Elevated levels of active Transforming Growth Factor ß1 in the subchondral bone relate spatially to cartilage loss and impaired bone quality in human knee osteoarthritis.

OBJECTIVE: The association between the spatially distributed level of active TGFß1 in human subchondral bone, and the characteristic structural and cellular parameters of human knee OA, was assessed. DESIGN: Paired subchondral bone samples from 35 OA arthroplasty patients, (15 men and 20 women, aged 69 ± 9 years) were obtained from beneath macroscopically present (CA+) or denuded cartilage (CA-) to determine the concentration of active TGFß1 (ELISA) and its relationship to bone quality (synchrotron micro-CT), cellularity, and vascularization (histology). RESULTS: Bone samples beneath (CA-) regions had significantly increased concentrations of active TGFß1 protein (mean difference: 26.4; 95% CI: [3.2, 49.7]), when compared to bone in CA + regions. Trabecular Bone below (CA-) regions had increased bone volume (median difference: 4.3; 96.49% CI: [-1.7, 17.8]), increased trabecular number (1.5 [0.006, 2.6], decreased trabecular separation (-0.05 [-0.1,-0.005]), and increased bone mineral density (394.5 [65.7, 723.3]) comparing to (CA+) regions. Further, (CA-) bone regions showed increased osteocyte density (0.012 [0.006, 0.018]), with larger osteocyte lacunae (39.8 [7.8, 71.7]) that were less spherical (-0.02 [-0.04, -0.003]), and increased bone matrix vascularity (12.4 [0.3, 24.5]) compared to (CA+). In addition, increased levels of active TGFß1 related to increased bone volume (0.04 [-0.11, 0.9]), while increased OARSI grade associated with lacunar volume (-44.1 [-71.1, -17.2]), and orientation (2.7 [0.8, 4.6]). CONCLUSION: Increased concentration of active TGFß1 in the subchondral bone of human knee OA associates spatially with impaired bone quality and disease severity, suggesting that TGFß1 is a potential therapeutic target to prevent or reduce human OA disease progression.

Bone marrow lesions in knee osteoarthritis: regional differences in tibial subchondral bone microstructure and their association with cartilage degeneration.

OBJECTIVE: The aim of this study was to investigate how bone microstructure within bone marrow lesions (BMLs) relates to the bone and cartilage across the whole human tibial plateau. DESIGN: Thirty-two tibial plateaus from patients with osteoarthritis (OA) at total knee arthroplasty and eleven age-matched non-OA controls, were scanned ex vivo by MRI to identify BMLs and by micro CT to quantitate the subchondral (plate and trabecular) bone microstructure. For cartilage evaluation, specimens were processed histologically. RESULTS: BMLs were detected in 75% of the OA samples (OA-BML), located predominantly in the anterior-medial (AM) region. In contrast to non-OA control and OA-no BML, in OA-BML differences in microstructure were significantly more evident between subregions. In OA-BML, the AM region contained the most prominent structural alterations. Between-group comparisons showed that the AM region of the OA-BML group had significantly higher histological degeneration (OARSI grade) (P < .0001, P < .05), thicker subchondral plate (P < .05, P < .05), trabeculae that are more anisotropic (P < .0001, P < .05), well connected (P < .05, P = n.s), and more plate-like (P < 0.05, P < 0.05), compared to controls and OA-no BML at this site. Compared to controls, OA-no BML had significantly higher OARSI grade (P < .0001), and lower trabecular number (P < .05). CONCLUSION: In established knee OA, both the extent of cartilage damage and microstructural degeneration of the subchondral bone were dependent on the presence of a BML. In OA-no BML, bone microstructural alterations are consistent with a bone attrition phase of the disease. Thus, the use of BMLs as MRI image-based biomarkers appear to inform on the degenerative state within the osteochondral unit.

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of ß-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released ß-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.

Semaphorin-3a, neuropilin-1 and plexin-A1 in prosthetic-particle induced bone loss.

