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.

Evidence for osteocyte-mediated bone-matrix degradation associated with periprosthetic joint infection (PJI)

Osteomyelitis associated with periprosthetic joint infection (PJI) signals a chronic infection and the need for revision surgery. An osteomyelitic bone exhibits distinct morphological features, including evidence for osteolysis and an accelerated bone remodelling into poorly organised, poor-quality bone. In addition to immune cells, various bone cell-types have been implicated in the pathology. The present study sought to determine the types of bone-cell activities in human PJI bones. Acetabular biopsies from peri-implant bone from patients undergoing revision total hip replacement (THR) for chronic PJI (with several identified pathogens) as well as control bone from the same patients and from patients undergoing primary THR were analysed. Histological analysis confirmed that PJI bone presented increased osteoclastic activity compared to control bone. Analysis of osteocyte parameters showed no differences in osteocyte lacunar area between the acetabular bone taken from PJI patients or primary THR controls. Analysis of bone matrix composition using Masson's trichrome staining and second-harmonic generation microscopy revealed widespread lack of mature collagen, commonly surrounding osteocytes, in PJI bone. Increased expression of known collagenases, such as matrix metallopeptidase (MMP) 13, MMP1 and cathepsin K (CTSK), was measured in infected bone compared to non-infected bone. Human bone and cultured osteocyte-like cells experimentally exposed to Staphylococcus aureus exhibited strongly upregulated expression of MMP1, MMP3 and MMP13 compared to non-exposed controls. In conclusion, the study identified previously unrecognised bone-matrix changes in PJI caused by multiple organisms deriving from osteocytes. Histological examination of bone collagen composition may provide a useful adjunct diagnostic measure of PJI.

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.

Evidence for the dysregulated expression of TWIST1, TGFß1 and SMAD3 in differentiating osteoblasts from primary hip osteoarthritis patients.

OBJECTIVE: This study compared human primary osteoblasts derived from hip osteoarthritis (OA) cases against controls (CTLs) to investigate candidate OA disease genes, twist homologue 1 (TWIST1), wingless MMTV integration site family member 5B (WNT5B), transforming growth factor-ß (TGFß1) and SMAD family member 3 (SMAD3), during osteoblast differentiation, relative to calcium apposition and elemental mineral composition. MATERIALS & METHODS: Primary osteoblast cultures were generated from intertrochanteric trabecular bone samples from five female primary hip OA cases and five age-matched female CTLs. During a 42-day differentiation time-course, alizarin red stains, energy-dispersive X-ray spectroscopy and real-time RT-polymerase chain reaction (PCR) were used to quantify calcium, elemental composition and gene expression, respectively. Data were analysed using linear mixed effects models and Pearson correlation matrices. RESULTS: Significant differences, correlations and associations were found in OA and CTL osteoblasts between gene and mineral measures. The calcium: phosphorous (Ca:P) ratio was significantly more varied in OA compared to CTL. Calcium apposition, mineral composition as well as TWIST1 and TGFß1 mRNA expression changed significantly over time. TWIST1 mRNA expression was elevated and correlated with SMAD3 mRNA levels in the OA cohort during the time-course. Associations were observed between tissue non-specific alkaline phosphatase (TNAP), osteocalcin (OCN), TWIST1, TGFß1, SMAD3 mRNA levels and mineral measures in OA against CTL. Temporal differences between SMAD3 mRNA expression and mineral composition were also found in OA. CONCLUSIONS: Dysregulated expression of TWIST1, TGFß1 and SMAD3 mRNA observed in OA bone is reflected in the functionality of the osteoblast when these cells are cultured ex vivo. The results presented here are consistent with at least part of the aetiology of primary hip OA deriving from altered intrinsic properties of the osteoblast.

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.

Changes in osteocyte density correspond with changes in osteoblast and osteoclast activity in an osteoporotic sheep model.

