Assessment of bone tissue cytoarchitectonics by 2D 1H NMR relaxometry maps.

Bone is a complex tissue that fulfills the role of a resistance structure. This quality is most commonly assessed by bone densitometry, but bone strength may not only be related to bone mineral density but also to the preservation of bone cytoarchitectonics. The study included two groups of rats, ovariectomized and non-ovariectomized. Each group was divided into three batches: control, simvastatin-treated, and fenofibrate-treated. In the ovariectomized group, hypolipidemic treatment was instituted at 12 weeks post ovariectomy. One rat from each of the 6 batches was sacrificed 8 weeks after the start of treatment in the group. The experimental study was performed using a Bruker Minispec mq 20 spectrometer operating at a frequency of 20 MHz, subsequently also performed by 1H T2-T2 molecular exchange maps. The results were represented by T2-T2 molecular exchange maps that showed, comparatively, both pore size and their interconnectivity at the level of the femoral epiphysis, being able to evaluate both the effect of estrogen on bone tissue biology and the effect of the lipid-lowering medication, simvastatin, and fenofibrate, in both the presence and absence of estrogen. T2-T2 molecular exchange maps showed that the absence of estrogen results in an increase in bone tissue pore size and interconnectivity. In the presence of estrogen, lipid-lowering medication, both simvastatin and fenofibrate alter bone tissue cytoarchitectonics by reducing pore interconnectivity. In the absence of estrogen, fenofibrate improves bone tissue cytoarchitectonics, the T2-T2 molecular exchange map being similar to that of non-osteoporotic bone tissue.

Evaluation of the Influence of Hypolipidemic Medication on Albino Wistar Rats' Bone Tissue by NMR Diffusiometry.

Introduction: The ongoing concern of the medical profession regarding chronic medication is related to increasing patient adherence and compliance to treatment and reducing medication side effects. In this respect, drugs represented by fixed-dose combinations of active substances within the same tablet have emerged. Such a principle can be extrapolated by following the potential beneficial effects that a chronic medication can have on chronic pathologies affecting different systems. Materials and Methods: The study included 48 female Albino Wistar rats, aged 16-18 months, which were divided into two groups: ovariectomized and non-ovariectomized rats. One batch of 12 non-ovariectomized rats received no treatment, becoming a control batch (NOVX-M). The ovariectomized (OVX) group was divided into 3 batches of 12 rats each: no treatment, control (OVX-M), fenofibrate-treated (OVX-F) and statin-treated (OVX-S) rats. At 12 weeks after ovariectomy, a femoral fracture occurred in the right hind limb of all animals included in the experiment To reveal the changes, at intervals of 2, 4, 6 and 8 weeks post-fracture, the proximal part of the femur was evaluated by NMR diffusiometry, which allows random motion of proton molecules expressed by self-diffusion coefficients, D, thus allowing analysis of the size and complexity of microscopic order cavities within biological structures, such as pores inside bones. Results: The effects of hypolipidemic medication in the absence of estrogen were evidenced, proving the beneficial effect that fenofibrate can have in preserving healthy tissue exposed to osteoporotic risk during the menopausal period. The effects of lipid-lowering medication are also influenced by the duration of administration. Conclusions: Osteoporosis and heart disease are two chronic pathologies that affect mainly female population in the second half of life, and proving the dual therapeutic potential of lipid-lowering medication may also have positive effects by increasing adherence and compliance to treatment.

Callus formation in albino Wistar rats after femur fracture assessed by visible spectroscopy.

