Left ventricular noncompaction cardiomyopathy is associated with heart failure, arrhythmia, and sudden cardiac death. The developmental mechanism underpinning noncompaction in the adult heart is still not fully understood, with lack of trabeculae compaction, hypertrabeculation, and loss of proliferation cited as possible causes. To study this, we utilised a mouse model of aberrant Rho kinase (ROCK) signalling in cardiomyocytes, which led to a noncompaction phenotype during embryogenesis, and monitored how this progressed after birth and into adulthood. The cause of the early noncompaction at E15.5 was attributed to a decrease in proliferation in the developing ventricular wall. By E18.5, the phenotype became patchy, with regions of noncompaction interspersed with thick compacted areas of ventricular wall. To study how this altered myoarchitecture of the heart influenced impulse propagation in the developing and adult heart, we used histology with immunohistochemistry for gap junction protein expression, optical mapping, and electrocardiography. At the prenatal stages, a clear reduction in left ventricular wall thickness, accompanied by abnormal conduction of the ectopically paced beat in that area, was observed in mutant hearts. This correlated with increased expression of connexin-40 and connexin-43 in noncompacted trabeculae. In postnatal stages, left ventricular noncompaction was resolved, but the right ventricular wall remained structurally abnormal through to adulthood with cardiomyocyte hypertrophy and retention of myocardial crypts. Thus, this is a novel model of self-correcting embryonic hypertrabeculation cardiomyopathy, but it highlights that remodelling potential differs between the left and right ventricles. We conclude that disruption of ROCK signalling induces both morphological and electrophysiological changes that evolve over time, highlighting the link between myocyte proliferation and noncompaction phenotypes and electrophysiological differentiation.

Magnesium is an excellent material in terms of biocompatibility and its corrosion products can serve as an active source for new bone formation. However, localized corrosion and H2 generation limit the potential of Mg-based implants. Utilizing low-alloyed Mg-Zn wires can strongly reduce problems with large H2 bubbles and improve the mechanical properties considerably while maintaining excellent long-term biocompatibility. Acidic pickling and a polymer coating can be effectively used to lower the rate of in vivo degradation. In this work, microstructural, mechanical, and in vitro characterization of 250 µm and 300 µm extruded wires made from ultra-pure Mg, commercially pure Mg, Mg-0.15Zn, Mg-0.4Zn and Mg-1Zn was performed. Additionally, Mg-0.4Zn wires together with a variant coated with a copolymer of l-lactide and ε-caprolactone were tested in vivo on artificially damaged Wistar rat femurs. Based on the observed Mg-induced osteogenesis, polymer-coated Mg wires with a small addition of Zn are a perspective material for bone-support applications, such as cerclage and fixation wires.

That the highly trabeculated ventricular walls of the developing embryos transforms to the arrangement during the fetal stages, when the mural architecture is dominated by the thickness of the compact myocardium, has been explained by coalescence of trabeculations, often erroneously described as “compaction”. Recent data, however, supports differential rates of growth of the trabecular and compact layers as the major driver of change. Here, these processes were assessed quantitatively and morphologically using a larger dataset than has previously been available of mouse hearts from embryonic day 10.5 to postnatal day 3, supported by images from human hearts. The volume of the trabecular layer increased throughout development, in contrast to what would be expected had there been “compaction”. During the embryonic-fetal transition, fast growth of the compact layer diminished the proportion of trabeculations. Similarly, great expansion of the central cavity reduced the proportion that intertrabecular recesses make of the total cavity. Using the median value of left ventricular trabeculation, we provided illustrations for each gestational day so as to provide pictorial evidence of the changes. The illustrations confirmed a pronounced growth of the compact wall, and prominence of the central cavity. This corresponds, in morphological terms, to a reduction in the extent of the trabecular layer. Similar observations were made in the human hearts. We conclude that it is a period of comparatively slow growth of the trabecular layer, rather than so-called compaction, that is the major determinant of the changing morphology of the ventricular walls of both mouse and human.

