The annulus fibrosus (AF) of the intervertebral disc is a multi-lamellar fibrocartilage that, together with the nucleus pulposus, confers mechanical support and flexibility to the spine. Function of the AF is predicated on a high degree of structural organization over multiple length scales: aligned collagen fibers reside within each lamella, and the direction of alignment alternates between adjacent lamellae from +30° to −30° with respect to the transverse axis of the spine. Electrospinning permits fabrication of scaffolds consisting of aligned arrays of nanofibers, and has proven effective for directing the alignment of both cells and extracellular matrix (ECM) deposition [1–3]. We recently employed electrospinning to engineer the primary functional unit of the AF, a single lamella [4]. However, it remains a challenge to engineer a multi-lamellar tissue that replicates the cross-ply fiber architecture of the native AF. Moreover, relatively few studies have considered functional properties of engineered AF, and, when measured, tensile properties of these constructs have been inferior to native AF [4]. In this study, mesenchymal stem cells (MSCs) were seeded onto aligned nanofibrous scaffolds organized into bi-lamellar constructs with opposing or parallel fiber orientations, and their functional maturation was evaluated with time. Additionally, we determined a novel role for inter-lamellar ECM in reinforcing the tensile response of bilayers, and confirmed this mechanism by testing acellular bilayers with controllable interface properties.

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