AbstractObjectiveDefects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril–associated proteins type IX collagen and cartilage oligomeric matrix protein (COMP) during cartilage matrix formation.MethodsPrimary chondrocytes from mice deficient in type IX collagen and COMP (double‐deficient) were cultured in monolayer or alginate beads. Anchorage of matrix proteins, proteoglycan and collagen content, collagen crosslinks, matrix metalloproteinase activity, and mechanical properties of the matrix were measured. Electron microscopy was used to study the formation of fibrillar structures.ResultsIn cartilage lacking both type IX collagen and COMP, matrilin 3 showed decreased matrix anchorage. Less matrilin 3 was deposited in the matrix of double‐deficient chondrocytes, while larger amounts were secreted into the medium. Proteoglycans were less well retained in the matrix formed in alginate cultures, while collagen deposition was not significantly affected. Electron microscopy revealed similar cartilage collagen fibril diameters in the cultures of double‐deficient and wild‐type chondrocytes. In contrast, a larger fibril diameter was observed in the matrix of chondrocytes deficient in only type IX collagen.ConclusionOur results show that type IX collagen and COMP are involved in matrix assembly by mediating the anchorage and regulating the distribution of other matrix macromolecules such as proteoglycans and matrilins and have counteracting effects on collagen fibril growth. Loss of type IX collagen and COMP leads to matrix aberrations that may make cartilage more susceptible to degeneration.