Discussion
To reinforce the abdominal wall with mesh material is the gold standard in hernia repair and has lead to a considerable reduction of recurrence rates, however their implantation is associated with acute and chronic side effects like seroma and as long-term complication in particular chronic pain. In particular long-term complications are supposed to be the consequence of a foreign body reaction (FBR), which is induced by every implantation of non-absorbable polymeric mesh material. FBR offers characteristics of a chronic inflammatory reaction, which is marked by an activation of cytokine cascades and proteases, such as the matrix metalloproteinases 2 (MMP-2). This well known continuous expression of MMP-2 in foreign body reaction can also be observed in our study (Figure 1) whereas MMP-2 expression after injury is normally restricted to the proliferative phase of wound healing.
For this reason the inhibition of inflammation and as a consequence the normalization of MMP-2 expression is suggested as a new therapeutical approach to optimize mesh integration and to reduce mesh related complications. By mesh modification with the aminoglycoside antibiotic gentamicin we found a time dependent reduction of the MMP-2 protein synthesis in a preceding study. Especially, the perifilamentary MMP-2 protein expression was significantly diminished by mesh coating with a concentration of 8 μg/mg gentamicin at each time point. In previously published studies this potential impact of gentamicin on MMP-2 expression was likewise demonstrated, but in contrast to these results our current findings provide evidence for a presumably dose-dependent down-regulation of the MMP-2. Our current survey confirms the previously proven beneficial effect of gentamicin supplemented polyvinylidenfluoride (PVDF) mesh materials on tissue integration and foreign body reaction due to an improved collagen type I/III ratio and reduced MMP-2 protein expression.
However, and in contrast to our previous findings the MMP-2 promoter activity increased reciprocally to the MMP-2 protein expression, thereby suggesting that gentamicin represses the MMP-2 promoter in F8del mice. This ostensible controversy with an increased ß-galactosidase expression in F8del mice may be explained most suitable as follows. It is known that the extent of MMP-2 expression and enzymatic activity is regulated at the transcriptional, translational, and post-translational levels. Specific regulatory elements governing MMP-2 gene transcription that reside up to -1686 base pairs (bps) relative of the translational start site have been identified. A strong enhancer element named response element-1 (RE-1) is located at -1282/-1322 bps of the mice MMP-2 gene, which is evolutionarily conserved and is similarly operative within the human gene at -1657/-1619 bps relative to the transcriptional start site. F8 mice harbor MMP-2 regulatory sequence -1686/+423, including the RE-1, showed both a reduced MMP-2 protein expression and promoter activity with presence of gentamcin. On the other hand the F8del mice that were investigated in the present study harbour MMP-2 regulatory sequence −1241/+423, excluding the RE-1. This mouse strain revealed a reduced MMP-2 protein expression but a rising MMP-2 promoter activity in all mesh groups. Taken into account the preceding and current results, RE-1 mostly acts as a repressor of mesh induced MMP-2 transcription. Though we could confirm that gentamicin is beneficial for tissue integration regarding reduced MMP-2 expression and enhanced type I/III collagen ratio, the same repressive function of RE-1 could be detected for both the coated and the uncoated mesh group.
It is known that the aminoglycosides like gentamicin are able to penetrate eukaryotic cell membrane and it is known that beside its antibacterial activity gentamicin interacts with intracellular molecules thereby influencing intracellular pathways, which might explain the reduced MMP-2 expression and gene regulation. Although the exact molecular mechanisms are not yet unraveled, our findings support the hypothesis that the observed down-regulation of MMP-2 is finally mediated by inhibition of the MMP-2 promoter.
Certainly it remains unclear which transcription factors mediate the down-regulation of mesh induced MMP-2 expression. At least distinct transcription factors have been shown to bind to the RE-1. These include activating protein-2 (AP 2),Y-box protein-1 (YB-1),signal transduction and activator of transcription factor 3 (Stat3), and p53. In a previously published study by Jansen et al. it could be shown that macrophages that are adjacent to the mesh filaments play a crucial role for mesh induced MMP-2 expression. Therefore, the gentamicin mediated down-regulation of MMP-2 transcription in our study might be due to a cellular effect, e.g. by reducing infiltration or activation of macrophages or other inflammatory cells that are involved in MMP-2 expression. The results of our current investigation further support the hypothesis of an intercellular crosstalk between inflammatory cell like macrophages and fibroblasts via MMP-2 regulation in the process of foreign body reaction.
Restrictively, it has to be noted that the collagen type I/III ratio was significantly elevated for each gentamicin concentration on day 90, whereas on postoperative day 7 and 21 merely the mesh with a coating of 8 μg/mg gentamicin constantly resulted in a significant improvement of the collagen ratio. As already mentioned a dose-dependent effect of gentamicin is possibly the reason for an insignificant or even negative effect on collagen formation and degradation. This is in line with a study by Asch and Farnham who demonstrated a dose-dependent effect of gentamicin on the protease activity of collagenase derived from Clostridium histolyticum.