Enhanced tumor regression and tissue repair when zoledronic acid is combined with ifosfamide in rat osteosarcoma.

The efficacy of zoledronic acid (ZOL), with or without the anticancer drug ifosfamide (IFO), was tested on primary bone tumor growth using a rat-transplantable model of osteosarcoma. The effects on bone remodeling and tumor growth were analyzed by radiography, micro-computed tomography (micro-CT), and histological staining. The in vitro effects of ZOL were studied by proliferation, apoptosis, and cell cycle analyses on the osteosarcoma cells OSRGA compared to rat primary osteoblasts. Treatment with ZOL was effective in preventing the formation of osteolytic lesions that developed in bone sites and in reducing the local tumor growth, as compared to the untreated rats. The combination of ZOL and IFO was more effective than each agent alone in preventing tumor recurrence, improving tissue repair, and increasing bone formation as revealed by the analysis of trabecular architecture. In vitro studies demonstrated that ZOL was more potent against the OSRGA cell line than osteoblasts (with a half-maximal inhibitory effect on proliferation seen at 0.2 and 20 microM, respectively), the ZOL-induced inhibition of OSRGA proliferation being due to cell cycle arrest in S-phase. No effect on OSRGA apoptosis could be observed in vitro, as assessed by Hoechst staining and caspase-1 and -3 activation. In situ cell death was determined by TUNEL staining on tumor tissue sections. No significant difference in TUNEL-positive cells could be observed between ZOL-treated and -untreated rats. This is the first report of the anti-bone resorption and antitumoral activities of zoledronic acid in a rat model of osteosarcoma, and its beneficial association with an antitumoral chemotherapeutic drug in preventing tumor recurrence.

Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities.

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.

Cellular activity and signaling induced by osteoprotegerin in osteoclasts: involvement of receptor activator of nuclear factor kappaB ligand and MAPK.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), an inducer of osteoclastogenesis via its receptor RANK. We recently demonstrated that OPG also exerts a direct effect in osteoclasts by regulating protease expression. Herein, we showed that OPG-induced pro-matrix metalloproteinase-9 activity was abolished by ras/MAPK inhibitors in purified osteoclasts. OPG induced the phosphorylation of p38 and ERK1/2 in RAW264.7 cells. Only p38 activation was totally abolished by a blocking anti-RANKL antibody or an excess of RANKL. Surface plasmon resonance experiments revealed that RANK, RANKL and OPG are able to form a tertiary complex. These results suggested a potential formation of a tertiary complex RANK-RANKL-OPG on osteoclasts. Thus, OPG is not only a soluble decoy receptor for RANKL but must be also considered as a direct effector of osteoclast functions.

Regulation of osteoclast protease expression by RANKL.

Receptor activator of NF-kappaB ligand (RANKL) is essential for osteoclast (OC) differentiation/activation and functions through its receptor RANK at the surface of the osteoclastic cells. This study investigated for the first time the direct effects of hRANKL on protease/protease inhibitor expressions and protease activities in purified rabbit osteoclast cultures, using semi-quantitative RT-PCR, gelatin zymography, and enzymatic assays. RANKL was shown to exert in vitro pro-resorptive effects by increasing osteoclast marker expressions (Tartrate resistant acid phosphatase (TRAP) and cathepsin K), MMP-9 expression, and pro-MMP-9 activity and by diminishing TIMP-1 expression, leading to an up-regulation of the MMP-9/TIMP-1 ratio.

Osteoprotegerin differentially regulates protease expression in osteoclast cultures.

Cysteine proteases and matrix metalloproteinases (MMPs) are important factors in the degradation of organic matrix components of bone. Osteoprotegerin (OPG) is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. This study investigated the direct effects of human OPG on cathepsin K, MMP-9, MMP-2, and tissue inhibitors of metalloproteinases (TIMP1 and TIMP2) expressed by purified rabbit osteoclasts. The expression of two osteoclast markers, namely tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was inhibited by 100 ng/mL hOPG, whereas MMP-9 expression was enhanced. Gelatinase activities were measured using a zymographic assay, and hOPG was shown to enhance both pro-MMP-9 and MMP-2 activities. Concomitantly, TIMP1 expression was greatly stimulated by hOPG, whereas TIMP2 mRNA levels were not modulated. Overall, these results show that hOPG regulates the proteases produced by purified osteoclasts differentially, producing a marked inhibitory effect on the expression of cathepsin K, the main enzyme involved in bone resorption.

Osteogenic potential in vitro of human bone marrow cells cultured on macroporous biphasic calcium phosphate ceramic.

Calcium phosphate ceramics are synthetic bone substitutes that promote bone formation by osteoconduction. However, they have shown an osteogenic potential in vivo in animal models when associated with bone marrow cells. In order to develop an osteogenic human "hybrid material," we studied the in vitro osteogenic potential of human bone marrow cells cultured on macroporous biphasic calcium phosphate (BCP) pellets in the presence of dexamethasone with or without 1alpha,25 dihydroxyvitamin D3. We were examining, in particular, their mesenchymal stem cell proliferation and hematopoietic potential. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and immunological characterization of the extracellular matrix formed by these cells. The specimens were observed by scanning and transmission electron microscopy. Human mesenchymal stem cells proliferated on macroporous BCP ceramic, with a doubling time delayed at the beginning of the culture as compared to the cultures realized on plastic. These cells maintained a hematopoietic activity identical to that of cultures performed in plastic wells. The proliferating mesenchymal stem cells expressed an osteoblastic phenotype, as shown by alkaline phosphatase activity and the synthesis in ceramic macropores of an extracellular matrix composed of fibronectin, osteocalcin, and collagen I (but not collagen III). In addition, numerous microcrystals of apatite precipitated on the extracellular matrix, producing a mineralized fibrillar network within the ceramic not observed with cell cultures realized on plastic. These results demonstrate that human mesenchymal stem cells cultured on macroporous BCP ceramic express and conserve their osteoblastic phenotype even after one month of culture and that these osteogenic cells are able to form new bone matrix in a BCP ceramic in vitro.

