Bone remodelling and tumour grade modifications induced by interactions between bone and swarm rat chondrosarcoma.

Chondrosarcoma is currently defined as a malignant cartilage tumour arising de novo or within a pre-existing benign cartilage tumour. Chondrosarcoma can be surgically resected, but all grades have significant rates of local recurrence. The purpose of the present study was to develop an animal intraosseous chondrosarcoma model simulating the progression of human chondrosarcoma and elucidating its behaviour and biology. An intraosseous Swarm rat model was designed to assess interactions between bone and chondrosarcoma. A comparison of tumour grading was carried out according to transplantation site. The effects of chondrosarcoma cells (SRC cells) on the mineralisation capacities of osteoblasts and on osteoclast differentiation were studied in relation to modifications observed in vivo at the cellular level. Transplantation of Swarm rat chondrosarcoma within bone marrow or contiguous to induced periosteal lesions led to extensive bone remodelling with trabecular bone rarefaction and periosteal apposition. Transplantation in close contact to bone but without any periosteal lesion had no effect on bone, suggesting that bone healing factors interact with tumour development. With the intramedullary model, the development of tumours of different grade confirms that bone environment is an important factor in malignancy. A decrease of bone nodule formation was noted after cocultures of SRC cells with rat bone marrow, but there was no modification of osteoclast differentiation after cultures of total rabbit bone cells with SRC cells. These data reveal the importance of interactions between bone environment and tumour in inducing bone remodelling and variations in tumour malignancy.

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells.

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.

Modulation by soluble factors of gelatinase activities released by osteoblastic cells.

This study investigated the ability of normal human osteoblasts (hOb) and osteogenic sarcoma cells (MG-63 and SaOS2) to produce gelatinases and undergo modulation by interleukin 1beta (IL-1beta), interleukin 6 (IL-6), oncostatin M (OSM), leukaemia inhibitory factor (LIF), growth hormone (GH) and insulin-like growth factor-I (IGF-I). Gelatinase activities were determined by zymogaphy, and a quantitative analysis was performed by ELISA. The MMP-2 activities of the three cell lines were significantly increased in the presence of IL-1beta and IL-6, but no modulation of MMP-2 activities was observed in the presence of OSM, LIF and GH. IGF-I increased the activity released by SaOS2 and hOb, but no modulation was detectable in MG-63 cell conditioned medium. An upmodulation of pro-MMP-2 secretion by SaOS2 and hOb was observed for all soluble factors used, whereas an upmodulation of pro-MMP-2 secretion by MG-63 was observed only in the presence of IL-1beta, IL-6 and IGF-I. Thus, osteoblastic cells modulated by cytokines can be involved in bone resorption as a result of the protease activities released.

Inhibition of apatite formation by vitronectin.

This study investigated the concentration-dependent effect of vitronectin (VN), a glycoprotein of the bone matrix, on apatite formation and growth. Precipitation trials in metastable solution and in a pH-controlled solution system showed an inhibition of apatite microcrystal formation by VN. In the presence of biphasic calcium-phosphate ceramic, transmission electron microscopy showed a reduction of precipitated microcrystal size: precipitates were significantly smaller than in ionic simulated body fluid without proteins or in the presence of type I collagen as a negative control. Moreover, the size of the precipitated microcrystals was reduced in a dose-dependent manner. Two indirect methods showed that calcium-phosphate precipitation was inhibited by VN. It would appear that VN prevents apatite formation by inhibiting the growth of apatite crystals rather than by secondary nucleation, as in the case of osteopontin, a bone-specific protein.

[In vitro effects of growth hormone on osteoclastic activity: clinical applications]. / Effets in vitro de l'hormone de croissance sur l'activité des ostéoclastes : intérêt clinique.

PURPOSE OF THE STUDY: This study was designed to investigate the in vitro effects of human growth hormone (hGH) on osteoclastic resorption in a nonfractionated rabbit bone cell model. MATERIAL AND METHODS: Rabbit bone cells were cultured on dentine slices in the presence of parathyroid hormone and vitamin D3. The percentage of dentine slice surface resorbed, number of lacunae per surface unit and mean area of lacunae were compared between cell cultures grown in the presence of graded concentrations of hGH and human insulin-like growth factor-1 (hIGF-1) and controls. RESULTS: After 4 days of culture, rabbit bone cells cultured on dentine slices in the presence of hGH and hIGF-1 showed significantly stimulated osteoclastic resorption activity. When neutralizing anti-hIGF-1 anti-serum (4 microg/l) was added to the starting culture, the stimulatory effects of hIGF-1 and hGH on osteoclastic resorption activity were totally abolished. DISCUSSION: These findings indicate that the effects of hGH stimulation on osteoclastic resorption in vitro are mediated via local hIGF-1 secretion by stromal cells such as osteoblasts. Proteases appear to play a role in the degradation of the organic matrix. Our experiments show that hIGF-1 and hGH stimulate the production of matrix metalloproteinases MMP-9 and MMP-2. Similar to the resorption activity, hGH stimulates protease activity via stromal cell production of hIGF-1. CONCLUSION: This study suggests that natural or synthetic MMP inhibitor modulation of protease activity could reduce the degradation of the organic matrix and then prevent, for example, inflammatory reactions subsequent to prosthetic loosening.

Cysteine protease production by human osteosarcoma cells (MG63, SAOS2) and its modulation by soluble factors.

The production of cysteine protease by two human osteosarcoma cell lines (MG-63 and SaOS2) was analyzed, as well as their modulation by interleukin 1beta (hIL-1 beta), interleukin 6 (hIL-6), insulin growth factor-1 (hIGF-1), oncostatin M (hOSM), leukemia inhibitory factor (hLIF) and growth hormone (hGH). Cysteine protease activities were detected using a synthetic substrate. The protease activities (especially cathepsin L activity) of both cell lines were increased significantly in the presence of hIL-1 beta, hIL-6 and hOSM. In contrast, hIGF-1 and hGH decreased these activities, and no effect was detectable in the presence of hLIF. The addition of antibodies against the gp-130 chain of the hIL-6 and hOSM receptors totally inhibited the stimulating effect of these two cytokines on cysteine protease activities. In increasing collagen type I degradation, hIL-1beta, hIL-6 and hOSM could be involved in bone resorption, whereas the inhibitory action of hIGF-1 and hGH on collagen type I degradation suggest that this factor could play a role in bone formation.