Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Abstract

BackgroundCollagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.Methodology/Principal FindingsThe investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.Conclusions/SignificanceIn this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.

The Role of Mechanical Loading in Tendon Development, Maintenance, Injury, and Repair

“…Normal, physiologic loads are required to maintain tendon homeostasis and prevent excessive degradation of the extracellular matrix [25][26][27] . Nabeshima et al found that culturing nontensioned rabbit patellar tendon explants in the presence of collagenase over a period of twenty hours significantly decreased linear stiffness (p < 0.0001), elongation to failure (p < 0.002), and maximum failure force (p < 0.002) by 80% compared with explants tensioned with constant 4% strain 26 .…”

“…Nabeshima et al found that culturing nontensioned rabbit patellar tendon explants in the presence of collagenase over a period of twenty hours significantly decreased linear stiffness (p < 0.0001), elongation to failure (p < 0.002), and maximum failure force (p < 0.002) by 80% compared with explants tensioned with constant 4% strain 26 . Further, Flynn et al found that reconstituted type-I collagen micronetworks, strained between micropipettes, degraded significantly slower (p < 0.05) than unloaded controls when exposed to mammalian collagenase matrix metalloproteinase 8 27 . These experiments support the beneficial effect of mechanical load and the need for its incorporation in both clinical postoperative rehabilitation protocols and in tissue-engineering applications 27 .…”

Interstitial Collagen Catabolism

“…These data suggested that heterotrimeric type I collagen was more unwound than homotrimeric type I collagen, and hence, the effect of strain on further unwinding the triple helix was less pronounced in the former case (84). Conversely, strain of reconstituted type I collagen fibrils increased degradation time by MMP-8 (90). The discrepancy between the single-molecule study (84) and the fibrillar collagen study (90) may be due to effects on diffusive transport of the MMP in fibrils (84).…”

“…Conversely, strain of reconstituted type I collagen fibrils increased degradation time by MMP-8 (90). The discrepancy between the single-molecule study (84) and the fibrillar collagen study (90) may be due to effects on diffusive transport of the MMP in fibrils (84).…”

Dynamics of enzymatic digestion of elastic fibers and networks under tension

“…Biological tissues in vivo are also under tension that may interfere with the enzymatic activity. Indeed, mechanical stretch accelerates the rate of degradation of native ECM during elastase-induced digestion of lung tissue (3,4), whereas it stabilizes type I collagen against in vitro digestion by collagenases (5,6).…”