A collagen based scaffold for the repair of annulus fibrosus defects

Abstract

The intervertebral disc (IVD) is the natural cartilaginous cushion found between the osseous vertebrae of the spinal column. It is an essential element for the flexibility of the spine but undergoes major degeneration with age, resulting in the loss of its functionality and in back and leg pain. Due to the large size of the IVD, its avascular nature and the difficulty for nutrients and waste particles to diffuse in or out of the IVD, damage and defects incurred by the IVD, be they natural or accidental, are very unlikely to heal and often trigger faster degeneration. The annulus fibrosus (AF) defines the outer boundaries of the disc and provides the structural integrity of the IVD. The purpose of this thesis was to evaluate the regenerative potential of a collagen type II scaffold on standardized defects of the AF. Specifically, this thesis evaluated in vitro the effects of the culture conditions, of a select group of growth factors, of the cross-linking method and of the biochemical composition of the scaffold, in the perspective of preparing a suitable autologous cell-seeded implant for a goat animal model. Cross-linking method and the presence of GAG dramatically affected cell proliferation and new tissue synthesis, with the most promising results obtained with dehydrothermal treatment (DHT) cross-linking and with scaffolds containing 5 to 10% of chrondroitin-6-sulfate.

(cont.) Autologous cells seeded in this promising scaffold and cultured in a serum-free environment supplemented with a mix of Transforming Growth Factor-[beta]1 and Fibroblast Growth Factor-2, maintained annulus cell morphology, GAG and type II': collagen synthesis, while exhibiting controlled contraction and a significant amount of new synthesized tissue just after a week in culture. Although culture in a rotating bioreactor showed encouraging results, lack of reliability in the replication of the experiments as well as uniformity in the resulting scaffold geometry and poor cell distribution in the scaffold, did not enable this culture condition to be a good candidate for implantation. To investigate the regenerative potential of this implant, an autologous AF cell-seeded scaffold was cultured for 10 days in a DHT cross-linked scaffold in the serum-free culture medium mentioned above. At this time, the implant consisted in a loose matrix of connective tissue mostly composed of fibrochondrocytic cells and type I and II collagens. When surgically implanting this scaffold in a standardized defect of the annulus fibrosus at the L4-L5 lumbar disc level, results showed slight reduction in fibrous tissue and increase in the fibrocartilaginous tissues 2 months postsurgery compared to controls or controls where a scaffold without cells was used.

(cont.) However, there was no significant improvement in the gross morphology of the disc. The results of this thesis recommend the use of an autologous AF cell-seeded type II collagen scaffold in the perspective of regeneration of defects of the annulus fibrosus. Future animal experiments should aim at locating the cells in the implant, evaluate the mechanical properties of the regenerated tissue and compare AF cells to stem cells that are easier to be harvested.