The micro-CT evaluation of structural parameter values is dependent on the processing of the image prior to its analysis. However, the degree of bias introduced by the image-processing process is generally unknown. The micro-CT analysis of three basic parameters (volume, surface, and pore size) was performed for 20 collagen-based porous scaffolds. The image data were processed using various approaches (noise reduction, binarization), analyzed and results were compared. A high degree of variability was observed in the values of the structural parameters. The differences between the mean values were as follows: 190% (volume), 160% (surface density), and 210% (pore size). Significant differences were observed with respect to the noise reduction in terms of the pore size values (frequently in excess of 300%). Our study proved the significant dependence of image processing on the results obtained. The data presented can be used as an estimate for the introduced bias. We suggest improvements in terms of the selection of the image-processing approach based on a combination of subjective selection (the standard method) and numerical analysis based on the consistency of the results. Although we focused on tissue engineering scaffolds, the principles are similar for all fields of the micro-CT application.

Purpose:Osteoporosis is a severe health problem with social and economic impacts on society. The standard treatment consists of the systemic administration of drugs such as bisphosphonates, with alendronate (ALN) being one of the most common. Nevertheless, complications of systemic administration occur with this drug. Therefore, it is necessary to develop new strategies, such as local administration.Methods:In this study, emulsion/dispersion scaffolds based on W/O emulsion of PCL and PF68 with ALN, containing hydroxyapatite (HA) nanoparticles as the dispersion phase were prepared using electrospinning. Scaffolds with different release kinetics were tested in vitro on the co-cultures of osteoblasts and osteoclast-like cells, isolated from adult osteoporotic and control rats. Cell viability, proliferation, ALP, TRAP and CA II activity were examined. A scaffold with a gradual release of ALN was tested in vivo in the bone defects of osteoporotic and control rats.Results:The release kinetics were dependent on the scaffold composition and the used system of the poloxamers. The ALN was released from the scaffolds for more than 22 days. The behavior of cells cultured in vitro on scaffolds with different release kinetics was comparable. The difference was evident between cell co-cultures isolated from osteoporotic and control animals. The PCL/HA scaffold show slow degradation in vivo and residual scaffold limited new bone formation inside the defects. Nevertheless, the released ALN supported bone formation in the areas surrounding the residual scaffold. Interestingly, a positive effect of systemic administration of ALN was not proved.Conclusion:The prepared scaffolds enabled tunable control release of ALN. The effect of ALN was proved in vitro and in in vivo study supported peri-implant bone formation.

Condylar hyperplasia is one of the causes of facial asymmetry and malocclusion, characterized by enlargement of the lower jaw due to excessive condyle growth activity. The aim of this study was to use micro-computed tomography (micro-CT) to evaluate the bone architecture of the condylar head and determine whether there are differences between patients with various forms of unilateral condylar hyperplasia (UCH): hemimandibular hyperplasia, elongation, and mixed form. The cohort consisted of 28 patients with a mean age of 21.9 years. All patients underwent surgical treatment (condylar shaving) for active pathological growth activity. The portion of the condylar head removed was imaged by micro-CT and subsequently evaluated. Micro-CT imaging and semiquantitative and quantitative evaluation of the bone structure (percentage bone volume, surface density, trabecular thickness, trabecular separation, degree of anisotropy, and porosity of the subchondral bone) did not reveal significant differences between the individual types of condylar hyperplasia (P > 0.05). There were no significant differences in bone structure between the anterior and posterior portions of the condylar head. No statistically significant differences between individual groups of UCH were found in the micro-CT evaluation of the condylar head bone architecture.

BACKGROUND:The ventricular trabeculae play a role, among others, in the impulse spreading in ectothermic hearts. Despite the morphological similarity with the early developing hearts of endotherms, this trabecular function in mammalian and avian embryos was poorly addressed. RESULTS:We simulated impulse propagation inside the looping ventricle and revealed delayed apical activation in the heart with inhibited trabecular growth. This finding was corroborated by direct imaging of the endocardial surface showing early activation within the trabeculae implying preferential spreading of depolarization along with them. Targeting two crucial pathways of trabecular formation (Neuregulin/ErbB and Nkx2.5), we showed that trabecular development is also essential for proper conduction patterning. Persistence of the slow isotropic conduction likely contributed to the pumping failure in the trabeculae-deficient hearts. CONCLUSIONS:Our results showed the essential role of trabeculae in intraventricular impulse spreading and conduction patterning in the early endothermic heart. Lack of trabeculae leads to the failure of conduction parameters differentiation resulting in primitive ventricular activation with consequent impact on the cardiac pumping function.

