Figure 8. Reduced collagen gel contraction by Mmp13^−/− mouse skin fibroblasts.

(A) Skin fibroblasts (MSF) established from wild type (WT) and MMP-13 knockout (Mmp13^−/−) mice were cultured in mechanically unloaded (floating) 3D collagen gel at density 2×10⁵/ml for 24 h in the presence of 0.5% FCS, 10% FCS or 0.5% FCS+TGF-β (5 ng/ml), as indicated. The cells were fixed, stained with fluorescently labeled phalloidin and Hoechst, and photographed with 20× magnification to observe morphological appearance. In contrast to Mmp13^−/− MSF, WT fibroblasts displayed stellate morphology with numerous thick cell extensions in response to TGF-β or 10% FCS (Scale bar = 10 µm). (B) WT and Mmp13^−/− MSF were cultured in mechanically unloaded 3D collagen gel at density 5×10⁵/ml for 24 and 48 h in the presence of 10% FCS. Contraction of collagen gels was measured from digital images of the gels and is shown as relative to the original gel size. (*P<0.005 compared to control, Independent samples T-test, n = 4) (C) WT and Mmp13^−/− MSF were cultured in attached 3D collagen gel at density 5×10⁵/ml for 72 h in the presence of 10% FCS. Subsequently the gels were detached from the well walls and contraction was quantified after 24 h. (*P<0.005 compared to control. Independent samples T-test, n = 3). (D) MSF were cultured for 72 h in 3D collagen gel in the presence 10% FCS. Equal aliquots of conditioned media were analyzed in gelatinase zymography.