G. Atkins

The osteoclastogenic molecules RANKL and RANK are associated with periprosthetic osteolysis

Extensive osteolysis adjacent to implants is often associated with wear particles of prosthetic material. We have investigated if RANKL, also known as osteoprotegerin ligand, osteoclast differentiation factor or TRANCE, and its natural inhibitor, osteoprotegerin (OPG), may be important in controlling this bone loss. Cells isolated from periprosthetic tissues containing wear particles expressed mRNA encoding for the pro-osteoclastogenic molecules, RANKL, its receptor RANK, monocyte colony-stimulating factor (M-CSF), interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, and soluble IL-6 receptor, as well as OPG. Osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells. When osteoclasts formed in the absence of osteoblastic cells, markedly higher levels of RANKL mRNA relative to OPG mRNA were expressed. Particles of prosthetic materials also stimulated human monocytes to express osteoclastogenic molecules in vitro. Our results suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast-differentiating molecules and the stimulation of macrophage differentiation into osteoclasts.

The Ratio of Messenger RNA Levels of Receptor Activator of Nuclear Factor κB 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 κB 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.

Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to apo2l/trail-induced apoptosis

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain‐containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 “decoy” receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK‐143, HOS, MG‐63, SJSA‐1, G‐292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK‐143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL‐resistant cells were susceptible to Apo2L/TRAIL‐mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL‐induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase‐8 and caspase‐3 activation. Furthermore, Apo2L/TRAIL‐induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase‐8 zIETD‐fmk and caspase‐3 zDEVD‐fmk protease inhibitors and by the pan‐caspase inhibitor zVAD‐fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE‐inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL‐induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug‐induced up‐regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL‐induced apoptosis. Stable over‐expression of a dominant negative form of the Fas‐associated death domain protein (FADD) in the Apo2L/TRAIL‐sensitive BTK‐143 cells completely inhibited Apo2L/TRAIL‐induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone‐derived osteoblast‐like cells, which has implications for future therapy of osteosarcoma.

Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy

Apo2 ligand (Apo2L, also known as TRAIL) is a member of the tumour necrosis factor (TNF) family of cytokines that selectively induces the death of cancer cells, but not of normal cells. We observed that recombinant Apo2L/TRAIL was proapoptotic in early-passage BTK-143 osteogenic sarcoma cells, inducing 80% cell death during a 24u2009h treatment period. Apo2L/TRAIL-induced apoptosis was blocked by caspase inhibition. With increasing passage in culture, BTK-143 cells became progressively resistant to the apoptotic effects of Apo2L/TRAIL. RNA and flow cytometric analysis demonstrated that resistance to Apo2L/TRAIL was paralleled by progressive acquisition of the decoy receptor, DcR2. Blocking of DcR2 function with a specific anti-DcR2 antibody restored sensitivity to Apo2L/TRAIL in a dose-dependent manner. Importantly, treatment of resistant cells with the chemotherapeutic agents doxorubicin, cisplatin and etoposide reversed the resistance to Apo2L/TRAIL, which was associated with drug-induced upregulation of mRNA encoding the death receptors DR4 and DR5. BTK-143 cells thus represent a useful model system to investigate both the mechanisms of acquisition of resistance of tumour cells to Apo2L/TRAIL and the use of conventional drugs and novel agents to overcome resistance to Apo2L/TRAIL.

