Decellularization of the human urethra for tissue engineering applications.

Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample (P < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.

Fabrication and In Vitro Characterization of Novel Hydroxyapatite Scaffolds 3D Printed Using Polyvinyl Alcohol as a Thermoplastic Binder.

This paper presents a proof-of-concept study on the biocolonization of 3D-printed hydroxyapatite scaffolds with mesenchymal stem cells (MSCs). Three-dimensional (3D) printed biomimetic bone structure made of calcium deficient hydroxyapatite (CDHA) intended as a future bone graft was made from newly developed composite material for FDM printing. The biopolymer polyvinyl alcohol serves in this material as a thermoplastic binder for 3D molding of the printed object with a passive function and is completely removed during sintering. The study presents the material, the process of fused deposition modeling (FDM) of CDHA scaffolds, and its post-processing at three temperatures (1200, 1300, and 1400 °C), as well it evaluates the cytotoxicity and biocompatibility of scaffolds with MTT and LDH release assays after 14 days. The study also includes a morphological evaluation of cellular colonization with scanning electron microscopy (SEM) in two different filament orientations (rectilinear and gyroid). The results of the MTT assay showed that the tested material was not toxic, and cells were preserved in both orientations, with most cells present on the material fired at 1300 °C. Results of the LDH release assay showed a slight increase in LDH leakage from all samples. Visual evaluation of SEM confirmed the ideal post-processing temperature of the 3D-printed FDM framework for samples fired at 1300 °C and 1400 °C, with a porosity of 0.3 mm between filaments. In conclusion, the presented fabrication and colonization of CDHA scaffolds have great potential to be used in the tissue engineering of bones.

Terminologia Histologica 10 years on: some disputable terms in need of discussion and recent developments.

At first sight, the issue of morphological terminology may seem to be a "closed and unchanging chapter", as many of the structures within the human body have been known for decades or even centuries. However, the exact opposite is true. The initial knowledge of the microscopic structure of the human body has been continuously broadening thanks to the development of new specialized staining techniques, discovery of the electron microscope, or later application of histochemical and immunohistochemical methods into routine tissue examination. Contrary to popular belief, histology has a status of constantly developing scientific discipline, with continuous influx of new knowledge, resulting in an unavoidable necessity to revise the histological nomenclature at regular intervals. The team of experts of the Federative International Programme on Anatomical Terminology, a working group of the International Federation of Associations of Anatomists, published in 2008 the First Edition of Terminologia Histologica. Terminologia Histologica (TH) is the best and most extensive of all the histological nomenclatures ever issued. However, here we suggest that several terms of important histological structures are still missing while several other terms are disputable. First, we present some clinically important terms of cells and tissue structures for inclusion in the next TH and, in a second part, we refer to some new terms in the current edition of the TH which are not yet mentioned in current histology textbooks (e.g., fusocellular connective tissue, bundle bone as the third type of bone tissue, spongy layer of vagina or arteria vaginata from the splenic white pulp). With this article we hope to start a wide scientific discussion which will lead to an inambiguous definition and demonstration of typical examples of all terms in the TH, with the result that the new edition of the Terminologia Histologica will become an internationally accepted communication tool for all practitioners and teachers of histology alike.

The end-stage failing human myocardium - Where changes in ultrastructure of human cardiac muscle cells do not appear to dictate clinical outcomes.

Heart failure is the end stage of cardiovascular abnormalities. Studies have primarily focused on the functional changes of cardiomyocytes in the failing heart from different animal models with very little information in the human condition. In addition little is known about the ultrastructural changes that proceed in cardiomyocytes in route to failure. The aim of this study was to examine the ultrastructural changes in the myocardium of human with end-stage heart failure. Left ventricular myocardial tissue samples from 7 patients with end-stage heart failure were examined with transmission and scanning electron microscopy. All heart failure patients were of New York Heart Association (NYHA) class III-IV. The data indicated normal three-dimensional arrangement of cardiac muscle cells in failing myocardium. The various organelles in cardiomyocytes including the nucleus, mitochondria, myofibrils, T-tubules and intercalated discs did not exhibit any remarkable morphological changes. We did observe the appearance of small membrane bound vesicles which appear to be associated with the intercalated discs. The nearly normal ultrastructure and arrangement of cardiomyocytes was remarkable in contrast to the dramatic clinical status of these patients in heart failure. These observations support the hypothesis, that there are no dramatic changes in the ultrastructure or three-dimensional architecture of cardiomyocytes in end-stage failing human myocardium.

The Non-cardiomyocyte Cells of the Heart. Their Possible Roles in Exercise-Induced Cardiac Regeneration and Remodeling.

