Anisotropic Tissue Research Articles

Mechanical Characterization of Human Fascia Lata: Uniaxial Tensile Tests from Fresh-Frozen Cadaver Samples and Constitutive Modelling

Human Fascia Lata (FL) is a connective tissue with a multilayered organization also known as aponeurotic fascia. FL biomechanics is influenced by its composite structure formed by fibrous layers (usually two) separated by loose connective tissue. In each layer, most of the collagen fibers run parallel in a distinct direction (with an interlayer angle that usually ranges from 75-80°), mirroring the fascia's ability to adapt and withstand specific tensile loads. Although FL is a key structure in several musculoskeletal dysfunctions and in tissue engineering, literature still lacks the evidence that proves tissue anisotropy according to predominant collagen fiber directions. For this purpose, this work aims to analyze the biomechanical properties of ex-vivo FL (collected from fresh-frozen human donors) by performing uniaxial tensile tests in order to highlight any differences with respect to loading directions. The experimental outcomes showed a strong anisotropic behavior in accordance with principal collagen fibers directions, which characterize the composite structure. These findings have been implemented to propose a first constitutive model able to mimic the intra- and interlayer interactions. Both approaches could potentially support surgeons in daily practices (such as graft preparation and placement), engineers during in silico simulation, and physiotherapists during musculoskeletal rehabilitation, to customize a medical intervention based on each specific patient and clinical condition.

Magnetic Resonance Acoustic Radiation Force Imaging (MR-ARFI) for the monitoring of High Intensity Focused Ultrasound (HIFU) ablation in anisotropic tissue.

We introduce a non-invasive MR-Acoustic Radiation Force Imaging (ARFI)-based elastography method that provides both the local shear modulus and temperature maps for the monitoring of High Intensity Focused Ultrasound (HIFU) therapy. To take tissue anisotropy into account, the local shear modulus μ is determined in selected radial directions around the focal spot by fitting the phase profiles to a linear viscoelastic model, including tissue-specific mechanical relaxation time τ. MR-ARFI was evaluated on a calibrated phantom, then applied to the monitoring of HIFU in a gel phantom, ex vivo and in vivo porcine muscle tissue, in parallel with MR-thermometry. As expected, the shear modulus polar maps reflected the isotropy of phantoms and the anisotropy of muscle. In the HIFU monitoring experiments, both the shear modulus polar map and the thermometry map were updated with every pair of MR-ARFI phase images acquired with opposite MR-ARFI-encoding. The shear modulus was found to decrease (phantom and ex vivo) or increase (in vivo) during heating, before remaining steady during the cooling phase. The mechanical relaxation time, estimated pre- and post-HIFU, was found to vary in muscle tissue. MR-ARFI allowed for monitoring of viscoelasticity changes around the HIFU focal spot even in anisotropic muscle tissue.

Imparting Multi‐Scalar Architectural Control into Silk Materials Using a Simple Multi‐Functional Ice‐Templating Fabrication Platform

AbstractHuman tissues and organs exhibit complex hierarchical and gradient structures that are essential to their function and should be recapitulated within biomaterial scaffolds targeting their regeneration. Unidirectional freezing, an ice templating technique where ice acts as a porogen, is uniquely suited to recapitulating the architectural anisotropy, gradients, and hierarchical transitions of human tissues, but ice templating of polymeric systems, including silk fibroin, remains less well understood than their colloidal counterparts. To address this, a versatile and accessible freezing setup for silk that allows tuning of freezing parameters including the polymer cooling rate (30 °C min−1 to 2 °C min−1) and ice solidification velocity (2.5 to 0.6 mm min−1) using liquid nitrogen, is developed. Real time visual and thermal monitoring of the freezing process for multiple silk concentrations (2–10% wt/v) and material states (liquid, hydrogels) is performed and the conditions are correlated with pore morphology. Unprecedented control over pore size (100–90 000 µm2) and pore morphology (cellular–lamellar), consistent pore alignment, and generation of gradient porosity in silk scaffolds are demonstrated. For the first time, impact of shear thinning behavior of silk in ice crystal formation is demonstrated, showing non‐linear and complex freezing phenomena in silk.

Mathematical Optimisation of Magnetic Nanoparticle Diffusion in the Brain White Matter.

