To determine the level of anabolic response when chondrocytes isolated from human osteoarthritic cartilage are stimulated with 2 doses of insulin-like growth factor-I (IGF-I) for extended culture periods.
Human chondrocytes were isolated from knee cartilage removed at the time of joint replacement surgery for osteoarthritis (OA). The cells were cultured in alginate beads under serum-free conditions and treated with 100 ng/ml or 1000 ng/ml of human recombinant IGF-I. Response was measured during culture periods of 1 to 28 days by determining the level of radiolabeled sulfate incorporated into alcian blue precipitable material and by measuring the level of total proteoglycan accumulation using the dimethylmethylene blue (DMB) assay. For the latter assay, cultures treated with osteogenic protein-1 (OP-1) were used for comparison to IGF-I. Results were normalized to cell numbers using DNA measurements.
The level of IGF-I stimulated sulfate incorporation relative to untreated controls increased with time in culture, with a peak response occurring between days 7 and 14 of culture. There was no significant difference between the 2 IGF-I doses. Despite the stimulation of sulfate incorporation, the DMB assay did not reveal a significant accumulation of proteoglycans in the cell-associated and further-removed matrix with either dose of IGF-I in cultures carried out to 21 days. In contrast, compared to controls, OP-1 at 100 ng/ml stimulated a 3-fold increase in matrix proteoglycan at day 21 of culture.
Prolonged IGF-I treatment of human OA chondrocytes in serum-free alginate cultures stimulated sulfate incorporation without significant accumulation of a proteoglycan matrix in longterm cultures. However, significant proteoglycan accumulation was seen in cultures treated with OP-1, suggesting it is a better stimulator of proteoglycan production by OA chondrocytes.