The intervertebral disc is composed of load-bearing fibrocartilage that may be subjected to compressive forces up to 10 times the body weight. The multilaminated outer layer, the annulus fibrosus (AF), is vulnerable to damage and its regenerative potential is limited, sometimes leading to nuclear herniation. Scaffold-based tissue engineering of AF using stem cell technology has enabled the development of bi-laminate constructs after 10 weeks of culture. It is difficult to know if these constructs are limited by the differentiation state of the stem cells or the culture system. In this study, we have characterized an expandable scaffold-free neoconstruct using autologous AF cells. The construct was prepared from pellet cultures derived from monolayer cultures of AF cells from mature pigs that became embedded in their own extracellular matrix. The pellet cultures were incubated for 24 h in a standardized conical tube and then carefully transferred intact to a culture flask and incubated for 21 days to allow continued matrix synthesis. Cell viability was maintained above 90% throughout the culture period. The engineered scaffold-free construct was compared with the native AF tissue by characterization of gene expression of representative markers, histological architecture, and biochemical composition. The morphological and biochemical characteristics of the cultured disc construct are very similar to that of native AF. The cell number per gram of construct was equal to that of native AF. Expression of aggrecan was elevated in the engineered construct compared with RNA extracted from the AF. The glycosaminoglycan content in the engineered construct showed no significant difference to that from native construct. These data indicate that scaffold-free tissue constructs prepared from AF cells using a pellet-culture format may be useful for in vitro expansion for transplantation into damaged discs.
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