Decellularization techniques of human foreskin for tissue engineering application.

The rapid development of tissue engineering (TE) and regenerative medicine brings an acute need for biocompatible and bioactive biological scaffolds to regenerate or restore damaged tissue. Great attention is focused on the decellularization of tissues or even whole organs, and the subsequent colonization of such decellularized extracellular matrices by recipient cells. The foreskin is an integral, normal part of the external genitalia that forms the anatomical covering of the glans penis and the urinary meatus of all human and non-human primates. It is mucocutaneous tissue that marks the boundary between mucosa and skin. In this work, we compared two innovative decellularization techniques for human foreskins obtained from donors. We compared the efficacy and feasibility of these protocols and the biosafety of prepared acellular dermal matrixes that can serve as a suitable scaffold for TE. The present study confirms the feasibility of foreskin decellularization based on enzymatic or detergent methods. Both techniques conserved the ultrastructure and composition of natural ECM while being DNA-free and non-toxic, making it an excellent scaffold for follow-up research and TE applications.

Our First Experience Implementing "Clinical Embryology and Reproductive Medicine" as a Curriculum Course of the Study Program General Medicine at the Faculty of Medicine in Bratislava, Slovakia.

There is no separate course in the medical curriculum summarizing all aspects of human reproduction in most medical school curricula. At the same time, such a course would logically connect knowledge from clinical embryology and assisted reproduction, encompassing the issue of female and male infertility, mechanisms of birth defect formation, their prenatal diagnosis and subsequent specialized neonatal care. The aim of a wide team of university teachers comprising embryologists, gynecologists, neonatologists, endocrinologists, geneticists and others was to create and implement a new course entitled "Clinical Embryology and Reproductive Medicine" into the fourth-year curriculum of the study program General Medicine at the Faculty of Medicine, Comenius University in Bratislava. There has been a great interest in the course, as evidenced by the number of medical students enrolled. The lecture syllabuses have been divided into several thematic areas: 1) Clinical embryology including a laboratory part of assisted reproduction, 2) Cause and treatment options of female and male infertility, 3) A comprehensive view of the issue of birth defects, 4) The issue of preconception education, prenatal and childbirth training, family planning, 5) Reproductive immunology and endocrinology. Despite the complexity of human reproduction being a mainstay of gynecology and obstetrics, it is underemphasized in the medical school curricula worldwide. It is often reflected in shorter hospital / practical trainings during undergraduate studies and lower requirements at the final exam. Therefore, as students almost unanimously valued, this new course is extremely helpful in preparing for the final state exam.

Immunohistochemical and Scanning Electron Microscopic Confirmation of the Lymphatic Lacunae in the Uterine Tube Mucosal Folds. What Are the Clinical Implications?

Uterine tubes (UTs) are essential during physiological reproduction. The most intriguing part of its wall is the mucosa. Apart from the epithelial cells vital for its normal function, the connective tissue lamina propria contains wide spaces whose function, morphology and structure are yet to be elucidated. The present study used bioptic samples from 25 premenopausal (mean age 48,33 years, ?=3,56) and 25 postmenopausal women (mean age 57,8 years, ?=7,79). In both study groups, samples were obtained from two anatomically distinct parts of the UT - ampulla and infundibulum with fimbriae. The specimens were processed for scanning electron microscopy (SEM) and immunohistochemical detection of podoplanin (clone D2-40) and VEGFR-3 - two markers of lymphatic endothelial cells. The results showed that specimens from premenopausal and postmenopausal women contain wide lymphatic spaces, also known as lymphatic lacunae. The most probable function of the lacunae in the fimbriae is oocyte pick-up upon ovulation thanks to their ability to get engorged with lymph, thus serving as an erectile-like tissue. The ampullary lacunae are probably responsible for tubal fluid maintenance and recirculation. These results indicate that they are vital for normal reproduction because tubal fluid dynamics are as important as fluid composition. Further research on this topic is highly warranted because more detailed insights into UT function have a great potential to refine the methods of reproductive medicine, e.g. in vitro fertilization (IVF), which are still far from optimal regarding fertility outcomes.

Scanning electron microscopic study of the human uterine tube epithelial lining: surgical biopsy samples and epithelial cell culture.

