Development of an in vitro outer annulus fibrosus-cartilage endplate interface model

Chong, Jasmine 1, 2 ; Kandel, Rita 1, 2, 3, 4 ; Santerre, Paul 1, 5;

 1.  Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2.  Lunenfeld-Tanenbaum Research Institute; 3.  Pathology and Laboratory Medicine, Sinai Health System; 4.  Laboratory Medicine  and Pathology, University of Toronto; 5.  Faculty of Dentistry, University of Toronto

INTRODUCTION: The intervertebral disc consists of annulus fibrosus (AF) tissue that surrounds a centrally placed nucleus pulposus. It is integrated to adjacent vertebral bodies through a cartilage endplate (CEP). Disc degeneration can lead to chronic neck or lower back pain. Current surgical treatments include discectomy, disc fusion and prosthetic disc implants; however, these treatments are not completely restorative. Attention has turned to developing a biological disc replacement that mimics native tissue and does not induce further degeneration. Integration of an engineered disc with adjacent vertebral bodies through a cartilage endplate is required to ensure mechanical stability of the implant replacement.

HYPOTHESIS: In vitro formed OAF tissues will integrate with in vitro formed cartilage to generate a mechanically stable interface model that resembles the composition of native OAF-CEP interface. This model will allow for investigation of mechanical factors that influence integration.

METHODS: OAF cells and articular chondrocytes were isolated from bovine caudal discs and synovial joints respectively. OAF cells were cultured on multilamellar electrospun nanofibrous polycarbonate urethane scaffolds in bioreactors for 2 weeks. Chondrocytes were placed onto collagen type II coated membranes and cultured for 3 days. Resulting OAF and cartilage tissues were co-cultured in direct contact for up to 4 weeks to form an OAF-cartilage interface. The interface was evaluated histologically by H&E and toluidine blue staining, and the composition was determined by immunohistochemical staining for collagen type I, collagen type II, and aggrecan. To determine if chondrocytes migrated into the OAF tissue, in select cultures chondrocytes were labelled with green fluorescent CFDA dye prior to culture. Interface strength was evaluated by a pull-apart test.

RESULTS: When 2-week old OAF tissues were co-cultured with 3-day old cartilage, integration occurred within one week for all cultures. OAF cells near the interface showed directional alignment parallel to the cartilage layer. The interface stained positively for collagen type I in the OAF and in the pericellular regions of the chondrocytes within the cartilage. Collagen type II and aggrecan were localized to the cartilage layer. Chondrocytes remained localized to the cartilage tissue and did not appear to migrate up into the OAF. This was similar to that seen for the native bovine OAF-CEP interface. Preliminary results demonstrated that the interface between the OAF and cartilage had mechanical strength.

CONCLUSION: This study demonstrated that it is possible to engineer an OAF-CEP interface model in vitro that has mechanical strength. Future study will focus on identifying factors (e.g. dynamic compressive loading) that may affect interface strength.