Characterization of ex vivo-generated bovine and human cartilage by immunohistochemical, biochemical, and MRI analyses


Ashleigh E Nugent, David A Reiter, Kenneth W Fishbein, Denise L McBurney, Travis Murray, Dorota Bartusik, Sharan Ramaswamy, Richard G Spencer, Walter E Horton


Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and MR endpoints similar to the original native cartilage. Chondrocytes were isolated from 1-3 week old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7mg/mL collagen I, and plated as 300µL spot cultures (SCs) with 5x10^6 cells each. Media formulations were varied including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the peri-cellular region. The ex vivo-formed bovine cartilage contained more chondroitin sulfate (CS) per dry-weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo-formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-SC model supports formation and maturation of 3-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.