Reconstruction of Fibrocartilage with Fibrous Alignment of Type I Collagen in Scaffold-Free Manner.

For functional reconstruction of fibrocartilage, it is necessary to reproduce the essential mechanical property exhibited by natural fibrocartilage. The distinctive mechanical property of fibrocartilage is originated from the specific histological features of fibrocartilage composed ofhighly-aligned type I collagen (Col I) and an abundant cartilaginous matrix. While the application of tensile stimulation induces highly aligned Col I, our study reveals that it also exerts an anti-chondrogenic effect on scaffold-free tissues constructed with meniscal chondrocytes (MCs) and induces down-regulation of Sox-9 expression and attenuated glycosaminoglycans (GAGs) production. Modulation of mechanotransduction by blocking nuclear translocation of Yes-associated protein (YAP) ameliorated anti-chondrogenic effect in the presence of tensile stimulation. Since MCs subjected to mechanical doses either by surface stiffness or tensile stimulation showed reversibility of YAP status even after a long-term exposure to mechanotransduction, fibrocartilage tissue was constructed by sequentially inducing tissue alignment by tensile stimulation followed by inducing cartilaginous matrix production in a tension-released state. The minimal tensile dose to constitute durable tissue alignment was screened by investigating the alignment of cytoskeleton and Col I after culturing the scaffold-free tissue constructs with various tensile doses (10% static tension for 1, 3, 7, 10 days) followed by maintaining in a released state for 5 days. Fluorescence-conjugated phalloidin binding and immunofluorescence of Col I indicated that the duration of static tension for more than 7 days resulted in durable tissue alignment for at least 5 days in tension-released state. The tissues subjected to tensile stimulation for 7 days followed by 14 days in a released state in chondrogenic media resulted in abundant cartilaginous matrix as well as uniaxial anisotropic alignment. Our results show that optimized tensile dose can facilitate the successful reconstruction of fibrocartilage by modulating the characteristics of matrix production by MCs.