Presence of leukaemia inhibitory factor (LIF) and LIF-receptor chain (gp190) in osteoclast-like cells cultured from human giant cell tumour of bone. Ultrastructural distribution.

The behaviour of multinucleated giant cells (MNC) obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized dentin slices was investigated using light and electron microscopy. Cells were studied in the presence or absence of LIF a cytokine known to be involved in bone turnover and to act as a growth factor in some solid tumours. The direct effect of LIF on MNC was examined by a post-embedding colloidal gold immunocytochemistry process using human anti-LIF and anti-LIF-receptor (chain gp190) antibodies. After 7 days of culture, the MNC obtained displayed osteoclast immunocytochemical features. Moreover, these MNC were able to resorb large amounts of dentin and presented typical features of active osteoclasts. Immunolocalization of LIF and LIF-receptor revealed the presence of this cytokine and its receptor within the cytoplasm and nucleus of active resorbing MNC. LIF upmodulated MNC number and nucleation but decreased their ability to resorb dentin. The present study suggests that MNC obtained from human GCT, currently considered as osteoclast-like cells, are targets for LIF and may be a source of LIF production in this pathological condition.

Expression of leukemia inhibitory factor by cartilage-forming tumors of bone: an immunohistochemical study.

Recent studies have implicated leukemia inhibitory factor in connective-tissue metabolism involving the remodeling of bone and the destruction of cartilage tissue. This cytokine, which has also been implicated in the proliferation of solid tumor, is expressed by osteotropic tumor cell lines. The present study investigated the presence of leukemia inhibitory factor in cartilage tissue harvested from cartilage-forming bone tumors. Immunohistochemical study showed that it was present in all benign enchondromas (n = 8) and malignant chondrosarcomas (n = 6) but not in control tissue (n = 3). The cytokine was localized in only cytoplasmic areas of cartilage cells. The number of stained cells ranged from less than 5% in enchondroma of the hand to more than 70% in grade-III chondrosarcoma. Moreover, high levels of leukemia inhibitory factor were found in the primary culture of tumor tissues (n = 7). These results question the significance of leukemia inhibitory factor in tumor-associated bone resorption and the potential role of this cytokine as a prognostic marker.

Growth hormone-loaded macroporous calcium phosphate ceramic: in vitro biopharmaceutical characterization and preliminary in vivo study.

Calcium phosphate ceramics recently have been used for administering therapeutic agents in bone. The present work investigated the efficacy of macroporous biphasic calcium phosphate (MBCP) implants as a matrix for local delivery of human growth hormone (hGH). An initial study showed that the release of 5 microg of hGH loaded onto MBCP cylinders was rapid during the first 48 h and sustained for a total of 11 days. The biological integrity of hGH (88.2%) was checked using a specific bioassay (cellular proliferation of hGH-sensitive Nb2 cells) in comparison with a radioimmunoassay to calculate the proportion of bioactive hGH released. MBCP cylinders then were loaded with 1, 10, and 100 microg of hGH and implanted into rabbit femurs (n = 16) to determine hGH effects on bone ingrowth and ceramic resorption, as evaluated by scanning electron microscopy and image analysis. Results indicated that hGH increased bone ingrowth and ceramic resorption significantly in comparison with contralateral and control implants. Biochemical parameters monitored in rabbit plasma showed that hGH did not produce detectable systemic effects. Thus the use of MBCP appears to be effective for local delivery of hGH and for increasing bone ingrowth.

Human growth hormone locally released in bone sites by calcium-phosphate biomaterial stimulates ceramic bone substitution without systemic effects: a rabbit study.

Calcium-phosphate bone replacement biomaterial has been used as a drug carrier for therapeutic agents. This study investigated the efficacy of local administration of human growth hormone (hGH) by macroporous biphasic calcium phosphate (MBCP) implants in improving the bone substitution qualities of ceramics. hGH release from MBCP implants loaded with 1 microg of hGH was rapid during the first 48 h and then sustained for a total of 9 days. Immunolocalization of hGH in vitro and in vivo by transmission electron microscopy showed its presence inside the material, indicating that it was able to penetrate within the porosity of the ceramic during the adsorption process. MBCP cylinders (6 x 6 mm) were loaded with 0.1, 1, and 10 microg of hGH and implanted into rabbit femurs (n = 40). The effects of locally released hGH on bone ingrowth and ceramic resorption were evaluated by scanning electron microscopy and image analysis. The results indicated that hGH increased bone ingrowth (+65%) and ceramic resorption (+140%) significantly in comparison with control implants and that the increase was dose dependent. Biochemical parameters monitored in rabbit plasma and urine, as well as the absence of any significant difference between contralateral implants and the control, indicated that hGH did not produce detectable systemic effects. Thus, the use of MBCP appears to be effective for local delivery of hGH, resulting in improved bone substitution.