Purpose: Cartilage erosion and ligamentous instability are common in advanced carpometacarpal (CMC) arthritis and often necessitate surgical intervention. An autogenous interpositional cartilage construct consisting of viable chondrocytes in an alginate matrix would be a useful adjunct for the surgical management of CMC arthritis. The purpose of this project was to optimize the proliferation and viability of rabbit chondrocytes in culture. We specifically examined the effects of rabbit serum and ascorbic acid supplementation on cell viability, cell retention, and cellular metabolism characteristics in an alginate-chondrocyte construct.
Methods: Rabbit chondrocytes were harvested and cultured in DMEM/F-12 media. The media was supplemented with 10% or 20% rabbit serum (RS) or, as control, 10% fetal bovine serum (FBS), with or without 200 ug/ml ascorbic acid. The chondrocytes were suspended in a 3% calcium-alginate matrix and the entire construct was maintained in vitro for up to 60 days in media plus either 20% RS or 20% FBS, with or without further supplementation from 200 ug/ml ascorbic acid. Gel matrices were dissolved to disperse the cells for assessment of viability and cell retention at 15, 30, 45, and 60 days. Collagen type II production within the construct was determined by immunohistochemical techniques and collagen infiltration within the alginate matrix architecture was examined by sirius red staining.
Results: Cells grown in RS demonstrated significantly higher rates of proliferation, viability, and resistance to growth crisis than cells in control FBS. 20% RS exhibited a 143% increase in cell count (P < 0.05) and showed increased viability, from 89.6% to 95%, as compared to control. In addition, cells grown in ascorbic acid-supplemented media demonstrated significantly higher rates of proliferation and viability than control, unsupplemented cultures (32.9% increase in cell count and 6.3% increase in viability; P < 0.05). Furthermore, 20% RS showed a substantial ability to reverse the quiescent growth arrest noted in late-passage senescent chondrocytes and promote metabolic activity. When examining matrices histologically and by cell count, alginate-chondrocyte constructs maintained in RS showed higher levels of cellular retention and higher levels of viable chondrocytes than constructs maintained in FBS (170% increase in cellular retention and 216% increase in viability; P < 0.05). Collagen type II production and the presence of a collagenous architecture were notably enhanced in constructs maintained in RS. Ascorbic acid supplementation significantly improved retention and viability.
Conclusion: The addition of both rabbit serum and ascorbic acid to chondrocyte culture media significantly increased cell proliferation and viability in our model. Furthermore, constructs maintained in media supplemented with rabbit serum and ascorbic acid demonstrated higher levels of cell viability and greater cell retention. Our optimization techniques also substantially enhanced the metabolic activity of chondrocytes in the alginate matrix as evidenced by marked increases in cellular collagen type II production, collagen infiltration within the alginate architecture, and reversal of metabolic growth arrest. Increases in collagen type II, a marker for a normal chondrocytic phenotype, suggests a method for preventing cellular dedifferentiation - a problem often encountered with in vitro cartilage constructs. The use of species-specific serum and ascorbic acid have optimized our in vitro conditions for the construction of alginate-chondrocyte matrices and hold obvious advantages for subsequent in vivo cartilage constructs addressing upper extremity joint pathologies.