Physicochemical, structural and biological characterisation of poly(3-hydroxyoctanoate) supplemented with diclofenac acid conjugates - Harnessing the potential in the construction of materials for skin regeneration processes.

This study involved creating oligomeric conjugates of 3-hydroxy fatty acids and diclofenac, named Dic-oligo(3HAs). Advanced NMR techniques confirmed no free diclofenac in the mix. We tested diclofenac release under conditions resembling healthy and chronic wound skin. These oligomers were used to make P(3HO) blends, forming patches for drug delivery. Their preparation used the solvent casting/porogen leaching (SCPL) method. The patches' properties like porosity, roughness, and wettability were thoroughly analysed. Antimicrobial assays showed that Dic-oligo(3HAs) exhibited antimicrobial activity against reference (S. aureus, S. epidermis, S. faecalis) and clinical (Staphylococcus spp.) strains. Human keratinocytes (HaCaT) cell line tests, as per ISO 10993-5, showed no toxicity. A clear link between material roughness and HaCaT cell adhesion was found. Deep cell infiltration was verified using DAPI and phalloidin staining, observed under confocal microscopy. SEM also confirmed HaCaT cell growth on these scaffolds. The strong adhesion and proliferation of HaCaT cells on these materials indicate their potential as wound dressing layers. Additionally, the successful diclofenac release tests point to their applicability in treating both normal and chronic wounds.

A morphological study and the variability in the number of infraorbital foramina in the African green monkey (Grivet) (Chlorocebus aethiops) using microcomputed tomography.

Knowledge of the nonhuman primate morphology and anatomy related to craniofacial mechanoreception is essential for a fundamental understanding of the incidents that have occurred during the evolution of craniofacial features. The present study focuses on the variability in the number of infraorbital foramina and associated anatomical structures such as the infraorbital canal (IOC) and the infraorbital groove (IOG), as they are considered to play an important role in the behavioral ecology of these animals. A total of 19 skulls of Chlorocebus aethiops were analyzed. The number of infraorbital foramina was assessed macroscopically using a magnifying glass and a small diameter probe. Three dimensional (3D) projections and morphometric analysis of the infraorbital foramina, IOCs, and IOGs were performed using microcomputed tomography (micro-CT) for two skulls that represent one of the most common morphological types. Regardless of sex and body side, the most common morphological type observed in the studied species is the presence of three infraorbital foramina. The IOC takes a funnel or pinched shape. 3D projections were made to assess the course of the infraorbital vascular and nerve bundles in selected individuals. The results indicate a high morphological diversity within the species, although there appears to be a consistent distribution pattern of infraorbital neurovascular bundles in species of the Cercopithecidae family. The use of X-ray micro-CT allowed 3D visualization of the maxillary region to determine the variability of the infraorbital foramina and to track the division of the infraorbital neurovascular bundle in the case of the most common macroscopic expression of the number of the infraorbital foramen in C. aethiops, as well as the morphometric of the IOCs and IOGs which are related to mechanoreception of the primate's snout.

Developing a novel positronium biomarker for cardiac myxoma imaging.

PURPOSE: Cardiac myxoma (CM), the most common cardiac tumor in adults, accounts for 50-75% of benign cardiac tumors. The diagnosis of CM is often elusive, especially in young stroke survivors and transthoracic echocardiography (TTE) is the initial technique for the differential diagnostics of CM. Less invasive cardiac computed tomography (CT) and magnetic resonance imaging (MRI) are not available for the majority of cardiac patients. Here, a robust imaging approach, ortho-Positronium (o-Ps) imaging, is presented to determine cardiac myxoma extracted from patients undergoing urgent cardiac surgery due to unexpected atrial masses. We aimed to assess if the o-Ps atom, produced copiously in intramolecular voids during the PET imaging, serves as a biomarker for CM diagnosing. METHODS: Six perioperative CM and normal (adipose) tissue samples from patients, with primary diagnosis confirmed by the histopathology examination, were examined using positron annihilation lifetime spectroscopy (PALS) and micro-CT. Additionally, cell cultures and confocal microscopy techniques were used to picture cell morphology and origin. RESULTS: We observed significant shortening in the mean o-Ps lifetime in tumor with compare to normal tissues: an average value of 1.92(02) ns and 2.72(05) ns for CM and the adipose tissue, respectively. Microscopic differences between tumor samples, confirmed in histopathology examination and micro-CT, did not influenced the major positronium imaging results. CONCLUSIONS: Our findings, combined with o-Ps lifetime analysis, revealed the novel emerging positronium imaging marker (o-PS) for cardiovascular imaging. This method opens the new perspective to facilitate the quantitative in vivo assessment of intracardiac masses on a molecular (nanoscale) level.