Magnetic nanoparticles (MNPs) are a promising drug delivery system to treat brain diseases, as the particle transport trajectory can be manipulated by an external magnetic field. However, due to the complex microstructure of brain tissues, particularly the arrangement of nerve fibres in the white matter (WM), how to achieve desired drug distribution patterns, e.g., uniform distribution, is largely unknown. In this study, by adopting a mathematical model capable of capturing the diffusion trajectories of MNPs, we conducted a pilot study to investigate the effects of key parameters in the MNP delivery on the particle diffusion behaviours in the brain WM microstructures. The results show that (i) a uniform distribution of MNPs can be achieved in anisotropic tissues by adjusting the particle size and magnetic field; (ii) particle size plays a key role in determining MNPs' diffusion behaviours. The magnitude of MNP equivalent diffusivity is reversely correlated to the particle size. The MNPs with a dimension greater than 90 nm cannot reach a uniform distribution in the brain WM even in an external magnitude field; (iii) axon tortuosity may lead to transversely anisotropic MNP transport in the brain WM; however, this effect can be mitigated by applying an external magnetic field perpendicular to the local axon track. This study not only advances understanding to answer the question of how to optimise MNP delivery, but also demonstrates the potential of mathematical modelling to help achieve desired drug distributions in biological tissues with a complex microstructure.

Fractional Dual-Phase-Lag Model for Nonlinear Viscoelastic Soft Tissues

The primary goal of this paper is to create a new fractional boundary element method (BEM) model for bio-thermomechanical problems in functionally graded anisotropic (FGA) nonlinear viscoelastic soft tissues. The governing equations of bio-thermomechanical problems are briefly presented, including the fractional dual-phase-lag (DPL) bioheat model and Biot’s model. The more complex shapes of nonlinear viscoelastic soft tissues can be handled by the boundary element method, which also avoids the need for the interior domain to be discretized. The fractional dual-phase-lag bioheat equation was solved using the general boundary element method (GBEM) based on the local radial basis function collocation method (LRBFCM). The poroelastic fields are then calculated using the convolution quadrature boundary element method (CQBEM) The numerical findings show that our proposed numerical model is valid, efficient, and accurate.

Resilience of Neural Cellularity to the Influence of Low Educational Level.

Education is believed to contribute positively to brain structure and function, as well as to cognitive reserve. One of the brain regions most impacted by education is the medial temporal lobe (MTL), a region that houses the hippocampus, which has an important role in learning processes and in consolidation of memories, and is also known to undergo neurogenesis in adulthood. We aimed to investigate the influence of education on the absolute cell numbers of the MTL (comprised by the hippocampal formation, amygdala, and parahippocampal gyrus) of men without cognitive impairment. The Isotropic Fractionator technique was used to allow the anisotropic brain tissue to be transformed into an isotropic suspension of nuclei, and therefore assess the absolute cell composition of the MTL. We dissected twenty-six brains from men aged 47 to 64 years, with either low or high education. A significant difference between groups was observed in brain mass, but not in MTL mass. No significant difference was found between groups in the number of total cells, number of neurons, and number of non-neuronal cells. Regression analysis showed that the total number of cells, number of neurons, and number of non-neuronal cells in MTL were not affected by education. The results indicate a resilience of the absolute cellular composition of the MTL of typical men to low schooling, suggesting that the cellularity of brain regions is not affected by formal education.

Transparent and Cell-Guiding Cellulose Nanofiber 3D Printing Bioinks.

For three-dimensional (3D) bioprinting to fulfill its promise and enable the automated fabrication of complex tissue-mimicking constructs, there is a need for developing bioinks that are not only printable and biocompatible but also have integrated cell-instructive properties. Toward this goal, we here present a scalable technique for generating nanofiber 3D printing inks with unique tissue-guiding capabilities. Our core methodology relies on tailoring the size and dispersibility of cellulose fibrils through a solvent-controlled partial carboxymethylation. This way, we generate partially negatively charged cellulose nanofibers with diameters of ∼250 nm and lengths spanning tens to hundreds of microns. In this range, the fibers structurally match the size and dimensions of natural collagen fibers making them sufficiently large to orient cells. Yet, they are simultaneously sufficiently thin to be optically transparent. By adjusting fiber concentration, 3D printing inks with excellent shear-thinning properties can be established. In addition, as the fibers are readily dispersible, composite inks with both carbohydrates and extracellular matrix (ECM)-derived proteins can easily be generated. We apply such composite inks for 3D printing cell-laden and cross-linkable structures, as well as tissue-guiding gel substrates. Interestingly, we find that the spatial organization of engineered tissues can be defined by the shear-induced alignment of fibers during the printing procedure. Specifically, we show how myotubes derived from human and murine skeletal myoblasts can be programmed into linear and complex nonlinear architectures on soft printed substrates with intermediate fiber contents. Our nanofibrillated cellulose inks can thus serve as a simple and scalable tool for engineering anisotropic human muscle tissues that mimic native structure and function.

