Abstract
BackgroundOsteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage—the remodeling of which is important in the osteoarthritic disease process—have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments.MethodsMature human osteoclasts were cultured on ECMs isolated from bovine knees—articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)—in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H+-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett’s T3 post hoc test, as applicable.ResultsOsteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)—similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p < 0.0001). Osteochondral C2M release was only inhibited by E-64 (49% of vehicle, p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16).ConclusionsOur study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.
Highlights
Osteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro
Osteochondral extracellular matrix (ECM) characterization and model optimization The osteochondral ECM was characterized by assessing the presence of calcified cartilage interspersed with subchondral bone, by safranin O-fast green staining of femoral condyle slices, and the overall calcification of the osteochondral matrix, by von Kossa staining of noncrushed strips of osteochondral matrix
Prior to removing the remaining articular cartilage from the partially denuded femoral condyles, the subchondral bone was still covered by some articular cartilage (Fig. 1a, stained red)
Summary
Osteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. Bone is resorbed by osteoclasts through secretion of hydrochloric acid dissolving the inorganic matrix [2] and proteases, mainly the cysteine protease cathepsin K [3,4,5] and matrix metalloproteinases (MMPs) [6,7,8], degrading the organic matrix. In addition to their well-established role in normal bone turnover, osteoclasts play important roles in diseases with progressive joint destruction, in particular in bone erosion in diarthrodial joints in rheumatoid arthritis (RA) [9,10,11,12,13]. The differentiation, activity and survival of osteoclasts is tightly regulated in healthy individuals [21] whereas monocytes isolated from patients with OA exhibit increased osteoclastogenesis, elevated resorption, and reduced osteoclast apoptosis [22]
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