Placental vascular alterations are associated with early neurodevelopmental and pulmonary impairment in the rabbit fetal growth restriction model.

Fetal growth restriction is one of the leading causes of perinatal mortality and morbidity and has consequences that extend well beyond the neonatal period. Current management relies on timely delivery rather than improving placental function. Several prenatal strategies have failed to show benefit in clinical trials after promising results in animal models. Most of these animal models have important developmental and structural differences compared to the human and/or are insufficiently characterized. We aimed to describe placental function and structure in an FGR rabbit model, and to characterize the early brain and lung developmental morbidity using a multimodal approach. FGR was induced in time-mated rabbits at gestational day 25 by partial uteroplacental vessel ligation in one horn. Umbilical artery Doppler was measured before caesarean delivery at gestational day 30, and placentas were harvested for computed microtomography and histology. Neonates underwent neurobehavioral or pulmonary functional assessment the day after delivery, followed by brain or lung harvesting, respectively. Neuropathological assessment included multiregional quantification of neuron density, apoptosis, astrogliosis, cellular proliferation, and oligodendrocyte progenitors. Brain region volumes and diffusion metrics were obtained from ex-vivo brain magnetic resonance imaging. Lung assessment included biomechanical tests and pulmonary histology. Fetal growth restriction was associated with labyrinth alterations in the placenta, driven by fetal capillary reduction, and overall reduced vessels volume. FGR caused altered neurobehavior paralleled by regional neuropathological deficits and reduced fractional anisotropy in the cortex, white matter, and hippocampus. In addition, FGR kittens presented functional alterations in the peripheral lung and structurally underdeveloped alveoli. In conclusion, in a uteroplacental insufficiency FGR rabbit model, placental vascular alterations coincide with neurodevelopmental and pulmonary disruption.

Micro-computed tomography for analysis of heavy metal accumulation in the opercula.

Micro-computed tomography (micro-CT) provides numerous opportunities in biomedical research. It allows the examination of samples in a non-destructive manner and visualization of the inner structures of various biological and nonbiological objects. This study was conducted to evaluate the potential of micro-CT scanner in the assessment of heavy metal accumulation in the opercula. The samples were taken from Prussian carp (Carassius gibelio) exposed to waterborne Cd (4.0 mg/L), Zn (4.0 mg/L), and the mixture of these two metals (4.0 mg Cd/L and 4.0 mg Zn/L) for 28 days. Heavy metal concentrations were determined using atomic absorption spectrometry. The results demonstrated higher concentrations of Cd and Zn in the treatment group opercula samples compared with the control group opercula samples. A simple micro-CT scan was performed to verify whether heavy metal accumulation could be determined in the reconstructed images. The results showed that micro-CT is potentially a powerful tool for metal accumulation detection. Moreover, it allowed visualization of the examined samples, revealing regions of heavy metal accumulation and providing the opportunity to compare samples exposed to different types of heavy metals.

Rehydration of the sleeping chironomid, Polypedilum vanderplanki Hinton, 1951 larvae from cryptobiotic state up to full physiological hydration (Diptera: Chironomidae).

