Quantitative measurement of mammographic density in breast-tissue explants using portable NMR: Precision and accuracy.

PURPOSE: Single-sided portable NMR (pNMR) has previously been demonstrated to be suitable for quantification of mammographic density (MD) in excised breast tissue samples. Here we investigate the precision and accuracy of pNMR measurements of MD ex vivo as compared with the gold standards. METHODS: Forty-five breast-tissue explants from 9 prophylactic mastectomy patients were measured. The relative tissue water content was taken as the MD-equivalent quantity. In each sample, the water content was measured using some combination of three pNMR techniques (apparent T2, diffusion, and T1 measurements) and two gold-standard techniques (computed microtomography [µCT] and hematoxylin and eosin [H&E] histology). Pairwise correlation plots and Bland-Altman analysis were used to quantify the degree of agreement between pNMR techniques and the gold standards. RESULTS: Relative water content measured from both apparent T2 relaxation spectra, and diffusion decays exhibited strong correlation with the H&E and µCT results. Bland-Altman analysis yielded average bias values of -0.4, -2.6, 2.6, and 2.8 water percentage points (pp) and 95% confidence intervals of 13.1, 7.5, 11.2, and 11.8 pp for the H&E - T2, µCT - T2, H&E - diffusion, and µCT - diffusion comparison pairs, respectively. T1-based measurements were found to be less reliable, with the Bland-Altman confidence intervals of 27.7 and 33.0 pp when compared with H&E and µCT, respectively. CONCLUSION: Apparent T2-based and diffusion-based pNMR measurements enable quantification of MD in breast-tissue explants with the precision of approximately 10 pp and accuracy of approximately 3 pp or better, making pNMR a promising measurement modality for radiation-free quantification of MD.

Mechanical Pressure Driving Proteoglycan Expression in Mammographic Density: a Self-perpetuating Cycle?

Regions of high mammographic density (MD) in the breast are characterised by a proteoglycan (PG)-rich fibrous stroma, where PGs mediate aligned collagen fibrils to control tissue stiffness and hence the response to mechanical forces. Literature is accumulating to support the notion that mechanical stiffness may drive PG synthesis in the breast contributing to MD. We review emerging patterns in MD and other biological settings, of a positive feedback cycle of force promoting PG synthesis, such as in articular cartilage, due to increased pressure on weight bearing joints. Furthermore, we present evidence to suggest a pro-tumorigenic effect of increased mechanical force on epithelial cells in contexts where PG-mediated, aligned collagen fibrous tissue abounds, with implications for breast cancer development attributable to high MD. Finally, we summarise means through which this positive feedback mechanism of PG synthesis may be intercepted to reduce mechanical force within tissues and thus reduce disease burden.

Transverse relaxation-based assessment of mammographic density and breast tissue composition by single-sided portable NMR.

PURPOSE: Elevated mammographic density (MD) is an independent risk factor for breast cancer (BC) as well as a source of masking in X-ray mammography. High-frequency longitudinal monitoring of MD could also be beneficial in hormonal BC prevention, where early MD changes herald the treatment's success. We present a novel approach to quantification of MD in breast tissue using single-sided portable NMR. Its development was motivated by the low cost of portable-NMR instrumentation, the suitability for measurements in vivo, and the absence of ionizing radiation. METHODS: Five breast slices were obtained from three patients undergoing prophylactic mastectomy or breast reduction surgery. Carr-Purcell-Meiboom-Gill (CPMG) relaxation curves were measured from (1) regions of high and low MD (HMD and LMD, respectively) in the full breast slices; (2) the same regions excised from the full slices; and (3) excised samples after H2 O-D2 O replacement. T2 distributions were reconstructed from the CPMG decays using inverse Laplace transform. RESULTS: Two major peaks, identified as fat and water, were consistently observed in the T2 distributions of HMD regions. The LMD T2 distributions were dominated by the fat peak. The relative areas of the two peaks exhibited statistically significant (P < .005) differences between HMD and LMD regions, enabling their classification as HMD or LMD. The relative-area distributions exhibited no statistically significant differences between full slices and excised samples. CONCLUSION: T2 -based portable-NMR analysis is a novel approach to MD quantification. The ability to quantify tissue composition, combined with the low cost of instrumentation, make this approach promising for clinical applications.