Classical histological methods such as hematoxylin-eosin staining, have been, and in some areas still are, an important benchmark for the evaluation of biological tissues. However, the current method of assessment is primarily a qualitative assessment of the tissue under investigation. The aim of this paper is to contribute to the improvement of classical histological methods, by applying physical techniques that allow objective, quantitative data to be added to qualitative assessments, especially in areas where conflicting results are available. To this end, the effect of hypolipidemic medication on the callus formation process of normal bone and pathological osteoporotic bone was investigated. The study allowed us to associate UV-VIS spectroscopy wave number with specific hematoxylin-eosin staining of different types of bone tissue structures, the evolving structures in the callus formation process. This association allowed the quantitative assessment of the callusing process in ovariectomized (associated with pathological, osteoporotic bone) and non-ovariectomized (associated with normal bone) rats, with three groups - the control group, simvastatin-treated group, and fenofibrate-treated group. The study showed that in the non-ovariectomized groups both treatments delayed callus formation. In the ovariectomized groups, simvastatin delayed and fenofibrate promoted callus formation.

Stimuli-responsive microgels with cationic moieties: characterization and interaction with E. coli cells.

Stimuli-responsive microgel copolymer networks with ionizable functional groups have important applications for encapsulation of drugs, peptides, enzymes, proteins, or cells. Rational design of such networks can be based on characterization of stimuli-induced volume phase transition and spatial distribution of neutral and charged monomer units in crosslinked polymer chains. In this work we successfully synthesized poly(N-vinylcaprolactam-co-1-vinyl-3-methylimidazolium) (poly(VCL-VIM+)) microgels carrying permanent positive charges and demonstrate that 1H high-resolution NMR spectroscopy in combination with transverse (T2) magnetization relaxometry allows investigating separately the behavior of each functional group in the microgel network. The information about comonomer transition temperatures, width of transition, and change in transition entropy were reported and correlated with the concentration of charged functional groups and resulting electrophoretic mobility. A two-state approach was used to describe the temperature-induced volume phase transition separately for neutral and charged polymer segments. The core-corona architecture specific to each functional group was detected revealing that the charged methylated vinylimidazolium groups (VIM+) are concentrated mainly in the corona of the microgel. These biocompatible PVCL-based microgels functionalized with permanent positive charges are shown to serve as an antibacterial system against Gram-negative E. coli strains, due to the positive charge of the incorporated VIM+ comonomer in the polymer network.

Poly(vinylamine-co-N-isopropylacrylamide) linear polymer and hydrogels with tuned thermoresponsivity.

The fabrication of functional hydrogels with tuned thermoresponsivity is a major challenge. To meet this challenge we copolymerize N-isopropylacrylamide (NIPAm) with N-vinylformamide (NVF) in different ratios with the formamide group being subsequently selectively hydrolyzed to the corresponding amine (VAm). The copolymers are crosslinked with phenylcarbonate telechelic glycol. The influence of the NIPAm : VAm ratio on the thermoresponsitiviy is investigated in terms of absorbance, rheology, NMR spectroscopy, relaxometry, and diffusometry. Phase transition temperatures, change in the entropy of the polymer-water system, and width of the transition in the process of coil-to-globule and swollen-to-collapsed network transitions were evaluated by a two state model and Boltzmann sigmoidal function.

Functional selenium modified microgels: temperature-induced phase transitions and network morphology.

Microgels that host selenium and mimic the structure of the enzyme glutathione peroxidase are of great interest for biotechnological and catalytic applications. For this purpose selenium-functionalized thermoresponsive poly(N-vinylcaprolactam) (PVCL) microgels with cleavable diselenide crosslinks have been investigated. Thermodynamic and morphological parameters characterizing the temperature-induced phase transitions of dual crosslinked PVCL microgels were obtained using dynamic light scattering (DLS), 1H high-resolution magic-angle sample-spinning (MAS) NMR spectroscopy, and transverse magnetization (T2) NMR relaxometry. Quantities obtained from Flory-Rehner theory, a two-state model and Boltzmann sigmoidal function were used to relate the phase transitions of the dual crosslinked microgels to the transition temperature, entropy, temperature width of the phase transition, Flory interaction parameters, average number of strands, polymer volume fraction of the collapsed microgels, core-corona fractions and chain dynamics. The morphology of the selenium modified microgels after the oxidation and reduction processes was investigated by 1H T2 NMR and further correlated with the crosslink density.

Diffusion of Gold Nanorods Functionalized with Thermoresponsive Polymer Brushes.

