BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus

Different approaches for disc regeneration are currently under investigation. Beside gene therapy and tissue engineering techniques, the application of growth and differentiation factors own promising potential. Studies using reduced intervertebral disc models, such as cell or tissue fragment cultures, have limited validity and show controversial results depending on the employed experimental model. Therefore, the goal of the current study was to investigate the effect of BMP-2 and TGF-beta 3 on intervertebral disc degeneration using an in vitro full-organ disc/endplate culture system. Intervertebral rabbit disc explants were cultured in the presence of 1 mu g/ml BMP-2 or TGF-beta 3 for 21 days in DMEM/F12 media. Nucleus and annulus were analyzed for gene expression of collagen type I and II (Col I/II), aggrecan, collagenases (MMP-1/MMP-13) with RT-qPCR, histological changes with bone and proteoglycan-specific staining (von Kossa, toluidine blue) and differences in cellularity (DNA) and proteoglycan content (alcian blue binding assay). The results demonstrate that disc proteoglycan concentration decreased with time in the TGF-beta 3 and BMP-2 groups. In the annulus fibrosus (AF), TGF-beta 3 and BMP-2 resulted in an up-regulation of Col I and type II, and of aggrecan gene expression. In contrast, MMP genes were inhibited. In the nucleus, the growth factors decreased gene expression of aggrecan and spontaneous Col I up-regulation was inhibited by TGF-beta 3, whereas expression of Col II was decreased with BMP-2. There was no effect on expression of MMP-1 and MMP-13 for most sampling points. However, TGF-beta 3 and BMP-2 induced ossification of the AF was demonstrated by histology. It can be concluded that both growth factors, at the tested concentrations, may not be suitable to regenerate the whole intervertebral disc organ but they are interesting candidates for being injected alone or in combination into a painful intervertebral disc to induce osseous fusion (spondylodesis).