Preferential sites for intramolecular glucosepane cross-link formation in type I collagen: A thermodynamic study

The extracellular matrix (ECM) undergoes progressive age-related stiffening and loss of proteolytic digestibility due to an increase in concentration of advanced glycation end products (AGEs). The most abundant AGE, glucosepane, accumulates in collagen with concentrations over 100 times greater than all other AGEs. Detrimental collagen stiffening properties are believed to play a significant role in several age-related diseases such as osteoporosis and cardiovascular disease. Currently little is known of the potential location of covalently cross-linked glucosepane formation within collagen molecules; neither are there reports on how the respective cross-link sites affect the physical and biochemical properties of collagen. Using fully atomistic molecular dynamics simulations (MD) we have identified six sites where the formation of a covalent intra-molecular glucosepane cross-link within a single collagen molecule in a fibrillar environment is energetically favourable. Identification of these favourable sites enables us to align collagen cross-linking with experimentally observed changes to the ECM. For example, formation of glucosepane was found to be energetically favourable within close proximity of the Matrix Metalloproteinase-1 (MMP1) binding site, which could potentially disrupt collagen degradation.     

ColBuilder: A server to build collagen fibril models

Type I collagen is the main structural component of many tissues in the human body. It provides excellent mechanical properties to connective tissue and acts as a protein interaction hub. There is thus a wide interest in understanding the properties and diverse functions of type I collagen at the molecular level. A precondition is an atomistic collagen I structure as it occurs in native tissue. To this end, we built full-atom models of cross-linked collagen fibrils by integrating the low-resolution structure of collagen fibril available from x-ray fiber diffraction with high-resolution structures of short collagen-like peptides from x-ray crystallography and mass spectrometry data. We created a Web resource of collagen models for 20 different species with a large variety of cross-link types and localization within the fibril to facilitate structure-based analyses and simulations of type I collagen in health and disease. To easily enable simulations, we provide parameters of the modeled cross-links for an Amber force field. The repository of collagen models is available at https://colbuilder.h-its.org.     

Computational study of glucosepane–water and hydrogen bond formation: an electron topology and orbital analysis

The collagen protein provides tensile strength to the extracellular matrix in addition to localising cells, proteins and protein cofactors. Collagen is susceptible to a build up of glycation modifications as a result of an exceptionally long half-life. Glucosepane is a collagen cross-linking advanced glycation end product; the structural and mechanical effects of glucosepane are still the subjects of much debate. With the prospect of an ageing population, the management and treatment of age-related diseases is becoming a pressing concern. One area of interest is the isolation of hydrated glucosepane, which has yet to be reported at an atomistic level. This study presents a series of glucosepane–water complexes within an implicit aqueous environment. Electronic structure calculations were performed using density functional theory and a high level basis set. Hydrogen bonds between glucosepane and explicit water were identified by monitoring changes to covalent bonds, calculating levels of electron donation from Natural Bonding Orbital analysis and the detection of bond critical points. Hydrogen bond strength was calculated using second-order perturbation calculations. The combined results suggest that glucosepane is very hydrophilic, with the imidazole feature being energetically more attractive to water than either hydroxyl group, although all hydrogen bonds, regardless of bond strength, were electrostatic in nature. Our results are in growing support of an earlier hypothesis that cross-links may result in an increase in interstitial water retention, which would permit the collagen fibril to swell, thereby potentially affecting the tensile and compression properties and biological function of connective tissues.     