Peri-prosthetic osteolysis (PPO) occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. Semaphorin-3a (SEM3A), neuropilin-1 (NRP1) and plexin-A1 (PLEXA1) are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This study investigated SEM3A, NRP1 and PLEXA1 protein and mRNA expression in human PPO tissue and polyethylene (PE) particle-stimulated human peripheral blood mononuclear cell (PBMC)-derived osteoclasts in vitro. In addition, the effects of tumour necrosis factor alpha (TNFα) on cultured osteoclasts was assessed. In PPO tissues, a granular staining pattern of SEM3A and NRP1 was observed within large multi-nucleated cells that contained prosthetic wear particles. Immunofluorescent staining confirmed the expression of SEM3A, NRP1 and PLEXA1 in large multi-nucleated human osteoclasts in vitro. Furthermore, SEM3A, NRP1 and PLEXA1 mRNA levels progressively increased throughout osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL), and the presence of PE particles further increased mRNA expression of all three molecules. Soluble SEM3A was detected in human osteoclast culture supernatant at days 7 and 17 of culture, as assessed by ELISA. TNFα treatment for 72h markedly decreased the mRNA expression of SEM3A, NRP1 and PLEXA1 by human osteoclasts in vitro. Our findings suggest that SEM3A, NRP1 and PLEXA1 may have important roles in PPO, and their interactions, alone or as a complex, may have a role in pathological bone loss progression. STATEMENT OF SIGNIFICANCE: Peri-prosthetic osteolysis occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. The rate of hip and knee arthroplasty is increasing by at least 5% per year. However, these joint replacements have a finite lifespan, with data from the National Joint Replacement Registry (Australia) showing that the major cause of failure of total hip replacements is aseptic loosening. In aseptic loosening, wear particles liberated from prostheses are phagocytosed by macrophages, leading to release of inflammatory cytokines and up-regulation of osteoclast formation and activity. Semaphorin-3a, neuropilin-1 and plexin-A1 are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This is the first report to show that these molecules may be involved in the implant failure.

Modulation of osteoclastic migration by metabolism of 25OH-vitamin D3.

We have reported the metabolism of 25(OH) vitamin D3 (25D) into active 1α,25(OH)2 vitamin D3 (1,25D) by osteoclasts derived from human peripheral blood mononuclear cells (PBMC), RAW 264.7cells or giant cell tumor of bone (GCT), which appears to optimize osteoclast differentiation but inhibit their activity. In this study, to elucidate the mechanism by which 25D reduces osteoclast resorption, we further examined the effect of 25D on osteoclast function by using GCT-derived osteoclasts. 25D treated cells on dentine slices resulted in decreased resorption volume and depth in 3D image analysis. Tartrate-resistant acid phosphatase (TRAP) has been reported to enhance the dephosphorylation of substrate binding proteins, resulting in reduced osteoclast attachment. Therefore, we next investigated the effect of 25D on cell migration. Treatment of GCT cells with 25D augmented cell migration, as determined by live cell imaging. These observations suggest that 25D metabolism by osteoclasts reduces their resorptive capacity, in part by modifying their surface adhesion and migration properties. This article is part of a Special Issue entitled "Vitamin D Workshop".

Polyethylene particles stimulate expression of ITAM-related molecules in peri-implant tissues and when stimulating osteoclastogenesis in vitro.

Wear particle-induced orthopaedic prosthesis loosening is associated with elevated osteoclast activity. The immunoreceptor tyrosine-based activation motif (ITAM)-related molecules OSCAR, FcRγ, TREM2 and DAP12 are important for osteoclast formation. The aim of this study was to determine if these molecules are involved in peri-implant loosening by investigating their expression in peri-implant tissues obtained at revision of joint replacement components containing polyethylene (PE) wear particles, and in osteoclasts formed in vitro in the presence of PE particles. The results showed that there was a marked and statistically significant increase in protein levels of the ITAM-related molecules in the revision tissues. The levels of OSCAR, FcRγ, TREM2 and DAP12 mRNA in the revision tissues were also increased. In vitro PE particles stimulated osteoclast resorption in the presence of 50 ng ml(-1) receptor activator NFκB (RANKL) and significantly elevated the expression of OSCAR, FcRγ, TREM2 and DAP12 during osteoclast formation. These findings suggest that the ITAM signalling molecules and their co-receptors have a role in pathogenic bone loss associated with implant PE wear.

