STIFFNESS VALUES OF RAT TAIL TENDONS MEASURED WITH SCANNING ACOUSTIC MICROSCOPY AND TENSILE TESTS Varsha Margrette (1, 3), Stéphane Blouin (1), Friedrich Reppe (2), Wolfgang Wagermaier (2), Philipp J. Thurner (3), Markus A. Hartmann (1) 1. Ludwig Boltzmann Institute of Osteology, Austria; 2. Max Planck Institute of Colloids and Interfaces, Germany; 3. TU Wien, Austria Introduction Cross-links in collagen are important factors in determining the mechanical properties of tendons and bones. However, non-enzymatic cross-links formed via glycation deteriorate bone toughness and post-yield strength in diabetes. The mechanical properties of collagen are also related to its hydration. Tensile test is the classical method to determine global mechanical properties by stretching the entire sample, while scanning acoustic microscopy (SAM) can provide a local map of mechanical properties on the sample at micrometer resolution [1]. In this study, we demonstrate the reliability of SAM measurements by correlating stiffness measurements of rat tail tendon (RTT) at the organ level through tensile test and at the micrometer scale with SAM. In this study, RTTs crosslinked with glutaraldehyde GAH [2] and dehydrated with ethanol eth [3] were used to obtain a broad range of stiffness values. Samples Tail tendons of approx. 1 cm length were extracted from 3 male ZDF +/FA (control rats from a strain that is a widely used model for diabetes mellitus type 2) rat tails and divided into 3 groups based on treatment (Table 1). Method Samples were stored in PBS until subsequent treatment with glutaraldehyde (GAH) or ethanol for 24 hours. Each tendon was washed thoroughly with distilled water and placed back in PBS. Until testing samples were kept at 4° C for 2 to 4 days. Tensile testing was conducted until failure with displacement control at a strain rate of 40 μm/sec using a micro-mechanical testing device. Optical images were used to obtain the cross-sectional area. Prior to tensile testing 2 mm long parts were cut from the ends of each tendon and embedded in 4% agarose in PBS. They were cryo-sectioned and measured longitudinally with SAM with 400 MHz lens and 2 μm pixel resolution. We measure the amplitude of ultrasound waves reflected from the sample surface, which is proportional to the stiffness of the sample. Results It was observed that samples that had undergone cross- linking and dehydration were stiffer compared to samples from the control group for both SAM and tensile measurements. The magnitude of the increase was different in the two types of experiments and higher in tensile measurements. Figure 1: (left) Optical and SAM image of cross-section of tendon in agarose gel. (right) Stress-strain curve of tendons obtained from tensile tests. Table 1: Amplitude of reflected acoustic waves and elastic moduli obtained by tensile tests of tendons. Given values are obtained by averaging over n samples. Discussion The observed difference in stiffness shows the influence of hydration and cross-links on the mechanical behavior of collagen. SAM and tensile tests both show similar trends in stiffness changes, indicating that SAM is a powerful tool to evaluate local mechanical properties at tissue level. Albeit the same trend is found for both measurement principles, a discrepancy is observed in the magnitude of the respective stiffness change between different groups. This may be due to the special shape of the tendon stress-strain curve (heel-toe-region) or to the high frequency of testing in the SAM. Further investigation is required to understand this effect. References 1. Blouin S et al. Microsc Microanal. 20(3):924-36, 2014. 2. Hansen et al. Connect Tissue Res. 50(4):211-222, 2009. 3. Gopinath A et al, Eur Biophys J. 43(12):643-652, 2014. Acknowledgements We thank Aleksandra Lebedeva (TU Wien) for her help in cryo-sectioning. Financial support from the Austrian Science Fund (FWF) in the framework of P 34608-N is gratefully acknowledged