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Three-dimensional (3D) culture in porous biomaterials and stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated if an initial boost with transforming growth factor-β1 (TGF-β1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells.Materials and Methods   Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of nonabsorbable poly vinylidene fluoride. An initial boost of TGF-β1 or TGF-β1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan, type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13.Results   An initial boost with TGF-β1 or TGF-β1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from, both, slightly degenerated or severely degenerated tissue, are capable of significant upregulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower upregulations in some matrix molecules such as aggrecan.Conclusion   We failed to show a supportive effect of an initial boost with TGF-β1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems. Disclosure of Interest A. A. Hegewald has no conflict of interest and C. Kaps is an employee of TransTissue Technologies GmbH, which develops products in the field of regenerative medicine.

Effects of Initial Boost with TGF-Beta 1 and Grade of Intervertebral Disc Degeneration on 3D Culture of Human Annulus Fibrosus Cells