Morphological characteristics on a Scanning Electron Microscope of generated hyaline cartilage tissue from adipose mesenchymal stem cells, on Polycaprolactone scaffolds

Cartilage regeneration is of great interest to the medical community, given the prevalence of osteocartilage defects in the population coupled with the tissue’s low intrinsic self-repair potential. A recently new FDA approved biomaterial and 3D-printing technology provided us the opportunity to fabricate tailor made scaffolds. We used collagen coated and uncoated scaffolds to develop hyaline cartilage from adipose mesenchymal stem cells (ADMSCs). The aim of this study is to present the scaffolds’ morphological characteristics, as observed on a Scanning Electron Microscope (SEM) of generated cartilage tissue and to compare the two types of scaffolds. Cylindrical shaped PCL scaffolds, 10mm in diameter, were fabricated. ADMSCs were harvested and were cultivated on PCL scaffolds. Half of the scaffolds were treated with collagen I by coating. After 26 days in culture, the scaffolds were examined by SEM. Visualization was succeeded on the top and bottom surfaces and on the cross sections of each scaffold. At day 26, scaffolds revealed extensive colonization and viability of ADMSCs, with concurrent depositions of extracellular matrix. SEM images show that surfaces were covered with a significant amount of material with a glossy, transparent appearance, indicating the development of regenerated cartilage, more apparent on the coated scaffolds. Cultured cells demonstrated aligned direction on the scaffolds' fibers and the ECM that was produced connected the pores of the scaffolds by building apparent bridges between them. The penetration of cells was limited in the coated scaffold. We used 3D printing technology for PCL scaffold production, towards a cartilaginous implant development. SEM images provide us visualization of the scaffolds with the newly developed cartilage tissue and demonstrate that the scaffolds’ purpose for chondrogenesis was served successfully in all cases and PCL displayed good biocompatibility. Collagenation of scaffolds led to a higher density of cells on the surfaces but also to a limited penetration within, not fully serving the purpose of a 3D culture

Ultrastructural study of hepatic stellate cells ( Ito cells) after teratogenic drug treatment in pregnancy: Experimental study in Balb/c mice

Introduction-Aim of the study: Ito cells are the hepatic stellate cells in the space of Disse. They play a key role in the development and regeneration of liver. Retinoid analogues are used therapeutically, while hydroxyurea (HU) is an antiretroviral and chemotherapeutic agent. The aim of this study is to evaluate the effect of the above substances on Ito cells’ ultrastructural morphology by TEM observations.Materials-Methods: Six groups of pregnant Balb/C mice were used for the study. All animals were treated on gestational days 10th, 11th, 12th. The first group was treated with All-Trans Retinoic Acid (RA), the second group with Retinyl Palmitate (RP), the third group with a combination of 13-cis RA and RP and the fourth group with HU. The fifth and the sixth groups were the control animals. Animals were sacrificed on the 19th gestational day. Livers from all animals were removed and were properly prepared for TEM observation.Results: Observation of the liver of All-Trans RA treated pregnant animals revealed Ito cell activation. Microbodies similar to peroxisomes and lysosomes were present in their cytoplasm. Similar findings were found in RP treated animals. In the activated stellate cells, the presence of microfibrils around their nuclei appeared with chromatin condensation in the periphery. In the liver of animals treated with a combination of RP and 13-Cis RA, microfibrils in the cytoplasm, fibrosis and extracellular edema were observed around the sinusoids. In the liver of HU treated animals, peroxisomes with different density were observed in the hepatocyte cytoplasm, surrounded by lipid droplets. In the cytoplasm of activated Ito cells, low density peroxisomes were observed. Fibrosis and extracellular edema were observed surrounding the sinusoids among the stellate cells and in the space of Disse.Conclusions: This ultrastructural study indicates that the drugs induced Ito cell activation and caused possibly irreversible damage to liver parenchyma

Design of a laboratory bioreactor for engineering articular cartilage based on 3D printed nasal septum-like scaffolds