This article summarizes the importance of the exact morphology of human uterine/fallopian tube epithelium at the scanning electron microscopy (SEM) level for the clinical outcome even nowadays. Visual referential micrographs from SEM reflect two ways to view human epithelial cell lining surfaces: the surface epithelial uterine tube from surgical tissue biopsy and human fallopian tube epithelial cells (HFTEC) culture monolayer surface. One colorized image visualizes ciliated cells, distinguishes them from non-ciliated cells, and provides an educational benefit. A detailed description of the ultrastructure in referential and pathologic human uterine tube epithelium is important in defining the morphological basis of high-grade carcinomas, in the mechanism of pathophysiology, and in discussing options for its prevention. Cell cultures of human fallopian tube epithelial cells offer new approaches in simulating the mechanisms of cancer genesis or may help to elucidate the genetic basis of several diagnoses. New technical approaches in SEM provide higher resolution and detailed surface images. The SEM modality is still one of the current options in diagnostics and may be useful for advancing human reproductive organ cancer research.

Ependymal cells surface of human third brain ventricle by scanning electron microscopy.

OBJECTIVES: The ependymal lining of the human brain ventricular system displays distinct structural differences and functional heterogeneity among individual ependymal cells (ECs). To date, multi-ciliated ECs (E1 cells), bi-ciliated ECs (E2 cells), uni-ciliated ECs (E3 cells), ECs without cilia, and ECs with cytoplasmic protrusions have been described in human brain ventricles. METHOD: Using scanning electron microscopy (SEM), we evaluated ependymal samples from 6 defined regions of the third ventricle from 9 human brains. These regions were strictly defined according to the periventricular structures they neighbour with. RESULTS: We observed different structures on the apical surface of the ECs. Various ECs differed from each other by the presence of microvilli, secretory bodies, and a variable number of cilia, which led us to divide the ECs into several exactly specified types according to their apical morphology. CONCLUSION: We found all types of ECs in every examined region with a predominance of particular types of apical surface of ECs in the individual areas (Tab. 4, Fig. 7, Ref. 22).

Cardiac telocytes as principal interstitial cells for myocardial reparation and regeneration after infarction - our hope.

According to our knowledge, this is the first research experiment that focuses on the study of the distribution of c-kit positive cells at the sites of myocardial infarction in human hearts (Fig. 3, Ref. 16).

Neuron-specific enolase in the intestinal wall in Crohn´s disease.

The authors described the localization of neuron-specific enolase in the intestinal wall in Crohn´s disease. We have used samples obtained by biopsy from the colon lining of five people affected by Crohn's disease for our examination. We have processed samples using the formol paraffin technique. From paraffin blocks, we have prepared histological sections approximately 5 µm thick. For immunohistochemic examinations, we have revitalised the sections by acquiring the heat-induced epitope. We detected NSE by monoclonal mouse antibodies against human neuron-specific enolase, clone BBS/NC/VI-H14 (DakoCytomation, Denmark) (Fig. 4, Ref. 7).

Iron-rich complexes in human spleen in hereditary spherocytosis.

OBJECTIVE: Tissue iron plays an important role in the development of certain diseases. Although it is one of biogenic elements, its excess induces the reactive oxygen species (ROS) formation. The aim of the present work is to examine the protection against free or loosely bound iron from the view of morphology and chemical composition of iron-rich complexes in human spleen tissues with hereditary spherocytosis (HS) by scanning and transmission electron microscope with energy-dispersive microanalysis (EDX). RESULTS: The examination of human spleen tissues by scanning and transmission electron microscope showed covering of iron-rich particles. EDX revealed many iron-rich complexes of multi-element composition in HS samples with sulphur and phosphorus as the major elements. Detection was negative in the reference samples. CONCLUSION: The covering of iron-rich particles can be explained by elimination and isolation of ferritin/iron complexes from surrounding environment to prevent the ROS formation. Sulphur, phosphorus and their compounds are probably the most significant elements that influence the ROS formation (Fig. 5, Ref. 16). Full Text in PDF www.elis.sk.

Iron accumulation in human spleen in autoimmune thrombocytopenia and hereditary spherocytosis.

BACKGROUND: Although the iron is an essential element for the physiological functions of cells, tissues and organs, it is also an important inductor of reactive oxygen species (ROS). MATERIAL AND METHODS: Three groups of human spleen with autoimmune thrombocytopenia (AITP), hereditary spherocytosis (HS) and reference samples stained by haematoxylin and eosin, Perls' reaction for nonheme Fe(III) iron and Alcian blue for glycoconjugates detection were studied. RESULTS: Positive Perls' reaction in both AITP and HS groups was seen. Higher positivity in the HS than in AITP group was observed. HS group showed a higher amount of acidic glycoconjugates deposits than AITP group. Iron overload in HS and AITP leads to overproduction of ROS. CONCLUSION: We suggest that acidic glycoconjugates deposits are involved in antioxidant defence by elimination and restriction of iron as a ROS inducer (Fig. 4, Ref. 19).

The thymus: picture review of human thymus prenatal development.