During desiccation the Polypedilum vanderplanki larva loses 97% of its body water, resulting in the shutdown of all metabolic and physiological processes. The larvae are able to resume active life when rehydrated. As dehydration process has already been largely understood, rehydration mechanisms are still poorly recognized. X-ray microtomograms and electron scanning microscopy images recorded during the hydration showed that the volume of the larva's head hardly changes, while the remaining parts of the body increase in volume. In the 1H-NMR spectrum, as recorded for active larvae, component characteristic of solid state matter is absent. The spectrum is superposition of components coming from tightly and loosely bound water fraction, as well as from lipids. The value of the c coefficient (0.66 ± 0.02) of the allometric function describing the hydration models means that the increase in the volume of rehydrated larvae over time is linear. The initial phase of hydration does not depend on the chemical composition of water, but the amount of ions affects the further process and the rate of return of larva's to active life. Diffusion and ion channels play a major role in the permeability of water through the larva's body integument.

Variability in the number of infraorbital foramina in rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis).

This study aimed to determine the number of infraorbital foramina in monkeys of the Papionini tribe. The authors performed a µCT analysis of the morphology of the infraorbital foramina. A total number of 52 simian skulls belonged to two macaque species: Macaca mulatta and Macaca fascicularis were used in the study. The number of infraorbital foramina was counted macroscopically and with the use of a magnifying glass. Next, the skull representing the most common morphological type was selected and scanned by micro-computed tomography (µCT). The Shapiro-Wilk normality test was used in the study. To compare the differences in the number of infraorbital foramen between species, sex and sides, the Mann-Whitney U test was applied. Three infraorbital foramina were present in most individuals from the test group. The Mann-Whitney test revealed no statistically significant difference between the number of foramina on the right- and left-hand side. Likewise, no statistically significant differences between the numbers of infraorbital foramina across sexes were observed. Volumetric reconstructions revealed the presence of separate infraorbital canals for each infraorbital foramen. Craniofacial innervation in macaques is formed by complex branching patterns of cranial nerves. Variability in the number of infraorbital foramina suggests a variable maxillary innervation pattern in these animals. Based on the analysis of volumetric projections, the presence of two labial branches and a single nasal branch of the infraorbital nerve is suggested. Detailed descriptions are supported by quantitative data and µCT evidence.

Morphology and serum and bone tissue calcium and magnesium concentrations in the bones of male rats chronically treated with letrozole, a nonsteroidal cytochrome P450 aromatase inhibitor.

Purpose/Aim of the study: The role of estrogen (E) in the regulation of bone turnover in women is well established, though the contributions of E versus testosterone (T) in the control of bone turnover in men are poorly understood. The aim of this study was to examine the association between chronic treatment with letrozole, a nonsteroidal inhibitor blocking the aromatase activity and thus the conversion of androgens into estrogens, and cortical bone morphology in the femur and humerus of male adult rats.Materials and Methods: Adult male rats were treated with letrozole for 6 months and the body and femur weight, morphology, collagen structure, blood serum, and bone tissue concentrations of calcium and magnesium were examined.Results: Long-term aromatase inhibition resulted in a decrease in femur mass, a wavelike arrangement of bone and lamellae with an altered organization of collagen in compact bone, a increased concentration of calcium in blood serum, and no change in calcium bone tissue concentration, magnesium serum, or bone tissue concentration. MicroCT study of the humerus revealed significant decreases of whole bone tissue volume, cortical bone thickness, cortical bone volume, and external cortical bone thickness with letrozole treatment.Conclusion: Chronic treatment with letrozole affected cortical bone structure and produced histomorphological changes in male rat bone similar to that observed in the aging processes.

Multivariate Design of 3D Printed Immediate-Release Tablets with Liquid Crystal-Forming Drug-Itraconazole.

The simplicity of object shape and composition modification make additive manufacturing a great option for customized dosage form production. To achieve this goal, the correlation between structural and functional attributes of the printed objects needs to be analyzed. So far, it has not been deeply investigated in 3D printing-related papers. The aim of our study was to modify the functionalities of printed tablets containing liquid crystal-forming drug itraconazole by introducing polyvinylpyrrolidone-based polymers into the filament-forming matrices composed predominantly of poly(vinyl alcohol). The effect of the molecular reorganization of the drug and improved tablets' disintegration was analyzed in terms of itraconazole dissolution. Micro-computed tomography was applied to analyze how the design of a printed object (in this case, a degree of an infill) affects its reproducibility during printing. It was also used to analyze the structure of the printed dosage forms. The results indicated that the improved disintegration obtained due to the use of Kollidon®CL-M was more beneficial for the dissolution of itraconazole than the molecular rearrangement and liquid crystal phase transitions. The lower infill density favored faster dissolution of the drug from printed tablets. However, it negatively affected the reproducibility of the 3D printed object.

