Scaffold-Free tissue engineering with aligned bone marrow stromal cell sheets to recapitulate the microstructural and biochemical composition of annulus fibrosus

Current tissue engineering strategies through scaffold-based approaches fail to recapitulate the complex three-dimensional microarchitecture and biochemical composition of the native Annulus Fibrosus tissue. Considering limited access to healthy annulus fibrosus cells from patients, this study explored the potential of bone marrow stromal cells (BMSC) to fabricate a scaffold-free multilamellar annulus fibrosus-like tissue by integrating micropatterning technologies into multi-layered BMSC engineering. BMSC sheet with cells and collagen fibres aligned at ~30° with respect to their longitudinal dimension were developed on a microgroove-patterned PDMS substrate. Two sheets were then stacked together in alternating directions to form an angle-ply bilayer tissue, which was rolled up, sliced to form a multi-lamellar angle-ply tissue and cultured in a customized medium. The development of the annulus fibrosus-like tissue was further characterized by histological, gene expression and microscopic and mechanical analysis. We demonstrated that the engineered annulus fibrosus-like tissue with aligned BMSC sheet showed parallel collagen fibrils, biochemical composition and microstructures that resemble the native disk. Furthermore, aligned cell sheet showed enhanced expression of annulus fibrosus associated extracellular matrix markers and higher mechanical strength than that of the non-aligned cell sheet. The present study provides a new strategy in annulus fibrosus tissue engineering methodology to develop a scaffold-free annulus fibrosus-like tissue that resembles the microarchitecture and biochemical attributes of a native tissue. This can potentially lead to a promising avenue for advancing BMSC-mediated annulus fibrosus regeneration towards future clinical applications.