Osteocyte regulation of bone mineral: a little give and take.

Osteocytes actively participate in almost every phase of mineral handling by bone. They regulate the mineralisation of osteoid during bone formation, and they are also a major RANKL-producing cell. Osteocytes are thus able to liberate bone mineral by regulating osteoclast differentiation and activity in response to a range of stimuli, including bone matrix damage, bone disuse and mechanical unloading, oestrogen deficiency, high-dose glucocorticoid and chemotherapeutic agents. At least some of these activities may be regulated by the osteocyte-secreted product, sclerostin. There is also mounting evidence that in addition to regulating phosphate homeostasis systemically, osteocytes contribute directly to calcium homeostasis in the mature skeleton. Osteocyte cell death and the local loss of control of bone mineralisation may be the cause of focal hypermineralisation of bone and osteopetrosis, as seen in aging and pathology. The sheer number of osteocytes in bone means that "a little give and take" in terms of regulation of bone mineral content translates into a powerful whole organism effect.

Relationship between serum RANKL and RANKL in bone.

It is now well accepted that the molecule receptor activator of NFκB ligand (RANKL) and osteoprotegerin play key roles in regulating physiological and pathological bone turnover. There are a large number of published reports of circulating RANKL levels in both health and pathology. However, interpretation of these data has been elusive, and the relationship between circulating RANKL and RANKL levels in bone is still not clear. This review explores this subject, documenting the possible origins of circulating RANKL and suggesting additional information that is required before serum RANKL levels can provide useful diagnostic or research information.

The metabolism of 25-(OH)vitamin D3 by osteoclasts and their precursors regulates the differentiation of osteoclasts.

Current evidence suggests that levels of 25-(OH)vitamin D3 (25D), rather than 1alpha,25-(OH)2vitamin D3 (1,25D), directly affect bone mineralization and that the skeleton is a site of extra-renal synthesis of 1,25D. Since cells of the monocyte lineage can also metabolise 25D, it is possible that osteoclasts participate in local production of, and the response to, 1,25D. In this study, we investigated the effects of vitamin D metabolism on osteoclastogenesis using both the murine RAW 264.7 cell line and the human peripheral blood mononuclear cell (PBMC) models. PBMC-derived osteoclasts expressed cytoplasmic cyp27b1 and nuclear vdr proteins. PBMC expressed CYP27B1 mRNA, levels of which increased during RANKL induced differentiation into osteoclasts in both cell types. While 1,25D elicited a robust CYP24 transcriptional response in PBMC, the response to 25D was approximately 100-fold less at the concentrations used. Using media devoid of pre-existing vitamin D metabolites, we found that 25D was metabolised by RAW 264.7 cells to 1,25D and resulted in significant elevation in the numbers of TRAP-positive, multinucleated osteoclasts when present in the cultures for the first 3-5 days. These results suggest that vitamin D metabolism by osteoclast lineage cells is an important regulator of osteoclast formation.

Gene expression profile of the bone microenvironment in human fragility fracture bone.