Multiaxial mechanical behavior of human fetal membranes and its relationship to microstructure

This study was directed to the measurement of the mechanical response of fetal membranes to physiologically relevant loading conditions. Characteristic mechanical parameters were determined and their relation to the microstructural constituents collagen and elastin as well as to the pyridinium cross-link concentrations analyzed. 51 samples from twelve fetal membranes were tested on a custom-built inflation device, which allows mechanical characterization within a multiaxial state of stress. Methods of nonlinear continuum mechanics were used to extract representative mechanical parameters. Established biochemical assays were applied for the determination of the collagen and elastin content. Collagen cross-link concentrations were determined by high-performance liquid chromatography measurements. The results indicate a distinct correlation between the mechanical parameters of high stretch stiffness and membrane tension at rupture and the biochemical data of collagen content and pyridinoline as well as deoxypyridinoline concentrations. No correlation was observed between the mechanical parameters and the elastin content. Moreover, the low stretch stiffness is, with a value of 105 ± 31 × 10^?3 N/ mm much higher for a biaxial state of stress compared to a uniaxial stress configuration. Determination of constitutive model equations leads to better predictive capabilities for a reduced polynomial hyperelastic model with only terms related to the second invariant, I ₂, of the right Cauchy-Green deformation tensor. Relevant insights were obtained on the mechanical behavior of fetal membranes. Collagen and its cross-linking were shown to determine membrane’s stiffness and strength for multiaxial stress states. Their nonlinear deformation behavior characterizes the fetal membranes as I ₂ material.     

Fracture Mechanics of Collagen Fibrils: Influence of Natural Cross-Links

Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within fibrils is limited. The presence of covalent enzymatic cross-links between collagen molecules is an important factor that has been shown to influence mechanical behavior of the tendons. To improve our understanding of how molecular bonds translate into tendon mechanics, we used an atomic force microscopy technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human collagen fibrils. There was an initial rise in modulus followed by a plateau with reduced modulus, which was finally followed by an even greater increase in stress and modulus before failure. The RTTs also displayed the initial increase and plateau phase, but the third region was virtually absent and the plateau continued until failure. The importance of cross-link lability was investigated by NaBH₄ reduction of the rat-tail fibrils, which did not alter their behavior. These findings shed light on the function of cross-links at the fibril level, but further studies will be required to establish the underlying mechanisms.     

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.     

Graphene-based pressure nano-sensors

We perform atomistic simulations to study the failure behavior of graphene-based pressure sensor, which is made of a graphene nanoflake suspended over a well in a silicon-carbide substrate and clamped on its surrounding edge by the covalent bonds between the graphene flake and the substrate. Two distinct types of mechanical failure are identified: the first one is characterized by complete detachment of the graphene nanoflake from the silicon-carbide substrate via breaking the covalent bonds between the carbon atoms of the graphene flake and the silicon atoms of the substrate; the second type is characterized by the rupture of the graphene nanoflake via breaking the carbon-carbon bonds within the graphene. The type of mechanical failure is determined by the clamped area between the graphene flake and the substrate. The failure pressure can be tuned by changing the clamped area and the well radius. A model is proposed to explain the transition between the two types of failure mode. The present work provides a quantitative framework for the design of graphene-based pressure sensors.     

Type III Collagen,a Fibril Network Modifier in Articular Cartilage

The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an α1(II) and an α1(III) telopeptide to the α1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.     

In Vitro Fracture Testing of Submicron Diameter Collagen Fibril Specimens

Mechanical testing of collagenous tissues at different length scales will provide improved understanding of the mechanical behavior of structures such as skin, tendon, and bone, and also guide the development of multiscale mechanical models. Using a microelectromechanical-systems (MEMS) platform, stress-strain response curves up to failure of type I collagen fibril specimens isolated from the dermis of sea cucumbers were obtained in vitro. A majority of the fibril specimens showed brittle fracture. Some displayed linear behavior up to failure, while others displayed some nonlinearity. The fibril specimens showed an elastic modulus of 470 ± 410 MPa, a fracture strength of 230 ± 160 MPa, and a fracture strain of 80% ± 44%. The fibril specimens displayed significantly lower elastic modulus in vitro than previously measured in air. Fracture strength/strain obtained in vitro and in air are both significantly larger than those obtained in vacuo, indicating that the difference arises from the lack of intrafibrillar water molecules produced by vacuum drying. Furthermore, fracture strength/strain of fibril specimens were different from those reported for collagenous tissues of higher hierarchical levels, indicating the importance of obtaining these properties at the fibrillar level for multiscale modeling.     

