Abstract
Objective
To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor β (TGFβ)–induced osteophyte formation.
Methods
In vivo, synovial lining macrophages were selectively depleted by injection of clodronate‐laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFβ into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP‐2) and BMP‐4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May‐Grünwald–Giemsa.
Results
Triple injections of 20 ng or 200 ng of TGFβ into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFβ injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFβ, respectively). Synovial lining cells produced BMP‐2 and BMP‐4 after TGFβ stimulation, whereas BMP‐2 and BMP‐4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFβ concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFβ was very effective in generating large spheroids, suggestive of macrophage‐derived (co)factors. In coculture supernatants, TGFβ concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation.
Conclusion
These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFβ, probably by inducing other chondrogenic signals.