9. THE EFFECT OF REMAINING AT CONFLUENCE ON THE CHONDROCYTIC PHENOTYPE

Abstract

Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and collagen II gene expression over time.

Method: Fresh normal human articular cartilage was collected from deceased donor patients. The matrix was digested and the chondrocytes were plated in monolayer to create two groups. The first group was cultured and passaged 2? at confluence seven times. The second group was cultured at confluence and left for seven weeks, with medium changes every 3–4 days without passaging. At weekly intervals RNA was extracted from cells in both groups and analyzed with real time PCR, probing specifically for the genes responsible for the production of collagen I, collagen II, aggrecan, and GAPDH. This was done in duplicate.

Results: Collagen II gene expression was maintained over seven weeks in cells left at confluence but was decreased in passaged cells. Collagen I gene expression decreased over seven weeks in cells left at confluence, but remained the same in passaged cells. Aggrecan gene expression remained the same in both groups.

Conclusion: Current culture and expansion techniques that employ passaging (as used in clinical scenarios) result in significant alterations in gene expression that are inconsistent with the current definition of a “chondrocyte”. Culturing chondrocytes at confluence can produce gene expression more similar to native chondrocytes but even these cells have expression of collagen type I that should not be present in chondrocytes. The results of this study suggest that further investigation is required to develop chondrocyte culture and expansion techniques that minimize the de-differentiation of chondrocytes by maintaining collagen II gene expression and eliminating/preventing collagen I gene expression.