Inhibition of Cartilage Degradation and Changes in Physical Properties Induced by IL-1β and Retinoic Acid Using Matrix Metalloproteinase Inhibitors

Abstract

Bovine cartilage explants were treated with 100 ng/ml recombinant human interleukin-1β (IL-1β) or 1 μmall-transretinoic acid (RA) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, samples cultured with IL-1β or RA were treated with 4 μmrecombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) or a synthetic metalloproteinase inhibitor (L-758,354) to inhibit this degradation. Treatment with IL-1β or RA each resulted in >90% GAG loss after 8 days in culture. Addition of TIMP or L-758,354 to the culture media inhibited IL-1β-induced loss of tissue GAG by 40 and 65%, respectively, and inhibited RA-induced GAG loss by 35 and 65%, respectively. Analysis of degradation products in the culture media using a G1 antibody indicated that IL-1β- and RA-treated plugs released 68-kDa fragments of aggrecan, corresponding to a segment of the aggrecan core protein from the G1 domain to the C-terminus NITEGE, consistent with “aggrecanase” activity. Release of the G1 fragment was inhibited by treatment with L-758,354. Both IL-1β and RA induced significant loss of hyaluronan from cartilage explants after 8 days of exposure and HA loss was also inhibited by addition of L-756,354 to the culture media. IL-1β, but not RA, induced a significant increase in swelling ratio (wet weight in 0.01mNaCl normalized to wet weight in DMEM) after 8 days in culture, consistent with degradation of the collagen network, and the increase in tissue swelling was inhibited by treatment with TIMP-1 or L-758,354. Exposure to IL-1β or RA resulted in significant changes in cartilage physical properties including streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient after 8 days in culture, and these changes were inhibited by 40–90% by exposure to TIMP and by 50–90% by exposure to L-758,354. Measurement of dynamic streaming potential showed that changes due to treatment with IL-1β alone were highly dependent in compression frequency, with dramatic changes seen at high frequency prior to changes in mechanical properties, and little initial change seen at low frequency. Streaming potential and equilibrium modulus of explants treated with RA decreased to 10% of their initial values after 8 days in culture, but decreased to only 40 and 90%, respectively, when treated with RA plus TIMP-1.