Development of a tensile testing device for individual collagen fibrils and other nanofibers

Abstract

Collagens constitute approximately 30 % of the total protein mass in the human body and serve as the primary structural proteins. They provide tensile strength and toughness to tissues such as tendons, ligaments, vessels, and bones. Collagens are also abundant in the extracellular matrix (ECM) of almost all tissue types, providing sites for cell attachment and playing a crucial role in cellular processes. The unique mechanical properties of these collagen rich tissues are achieved through their complex nano- and microstructure, in which they form collagen fibrils. Collagen fibrils are nanoscale fibers, formed by fibril-forming collagen molecules in a self-assembly process. They are the smallest discernable structural unit of collagenous tissue that can be visualized with high-power microscopes. Collagen fibrils have diameters ranging from tens to hundreds of nanometers and lengths of up to several millimeters. It is debated, whether altered mechanical properties of collagen fibrils contribute to ageing and disease progression (or vice versa). In this context, there is increasing evidence that understanding changes in the viscoelasticity of collagenous tissues is paramount to gaining a better knowledge of disease progression and altered cell behavior. However, current methods for conducting tensile tests on collagen fibrils are very time-consuming and/or lack the capability to precisely determine their viscoelastic properties.The first objective of this thesis was to provide a novel method to overcome current technical limitations and achieve higher sample throughput. This was accomplished by developing the NanoTens, a nano tensile testing instrument that uses a 3D-printed gripper attached to a fiber-optic force probe. Reversible sample attachment to the force probe was achieved through magnetic manipulation. This increases sample throughput at least 25-fold. The NanoTens was utilized to explore the mechanical behavior of collagen fibrils from a mouse model of osteogenesis imperfecta, or brittle bone disease, manifested by a genetic defect that affects the synthesis of collagen molecules. As a result, the synthesized collagen molecules have an impaired structure. The study, surprisingly, revealed that collagen fibrils from the osteogenesis imperfecta mouse model have superior mechanical properties compared to collagen fibrils from wild-type mice, despite the defective collagen molecules. Fibrils from the tail tendons of the osteogenesis mouse model exhibited the characteristics of densely cross-linked collagen fibrils, which hints at elevated cross-linking in the osteogenesis imperfecta mouse model.The second objective was to enhance NanoTens with the capability to perform force-controlled tensile tests. This was done to quantify viscoelastic material properties. Following successful implementation, force-controlled tensile tests were utilized in two studies. The first study compared the viscoelastic properties of collagen fibrils to those of individual electrospun nanofibers. These nanofibers are used in tissue grafts that mimic the mechanical properties of native tendons. It was discovered that collagen fibrils exhibit lower energy dissipation while demonstrating similar elastic properties. In the second study, collagen fibrils were artificially cross-linked using methylglyoxal (MGO), a fast-reacting sugar. Subsequently, creep experiments were performed at relatively low fibril strains (below 10 %) to determine the effect of advanced glycation endproducts (AGEs), i.e. cross-links (and adducts), on viscoelasticity in the physiological strain range. It appears that AGE cross-links reduce molecular sliding even at physiological strains, as determined by reduced creep and residual strain in the MGO cross-linked fibrils. Interestingly, the same fibrils do not display significant differences in transient viscoelasticity.