Unraveling the role of Calcium ions in the mechanical properties of individual collagen fibrils

By. CBHI Research Team

Unraveling-the-role-of-Calcium-ions-in-the-mechanical-properties-of-individual-collagen-fibrils

Abstract

Collagen, the dominating material in the extracellular matrix, provides the strength, elasticity and mechanical stability to the organisms. The mechanical property of collagen is mainly dominated by its surrounding environments. However, the variation and origin of the mechanics of collagen fibril under different concentrations of calcium ions (χCa) remains unknown. By using the atomic force microscopy based nanoindentation, the mechanics and structure of individual type II collagen fibril were first investigated under different χCa in this study. The results demonstrate that both of the mechanical and structural properties of the collagen fibril show a prominent dependence on χCa. The mechanism of χCa-dependence of the collagen fibril was attributed to the chelation between collagen molecules and the calcium ions. Given the role of calcium in the pathology of osteoarthritis, the current study may cast new light on the understanding of osteoarthritis and other soft tissue hardening related diseases in the future.

 

Introduction

Calcium is one of the most important ions in human body and plays a critical role in certain physiological functions. It has been proven that several human diseases are related with the disturbance of calcium, e.g., osteoarthritis, cardiac arrest, respiratory arrest, cataract and asteroid hyalosis1. According to the compositional analysis, Jubeck et al. found that the abnormal calcium enrichment was in up to 60% osteoarthritic articular cartilage (AC)2. In order to reveal the relationship between the calcium and osteoarthritic AC, researchers have investigated the effect of calcium on the proteoglycans, collagen and chondrocytes. These studies indicated that calcium plays a pivotal role in the death of chondrocytes, proteoglycans can be treated as a calcium-concentrating agent, and collagen also can bind calcium ions3,4. Although the role of calcium in the AC is extensively studied, there is a scarcity of studies about the influence of calcium on the mechanics of individual collagen fibril, which is the main composition of AC.

Collagen is one of the most abundant proteins in human body, which holds the whole body together5,6. It extensively exists in the bones, muscles, skin, blood vessels and connective tissues, and provides the strength, elasticity and mechanical stability to the body. For its key role in the body, collagen has attracted huge interests from both theoretical and practical points of view. Collagen fibril, the main form of collagen, consists of collagen molecules. Collagen molecules has a molecular structure that is composed of triple helix of left-handed polypeptide chain known as tropocollagen. In other words, collagen fibril can be treated as the linear aggregates of tropocollagen. The typical diameter and length of collagen molecule are ~1.5 nm and ~300 nm, respectively7. They generally self-assemble into a cross-striated collagen fibril with a D-band periodicity of about 67 nm. Such a so-called “quarter-staggered” structure creates the periodic gap (about 0.54 D) and overlap (about 0.46 D) regions where the gap regions have 20% less packing density than that of the overlap regions8,9. More than 20 genetically distinct collagens are found in animal tissue. Type I, II, III, V and XI collagens can form fibrils, and have the D-band periodicity10. Type I collagen, consisted of two α1(I) chains and one α2(I) chain, is found throughout the body except in cartilaginous tissues. Type II collagen, consisted of three identical α1(II) chains, is found in the cartilage, developing cornea and vitreous humour. There are some differences in the amino acid composition of α1 and α2, but they are minor11. The mechanics of collagen fibril as well as collagen molecules have been also extensively investigated7,12,13,14,15. However, how external ions (i.e., calcium ions) affect both structural and mechanical properties of individual collagen fibril is still ambiguous, especially under the nanoscale level.

Atomic force microscopy (AFM) based nanoindentation has been well established as a powerful technique for the investigation of the mechanics at the nanoscale level9,16,17,18,19. To have a better understanding of the effect of abnormal calcium homeostasis on soft tissues’ mechanics, we attempted to employ AFM-based nanoindentation to study the mechanical behavior of type II collagen fibril extracted from AC. The structural properties (D-band periodicity and the height difference between the gap and overlap regions) of type II collagen fibril were also investigated. It was found that the structural and mechanical behaviors of individual type II collagen fibril are significantly depending on the concentration of calcium ions (χCa). When 2 mM < χCa <= 5 mM, the elastic modulus of individual collagen fibril increases with the increasing χCa; the structural characteristic of the collagen fibril, D-band periodicity, decreases with the increasing χCa; and another structural characteristic, the height difference between overlap and gap regions, increases with the increasing χCa. With further increase of χCa (>5 mM), the elastic modulus and D-band periodicity of the collagen fibril level off; while the height difference between overlap and gap regions remains increasing. The mechanism of χCa-dependence of the collagen fibril was attributed to the chelation between collagen molecules and the calcium ions. Given the relationship between the disturbance of calcium and the tissue stiffening, the presented study strongly suggests that the tissue stiffening found in various diseases should be due to the interaction between calcium ions and collagen fibril. To the best of our knowledge, the presented work should be the first systematic study on the role of calcium ions on mechanical and structural properties of individual type II collagen fibril. This study will provide fundamental understanding of the influence of calcium ions on the collagen fibril as well as the pathology of related diseases, and so possibly lead to new diagnosis techniques and clinical treatments.

Results

Mechanics of individual collagen fibril under different χCa

It is reported that five kinds of collagen (II, VI, IX, X and XI) have been identified in AC20. Type II collagen, a homotrimer composed of α1(II) chains, is the most abundant, which makes up about 90% of the total collagen in AC21. Therefore, collagen mainly investigated in this study is the type II collagen. In order to locate the collagen fibril, it is necessary to image the sample in advance of the nanoindentation measurement. Moreover, AFM images can provide a cross-check of the collagen fibril investigated in this study. Figure 1 shows a typical image of the collagen fibril obtained under the tapping model at room temperature (RT, 25 °C). The morphology of the type II collagen fibril shown in Fig. 1 is consistent with previous studies, especially the regular D-band periodicity, which is the typical characteristics of collagen fibril. The length of the D-band periodicity is also in agreement with previous studies on the type I collagen fibril17,20.

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