Heterogeneous Structure and Dynamics of Water in a Hydrated Collagen Microfibril.

Collagen type I is well-known for its outstanding mechanical properties which it inherits from its hierarchical structure. Collagen type I fibrils may be viewed as a heterogeneous material made of protein, macromolecules (such as glycosaminoglycans and proteoglycans) and water. Water content modulates the properties of these fibrils. Yet, the properties of water and the fine interactions of water with the protein constituent of these heterofibrils have only received limited attention. Here, we propose to model collagen type I fibrils as a hydrated structure made of tropocollagen molecules assembled in a microfibril crystal. We perform large-scale all-atom molecular dynamics simulations of the hydration of collagen fibrils beyond the onset of disassembly. We found that the structural and dynamic properties of water vary strongly with the level of hydration of the microfibril. More importantly, we found that the properties vary spatially within the 67 nm D-spacing periodic structure. Alteration of the structural and dynamical properties of the collagen microfibril occur first in the gap region. Overall, we identify that the change in the role of water molecules from glue to lubricant between tropocollagen molecules arises around 100% hydration while the microfibril begins to disassemble beyond 130% water content. Our findings are supported by a decrease in hydrogen bonding, recovery of bulk water properties and amorphization of the tropocollagen molecules packing. Our simulations reveal the structure and dynamics of hydrated collagen fibrils with unprecedented spatial resolution from physiological conditions to disassembly. Beyond the process of self-assembly and the emergence of mechanical properties of collagen type I fibrils, our results may also provide new insights into mineralization of collagen fibrils.

Self-Assembled Hydrogel Membranes with Structurally Tunable Mechanical and Biological Properties.

Using supramolecular self-assembled nanocomposite materials made from protein and polysaccharide components is becoming more popular because of their unique properties, such as biodegradability, hierarchical structures, and tunable multifunctionality. However, the fabrication of these materials in a reproducible way remains a challenge. This study presents a new evaporation-induced self-assembly method producing layered hydrogel membranes (LHMs) using tropocollagen grafted by partially deacetylated chitin nanocrystals (CO-g-ChNCs). ChNCs help stabilize tropocollagen's helical conformation and fibrillar structure by forming a hierarchical microstructure through chemical and physical interactions. The LHMs show improved mechanical properties, cytocompatibility, and the ability to control drug release using octenidine dihydrochloride (OCT) as a drug model. Because of the high synergetic performance between CO and ChNCs, the modulus, strength, and toughness increased significantly compared to native CO. The biocompatibility of LHM was tested using the normal human dermal fibroblast (NHDF) and the human osteosarcoma cell line (Saos-2). Cytocompatibility and cell adhesion improved with the introduction of ChNCs. The extracted ChNCs are used as a reinforcing nanofiller to enhance the performance properties of tropocollagen hydrogel membranes and provide new insights into the design of novel LHMs that could be used for various medical applications, such as control of drug release in the skin and bone tissue regeneration.

Finite element modeling of α-helices and tropocollagen molecules referring to spike of SARS-CoV-2.

The newly developed finite element (FE) modeling at the atomic scale was used to predict the static and dynamic response of the α-helix (AH) and tropocollagen (TC) protein fragments, the main building blocks of the spike of the SARS-CoV-2. The geometry and morphology of the spike's stalk and its connection to the viral envelope were determined from the combination of most recent molecular dynamics (MD) simulation and images of cryoelectron microscopy. The stiffness parameters of the covalent bonds in the main chain of the helix were taken from the literature. The AH and TC were modeled using both beam elements (wire model) and shell elements (ribbon model) in FE analysis to predict their mechanical properties under tension. The asymptotic stiffening features of AH and TC under tensile loading were revealed and compared with a new analytical solution. The mechanical stiffnesses under other loading conditions, including compression, torsion, and bending, were also predicted numerically and correlated with the results of the existing MD simulations and tests. The mode shapes and natural frequencies of the spike were predicted using the built FE model. The frequencies were shown to be within the safe range of 1-20 MHz routinely used for medical imaging and diagnosis by means of ultrasound. These results provide a solid theoretical basis for using ultrasound to study damaging coronavirus through transient and resonant vibration at large deformations.

Nanomechanical mapping of hydrated rat tail tendon collagen I fibrils.

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 µm in length, were acquired at indentation speeds around 10(5) nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10(5) nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.

