## 3 Results  
### Prime PCR of DISH and Traumatic/Degenerate Discs  
PrimePCR(tm) screening for 90 genes of the BMP pathway was initially run. Most strikingly, these data revealed a significant mean up-regulation of \(EGR2\) (12.8 \(\pm\) 7.6-fold, \(p\) = 0.0068, overall mean \(\pm\) SEM of all comparisons) and of interleukin 6 (IL6) (54.0 \(\pm\) 46.5-fold, \(p\) = 0.0005) in DISH-IVDs relative to the \"control\" discs (Figure 1, Supplementary Figure S1). Furthermore, \(IGF1\) tended to be up-regulated in DISH-IVD donors (174.1 \(\pm\) 120.6-fold, \(p\) = 0.1704), although this was non-significant. Growth and Differentiation Factor 5 (\(GDF5\)) and \(GDF6\) both tended to be down-regulated in the DISH-IVDs (i.e., \(-\)11.5 \(\pm\) 10.0, \(p\) = 0.26 and \(-\)3.7 \(\pm\) 3.1 \(p\) = 0.30, respectively, Figure 1, Supplementary Figure S1). However, these differences were not significant.  
Figure 1: Normalised relative gene expression in DISH-IVDs relative to IVDs of “control” patients. The relative gene expression of IL6 and EGR2 was significantly up-regulated: ***\(p\) < 0.01. The scatterplots of the pairwise comparisons of the 90 PrimePCR(tm) gene array is given in Supplementary Figure S1. Means \(\pm\) SEM.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Primary AB** & **Source** & **Concentration ICC** \\ \hline Mouse GDF5 & Polyclonal Goat IgG (Biotechne, Zug, Switzerland) & 5 \(\upmu\)g/mL \\ Human IL6 & Monoclonal mouse IgG (Biotechne) & 16 \(\upmu\)g/mL \\ Human IGF1 & Polyclonal Goat IgG (Biotechne) & 5 \(\upmu\)g/mL \\ Human BMP2 & Polyclonal Rabbit (Novusbio, Zug, Switzerland) & 5.75 \(\upmu\)g/mL \\ Human EGR2 & Polyclonal Rabbit (Novusbio) & 5 \(\upmu\)g/mL \\ Human GDF6 & Rabbit polyclonal–C terminal (Abcam, Cambridge, UK) & 1:100 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Primary antibodies used for immunohistochemistry.  
Clustering the gene expression profiles of the 19 analysed samples of the three control patients and the six DISH patients revealed that not all samples clustered together as expected (Figure 2). However, it seems also evident that the hierarchical phenogram generated two main clusters of phenotypes with a similar TGFB pathway pattern (Figure 2). Clade 1 consisted of mainly DISH samples, and clade 2 consisted mainly of control samples. However, samples of DISH patients 3 and 5 (i.e., D P3 IVDC and D P5 AFC and EPC) showed a phenotype more similar to the cells of the included trauma patients. On the other hand, the dendrogram confirmed that cells originating from different tissue types of the IVD from the same patient were grouped closely together, which was expected.  
Figure 2: Clustergram displaying hierarchy based on the degree of similarity of expression for different samples and normalised gene expression heat map of relative gene expression at 88 genes of PrimePCRTM of the TGFB-BMP Signalling Pathway Plus H96. Red indicates up-regulations, green indicates down-regulations and black indicates no regulations. The lighter the shade of colour, the greater the relative normalised expression difference. If no normalised expression value could be calculated, this is shown in black with a white X. Relative gene expression was estimated using ACTB and GAPDH as the two reference genes. Samples encoded in orange are samples categorised as trauma patients = control (C), and samples in light blue are from patients that were diagnosed with DISH (D). Tissue types were colour-coded in dark blue for CEP, in yellow for NP, and in red for AF.