Understanding the diffusion of gold nanorods (AuNRs) and their composites in dispersion is important at fundamental level and in fields as diverse as material science, nanobiotechnology to drug delivery. The translational and rotational diffusion of AuNRs decorated with thermoresponsive poly( N-isopropylacrylamide) brushes having hydrophilic and hydrophobic end groups was investigated in the dilute regime by dynamic light scattering. The same series of functionalized AuNRs were studied in the isotropic concentrated dispersions by high-resolution NMR diffusometry. The dependence of translational and rotational diffusivity upon molecular weight and polymer end group were measured as a function of temperature in the region of the brush phase transition. The effective hydrodynamic radius of AuNR composites proved to be the most sensitive quantity to the temperature-induced phase transition of brushes, allowing the evaluation of the brush thickness in the swollen and collapsed states.

Peptizing Mechanism at the Molecular Level of Laponite Nanoclay Gels.

In the presence of additives such as etidronic acid (1-hydroxyethane-1,1-diphosphonic acid, HEDP), a process of peptizing of Laponite clay gels takes place. The peptizing process at the molecular level was directly revealed by 31P and 1H high-resolution magic-angle sample spinning (HRMAS) NMR spectroscopy. Two NMR spectral components were detected and assigned to free etidronic acid and bound to the Laponite disk edges. Furthermore, with increase of temperature the ratio of bound-to-free etidronic acid increases. This thermal activation process could be explained by the increase in electrical polarization of the hydroxyl group at the edges and by the exfoliation of the tactoids that leads to more access to the additive molecules to the electrical charges of platelet edges. 31P HRNMR spectroscopy on sodium fluorohectorite with an aspect ratio of ∼750 shows a reduction of the bound etidronic acid molecules. Transmission electron microscopy (TEM), field-emission scanning microscopy (FESEM), UV-vis spectrophotometry, dynamic light scattering (DLS), and zeta potential results support the proposed peptizing mechanisms.

Thermodynamic Parameters of Temperature-Induced Phase Transition for Brushes onto Nanoparticles: Hydrophilic versus Hydrophobic End-Groups Functionalization.

Quantification of the stimuli-responsive phase transition in polymers is topical and important for the understanding and development of novel stimuli-responsive materials. The temperature-induced phase transition of poly(N-isopropylacrylamide) (PNIPAm) with one thiol end group depends on the confinement-free polymer or polymer brush-on the molecular weight and on the nature of the second end. This paper describes the synthesis of heterotelechelic PNIPAm of different molecular weights with a thiol end group-that specifically binds to gold nanorods and a hydrophilic NIPAm end group by reversible addition-fragmentation chain-transfer polymerization. Proton high-resolution magic angle sample spinning NMR spectra are used as an indicator of the polymer chain conformations. The characteristics of phase transition given by the transition temperature, entropy, and width of transition are obtained by a two-state model. The dependence of thermodynamic parameters on molecular weight is compared for hydrophilic and hydrophobic end functional-free polymers and brushes.

Aggregation behaviour of biohybrid microgels from elastin-like recombinamers.

Investigation of the aggregation behavior of biohybrid microgels, which can potentially be used as drug carriers, is an important topic, because aggregation not only causes loss of activity, but also toxicity and immunogenicity. To study this effect we synthesized microgels from elastin-like recombinamers (ELRs) using the miniemulsion technique. The existence of aggregation for such biohybrid microgels at different concentrations and temperatures was studied by different methods which include dynamic light scattering (DLS), (1)H high-resolution magic angle sample spinning (HRMAS) NMR spectroscopy, relaxometry and diffusometry. A hysteresis effect was detected in the process of aggregation by DLS as a function of temperature that strongly depends on ELR microgel concentration. The aggregation process was further quantitatively analyzed by the concentration dependence of the (1)H amino-acid residue chemical shifts and microgel diffusivity measured by NMR methods using the population balance kinetic aggregation model.

Coacervation of Elastin-Like Recombinamer Microgels.