Strain-induced collagen denaturation is rate dependent in failure of cerebral arteries.

While soft tissues are commonly damaged by mechanical loading, the manifestation of this damage at the microstructural level is not fully understood. Specifically, while rate-induced stiffening has been previously observed in cerebral arteries, associated changes in microstructural damage patterns following high-rate loading are largely undefined. In this study, we stretched porcine middle cerebral arteries to failure at 0.01 and >150 s-1, both axially and circumferentially, followed by probing for denatured tropocollagen using collagen hybridizing peptide (CHP). We found that collagen fibrils aligned with the loading direction experienced less denaturation following failure tests at high than low rates. Others have demonstrated similar rate dependence in tropocollagen denaturation during soft tissue failure, but this is the first study to quantify this behavior using CHP and to report it for cerebral arteries. These findings may have significant implications for traumatic brain injury and intracranial balloon angioplasty. We additionally observed possible tropocollagen denaturation in vessel layers primarily composed of fibrils transversely aligned to the loading axis. To our knowledge, this is the first observation of collagen denaturation due to transverse loading, but further research is needed to confirm this finding. STATEMENT OF SIGNIFICANCE: Previous work shows that collagen hybridizing peptide (CHP) can be used to identify collagen molecule unfolding and denaturation in mechanically overloaded soft tissues, including the cerebral arteries. But experiments have not explored collagen damage at rates relevant to traumatic brain injury. In this work, we quantified collagen damage in cerebral arteries stretched to failure at both high and low rates. We found that the collagen molecule is less damaged at high than at low rates, suggesting that damage mechanisms of either the collagen molecule or other elements of the collagen superstructure are rate dependent. This work implies that arteries failed at high rates, such as in traumatic brain injury, will have different molecular-level damage patterns than arteries failed at low rates. Consequently, improved understanding of damage characteristics may be expanded in the future to better inform clinically relevant cases of collagen damage such as angioplasty and injury healing.

Multiscale approach incorporating tropocollagen scale to assess the effect of molecular age-related modifications on elastic constants of cortical bone based on finite element and homogenization methods.

With osteoporosis and aging, structural changes occur at all hierarchical levels of bone from the molecular scale to the whole tissue, which requires multiscale modeling to analyze the effect of these modifications on the mechanical behavior of bone and its remodeling process. In this paper, a novel hybrid multiscale model for cortical bone incorporating the tropocollagen molecule based on the combination of finite element method and different homogenization techniques was developed. The objective was to investigate the influence of age-related structural alterations that occur at the molecular level, namely the decrease in both molecular diameter (due to the loss of hydration) and number of hydrogen bonds, on mechanical properties of the bone tissue. The proposed multiscale hierarchical approach is divided in two phases: (i) in Step 0, a realistic 3D finite element model for tropocollagen was used to estimate the effective elastic properties at the molecular scale as a function of the collagen molecule's degree of hydration (represented by its external diameter) and the number of its intramolecular hydrogen bonds, and (ii) in Steps 1-10, the effective elastic constants at the higher scales from mineralized fibril to continuum cortical bone tissue were predicted analytically using homogenization equations. The results obtained in healthy mature cortical bone at different scales are in good agreement with the experimental data and multiscale models reported in the literature. Moreover, our model made it possible to visualize the influence of the two parameters (molecular diameter and number of hydrogen bonds) that represent the main age-related alterations at the molecular scale on the mechanical properties of cortical bone, at its different hierarchical levels. Keywords: Bone aging, multiscale model, tropocollagen, cortical bone, finite element modeling, homogenization method.

Stability of the hydration layer of tropocollagen: A QM study.

Collagen is a triple helical protein, highly hydrated in nature. Bella and Berman (J Mol Biol 1996, 264, 734) have reported the structure of the first hydration layer. Water molecules form bridges of different length around the POG repeats and self assemble into left-handed helical water threads. To explore the stability of these specifically hydrated places, we have designed suitable QM models: each comprises a triple helix formed by 18 residues surrounded by 8 to 12 explicit waters. Two sets of amino acids were used, one standing for the core structural subunit of tropocollagen (POG-model) and one for its natural enzyme recognition sites (AAG-model). We have determined the stability order of the water binding places, the strongest being -8.1 kcal mol(-1), while the weakest -6.1 kcal mol(-1) per hydrogen bond. In X-ray structures, each triplet of tropocollagen is shielded by six to nine water molecules. Beside the mandatory six, the "surplus" three water molecules further strengthen the binding of all the others. However, the displacement of selected water molecules turns out to be energy neutral. These water binding places on the surface of the triple helix can provide explanation on how an almost liquid-like hydration environment exists between the closely packed tropocollagens (Henkelman et al., Magn Reson Med 1994, 32, 592). It seems that these water reservoirs or buffers can provide space for "hole conduction" of water molecules and thus contribute to the elasticity of collagen.