Load-induced changes in the diffusion tensor of ovine anulus fibrosus: A pilot MRI study.

PURPOSE: To assess the feasibility of diffusion tensor imaging (DTI) for evaluating changes in anulus fibrosus (AF) microstructure following uniaxial compression. MATERIALS AND METHODS: Six axially aligned samples of AF were obtained from a merino sheep disc; two each from the anterior, lateral, and posterior regions. The samples were mechanically loaded in axial compression during five cycles at a rate and maximum compressive strain that reflected physiological conditions. DTI was conducted at 7T for each sample before and after mechanical testing. RESULTS: The mechanical response of all samples in unconfined compression was nonlinear. A stiffer response during the first loading cycle, compared to the remaining cycles, was observed. Change in diffusion parameters appeared to be region-dependent. The mean fractional anisotropy increased following mechanical testing. This was smallest in the lateral (2% and 9%) and largest in the anterior and posterior samples (17-25%). The mean average diffusivity remained relatively constant (<2%) after mechanical testing in the lateral and posterior samples, but increased (by 5%) in the anterior samples. The mean angle made by the principal eigenvector with the spine axis in the lateral samples was 73° and remained relatively constant (<2%) following mechanical testing. This angle was smaller in the anterior (55°) and posterior (47°) regions and increased by 6-16° following mechanical testing. CONCLUSION: These preliminary results suggest that axial compression reorients the collagen fibers, such that they become more consistently aligned parallel to the plane of the endplates. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;45:1723-1735.

Anisotropic diffusion in stretched hydrogels containing erythrocytes: evidence of cell-shape distortion recorded by PGSE NMR spectroscopy.

The remarkable flexibility of human red blood cells (RBCs) allows them to assume a range of shapes in normal and disease states. Biochemical mechanisms and energetic requirements associated with changes in RBC geometry are not well understood because of a lack of experimental procedures to fix and study cells in different morphological forms. By incorporating RBCs into stretchable gelatin hydrogels, we created conditions for adjustable elongation of their normal discocytic shape in all orientations. As the RBC-containing gels were stretched or compressed, the changes in the cell morphology were studied by using 1 H-PGSE-NMR spectroscopy. Measurements of the apparent diffusion coefficient of water along the three orthogonal directions revealed tuneable anisotropy in the environment of the hydrogel samples. Light microscopy was also used for recording the extent to which RBCs were distorted in a stretched gel that had been set around them. Having demonstrated the applicability of NMR diffusometry to detect morphological changes of immobilised cells, we have laid the groundwork for future investigations of controllably distorted RBCs. Specifically, we expect studies of metabolic and biophysical properties of the physically deformed cells, thus inferring the connection between intracellular physico-chemical processes and RBC morphology. Copyright © 2016 John Wiley & Sons, Ltd.

Na+ and solute diffusion in aqueous channels of Myverol bicontinuous cubic phase: PGSE NMR and computer modelling.

The apparent diffusion coefficients of 23 Na+ ions and the solute 2-fluoroethylamine present in the aqueous domain of a Myverol/water bulk bicontinuous cubic phase (BCP) were measured using pulsed field-gradient spin echo (PGSE) NMR spectroscopy. The measured values were dependent on the diffusion time interval, which is a characteristic of restricted diffusion. The translational motion of 23 Na+ and water in the aqueous channels of a cubic phase were simulated using a Monte-Carlo random walk algorithm, and the simulation results were compared with those from real PGSE NMR experiments. The simulations indicated that diffusion of 23 Na+ ions and water would appear to be restricted even on the shortest timescales available to PGSE NMR experiments. The micro-viscosity of the aqueous domain of the BCPs was estimated from the longitudinal relaxation times of 23 Na+ and 2-fluoroethylamine; this was three times higher than in free solution and suggests one of (but not the only) likely impediments to the release of hydrophilic drugs from stabilised aqueous dispersions of BCPs (cubosomes) when they are used therapeutically in vivo. Monte Carlo simulations of diffusive efflux from cubosomes suggest that the principal impediment to drug release is presented by a surfactant or lipid barrier at the cubosome surface, which separates the BCP aqueous channels from the bulk solution. The dynamics inferred from these studies informs quantitative predictions of drug delivery from cubosomes. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous magnetic resonance imaging and consolidation measurement of articular cartilage.