Optical encryption using structural uncorrelated characteristics of biological scattering media

ABSTRACT Inspired by the unique internal structures with inherent anisotropy in biological tissues and their dynamic scattering characteristic, a simple and easy-to-fabricate optical encryption technique using structural uncorrelated characteristics of biological tissues was proposed. It can generate unique keys with the random characteristics of the tissues and transforms multiple image plaintexts into speckle-like ciphertexts. The optical encryptions of grayscale images are demonstrated utilizing the uncorrelated characteristics of shallot and chicken breast respectively. Ciphertext-only-attack is resisted by phase retrieval due to the inherent dynamic randomness of biological tissues and the complexity of the plaintexts. Besides, once the unique PSF keys is recorded by the optical setup, the encryption can be accomplished, either online using the setup, or offline through performing convolutions on the plaintexts with pre-recorded uncorrelated PSFs to yield ciphertexts. Therefore, it is very secure, simple, and flexible to guarantee the promising potentials for information encryption of grayscale images and/or videos.

Meningioma microstructure assessed by diffusion MRI: An investigation of the source of mean diffusivity and fractional anisotropy by quantitative histology

BackgroundMean diffusivity (MD) and fractional anisotropy (FA) from diffusion MRI (dMRI) have been associated with cell density and tissue anisotropy across tumors, but it is unknown whether these associations persist at the microscopic level. PurposeTo quantify the degree to which cell density and anisotropy, as determined from histology, account for the intra-tumor variability of MD and FA in meningioma tumors. Furthermore, to clarify whether other histological features account for additional intra-tumor variability of dMRI parameters. Materials and MethodsWe performed ex-vivo dMRI at 200 μm isotropic resolution and histological imaging of 16 excised meningioma tumor samples. Diffusion tensor imaging (DTI) was used to map MD and FA, as well as the in-plane FA (FAIP). Histology images were analyzed in terms of cell nuclei density (CD) and structure anisotropy (SA; obtained from structure tensor analysis) and were used separately in a regression analysis to predict MD and FAIP, respectively. A convolutional neural network (CNN) was also trained to predict the dMRI parameters from histology patches. The association between MRI and histology was analyzed in terms of out-of-sample (R2OS) on the intra-tumor level and within-sample R2 across tumors. Regions where the dMRI parameters were poorly predicted from histology were analyzed to identify features apart from CD and SA that could influence MD and FAIP, respectively. ResultsCell density assessed by histology poorly explained intra-tumor variability of MD at the mesoscopic level (200 μm), as median R2OS = 0.04 (interquartile range 0.01–0.26). Structure anisotropy explained more of the variation in FAIP (median R2OS = 0.31, 0.20–0.42). Samples with low R2OS for FAIP exhibited low variations throughout the samples and thus low explainable variability, however, this was not the case for MD. Across tumors, CD and SA were clearly associated with MD (R2 = 0.60) and FAIP (R2 = 0.81), respectively. In 37% of the samples (6 out of 16), cell density did not explain intra-tumor variability of MD when compared to the degree explained by the CNN. Tumor vascularization, psammoma bodies, microcysts, and tissue cohesivity were associated with bias in MD prediction based solely on CD. Our results support that FAIP is high in the presence of elongated and aligned cell structures, but low otherwise. ConclusionCell density and structure anisotropy account for variability in MD and FAIP across tumors but cell density does not explain MD variations within the tumor, which means that low or high values of MD locally may not always reflect high or low tumor cell density. Features beyond cell density need to be considered when interpreting MD.

Engineering Large Airways.