Effect of changes in tropocollagen residue sequence and hydroxyapatite mineral texture on the strength of ideal nanoscale tropocollagen-hydroxyapatite biomaterials.

Changes in mineral texture (e.g. hydroxyapatite (HAP) or aragonite) and polypeptide (e.g. tropocollagen (TC)) residue sequence are characteristic features of a disease known as osteogenesis imperfecta (OI). In OI, different possibilities of changes in polypeptide residue sequence as well as changes in polypeptide helix replacement (e.g. 3 alpha1 chains instead of 2 alpha1 and 1 alpha2 chain in OI murine) exist. The cross section of the HAP crystals could be needle like or plate like. Such texture and residue sequence related changes can significantly affect the material strength at the nanoscale. In this work, a mechanistic understanding of such factors in determining strength of nanoscale TC-HAP biomaterials is presented using three dimensional molecular dynamics (MD) simulations. Analyses point out that the peak interfacial strength for failure is the highest for supercells with plate shaped HAP crystals. TC molecules with higher number of side chain functional groups impart higher strength to the TC-HAP biomaterials at the nanoscale. Overall, HAP crystal shape variation, the direction of applied loading with respect to the relative TC-HAP orientation, and the number of side chain functional groups in TC molecules are the factor that affect TC-HAP biomaterial strength in a significant manner.

Numerical modeling of the effects hydration and number of hydrogen bonds on the mechanical properties of the tropocollagen molecule.

Bone aging involves structural and molecular modifications, especially at the level of type I tropocollagen. This macromolecule shows two main age-related alterations, which are the decrease of both molecular diameter (due to the loss of hydration) and number of hydrogen bonds. In this work, it is proposed to investigate the influence of these two parameters (molecular diameter and number of hydrogen bonds) on the mechanical behavior of tropocollagen using finite element method. To this end, a novel three-dimensional finite element model of collagen molecule accounting for hydrogen bonds was developed. Then, a numerical design of experiments for the diameter of tropocollagen and variations in the number of hydrogen bonds has been established. The mechanical properties ("load-strain" curve and apparent Young's modulus) of the collagen molecule were obtained by employing the proposed model to uniaxial tensile tests. The parametric study demonstrates that the mechanical properties of tropocollagen are slightly affected by the rate of hydration but considerably affected by variation of the number of hydrogen bonds. Finally, a fitted analytical function was deduced from the above results showing effects of the two parameters (hydration rate and hydrogen bonds) on the apparent Young's modulus of tropocollagen. This study could be useful to understand the influence of structural age modifications of tropocollagen on the macroscopic mechanical properties of bone.

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.

Steered molecular dynamics characterization of the elastic modulus and deformation mechanisms of single natural tropocollagen molecules.

Collagen is a common structural protein, providing mechanical integrity for various vertebrate connective tissues such as cartilage and bone. The mechanical behaviours of these tissues under physical stimulations are controlled by the hierarchical structure of collagen and its interactions with other extracellular matrix molecules. However, the mechanical properties and deformation mechanisms of natural collagen under physiological loading rates at the molecular level are not fully understood. In this study, comprehensive steered molecular dynamics (SMD) simulations were performed on the 2nd intact overlap region (d2ol) and the 2nd intact D-period (d2olgp) of an in-situ characterized collagen molecule, under a large range of strain rates (6.5 × 106% s-1 to 1.3 × 1012% s-1). The results show that, depending on the applied strain rates, tropocollagen molecules unfold in different ways. Particularly, at high and intermediate strain rates, the number of inter-chain hydrogen bonds decreases rapidly even at small deformations, leading to a dramatic increase in the force. This results in an increase in the estimated Young's modulus of collagen triple helices as the deformation rate goes up, which, together with the nonlinear mechanical behaviour, explains the broad range of the Young's modulus for collagen model peptides reported in earlier SMD studies. Atomistic-level analyses indicate that the elastic modulus of single tropocollagen molecules decreases as the strain rate becomes smaller. However, for strain rates below 1.3 × 108% s-1, the tangent Young's modulus of d2ol (d2olgp) converges to approximately 3.2 GPa (3.4 GPa), at the strain of 10.5% (12%) when the segment is fully uncrimped. Furthermore, for strain rates under 1.3 × 108% s-1, d2ol and d2olgp show identical deformation mechanisms (unwinding, uncoiling and backbone stretching), but the corresponding strain ranges are different. This study will aid in future studies on characterizing the mechanical properties of collagen molecules and collagen-like peptides by indicating the proper pulling strain rates and how to determine the suitable strain range used for evaluating the elastic modulus.

