RGTA modulates the healing pattern of a defect in a monolayer of osteoblastic cells by acting on both proliferation and migration.

ABSTRACT

A family of heparan-like polymers, RGTAs, was shown to promote repair of various tissues. Like heparin and heparan-sulfates, RGTAs potentiate in vitro the biological activities of heparin-binding growth factors (HBGFs) and protect them against proteolytic degradation. It was postulated that RGTAs stimulate bone healing by interacting with HBGFs released in the wound site and, subsequently, by promoting the proliferation of cells implicated in this process. In a previous report, we examined how RGTA can modulate the proliferation of MC3T3-E1 osteoblastic cells. To further complete this study and to support this hypothesis, we developed an in vitro model of bone repair and examined the effects of RGTA alone or in association with FGF2, BMP-2, and TGF-beta1 which are representative of HBGFs known to stimulate bone repair. The model consisted of a 6-mm reproducible defect created on a MC3T3-E1 cell monolayer. In the presence of the different products added to the medium, the process of wound repair was measured through the filling of the acellular defect. We show that in 8 days, RGTA slightly inhibits repair alone compared to the control (2% FBS), that it inhibits the mitogenic effect of FGF2, and that it amplifies the inhibitory effect of BMP-2 and TGF-beta1. Repair was realized by an association of cell migration and cell proliferation mechanisms. To determine the part played by each process, DNA synthesis was evaluated for cell proliferation using an immunodetection technique [to measure incorporation of 5-bromo-2-deoxyuridine (BrdU)], coupled with a computer-assisted image analysis. The results show that the presence of RGTA (1) amplified the number of labeled nuclei compared to the control, (2) added to FGF2 or TGF-beta1, it reduced the number of labeled nuclei compared to FGF2 or TGF-beta1 alone, and (3) in the presence of BMP-2, it amplified the number of labeled nuclei compared to BMP-2 alone. Proper interpretation of these data requires a better understanding of the mechanism of action of RGTA on bone healing.