Investigation of relaxation behaviour of individual collagen fibrils through AFM nanoindentation

Abstract

Collagen plays an integral part for the biomechanical properties of many tissues, like bones, blood vessels, ligaments and tendons. The mechanical assessment has moved, with help of technical advancements, from tendons to individual collagen fibrils. Their biomechanical properties can nowadays be analyzed under hydration with tools such as the atomic force microscopy (AFM). The effects of sample preparation and hydration itself are not well studied so far. Therefore this thesis was aimed to determine the behavior of collagen fibril (CF) in typical phosphate-buffered saline (PBS) test setups with a series of long and short incubations and intermittent drying. Collagen fibrils came from a thawed mice tail tendon. Height and mechanical properties of 27 CF and 16 reconstituted collagen protein (RCP) fibrils were assessed with AFM nanoindentation. Eight CF were incubated two times for 9 days in 37 PBS with in between testing. The mean indentation modulus (IM) was reduced significantly after both incubations (1D vs 9D p=0.005, 9D vs 18D p=0.002). Swelling was significant (p=0) on every individual fibril after 9D. Further incubation did not increase the observed swelling. A sequence of short incubations of 2 hours on eight CF for determining the onset of height and IM changes indicated the first 2 hours as instance of most changes. However, significantchanges were only on individual fibril level. 6 of 8 CF swell (p0.01) and 4 reducedtheir IM (p0.36) significantly. RCP fibrils behaved very differently from native CF.They were generally smaller and showed neither swelling nor IM changes. Intermittent drying of native CF incubated in PBS increased their IM significantly compared to before drying (p=0.007) and to distilled water incubation (p=0.002). D-Banding spacing showed mostly no changes during incubation and drying while imprinting with force of 500-6000 nN recovered initial surface after hydration. The findings indicate combinations of tropocollagen (TC) and cross-link changes due to incubation. Debonding of cross-links and local melting of TC could explain the swelling and IM reduction due to less structural integrity. Salt concentration elevation and cross-link rigidity increase due to residual irons after washing were assumed as influencing factors for the IM increase after drying. The handling of CF in nanoindentation experiments should incorporate a preselection of similar fibrils for better statistical evaluation, protease inhibitors and avoidance of intermittent drying if testing mechanical properties.