Dedifferentiation and redifferentiation of articular chondrocytes from surface and middle zones: changes in microRNAs-221/-222, -140, and -143/145 expression.

Articular cartilage contains three functional zones (superficial, middle, and deep) characterized by distinct structure, composition, and biomechanical properties. One of the unsolved major challenges in cartilage tissue engineering is to produce tissue that mimics the zonal organization of the native articular cartilage. An increasing number of studies aim to design zonal organization into tissue-engineered cartilage by forming a stratified construct using zonal cell subpopulations. However, in vitro monolayer expansion of chondrocytes, which is generally required to obtain high cell numbers necessary for tissue engineering and autologous chondrocyte implantation, leads to dedifferentiation of chondrocytes into fibroblast-like cells, resulting in loss of zonal markers, such as the superficial zone protein (SZP) of the superficial zone as well as chondrocytic phenotype markers, such as type II collagen and aggrecan. Several microRNAs (miRNAs), including miR-221, miR-222, miR-143, and miR-145, have been identified from bovine articular cartilage as superficial zone-enriched miRNAs. miR-140 has been known as a cartilage-specific miRNA whose expression is implicated in chondrocyte differentiation and cartilage tissue homeostasis. As miRNAs play an important role in regulating gene expression during cell differentiation and maintaining tissue homeostasis, we determined the expression of the miRNAs with zonal differentiation and homeostasis. We investigated how chondrocyte dedifferentiation during multiple passages and redifferentiation in a three-dimensional (3D) agarose culture regulates the expression of these miRNAs by quantitative reverse transcription-polymerase chain reaction. Additionally, the effect of transforming growth factor beta 1 (TGF-β1), which is known to enhance chondrocytic differentiation and SZP expression, on these miRNAs was evaluated. The expression of miR-221 and miR-222 increased during dedifferentiation and during redifferentiation in a 3D culture and TGF-β1 restored them to normalcy. miR-140 dramatically decreased during dedifferentiation and its expression is partially recovered in a 3D culture with TGF-β1. miR-143 and miR-145 in the superficial chondrocytes decreased during dedifferentiation and further decreased in a 3D culture, but TGF-β1 partially recovered their expression in a 3D culture. In conclusion, the expression patterns of the miRNAs will be of functional utility for strategies and approaches to tissue engineering of the articular cartilage.