Tendon collagen fibril morphology and nano-mechanics via atomic force microscopy

Abstract

Due to the hierarchical structure of tendon tissue (fascicles→fibers→fibrils), it is essential to investigate morphology and mechanics at the nano-scopic level to better understand whole body function. Previous studies investigating changes in tendon mechanics after exercise/disease/unloading have focused on the whole tendon and fascicle levels, but limited data exists on adaptations at the collagen fibril level. Objective: Provide a new method to study previously frozen tendon tissue by investigating collagen fibril 1) morphology, 2) nano-mechanical properties, and 3) elasticity maps using atomic force microscopy (AFM). Hypothesis: This method will provide a reliable way to assess tendon structure and function at the nano-scale. Methods: C57BL/6J mouse tendons from NASA Rodent Research-1 (-80°C) were chemically separated and mounted onto glass slides then mechanically separated into tendon subunit sheets and left to air-dry. Collagen fibril morphology was tested with a JPK Nanowizard 4a in tapping mode with a Tap300Al-G cantilever (tip radius=10nm, k=40N/m). Samples were then rehydrated for mechanical testing in Quantitative Imaging (QI) mode with an SNL-10D cantilever (tip radius=2nm, k=0.06N/m). Force data acquisition took place in liquid with a set point of 2nN and Z-speed of 40μm/s. Force-microscopy (FM) and structural data were processed using JPK Data Processing Software and Gwyddion, respectively. Data: Tendon collagen fibrils exhibited uniform D-periods across 38 fibril sections (D-period: 66nm±2.1, diameter: 63nm±23.5). Quantitative analysis of the force curves (n=30) along the highest point of the fibril revealed an average Young’s Modulus (YM) of 2.53MPa±0.31. Elasticity maps (n=3) highlighting the distribution of mechanical properties were generated using FM data. Results Summary: Our tissue preparation protocol combined with AFM reliably determines collagen fibril morphology and nano-mechanical properties of mouse tendons that were previously frozen. As evidence, we present systematic YM within and across different data sets. In addition, the YM values were found to be consistent with previous measurements in tendon tissue under similar conditions. Elasticity maps produced by analysis of FM data indicate the similarity of YM values across the tendon surface. Tendon fibrils were found to have similar D-periodicity despite the variation in fibril size, which is consistent with previous literature. Conclusions: This research provides a new method to test tendon collagen fibril structure and mechanical properties at the nano-scale, presenting a novel analysis (elasticity maps) showing contrast in YM across the fibril surface. Funding: CSUPERB New Investigator Grant to JRB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.