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1 In the format provided by the authors and unedited. DOI: /NMAT4863 The microstructure and micromechanics of the tendon-bone insertion Supplementary Notes and Figures L. Rossetti a,, L.A. Kuntz a,b,, E. Kunold c, J. Schock d, K.W. Müller e, H. Grabmayr a,f, J. Stolberg-Stolberg b,g, F. Pfeiffer d,h, S.A. Sieber c, R. Burgkart b,*, and A.R. Bausch a,* Authors contributed equally a Lehrstuhl für Zellbiophysik,Technische Universität München,D Garching, Germany b Klinik für Orthopaedie und Sportorthopaedie, Klinikum rechts der Isar, Technische Universität München, D München, Germany c Center for Integrated Protein Science (CIPSM), Department of Chemistry, Technische Universität München, D Garching, Germany d Lehrstuhl für Biomedizinische Physik, Physik-Department & Institut für Medizintechnik, Technische Universität München, D Garching, Germany e Institute for Computational Mechanics, Technische Universität München, D Garching, Germany f Department of Physics and Center for Nanoscience, Ludwig Maximilian University, D Munich, Germany g University Hospital Münster, Department of Trauma-, Hand- and Reconstructive Surgery, Albert-Schweitzer-Campus 1, Building W1, D Münster, Germany h Institut für diagnostische und interventionelle Radiologie, Klinikum rechts der Isar, Technische Universität München, D München, Germany * To whom correspondence should be addressed. rainer.burgkart@tum.de, abausch@mytum.de S.1 Displacement fields and strain distribution in micromechanically tested entheses The PIV algorithm used for obtaining displacement fields was validated on pregenerated data. Specifically, a confocal image coming from one of the micromechanical testing datasets was displaced by 30 px (in x and y) and rotated by 3. The same image was also displaced by 1 px (in x and y) and rotated by 0.1. The PIV algorithm was run on both these artificially transformed images (comparing them to the untransformed image) and the results were tested for accuracy, yielding the same average error of ±0.16 µm on the magnitude of the displacement vectors in both cases. As a further validation, a pair of consecutive images from one of the micromechanical tests was analyzed by manually tracking the displacements of 30 points belonging to a region of high strain heterogeneity. The manually tracked data was compared to the displacement vectors obtained for the same points via the PIV algorithm, and the similarity between the two data sets was quantified. 1 NATURE MATERIALS 1

2 This was done for different sizes of the PIV search window, showing that the larger the search window, the worse the agreement between the two datasets and that a search window that is below 30 px also yields imprecise PIV vectors. The optimal search window size reported in the Materials and Methods section was identified in this way. To avoid mistracking artifacts several control steps were applied, as reported in the Material and Methods section. The local longitudinal Lagrangian strain ε was calculated as stated in the main text and the ratio between ε and the macroscopic strain ε 0 was calculated and mapped for the n =5mechanically tested entheses. All strain steps belonging to the linear response region of the force-strain curve were analyzed. Heterogeneous distributions of ε/ε 0 were consistently observed across all samples and for all analyzed strain steps (supplementary figure S2). Adjacent regions of positive strain and negative strain were observed in all samples. At a first glance these might be misinterpreted as stemming from spurious peaks in the displacement fields caused by mistracked vectors. In fact, they are originating from well defined peaks composed of several tens of tracked points (supplementary figure S1). Furthermore, we verified that the regions of negative strain were not originating from the tracking of counteracting displacements of interface fibers on different planes. This was ruled out via PIV analysis of the bottom 10 µm of the compression areas. This corresponds to the thickness of a single interface fiber. The analysis of these thin z-stacks consistently yielded compression areas equivalent to those observed with the full z-stack projections(supplementary figure S3). Crimping occurs within the tendon fibers only far away from the attachment site and not within the interface fibers that we analyze, therefore we do not expect out-of-plane movements due to it. Within the interface region, possible out of plane movements can result in only a minor underestimation of the true displacement ( 4%), as estimated from the observed maximal splaying angle of 15. To assess the effect of incubation in PBS on the mechanical response of the samples, one enthesis was not stained and micromechanically tested while covered in tissues soaked in DMEM supplemented with 10% FBS to prevent drying. These data show the same behavior observed for the other samples (supplementary figure S4). To test the mechanical response of enthesis on the force application from two different angles was unclamped at both ends and rotated by 45 around an axis perpendicular to the plane of the original loading. Comparison of the ε/ε 0 distributions shows that the strain response of the same 2 NATURE MATERIALS 2