The investigation of the coacervation (self-aggregation) behavior of biomicrogels which can potentially be used as drug carriers is an important topic, because self-aggregation can not only cause loss of activity, but also toxicity and immunogenicity. To study this effect microgels from elastin-like recombinamer are synthesized using miniemulsion technique. The existence of coacervation for such microgels, at different concentrations and temperatures, is studied and proved by cryo-field emission scanning clectron microscopy (cryo-FESEM), cryo-transmission electron microscopy (cryo-TEM), and by a novel (1) H high-resolution magic angle sample spinning (HRMAS), nuclear magnetic resonance (NMR) spectroscopy, and relaxometry methods. The findings by (1) H HRMAS NMR spectroscopy and relaxometry show simultaneous processes of volume phase temperature transition and coacervation with different sensitivity for hydrophobic and hydrophilic amino acid side-chains in the microgel. The coacervation process is more evidential by the behavior of glycine α-CH2 , (1) H NMR peak as compared to the proline ß-CH2 .

The Influence of Lyophilization Pretreatment and Whey Content on Whey and Gelatin-Based Hydrogels.

Whey and gelatin, natural polymers within the protein category, find widespread use in hydrogel formulations applied across the food, medical, and pharmaceutical industries. This study presents new characteristics of hydrogels based on whey, gelatin, and copper sulfate as a consequence of the additional steps in the preparation method, specifically refrigeration and freezing storage followed by lyophilization. The water state in hydrogels prior to lyophilization impacts the morphological appearance, with refrigerated hydrogels exhibiting a more regular and dense pore distribution, as shown by the Scanning Electron Microscopy (SEM) images. This observation aligns with the higher mobility of polymer chains indicated by T2 distributions in 1H nuclear magnetic resonance (RMN) relaxometry measurements. Changes in the intensity and amide-specific wavenumbers of the FTIR spectra of whey and gelatin proteins are evident in the Fourier Transformed Infrared (FTIR) spectra of crosslinked and frozen hydrogels before lyophilization. Moreover, the reinforcing effect in the hydrogel matrix, noted in mechanical tests, is attributed to increased polymer chain content and copper sulfate crosslinking.

Characterization of a Graphene Oxide-Reinforced Whey Hydrogel as an Eco-Friendly Absorbent for Food Packaging.

This study presents a structural analysis of a whey and gelatin-based hydrogel reinforced with graphene oxide (GO) by ultraviolet and visible (UV-VIS) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD). The results revealed barrier properties in the UV range for the reference sample (containing no graphene oxide) and the samples with minimal GO content of 0.66×10-3% and 3.33×10-3%, respectively, in the UV-VIS and near-IR range; for the samples with higher GO content, this was 6.67×10-3% and 33.33×10-3% as an effect of the introduction of GO into the hydrogel composite. The changes in the position of diffraction angles 2θ from the X-ray diffraction patterns of GO-reinforced hydrogels indicated a decrease in the distances between the turns of the protein helix structure due to the GO cross-linking effect. Transmission electron spectroscopy (TEM) was used for GO, whilst scanning electron microscopy (SEM) was used for the composite characterization. A novel technique for investigating the swelling rate was presented by performing electrical conductivity measurements, the results of which led to the identification of a potential hydrogel with sensor properties.

New Composite Hydrogel Based on Whey and Gelatin Crosslinked with Copper Sulphate.

By-products from the meat and dairy industries are important sources of high biological value proteins. This paper explores possibilities for improving the swelling and integrity of a cross-linked whey and gelatin hydrogel with different amounts of CuSO4 × 5H2O. Overall, swelling tests demonstrate that cross-linked samples show a better hydration capacity and stability in the hydration medium, but different copper concentrations lead to different swelling behavior. At concentrations smaller than 0.39%, the sample lasts for 75 h in a water environment before beginning to disintegrate. At a concentration of copper sulphate higher than 0.55%, the stability of the sample increased substantially. The swelling kinetics has been investigated. The diffusion constant values increased with the increase in copper concentration, but, at the highest concentration of copper (0.86%), its value has decreased. Spectroscopy analyses such as Fourier transform infrared (FT-IR), X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-VIS), and nuclear magnetic resonance (NMR) relaxometry analyses revealed changes in the secondary and tertiary structure of proteins as a result of the interaction of Cu2+ ions with functional groups of protein chains. In addition to its cross-linking ability, CuSO4 × 5H2O has also shown excellent antibacterial properties over common bacterial strains responsible for food spoilage. The result of this research demonstrates the potential of this hydrogel system as a unique material for food packaging.