Molecular and Mesoscale Mechanisms of Osteogenesis Imperfecta Disease in Collagen Fibrils

Osteogenesis imperfecta (OI) is a genetic disorder in collagen characterized by mechanically weakened tendon, fragile bones, skeletal deformities, and in severe cases, prenatal death. Although many studies have attempted to associate specific mutation types with phenotypic severity, the molecular and mesoscale mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length scales remain unknown. We show by a hierarchy of full atomistic and mesoscale simulation that OI mutations severely compromise the mechanical properties of collagenous tissues at multiple scales, from single molecules to collagen fibrils. Mutations that lead to the most severe OI phenotype correlate with the strongest effects, leading to weakened intermolecular adhesion, increased intermolecular spacing, reduced stiffness, as well as a reduced failure strength of collagen fibrils. We find that these molecular-level changes lead to an alteration of the stress distribution in mutated collagen fibrils, causing the formation of stress concentrations that induce material failure via intermolecular slip. We believe that our findings provide insight into the microscopic mechanisms of this disease and lead to explanations of characteristic OI tissue features such as reduced mechanical strength and a lower cross-link density. Our study explains how single point mutations can control the breakdown of tissue at much larger length scales, a question of great relevance for a broad class of genetic diseases.     

Direct measurement of the rupture force of single pair of decorin interactions

Decorin is one important member of the family of small leucine-rich proteoglycans, which are widely distributed in connective tissues in the body such as tendon and ligament. Decorin may be responsible for collagen fibril connection in those tissues. A recent hypothesis suggests that decorin may bind to collagen with its core protein while binding to another decorin through the interaction with their glycosaminoglycan (GAG) chains. However, there is no direct evidence supporting this hypothesis to date. In this study, the interaction of decorin GAG chains was directly determined for the first time. The rupture force of single bonds between decorins (GAG chains interaction) was determined directly as 16.5+/-5.1 pN using a laser tweezers/interferometer single molecular nanomechanical testing system. This information can improve our understanding of the mechanical properties of connective tissues at the molecular level.     

Multiscale mechanobiology: Coupling models of adhesion kinetics and nonlinear tissue mechanics

The mechanical behavior of tissues at the macroscale is tightly coupled to cellular activity at the microscale. Dermal wound healing is a prominent example of a complex system in which multiscale mechanics regulate restoration of tissue form and function. In cutaneous wound healing, a fibrin matrix is populated by fibroblasts migrating in from a surrounding tissue made mostly out of collagen. Fibroblasts both respond to mechanical cues, such as fiber alignment and stiffness, as well as exert active stresses needed for wound closure.Here, we develop a multiscale model with a two-way coupling between a microscale cell adhesion model and a macroscale tissue mechanics model. Starting from the well-known model of adhesion kinetics proposed by Bell, we extend the formulation to account for nonlinear mechanics of fibrin and collagen and show how this nonlinear response naturally captures stretch-driven mechanosensing. We then embed the new nonlinear adhesion model into a custom finite element implementation of tissue mechanical equilibrium. Strains and stresses at the tissue level are coupled with the solution of the microscale adhesion model at each integration point of the finite element mesh. In addition, solution of the adhesion model is coupled with the active contractile stress of the cell population. The multiscale model successfully captures the mechanical response of biopolymer fibers and gels, contractile stresses generated by fibroblasts, and stress-strain contours observed during wound healing. We anticipate that this framework will not only increase our understanding of how mechanical cues guide cellular behavior in cutaneous wound healing, but will also be helpful in the study of mechanobiology, growth, and remodeling in other tissues.     