High pressure Raman study of type-I collagen.

The high pressure response of type-I collagen from bovine Achilles tendon is investigated with micro-Raman spectroscopy. Fluorinert™ and methanol-ethanol mixtures were used as pressure transmitting media (PTM) in a diamond anvil cell. The Raman spectrum of collagen is dominated by three bands centred at approximately 1450, 1660 and 2930 cm-1 , attributed to C-H deformation, C=O stretching of the peptide bond (amide-I band) and C-H stretching modes respectively. Upon pressure increase, using Fluorinert™ as PTM, a shift towards higher frequencies of the C-H stretching and deformation peaks is observed. Contrary, the amide-I band peaks are shifted to lower frequencies with moderate pressure slopes. On the other hand, when using the alcohol mixture as PTM, the amide-I band exhibits more pronounced C=O bond softening, deduced from the shift to lower frequencies, suggesting a strengthening of the hydrogen bonds between glycine and proline residues of different collagen chains due to the presence of the polar alcohol molecules. Furthermore, some of the peaks exhibit abrupt changes in their pressure slopes at approximately 2 GPa, implying a variation in the compressibility of the collagen fibres. This could be attributed to a pitch change from 10/3 to 7/2, sliding of the tropocollagen molecules, twisting variation at the molecular level and/or elimination of the D-gaps induced by kink compression. All spectral changes are reversible upon pressure release, which indicates that denaturation has not taken place. Finally, a minor lipid phase contamination was detected in some sample spots. Its pressure response is also monitored.

In vitro fibrillogenesis of tropocollagen type III in collagen type I affects its relative fibrillar topology and mechanics.

Tropocollagen types I and III were simultaneously fibrilized in vitro, and the differences between the geometric and mechanical properties of the heterotypic fibrils with different mixing ratios of tropocollagen III to I were investigated. Transmission electron microscopy was used to confirm the simultaneous presence of both tropocollagen types within the heterotypic fibrils. The incorporation of collagen III in I caused the fibrils to be thinner with a shorter D-banding than pure collagen I. Hertzian contact model was used to obtain the elastic moduli from atomic force microscope indentation testing using a force volume analysis. The results indicated that an increase in the percentage of tropocollagen III reduced the mechanical stiffness of the obtained fibrils. The mechanical stiffness of the collagen fibrils was found to be greater at higher loading frequencies. This observation might explain the dominance of collagen III over I in soft distensible organs such as human vocal folds.

New three-dimensional model based on finite element method of bone nanostructure: single TC molecule scale level.

At the macroscopic scale, the bone mechanical behavior (fracture, elastic) depends mainly on its components' nature at the nanoscopic scale (collagen, mineral). Thus, an understanding of the mechanical behavior of the elementary components is demanded to understand the phenomena that can be observed at the macroscopic scale. In this article, a new numerical model based on finite element method is proposed in order to describe the mechanical behavior of a single Tropocollagen molecule. Furthermore, a parametric study with different geometric properties covering the molecular composition and the rate hydration influence is presented. The proposed model has been tested under tensile loading. While focusing on the entropic response, the geometric parameter variation effect on the mechanical behavior of Tropocollagen molecule has been revealed using the model. Using numerical and experimental testing, the obtained numerical simulation results seem to be acceptable, showing a good agreement with those found in literature.

A study of collagen and gelatin solutions by optical self beat spectroscopy.

