M2-macrophages modulate the cartilage-forming capacity of human bone marrow-derived mesenchymal stromal cells

Purpose: Monocytes play a pivotal role in controlling tissue inflammation and repair. They infiltrate the tissues and develop into two main subsets of macrophages in response to local signals: inflammatory (M1-) and tissue-repair (M2-) macrophages (Mφ). Here we investigated whether M1 and M2φ differentially modulate chondrogenesis of mesenchymal stem/stromal cells (MSC). Methods: Monocytes isolated from buffy-coats were cultured for 5 days with GM-CSF or MCSF to induce respectively M1- or M2φ-polarization. MSC were isolated from the bone marrow of a total of 9 patients and expanded for two passages. MSC or skin fibroblasts (as control cells) and polarized macrophages were cultured in collagen scaffolds up to 3 weeks alone or after being mixed (at the ratio 1:1). In order to determine the reached cell number, selected constructs were generated by combining carboxyfluorescein succinimidyl ester (CFSE) labeled MSC with macrophages. To investigate the role of soluble factors in modulating MSC differentiation, conditioned media harvested from M1φ and M2φ were supplemented during the chondrogenic culture of MSC. Resulting tissues and/or isolate cells were assessed histologically (glycosaminoglycan, GAG), biochemically (GAG contents), cytofluorimetrically (percentage of cells expressing or not CD45, and CFSE) and by RT-PCR (Sox9 and collagen-II) Results: Coculture of MSC/M2φ in scaffolds resulted in statistically significant 1.9-fold higher GAG content than what would be expected (defined as chondro-induction). Chondro-induction was lower (1.3±0.4) and less reproducible when coculture was performed with M1φ and did not occur with skin fibroblasts. GAG contents of constructs generated by solely macrophages were undetectable. Histological analyses of constructs confirmed the biochemical results. In the coculture there was no modulation of the chondrogenic genes. As compared to monocultures, in co-culture MSC and M2φ numbers decreased less markedly (at day 7, MSC were 84% and 42% of the initial number, M2φ were 26% of the initial number and undetectable, respectively for co-cultured and monocultured cells). Conclusions: We have demonstrated that coculture MSC/Mφ2 results in synergistic cartilage tissue formation, which is not mediated by soluble factors alone. Further studies are envisioned to investigate whether Mφ2 modulate the survival of specific MSC sub-populations. Finally, in vivo studies are necessary to assess the clinical relevance of our findings in the context of cartilage repair.