Tissue-Engineered Human Auricular Cartilage Demonstrates Euploidy by Flow Cytometry

Abstract

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.

Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage

“…The medium is also supplemented with 10% serum and growth factors which are necessary for efficient cell number expansion in the shortest period of time [18][19][20][21]. Monolayer culture system has been recommended for human chondrocyte expansion [22,23] and the passaged chondrocytes were proven to retain the diploidy feature, hence are safe for cartilage regeneration [24,25]. However, cell expansion in monolayer system led to a problem in dedifferentiation due to failure in producing functional cartilaginous extracellular matrix (ECM) [28].…”

“…In previous studies, human auricular chondrocytes were cultured in Ham's F12 medium for tissue engineering research [24][25][26][27]. It is known that culture medium plays an important role in supporting the chondrocytes proliferation rate and cell characteristic; yet comparison on the types of basal media which promote higher chondrocyte growth and preserve phenotype expression has not been reported.…”

Formation of in vivo tissue engineered human hyaline cartilage in the shape of a trachea with internal support

“…The covering of HDP with tissue engineered cartilage would make it more bioinert in autologous transplantation thus solving the problem of rejection [23][24][25]. The safety of using long-term cultured human chondrocytes has been proven by Kamil and Aminuddin that human chondrocytes cultures in multiple passages have not shown aneuploidy by DNA analysis [26,27]. The quality of cultured human chondrocytes for trachea reconstruction was clarified by RT-PCR analysis that the cultured chondrocytes showed high expression of type II collagen gene, a specific type of collagen in mature cartilage matrix.…”

Tissue engineering possibilities in articular cartilage regeneration

“…В качестве стимуляторов роста хряща в разное время с разным успехом были использованы ИЛ-4 [18,33], поли-гликоевая кислота, активин-А, фактор роста хондроцитов, ретиноевая кислота [52], ядерный фактор активирования Т-клеток, фактор роста фибробластов [50], хрящевой морфо генетический белок [15].…”