Effect of tissue expansion on chondrocyte sheets in cartilage composite reconstruction

Flap prelamination has been successfully established in tissue engineering; however, cartilage generation through combination of an expanded flap and chondrocyte sheets has not been reported. Herein, we investigate the effect of tissue expansion on chondrocyte sheets in prelaminating an expanded chondrocutaneous flap. Chondrocyte sheets were implanted into a tissue expander capsule following which capsule inflation was performed weekly. At 4 and 12 weeks post implantation, the specimens were examined with histology and immunohistochemistry analyses. Extracellular matrix (ECM) formation and type II collagen deposition in the regenerated cartilage tissue in vivo were also examined. After 4 weeks of implantation, the generated cartilage was phenotypically stable with minimal hypertrophy, while that formed after the 12-week expansion showed visible hypertrophic differentiation. To evaluate the effect of static pressure and/or hypoxic conditions generated by the expanding tissue, static pressure and/or hypoxic conditions were reproduced in vitro. The chondrocyte sheets stimulated by mechanical static pressure and hypoxia maintained their chondrogenic phenotype. The expression of aggrecan, collagen II, Sox-9, and HIF-1α was increased in chondrocyte sheets cultured in 2% oxygen (hypoxia); however, aggrecan, collagen II, and Sox-9 were downregulated in the static pressure/normoxia group. These results suggest that the expanded environment promoted cartilage formation by the chondrocyte cell sheets, while mechanical forces and hypoxic conditions in vitro allowed chondrocyte cell sheets to retain their chondrogenic phenotype.