Characterisation of the osteoclastogenic potential of human osteoblastic and fibroblastic conditioned media

Abstract

Although M-CSF and RANKL are sufficient to promote in vitro osteoclastogenesis, in vivo this is a complex process which requires the action of many signalling molecules and cellular crosstalks. In this work, isolated or combined conditioned media, obtained from human adult skin fibroblast and bone marrow cells, were tested for their osteoclastogenic potential, through an indirect co-culture system, in the absence of recombinant M-CSF and RANKL. Osteoclastogenesis was assessed on human peripheral blood mononuclear cells (PBMC) and CD14+ cell cultures by quantification of total protein content, tartrate-resistant acid phosphatase (TRAP) activity, presence of multinucleated cells positive for TRAP, RT-PCR of TRAP, CATK, CA2, c-myc and c-src and presence of multinucleated cells displaying actin rings, vitronectin and calcitonin receptors. Cultures supplemented with M-CSF and RANKL were used as positive controls. It was observed that the conditioned medium from dexamethasone osteogenic-induced bone marrow cell cultures displayed the highest osteoclastogenic potential, with similar behaviour to that observed in the presence of both M-CSF and RANKL. Comparatively, fibroblastic conditioned medium elicited a slightly lower osteoclastogenic response. Combination of both conditioned media resulted in a significant increase of TRAP activity. On the other hand, conditioned medium from non-osteogenic-induced bone marrow cell cultures presented the lowest osteoclastogenic potential. These results were observed for both PBMC and CD14+ cell cultures, suggesting that fibroblast and osteoblast cells are able to modulate osteoclastogenesis in the absence of physical cell-cell interactions. In addition, osteoclastogenic potential of bone marrow cells increases with their osteoblastic differentiation.

Reciprocal osteoblastic and osteoclastic modulation in co-cultured MG63 osteosarcoma cells and human osteoclast precursors

“…PBMC expressed the osteoclast differentiation and activation genes c-myc and c-src [Zhao et al, 2007], and, also, the functional related genes TRAP, CATK, and CA2 [Zhao et al, 2007;Datta et al, 2008]. However, in spite of the increasing values of TRAP activity throughout the culture time, this cell population displayed low number of TRAP-positive multinucleated cells and limited calcium phosphate resorbing activity, which is in line with previous studies showing that development of osteoclast cells in vitro is low in the absence of osteoclastogenic inducing factors [Costa-Rodrigues et al, 2010]. Co-cultured MG63 I þ PBMC revealed total protein content higher than that found in the corresponding monocultures, as expected due to the presence of the two-cell populations.…”

“…Osteoblast-osteoclast communications, involving cell-cell contacts and paracrine mechanisms, have a central role in the maintenance of a proper bone structure [Matsuo and Irie, 2008;Costa-Rodrigues et al, 2010], and the presence of osteosarcoma cells usually led to disturbed bone metabolism [San-Julian et al, 2003;Avnet et al, 2008]. In the present work, osteosarcoma MG63 cells were cocultured with osteoclast precursor cells aiming to assess the reciprocal cellular interactions.…”

Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

“…Human peripheral blood mononuclear cell cultures Peripheral blood mononuclear cells (PBMC) were isolated from blood of healthy male donors with 25-35 years old, as described previously [25]. After dilution with PBS (1:1), blood was applied on top of Ficoll-Paque TM PREMIUM (GE Healthcare Bio-Sciences) and centrifuged at 400g for 30 min.…”

“…This cell line was selected because MG63 cells are believed to be relatively immature osteoblast-like cells [19] and are known to express low amounts of RANKL [20][21][22][23][24]. Both are relevant issues, as the modulation of osteoclastogenesis by osteoblasts is reported to be dependent on the degree of osteoblast differentiation [25] and RANKL is a key player in this process [3,5]. In addition, the osteoclastogenic effects induced by human MG63 osteosarcoma cell line through paracrine mechanisms are scarcely documented.…”

Variability of the paracrine‐induced osteoclastogenesis by human breast cancer cell lines

“…Cell cultures were performed in the absence or presence of recombinant 25 ng/ml M-CSF and 40 ng/ml RANKL [Costa-Rodrigues et al, 2010] (negative and positive control, respectively), or in the presence of 10% or 20% (v/v) of CM from the human breast cancer cell lines. Cell cultures were incubated for 21 days at 378C in a 5% CO 2 humidified atmosphere.…”