Effect of Passage Number and Collagen Type on the Proliferative, Biosynthetic, and Contractile Activity of Adult Canine Articular Chondrocytes in Type I and II Collagen-Glycosaminoglycan Matrices in Vitro

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Abstract
One tissue-engineering approach being investigated for the treatment of defects in articular cartilage involves the implantation of autologous chondrocyte-seeded absorbable scaffolds. The present study evaluated the effects of passage number (freshly isolated and passages 1 and 2) and collagen type on the proliferative, biosynthetic, and contractile activity of adult canine articular chondrocytes grown in type I and II collagen–glycosaminoglycan (GAG) matrices that were cross-linked by dehydrothermal/carbodiimide treatment. P0, P1, and P2 cells seeded in the type II matrices continued to proliferate over a 4-week period, but thereafter the P0 and P1 cells continued to increase in number and the P2 cells decreased. At 4 weeks the DNA contents of the type I and II matrices seeded with P1 and P2 cells were comparable, and higher than the values for matrices seeded with freshly isolated chondrocytes. The rates of protein and GAG synthesis by the P1 and P2 cells were comparable, and higher than the rates for the P0 chondrocytes, after 1 week, and the rates were generally higher in the type II than in the type I collagen scaffolds. Western blot analysis demonstrated the presence of newly synthesized type II collagen in type II matrices in which P1 and P2 cells were grown. The cross-linking treatment imparted a sufficient degree of mechanical stiffness to both types of matrices to resist cell-mediated contraction. This study demonstrated that adult articular chondrocytes expanded in number through two passages in monolayer culture can be expected to provide behavior comparable to or better than freshly isolated cells with respect to proliferation and biosynthesis through 4 weeks of culture in collagen–GAG matrices, and these cells retain the capability to synthesize type II collagen. The results of this investigation further commend the use of a type II collagen–GAG matrix, based on the higher biosynthetic rates of the cells grown in the matrices, for the preparation of chondrocyte-seeded scaffolds for articular cartilage tissue engineering.