A study of the conformation of collagen and gelatin in aqueous solution by Optical Self Beat Spectroscopy is reported. The translational diffusion coefficient of monomeric tropocollagen was experimentally measured from the half-width of the Rayleigh scattered radiation and the value obtained is shown to be in good agreement with that calculated from hydrodynamical theory for the tropocollagen rod. The Self Beat Spectrometer was also used to investigate the factors affecting the aggregation and flexing of molecules in dilute gelatin solutions and the gel-sol transition in more concentrated gelatin solutions.

Elastic properties, Young's modulus determination and structural stability of the tropocollagen molecule: a computational study by steered molecular dynamics.

The aim of this report is to investigate at microscopic level the elastic properties of a tropocollagen-like molecule submitted to linear traction along its longitudinal axis. For this purpose, we performed steered molecular dynamics (SMD) simulations for a wide range of spring constants in order to test the molecular response based on a two-spring model connected in series. An elastic behavior was observed in an elongation range of 2.5-4% of the molecular length, estimating an "effective molecular elastic constant" of 1.02+/-0.20 kcal/mol A2 in this region. Accordingly, a Young's modulus for the tropocollagen molecule of Y=4.8+/-1.0 GPa was calculated. The complex hydrogen bond network was traced along molecular dynamics (MD) and SMD simulations revealing a rearrangement of these interactions preserving the integrity of the molecular structure when submitted to traction. No evidence of the significant role attributed to water bridges for structural stability was detected, on the contrary facts pointed out that the hydrogen bond network might be the responsible.

Functional anatomy and physiology of tendons.

Tendons vary in type, shape, and size. The anatomy, histology, and physiology of tendons are described in this article, with particular reference to those tendons that are vulnerable in sports medicine.

[Nutritive fibers of animal origin. Collagen and its fractions as essential components of new and efficient food products]. / Pishchevye volokna zhivotnogo proiskhozhdeniia. Kollagen i ego fraktsii kak neobkhodimye komponenty novykh i éffektivnykh pishchevykh produktov.

Data on the production of collagen and its fractions (which in many cases surpass the parent protein in functional properties), reported in the literature throughout the past two decades, are reviewed. The material presented elucidates the role played by collagen and its fractions in the development of new, ecologically pure foodstuffs containing nutritive fibers. As follows from the analysis of the data, collagen fractions with molecular weights in excess of 120 kDa offer the greatest promise for producing useful foodstuffs.

Nanomechanical properties of mineralised collagen microfibrils based on finite elements method: biomechanical role of cross-links.

Hierarchical structures in bio-composites such as bone tissue have many scales or levels and synergic interactions between the different levels. They also have a highly complex architecture in order to fulfil their biological and mechanical functions. In this study, a new three-dimensional (3D) model based on the finite elements (FEs) method was used to model the relationship between the hierarchical structure and the properties of the constituents at the sub-structure scale (mineralised collagen microfibrils) and to investigate their apparent nanomechanical properties. The results of the proposed FE simulations show that the elastic properties of microfibrils depend on different factors such as the number of cross-links, the mechanical properties and the volume fraction of phases. The results obtained under compression loading at a small deformation < 2% show that the microfibrils have a Young's modulus (Ef) ranging from 0.4 to 1.16 GPa and a Poisson's ratio ranging from 0.26 to 0.3. These results are in excellent agreement with experimental data (X-ray, AFM and MEMS) and molecular simulations.

An analysis of the effects of temperature and structural arrangements on the thermal conductivity and thermal diffusivity of tropocollagen-hydroxyapatite interfaces.

The ability of a biomaterial to transport energy by conduction is best characterized in the steady state by its thermal conductivity and in the non-steady state by its thermal diffusivity. The complex hierarchical structure of most biomaterials makes the direct determination of the thermal diffusivity and thermal conductivity difficult using experimental methods. This study presents a classical molecular simulation based approach for the thermal diffusivity and thermal conductivity prediction for a set of tropocollagen and hydroxyapatite based idealized biomaterial interfaces. The thermal diffusivity and thermal conductivity values are calculated using the presented approach at three different temperatures (300 K, 500 K and 700 K). The effects of temperature, structural arrangements, and size of simulated systems on the thermal properties are analyzed. Analyses point out important role played by the interface orientation, interface area, and structural hierarchy. Ensuing discussions establish that the interface structural arrangement and interface orientation combined with biomimetic structural hierarchy can lead to non-intuitive thermal property variations as a function of structural features.