* In Vitro Generated Intervertebral Discs: Toward Engineering Tissue Integration.

Abstract

The intervertebral disc (IVD) is composed of nucleus pulposus (NP) surrounded by multilamellated annulus fibrosus (AF), and is located between the vertebral bodies. Current treatments for chronic neck or low back pain do not completely restore the functionality of degenerated IVDs. Thus, developing biological disc replacements is an approach of great interest. Given the complex structure of the IVD, tissue engineering of the individual IVD components and then combining them together may be the only way to achieve this. The engineered disc must then be able to integrate into the host spine to ensure mechanical stability. The goal of this study was to generate an integrated model of an IVD in vitro. Multilamellated AF tissues were generated in vitro using aligned nanofibrous polycarbonate urethane scaffolds and AF cells. After 3 weeks in culture, it was placed around NP tissue formed on and integrated with a porous bone substitute material (calcium polyphosphate). The two tissues were cocultured to fabricate the IVD model. The AF tissue composed of six lamellae containing type I collagen-rich extracellular matrix (ECM) and the NP tissue had type II collagen- and aggrecan-rich ECM. Immunofluorescence studies showed both type I and II collagen at the AF-NP interface. There was evidence of integration of the tissues. The peel test for AF lamellae showed an interlamellar shear stress of 0.03 N/mm. The AF and NP were integrated as the pushout test demonstrated that the AF-NP interface had significantly increased mechanical stability by 2 weeks of coculture. To evaluate if these tissues remained integrated, allogeneic IVD model constructs were implanted into defects freshly made in the NP-inner AF and bone of the bovine coccygeal spine. One month postimplantation, the interfaces between the AF lamellae remained intact and there was integration with the host AF tissue. No inflammatory reaction was noted at this time period. In summary, an engineered IVD implant with mechanically stable integration between AF lamellae and AF-NP can be generated in vitro. Further study is required to scale up the size of this construct and evaluate its ability to serve as a biological disc replacement.