Swelling and Antimicrobial Activity Characterization of a GO-Reinforced Gelatin-Whey Hydrogel.

Whey-based hydrogel samples with increasing concentrations of graphene oxide (GO) were studied, against a control sample (M), for swelling behavior in light of nuclear magnetic resonance (NMR) and mathematical models of the diffusion process and for antibacterial activity. Graphene oxide (GO) is an optimal filler for whey-based hydrogels, giving them improved mechanical and swelling properties at low concentrations. Crosslinking induces a certain stiffness of the hydrogels, which is why only the first part of the swelling process (<60%) follows the first-order model, while during the whole time interval, the swelling process follows the second-order diffusion model. The NMR relaxometry results are consistent with the swelling behavior of GO-reinforced whey−gelatin composite hydrogels, showing that higher GO concentrations induce a higher degree of cross-linking and, therefore, lower swelling capacity. Only hydrogel samples with higher GO concentrations demonstrated antibacterial activity.

Predicting the Early Response to Neoadjuvant Therapy with Breast MR Morphological, Functional and Relaxometry Features-A Pilot Study.

Aim: To evaluate the role of MR relaxometry and derived proton density analysis in the prediction of early treatment response after two cycles of neoadjuvant therapy (NAT), in patients with breast cancer. Methods: This was a prospective study that included 59 patients with breast cancer, who underwent breast MRI prior (MRI1) and after two cycles of NAT (MRI2). The MRI1 included a sequential acquisition with five different TE's (50, 100, 150, 200 and 250 ms) and a TR of 5000 ms. Post-processing was used to obtain the T2 relaxometry map from the MR acquisition. The tumor was delineated and seven relaxometry and proton density parameters were extracted. Additional histopathology data, T2 features and ADC were included. The response to NAT was reported based on the MRI2 as responders: partial response (>30% decreased size) and complete response (no visible tumor stable disease (SD); and non-responders: stable disease or progression (>20% increased size). Statistics was done using Medcalc software. Results: There were 50 (79.3%) patients with response and 13 (20.7%) non-responders to NAT. Age, histologic type, "in situ" component, tumor grade, estrogen and progesterone receptors, ki67% proliferation index and HER2 status were not associated with NAT response (all p > 0.05). The nodal status (N) 0 was associated with early response, while N2 was associated with non-response (p = 0.005). The tumor (T) and metastatic (M) stage were not statistically significant associated with response (p > 0.05). The margins, size and ADC values were not associated with NAT response (p-value > 0.05). The T2 min relaxometry value was associated with response (p = 0.017); a cut-off value of 53.58 obtained 86% sensitivity (95% CI 73.3−94.2), 69.23 specificity (95% CI 38.6−90.9), with an AUC = 0.715 (p = 0.038). The combined model (T2 min and N stage) achieved an AUC of 0.826 [95% CI: 0.66−0.90, p-value < 0.001]. Conclusions: MR relaxometry may be a useful tool in predicting early treatment response to NAT in breast cancer patients.

Bimodal 1H Double Quantum Build-Up Curves by Fourier and Laplace-like Transforms on Aged Cross-Linked Natural Rubber.

