Figure 2.

Fibronectin fragment (FN-f)-stimulated matrix metalloproteinase-13 (MMP-13) production in ankle chondrocytes from young and old donors. A, MMP-13 was assessed by immunoblotting culture media samples from cells isolated from donors of different ages and stimulated in serum-free media with FN-f as described in Figure 1. Media were collected from wells with equal cell numbers (2 × 10⁶ cells/well) and normalized for equal total protein loaded per lane (20 μg/lane). B, MMP-13 immunoblots from 89 different donors were scanned, and densitometric data were obtained for control (unstimulated) and FN-f-stimulated MMP-13 bands and expressed as the ratio of proMMP-13 after FN-f treatment/baseline proMMP-13. These results were regressed versus donor age (r = 0.38, p = .003). C, A full-length MMP-13 promoter–luciferase reporter and a control Renilla luciferase reporter were transiently cotransfected into cells from a young and an old donor followed by treatment with 1 μM FN-f or interleukin-1β (IL-1β) at 5 ng/ml or left untreated (basal activity). After 24 hours, the cells were lysed and subjected to dual luciferase assay. Results are means of duplicate transfections and are representative of experiments performed with two sets of young and old donors with each set performed in parallel.