Osteoporosis (OP) is a common age-related systemic skeletal disease, with a strong genetic component, characterised by loss of bone mass and strength, which leads to increased bone fragility and susceptibility to fracture. Although some progress has been made in identifying genes that may contribute to OP disease, much of the genetic component of OP has yet to be accounted for. Therefore, to investigate the molecular basis for the changes in bone causally involved in OP and fragility fracture, we have used a microarray approach. We have analysed altered gene expression in human OP fracture bone by comparing mRNA in bone from individuals with fracture of the neck of the proximal femur (OP) with that from age-matched individuals with osteoarthritis (OA), and control (CTL) individuals with no known bone pathology. The OA sample set was included because an inverse association, with respect to bone density, has been reported between OA and the OP individuals. Compugen H19K oligo human microarray slides were used to compare the gene expression profiles of three sets of female samples comprising, 10 OP-CTL, 10 OP-OA, and 10 OA-CTL sample pairs. Using linear models for microarray analysis (Limma), 150 differentially expressed genes in OP bone with t scores >5 were identified. Differential expression of 32 genes in OP bone was confirmed by real time PCR analysis (p<0.01). Many of the genes identified have known or suspected roles in bone metabolism and in some cases have been implicated previously in OP pathogenesis. Three major sets of differentially expressed genes in OP bone were identified with known or suspected roles in either osteoblast maturation (PRRX1, ANXA2, ST14, CTSB, SPARC, FST, LGALS1, SPP1, ADM, and COL4A1), myelomonocytic differentiation and osteoclastogenesis (TREM2, ANXA2, IL10, CD14, CCR1, ADAM9, CCL2, CTGF, and KLF10), or adipogenesis, lipid and/or glucose metabolism (IL10, MARCO, CD14, AEBP1, FST, CCL2, CTGF, SLC14A1, ANGPTL4, ADM, TAZ, PEA15, and DOK4). Altered expression of these genes and others in these groups is consistent with previously suggested underlying molecular mechanisms for OP that include altered osteoblast and osteoclast differentiation and function, and an imbalance between osteoblastogenesis and adipogenesis.

Strontium ranelate treatment of human primary osteoblasts promotes an osteocyte-like phenotype while eliciting an osteoprotegerin response.

SUMMARY: The effect of strontium ranelate (SR) on human osteoblast differentiation was tested. SR induced osteoblastic proliferation, in vitro mineralization, and increased the expression of osteocyte markers. SR also elicited an osteoprotegerin (OPG) secretory response. We conclude that SR promotes the osteoblast maturation and osteocyte differentiation while promoting an additional antiresorptive effect. INTRODUCTION: SR is a new treatment for osteoporosis that reduces the risk of hip and vertebral fractures in postmenopausal women. This study sought to investigate the extent, to which SR modulates human osteoblast differentiation. METHODS: Adult human primary osteoblasts (NHBC) were exposed to SR under mineralizing conditions in long-term cultures. Osteoblast differentiation status was investigated by cell-surface phenotypic analysis. Expression of genes associated with osteoblast/osteocyte differentiation was examined using real-time RT-PCR. Secreted OPG was assayed by enzyme-linked immunosorbent assay. RESULTS: SR significantly increased osteoblast replication. SR time- and dose-dependently induced an osteocyte-like phenotype, as determined by cell surface alkaline phosphatase and STRO-1 expression. SR at 5 mM or greater dramatically increased in vitro mineralization. In parallel, mRNA levels of dentin matrix protein (DMP)-1 and sclerostin were higher under SR treatment, strongly suggestive of the presence of osteocytes. SR also increased the OPG/RANKL ratio throughout the culture period, consistent with an effect to inhibit osteoblast-induced osteoclastogenesis. CONCLUSIONS: This study suggests that SR can promote osteoblast maturation and an osteocyte-like phenotype. Coupled with its effect on the OPG/RANKL system, these findings are consistent with in vivo effects in patients receiving SR for the treatment of osteoporosis.

Vascular pathology and osteoarthritis.

