Characterization of Chondrocyte Behaviors Affecting Quality of Cultured Cartilage

Khoshfetrat A.B *, Kino-Oka M , Taya M ,


Objective: Chondrocyte cells exhibited different behaviors when cultured at low seeding density (X0=2.0×105 cells/ cm3), as compared to high seeding density (X0=2.0×106 cells/ cm3) in collagen gels to generate tissue-engineered cartilage (1). The cells in the low-density culture migrated to form loose aggregates, showing less production of collagen type II. In this research, we examined morphological behaviors of chondrocyte cells in more detail and evaluated quantitatively the effect of seeding density on the migration of chondrocytes in collagen gels. Since communications among chondrocytes via autocrine/paracrine signaling can affect behaviors of the cells, we also evaluated the effect of a well-known chondrogenic growth factor, transforming growth factor-beta1 (TGF-beta1), on the migration of chondrocytes in collagen gels and architecture of cultured cartilage. Materials and Methods: Rabbit chondrocyte cells were cultured at different seeding densities, ranging from X0=1.0×105 to X0=1.6×106 cells/cm3, in collagen gels. Cytoplasm and collagen type II were stained, and stereoscopic observation was performed by using confocal laser scanning microscopy (CLSM) to define the migrating cells by sphericity values (Sc) of individual cells at 5 days. The cultured cartilages were observed at 10 days after seeding using CLSM in terms of spatial distribution, morphology and collagen type II formation. To study the effect of TGF-beta1 on chondrocyte behaviors and architecture of cell aggregates, the cells were also cultured at low seeding density (X0=2.0×105 cells/cm3) for a 14-day period and exposed to various TGFβ1 concentrations in range of 0 to 10 ng/ml. The quantitative evaluation of morphology and the histological observation of cultured gel were carried out at culture days of 5 and 14, respectively. Gene expression analysis of migration- and differentiation-related genes was also performed by real time RT-PCR to support the morphological evaluation of the cell behaviors in the research. Results: The chondrocytes underwent a transition to a spindle-shaped morphology, then started to transform again to original phenotype after migration and gathering in the starburst aggregates which was accompanied by poor production of collagen type II in the culture seeded at X0=1.0×105 cells/cm3 during a 10-day culture period. In contrast, the cells proliferated normally in the dense aggregates of semilunar-shaped cells with rich excretion of collagen type II in the culture seeded at X0=1.6×106 cells/cm3. Quantitative evaluation of morphology at 5 days also revealed that the frequency of migrating cells (Sc