UNLABELLED: Histomorphometric assessment of trabecular bone in osteoporotic sheep showed that bone volume, osteoid surface area, bone formation rate, and osteocyte density were reduced. In contrast, eroded surface area and empty lacunae density were increased. Changes in osteocyte density correlated with changes in osteoblast and osteoclast activity. INTRODUCTION: Osteocytes contribute to the regulation of the activity of osteoclasts and osteoblasts that together control bone mass. Osteocytes therefore likely play a role in the loss of bone mass associated with osteoporosis. The purpose of this study was to investigate the relationships between osteocyte lacunar density and other bone histomorphometric parameters in the iliac crest (IC) and lumbar spine (LS) of osteoporotic sheep. METHODS: Osteoporosis was induced in ten mature ewes by an established protocol involving a combination of ovariectomy, dexamethasone injection, and low calcium diet for 6 months. Five ewes were used as controls. Post-mortem IC and LS biopsies were collected and processed for further histomorphometric assessment. RESULTS: Bone volume, osteoid surface, and bone formation rate in the IC and LS of osteoporotic sheep were reduced compared to those of the controls. In contrast, eroded surface area was increased in osteoporotic sheep. In the osteoporotic group, osteocyte density was reduced in the LS region and to a greater extent in the IC region. The empty osteocyte lacunae were increased 1.7-fold in LS and 2.1-fold in IC in the osteoporotic group. The osteocyte density correlated positively with markers of osteoblast activity and negatively with those of osteoclast activity. CONCLUSIONS: Depletion of osteocytes and an increase in the empty lacunae could be important factors contributing to bone loss in this model since they may adversely affect intercellular communication between osteoblasts and osteoclasts. The regional differences in histology suggest that there may be different pathological mechanisms operating at different anatomical sites.

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.

Critical molecular regulators, histomorphometric indices and their correlations in the trabecular bone in primary hip osteoarthritis.

OBJECTIVE: This study examined differential gene expression, histomorphometric indices and relationships between these, in femoral trabecular bone from osteoarthritis (OA) patients and control (CTL) subjects, with the aim of identifying potential molecular drivers consistent with changes in structural and remodelling indices in the OA pathology. MATERIALS AND METHODS: Bone samples from the intertrochanteric (IT) region were obtained from age and sex-matched cohorts of 23 primary hip OA patients and 21 CTL subjects. Real-time polymerase chain reaction (PCR) and histomorphometric analysis were performed on each sample and correlations between gene expression and histomorphometric variables determined. RESULTS: Alterations in gene expression, structural indices and correlations between these were found in OA bone compared to CTL. In OA bone, expression of critical regulators of osteoblast differentiation (TWIST1) and function (PTEN, TIMP4) were decreased, while genes associated with inflammation (SMAD3, CD14) were increased. Bone structural and formation indices (BV/TV, Tb.N, OS/BS) were increased, whereas resorption indices (ES/BS, ES/BV) were decreased. Importantly, significant correlations in CTL bone between CTNNB1 expression and formation indices (OS/BS, OS/BV, OV/BV) were absent in OA bone, indicating altered WNT/ß-catenin signalling. TWIST1 expression and BV/TV were correlated in CTL bone, but not in OA bone, consistent with altered osteoblastogenesis in OA. Matrix metalloproteinase 25 (MMP25) expression and remodelling indices (ES/BS, ES/BV, ES/TV) were correlated only in OA pointing to aberrant bone remodelling in this pathology. CONCLUSIONS: These findings indicate an altered state of osteoblast differentiation and function in OA driven by several key molecular regulators. In association with this differential gene expression, an altered state of both trabecular bone remodelling and resulting microarchitecture were also observed, further characterising the pathogenesis of primary hip OA.

Semliki Forest virus-mediated gene therapy of the RG2 rat glioma.

AIMS: Glioblastoma multiforme is the most common and most malignant adult brain tumour. Despite numerous advances in cancer therapy there has been little change in the prognosis of glioblastoma multiforme, which remains invariably fatal. We examined the Semliki Forest virus virus-like particle (SFV VLP) expression system encoding interleukin-12 (IL-12) as a therapeutic intervention against the syngeneic RG2 rat glioma model. METHODS: Glioma-bearing rats were treated with IL-12-encoding SFV VLPs via an implanted cannula. Animals were treated with 5 × 107 (low-dose) or 5 × 108 (high-dose) VLPs per treatment and the effect on glioma growth and survival was assessed. RESULTS: Low-dose treatment produced a 70% reduction in tumour volume, associated with a significant extension (20.45%) in survival that was dependent upon IL-12 expression. High-dose treatment resulted in an 87% reduction in tumour volume, related to the oncolytic capacity of the SFV VLP system. VLP delivery to the central nervous system (CNS) demonstrated the potential of the vector system to induce lethal pathology that was unrelated to replication-competent virus or high-level IL-12 expression. Treatment-related death was pronounced in high dose-treated animals and appeared to be the result of inflammation, necrosis and oedema at the inoculation site. CONCLUSION: The efficacy of an IL-12 gene therapy approach for the treatment of the RG2 glioma model has been demonstrated in addition to the oncolytic capacity of the VLP vector system. Despite this, the broad tropism of the SFV-based expression vector may limit use as a CNS gene therapy vector unless this inherent limitation can be overcome.

Expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 protein (Fn14), in healthy tissues and in tissues affected by periodontitis.

BACKGROUND AND OBJECTIVE: Host-derived enzymes, cytokines and other proinflammatory mediators play an integral role in periodontal destruction. The levels of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 protein (Fn14), are elevated in tissues from a number of chronic inflammatory diseases. The aim of the present study was to investigate the expression of TWEAK and Fn14 at the protein and mRNA levels in gingival biopsies from periodontitis patients and from clinically healthy patients. MATERIALS AND METHODS: Gingival biopsies were obtained from healthy sites (n = 7) and from sites affected by periodontitis (n = 27). The expression of TWEAK and Fn14 was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissues. The levels of mRNA for TWEAK and Fn14 were also investigated by RT-PCR. RESULTS: The expression of TWEAK and Fn14 proteins was significantly higher in periodontitis tissue than in healthy tissue. In periodontitis tissues, TWEAK and Fn14 proteins were mainly expressed by mononuclear leukocytes (morphologically resembling lymphocytes and plasma cells), by cells lining blood vessels, by spindle-shaped cells resembling fibroblasts and by multinucleated cells. The Fn14 mRNA level in periodontitis tissue was significantly higher than that in healthy tissue. A moderate correlation between TWEAK/Fn14 expression and inflammation and bone loss, but not pocket depth, was noted. CONCLUSION: This study demonstrates higher expression of TWEAK protein and of Fn14 mRNA and protein in periodontitis tissues than in clinically healthy controls. Our data support the concept that TWEAK/Fn14 signaling is an additional player in the pathogenesis of periodontitis and adds to the increasing number of cytokine networks involved in periodontal inflammation.

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.

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.

Inhibition of murine K-BALB and CT26 tumour growth using a Semliki Forest virus vector with enhanced expression of IL-18.

The enhanced Semliki Forest virus vector (SFV10-E), an RNA-based suicide expression vector system, expresses foreign genes at levels up to 10x higher than the original SFV10 vector. This vector has been used previously to express interleukin-12 for a tumour treatment study in a BALB/c murine model. Interleukin-18, an IFN-gamma-inducing cytokine, plays a key role in the early induction of T helper1 (Th1) cell-mediated immune responses in addition to anti-angiogenic activity. In this study, the murine IL-18 gene along with an Ig-kappa leader sequence was cloned into the SFV10-E vector. The pSFV10-E-IL-18 construct was characterised in vitro for levels of expression and secretion, and the production of biologically active IL-18 was confirmed. An in vivo tumour treatment study using high titre rSFV10-E-IL-18 virus-like particles to treat subcutaneous K-BALB and CT26 tumours in BALB/c mice demonstrated therapeutic efficacy including the disappearance of tumour cells in a minority of treated animals. Tumour regression was associated with induction of avascular and suppurative necrosis.

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.

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.

Enhanced expression of osteocalcin mRNA in human osteoarthritic trabecular bone of the proximal femur is associated with decreased expression of interleukin-6 and interleukin-11 mRNA.

Few studies have investigated the factors or mechanisms that may lead to structural changes in OA bone. This study examines the in vivo expression of messenger RNA encoding the osteoclastogenic cytokines interleukin-6 (IL-6) and interleukin-11 (IL-11), together with the osteoblastic marker osteocalcin (OCN) and the calcitonin receptor (CTR), which in bone is exclusively expressed by osteoclasts. Total RNA was isolated from intertrochanteric trabecular bone from OA patients, and from controls taken at autopsy. The patterns of mRNA expression of IL-6, IL-11, OCN, and CTR were examined using reverse-transcription polymerase chain reaction (RT-PCR) by determining the relative ratios of the amplified products with respect to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both IL-6 and IL-11 mRNA were significantly less abundant in OA than in the control group. Expression of IL-11 mRNA decreased significantly with age for both groups. OCN mRNA expression was significantly more abundant in OA, and there was no significant difference for CTR mRNA between the two groups. For both OCN and CTR in OA, expression increased significantly with increasing age. These differences in expression between the OA and control groups are consistent with an hypothesis that biochemical and genetic factors in bone can contribute or perhaps underlie the degenerative joint changes seen in OA.