There is mounting evidence that vascular pathology plays a role in the initiation and/or progression of the major disease of joints: osteoarthritis (OA). Potential mechanisms are: episodically reduced blood flow through the small vessels in the subchondral bone at the ends of long bones, and related to this, reduced interstitial fluid flow in subchondral bone. Blood flow may be reduced by venous occlusion and stasis or by the development of microemboli in the subchondral vessels. There are several likely effects of subchondral ischaemia: the first of these is compromised nutrient and gas exchange into the articular cartilage, a potential initiator of degradative changes in the cartilage. The second is apoptosis of osteocytes in regions of the subchondral bone, which would initiate osteoclastic resorption of that bone and at least temporarily reduce the bony support for the overlying cartilage. It may be important to recognize these potential aetiological factors in order to develop more effective treatments to inhibit the progression of OA.

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells.

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.

Regulation of cell growth mediated by the calcitonin receptor.

Calcitonin (CT) is a 32 amino acid peptide hormone of thyroidal origin, whose main recognised physiological role is the inhibition of osteoclast--mediated bone resorption. There is also evidence that CT might modulate bone formation. However, both CT and its receptors (CTR) have also been identified in a large number of other cell types and tissue sites, suggesting roles for the CT/CTR system distinct from those involving calcium homeostasis. Evidence has accumulated consistent with the involvement of CT in cell growth and differentiation and in tissue development and remodelling. The close proximity of cells expressing CT, or CT receptors (CTR), during development, and during pregnancy and lactation, is consistent with important roles for CT in morphogenesis. It thus appears that, in tissues such as the uterus, breast and pituitary, CT acts in a paracrine manner to Influence cell proliferation and function, as distinct from its endocrine actions to regulate calcium stores in the skeleton. In vitro studies have shown that CT can be either mitogenic or can inhibit cell proliferation, depending on the cell type and the conditions of the experiment. More recently, evidence has also been obtained for a role for CT in cell survival, in cells as diverse as osteoblast--like and osteocyte--like cells, osteoclasts and neurons.

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor.

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.

Identification of differentially expressed genes between osteoarthritic and normal trabecular bone from the intertrochanteric region of the proximal femur using cDNA microarray analysis.

Osteoarthritis (OA) is a common age-related joint disease resulting in progressive degenerative damage to articular cartilage. The etiology of primary OA has not yet been determined. However, there is evidence supporting the hypothesis that primary OA is a disease affecting bone remodeling in addition to articular cartilage. In this study, we have used cDNA microarray analysis to compare gene expression in bone between normal (CTL) and OA individuals. Trabecular bone was sampled from the intertrochanteric region of the proximal femur, a site distal to the diseased hip joint. Total RNA was extracted from three pairs of age- and sex-matched CTL and OA bone samples, reverse-transcribed and radioactively labeled to generate cDNA probes, before hybridization with the Research Genetics GF211 human gene microarray filter. The CTL and OA samples were found to have similar levels of gene expression for more than 4000 known human genes. However, forty-one genes were identified that were differentially expressed, twofold or more, between all three CTL-OA sample pairs. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, three genes, fms-like tyrosine kinase 1 (FLT1), plexin B1 (PLXNB1), and small inducible cytokine A2 (SCYA2), were confirmed to be consistently expressed at lower levels in OA, in a majority of twenty age- and sex-matched CTL-OA bone sample pairs tested. FLT1, PLXNB1, and SCYA2 have known or potential roles in angiogenesis and bone remodeling. Down-regulation of these genes is consistent with a role for bone in the pathogenesis of OA.

Death to the bad guys: targeting cancer via Apo2L/TRAIL.

All higher organisms consist of an ordered society of individual cells that must communicate to maintain and regulate their functions. This is achieved through a complex but highly regulated network of hormones, chemical mediators, chemokines and other cytokines, acting as ligands for intra or extra-cellular receptors. Ligands and receptors of the tumor necrosis factor (TNF) superfamilies are examples of signal transducers, whose integrated actions influence the development, homeostasis and adaptive responses of many cells and tissue types. Apo2L/TRAIL is one of several members of the tumour necrosis factor superfamily that induce apoptosis through the engagement of death receptors. Apo2L/TRAIL interacts with an unusually complex receptor system, which in humans comprises two death receptors and three decoy receptors. This molecule has received considerable attention recently because of the finding that many cancer cell types are sensitive to Apo2L/TRAIL-induced apoptosis, while most normal cells appear to be resistant to this action of Apo2L/TRAIL. In this review, we specifically emphasise on the actions of Apo2L/TRAIL with respect to its apoptotic signaling pathways and summarise what is known about its physiological role. The potential therapeutic usefulness of Apo2L/TRAIL, especially in combination with chemotherapeutic agents, is also discussed in some detail.

