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.

中文翻译：

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无支架的组织工程，具有对齐的骨髓基质细胞表层，以概括纤维环的微结构和生化成分。

通过基于支架的方法的当前组织工程策略未能概括天然纤维环组织的复杂三维微结构和生化组成。考虑到患者对健康的环状纤维细胞的访问有限，本研究探索了通过将微模式技术整合到多层BMSC工程中，骨髓基质细胞（BMSC）制造无支架的多层环状纤维样组织的潜力。在微沟槽图案的PDMS基板上显影出具有细胞和胶原纤维（相对于其纵向尺寸在约30°方向）排列的BMSC板。然后将两张纸按交替的方向堆叠在一起，形成一层斜角双层薄纸，将其卷起，切片以形成多层角膜组织并在定制培养基中培养。通过组织学，基因表达以及显微和机械分析进一步表征纤维环样组织的发育。我们证明，具有对齐的BMSC片的工程化的纤维环样组织显示出平行的胶原纤维，生化成分和类似于天然椎间盘的微结构。此外，与未对齐的细胞片相比，对齐的细胞片显示增强的纤维环相关细胞外基质标记的表达和更高的机械强度。本研究为纤维环组织工程学方法的开发提供了一种新的策略，以开发无支架的纤维环样组织，该组织类似于天然组织的微结构和生化特性。