From Mouse to Man and Back: Closing the Correlation Gap between Imaging and Histopathology for Lung Diseases.

Lung diseases such as fibrosis, asthma, cystic fibrosis, infection and cancer are life-threatening conditions that slowly deteriorate quality of life and for which our diagnostic power is high, but our knowledge on etiology and/or effective treatment options still contains important gaps. In the context of day-to-day practice, clinical and preclinical studies, clinicians and basic researchers team up and continuously strive to increase insights into lung disease progression, diagnostic and treatment options. To unravel disease processes and to test novel therapeutic approaches, investigators typically rely on end-stage procedures such as serum analysis, cyto-/chemokine profiles and selective tissue histology from animal models. These techniques are useful but provide only a snapshot of disease processes that are essentially dynamic in time and space. Technology allowing evaluation of live animals repeatedly is indispensable to gain a better insight into the dynamics of lung disease progression and treatment effects. Computed tomography (CT) is a clinical diagnostic imaging technique that can have enormous benefits in a research context too. Yet, the implementation of imaging techniques in laboratories lags behind. In this review we want to showcase the integrated approaches and novel developments in imaging, lung functional testing and pathological techniques that are used to assess, diagnose, quantify and treat lung disease and that may be employed in research on patients and animals. Imaging approaches result in often novel anatomical and functional biomarkers, resulting in many advantages, such as better insight in disease progression and a reduction in the numbers of animals necessary. We here showcase integrated assessment of lung disease with imaging and histopathological technologies, applied to the example of lung fibrosis. Better integration of clinical and preclinical imaging technologies with pathology will ultimately result in improved clinical translation of (therapy) study results.

X-ray microtomography as a new approach for imaging and analysis of tumor spheroids.

Three-dimensional (3D) spheroids mimic important properties of tumors and may soon become a reasonable substitute for animal models and human tissue, eliminating numerous problems related to in vivo and ex vivo experiments and pre-clinical drug trials. Currently, various imaging methods including X-ray microtomography (micro-CT), exist but their spatial resolution is limited. Here, we visualized and provided a morphological analysis of spheroid cell cultures using micro-CT and compared it to that of confocal microscopy. An approach is proposed that can potentially open new diagnostic opportunities to determine the morphology of cancer cells cultured in 3D structures instead of using actual tumors. Spheroids were formed from human melanoma cell lines WM266-4 and WM115 seeded at different cell densities using the hanging drop method. Micro-CT analysis of spheroid showed that spheroid size and shape differed depending on the cell line, initial cell number, and duration of culture. The melanoma cell lines used in this study can successfully be cultured as 3D spheroids and used to substitute human and animal models in pre-clinical studies. The micro-CT allows for high-resolution visualization of the spheroids structure.

Speed it up, slow it down An issue of bicalutamide release from 3D printed tablets.

The article describes the preparation and characterization of 3D-printed tablets with bicalutamide obtained using two-material co-extrusion-based fused deposition modeling (FDM). This method is a modification of typical two-material FDM where separate nozzles are used to print from two filaments. In this work we used a ZMorph® 3D printer with DualPro printhead which allows us to co-extrude two filaments through a single nozzle. This approach gives the opportunity to modify tablet properties in a wide range, especially the dissolution rate, by producing dosage forms with a complex design. The great advantage of this method is that switching between immediate dosage form and controlled release does not require any change in the 3D-printer set-up. We checked the accuracy of co-extrusion printing simply by weighing the amounts of soluble and insoluble material in the printed object as well as calculating the volumes of the printed objects from micro computed tomography (µ-CT) images. We printed several tablets with a different design including simple one-material tablets, two- and three-compartment tablets with various internal structure and composition of the printing path. The dissolution tests were conducted in sink and non-sink conditions. We obtained tablets with desired bicalutamide dissolution profiles, i.e. immediate, controlled, and combined. The formation of spatial matrix slows down the dissolution in controlled and combined release bicalutamide tablets what was confirmed by µ-CT analysis before and after dissolution.

