs) may contribute to the biological activity of the wear particles. Although pathogen-associated molecular patterns (PAMPs) produced by Gram-positive bacteria are likely to be more common in patients with aseptic loosening, prior studies have focused on LPS, a TLR4-specific PAMP produced by Gram-negative bacteria. Here we show that both TLR2 and TLR4 contribute to the biological activity of titanium particles with adherent bacterial debris. In addition, lipoteichoic acid, a PAMP produced by Gram-positive bacteria that activates TLR2, can, like LPS, adhere to the particles and increase their biological activity, and the increased biological activity requires the presence of the cognate TLR. Moreover, three lines of evidence support the conclusion that TLR activation requires bacterially derived PAMPs and that endogenously produced alarmins are not sufficient. First, neither TLR2 nor TLR4 contribute to the activity of “endotoxin-free” particles as would be expected if alarmins are sufficient to activate the TLRs. Second, noncognate TLRs do not contribute to the activity of particles with adherent LPS or lipoteichoic acid as would be expected if alarmins are sufficient to activate the TLRs. Third, polymyxin B, which inactivates LPS, blocks the activity of particles with adherent LPS. These results support the hypothesis that PAMPs produced by low levels of bacterial colonization may contribute to aseptic loosening of orthopaedic implants.

Intermittent parathyroid hormone (iPTH) is the only FDA-approved therapy for bone loss due to conditions such as osteoporosis that increases bone formation by osteoblasts; all other therapies approved for osteoporosis block bone resorption by osteoclasts. The anabolic effects of iPTH are likely due to a combination of multiple mechanisms, including induction of immediate-early genes, increased expression and/or activity of essential osteoblast transcription factors, and downregulation of anti-osteogenic proteins, such as sclerostin. In contrast, continuous administration of PTH induces bone loss primarily due to up-regulation of RANKL expression and inhibition of osteoprotegerin expression.

Susceptibility to osteolysis after total hip arthroplasty (THA) varies between individuals. We examined whether patients susceptible to osteolysis (group I, n = 34 subjects) after cemented Charnley THA have quantitatively different innate immune responses to pro-inflammatory stimuli versus patients without this susceptibility (group II, n = 28 subjects) at a mean of 14 years after primary surgery. Extracted peripheral blood mononuclear cells were stimulated for 3 h using endotoxin (lipopolysaccharide-LPS, 100 ng/mL), endotoxin-stripped titanium particles (Ti) or endotoxin-stripped particles with adherent LPS added-back (TI + LPS). Subjects returned 1 week later and the experimental protocol was repeated. Assays for mRNA induction for interleukin (IL)-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, IL-18, and tumor necrosis factor (TNF) were made using quantitative real-time PCR. Although baseline levels of mRNA expression were slightly lower in group I, inducibility of mRNA expression was markedly greater in group I versus group II for all cytokines in response to LPS or Ti + LPS, and for IL-1alpha in response to Ti (P0.05). LPS or Ti + LPS stimulation also resulted in an increase in the IL-1/IL-1Ra mRNA ratio in group I versus group II (P0.05). mRNA induction was highly reproducible between subject visits (r0.7, P0.001). Osteolysis-susceptible patients show repeatable, quantitatively different patterns of innate cytokine gene expression in response to pro-inflammatory stimuli versus THA patients who do not display this susceptibility. These innate immune differences may contribute to the variation in osteolysis-susceptibility observed clinically between individuals.