Implications of AGE cross-linking on the viscoelastic behavior in individual collagen Fibrils

Abstract

Collagenous tissues are essential and have great importance to the biomechanical integrity of the body. Collagen type I, among other collagen types of the collagen super family, is the major protein found in vertebrates and in higher invertebrates. Most of collagen-rich tissues exhibit a hierarchical structure with different mechanical behavior at each individual level. At the nano level, collagen fibrils are the functional and basic building blocks in these tissues. Collagen cross-linking affects the mechanical properties of the fibrils. Advanced Glycation End products (AGEs) are products resulting from non-enzymatic cross-linking, a process known as glycation and leads to stiffening of the fibrils, which may cause whole tissue deterioration. In this thesis a study is performed on collagen fibrils extracted from a wild type (WT) mouse tail tendon to examine the effect of glycation on their viscoelastic behavior. Two types of mechanical measurements were accomplished: Atomic Force Microscopy (AFM) dynamic nanoindentation and dynamic tensile tests using a novel instrument with quick sample loading and unloading. Both measurements were executed on the same fibrils from the same tendon fascicle, in phosphate buffered saline (PBS) to simulate the physiological environment. A test group was prepared by glycating collagen fibrils with Methylglyoxal (MGO) to mimic the physiological effects of the AGEs in aging and pathology. Another group was prepared from the same tendon fascicle as a control group to reduce any systematic errors or any differences that are not related to glycation. AFM dynamic nanoindentation measurements in PBS were performed on both groups (N= 10 in each group) before and after incubation. Afterwards dynamic tensile tests were conducted on both groups also in PBS to investigate the viscoelastic behavior of both groups in their normal loading direction (the long axis).Through the combination of the two methods, different parameters such as loss and storage modulus, loss tangent, dynamic modulus and ultimate strength could be measured and investigated to assess the effect of AGEs on nanomechanics at the collagen fibril-level. Comparing both methods, the AFM dynamic nanoindentation is a powerful method, in which different experiments can be conducted using different protocols (e.g., stress relaxation experiments, creep experiments, etc.) with a large range of forces and frequencies. The measurements on the fibrils are reproducible and can be conducted several times with different configurations on the very same spot on the fibril. Despite these benefits one of the shortcomings of this method is the low acquisition speed resulting in long measurement times that can last for hours. Also, an important range of frequencies less than 0.5 Hz, which simulates quasi-static tests and reveals more the viscous behavior of the sample is hard to be investigated using the AFM nanoindentation as the measurements at this range are very noisy.On the other hand, the dynamic tensile testing measurement using NanoTens device is easy to conduct with fast sample loading and unloading. It can be performed in a very short time (5 minutes per fibril) compared to AFM. The measurements are also reproducible unless the fibril ruptures, which needs some expertise to be avoided. The shortcoming of this method is the extra sample preparation, which must be conducted with certain conditions to make the loading of the fibril on the microgripper easy and possible. While the lower frequencies (< 0.5 Hz) are more feasible than with AFM, higher frequencies (> 2 Hz) cannot be examined using this method.The results of the analyzed data of both experiments correlated but could not give clear evidence on the influences of glycation on the viscoelastic behavior at the nanoscale of collagen fibrils. Although the glycated fibrils seemed to be less viscous than the control group and the ultimate strength of the glycated fibrils showed higher values at lower strain compared to the untreated ones, this may not be directly related to glycation.