Local Strain Measurement Reveals a Varied Regional Dependence of Tensile Tendon Properties on Glycosaminoglycan Content

INTRODUCTION: Tendon is a connective tissue composed mainly of Type I collagen, embedded in a proteoglycan (PG) rich extracellular matrix (ECM) [1,2]. From a mechanical point of view, tendon can be considered as a composite material in which every component has a specific mechanical role in bearing tensile load. Proteoglycans with their associated glycosaminoglycan (GAG) chains may have an important function. Other studies have shown that there is a difference in GAG concentration between the tensile and the insertion regions of tendon. Specifically, the concentration of GAGs in the insertion region can be up to 25 times higher than in the midsubstance [3]. This implies that the mechanical properties along the tendon might be mediated by GAG content. The aim of this study was therefore to develop and validate a method to locally measure the strain of the tendon under load and quantify the contribution of proteoglycans to the tensile mechanical properties. GAG depleted tendon were analyzed under load and compared to natural tendon. METHODS: Two groups of ten Achilles tendons from 16-week-old C57/BL6 wild type mice were dissected. The control group of tendons was incubated for 24 hours in phosphate buffered saline solution (PBS), while the treated group of tendons was incubated for 24 hours in PBS + 1U/ml chondroitinase ABC in order to partially digest the GAGs. Tendons were tensile tested to failure in physiological saline at 37.5°C after 10cycles of preconditioning to 10% nominal strain. Local strain in the tendon midsubstance was optically measured using high-speed video. In order to improve image contrast, the tendon was partially stained with indigo ink. By measuring midsubstance strain, the midsubstance stiffness in the tendon was calculated and compared with the structural stiffness of the bone-tendon-muscle unit over identical force-displacement intervals corresponding to the linear region of the stress-strain curve. Mechanical tests were performed on both groups, and experimental results were analyzed for strain at failure (the measured strain when the tendon reaches its maximal load), structural stiffness (the change in force per change in normalized length of the tendon complex in the linear region of the force displacement curve), midsubstance stiffness (the change in force per change in midsubstance strain as measured from the video) and maximal force of the bone-tendon-muscle unit. RESULTS SECTION: Results after 30% of GAG digestion on the entire tendon indicated that the complex exhibited approximately 28% reduced maximal load and 42% reduced structural stiffness (p<0.05), and a reduction in the failure strain that was not significant. Localized optical strain measurement of the tendon midsubstance indicated a 20% reduction in local stiffness after digestion, which was significant at p=0.06 only. The correlation between structural tendon stiffness and localized midsubstance stiffness is summarized in Figure 1. Here it can be seen that there is a poor correlation between the whole tendon complex and the midsubstance stiffness in the control group, and no apparent correlation in the digested group. Also evident in this figure is that GAG digestion results in a large drop in structural tendon stiffness with apparently less effect in the midsubstance region. Additional localized measurements were performed on the proximal, middle, and distal tendon midsubstance subregions (see Figure 2). Twenty strains were measured for each region on randomly chosen tendons from the digested group. Local strains were calculated using two points forming a line segment that did not deviate more than 45° from vertical. Since the lengths of the three regions were the same, the average of the three calculated strains should correspond to the previously calculated midsubstance strain. In fact, the mean value for the midsubstance strain was 16.8 % while the average of the three subregions was 17.6 % (p=0.81).