Magnetic resonance imaging (MRI) offers the opportunity to study biological tissues and processes in a non-disruptive manner. The technique shows promise for the study of the load-bearing performance (consolidation) of articular cartilage and changes in articular cartilage accompanying osteoarthritis. Consolidation of articular cartilage involves the recording of two transient characteristics: the change over time of strain and the hydrostatic excess pore pressure (HEPP). MRI study of cartilage consolidation under mechanical load is limited by difficulties in measuring the HEPP in the presence of the strong magnetic fields associated with the MRI technique. Here we describe the use of MRI to image and characterize bovine articular cartilage deforming under load in an MRI compatible consolidometer while monitoring pressure with a Fabry-Perot interferometer-based fiber-optic pressure transducer.

An investigation of water status in gelatin methacrylate hydrogels by means of water relaxometry and differential scanning calorimetry.

The relationship between molecular structure and water dynamics is a fundamental yet often neglected subject in the field of hydrogels for drug delivery, bioprinting, as well as biomaterial science and tissue engineering & regenerative medicine (TE&RM). Water is a fundamental constituent of hydrogel systems and engages via hydrogen bonding with the macromolecular network. The methods and techniques to measure and reveal the phenomena and dynamics of water within hydrogels are still limited. In this work, differential scanning calorimetry (DSC) was used as a quantitative method to analyze freezable (including free and freezable bound) and non-freezable bound water within gelatin methacrylate (GelMA) hydrogels. Nuclear magnetic resonance (NMR) is a complementary method for the study of water behavior and can be used to measure the spin-relaxation of water hydrogen nuclei, which is related to water dynamics. In this research, nuclear magnetic resonance relaxometry was employed to investigate the molecular state of water in GelMA hydrogels using spin-lattice (T1) and spin-spin (T2) spin-relaxation time constants. The data displays a trend of increasing bound water content with increasing GelMA concentration. In addition, T2 values were further applied to calculate microviscosity and translational diffusion coefficients. Water relaxation under various chemical environments, including different media, temperatures, gelatin sources, as well as crosslinking effects, were also examined. These comprehensive physical data sets offer fundamental insight into biomolecule transport within the GelMA hydrogel system, which ultimately are important for drug delivery, bioprinting, as well as biomaterial science and TE&RM communities.

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.

Sensing mammographic density using single-sided portable Nuclear Magnetic Resonance.

This research paper presents a quantitative approach to sensing mammographic density (MD) using single-sided portable Nuclear Magnetic Resonance (NMR). It focuses on three main techniques: spin-lattice relaxation (recovery) time (T1), spin-spin relaxation (decay) time (T2), and Diffusion (D) techniques by testing whether or not the aforementioned techniques are in agreement with the gold standard and with each other when used for scanning breast tissue specimens with a variety of mammographic densities (MDs). The high mammographic density (HMD), intermediate MD, and low mammographic density (LMD) regions of each slice were identified according to the mammogram images. Subsequently, the grayscale values for these regions were quantified. One region was measured from the first sample while the remaining ones were measured from the second sample. The same areas were then exposed to portable NMR, and the sequences used as following: the stimulated echo sequence for diffusion (D), the Carr-Purcell-Meiboom-Gill (CPMG) sequence for T2, and saturation recovery sequence for T1. The correlations between the grayscale values and NMR techniques were strongly correlated. The Pearson correlation coefficient, R, of T1 (%) versus grayscale value, D (%) versus grayscale value, and T2 (%) versus grayscale value, was 0.91, 0.91, and 0.93, respectively. Furthermore, the relative water content of the breast slices based on T1, T2, and diffusion (D) measurements were strongly in agreement with each other. The Pearson correlation coefficient, R, of D (%) versus T1 (%), D (%) versus T2 (%), and T1 (%) versus T2 (%), was 0.984, 0.966, and 0.9868, respectively. The three pulse sequences can be employed in a portable NMR device to deliver continuous quantitative measurements of MD in breast tissue samples. As a result, the method demonstrated to be acceptable for determining the distribution of MDs among breast tissue samples without the need for additional qualitative analysis.

