Tissue engineered cartilage: utilization of autologous serum and serum-free media for chondrocyte culture.

BACKGROUND: Standard culture medium contains bovine serum. If standard culture methodology is used for future human tissue-engineering, unknown risks of infection from bovine disease or immune reaction to foreign proteins theoretically might occur. In this study we wished to evaluate the potential of chondrocyte expansion using autologous and serum free media. METHODS: Autologous auricular cartilage was harvested in a swine model. An initial concentration of 100x10(3) cells per group were expanded in three groups. Group A, F-12 with 10% fetal calf serum; Group B, F-12 supplemented with 10% autologous serum; Group C, F-12 supplemented with growth factors. Cell numbers were counted at days 3, 6, 9 and 12. RESULTS: The cells in all the three groups exhibited normal chondrocyte morphology. At early time points there was a statistically significant difference in the number of cells between Group A and the two other groups (p<0.05). By day 12, both Groups A and C demonstrated greater cell number as compared to Group B (p<0.05). CONCLUSION: The results suggest that both autologous serum as well as serum free media might be substituted for the expansion of the number of chondrocytes, thus avoiding the potential need for a bovine serum supplement.

Tissue engineering of a human sized and shaped auricle using a mold.

OBJECTIVES: The creation of a tissue-engineered auricle was initially successful in an immunocompromised nude mouse model. Subsequently, an immunocompetent porcine model successfully generated a helical construct. We wished to evaluate the novel technique of using a mold to create a complete, anatomically refined auricle in a large animal model. METHODS: Mixtures of autogenous chondrocytes and biodegradable polymers were used inside a perforated, auricle shaped hollow gold mold. Three biodegradable polymers (calcium alginate, pluronic F-127, and polyglycolic acid) were used to retain the seeded chondrocytes inside the mold. These molds, along with a control, were implanted subcutaneously in the abdominal area of 10 animals (pigs and sheep). The constructs were removed after 8 to 20 weeks and were assessed by gross morphology and histology. RESULTS: All the gold implants were well tolerated by the animals. The implants using calcium alginate (n = 3) generated constructs of the exact shape and size of a normal human ear; the histology demonstrated mostly normal cartilage with some persistent alginate. The implants with pluronic F-127 (n = 3) resulted in cartilage with essentially normal histology, although leakage outside the molds and external cartilage generation was noted. Polyglycolic acid implants (n = 3) produced no useful cartilage because of an inflammatory reaction with fibrosis. The empty control mold (n = 1) demonstrated only a very small amount of fibrous tissue inside. CONCLUSION: A tissue-engineered human sized auricle of normal anatomic definition can be generated in an immunocompetent large-animal model using a mold technique. Although further refinements will be necessary, the technique appears promising for potential use in patients with microtia.

Expansion of the number of human auricular chondrocytes: recycling of culture media containing floating cells.

To grow a complete human size auricle by utilizing the principles of tissue engineering, a large number of chondrocytes is required for initial implantation. The number of chondrocytes can be increased by repeated passaging or by incubation with different growth factors, both of which can promote dedifferentiation. New methods of chondrocyte expansion over a relatively brief time period for potential practical application are required. In this study auricular chondrocytes were obtained from patients and cultured in vitro. Two groups of cells were created. Group A chondrocyte number was increased by repeated passaging. Group B cells were grown from floating culture medium and their number was increased both by passaging and by repeated recycling of the culture medium. Chondrocytes from both groups were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Flow cytometry studies performed on both groups confirmed the presence of two distinct populations of structures as the source of chondrocytes from the recycled medium. Repeated recycling of the culture medium demonstrates a promising method to increase the number of chondrocytes in vitro for clinical application.

Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry.

Transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are known to stimulate the rate of chondrocyte proliferation. The theoretical risk of malignant transformation associated with growth factor stimulation of chondrocytes should be addressed; aneuploidy has been found to occur in human cartilaginous tumors. In this study, chondrocytes were obtained from six human auricles and cultured in vitro for 6 weeks in the presence or absence of TGF-beta and bFGF. Cells were analyzed for DNA at 3-, 4-, 5-, and 6-week intervals by flow cytometry (FACScan), which demonstrated no evidence of aneuploidy. A persistent increase in S-phase was noted in cells cultured only with TGF-beta. Cells were implanted in athymic mice, and after 8 weeks of implantation, the cartilage constructs formed were examined histologically. The tissue-engineered cartilage cultured originally in bFGF most resembled normal, native cartilage. Specimens cultured in TGF-beta produced suboptimal cartilage morphology. Flow cytometry shows no evidence of aneuploidy, with chondrocytes maintaining their normal diploid state. Further studies incorporating additional methods of analysis need to be done.