Heterogeneity in proline hydroxylation of fibrillar collagens observed by mass spectrometry

Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.

Mechanical stretching changes crosslinking and glycation levels in the collagen of mouse tail tendon

Collagen I is a major tendon protein whose polypeptide chains are linked by covalent crosslinks. It is unknown how the crosslinking contributes to the mechanical properties of tendon or whether crosslinking changes in response to stretching or relaxation. Since their discovery, imine bonds within collagen have been recognized as being important in both crosslink formation and collagen structure. They are often described as acidic or thermally labile, but no evidence is available from direct measurements of crosslink levels whether these bonds contribute to the mechanical properties of collagen. Here, we used MS to analyze these imine bonds after reduction with sodium borohydride while under tension and found that their levels are altered in stretched tendon. We studied the changes in crosslink bonding in tail tendon from 11-week-old C57Bl/6 mice at 4% physical strain, at 10% strain, and at breaking point. The crosslinks hydroxy-lysino-norleucine (HLNL), dihydroxy-lysino-norleucine (DHLNL), and lysino-norleucine (LNL) in-creased or decreased depending on the specific crosslink and amount of mechanical strain. We also noted a decrease in glycated lysine residues in collagen, indicating that the imine formed between circulating glucose and lysine is also stress labile. We also carried out mechanical testing, including cyclic testing at 4% strain, stress relaxation tests, and stress-strain profiles taken at breaking point, both with and without sodium borohydride reduction. The results from both the MS studies and mechanical testing provide insights into the chemical changes during tendon stretching and directly link these chemical changes to functional collagen properties.

Age-related changes in the physical properties, cross-linking, and glycation of collagen from mouse tail tendon

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrated that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decreased with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20-week-old WT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes.

A damage model for collagen fibres with an application to collagenous soft tissues

We propose a mechanical model to account for progressive damage in collagen fibres within fibrous soft tissues. The model has a similar basis to the pseudoelastic model that describes the Mullins effect in rubber but it also accounts for the effect of cross-links between collagen fibres. We show that the model is able to capture experimental data obtained from rat tail tendon fibres, and the combined effect of damage and collagen cross-links is illustrated for a simple shear test. The proposed three-dimensional framework allows a straightforward implementation in finite-element codes, which are needed to analyse more complex boundary-value problems for soft tissues under supra-physiological loading or tissues weakened by disease.

A Recruitment Model of Tendon Viscoelasticity That Incorporates Fibril Creep and Explains Strain-Dependent Relaxation

Soft tissues exhibit complex viscoelastic behavior, including strain-rate dependence, hysteresis, and strain-dependent relaxation. In this paper, a model for soft tissue viscoelasticity is developed that captures all of these features and is based upon collagen recruitment, whereby fibrils contribute to tissue stiffness only when taut. We build upon existing recruitment models by additionally accounting for fibril creep and by explicitly modeling the contribution of the matrix to the overall tissue viscoelasticity. The fibrils and matrix are modeled as linear viscoelastic and each fibril has an associated critical strain (corresponding to its length) at which it becomes taut. The model is used to fit relaxation tests on three rat tail tendon fascicles and predict their response to cyclic loading. It is shown that all of these mechanical tests can be reproduced accurately with a single set of constitutive parameters, the only difference between each fascicle being the distribution of their fibril crimp lengths. By accounting for fibril creep, we are able to predict how the fibril length distribution of a fascicle changes over time under a given deformation. Furthermore, the phenomenon of strain-dependent relaxation is explained as arising from the competition between the fibril and matrix relaxation functions.

Experimental investigation of temperature-dependent denaturation behavior of type-I Collagen

Precise investigation of the temperature and the duration for collagen denaturation is critical for a number of applications, such as adjustment of temperature and duration during a laser-assisted tissue welding or collagen-based tissue repair products (films, implants, cross-linkers) preparation procedures. The result of such studies can serve as a guideline to mitigate potential side effects while maintaining the functionality of the collagen. Though a variety of collagen denaturation temperatures have been reported, there has not been a systematic study to report temperature-dependent denaturation rates. In this study, we perform a set of experiments on type-I collagen fiber bundles, extracted from the rat-tail tendon, and provide an Arrhenius model based on the acquired data. The tendons are introduced to buffer solutions having different temperatures, while monitoring the contrast in the crimp sights with a wide field microscope, where collagen fibers bend with respect to their original orientation. For all tested temperatures of 50°C–70 °C and tissues that were extracted from 5 rats, increasing the temperature reduced the contrast. On the average, we observed a decay of the contrast to half of its initial value at 37, 157, and 266 seconds when the collagen was introduced to 70 °C, 65 °C, and 60 °C buffer solutions, respectively. For the lower temperatures tested we only observed to be only about 20% and 2 % decay in the crimp contrast after > 2 hours at 55 °C and 50 °C, respectively. The observed denaturation behavior is also in line with Arrhenius Law, as expected. We are looking forward to expand this study to other types of collagen as a future work. Overall, with further development the data and model we present here could potentially serve as a guideline to determine limits for welding and manufacturing process of collagen-based tissue repair agents.

3D Microstructure of Tendon Collagen Fibrils using Serial Block-Face SEM and a Mechanical Model for Load Transfer

Tendon’s hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer, however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy (SBF-SEM) to investigate the threedimensional microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite element modeling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.