The present study investigates the effect of an oxidized nanocrystalline diamond (O-NCD) coating functionalized with bone morphogenetic protein 7 (BMP-7) on human osteoblast maturation and extracellular matrix mineralization in vitro and on new bone formation in vivo. The chemical structure and the morphology of the NCD coating and the adhesion, thickness and morphology of the superimposed BMP-7 layer have also been assessed. The material analysis proved synthesis of a conformal diamond coating with a fine nanostructured morphology on the Ti6Al4V samples. The homogeneous nanostructured layer of BMP-7 on the NCD coating created by a physisorption method was confirmed by AFM. The osteogenic maturation of hFOB 1.19 cells in vitro was only slightly enhanced by the O-NCD coating alone without any increase in the mineralization of the matrix. Functionalization of the coating with BMP-7 resulted in more pronounced cell osteogenic maturation and increased extracellular matrix mineralization. Similar results were obtained in vivo from micro-CT and histological analyses of rabbit distal femurs with screws implanted for 4 or 12 weeks. While the O-NCD-coated implants alone promoted greater thickness of newly-formed bone in direct contact with the implant surface than the bare material, a further increase was induced by BMP-7. It can be therefore concluded that O-NCD coating functionalized with BMP-7 is a promising surface modification of metallic bone implants in order to improve their osseointegration.

This study is aimed at proving the clinical benefit of the MELISA® test in the minimization or complete elimination of health problems in patients with confirmed hypersensitivity to metals used for tissue replacements. A group of 305 patients aged 20-75 years with previously proven metal hypersensitivity (initial MELISA® test), mainly to titanium and then to another fifteen metals, was chosen from the database at the Institute of Dental Medicine. From these patients, a final group of 42 patients agreed to participate in the study, 35 of which were female and 7 were male. The patients completed a special questionnaire aimed at information regarding change of health status from their last visit and determining whether the results of the initial MELISA® test and recommendations based on it were beneficial for patients or not. They were clinically examined, and peripheral blood samples were taken to perform follow-up MELISA® tests. Questionnaire data was processed, and the follow-up MELISA® test results were compared with the results of the initial MELISA® tests. For statistical analysis, the Fisher’s exact test and paired T -test were used. Thirty-two patients reported that they followed the recommendations based on the results of the initial MELISA® tests, and of these, 30 patients (94%) confirmed significant health improvement. Six patients did not follow the recommendation, and from these, only one patient reported an improvement in his health problems. By comparison of the initial and follow-up MELISA® test results, it can be stated that the hypersensitivity to the given metal decreased or disappeared after the therapeutic interventions performed based on the initial MELISA® test results. The evaluation of the data obtained from patients in this study confirmed a significant clinical benefit of MELISA® test.

BACKGROUND:During amphibian metamorphosis, the crucial moment lies in the rearrangement of the heart, reflecting the changes in circulatory demands. However, little is known about the exact shifts linked with this rearrangement. Here, we demonstrate such myocardial changes in axolotl (Ambystoma mexicanum) from the morphological and physiological point of view.RESULTS:Micro-CT and histological analysis showed changes in ventricular trabeculae organization, completion of the atrial septum and its connection to the atrioventricular valve. Based on Myosin Heavy Chain and Smooth Muscle Actin expression we distinguished metamorphosis-induced changes in myocardial differentiation at the ventricular trabeculae and atrioventricular canal. Using optical mapping, faster speed of conduction through the atrioventricular canal was demonstrated in metamorphic animals. No differences between the groups were observed in the heart rates, ventricular activation times, and activation patterns.CONCLUSIONS:Transition from aquatic to terrestrial life-style is reflected in the heart morphology and function. Rebuilding of the axolotl heart during metamorphosis was connected with reorganization of ventricular trabeculae, completion of the atrial septum and its connection to the atrioventricular valve, and acceleration of AV conduction.

