Structural and mechanical properties of collagen during embryonic development

Abstract

The mechanical properties of collagen fibrils in tendon tissue have been a focus of scientific interest as their function to transfer loads from muscle to bone are a critical aspect of locomotion. However, efforts to heal and regenerate tendon are still challenged by the limited knowledge on its natural development within the body. Especially the effects of intrafibrillar interactions on the mechanical properties of individual collagen fibrils is not yet fully understood. The functional characterisation of individual collagen fibrils during embryonic development can provide new perspectives on collagen structure, mechanics and maturation. Understanding the assembly and mechanics of individual early collagen fibrils has major implications for tissue morphogenesis and repair. The goal of this thesis was to investigate changes in mechanical properties of individual tendon collagen fibrils during embryonic chick development. In this context enzymatic cross-linking is one point of focus as the chemical modification in similar chicks is known as it has been studied before. Nanomechanical assessment of individual collagen fibrils was conducted in phosphate buffered saline through cantilever-based nanoindentation via atomic force microscopy which proved to be a promising tool for research on tissue biomechanics. With this technique collagen fibrils of less than 30 nm during embryonic development in chicks from stage HH37 to HH41 were mechanically characterised. The recorded data was analysed with the Oliver-Pharr method to calculate the indentation moduli of individual collagen fibrils at the mentioned developmental stages. The experiments showed that the indentation moduli of single collagen fibrils during embryonic chick development increase non-linearly from 1.63 MPa to 2.74 MPa from development stage HH38 to HH41, respectively (p < 0.05). This indicates an accumulation of cross-links during continued embryonic development. Also, small but significant changes in fibril diameter after HH39 could be determined (p < 0.05). Furthermore, the effect of enzymatic cross-linking on the indentation modulus of collagen fibrils harvested from chick embryos that had been treated in ovo with β-aminopropionitrile to inhibit lysyl-oxidase-mediated cross-linking were investigated. The treated collagen fibrils showed a significantly lower indentation modulus than fibrils treated with saline in a comparison group (p < 0.05). This suggests that lysyl-oxidase-mediated cross-linking is partly, if not completely, responsible for the non-linearly increasing indentation modulus of collagen fibrils during embryonic development and therefore contributes to changes in mechanical properties of tendon during development.