NMR IN BIOMEDICINE NMR Biomed. 2005;18:577–586 Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/nbm.995 Self-diffusion anisotropy of water in sheep Achilles tendon R. Fechete, 1,2 D. E. Demco, 1 U. Eliav, 3 B. Blu ¨ mich 1 * and G. Navon 3 1 Institut fu ¨ r Technische Chemie und Makromolekulare Chemie, Rheinisch-Westfa ¨ lische Technische Hochschule, Worringerweg 1, D-52056 Aachen, Germany 2 Technical University Cluj-Napoca, Daicoviciu 1, R-400020 Cluj-Napoca, Romania 3 School of Chemistry, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel Received 17 March 2005; Revised 16 August 2005; Accepted 6 September 2005 ABSTRACT: The principal values of the diffusion tensor of free water in the pores of sheep Achilles tendon were determined. For this purpose, the azimuthally angular dependence of the self-diffusion coefficient was measured using a radiofrequency tilt coil and pulsed-field-gradient stimulated-echo (PFGSE) NMR. Combining the PFGSE with multiple acquisitions of Hahn echoes using the Carr–Purcell–Meiboom–Gill pulse sequence reduced the measuring time. The diffusion measurements revealed two diffusion process characterized by a fast and a slow effective diffusion coefficient. A model which describes the stimulated-echo amplitude, encoded by the water diffusion and magnetization transfer, was used for evaluation of the fast diffusion coefficients. The fast diffusion process characterizes the water molecules in pores surrounding the collagen fibrils. The diffusion coefficients characterizing the fast process show a well-defined anisotropy. The principal values of the diffusion tensors were determined assuming the elongated pores to be oriented parallel to the tendon fibrils and thus the orientation distribution function of the pores followed that of the collagen fibrils. The average aspect ratio of pores was estimated from the principal values of the water diffusion tensor and is suitable to characterize quantitatively the changes in tendon morphology due to healing or aging. The methods in this investigation can also be applied to measurements of the diffusion anisotropy using ex situ NMR sensors. Copyright # 2005 John Wiley & Sons, Ltd. KEYWORDS: water self-diffusion anisotropy; diffusion tensor; propagators; sheep Achilles tendon; collagen orientation distribution function; collagen–water proton exchange INTRODUCTION Self-diffusion of water in ordered biological tissues is a topic studied most comprehensively by NMR measure- ments that are sensitive to macroscopic anisotropy. Ani- sotropic water diffusion was first reported by Cleveland et al. in skeletal muscle (1). More recently, it was described in the highly anisotropic white matter and also in the more isotropic gray matter (2–5). In many tissues, anisotropic diffusion is caused by the restriction of the translational water motion in certain directions by the biological membranes. It is, therefore, a macroscopic measure of neural fiber tract orientation (6,7). Diffusion- weighted magnetic resonance imaging of the nervous tissue underwent rapid development during last decade, owing to the possibility of visualizing pathological changes of the brain tissue such as demyelination, ischemia and stroke (4). Considerable effort has been made in recent years to characterize non-exponential diffusion in the brain and the spinal cord tissues and to correlate its components with pathological water pools. The self-diffusion of free water in bovine Achilles tendon has been measured by pulsed gradient spin-echo NMR, but no anisotropy was detected (6). Nevertheless, the presence of self-diffusion anisotropy is plausible owing to the existence of free water confined in the mesoscopic regions between the collagen fibrils that are well oriented along the macroscopic axis of the tendon. Recently, the constant relaxation method and a unilateral NMR sensor were employed for measuring DðÂÞ coeffi- cients of free water in bovine Achilles tendon for three orientation angles,  ¼ 0  , 35  and 90  of the axis of a tendon plug relative to the symmetry axis of the sensor (8). The measured anisotropy ratio Dð ¼ 0  Þ= Dð ¼ 90  Þ in the tendon seems to be larger by a factor of 1.5 than the values measured for both white matter and the optic nerve (6). The water apparent diffusion coefficient in rabbit Achilles tendon was shown to be anisotropic, diffusion- time dependent and to change as a function of tensile load (9,10). The short diffusion-time behavior of the resulting time-dependent diffusion curves was used to infer indir- ectly information regarding the average surface area-to- volume ratio of the space available for water diffusion. Copyright # 2005 John Wiley & Sons, Ltd. NMR Biomed. 2005;18:577–586 *Correspondence to: B. Blu ¨mich, Institut fu ¨r Technische Chemie und Makromolekulare Chemie, Rheinisch-Westfa ¨lische Technische Hochschule, Worringerweg 1, D-52056 Aachen, Germany. E-mail: bluemich@mc.rwth-aachen.de Contract/grant sponsor: German Federal Ministry of Education and Research (BMBF). Contract/grant sponsor: Deutsche Forschungsgemeinschaft; contract/ grant number: DE 780/1-2. Abbreviations used: DQ, double quantum; LF, laboratory reference frame; NOE, nuclear Overhauser effect; PAS, principal axis frame; PFGSE, pulsed-field-gradient stimulated-echo; TF , tendon frame.