Human adipose stem cells in chondrogenic differentiation medium without growth factors differentiate towards annulus fibrosus phenotype in vitro

Intervertebral disc degeneration is the main cause of chronic back pain. Disc degeneration mainly leads to tearing of annulus fibrosus (AF), which is with current methods difficult to restore and impossible to regenerate. Stem cell technology offers a potential technique to repair the ruptured AF by enabling new matrix synthesis at the defect site. Previous studies have shown that human adipose stem cells (hASCs) are able to differentiate towards AF phenotype when treated with suitable growth factors. There are concerns about the use of growth factors in clinical applications because of their short half-lives, high costs and low effectiveness. The main aim of this research project was to regenerate AF tissue in vitro using hASCs and serum free chondrogenic medium without supplementation of growth factors. Differentiation of hASCs was induced by using the micromass culture technique. Human annulus fibrosus cell (hAFCs) cultured in commercial AF growth medium were used as positive control. Assessment of AF phenotype of hASCs and hAFCs was done at 14 and 21 days of culture. Quantification of sulphated glycosaminoglycan (GAG) content showed that hASCs cultured in chondrogenic medium expressed similar levels of sulphated GAGs as hAFCs. qRT-PCR confirmed the similarity of the differentiated hASCs with AF phenotype. Several markers for AF phenotype (aggrecan, collagen type I and glypican-3) were expressed in both hAFCs and differentiated hASCs. This is the first study that demonstrated that hASCs can be differentiated towards AF phenotype using serum free chondrogenic medium without growth factors. In a next step, scaffolds manufactured from biodegradable polymers will be used in combination with ASCs to find an optimal construct to repair AF defects.