Human osteoblasts are resistant to Apo2L/TRAIL-mediated apoptosis

Apo2 ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. Apo2L/TRAIL can selectively induce programmed cell death in transformed cells, although its wide tissue distribution suggests potential physiological roles. We have investigated the expression, in human osteoblast-like cells (NHBC), of Apo2L/TRAIL and the known Apo2L/TRAIL death receptors, DR4 and DR5, and the Apo2L/TRAIL decoy receptors, DcR-1, DcR-2, and osteoprotegerin (OPG). NHBC expressed abundant mRNA corresponding to each of these molecular species. Immunofluorescence staining demonstrated that Apo2L/TRAIL protein was abundant within the cytoplasm of NHBC and OPG was strongly expressed at the cell surface. DR5 and DcR-2 were present in the cell membrane and cytoplasm and DcR-1 was confined to the nucleus. DR4 staining was weak. Neither Apo2L/TRAIL alone, nor in combination with chemotherapeutic agents of clinical relevance to treatment of osteogenic sarcoma, induced cell death in NHBC, as assessed morphologically and by activation of caspase-3. In contrast, the human osteogenic sarcoma cell lines, BTK-143 and G-292, were sensitive to exogenous Apo2L/TRAIL alone, and to the combined effect of Apo2L/TRAIL/cisplatin and Apo2L/TRAIL/doxorubicin treatments, respectively. In NHBC, we observed strong associations between the levels of mRNA corresponding to the pro-apoptotic molecules, Apo2L/TRAIL, DR4, and DR5, and those corresponding to pro-survival molecules, DcR-1, DcR-2, OPG, and FLIP, suggesting that the balance between pro-survival and pro-apoptotic molecules is a mechanism by which NHBC can resist Apo2L/TRAIL-mediated apoptosis. In contrast, osteogenic sarcoma cells had low or absent levels of DcR-1 and DcR-2. These results provide a foundation to explore the role of Apo2L/TRAIL in osteoblast physiology. In addition, they predict that therapeutic use of recombinant Apo2L/TRAIL, in combination with chemotherapeutic agents to treat skeletal malignancies, would have limited toxic effects on normal osteoblastic cells.

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. (J Bone Miner Res 2000;15:332–341)

Postoperative weight bearing and patient reported outcomes at one year following tibial plateau fractures

Tibial plateau fractures are complex and the current evidence for postoperative rehabilitation is weak, especially related to the recommended postoperative weight bearing. The primary aim of this study was to investigate if loading in the first 12 weeks of recovery is associated with patient reported outcome measures at 26 and 52 weeks postoperative. We hypothesized that there would be no association between loading and patient reported outcome measures. Seventeen patients, with a minimum of 52-week follow-up following fragment-specific open reduction and internal fixation for tibial plateau fracture, were selected for this retrospective analysis. Postoperatively, patients were advised to load their limb to a maximum of 20kg during the first 6 weeks. Loading data were collected during walking using force platforms. A ratio of limb loading (affected to unaffected) was calculated at 2, 6 and 12 weeks postoperative. Knee Injury and Osteoarthritis Scores were collected at 6, 12, 26 and 52 weeks postoperative. The association between loading ratios and patient reported outcomes were investigated. Compliance with weight bearing recommendations and changes in the patient reported outcome measures are described. Fracture reduction and migration were assessed on plain radiographs. No fractures demonstrated any measurable postoperative migration at 52 weeks. Significant improvements were seen in all patient reported outcome measures over the first 52 weeks, despite poor adherence to postoperative weight bearing restrictions. There were no associations between weight bearing ratio and patient reported outcomes at 52 weeks postoperative. Significant associations were identified between the loading ratio at 2 weeks and knee-related quality of life at six months (R2=0.392), and between the loading ratio at 6 weeks combined with injury severity and knee-related quality of life at 26 weeks (R2=0.441). In summary, weight bearing as tolerated does not negatively affect the results of tibial plateau fracture and may therefore be safe for postoperative management. These findings should be taken in context of the sample size, which was not sufficient for sub-group analysis to investigate the role of impaction grafting.

A Fluorometric Method for the Quantification of Cell Number in Complex Differentiating Osteoblast-Osteocyte Cultures

Osteoblast/osteocyte cultures continue to emerge as essential tools for bone biology research in vitro. The change in cell number is an important parameter to be considered for investigating osteogenic differentiation. However, there is no reliable method for quantifying absolute cell count in differentiating osteoblast/osteocyte cultures because of their strongly adherent, multi-layered, super-confluent nature, and their accumulated extracellular matrix production which progressively mineralises in vitro. We developed a practical, simple and cost-effective method based on the fluorometric quantification of a nucleic dye, GelRed™, to enumerate cell number in osteoblast/osteocyte cultures. This method may also be suitable for quantifying cell numbers on other mammalian adherent cell types.