A model for the stability and creep of organic materials

A model is presented for the thermally assisted breaking of a number of bonds arranged in parallel and stressed by an individual soft spring each. Using a simplified potential for the bond it is shown that in equilibrium there are two definite regions of elastic behavior: one with all bonds intact, the other with a variable fraction of bonds broken, therefore with a tangent modulus steadily decreasing with applied stress. Criteria are given for the existence of these regions. Beyond these regions time-dependent creep to rupture is found, limited, in turn, by the theoretical fracture strength, the stress necessary for fracture without any thermal assistance, beyond which a bound state is impossible. The time-to-fracture for creep rupture is calculated and an example of the time evolution of the accelerating creep given. The results of the calculations are applied to experimental data on Wallaby tendons by Wang and Ker (J. Exp. Biol. 198 (1995) 831) and data estimated for the bond potential depth, the theoretical fracture strength and the number density of bonds involved as well as the elastic modulus of the ensemble. Values are derived under the assumption of one deformation mechanism being dominant--e.g., (sub-)fibril sliding or sliding of collagen molecules along one another--but the model cannot definitely distinguish between mechanisms.     

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1^+/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.     

Biochemical and biophysical characterization of EDC treated rattus type I collagen

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) treatment changes the biophysical and biochemical properties of collagen. EDC is known to be able to form an isopeptide bond between carboxyl and amine reactive groups. Surprisingly EDC did not form significant number of isopeptide bonds between intra-collagen and inter-collagen. EDC treatment enhances the helicity of the polyproline II conformation and fibril assembly. The treatment increases the hydrothermal stability, enhances cell viability and inhibit breakdown by bacterial collagenases. EDC treatment is a simple method to manufacture durable biomatrix with desirable properties.     

Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage

From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the alpha1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the alpha1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of alpha1(IX) and the known cross-linking lysine at residue 930 of the alpha1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network.     

Molecular assessment of the elastic properties of collagen-like homotrimer sequences

Knowledge of the mechanical behavior of collagen molecules is critical for understanding the mechanical properties of collagen fibrils that constitute the main architectural building block of a number of connective tissues. In this study, the elastic properties of four different type I collagen 30-residue long molecular sequences, were studied by performing stretching simulations using the molecular mechanics approach. The energy–molecular length relationship was achieved by means of the geometry optimization procedure for collagen molecule strains up to 10%. The energy was interpolated by a second order function, and the second order of the derivative with respect to the mean length corresponded to the molecule stiffness. According to the hypothesis of linear elastic behavior, except for one sequence, the elastic modulus was around 2.40 GPa. These values are larger than fibril values, and they confirm the hypothesis that tendon mechanical properties are deeply related to tendon hierarchical structure. A possible explanation of the lowest values obtained for one sequence (1.33–1.53 GPa) is provided and discussed.     

Modeling the effect of collagen fibril alignment on ligament mechanical behavior

Ligament mechanical behavior is primarily regulated by fibrous networks of type I collagen. Although these fibrous networks are typically highly aligned, healthy and injured ligament can also exhibit disorganized collagen architecture. The objective of this study was to determine whether variations in the collagen fibril network between neighboring ligaments can predict observed differences in mechanical behavior. Ligament specimens from two regions of bovine fetlock joints, which either exhibited highly aligned or disorganized collagen fibril networks, were mechanically tested in uniaxial tension. Confocal microscopy and FiberFit software were used to quantify the collagen fibril dispersion and mean fibril orientation in the mechanically tested specimens. These two structural parameters served as inputs into an established hyperelastic constitutive model that accounts for a continuous distribution of planar fibril orientations. The ability of the model to predict differences in the mechanical behavior between neighboring ligaments was tested by (1) curve fitting the model parameters to the stress response of the ligament with highly aligned fibrils and then (2) using this model to predict the stress response of the ligament with disorganized fibrils by only changing the parameter values for fibril dispersion and mean fibril orientation. This study found that when using parameter values for fibril dispersion and mean fibril orientation based on confocal imaging data, the model strongly predicted the average stress response of ligaments with disorganized fibrils (\(R^{2}=0.97\)); however, the model only successfully predicted the individual stress response of ligaments with disorganized fibrils in half the specimens tested. Model predictions became worse when parameters for fibril dispersion and mean fibril orientation were not based on confocal imaging data. These findings emphasize the importance of collagen fibril alignment in ligament mechanics and help advance a mechanistic understanding of fibrillar networks in healthy and injured ligament.