A key issue facing trachea replacement attempts has been the discrepancy of the mechanical properties between the native tracheal tissue and that of the replacement construct; this difference is often one of the major causes for implant failure in vivo and within clinical efforts. The trachea is composed of distinct structural regions, with each component fulfilling a different role in maintaining overall tracheal stability. The trachea's horseshoe-shaped hyaline cartilage rings, smooth muscle and annular ligament collectively produce an anisotropic tissue that allows for longitudinal extensibility and lateral rigidity. Therefore, any tracheal substitute must be mechanically robust in order to withstand intra-thoracic pressure changes that occur during respiration. Conversely, they must also be able to deform radially to allow for changes in the cross-sectional area during coughing and swallowing. These complicated native tissue characteristics, coupled with a lack of standardised protocols to accurately quantify tracheal biomechanics as guidance for implant design, constitute a significant hurdle for tracheal biomaterial scaffold fabrication. This chapter aims to highlight the pressure forces exerted on the trachea and how they can influence tracheal construct design and also the biomechanical properties of the three main components of the trachea and how to mechanically assess them.

Inspired by plants: Carbonization mechanism, micro architecture and electrochemical applications of corn stalks

Plants possess the natural organic composite frameworks and the desirable micro-architecture, which are one kind of the promising precursors of porous carbons. In this work, corn stalks are studied as carbon precursors for the efficient utilization of their sponge-like tissue. Unlike other reported works, this study focuses on the carbonization mechanism of corn stalks and the inherited process from the organic composite tissues to carbon frameworks. After three carbonization stages, the cellular-like porous biocarbons with the replicated biological structures were generated from corn stalks. It is found that the lignin-networks wrapped on cellulose and hemicellulose will become into the main composition of the carbon-skeletons. The gas-phase products derived from carbohydrates promoted the lignin carbonization into porous carbon frameworks, which inherit the cellular structure and the anisotropy of plant tissues. Based on our discovery, the ruthenium nanoparticles are prepared by using the spontaneous capillary action and the steric hindrance of plant tissues. Due to the high-dispersion active particles and the accelerated mass transfer, the in-situ prepared composite electrocatalysts exhibit high performance towards the hydrogen evolution reaction. This study provides a new idea to establish an environmentally sustainable solution of agricultural waste and the utilization of plants natural structures for high-performance applications.

On a three–dimensional model for the description of the passive characteristics of skeletal muscle tissue

In this work, a three–dimensional model was developed to describe the passive mechanical behaviour of anisotropic skeletal muscle tissue. To validate the model, orientation–dependent axial (0^circ, 45^circ, 90^circ) and semi–confined compression experiments (mode I, II, III) were performed on soleus muscle tissue from rabbits. In the latter experiments, specimen deformation is prescribed in the loading direction and prevented in an additional spatial direction, fibre compression at 0^circ (mode I), fibre elongation at 90^circ (mode II) and a neutral state of the fibres at 90^circ where their length is kept constant (mode III). Overall, the model can adequately describe the mechanical behaviour with a relatively small number of model parameters. The stiffest tissue response during orientation–dependent axial compression (-,7.7,pm ,1.3 kPa) occurs when the fibres are oriented perpendicular to the loading direction (90^circ) and are thus stretched during loading. Semi–confined compression experiments yielded the stiffest tissue (-,36.7,pm ,11.2 kPa) in mode II when the muscle fibres are stretched. The extensive data set collected in this study allows to study the different error measures depending on the deformation state or the combination of deformation states.

Spatial frequency domain Mueller matrix imaging.

Mueller matrix polarimetry (MMP) and spatial frequency domain imaging (SFDI) are wide-field optical imaging modalities that differentiate tissue primarily by structure alignment and photon transport coefficient, respectively. Because these effects can be related, combining MMP and SFDI may enhance tissue differentiation beyond the capability of each modality alone. An instrument was developed to combine MMP and SFDI with the goal of testing whether it enhances contrast of features in reflection mode. The instrument was constructed using liquid crystal elements for polarization control, a digital light processing projector for generating sinusoidal illumination patterns, and a digital camera for imaging. A theoretical analysis shows that the SFD Mueller matrix is complex-valued and does not follow the same behavior as a regular Mueller matrix. Images were acquired from an anisotropic tissue phantom, an optical fiber bundle, and cerebellum, thalamus, and cerebrum tissues. The measurement results suggest that singly scattered, few scattered, and diffusely scattered photon paths can be distinguished in some of the samples investigated. The combined imaging modality yields additional spatial frequency phase information, which highlights paths having only a few scattering events. The combination of MMP and SFDI offers contrast mechanisms inaccessible by each modality used alone.

