Adenovirus mediated transfer of SOX-9 and bone morphogenetic protein–2 genes to human degenerative disc chondrocytes

Abstract

Ronjon Paul, MD, T.C. He, MD, PhD, Frank M. Phillips, MD, Chicago, IL, USABackground: Human intervertebral disc degeneration is thought to be an important early event in the spinal “degenerative cascade” and is characterized by cell death with loss of water, proteoglycans and type 2 collagen. Transfer of genes encoding for growth factors that might inhibit or reverse these biologic processes may afford an opportunity to promote disc regeneration. The Sox-9 gene has been shown to be an essential transcription factor for type 2 collagen synthesis as well as for chondrogenesis. In humans, Sox-9 deficiency is associated with skeletal malformations. Bone morphogenetic protein (BMP)-2 has also been shown to stimulate type 2 collagen production as well as chondrogenesis. The goals of this study were to determine whether human degenerative intervertebral disc tissue and cells could be transduced with adenoviral vectors carrying Sox-9 or BMP-2 genes and as a result increase their synthesis of type 2 collagen.Materials and methods: Genes encoding for growth factors BMP-2 and Sox-9 were incorporated into defective recombinant adenovirus in tandem with a green fluorescent protein (GFP) marker. Intervertebral disc tissue was removed from six patients during surgery for a diagnosis of degenerative disc disease (institutional review board approval was obtained). In certain patients, nucleus pulposus tissue was cultured in DMEM medium for 48 hours. In other cases, disc cells were extracted from tissues using collagenase A (.1 μg/ml) dissolved in PBS for a period of 20 minutes. Cells were cultured at 37° C in standard DMEM complete medium for 14 to 25 days with media exchange every 3 to 4 days. Cell or tissue cultures were then transfected with adenovirus containing GFP marker, BMP-2 gene with GFP marker, or Sox-9 gene with GFP marker. Control cultures were treated with saline. Transfections were done at an MOI of 300. The medium of cell and tissue cultures was exchanged at 18 hours after exposure to the virus. Cell and tissue cultures were then washed in DMEM complete medium at 24 hours and then allowed to incubate. Samples were then evaluated at 36 hours and 1 week with fluorescent microscopy for signs of green fluorescent protein expression. At 1 week after viral exposure, tissue samples were sectioned and stained for type 2 collagen using an anti–type 2 collagen antibody. A secondary antibody and HRP indicator was used to complete the immunohistochemical staining process.Results: Viable chondrocytes were isolated and cultured from degenerative disc tissues. Cell viability remained high throughout the incubation and transfection periods (greater than 95%). In human degenerative disc cell cultures, fluorescent light microscopy documented 73% to 100% marker gene transfection. No cells were found to fluoresce in the uninfected control group. The tissue cultures transfected with virus showed evidence of sustained GFP expression as documented by fluorescence microscopy at 48 hours and 7 days. After transfection with the Sox-9 gene, disc tissue showed significantly increased levels of type 2 collagen using immunohistochemical staining up to 1 week after transfection (Table 1)Table 1Human degenerative disc transfection ratesGFP onlyBMP-2 + GFPSOX-9 + GFPControl48 hours93 + 12%91 + 15%93 + 12%0%7 days86 + 14%87 + 16%88 + 13%0%.Conclusion: This study confirms that degenerative human intervertebral disc chondrocytes can be transfected with potentially therapeutic genes. Previous studies of gene transfer in the intervertebral disc have used marker genes only or TGF-B1 for its wide range of potentially therapeutic effects. In this study, we used a gene encoding for Sox-9, a factor known to be critical for type 2 collagen synthesis and chondrogenesis. We were able to demonstrate Sox-9–mediated upregulation of type 2 collagen synthesis in degenerative human disc tissue. These observations suggest that this approach has potential applications for altering intervertebral disc degeneration.