Jonathan W. Bourne

Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagens stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

Deep Tissue Injury in Development of Pressure Ulcers: A Decrease of Inflammasome Activation and Changes in Human Skin Morphology in Response to Aging and Mechanical Load

Molecular mechanisms leading to pressure ulcer development are scarce in spite of high mortality of patients. Development of pressure ulcers that is initially observed as deep tissue injury is multifactorial. We postulate that biomechanical forces and inflammasome activation, together with ischemia and aging, may play a role in pressure ulcer development. To test this we used a newly-developed bio-mechanical model in which ischemic young and aged human skin was subjected to a constant physiological compressive stress (load) of 300 kPa (determined by pressure plate analyses of a person in a reclining position) for 0.5–4 hours. Collagen orientation was assessed using polarized light, whereas inflammasome proteins were quantified by immunoblotting. Loaded skin showed marked changes in morphology and NLRP3 inflammasome protein expression. Sub-epidermal separations and altered orientation of collagen fibers were observed in aged skin at earlier time points. Aged skin showed significant decreases in the levels of NLRP3 inflammasome proteins. Loading did not alter NLRP3 inflammasome proteins expression in aged skin, whereas it significantly increased their levels in young skin. We conclude that aging contributes to rapid morphological changes and decrease in inflammasome proteins in response to tissue damage, suggesting that a decline in the innate inflammatory response in elderly skin could contribute to pressure ulcer pathogenesis. Observed morphological changes suggest that tissue damage upon loading may not be entirely preventable. Furthermore, newly developed model described here may be very useful in understanding the mechanisms of deep tissue injury that may lead towards development of pressure ulcers.

A New Paradigm for Mechanobiological Mechanisms in Tumor Metastasis

Tumor metastases and epithelial to mesenchymal transition (EMT) involve tumor cell invasion and migration through the dense collagen-rich extracellular matrix surrounding the tumor. Little is neither known about the mechanobiological mechanisms involved in this process, nor the role of the mechanical forces generated by the cells in their effort to invade and migrate through the stroma. In this paper we propose a new fundamental mechanobiological mechanism involved in cancer growth and metastasis, which can be both protective or destructive depending on the magnitude of the forces generated by the cells. This new mechanobiological mechanism directly challenges current paradigms that are focused mainly on biological and biochemical mechanisms associated with tumor metastasis. Our new mechanobiological mechanism describes how tumor expansion generates mechanical forces within the stroma to not only resist tumor expansion but also inhibit or enhance tumor invasion by, respectively, inhibiting or enhancing matrix metalloproteinase (MMP) degradation of the tensed interstitial collagen. While this mechanobiological mechanism has not been previously applied to the study of tumor metastasis and EMT, it may have the potential to broaden our understanding of the tumor invasive process and assist in developing new strategies for preventing or treating cancer metastasis.

Molecular simulations predict novel collagen conformations during cross-link loading

Collagen cross-linking mechanically strengthens tissues during development and aging, but there is limited data describing how force transmitted across cross-links affects molecular conformation. We used Steered Molecular Dynamics (SMD) to model perpendicular force through a side chain. Results predicted that collagen peptides have negligible bending resistance and that mechanical force causes helix disruption below covalent bond failure strength, suggesting alternative molecular conformations precede cross-link rupture and macroscopic damage during mechanical loading.

Glycation Cross-Linking Induced Mechanical-Enzymatic Cleavage of Microscale Tendon Fibers

Recent molecular modeling data using collagen peptides predicted that mechanical force transmitted through intermolecular cross-links resulted in collagen triple helix unwinding. These simulations further predicted that this unwinding, referred to as triple helical microunfolding, occurred at forces well below canonical collagen damage mechanisms. Based in large part on these data, we hypothesized that mechanical loading of glycation cross-linked tendon microfibers would result in accelerated collagenolytic enzyme damage. This hypothesis is in stark contrast to reports in literature that indicated that individually mechanical loading or cross-linking each retards enzymatic degradation of collagen substrates. Using our Collagen Enzyme Mechano-Kinetic Automated Testing (CEMKAT) System we mechanically loaded collagen-rich tendon microfibers that had been chemically cross-linked with sugar and tested for degrading enzyme susceptibility. Our results indicated that cross-linked fibers were >5 times more resistant to enzymatic degradation while unloaded but became highly susceptible to enzyme cleavage when they were stretched by an applied mechanical deformation.

