Scaffolds composed of synthetic, natural, and hybrid materials have been investigated as options to restore intervertebral disk (IVD) tissue function. These systems fall short of the lamellar features of the native annulus fibrosus (AF) tissue or focus only on the nucleus pulposus (NP) tissue. However, successful regeneration of the entire IVD requires a combination approach to restore functions of both the AF and NP. To address this need, a biphasic biomaterial structure was generated by using silk protein for the AF and fibrin/hyaluronic acid (HA) gels for the NP. Two cell types, porcine AF cells and chondrocytes, were utilized. For the AF tissue, two types of scaffold morphologies, lamellar and porous, were studied with the porous system serving as a control. Toroidal scaffolds formed out of the lamellar, and porous silk materials were used to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm, and a height of 3 mm (the interlamellar distance in the lamellar scaffold was 150-250 μm, and the average pore sizes in the porous scaffolds were 100-250 μm). The scaffolds were seeded with porcine AF cells to form AF tissue, whereas porcine chondrocytes were encapsulated in fibrin/HA hydrogels for the NP tissue and embedded in the center of the toroidal disk. Histology, biochemical assays, and gene expression indicated that the lamellar scaffolds supported AF-like tissue over 2 weeks. Porcine chondrocytes formed the NP phenotype within the hydrogel after 4 weeks of culture with the AF tissue that had been previously cultured for 2 weeks, for a total of 6 weeks of cultivation. This biphasic scaffold simulating in combination of both AF and NP tissues was effective in the formation of the total IVD in vitro.
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