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.

Demonstrating collagen tendon fibril segments involvement in intrinsic tendon repair.

Severed tendons can undergo regenerative healing, intrinsic tendon repair. Fibrillogenesis of chick tendon involves "collagen fibril segments" (CFS), which are the building blocks of collagen fibers that make up tendon fascicles. The CFS are 10.5 micron in length, composed of tropocollagen monomers arranged in parallel arrays. Rather than incorporating single tropocollagen molecules into growing collagen fibers, incorporating large CFS units is the mechanism for generating collagen fibers. Is intrinsic tendon repair through the reestablishment of tendon embryogenesis? Gentamicin treated 10-day-old chick embryo tendons released CFS were fluorescently tagged with Rhodamine (Rh). Organ cultured severed 14-day-old embryo tendon explants received Rh tagged CFS. At day 4 auto fluorescent tagged CFS were identified at the severed tendon ends by fluorescent microscopy. Accumulation of fluorescent tagged CFS was exclusively localized to the severed ends of tendon explants. Parallels between collagen fiber growth during embryonic fibrillogenesis and tendon repair reveal CFS incorporation is responsible for collagen fibers growth. CFS incorporation into frayed collagen fibers from severed tendons is the proposed mechanism for intrinsic tendon repair, which is an example of regenerative repair.

Nanomechanical sequencing of collagen: tropocollagen features heterogeneous elastic properties at the nanoscale.

Collagen is the most important structural protein in biology and is responsible for the strength and integrity of tissues such as bone, teeth, cartilage and tendon. Here we report a systematic computational sequencing of the effect of amino acid motif variations on the mechanical properties of single tropocollagen molecules, with a particular focus on elastic deformation at varying applied strains. By utilizing a bottom-up computational materiomics approach applied to four model sequence motifs found in human type I collagen, we show that variations in the amino acid motif severely influence the elastic behavior of tropocollagen molecules, leading to softening or stiffening behavior. We also show that interpeptide interactions via H-bonds vary strongly with the type of motif, which implies that it plays a distinct role in the molecule's stability. The most important implication of our results is that deformation in tropocollagen molecules is highly inhomogeneous, since softer regions deform more than stiffer regions, potentially leading to strain and stress concentrations within collagen fibrils. We confirm the hypothesis of inhomogeneous molecular deformation through direct simulation of stretching of a segment of tropocollagen from human type I collagen that features the physiological amino acid sequence. Our results show that the biomechanical properties of tropocollagen must be understood in the context of the specific amino acid sequence as well as the state of deformation, since the elastic properties depend strongly on the amount of deformation applied to a molecule.

Ultrastructural and immunohistochemical analysis of fibrous long-spacing collagen fibrils in malignant mesothelioma.

Three cases of biphasic mesothelioma and 2 cases of sarcomatoid mesothelioma were investigated using light and electron microscopy. In 2 of the 3 cases of biphasic mesotheliomas, fibrous long-spacing (FLS) collagen fibrils were discovered with a symmetrical cross-striation of 130 nm in periodicity. However, no connection between the FLS fibrils and usual collagen fibrils were observed. Periodic acid silver methenamine stain revealed unstained bands with periods of 130 nm in FLS fibrils, whereas the usual collagen fibrils showed continuous positive staining. All 3 cases of biphasic mesotheliomas showed deposits of hyaluronic acid, whereas both cases of sarcomatoid mesotheliomas showed little hyaluronic acid. As a high concentration of hyaluronic acid induces the formation of FLS collagen fibrils in vitro, the authors propose that FLS fibrils from mesothelioma may be special structures that occur as the tropocollagens are assembled into new collagen fibrils in the presence of hyaluronic acid.

Entropic elasticity controls nanomechanics of single tropocollagen molecules.

We report molecular modeling of stretching single molecules of tropocollagen, the building block of collagen fibrils and fibers that provide mechanical support in connective tissues. For small deformation, we observe a dominance of entropic elasticity. At larger deformation, we find a transition to energetic elasticity, which is characterized by first stretching and breaking of hydrogen bonds, followed by deformation of covalent bonds in the protein backbone, eventually leading to molecular fracture. Our force-displacement curves at small forces show excellent quantitative agreement with optical tweezer experiments. Our model predicts a persistence length xi(p) approximately 16 nm, confirming experimental results suggesting that tropocollagen molecules are very flexible elastic entities. We demonstrate that assembly of single tropocollagen molecules into fibrils significantly decreases their bending flexibility, leading to decreased contributions of entropic effects during deformation. The molecular simulation results are used to develop a simple continuum model capable of describing an entire deformation range of tropocollagen molecules. Our molecular model is capable of describing different regimes of elastic and permanent deformation, without relying on empirical parameters, including a transition from entropic to energetic elasticity.

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.

Topography and mechanical properties of single molecules of type I collagen using atomic force microscopy.

Although the mechanical behavior of tendon and bone has been studied for decades, there is still relatively little understanding of the molecular basis for their specific properties. Thus, despite consisting structurally of the same type I collagen, bones and tendons have evolved to fulfill quite different functions in living organisms. In an attempt to understand the links between the mechanical properties of these collageneous structures at the macro- and nanoscale, we studied trimeric type I tropocollagen molecules by atomic force microscopy, both topologically and by force spectroscopy. High-resolution imaging demonstrated a mean (+/- SD) contour length of (287 +/- 35) nm and height of (0.21 +/- 0.03) nm. Submolecular features, namely the coil-pitch of the molecule, were also observed, appearing as a repeat pattern along the length of the molecule, with a length of approximately 8 nm that is comparable to the theoretical value. Using force spectroscopy, we established the stretching pattern of the molecule, where both the mechanical response of the molecule and pull-off peak are convoluted in a single feature. By interpreting this response with a wormlike chain model, we extracted the value of the effective contour length of the molecule at (202 +/- 5) nm. This value was smaller than that given by direct measurement, suggesting that the entire molecule was not being stretched during the force measurements; this is likely to be related to the absence of covalent binding between probe, sample, and substrate in our experimental procedure.