Portable NMR for quantification of breast density in vivo: Proof-of-concept measurements and comparison with quantitative MRI.

Mammographic Density (MD) is the degree of radio-opacity of the breast in an X-ray mammogram. It is determined by the Fibroglandular: Adipose tissue ratio. MD has major implications in breast cancer risk and breast cancer chemoprevention. This study aimed to investigate the feasibility of accurate, low-cost quantification of MD in vivo without ionising radiation. We used single-sided portable nuclear magnetic resonance ("Portable NMR") due to its low cost and the absence of radiation-related safety concerns. Fifteen (N = 15) healthy female volunteers were selected for the study and underwent an imaging routine consisting of 2D X-ray mammography, quantitative breast 3T MRI (Dixon and T1-based 3D compositional breast imaging), and 1D compositional depth profiling of the right breast using Portable NMR. For each participant, all the measurements were made within 3-4 h of each other. MRI-determined tissue water content was used as the MD-equivalent quantity. Portable NMR depth profiles of tissue water were compared with the equivalent depth profiles reconstructed from Dixon and T1-based MR images, which were used as the MD-equivalent reference standard. The agreement between the depth profiles acquired using Portable NMR and the reconstructed reference-standard profiles was variable but overall encouraging. The agreement was somewhat inferior to that seen in breast tissue explant measurements conducted in vitro, where quantitative micro-CT was used as the reference standard. The lower agreement in vivo can be attributed to an uncertainty in the positioning of the Portable NMR sensor on the breast surface and breast compression in Portable NMR measurements. The degree of agreement between Portable NMR and quantitative MRI is encouraging. While the results call for further development of quantitative Portable NMR, they demonstrate the in-principle feasibility of Portable NMR-based quantitative compositional imaging in vivo and show promise for the development of safe and low-cost protocols for quantification of MD suitable for clinical applications.

Diffusion tensor of water in model articular cartilage.

We used Monte Carlo simulations of Brownian dynamics of water to study anisotropic water diffusion in an idealised model of articular cartilage. The main aim was to use the simulations as a tool for translation of the fractional anisotropy of the water diffusion tensor in cartilage into quantitative characteristics of its collagen fibre network. The key finding was a linear empirical relationship between the collagen volume fraction and the fractional anisotropy of the diffusion tensor. Fractional anisotropy of the diffusion tensor is potentially a robust indicator of the microstructure of the tissue because, to a first approximation, it is invariant to inclusion of proteoglycans or chemical exchange between free and collagen-bound water in the model. We discuss potential applications of Monte Carlo diffusion-tensor simulations for quantitative biophysical interpretation of magnetic resonance diffusion-tensor images of cartilage. Extension of the model to include collagen fibre disorder is also discussed.

RASSF1A Suppression as a Potential Regulator of Mechano-Pathobiology Associated with Mammographic Density in BRCA Mutation Carriers.