Open macroporosity is crucial for scaffolds in tissue engineering. Porogen-templating method is an attractive approach for fabrication of macroporous hydrogels, however, the effect of shape and amount of template particles on imprinted structure has not yet been quantitatively established. We present a mathematical model for simulating the formation of paths percolating through distributed cubical particles as a function of the filling volume. The model was used to select the fraction of NaCl particles as templates for preparation of hydrogels with communicating pores. Hydrogels were prepared from 2-hydroxyethyl methacrylate (HEMA) copolymerized with 2-ethoxyethyl methacrylate (EOEMA), [2-methacryloyloxy)ethyl]trimethylammonium chloride (MOETACl) or ionizable methacrylic acid (MANa) to modulate swelling, surface and mechanical properties of gels. Micro-CT analysis of swollen samples proved a highly-interconnected pore structure. Charged hydrogels swelled more and their apparent elastic modulus G′ was below 1 kPa. For PHEMA and P(HEMA/EOEMA) hydrogels, G′ was 5 and 80 kPa, respectively. Within two-week in vitro studies, MG63 osteoblasts proliferated fastest on P(HEMA/EOEMA) showing the lowest swelling and the highest elastic modulus, whereas cell growth was impaired on positively charged P(HEMA/MOETACl). The mathematical simulation of cubical particle packing in hydrogels and micro-CT data in swollen state provided evidence of an extensive void communication in 3D.

The aim of this study was to compare the effect of several commercially available disinfectants on the accuracy of various types of impression materials and their compatibility with gypsum including surface quality and structure evaluation. Four alginate and three elastomeric impression materials in combination with disinfectants Aseptoprint Liquid, Zeta 7 solution, Silosept and Dentaclean Form were tested. The dimensional changes, detail reproduction, the compatibility with gypsum and surface/subsurface morphology were evaluated using light microscopy, scanning electron microscopy and micro computed tomography. Two alginate materials disinfected in Dentaclean Form exhibited the most significant differences (p<0.0001). The loss of detail on some alginate impressions in combination with this disinfectant including deterioration and change of morphology of gypsum surfaces was observed. Porosity in subsurface area and exposed large particles were detected. It was confirmed that the desired properties of impressions may be negatively affected in combination with some disinfectants.

The oral cavity is an area of numerous pathologies present not only within, but also outside the dental arch. The presence of supernumerary ectopically located teeth in non-dental areas or pathologies with dental tissue formation in non-dental areas evokes the questions how these pathologies are formed and what their origin is. One of the tissues maintaining an odontogenic potential is the prospective oral vestibule located externally to the developing dentition. In case the signaling is awoken, it might explain the presence of pathologies externally to the dentition with signs of possible tooth-associated structure formation. Also during successional dental lamina disintegration, some lamina cells remain in small islands known as the rests of Serres or as epithelial pearls. These epithelial remnants are of general interest for human clinicians because they can give rise to cysts or odontogenic tumors. Odontogenic cysts are enclosed epithelial sacs with a distinct membrane derived from the rests of the odontogenic epithelium. Odontogenic tumors are pathological lesions derived from the epithelial and/or mesenchymal remnants of tooth germs. In addition, dental hard tissue may or may not be included in these lesions. The precise mechanisms controlling the initiation of odontogenic cysts or tumors are however still unknown. Uncovering the signaling pathologies and altered cell communication between epithelial and mesenchymal components could explain the mechanisms driving the initiation of tumors or cysts in our patients. As this field is largely unexplored, an association of clinical findings with molecular events in individual patients could help us to predict the patient's prognosis and to plan directly targeted treatment.

Significance The sympathetic nervous system plays a critical role in stimulating heart rate and contractility to increase cardiac output and thereby insure adequate tissue O 2 delivery. Altered sympathetic output contributes to cardiac pathologies, such as sudden cardiac death and heart failure. Our data provide insight into the role of the transcription factor hypoxia-inducible factor 1α (HIF-1α) in the development of sympathetic ganglion neurons and cardiac sympathetic innervation. We found that genetic deletion of HIF-1α resulted in increased cell death and decreased proliferation of neuronal progenitors of the sympathetic system. These findings suggest that dysregulated HIF-1α expression may contribute to cardiac dysfunction and disease associated with defects in the cardiac sympathetic system by affecting the function and survival of sympathetic neurons.