Novel bioresorbable tricalcium phosphate/polyhydroxyoctanoate (TCP/PHO) composites as scaffolds for bone tissue engineering applications.

Development of new composite materials for bone tissue engineering is a constantly growing field of medicine. Therefore there is a continuous need in creating novel materials that can not only regenerate the defected tissue but also nourish it while the healing process progresses. Here we present a novel type of composite material that fulfils these requirements. The study describes creation of a composite with macroporous bioceramic core that is infiltrated with a thin biopolymer layer. The ceramic component, namely tricalcium phosphate (TCP), due to its mechanistic and bioactive properties may promote new bone creation as shown through the in vitro studies. To the best of our knowledge the coating layer was created for the first time from a representative of bacterially derived medium chain length polyhydroxyalkanoate polymers (mcl-PHA), namely polyhydroxyoctanoate (PHO). This polymer layer not only profoundly changed the stress-strain characteristics of the bioceramic foam but also released (R)-3-hydroxyacids and their dimers/trimers to the investigated environment. In the manuscript we have in depth characterised these materials employing a set of basic procedures, through 3D structure reconstruction and finishing with prolonged in vitro experiments.

Oval Window Size and Shape: a Micro-CT Anatomical Study With Considerations for Stapes Surgery.

BACKGROUND: The oval window is an important structure with regard to stapes surgeries, including stapedotomy for the treatment of otosclerosis. Recent study of perioperative imaging of the oval window has revealed that oval window niche height can indicate both operative difficulty and subjective discomfort during otosclerosis surgery. With regard to shape, structures incorporated into the oval window niche, such as cartilage grafts, must be compatible with the shape of the oval window. Despite the clinical importance of the oval window, there is little information regarding its size and shape. METHODS: This study assessed oval window size and shape via micro-computed tomography paired with modern morphometric methodology in the fetal, infant, child, and adult populations. Additionally, the study compared oval window size and shape between sexes and between left- and right-sided ears. RESULTS: No significant differences were found among traditional morphometric parameters among age groups, sides, or sexes. However, geometric morphometric methods revealed shape differences between age groups. Further, geometric morphometric methods provided the average oval window shape and most-likely shape variance. CONCLUSION: Beyond demonstrating oval window size and shape variation, the results of this report will aid in identifying patients among whom anatomical variation may contribute to surgical difficulty and surgeon discomfort, or otherwise warrant preoperative adaptations for the incorporation of materials into and around the oval window.

Visualization and Quantitative 3D Analysis of Intraocular Melanoma and Its Vascularization in a Hamster Eye.

A tumor vasculature network undergoes intense growth and rebuilding during tumor growth. Traditionally, vascular networks are histologically examined using parameters such as vessel density determined from two-dimensional slices of the tumor. Two-dimensional probing of a complicated three-dimensional (3D) structure only provides partial information. Therefore, we propose the use of microcomputed tomography (micro-CT) imaging to analyze the evolution of a tumor vasculature in an experimental ocular tumor model. A Bomirski Hamster Melanoma was implanted in the anterior chamber of a hamster eye. Ultrasound (US) imaging of the same tumor was performed in vivo, and the vascular results obtained using the two methods were compared. Normal ocular tissues, a tumor, and a tumor vascular structure were revealed with high accuracy using micro-CT. The vessels that grew within the tumor were chaotic, leaky, and contained many convoluted micro-vessels and embolizations. They comprised 20-38% of the tumor mass. The blood flow in the larger functional vessels was in the range from 10 to 25 mm/s, as determined by in vivo Doppler US. The micro-CT imaging of the hamster eyeball enabled both qualitative and quantitative 3D analyses of the globe at a histological level. Although the presented images were obtained ex vivo, micro-CT noninvasive imaging is being developed intensively, and high-resolution in vivo imaging is feasible.

