Comparison of matrix metalloproteinase-13 (MMP-13) production in knee and ankle chondrocytes from the same donors. Culture media from equal numbers of normal knee and ankle chondrocytes from the same 58-year-old donor (A) or from young (35-year-old) and old (67-year-old) donors (B) were assessed for MMP-13 production by immunoblotting. Cells were made serum-free for 18 hours prior to each experiment. Samples are from concentrated (10X) serum-free culture media without (–) and with fibronectin fragment (FN-f) treatment for 24 hours with 1 μM 110 kd fibronectin fragment (FN-f) or interleukin-1β (IL-1β) at 5 ng/ml and were equalized for total protein loaded per lane. Cells preincubated with the protein kinase C inhibitor rottlerin at 4 μm (PKCi) were also tested.

Interleukin-1β (IL-1β)–stimulated matrix metalloproteinase-13 (MMP-13) production increases with donor age. Experiments were performed as described in Figure 2B, except that cells were stimulated with IL-1β at 5 ng/ml.

Chondrocyte production of interleukin-1β (IL-1β) increases with donor age. Serum-free culture media from unstimulated primary human chondrocytes cultured as described in Figure 1 were assessed for constitutive (baseline) IL-1β production over 24 hours using 200 μl of unconcentrated medium and an ultrasensitive IL-1β enzyme-linked immunosorbent assay (ELISA). Diamonds represent the means of duplicate wells for each donor.

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.

Inhibition of fibronectin fragment (FN-f)-stimulated matrix metalloproteinase-13 (MMP-13) production by a nuclear factor- kB (NFkB) inhibitor peptide. Serum-free, 24-hour unconcentrated media from primary human chondrocytes of a 76-year-old donor, prepared as in Figure 1, were assessed for MMP-13 production by immunoblot. Cells were treated as follows: untreated, 110 kd FN-f (1 μM) alone, FN-f with NKkB inhibitory peptide (12 μM), FN-f with manufacturer-supplied control peptide (12 μM). Cells were counted and judged >95% viable by trypan blue, and loading in each lane was normalized for total protein loaded. Data are representative of experiments performed using three different donor cultures.