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.

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.

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.

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.

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.

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.

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.].

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.

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.

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.

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.

Progression of Post-Traumatic Osteoarthritis in rat meniscectomy models: Comprehensive monitoring using MRI.

Knee injury often triggers post-traumatic osteoarthritis (PTOA) that affects articular cartilage (AC), subchondral bone, meniscus and the synovial membrane. The available treatments for PTOA are largely ineffective due to late diagnosis past the "treatment window". This study aimed to develop a detailed understanding of the time line of the progression of PTOA in murine models through longitudinal observation of the femorotibial joint from the onset of the disease to the advanced stage. Quantitative magnetic resonance microimaging (µMRI) and histology were used to evaluate PTOA-associated changes in the knee joints of rats subjected to knee meniscectomy. Systematic longitudinal changes in the articular cartilage thickness, cartilage T2 and the T2 of epiphysis within medial condyles of the tibia were all found to be associated with the development of PTOA in the animals. The following pathogenesis cascade was found to precede advanced PTOA: meniscal injury → AC swelling → subchondral bone remodelling → proteoglycan depletion → free water influx → cartilage erosion. Importantly, the imaging protocol used was entirely MRI-based. This protocol is potentially suitable for whole-knee longitudinal, non-invasive assessment of the development of OA. The results of this work will inform the improvement of the imaging methods for early diagnosis of PTOA.

Assessment of collagen fiber orientation dispersion in articular cartilage by small-angle X-ray scattering and diffusion tensor imaging: Preliminary results.

Measurements of the orientational dispersion of collagen fibers in articular cartilage were made using diffusion tensor imaging (DTI) and small-angle X-ray scattering (SAXS) on matched bovine articular cartilage samples. Thirteen pairs of samples were excised from bovine knee joints; each pair was taken from neighboring locations in the same bone. One sample from each pair was used for DTI measurements and the other for SAXS measurements. Fractional anisotropy (FA) values were calculated from the DTI data both for the individual imaging voxels and for whole regions of interest (ROI). The FA values were used as a measure of fiber dispersion and compared to the ellipticities of the fiber orientation distributions obtained from SAXS. Neither the spatially-resolved FA values nor whole-ROI FA values showed any correlation with SAXS ellipticities. We attribute the lack of DTI-SAXS correlation to two principal factors: (1) the significant difference in the imaging resolution of the two techniques; and (2) the inherent limitations of both the SAXS data analysis methodology and the diffusion tensor model in the case of multi-modal fiber orientation distributions. We discuss how these factors could be overcome in future work.

Rotational-Diffusion Propagator of the Intramolecular Proton-Proton Vector in Liquid Water: A Molecular Dynamics Study.

The rotational motion of water molecules plays the dominant role in determining NMR spin-relaxation properties of liquid water and many biological tissues. The traditional theory of NMR spin relaxation predominantly uses the assumption that the reorientational dynamics of water molecules is described by a continuous-time rotational-diffusion random walk with a single rotational-diffusion coefficient. However, recent experimental and theoretical studies have demonstrated that water reorientation occurs by large, discrete angular jumps superimposed on a continuous-time rotational-diffusion process. We have investigated the rotational-diffusion propagator of the proton-proton (H-H) vector of water molecules in liquid water at 298 K using molecular dynamics (MD) simulations. Analysis of the MD-simulated reorientational trajectories reveals that reorientation of the intramolecular H-H vector occurs through a combination of the two mechanisms: rotational diffusion proper and discrete large-angle jumps. We demonstrate that, empirically, the rotational-diffusion propagator of the intramolecular H-H vector in liquid water can be described in terms of multiple rotational-diffusion coefficients. A model with two rotational-diffusion coefficients was found to provide a reasonable (albeit imperfect) fit of the MD-simulated propagator on the time scales relevant to NMR spin relaxation near room or physiological temperature (picoseconds to nanoseconds). We report the apparent values of the two rotational-diffusion coefficients determined from the propagator analysis at 298 K and discuss their physical meaning.

MRI magic-angle effect in femorotibial cartilages of the red kangaroo.

OBJECTIVE: Kangaroo knee cartilages are robust tissues that can support knee flexion and endure high levels of compressive stress. This study aimed to develop a detailed understanding of the collagen architecture in kangaroo knee cartilages and thus obtain insights into the biophysical basis of their function. DESIGN: Cylindrical/square plugs from femoral and tibial hyaline cartilage and tibial fibrocartilage were excised from the knees of three adult red kangaroos. Multi-slice, multi-echo MR images were acquired at the sample orientations 0° and 55° ("magic angle") with respect to the static magnetic field. Maps of the transverse relaxation rate constant (R2) and depth profiles of R2 and its anisotropic component (R2A) were constructed from the data. RESULTS: The R2A profiles confirmed the classic three-zone organisation of all cartilage samples. Femoral hyaline cartilage possessed a well-developed, thick superficial zone. Tibial hyaline cartilage possessed a very thick radial zone (80% relative thickness) that exhibited large R2A values consistent with highly ordered collagen. The R2A profile of tibial fibrocartilage exhibited a unique region near the bone (bottom 5-10%) consistent with elevated proteoglycan content ("attachment sub-zone"). CONCLUSIONS: Our observations suggest that the well-developed superficial zone of femoral hyaline cartilage is suitable for supporting knee flexion; the thick and well-aligned radial zone of tibial hyaline cartilage is adapted to endure high compressive stress; while the innermost part of the radial zone of tibial fibrocartilage may facilitate anchoring of the collagen fibres to withstand high shear deformation. These findings may inspire new designs for cartilage tissue engineering.

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.

The distribution of the apparent diffusion coefficient as an indicator of the response to chemotherapeutics in ovarian tumour xenografts.

Diffusion-weighted magnetic resonance imaging (DW-MRI) was used to evaluate the effects of single-agent and combination treatment regimens in a spheroid-based animal model of ovarian cancer. Ovarian tumour xenografts grown in non-obese diabetic/severe-combined-immunodeficiency (NOD/SCID) mice were treated with carboplatin or paclitaxel, or combination carboplatin/paclitaxel chemotherapy regimens. After 4 weeks of treatment, tumours were extracted and underwent DW-MRI, mechanical testing, immunohistochemical and gene expression analyses. The distribution of the apparent diffusion coefficient (ADC) exhibited an upward shift as a result of each treatment regimen. The 99-th percentile of the ADC distribution ("maximum ADC") exhibited a strong correlation with the tumour size (r2 = 0.90) and with the inverse of the elastic modulus (r2 = 0.96). Single-agent paclitaxel (n = 5) and combination carboplatin/paclitaxel (n = 2) treatment regimens were more effective in inducing changes in regions of higher cell density than single-agent carboplatin (n = 3) or the no-treatment control (n = 5). The maximum ADC was a good indicator of treatment-induced cell death and changes in the extracellular matrix (ECM). Comparative analysis of the tumours' ADC distribution, mechanical properties and ECM constituents provides insights into the molecular and cellular response of the ovarian tumour xenografts to chemotherapy. Increased sample sizes are recommended for future studies. We propose experimental approaches to evaluation of the timeline of the tumour's response to treatment.

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.