Collagen structures from cell culture to intact tendon

Abstract

This study grasps the complexity of connective tissue and its major component type I collagen. The extended introduction offers the historical overview on collagen investigation. It summarizes the experimental insights into the triple helix organisation thanks to the invention of X-ray crystallography. The following sections target the collagen synthesis and formation of collagen in the extracellular matrix. The different types of collagen are introduced in respect of function and location. A short section is also devoted to collagen tissue aging due to cross-linking. The introduction is concluded with the sections on mechanical properties of tendon and common methods used for visualisation of connective tissue, especially the non-linear methods such as second harmonic generation (SHG). At the beginning, the in vitro model was constructed to measure the production of type I collagen and visualise collagen fibres. The aim was to label only the selected extracellular proteins and to preserve morphology of the cell culture. As immunostaining (IS) offers a wide pallet of labelling protocols150,211, several modifications were implemented to meet this target. The modifications included placement of the cell culture on ice while applying the primary antibody and fixation of the cell culture after applying the primary antibody and washing. When comparing the cell culture with and without ascorbic acid (AA) treatment, the results showed higher cell growth and collagen deposition in culture with AA treatment, but no significant difference was found between expression levels of type I collagen gene. Besides, the cell culture was studied by label-free SHG microscopy. This measurement experimentally proved that the absence of AA in the cell culture, which directly affects the hydroxylation of proline, results in the loss of SHG signal. This validates the second-order susceptibility tensor model107 , in which hydroxyproline plays a crucial role while generating the second harmonic signal. As the presence of the SHG signal depends on the inner structure, it offers not only a quantitative but also qualitative tool for type I collagen investigation. This is the main advantage of non-linear SHG microscopy over the classic IS methods when revealing pathological conditions146 because IS scoring is often based on subjective expertise and returns results with high variability212. The advantages of SHG microscopy were even more pronounced when examining microtome section of tendon. The samples did not have to undergo multiple preparation steps160,213 that affect morphology135 and could be optically sectioned into 3-D label-free reconstruction. Thanks to the near infrared laser source, SHG microscopy also reduces scattering, photodamage and increases penetration depth of observation. The observation can be also directly enriched for the information about the orientation of collagen fibres. This approach relies on the fact that the SHG signal depends on the molecular orientation and incident beam polarization, respectively123. But sometimes, the sample cannot be scanned under different polarization angles, and therefore the fibre orientation must be retrieve from a single image. For that reason, the algorithm was developed to quantitatively determine the orientation regularity of collagen fibres. The results showed that there is a significant difference in the orientation regularity of fibres between the microtome sections of the tendons from the young and old animals. The microtome sections from the young animals possessed high isotropy of fibres. On the other hand, the microtome sections from the old animals contained anisotropic fibres. The experiment on the intact tendons followed to investigate whether the differences of the orientation regularity had been caused by sectioning or by senescence. In this case, the load-free intact tendons had strong orientation regularity of the fibers within the main load-bearing axis. This fibrillar regularity was represented by periodically undulating pattern – the crimp pattern. This pattern developed with age until it almost disappeared in the tendons from the old animals. The comparison of the results proved that the microtome sectioning modified the morphology of tendons, especially in the tendons from young animals. Although there is not a generally accepted explanation for the cause and purpose of the crimp pattern, it is supposed that it reflects both the inner physiological and morphological status and provides an optimum mechanical response. The intact tendons were mechanically tested by the uniaxial tensile test but the data had high variability, and therefore, it was impossible to decide on the significance of the crimp pattern from the mechanical point of view. This problem was mainly caused by the tendon-grip interface, extensometry, and identification of the tendon geometry. For this reason, the tendon was stretched and simultaneously imaged by SHG microscopy. It was discovered that the crimp pattern had disappeared before reaching minimum resolution of the force sensors and reappeared immediately after returning to the initial elongation. This behaviour indicated a preload function of the crimp pattern which would lead to a lower increment of stress when activating muscles. After conducting the mechanical test with SHG microscopy, the visualization method was upgraded to polarized SHG (PSHG) microscopy. As the result, the helical pitch angle (HPA) of collagen molecule was detected for the intact tendons from animals of different age at the load-free state. This pixel resolution approach showed no change in the HPA in respect to age or mechanical loading. This finding supports the theoretical framework about the mechanical properties and stability of collagen molecule. On the other hand, fluorescence lifetime microscopy revealed significant changes in the micro-environment of tendon. These changes in fluorescence lifetime were probably caused by collagen cross-linkers which act as a reinforcement component that directly affect the mechanical properties of tendon.