Sensitivity of fresh isolates of soft tissue sarcoma, osteosarcoma and giant cell tumour cells to Apo2L/TRAIL and doxorubicin.

Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). However, the use of chemotherapy in high-grade soft tissue sarcomas remains controversial, with the most active chemotherapeutic agent, doxorubicin (DOX), reported to have a response rate of, at best only 34% and most studies reporting lower response rates. Apo2L/TRAIL is a member of the tumour necrosis factor (TNF) family of cytokines and induces death of tumour cells, but not normal cells. Its potent apoptotic activity is mediated through cell surface death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. We investigated the efficacy of Apo2L/TRAIL as a single agent, and in combination with clinically relevant chemotherapeutic drugs, in fresh isolates of primary malignant cells obtained from biopsy material. The data presented here demonstrate that, in a range of primary bone related tumours, as well as soft tissue sarcomas, chemotherapeutic agents were only moderately effective, in terms of induction of cell death. Apo2L/TRAIL alone had little or no effect on any bone-related tumour or sarcoma in culture. In contrast, the combination of Apo2L/TRAIL and chemotherapeutic drugs produced a significant increase in tumour cell death, with DOX and Apo2L/TRAIL proving to be the most effective combination. These data suggest the potential for Apo2L/TRAIL to increase the effectiveness of chemotherapeutic drugs in bone and soft tissue sarcomas, while perhaps concurrently allowing a reduction in the exposure to drugs such as DOX, and a consequent reduction in toxicity. The synergistic action between these two different classes of agents has yet to be tested in vivo but may prove clinically relevant in the treatment of this refractive class of malignancies.

Factors regulating osteoclast formation in human tissues adjacent to peri-implant bone loss: expression of receptor activator NFkappaB, RANK ligand and osteoprotegerin.

Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.

Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis.

The aim of this study was to investigate the cytotoxic activity of the third-generation nitrogen-containing bisphosphonate zoledronic acid (ZOL) as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines (HOS, BTK-143, MG-63, SJSA-1, G-292, and SAOS2). We found that ZOL, when used alone, reduced cell number in a dose- and time-dependent manner, due either to cell cycle arrest in S-phase or to the induction of apoptosis. In the sensitive HOS, BTK-143, and G-292 cell lines, genomic DNA fragmentation and morphological changes characteristic of apoptosis were evident, and cells became nonadherent. Induction of apoptosis in osteosarcoma cells by ZOL was associated with caspase activation. However, coaddition of the broad-spectrum caspase inhibitors, z-VAD-fmk, Boc-D-fmk, or the caspase-3-specific inhibitor z-DEVD fmk, failed to protect these cells from ZOL-induced apoptosis. Our data support a ZOL-specific induction of cell apoptosis that involves cell detachment (anoikis), and in which caspase activation occurs secondarily to, and is redundant as a mediator of cell death. The addition of geranylgeraniol, an intermediate of the mevalonate pathway, suppressed the ZOL-induced apoptosis, suggesting that the cytotoxic effects of ZOL in osteosarcoma cells were mediated by the mevalonate pathway. While treatment of osteosarcoma cells with the chemotherapeutic agents doxorubicin or etoposide decreased cell viability, combination of these agents with ZOL did not significantly augment apoptosis in any of the cell lines tested. These observations suggest that ZOL has direct effects on the proliferation and survival of osteosarcoma cells in vitro, which has implications for future therapy of osteosarcoma.