Modeling large-volume subcutaneous injection of monoclonal antibodies with anisotropic porohyperelastic models and data-driven tissue layer geometries

Subcutaneous injection of therapeutic monoclonal antibodies (mAbs) has become one of the fastest-growing fields in the pharmaceutical industry. The transport and mechanical processes behind large volume injections are poorly understood. Here, we leverage a large-deformation poroelastic model to study high-dose, high-speed subcutaneous injection. We account for the anisotropy of subcutaneous tissue using of a fibril-reinforced porohyperelastic model. We also incorporate the multi-layer structure of the skin tissue, generating data-driven geometrical models of the tissue layers using histological data. We analyze the impact of handheld autoinjectors on the injection dynamics for different patient forces. Our simulations show the importance of considering the large deformation approach to model large injection volumes. This work opens opportunities to better understand the mechanics and transport processes that occur in large-volume subcutaneous injections of mAbs.

Hydrated Collagen: Where Physical Chemistry, Medical Imaging, and Bioengineering Meet.

It is well-known that collagen is the most abundant protein in the human body; however, what is not often appreciated is its fascinating physical chemistry and molecular physics. In this Perspective, we aim to expose some of the physicochemical phenomena associated with the hydration of collagen and to examine the role collagen's hydration water plays in determining its biological function as well as applications ranging from radiology to bioengineering. The main focus is on the Magic-Angle Effect, a phenomenon observed in Nuclear Magnetic Resonance (NMR) spectroscopy and Magnetic Resonance Imaging (MRI) of anisotropic collagenous tissues such as articular cartilage and tendon. While the effect has been known in NMR and MRI for decades, its exact molecular mechanism remains a topic of debate and continuing research in scientific literature. We survey some of the latest research aiming to develop a comprehensive molecular-level model of the Magic-Angle Effect. We also touch on other fields where understanding of collagen hydration is important, particularly nanomechanics and mechanobiology, biomaterials, and piezoelectric sensors.

Human pluripotent stem cell-derived cardiomyocytes align under cyclic strain when guided by cardiac fibroblasts.

The myocardium is a mechanically active tissue typified by anisotropy of the resident cells [cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)] and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding of how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a two-dimensional human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain-mimicking the local anisotropic deformation of beating myocardium-causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs was no contributing factor. Our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study, therefore, highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium.

Magnetic Systems for Regenerative Medicine

Magnetic Systems for Regenerative Medicine

Supine magnetic resonance image registration for breast surgery: insights on material mechanics.

Breast conserving surgery (BCS) is a common procedure for early-stage breast cancer patients. Supine preoperative magnetic resonance (MR) breast imaging for visualizing tumor location and extent, while not standard for procedural guidance, is being explored since it more closely represents the surgical presentation compared to conventional diagnostic imaging positions. Despite this preoperative imaging position, deformation is still present between the supine imaging and surgical state. As a result, a fast and accurate image-to-physical registration approach is needed to realize image-guided breast surgery. In this study, three registration methods were investigated on healthy volunteers' breasts ( ) with the supine arm-down position simulating preoperative imaging and supine arm-up position simulating intraoperative presentation. The registration methods included (1)point-based rigid registration using synthetic fiducials, (2)nonrigid biomechanical model-based registration using sparse data, and (3)a data-dense three-dimensional diffeomorphic image-based registration from the Advanced Normalization Tools (ANTs) repository. Additionally, deformation metrics (volume change and anisotropy) were calculated from the ANTs deformation field to better understand breast material mechanics. The average target registration errors (TRE) were , , and (mean ± standard deviation) and the average fiducial registration errors (FRE) were , , and for the point-based rigid, nonrigid biomechanical, and ANTs registrations, respectively. The mechanics-based deformation metrics revealed an overall anisotropic tissue behavior and a statistically significant difference in volume change between glandular and adipose tissue, suggesting that nonrigid modeling methods may be improved by incorporating material heterogeneity and anisotropy. Overall, registration accuracy significantly improved with increasingly flexible and data-dense registration methods. Analysis of these outcomes may inform the future development of image guidance systems for lumpectomy procedures.