3 sample changes depending on the angle at which the strain is applied (supplementary figure S5). Notice that during the the 45 pull some parts of the tendon experience compression against the bone. This can be determined straight away from the plots of u y, where a positive value denotes movement in the opposite direction to that of strain application, which results in compression against the bone. The representative plots of u y shown in fig. 2-f in the main text are taken from a position in the sample where extension was experienced for both angles of strain application. S.2 Micromechanical testing device A loading chamber was designed to fit on a Leica TCS SP5 inverted microscope. A micrometre precision motor (PI M-404.1PD, Physik Instrumente GmbH & Co., Germany) was used to apply known deformations and a sensor (mounted on a mobile platform moved by the motor) simultaneously measured the force (LCF300 50lb, FUTEK Advanced Sensor Technology Inc.,USA). Both were controlled and monitored by a custom-written software (LabView 2012, National Instruments, USA). Samples in the chamber were blocked with a screw-on clamp at the bone side. The tendon extremity was locked between two metal plates, fixed together by screws. One of these plates was connected to a steel wire whose other extremity was clamped to the force sensor, after running over a lubricated ball-bearing pulley wheel(supplementary figure S6). Samples were loaded in the chamber and some slack was left in steel wire, to ensure not to create any strain before the imaging began. Calibration measurements with an immobilized clamp were performed to calculate and remove the effect of the slack from the measurement of the total macroscopic strain. This introduces a systematic error of ±1%on the macroscopic strain measurements. S.3 Image analysis The confocal reflectance z-stacks were passed through a background removal procedure based on the ImageJ Subtract Background tool. To remove the effect of coverslip reflections, slices that contained more than 50 % saturated pixels were discarded. The interface between bone and tendon was easily traceable by hand using an overlaid image of the reflectance channel and the NHS-Aberrior 635 channel (supplementary figure S7). 3 NATURE MATERIALS 3

4 Figure S1: Validation of displacement fields obtained by PIV a Displacement field (u y (x, y) of an enthesis under longitudinal strain. b Local strain fields at the interface, normalized by the applied macroscopic strain of (1.5 ± 0.1) %. The numbered gray lines show the location of longitudinal profiles cutting through areas where high positive strains are found adjacent to high negative strains. c Plots of longitudinal displacement as a function of distance along the gray lines in a and b. The profiles are composed of a dense array of points (spacing is 15 µm), confirming that the aforementioned features of the strain fields arise from well substantiated data. 4 NATURE MATERIALS 4

5 Figure S2: Plots of ε/ε 0 for the n =5micromechanically tested samples. The contourplots refer to the strain step marked in red on the adjacent force-strain plots. All samples show a heterogeneous distribution of ε/ε 0. Localized strain amplifications correspond to areas of the interface region behaving as if more compliant than bone and the remaining tendon. All scale bars are 2000 µm. The adjacent plots show force-strain curves for the tested samples, highlighting in red the step shown in the heat maps. The x axis values represent the applied strain, originating from steps of mm. 5 NATURE MATERIALS 5

6 Figure S3: Compressive strain does not depend on the depth of the z-stack used for strain calculation. A region of the sample from fig. 2b is selected (green box). Specifically, a region where compressive strain occurs is chosen (as seen in fig. 2c, which for ease of comparison is repeated at the bottom of this figure.) A z-projection of the complete data (left) and a z-projection of the lower three slices (corresponding to 12 µm, the thickness of a single interface fiber) are obtained. PIV analysis and subsequent strain calculations are performed on this subregion, in the manner detailed in the Materials and Methods section. The distributions of ε/ε 0 obtained are equivalent. The colourmap at the bottom applies to all three contourplots.white arrows indicate the region of compressive strain. 6 NATURE MATERIALS 6

7 Figure S4: Plots of ε/ε 0 for the a micromechanically tested samples without PBS incubation. The contourplot refers to the strain step marked in red on the inset force-strain plot. The sample was not incubated in PBS. It was prevented from drying by being covered in tissues soaked in DMEM supplemented with 10% FBS. The sample shows a heterogeneous distribution of ε/ε 0 analogous to that seen in the samples incubated in PBS (supplementary figure S2). 7 NATURE MATERIALS 7

8 Figure S5: Enthesis under strain at two different angles. a,b, Confocal reflectance images of the sample in the two orientations. Inset: force-strain plot for the the sample strained at 45. The equivalent plot for the 0 strain is reported in supplementary figure S2d. c, comparison between ε/ε 0 distributions for the two samples. For better clarity the 45 data has been rotated and overlayed with a vertical offset of 800 µm. The blue line marks the part of the sample where compression against the bone was occurring during 45 straining. 8 NATURE MATERIALS 8

9 Figure S6: Custom built loading chamber. a, motor. b, force sensor. c, bone clamp. d tendon clamp. e steel wire. Inset: a sample clamped in the loading chamber. 9 NATURE MATERIALS 9

10 Figure S7: Confocal image of one of a micromechanically tested samples. Cyan - protein content labelled via an NHS-coupled fluorophore. Magenta - confocal reflectance. The border between bone (top hlf of the image) and the interface region of the tendon (bottom half of the image) is clearly visible. The image was obtained by stitching together a grid of 13 5 z projections of confocal stacks. The stacks were 512 px 512 px and each covered areas of 1550 µm 1550 µm. Scale bar 2000 µm. 10 NATURE MATERIALS 10