The non-cardiomyocyte cellular microenvironment of the heart includes diverse types of cells of mesenchymal origin. During development, the majority of these cells derive from the epicardium, while a subset derives from the endothelium/endocardium and neural crest derived mesenchyme. This subset includes cardiac fibroblasts and telocytes, the latter of which are a controversial type of "connecting cell" that support resident cardiac progenitors in the postnatal heart. Smooth muscle cells, pericytes, and endothelial cells are also present, in addition to adipocytes, which accumulate as epicardial adipose connective tissue. Furthermore, the heart harbors many cells of hematopoietic origin, such as mast cells, macrophages, and other immune cell populations. Most of these control immune reactions and inflammation. All of the above-mentioned non-cardiomyocyte cells of the heart contribute to this organ's well-orchestrated physiology. These cells also contribute to regeneration as a result of injury or age, in addition to tissue remodeling triggered by chronic disease or increased physical activity (exercise-induced cardiac growth). These processes in the heart, the most important vital organ in the human body, are not only fascinating from a scientific standpoint, but they are also clinically important. It is well-known that regular exercise can help prevent many cardiovascular diseases. However, the precise mechanisms underpinning myocardial remodeling triggered by physical activity are still unknown. Surprisingly, exercise-induced adaptation mechanisms are often identical or very similar to tissue remodeling caused by pathological conditions, such as hypertension, cardiac hypertrophy, and cardiac fibrosis. This review provides a summary of our current knowledge regarding the cardiac cellular microenvironment, focusing on the clinical applications this information to the study of heart remodeling during regular physical exercise.

Congenital anomalies of the spleen from an embryological point of view.

The spleen is the major accumulation of lymphoid tissue in the human body, an organ which prenatally produces and postnatally controls blood cells. Normally, a developed spleen lies in the upper left quadrant in parallel with the long axis of the 10th rib. It is a mesodermal derivate which first appears as a condensation of mesenchymal cells inside the dorsal mesogastrium at the end of the fourth embryonic week. Some congenital anomalies of the spleen are common, such as splenic lobulation and accessory spleen, while other conditions are rare, such as wandering spleen and polysplenia. Splenogonadal fusion is also a rare developmental anomaly, resulting from abnormal fusion of the splenic and gonadal primordia during prenatal development. The purpose of this article is to describe the normal development of the human spleen, supplemented with our own photomicrographs and a review of congenital anomalies of the spleen with their possible embryonic basis.

Morphologic heterogeneity of human thymic nonlymphocytic cells.

The thymus is the central organ of the immune system. It is essential for the development and maintenance of normal immune system, especially cell-mediated immunity. From the morphological point of view, the thymus is divided into two main compartments, cortex and medulla. The thymic microenvironment consists of a network of reticular epithelial cells and other fixed and free cells. The microenvironment of thymus is very important for the selection and maturation of T cells. T cell differentiation occurs via T cell receptors. The major histocompatibility complex participates in interactions between T cells and thymic epithelial cells, in addition to interactions between T cells and dendritic cells, macrophages and myoid cells. The neuroendocrine system regulates early T cell differentiation by the transcription of neuroendocrine genes in the stromal network and expression of cognitive receptors by immature T cells. This work briefly summarizes morphological and ultrastructural characteristics of thymic epithelial cells, dendritic cells, macrophages and myoid cells. It is accompanied by the authors' own photomicrographs and electronmicrograph from a transmission electron microscope. All of these cells play a critical role in the proliferation, differentiation and selection of precursor cells in the T-cell lineage, but the precise mechanisms not well understudood.

The phylogenesis and ontogenesis of the human pharyngeal region focused on the thymus, parathyroid, and thyroid glands.

The pharyngeal (branchial) region represents a classic example where the relationship between ontogenesis and phylogenesis has been demonstrated. It is a region where the development of gills during ontogenesis of all chordates has been recapitulated. In the process of evolution the pharyngeal region has undergone marked changes. While it functioned to ensure blood oxygenation and regulation of a constant internal environment in aquatic animals, it had to adapt to new and more complex functions in terrestrial vertebrates. The lungs have taken on the main role of blood oxygenation and the salivary glands now regulate ionic balance. The immune organs in mammals such as the thymus and the palatine tonsil, endocrine organs such as the parathyroid glands and the parafollicular cells of the thyroid gland, which produces calcitonin (originally as independent ultimobranchial bodies), as well as a part of the ear developed from the pharyngeal region. This article briefly summarizes the current knowledge regarding the phylogenesis and development of the human thymus, parathyroids, and the thyroid gland with a focus on the influence of neural crest cells during development.