W.M. Oxner, MD, C. Latterman, MD, Lars Gilbertson, PhD, P.D. Robbins, James D. Kang, MD, Pittsburgh, PA, USADegenerative disc disease (DDD) poses a substantial clinical problem. At present, therapeutic options include highly invasive surgical procedures, such as the insertion of interbody fusion devices. Such approaches may put the adjacent discs at risk for accelerated degeneration. A novel approach to slow DDD is to introduce high levels of growth factors into the degenerating disc by delivering the gene coding for the appropriate growth factor. Previous work has shown that gene therapy using adenovirus coding for TGF-b, IGF-1 and BMP-2 results in an increase in the production of matrix by the nucleus pulposus cells. To date, no investigators have looked at gene therapy and the catabolic cascade. IL-1, stromelysin and chondroitinase ABC (CABC) are substances that have a known role in the catabolic cascade, which leads to degeneration within the intervertebral disc. The purpose of this study is to evaluate gene therapy using adenovirus coding for IGF-1, TGF-b, BMP-2 and TIMP-1 to determine their potential for inhibiting the catabolic effects of IL-1, stromelysin and CABC in vivo using a previously validated pellet culture system for human nucleus pulposus cells.Materials and methods: Human nucleus cells were isolated and cultured. The first passage was passed into T25 flasks and allowed to propagate to 80% confluency. The cells were transduced in monolayer by adding 100 MOI of viral particles in serumless media and allowing a 4-hour transfection period, after which the cells were covered with fresh media. Groups included a negative control, positive control (adeno-LacZ), IGF-1, TGF-b, BMP-2 and TIMP-1. Twenty-four hours after transfection, the cells were pelletized into pellets of 200,000 cells each and transferred into 24 well plates. Four days later, each of the groups were challenged by adding IL-1, CABC and stomelysin to the culture media, and then the culture media was changed for another 4 days. Outcomes were measured by analyzing pellet size, total proteoglycan content and histology with Safranin O staining for qualitative proteoglycan content.Results: Nucleus pulposus cells transduced with BMP-2, TGF-b, TIMP-1 and IGF-1 all produce significantly more total proteoglycans that both the positive and negative controls. In response to CABC (0.001 units/ml), all pellets were depleted of more than 70% of their proteoglycans. Pellets transduced with the above-mentioned cytokines were able to produce more proteoglycans after removal of the CABC than were the controls. In response to IL-1 and stomelysin, the proteoglycans of the pellets transduced with the cytokines showed significantly less proteoglycan depletion and less loss in their size than did both the positive and negative control pellets.Conclusion: CABC, IL-1 and stromelysin all deplete proteoglycans of nucleus pulposus pellets in vivo. Pellets tranduced with adenovirus coding for IGF-1, TGF-b, TIMP-1 and BMP-2 all were less severely affected by the addition of IL-1 and stromelysin. Whereas none of the cytokines protected the pellets from the deleterious effects of CABC, the pellets transduced with the cytokines were able to regenerate proteoglycans more quickly than control.
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Jan 1, 2003
Thomas Pap 
Open Access
