Intervertebral disc degeneration is characterized by a number of changes linked to the degradation of the extracellular matrix [20–22]. This loss of tissue integrity has been associated with an increase in the expression of MMPs of disc's cells. Indeed, a number of MMPs, such as MMP-1, -3, -13 has been reported to be expressed in aged and degenerated discs. Among them, MMP-1 seems to be crucial as it is expressed by the majority of the disc's cells  and can degrade several extracellular matrix components, such as, fibrillar collagens, gelatines, proteoglycans, fibronectin, laminin, etc. . Roberts et al. by using immunohistochemical techniques have shown that MMP-1 is localized in the disc's cells . MMP-1 was also found to be expressed in some cells in non-degenerated discs, indicating that it plays a role in normal tissue homeostasis, such in normal turnover of extracellulatr matrix components . Its expression seems to be differentially regulated in humans in the various stages of development and during ageing. Weiler et al. have shown that MMP-1 is vaguely expressed in the discs of foetuses, infants and adolescents, while it is found in the discs of young adults, usually in tissue clefts and chondrocyte-like cells . In older adults the expression of MMP-1 was also intense; however a reduced number of positive cells were found in people over 60 years of age. In addition, they have reported an increased expression in degenerated (and especially herniated) discs, and particularly in clustered cells. Similarly, a number of studies from other groups have also shown that MMP-1 is over-expressed in degenerated discs [24, 26, 27]. MMPs are secreted from the cells as proenzymes or zymogens that can be activated by proteolytic degradation. Plasmin, a broad-spectrum serine protease can activate pro-MMPs to fully active MMPs. Interestingly, in traumatized and degenerated porcine intervertebral discs MMP-1 over-expression was accompanied by increased expression of plasmin , indicating that cascades of enzymatic activations can lead to local tissue degradation. Furthermore, collagen fragments that accumulate in the degenerated intervertebral disc can enhance the expression of MMP-1 , probably forming a self perpetuating cycle affecting tissue homeostasis. The same mechanism has also been proposed for aged skin, where collagen fragments can provoke an oxidative stress leading to MMP-1 overproduction that can be inhibited by antioxidant compounds .
In our study, an over-expression of MMP-1 has been observed after mechanical deformation of intervertebral discs, by a process simulating the scoliotic conditions. Interestingly, the increase in MMP-1 expression presented in our study is observed mostly in fibroblastic, annulus fibrosus cells. This is in contrast to previous studies indicating that the main increase in MMP-1 expression in degenerated discs is observed in nucleus pulposus cells [21, 26]. This controversy can be due to species differences (human vs. rat), or can probably indicate different mechanisms in scoliosis and in disc degeneration disease. A positive effect of mechanical forces on MMP-1 expression or secretion has been reported also in various other models, from periodontal tissues to osteoblastic cells [30–32]. The increase of MMP-1 expression observed in this study seems to be dependent on the intensity of deformation and on the site of the tissue affected, as convex areas are more affected compared to concave ones. Similarly, Crean et al reported increased expression of MMP-2 and MMP-9 in the convexity of the curve of scoliotic discs . Previous studies have shown that the intensity of mechanical forces can regulate the expression and activity of MMP-1. As an example, porcine aortic valve cups have been subjected to cyclic stretch and has been found that a physiological stretching, i.e. 10%, there was no increase of MMP-1. However, pathological stretch of 15% causes an intense increase of MMP-1 expression, also 20% extensive over-stretch still upregulated MMP-1 but in a lesser amount in comparison to 15% stretch . In the same direction, it has been recently shown that circumferential constrain of in vitro formed nucleus pulposus tissues can lead to a decrease gene expression of aggrecan and collagen II and an increase expression of MMP-1 and ADAMTS-5, that can play a role towards remodelling and degradation of the tissue . On the other hand, the expression of other MMPs (i.e. (MMP-2 and MMP-9) seem to be decreased after exposure of disc cells to mechanical forces . Regarding the role of MMPs family in idiopathic scoliosis, there are some contradictory findings in the literature about the role of gene variants of IL-6 and MMP-3 and whether MMP-3 and IL-6 promoter polymorphisms constitute important factors for the genetic predisposition to idiopathic scoliosis [36, 37].
In this study we observed changes in the numbers and types of cells in the various areas of the discs after a long-term deformation. In particular, a reduction of the number of cells was observed in discs subjected to intense force. The mechanism leading to the reduction of the number of cells (i.e. apoptosis) needs to be further investigated with the use of specific markers. Analogous data have been reported in mouse tails subjected to static bending . Interestingly, in the areas of reduced cellularity a severe degradation of matrix microstructure has also been found. A plausible explanation can be the leakage of proteases from dying cells, which can catabolise extracellular matrix components. In addition, we observed that at the margins of intervertebral discs adjacent to the vertebral end plates a reduction of fibroblast-like cells and an increased number of chondrocytes positive in MMP-1 expression. Similarly, Court et al. have reported that in bended mouse tails and specifically in the inner concave annulus a change from a fibroblastic to a chondrocytic phenotype . The authors provide three alternative explanations for this phenomenon: first, a preferential fibroblast cell death; second, a chondrocytic migration in this area; and third, a differentiation of fibroblasts to chondrocytes. Obviously, these possibilities remain to be experimentally validated. The former hypothesis is partly supported by the increased apoptosis found in scoliotic discs ; however, differences in apoptotic rates between fibrocyte-like and chondrocyte-like cells need to be investigated.
Mechanical forces affect several aspects of tissue homeostasis by activating numerous intracellular signalling pathways. Static mechanical deformation can stimulate the proliferation and expression of transcription factors regulating the differentiation of periodontal ligament fibroblasts [40, 41]. This is achieved by the activation of members of the MAPK family (ERK, JNK and p38) and the upregulation of the components of the AP-1 transcription factor, i.e. c-Fos and c-Jun [41, 42]. On the other hand, interstitial flow stimulates the expression of MMP-1 in smooth muscle cells via ERK and JNK activation , while in monocytes MMP-1 induction is regulated by AP-1 . However, in intervertebral disc cells MMP-1 can also be overexpressed by the pro-inflammatory cytokine TNF-α, via the p38 and JNK pathways [45, 46]. Accordingly, the involvement of the above mentioned pathways in the regulation of MMP-1 in scoliotic disc needs further investigation in vitro and in vivo.