The 1H DQ Fourier and Laplace-like spectra for a series of cross-linked natural rubber (NR) samples naturally aged during six years are presented and characterized. The DQ build-up curves of these samples present two peaks which cannot be described by classical functions. The DQ Fourier spectra can be obtained after a numeric procedure which introduces a correction time which depends less on the chosen approximation, spin-½ and isolated CH2 and CH3 functional groups. The DQ Fourier spectra are well described by the distributions of the residual dipolar coupling correlated with the distribution of the end-to-end vector of the polymer network, and with the second and fourth van Vleck moments. The deconvolution of DQ Fourier spectra with a sum of four Gaussian variates show that the center and the width of Gaussian functions increase linearly with the increase in the cross-link density. The Laplace-like spectra for the natural aged NR DQ build-up curves are presented. The centers of four Gaussian distributions obtained via both methods are consistent. The differences between the Fourier and Laplace-like spectra consist mainly of the spectral resolution in the favor of Laplace-like spectra. The last one was used to discuss the effect of natural aging for cross-linked NR.

Fourier and Laplace-like low-field NMR spectroscopy: The perspectives of multivariate and artificial neural networks analyses.

Low field Nuclear Magnetic Resonance (LF-NMR) is a rich source of information for a wide range of samples types. These can be hard or soft solids, such as plastics or elastomers; bulk liquids or liquids absorbed in porous materials, and can come from biomaterials, biological tissues, archaeological artifacts, cultural heritage objects. LF-NMR instruments present a significant advance especially for in situ, ex situ and in vivo measurement of relaxation and diffusion. Moreover, high resolution 1D and 2D spectroscopy, as well as magnetic resonance (MR) imaging are available in these fields. In this work we discuss the advanced analysis of the data measured in LF-NMR from the perspectives of tertiary level that implies the analysis on principal components (PCA), and on the quaternary analysis that uses an artificial neural network (ANN). The principles of PCA and ANN are largely discussed. For the PCA analysis, a series of 52 spectra were analyzed, having been recorded in vivo by LF-NMR. Of these spectra, 38 were generated from normal uterus, 7 by uterus tissue with endometrial cancer, and another 7 were obtained from tissues of women with uterine cervical cancer. The PC1 vs PC2 plot was further analyzed using an artificial neural network, and the results are presented as 2D maps of probability. Furthermore, the perspectives of applying an ANN to solve the problem of Laplace-like inversion are discussed. An example of such ANN was presented and the performance was discussed. Finally, a model of complex ANN, capable to sequentially solve this kind of problems specific to LF-NMR is proposed and discussed.

Visualization of the shear wave on gray-scale ultrasound: experience with a complex breast cyst.

Shear wave elastography (SWE) is the newer of the two methods of measuring tissue stiffness using ultrasound. We present the case of a 39-year-old patient with fibrocystic breast disease who presented with a left painful breast lump. The patient underwent a breast ultrasound that depicted a complicated cyst with a "pseudo-solid" content. When the SWE mode was turned on, the ultrasound share wave was observed on the gray-scale image, as surface undulations of the "pseudo-solid" component. To the best of our knowledge, this is the first report of the share wave visualization on a gray-scale image. The impact on patient management is discussed.

Thermal denaturation of hydrated wool keratin by 1H solid-state NMR.

Thermal denaturation of hydrated keratin in wool was investigated by NMR using 1H wide-line spectra to obtain the phase composition and 1H spin-diffusion experiments using a double-quantum filter to obtain the domain sizes for the wool fibers. The denaturation process detected by DSC takes place for wool fibers in deuterated water in the temperature range 140-144 degreeC. The phase composition measured by 1H wide line NMR spectra reveals a rigid, semirigid and an amorphous phase for temperatures in the range 25-160 degreeC. A dramatic change in the phase composition was detected around 142 degreeC, corresponding to the denaturation temperature. The morphological domain sizes measured by 1H spin-diffusion NMR experiments were obtain from the solutions of the spin-diffusion equations for two-dimensional rectangular and cylindrical morphologies. The keratin mobility gradient in the interfacial region at different denaturation temperatures was measured from the 1H spin-diffusion data. A qualitative model describing the denaturation process of hydrated keratin protein was developed that explains the changes in domain thickness, spin diffusivities, phase composition, and thermodynamic parameters.