gms | German Medical Science

Objectives: Intervertebral disc (IVD) degeneration and herniation are major sources of back pain. Based on scoring systems, IVDs are categorized in different degeneration grades. In order to initiate cell based regeneration strategies for the closure of ruptures in the annulus fibrosus (AF), genome-wide gene expression of AF-tissues and cultured cells of mild and severe degenerated lumbar annulus fibrosus were analyzed and compared. Furthermore, stimulation of AF cells towards expression of extracellular matrix (ECM) genes has to be examined.

Methods: For cell cultivation, cells were isolated from lumbar AF-tissue samples using enzymatic digestion. In order to analyze gene expression of mild and severe degenerated native AF-tissues and cells (n=3 each), genome-wide Affymetrix HG-U133plus2.0 microarrays were performed and analyzed using the Affymetrix GCOS 1.4 software. For verification of genes differentially expressed, real time detection PCRs (RTD-PCRs) were accomplished. The stimulation of AF-cells towards ECM induction or inflammation was induced using bone morphogenetic protein 2 (BMP2), transforming growth factor β 1 (TGF β 1) and tumor necrosis factor α (TNF α) in a 6-well plate assay. Cells were starved with a minimal medium for 3h and then induced for 24h and 48h with one of the stimulants. Gene expression was analyzed using RTD-PCRs genes of interest. Statistical significance between mild and severe group was determined using t-test.

Results and conclusion: Genome-wide microarray analysis of native AF-tissues showed 788 genes with a significantly different gene expression (detection 100% in cells with higher expression; fold change > 2) with 213 genes higher expressed in mild (including cartilage associated genes e.g. aggrecan, collagen type II, cartilage oligomeric protein) and 575 genes in severe (including matrix metalloproteinases (MMP) and bone associated genes e.g. osteocalcin, RUNX2) degenerated AF-tissue. Cell culture of these tissues in passage 2 demonstrated only 164 differentially expressed genes with none of the common cartilage and bone associated genes differentially expressed. RTD-PCR analyses of BMP2 and TGF β 1 stimulated cells from mild and severe degenerated AF-tissue after 24 and 48h showed a successful stimulation regarding expression of cartilage associated genes e.g. aggrecan, collagen type II. TNF α stimulation led to an increase of MMP1, -3, and -13 expression.

The study revealed that mild degenerated native AF-tissues showed a higher gene expression of common ECM genes, whereas severe degenerated AF-tissues expressed genes known from degenerative processes and bone associated genes. During cell cultivation, the cells dedifferentiated and adjusted their gene expression profile. Moreover, cells derived from mild and severe degenerated tissues could be stimulated in a similar way. Therefore, a cell-based therapy for AF regeneration could be a possible treatment option for both patients suffering from mild or severe disc degeneration.