High mammographic density (MD) increases breast cancer (BC) risk and creates a stiff tissue environment. BC risk is also increased in BRCA1/2 gene mutation carriers, which may be in part due to genetic disruption of the tumour suppressor gene Ras association domain family member 1 (RASSF1A), a gene that is also directly regulated by tissue stiffness. High MD combined with BRCA1/2 mutations further increase breast cancer risk, yet BRCA1/2 mutations alone or in combination do not increase MD. The molecular basis for this additive effect therefore remains unclear. We studied the interplay between MD, stiffness, and BRCA1/2 mutation status in human mammary tissue obtained after prophylactic mastectomy from women at risk of developing BC. Our results demonstrate that RASSF1A expression increased in MCF10DCIS.com cell cultures with matrix stiffness up until ranges corresponding with BiRADs 4 stiffnesses (~16 kPa), but decreased in higher stiffnesses approaching malignancy levels (>50 kPa). Similarly, higher RASSF1A protein was seen in these cells when co-cultivated with high MD tissue in murine biochambers. Conversely, local stiffness, as measured by collagen I versus III abundance, repressed RASSF1A protein expression in BRCA1, but not BRCA2 gene mutated tissues; regional density as measured radiographically repressed RASSF1A in both BRCA1/2 mutated tissues. The combinatory effect of high MD and BRCA mutations on breast cancer risk may be due to RASSF1A gene repression in regions of increased tissue stiffness.

Corrigendum: Heparanase Promotes Syndecan-1 Expression to Mediate Fibrillar Collagen and Mammographic Density in Human Breast Tissue Cultured ex vivo.

[This corrects the article DOI: 10.3389/fcell.2020.00599.].

Anisotropy of spin relaxation of water protons in cartilage and tendon.

Transverse spin relaxation rates of water protons in articular cartilage and tendon depend on the orientation of the tissue relative to the applied static magnetic field. This complicates the interpretation of magnetic resonance images of these tissues. At the same time, relaxation data can provide information about their organisation and microstructure. We present a theoretical analysis of the anisotropy of spin relaxation of water protons observed in fully hydrated cartilage. We demonstrate that the anisotropy of transverse relaxation is due almost entirely to intramolecular dipolar coupling modulated by a specific mode of slow molecular motion: the diffusion of water molecules in the hydration shell of a collagen fibre around the fibre, such that the molecular director remains perpendicular to the fibre. The theoretical anisotropy arising from this mechanism follows the 'magic-angle' dependence observed in magnetic-resonance measurements of cartilage and tendon and is in good agreement with the available experimental results. We discuss the implications of the theoretical findings for MRI of ordered collagenous tissues.

Heparanase Promotes Syndecan-1 Expression to Mediate Fibrillar Collagen and Mammographic Density in Human Breast Tissue Cultured ex vivo.

Mammographic density (MD) is a strong and independent factor for breast cancer (BC) risk and is increasingly associated with BC progression. We have previously shown in mice that high MD, which is characterized by the preponderance of a fibrous stroma, facilitates BC xenograft growth and metastasis. This stroma is rich in extracellular matrix (ECM) factors, including heparan sulfate proteoglycans (HSPGs), such as the BC-associated syndecan-1 (SDC1). These proteoglycans tether growth factors, which are released by heparanase (HPSE). MD is positively associated with estrogen exposure and, in cell models, estrogen has been implicated in the upregulation of HPSE, the activity of which promotes SDC expression. Herein we describe a novel measurement approach (single-sided NMR) using a patient-derived explant (PDE) model of normal human (female) mammary tissue cultured ex vivo to investigate the role(s) of HPSE and SDC1 on MD. Relative HSPG gene and protein analyses determined in patient-paired high vs. low MD tissues identified SDC1 and SDC4 as potential mediators of MD. Using the PDE model we demonstrate that HPSE promotes SDC1 rather than SDC4 expression and cleavage, leading to increased MD. In this model system, synstatin (SSTN), an SDC1 inhibitory peptide designed to decouple SDC1-ITGαvß3 parallel collagen alignment, reduced the abundance of fibrillar collagen as assessed by picrosirius red viewed under polarized light, and reduced MD. Our results reveal a potential role for HPSE in maintaining MD via its direct regulation of SDC1, which in turn physically tethers collagen into aligned fibers characteristic of MD. We propose that inhibitors of HPSE and/or SDC1 may afford an opportunity to reduce MD in high BC risk individuals and reduce MD-associated BC progression in conjunction with established BC therapies.

Structure and Dynamics of Collagen Hydration Water from Molecular Dynamics Simulations: Implications of Temperature and Pressure.

