Background/Purpose: Osteoarthritis (OA) is associated with cartilage breakdown, where degradation of aggrecan by ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) is thought to be an early event of the breakdown. Animal studies have suggested that inhibition of aggrecan degradation inhibits cartilage breakdown, whereas inhibition of the later collagen degradation has limited effect on cartilage preservation. We investigated the effect of M6495, an anti-ADAMTS-5 inhibiting Nanobody®, on cartilage turnover in explant cultures.

Methods: Bovine cartilage explants (BEX, N=4), human OA cartilage explants from replaced knee joints (HEX, N=8), and from 1 healthy human knee joint (hHEX) were cultured for 21 days in medium alone (w/o), in pro-inflammatory cytokines (oncostatin M [10 ng/mL] + TNFα [20 ng/mL] (O+T)) or O+T with M6495 [1 µM–1 nM]. Cartilage and synovium from cows (bCC) and OA human knee joints (hCC, N=4) were co-cultured for 28 days in w/o, with O+T or O+T plus M6495 [1 μM–0.6 nM]. Metabolic activity was assessed by Alamar Blue. Cartilage tissue turnover was assessed by ELISA (huARGS, AGNxI, C2M and ProC2) in conditioned medium, which are measurement of type II collagen and aggrecan degradation and type II collagen formation. Data was analyzed by 1-way and 2-way ANOVA.

Results: Metabolic activity of BEX, HEX, and bCC was stable throughout the culture period, whereas the metabolic activity in hCC and hHEX dropped markedly from day 14 in O+T treated conditions compared to w/o. In cultures stimulated with O+T, metabolites of ADAMTS-5 degraded aggrecan peaked within the first week of the culture, except for hHEX in which huARGS and exAGNxI increased slightly later. Type II collagen degradation, C2M, by O+T peaked after day 19. Type II collagen formation, ProC2, remained relatively stable throughout the cultures, compared to the w/o control. In BEX, treatment with M6495 in combination with O+T decreased huARGS with the highest doses on day 5 (8% of O+T); in HEX (40% of O+T), bCC (10% of O+T), hCC (40% of O+T), and hHEX (24% of O+T) (Fig.). The effect of M6495 on exAGNxI was similar to huARGS in the cultures tested. M6495 also reduced C2M (marker for type II collagen degradation) significantly, albeit the effect was less than for aggrecan degradation markers. M6495 had no effect on on ProC2.

Conclusion: Here, we have shown that the Nanobody® M6495 has cartilage protective effects due to its inhibition of ADAMTS-5-mediated aggrecan degradation and MMP-mediated type II collagen degradation in pro-inflammatory conditions of bovine and human cartilage cultures and in co-cultures of cartilage and synovium.