The ratio of messenger RNA levels of receptor activator of nuclear factor kappaB ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis.

The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor kappaB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Expression of osteoclast differentiation signals by stromal elements of giant cell tumors.

The mechanisms by which primary tumors of the bone cause bone destruction have not been elucidated. Unlike most other lytic bone tumors, osteoclastomas, otherwise known as giant cell tumors (GCT), contain osteoclast-like cells within the tumor stroma. A new member of the TNF-ligand superfamily member, osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE), was recently identified. ODF was shown to directly stimulate osteoclastogenesis, in the presence of M-CSF. In this study, the expression of ODF was examined in a number of tumor samples associated with bone lysis in vivo. In addition, we investigated expression of the ODF receptor on osteoclast precursors, RANK, as well as the ODF inhibitor osteoprotegerin (OPG), and another TNF-ligand superfamily member, TRAIL, previously shown to abrogate the inhibitory effects of OPG. We report here the novel finding that GCT stromal cells contain abundant ODF mRNA, whereas the giant cell population exclusively expresses RANK mRNA. These results are consistent with the osteoclast-mediated bone destruction by these tumors. We also report the expression of OPG and TRAIL mRNA in GCT samples. A comparison with other lytic and nonlytic tumors of bone showed that GCT express more ODF and TRAIL mRNA relative to OPG mRNA. In addition, GCT were found to express a number of cytokines previously reported to play central roles in osteoclastogenesis, namely, IL-1, -6, -11, -17, as well as TNF-alpha. Importantly, GCT were also found to express high levels of M-CSF mRNA, a cytokine shown to be an essential cofactor of ODF, and a survival factor for mature and developing osteoclasts. Furthermore, expression of these molecules by stromal cells isolated from GCT continued in vitro. Thus GCT constitutively express all of the signals that are currently understood to be necessary for the differentiation of osteoclasts from precursor cells.

Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation.

An in vitro culture system to generate human osteoclasts (OC) was recently described in which OC precursors in the human peripheral blood mononuclear cell (PBMC) population differentiate in the presence of murine ST-2 stromal cells. We used this culture system to define the cytokine environment in which human OC form and to determine the separate contributions of the stromal and hematopoietic elements. We designed a panel of reverse transcriptase-polymerase chain reaction (RT-PCR) primers that specifically amplify the respective murine or human mRNA species that correspond to cytokines and their cognate receptors previously shown to promote or inhibit OC differentiation. ST-2 cells were cocultured with human PBMC for up to 21 days in the presence of 1alpha,25(OH)(2) vitamin D(3), dexamethasone, and recombinant human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate-resistant acid phosphatase (TRAP) and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures express messenger RNA (mRNA) encoding a repertoire of many of the reported osteoclastogenic factors (interleukins [IL]-1/IL-1R1, IL-11, IL-6/IL-6R, and IL-17 transforming growth factor [TGF]-beta), as well as the recently described OC differentiation factor (ODF/TRANCE/RANKL). The stromal cells also expressed mRNA encoding two molecules shown to be inhibitory to osteoclastogenesis, osteoprotegerin (OPG) and IL-18. OPG, IL-1, IL-1R1, IL-6, IL-6R, IL-11R, IL-17, IL-18, IL-18R, TGF-beta, and M-CSF were expressed by both the stromal cells and the PBMC. Expression of mRNA encoding RANK, IL-1R2, and c-fms, was specific for the PBMC. In addition, PBMC were found to express sIL-6R, granulocyte macrophage (GM)-CSF, GM-CSFRalpha, and tumor necrosis factor (TNF)-alpha. Whereas this indicated that human OC formation occurs in a complex environment of many positive and negative influences, we identified three apparent features of the cytokine environment that may be a characteristic of normal osteoclast formation. First, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Second, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Third, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6, and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. We conclude that human OC formation occurs in a complex environment of many positive and negative influences; however, these are likely to be strictly regulated by a coordinated cytokine response of both stromal and hematopoietic cells.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells.

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.