Abstract
Introduction
The etiology of the spinal deformity in idiopathic scoliosis is unclear to date, both with respect to initiation and progression of the disease. While the influence of certain genetic factors has been established, the role of the intervertebral disc (IVD) in the development of idiopathic scoliosis has scarcely been investigated. The aim of this study was to identify molecular differences between disc cells from patients with idiopathic scoliosis in comparison with trauma patients and healthy individuals. To address this aim, cellular gene expression profiles were analyzed by microarray and quantitative RT-PCR.
Material and Methods
Surgical samples from IVDs of patients with idiopathic scoliosis were obtained after informed consent and approval of the local ethical commission at the time of discectomy during spinal fusion surgery. The disorder and exact curve pattern were documented for further records. Control disc samples were obtained from trauma fusion cases and from organ donors with no known disc disorders according to local and institutional ethical guidelines. Annulus fibrosus (AF) and nucleus pulposus (NP) tissues were separated and cells were isolated by enzymatic digestion within 24 hours. Total RNA was extracted from the cells and subjected to Affymetrix GeneChip® expression profiling. Genes with significant differences between scoliotic and control samples were further analyzed using real time RT-PCR.
Results
After exclusion of RNA samples with insufficient quality or quantity, the following numbers of samples were used for microarray profiling: 10 AF and 6 NP samples from scoliotic discs; 5 AF and 4 NP samples from traumatic discs; 4 AF and 4 NP samples from healthy discs of organ donors. Microarray data revealed that 52 genes were more highly expressed in scoliotic vs. healthy AF and 26 genes in scoliotic vs. traumatic AF, whereby 21 genes showed higher expression in scoliotic AF compared with both control groups. In addition, 116 genes were more highly expressed in scoliotic vs. healthy NP, 45 genes in scoliotic vs. traumatic NP, and 40 of those in scoliotic NP compared with both control groups. Quantitative gene expression analysis by real time RT-PCR (n = 6 per group) confirmed significantly increased mRNA levels of S100A8, S100A12, MMP8, MMP13 and Collagen X in annulus fibrosus cells from scoliotic discs (p < 0.05; Kruskal-Wallis test of log2 transformed data).
Conclusion
Results of this study reveal significant changes in the gene expression profile of IVD cells from patients with idiopathic scoliosis compared with patients with traumatic disc damage or donors with no known disc disorders. MMP8 and MMP13 are important collagenases involved in disc matrix degradation; while elevated S100 calcium binding proteins may indicate an inflammatory reaction. Interestingly, MMP13 has also been up-regulated by imbalanced loading in an IVD organ culture model. Better knowledge of the dysregulation of structural or regulatory molecules may identify underlying mechanisms of spinal deformities, which will help defining new targets for early therapeutic intervention.
Acknowledgment
This study is supported by AOSpine International.