Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades

Abstract

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor β1 (TGFβ1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFβ1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

Keywords: anulus fibrosus; degeneration; genome-wide microarray; intervertebral disc.

Figure 1

Genesis cluster analysis of native anulus fibrosus (AF) microarray signals from mild and severe degenerated AF.

Figure 2

Real-time detection polymerase chain reaction (RTD-PCR) of chosen genes for validation microarray results of native AF tissue and monolayer cells from mild and severe degenerated intervertebral discs (IVDs). Significant differences in aggrecan (ACAN) and cartilage oligomeric matrix protein (COMP) gene expression (*¹ p = 0.0260, *² p = 0.0141, *³ p = 0.0141, *⁴ p = 0.0443, *⁵ p = 0.0114, *⁶ p = 0.0114) (error bars SEM).

Figure 3

Glycosaminoglycan (GAG) and collagen content of mild (Pfirrmann score 2–3) and severe (Pfirrmann score 4) degenerated native annulus fibrosus (AF) tissues (error bars SEM).

Figure 4

Gene expression of bone morphogenetic protein 2 (BMP2)-, transforming growth factor β1 (TGFβ1)-, and tumor necrosis factor α (TNFα)-stimulated cells and unstimulated cells after 24 h given as fold induction of gene expression at the starting point of the stimulation 0 h (error bars SEM); significant increase after 24 h indicated for * p < 0.05 and exact values given in the figure.

Figure 5

Mean normalized expression of bone morphogenetic protein 2 (BMP2)-, transforming growth factor β1 (TGFβ1)-, and tumor necrosis factor α (TNF α)-stimulated cells after 24 h and unstimulated cells at the starting point of 0 h and after 24 h (error bars SEM). * p < 0.05.

Figure 6

Experimental design for gene expression in differentially degenerated anulus fibrosus (AF) and isolated cells, and for the stimulation of isolated cells from differentially degenerated AF.