## 4 Discussion  
In this study, we report, for the first time, on the phenotypic changes in IVD cells from DISH patients compared to IVD cells from supposedly \"healthier\" cells from younger and mid-aged trauma patients (Table 1). We observed the consistent up-regulation of IGF-1, and EGR2, which seems to be a hallmark of the DISH-IVD cells [4; 29], and cells from degenerative discs showed a different BMP gene-related transcript and protein expression (Figures 1-3). One of the main limitations of this study was the relatively small sample size. Thus, sample collection from a single hospital was a challenge, and future studies should recruit IVD material from bigger cohorts and multiple populations and etiologies. Additionally, the \"controls\" used in the study were not truly healthy IVDs, as they were either degenerative or traumatic discs. In this respect, a comparison of fresh cadaveric IVD material of undegenerated nature would be essential for any future investigations. Acquiring more DISH and matching healthy IVDs are required to support our first findings regarding the phenotypic status of the IVD during DISH disease. Moreover, it should be investigated whether DISH affects the overall phenotype of IVD cells and their tissues. To answer this particular question, complete transcriptome analyses using bulk RNA next-generation sequencing would definitively shed new light on the better understanding of DISH pathology and into yet-unidentified modified pathways of this disease. Since the discovery of DISH, various studies, especially concerning the metabolic conditions of these patients, were conducted. Nevertheless, this disease is still poorly understood. In the current study, we investigated the changes at the transcript and the protein expression level of IVDs from patients diagnosed with DISH. We observed significant up-regulation of EGR2 and IL6 and a trend of higher IGF1 expression at the transcript level. These changes could be confirmed qualitatively at the protein level using immunohistochemistry. For GDF5 and GDF6, we noticed, for both genes, a down-regulation in gene expression in the IVD of those patients, though this was not statistically significant. However, these differences were not apparent at the protein level (Figure 3). This gene expression profile points to an involvement in inflammation in DISH, recently identified in a review by [PERSON] et al. (2021) [29]. Previous research by [PERSON] et al. (1996) [32] has shown that EGR2-/- mice develop skeletal abnormalities, i.e., reducing length and thickness in newly formed bone and calcified trabeculae. These phenomena were explained by the activation of EGR2 in a subpopulation of hypertrophic chondrocytes of the growth plate and differentiating osteoblasts (OBs) [32]. Furthermore, [PERSON] et al. (2008) [33] showed up-regulation of EGR2 expression during the late stage of OB differentiation in-vitro. Furthermore, [PERSON] et al. (2012) [34] found that EGR2 expression was up-regulated in the osteosarcoma-derived UMR106 cell line through IGF1 stimulation. The cause of the observed up-regulation of EGR2 and its role in DISH-IVD cells would need to be further explored. DISH has been associated with the change in inflammatory mediators [35]. Interleukin 1 (IL1) and IL6 are increased in DISH patients through the activation of nuclear factor kappa-B (NF-kB) ligand, and these cytokines stimulate the proliferation of OBs and bone deposition [29; 35; 36]. Two other studies [37; 38] have also suggested the stimulating effect of IL6 on bone formation and turnover. [PERSON] et al. (1996) [39] further demonstrated an increased concentration of IL6 in herniated lumbar discs, which may indicate an association with degradation of the IVD. We could most interestingly confirm the involvement of IL6 in our data, as it was up-regulated in all analysed DISH-IVDs (Figure 1, and Supplementary Figure S1). Several studies have shown the possible overlap between DISH, obesity and Diabetes mellitus, with a pathogenic effect of insulin, IGF1 and GH [40; 41; 42; 43].