Dynamics of water molecules in hydrated collagen plays an important role in determining the structural and functional properties of collagenous tissues. Experimental results suggest that collagen-bridging water molecules exhibit dynamic and thermodynamic properties of one-dimensional ice. However, molecular dynamics (MD) studies performed to date have failed to identify icelike water bridges. It has been hypothesized that this discrepancy is due to the experimental measurements and computational MD analysis having been performed on very different systems: complete tissues with large-scale collagen fiber assemblies and individual tropocollagen fragments, respectively. In this work, we explore ways of emulating a tissuelike macromolecular environment in MD simulations of hydrated collagen without increasing the size of the system to computationally prohibitive levels. We have investigated the effects of temperature and pressure on the dynamics of a small hydrated tropocollagen fragment. The occupancy and bond energies of interchain hydrogen bonds were relatively insensitive to temperature, suggesting that they play a key role in the stability of the collagen triple helix. The lifetimes of water bridges lengthened with decreasing temperature, but even at 280 K, no bridging water molecules exhibited icelike dynamics. We discuss the implications of these findings for the ability to emulate tissuelike conditions in hydrated collagen.

Quantification of breast tissue density: Correlation between single-sided portable NMR and micro-CT measurements.

Mammographic density (MD) is a strong independent risk factor for breast cancer. Traditional screening for MD using X-ray mammography involves ionising radiation, which is not suitable for young women, those with previous radiation exposure, or those having undergone a partial mastectomy. Therefore, alternative approaches for MD screening that do not involve ionising radiation will be important as the clinical use of MD increases, and as more frequent MD testing becomes desirable for research purposes. We have previously demonstrated the potential utility of spin relaxation-based, single-sided portable-NMR measurements for the purpose of MD quantification. We present here a more refined analysis by quantifying breast tissue density in excised samples on a continuous scale (0% to 100% fibroglandular tissue content) using micro-CT (µCT), and comparing the results to spin-relaxation and diffusion portable-NMR measurements of the same samples. µCT analysis of mammary tissues containing high- and low-MD (HMD and LMD, respectively) regions had Hounsfield Unit (HU) histograms with a bimodal pattern, with HMD regions exhibiting significantly higher HU values than LMD regions. Quantitative MD (%HMD) values obtained using µCT exhibited an excellent correlation with portable-NMR results, namely longitudinal spin-relaxation time constants (T1) and the relative tissue water content obtained from portable-NMR diffusion measurements (R2 = 0.92, p < 0.0001 and R2 = 0.96, p < 0.0001, respectively). These findings are consistent with our previous results demonstrating relatively high water content in HMD breast tissue, consistent with the high proportion of fibroglandular tissue, FGT, which in turn contains more abundant water-carrying HSPG proteins. We observed an excellent correlation between the T1 values and diffusion NMR-measured relative tissue water content (R2 = 0.94, p < 0.0001). These findings demonstrate, for the first time, the ability of single-sided portable NMR to accurately quantify MD in vitro on a continuous scale. The results also indicate that portable-NMR analysis can assist in the identification of features underpinning MD, namely FGT and adipose tissue content. Future work will involve application of portable NMR to quantifying MD in vivo.

Inhomogeneous NMR line shape as a probe of microscopic organization of bicontinuous cubic phases.

NMR line shapes of the lipid and aqueous species in bicontinuous cubic phase (BCP) samples prepared by centrifugation are inhomogeneously broadened. The broadening of the lipid peaks is removed by magic-angle spinning (MAS). In this work, we studied the mechanism of this broadening using (1)H and (13)C NMR spectroscopy of a myverol/water BCP. It is demonstrated that the inhomogeneity possesses an intrinsic contribution that is independent of instrumental or setup factors and can be attributed to the microscopic organization of the BCP bilayer. A mechanism of the inhomogeneous broadening is proposed, which involves a spatially nonuniform diamagnetically induced magnetic field determined by the mesoscopic structure and the diamagnetic susceptibilities of the two BCP domains. The proposed mechanism does not require that molecular reorientation of the lipid be slow for the inhomogeneous broadening to survive. We discuss how this inhomogeneous broadening can be employed as a probe of compositional uniformity and microscopic organization of BCP samples.