Abstract 15845: Microfabricated Anisotropic Tissue Bundles for the Production of 3D Cardiac Tissue at Scale

Rationale: Following myocardial infarct, tissue ischemia and cardiomyocyte (CM) death trigger inflammation followed by extracellular matrix (ECM) remodeling mediated by cardiac fibroblasts (CF). The cardiac tissue engineering field aims to replace damaged myocardium and restore normal cardiac function. The ECM’s high degree of structural anisotropy and the organization of resident cell populations are critical features necessary to recapitulate healthy myocardial function. Thus, there is a critical need for biomanufacturing technologies that can organize ECM and multiple, distinct cell types with microscale precision in tissue grafts of translationally relevant scale. Here, we propose a scalable material strategy for fabricating individual myofiber-like tissues with anisotropic organization and relevant cell populations comparable to native myocardium. Methods and Results: Microchannels of varying widths were perfused with fibrin (20mg/mL fibrinogen with 1U/mL thrombin supplemented with 0.05mg/mL aprotinin for the first 3 days of culture) or a mixture of fibrin and collagen (10mg/ml fibrinogen with 1U/mL thrombin and 2mg/ml collagen). Hydrogels contained up to 40 million/mL cells (25% CFs and 75% CMs) and 8 vol% RGD-functionalized dextran-vinyl sulfone (DVS) electrospun fibers. Microchannel perfusion of composite hydrogel solutions resulted in flow-induced alignment of DVS fibers across all channel sizes (200, 300, 400, and 500 μm), although fiber alignment decreased with increasing channel size. Tissue size, cell spreading, CM contraction, and myofibril development and alignment varied with channel size, cell density, and hydrogel formulation over two weeks inculture. Overall, hydrogel composites with aligned fibers and a combination of CMs and CFs resulted in anisotropic, myofiber-like tissue structures. Conclusion: In this study, we developed a microfabrication approach to produce anisotropic myofiber-like bundles using composites of naturally derived ECM and synthetic polymeric fibers that can be flow-aligned within microfabricated microchannels. This approach allows us to fabricate unit cell tissues of desired size and alignment that can be combined into a larger tissue construct for therapeutic applications.

Intersession Repeatability of Diffusion-Tensor Imaging in the Supraspinatus and the Infraspinatus Muscles of Volunteers.

Quantifying the rotator cuff (RC) muscles' viscoelasticity could provide outcome relevant information in patients with RC tears. MR-elastography requires robust diffusion-tensor imaging (DTI) to account for tissue anisotropy in muscles stiffness computation. To assess the repeatability of DTI parameters in the supraspinatus and infraspinatus muscles and to explore DTI tractography conformity with the muscles' anatomy. Prospective. Six healthy volunteers underwent three consecutive shoulder MRI sessions about 10 minutes apart. 3T/T1-vibe Dixon and Spin echo EPI DTI (12 gradient encoding directions, b-values 500 and 800 sec/mm2 ). Supraspinatus and infraspinatus muscles were segmented on the T1-vibe Dixon sequence. DTI image quality was assessed using a quantitative threshold based on the signal-to-noise ratio (SNR). The eigenvalues ( ), fractional anisotropy (FA) and mean diffusivity were calculated. DTI tractography was visually assessed. DTI parameters within-subject intersession repeatability was assessed with Bland-Altman analysis and the coefficient of variation (CV). Repeatability was considered good for CV < 10%. The SNR between diffusion-weighted and non-diffusion-weighted images was greater than 3, which aligns with standards for estimating DTI parameters. The FA showed the lowest mean bias (-0.007; 95% confidence interval [CI] -0.031 to 0.018) whereas the λ1 had the highest mean bias (0.146 × 10-3 mm2 /sec; CI -0.034 to 0.326 × 10-3 mm2 /sec). CVs of the DTI parameters varied between 3.5% (FA) and 8.4% (λ3 ) for the supraspinatus and between 3.2% (λ1 ) and 6.8% (λ3 ) for the infraspinatus. Tractography provided muscle fiber representations in three-dimensional space concordant with RC anatomy. DTI of the supraspinatus and infraspinatus muscles achieved an adequate SNR, allowing the measurement of the DTI metrics with good repeatability, and thus can be used for optimizing stiffness estimation in these anisotropic tissues. 2 TECHNICAL EFFICACY: Stage 2.