Our study presents a novel collagen wound dressing prepared from freshwater fish skin (Cyprinus Carpio) collagen type I. Half of the sponges were cross-linked with carbodiimide. The cross-linked and non-cross-linked collagen sponges were subsequently impregnated with gentamicin and lyophilized thus allowing for the attainment of the appropriate gentamicin content without the removal thereof during the cross-linking stage. The structure was evaluated via micro-CT and infrared spectrometry and the structural stability and gentamicin release properties were evaluated in phosphate buffer solution. The sponges were further tested via a rat model of an infected wound with Pseudomonas aeruginosa inoculation and compared with a reference commercial product. The sponges thus prepared provided a degree of open porosity that was comparable to or higher than that of the reference commercial product. Spectrometry analysis revealed that the cross-linked collagen sponge and reference commercial product sponge preserved their secondary collagen structure after 168h while early accelerated degradation was observed with respect to the non-cross-linked collagen sponge. Gentamicin was released rapidly from all the sponges. Compared to those animals with gentamicin-containing sponges or gentamicin administered intramuscularly, the animals with the cross-linked collagen sponge without gentamicin exhibited marked clinical and laboratory infection signs. Both the administration routes (intramuscular and via gentamicin-containing sponges) provided similar gentamicin plasma levels. The resulting highly homogeneous product which was characterized by excellent structural and clinical properties proved effective in terms of the treatment of a surgical wound infection in a rat model. We demonstrated that all the gentamicin was released from the sponge and was absorbed in the systemic circulation. This is the first time that Cyprinus Carpio collagen has been used in the preparation of wound dressings. Thus, gentamicin-containing sponges provide a promising tool for the treatment and prevention of surgical site infections.

One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.

Micro-CT (micro-computed tomography) is a modern preclinical imaging method allowing non-destructive visualizations and structure analysis yielding at a resolution of a few micrometres. Tissue engineering scaffolds are a promising treatment for bone defects. Micro-CT application for the evaluation of tissue engineering scaffolds for bone surgery and traumatology application is increasing, which comprises in vitro, in vivo and ex vivo studies. Micro-CT itself is not able to replace conventional approaches completely, such as scanning electron microscopy (SEM) or histological examination, but it may offer important benefits regarding non-destructive approach, direct 3D model structure analysis, and visualization and time efficacy. The overview presented herein focuses on a micro-CT application in the field of tissue engineering scaffolds aimed at bone regeneration.

Most people worldwide suffer from dental caries. Only a small part of the population is cariesresistant and the reason for this resistance in unknown. Only a few studies compared the saliva protein composition of persons with carious teeth and persons with no caries. Our study is the first to relate proteomic analysis of the caries aetiology with gender. In this study, we compared the differences in the abundances of proteins in the saliva between cariesresistant and caries-susceptible females and males by nano-liquid chromatography-tandem mass spectrometry (Label-Free Quantitative Proteomics). Our results demonstrate that the observed differences in the protein levels might have an influence on anticaries resistance. A total of 19 potential markers of tooth caries were found, for example proteins S100A8 and annexin A1 with higher expression in the cariessusceptible group in comparison with the caries-free group and mucin-5B, lactoferrin, lysozyme C with higher expression in the caries-free group in comparison with the caries-susceptible group. The presented study is the first complex proteomic and gender project where the saliva protein content of caries-free and caries-susceptible persons were compared by label-free MS. The newly detected potential protein markers of dental caries can be a good basis for further research and for possible future therapeutic use.

Background : Coronary artery development is an intensely studied field. Mice are a popular genetic model for developmental studies, but there is no widely accepted protocol for high‐throughput, high‐resolution imaging of their developmental and adult coronary artery anatomy. Results : Using tissue‐clearing protocols and confocal microscopy, we have analyzed embryonic and juvenile mouse hearts in Cx40:GFP knock‐in models with a special focus on septal artery development. We found that the septal artery, which supplies the interventricular septum, was initially formed as an arterial plexus that connected to both the left and right coronary arteries. During development, the plexus was remodeled into a single tube, which then remained connected only to the right coronary artery. Since optical imaging became limited at postnatal stages, it was supplemented with injection techniques using India ink and Microfil; the latter was subsequently analyzed with micro‐CT to visualize the anatomy of coronary vessels in 3D. Conclusions : The techniques described here enable us to study the finer details of coronary artery development in mice and can, therefore, be implemented to study the pathogenesis of coronary malformations in various mouse models. Developmental Dynamics 247:1018‐1027, 2018 . © 2018 Wiley Periodicals, Inc.