Mean values ± S.D. of unnormalized complex modulus (n = 7).

<p>Mean values ± S.D. of unnormalized complex modulus (n = 7).</p

Schematic representation of the CDL dissection.

<p>Schematic representation of the CDL dissection.</p

Reversibility of MMP inhibition.

<p>(A–C) Examples of recordings showing the reversibility of the galardin effect on (A) standard, (B) stiff and (C) compliant CDLs. (D) Normalized results. Tissues in compliant, standard and stiff states were stimulated with 50 µM galardin, washed and treated again with galardin. Data are expressed as means ± SD.</p

MMPs of CDLs in compliant, standard and stiff conditions visualized by gelatin zymography.

<p>(A) Zymogram showing pro-enzyme and active MMP band profile of CDLs in the three mechanical states. (B) Zymogram comparing the band profiles of CDLs, compared with human cell line showing MMP-2 and MMP-9 activity; this zymogram was included because of its very good band separation. (C) Optical density of MMP activity in CDLs in the three mechanical states. (D) Comparative densitometric analysis of scanned gels of CDLs in the different mechanical states with and without 50 µM galardin. Data are expressed as means ± SD. The asterisk (*) represents statistically significant difference P<0.05. MMPs were detected in more than six animals for each of the three mechanical states. (E) Zymogram comparing standard CDLs with and without galardin treatment.</p

Mechanical properties of CDL.

<p>(A) Representative stress vs. strain curves of CDLs from one animal in the three mechanical states. (B) Effect of frequency on the complex modulus of CDLs from one animal in the three mechanical states tested at 13% strain.</p

Effects of propylene phenoxetol (PPSW) and acetylcholine (AChSW) on the mechanical properties of the CDL.

<p>(A) Immediate effect of PPSW on a standard CDL. (B) Immediate effect of AChSW on a standard CDL. (C) Comparison of the normalized complex modulus of compliant (PPSW-treated), standard (untreated) and stiff (AChSW-treated) CDLs (n = 7).</p

Effect of MMP inhibition on CDL viscoelasticity.

<p>(A) complex modulus, (B) storage modulus, (C) loss modulus and (D) tan delta of compliant, standard and stiff CDLs treated with 50 µM galardin in PPSW, SW, and AChSW respectively. The action of galardin was quantified by normalizing the maximum E* reached after galardin addition against the value just before the application of the chemical. The asterisk (*) represents statistically significant difference P<0.05 and the double asterisk (**) P<0.01.</p

Hypothetical model of the involvement of MMPs in MCT mutability.

<p>It is known that MCTs consist of discontinuous collagen fibrils crosslinked by complexes of molecular components, and that changes in the mechanical properties of MCTs result from rapid changes in the strength of the interfibrillar cohesion that is mediated by these crosslink complexes. We found that the synthetic MMP inhibitor, galardin, increased the stiffness of CDLs in all three mechanical states, which suggests that in all three states there is ongoing MMP activity that has the potential to degrade components already incorporated into existing crosslink complexes and components that have been secreted but not yet incorporated, and ongoing synthesis and release of new crosslink components. The model acknowledges that MMPs are synthesized and secreted as inactive pro-enzymes, then activated extracellularly by proteolytic removal of the pro-peptide domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049016#pone.0049016-SnoekvanBeurden1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049016#pone.0049016-Motokawa3" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049016#pone.0049016-Lee1" target="_blank">[52]</a>. It is envisaged that crosslink components are synthesized and secreted separately, then assembled extracellularly to form functional complexes. The black boxes represent cells, although it should be noted that the three processes do not necessarily occur in different cell-types. The red box represents the process by which MMP-TIMP complexes are removed and degraded. For the sake of simplicity, the model assumes that activated MMPs and new crosslink components reach the extracellular environment at a constant rate. It is hypothesized that interfibrillar cohesion is regulated only through changes in the rate at which an endogenous MMP inhibitor (which we assume is a TIMP-like molecule) is released into the extracellular environment. In the stiff state there are high levels of TIMP secretion (1), MMP inhibition and crosslinking. In the standard state there are intermediate levels of TIMP secretion (2), MMP inhibition and crosslinking. In the compliant state there are low levels of TIMP secretion (3), MMP inhibition and crosslinking. Also represented is the possibility that an endogenous inhibitor could function as a component of the crosslink complex (red arrows) and thus have a dual function (which may apply to TIMP-like tensilin). The model also assumes that the production of MMP-TIMP complexes exceeds the rate of removal and degradation of MMP-TIMP complexes, which would account for the positive correlation between degree of CDL stiffness and total gelatinolytic activity. The components marked with a red asterisk contribute to the gelatinolytic activity of CDLs as quantified by gelatin zymography.</p