A micro-computed tomographic (micro-CT) analysis of the root canal morphology of maxillary third molar teeth.

INTRODUCTION: The aim of this study was to analyze the root canal morphology of maxillary third molars (MTMs) using micro-computed tomography (micro-CT). MATERIALS AND METHODS: Seventy-eight consecutively-extracted human MTMs were scanned using micro-CT (spatial resolution=13.68µm per pixel). Dedicated software (SkyScan®) was used to create virtual reconstructions and perform 3D-analysis. A range of anatomical features were assessed; externally (root number, length, fusion, curvature, apex), within the pulp chamber (distance between canal orifices, floor thickness) and within the root canal system (root canal number, classification, ramifications, isthmuses, apical constriction). RESULTS: The donor age ranged from 19 to 73 years (mean±SD 32.3±16.5years). MTMs possessed one or three roots, which principally curved buccally/palatally (75.9%), had 1-4 root canals and typically no apical constriction (84.4%). The average external root length was 11.89±1.53mm, while root canal length was 10.18±0.35mm. The root canal diameter 1mm from the apex was 0.37±0.23mm and negatively correlated with donor's age (r=-0.76; p=0.01), while pulp chamber thickness positively correlated with age (r=0.58; p=0.035). Significantly, furcation canals, canal loops and root canal calcifications were sporadic findings. CONCLUSIONS: In some cases the anatomy of MTMs may not be as complicated as previously documented, being similar to the reported anatomy of other maxillary molars. During root canal treatment of MTMs, the frequent deviation of the apical foramen from the radiographic apex should be considered, as should the absence of an apical constriction in the majority of cases. In addition, buccal/palatal root curvature, often undiagnosed radiographically, is the most common root curvature in MTMs.

Visualization of porcine eye anatomy by X-ray microtomography.

The aim of our study is to obtain, as accurately as possible, porcine ocular tissue visualization using microtomography (micro-CT) method. We propose image contrast enhancement by different staining procedures with combination of micro-CT scanning. Porcine eye globes were investigated with Bruker-SkyScan 1172 micro-CT. We used 4F1G and Bouin's as sample fixation solutions and tincture of iodine, 100% Lugol, phosphotungstic acid and 1% osmium tetroxide solutions for staining. Quantitative and qualitative analysis was performed based on micro-CT reconstruction images histograms and 3D volume rendering models of investigated samples. This investigation showed that staining methods improved micro-CT image quality in case of ocular anatomy visualization. Characteristic profiles of the grey level distributions and quality of the cross-section and 3D volume rendering images confirmed the staining effect. Most significant contrast enhancement was obtained after 96 h staining in osmium tetroxide and Lugol solutions. The images of eye anatomical structures were characterized: cornea, lens, iris, ciliary body, vitreous, retina, choroid and sclera, vasculature and optic nerve. Staining of porcine eye globes used in this work leads to quality improvement of the micro-CT imaging. The most contrast images were obtained for Lugol and osmium tetroxide solutions. Different affinity of staining solutions to eye anatomical structures has been observed in the obtained images. Osmium tetroxide provides sharper image of conjunctiva, sclera, choroid, retina, iris and ciliary body structure. Lugol staining leads to more accurate vessels, cornea and optic nerve imagining.

Imaging of the Wormian bones using microcomputed tomography.

Wormian bones are irregular ossicles of small size and reveal fractal pattern of their edges. Their anatomy was visualized in volumetric reconstructions obtained from a series of micro-CT scans. In visual evaluation Wormian bones showed typical anatomy for the calvarial bones. They revealed three-layer composition: the outer and the inner table of the compact bone intervening with the table of the spongy bone. Microcomputed tomography captured all details of the interdigitation of the edge being incorporated into the lambdoid suture and interlocked between opposing edges of the occipital and parietal bones. This modality provided accurate images which allowed delineating morphological differences between the compact bone and the diploe, including vascular channels.