Covalent Cross-Linking Accelerates Collagen Enzyme Mechano-Kinetic Cleavage: Nanomechanics Predicts Microscale Behavior

Fibrillar collagens are an integral structural element of tissues throughout the body and help provide tensile strength. These collagens are highly resistant to degradation other than by a small number of collagenolytic enzymes. Examples of tensile mechanical forces in vivo include expansion and contraction of blood vessels, tension on tendons and ligaments, and compression and swelling of cartilage.Copyright

Computational Modeling Predicts Novel Collagen Conformations During Mechanical Loading

Collagens contribute to the mechanical strength of many tissues throughout the body and are generally resistant to ezymatic degradation. As structural proteins, collagens often experience in vivo mechanical forces such as expansion and contraction of blood vessels and tension on tendons and ligaments. Collagen crosslinking, which enhances the strength of these structural networks, occurs during tissue development and aging. While much is known about the structure of collagen, there is a paucity of data describing how mechanical force transmitted across these crosslinks affects molecular conformation. We hypothesized that mechanical force applied perpendicular to the long axis of the collagen triple helix will result in bending and microunfolding of the triple helix structure. To test this we used Steered Molecular Dynamics to model the conformation of a collagen peptide when subjected to perpendicular forces. In silico loading predicted that the collagen peptide had minimal resistance to bending, and exhibited increased curvature with no distinct disruption of the characteristic triple helix at low forces. As force increased, we observed that the helix began to fail and underwent a microunfolding event, where a loop pulled out from the complex. This local triple helix disruption was predicted to occur below covalent bond failure strength, suggesting that alternative molecular conformations occur within the molecule as structures are loaded before the onset of structural failure. We speculate that these changes may represent nano-damage to the structure and be a mechanism for energy storage and dissipation. Furthermore, these predicted conformational changes would precede macroscopic damage mechanisms.

In Silico Molecular Modeling of Collagen Crosslink Loading

Collagens strengthen tissues throughout the body and are generally resistant to degradation, except by a small number of collagenolytic enzymes. As structural proteins, collagens often experience mechanical forces in vivo including expansion and contraction of blood vessels, tension on tendons and ligaments, and compression of cartilage.© 2011 ASME

Collagen Molecular Conformation Exhibits Strain-Rate Dependent Response to Axial Deformation in Silico

Fibrillar collagens are a group of structural proteins that self assemble into a complex ordered structure of interconnected molecules to form supramolecular fibril structures. These collagens have three subgroups based on sequence similarity, of which clade A includes the most abundant fibrillar collagens, types I, II, and III. The fibrous hierarchical structures starts with individual collagen molecules that are crosslinked by enzymes to other collagen molecules to form micro fibrils, which aggregate and link to form sub fibrils, then larger fibrils and in some tissues continuing to assemble into larger levels of fascicles and above.Copyright

Deformation-Dependent Enzyme Cleavage of Collagen

Collagen degradation is a mechanism for normal musculoskeletal development and extracellular matrix (ECM) maintenance, and in response to trauma, disease and inflammation. Matrix metalloproteinases (MMP-1, 8, and 13, the collagenases) are the primary enzymes that act to degrade collagen. These MMPs gain access to the collagen triple helix by binding to the enzyme’s attachment domain along the α-chains, followed by separation (unwinding) of the α-chains to expose the 3/4–1/4 cleavage site, and then cleavage of the α-chain by the enzyme’s catalytic domain [3, 5].Copyright