The thymus is derived from pharyngeal region; a region from which, in case of aquatic vertebrates, the gills develop. According to the classical hypothesis, the epithelial thymus stroma of human embryos is derived from the endodermal cells of the left and right ventral parts of the third pharyngeal pouches. But a close contact of the third pharyngeal pouch with its corresponding third pharyngeal clefts ectoderm plays an important role. Also an epithelial-mesenchymal interaction between the third pharyngeal pouch endoderm and surrounding neural crest derived-cells ectomesenchyme is necessary for the proliferation and differentiation of thymic epithelial cells. In our work we photographically presented the development of thymus from the 6th up to the 25th week of development. The first primordia of the thymus and parathyroid glands within the endoderm of third pharyngeal pouches can be seen in 8 to 9 mm stages. We found also an epithelial proliferation in the second pharyngeal pouches, but this "thymus secundus" stopped their differentiation. The thymus primordia at the 7th and 8th week of development contain almost exclusively epithelial cells. These cells are arranged at the periphery as a row of prismatic cells. The mesenchyme accumulates around the epithelial thymic primordium, and during 9th to 12th weeks of development, septa from mesenchyme fold between the epithelial cells and create the "openings" in the capsular surround. According to our observations, in the 13th week of development the differentiation of cortex and medulla becomes obvious and is completed from the 17th up to 18th week of development onward. The first developing Hassall's corpuscle was detected in the 13th week of development. The striking increase in the number of the Hassall's bodies was observed between the 16th and the 18th week of development, as well as between the 22nd and the 25th week of development (Fig. 14, Ref. 35).

Thymic Hassall's bodies of children with congenital heart defects.

The development of the thymus and heart are closely related while in both, the neural crest cells play an important role. In our preliminary study, the thymic microscopic structures of the infant's thymuses with the congenital heart defects were observed. The study was conducted on 36 specimens of newborn thymuses removed due to surgery for cardiovascular malformations. Standard formalin-fixed paraffin-embedded tissue technique was used. Five-microm-thick sections were stained with hematoxylin and eosin and the microscopic examination was focused on the structure of Hassall's bodies. The Hassall's bodies showed considerable variations in size as well as in quantity. In most cases, the Hassall's bodies were large with the heterogeneous amorphous material enclosed in cystic dilatations. This type of Hassall's bodies is typical for adult thymuses. The most conspicuous changes (huge Hassall's bodies with the cystic dilatation filled up with cell detritus) were observed in patients with ventricular septal defect, atrioventricular septal defect and tetralogy of Fallot. Small-sized Hassall's bodies corresponding with infant age, were observed in cases with pulmonary valve atresia, atrial septal defect and in some cases of transposition of great arteries. We assume that the changes of microenvironment of the thymic medulla are associated with disrupted migration of the neural crest cells which are essential in the normogenesis of both heart and thymus (Tab. 1, Fig. 12, Ref. 33).

Endocrine organs and laser scanning confocal microscopy (LSCM) imaging: vascular bed in human spleen.

OBJECTIVE: This work was aimed to utilize the precise method of laser confocal microscopy (LSCM) to depict the image of spatial relationships of the vessel network in the tissue structures of the human spleen. METHODS: With the use of serial paraffin or vibratome sections of more than 20 µm thickness infiltrated with eosin fluorescence dye the images of arterial and venous walls of different calibres, capillaries, and venous sinuses were morphologically revealed. RESULTS: Venous sinuses were frequently found to create mutually communicating branches and their lining projected into the lumen protruding cells with distinct spherically or ovally shaped nuclei, positioned on the brightly fluorescent and fragmented lamina basalis. The presence of lymphocytes was distinct in periarteriolar lymphoid sheath (PALS) and lymphatic follicles. Lining cells of the red pulp veins sporadically contained marked eosinophilic granules. CONCLUSION: The method of LSCM allowed: 1. to reveal two-dimensional and sharp image of the human spleen structures, 2. to investigate the vertical course of venous structures in the tissue, 3. to obtain serial optic sections in z axis to their maximum spatial projections. These data will also serve for the creation of three-dimensional images of vessel network in the human spleen in the future studies.

Perspectives and complexity of an experimental cancer study. the secrets of tumorigenesis. (To the Gupta's, Chaffer's and Weinberg's "perspectives" and to the nurse's "horizons").

We would like to add to the "mysteriousness", our observations from the application of "identical" BP6 cells either intraperitoneally or subcutaneously. In connection with the concept that tumor development is not only a portrayal of cells proliferation, we could presume that different "environment" will result in structurally different tumors. Morphological differences observed are not significant, but they are present (Fig. 2, Ref. 12). Full Text (Free, PDF) www.bmj.sk.