Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks

Objective-To produce tissue engineered cartilage by human articular chondro cytes in vitro for further use in in vivo manipulations for the treatment o f cartilage defects. Methods-Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal concentration of an alginate matrix for cell replication and for aggrecan synthesis by chondrocytes was determined. DNA content in the different culture conditions was measured af ter two and four weeks. Aggrecan synthesis rates and accumulation in the su rrounding extracellular matrix were assessed by [S-35]sulphate incorporatio n after the same periods of culture. To follow the outgrowth of chondrocyte s from the alginate beads, chondrocytes were cultured for four weeks in 0.5 or 1.0% alginate surrounded by 0.25 or 0.5% fibrin gel. DNA content of eac h culture was measured after different culture periods. Finally, human chon drocytes in 1.0% alginate beads were embedded in 0.5% fibrin gel for eight weeks. Immunohistochemical analysis for aggrecan, type I and II collagen wa s performed weekly. Results-At two weeks the DNA content in each culture significantly increase d in 0.5 and 1.0% alginate cultures in comparison with baseline values. Thi s increase continued until week 4 at the three alginate concentrations. Agg recan synthesis at two weeks was highest in 0.5 and 1.0% alginate cell cult ures. At four weeks aggrecan synthesis rates decreased independently of the alginate concentrations. Aggrecan mainly accumulated in the interterritori al matrix. Proliferation of chondrocytes in alginate and outgrowth of these cells in the surrounding fibrin gel were evident throughout the culture pe riod. The accumulation of aggrecan and type II collagen around the cells, i n alginate as well as in fibrin gel, gradually increased over the culture p eriod. Type I collagen appeared after six weeks in alginate and in the surr ounding fibrin. Conclusion-Human chondrocytes proliferate in this culture system, show an o utgrowth into the surrounding fibrin, and synthesise a cartilage-like matri x for up to eight weeks.