«Η εκφύλιση των χόνδρων είναι μια σοβαρή πάθηση που επηρεάζει μεγάλο μέρος του πληθυσμού σε όλο το ηλικιακό φάσμα. Επί του παρόντος χρησιμοποιούνται διάφορες τεχνικές αποκατάστασης για μικρής έκτασης βλάβες όπως η αρθροπλαστική απόξεσης και ο υποχονδρικός τρυπανισμός, οι οποίες δεν μπορούν να επιδιορθώσουν βλάβες μεγαλύτερης έκτασης. Η Αναγεννητική Ιατρική προωθεί την Μηχανική Ιστών στο προσκήνιο των σύγχρονων μηχανικών τεχνικών, συνδυάζοντας καινοτόμα βιοσυμβατά υλικά, νέες μεθόδους μηχανικής ιστών όπως η τεχνολογία 3D εκτύπωσης και βιοδιαδικασίες που αποσκοπούν στην δημιουργία ποιοτικών μοσχευμάτων για εκτεταμένες βλάβες των χόνδρων. Κατάλληλο κυτταρικό περιβάλλον για δημιουργία ιστών μπορεί να επιτευχθεί με την ανάπτυξη αυτών των μοσχευμάτων σε βιοαντιδραστήρες. O κάθε βιοαντιδραστήρας χρησιμοποιεί διαφορετικές αρχές καλλιέργειας, και ορισμένοι από αυτούς όπως οι μικτού τύπου και οι βιοαντιδραστήρες διαπότισης επιστρατεύουν την άσκηση μηχανικών δυνάμεων επί του ικριώματος ώστε να επιτευχθεί μεγάλη κυτταρική πυκνότητα και ενισχυμένες μηχανικές ιδιότητες που οδηγούν στην δημιουργία καλύτερης ποιότητας χόνδρου. Αυτές οι ιδιαιτερότητες των βιοαντιδραστήρων μπορούν να αποτελέσουν εφαλτήριο κατασκευής εργαστηριακών βιοαντιδραστήρων, για την καλλιέργεια 3D εκτυπωμένων ρινικών διαφραγμάτων ως ένα λειτουργικό παράδειγμα υαλώδους χόνδρου».Cartilage degeneration is a severe disease affecting a significant part of the population at all ages. Various treatment modalities are currently used for small-sized cartilage defects, such as abrasion arthroplasty and subchondral drilling, but fail to repair larger-scale damages. Regenerative Medicine pushes Tissue Engineering (TE) to the forefront of modern engineering techniques combining novel biocompatible materials, new tissue engineering methods, like 3D printing technology and bioprocesses trying to create quality transplants for large cartilage defects. The appropriate cell environment for engineered tissues can be achieved through growth of the tissue-engineered constructs into bioreactors. Each bioreactor uses different principles for culturing processes, and some of them mostly mixed and perfusion bioreactors, use different kind of mechanical forces on the scaffold to achieve high cell densities, enhanced mechanical properties leading to better quality of engineered cartilage. These advantageous particularities can be used to create a laboratory bioreactor design, for culturing 3D printed nasal septum cartilage as a working example of hyaline cartilage

Translational research for nasal septum cartilage regeneration with chondrocytes derived from differentiated human adipose mesenchymal stem cells

Η εργασία αφορά στη μεταφραστική έρευνα ιστοτεχνολογίας και συγκεκριμένα στη δημιουργία ανθρώπινου ρινικού διαφράγματος με τη χρήση ηλεκτρονικά υποβοηθούμενου σχεδιασμού και τρισδιάστατης εκτύπωσης τρισδιάστατου (3D) πορώδους ικριώματος χιτοζάνης/ζελατίνης (CAD/CAM). Το ικρίωμα θα χρησιμοποιηθεί για να αποικιστεί από χρονδροκύτταρα που προκύπτουν από διαφοροποιημένα μεσεγχυματικά κύτταρα ανθρώπου προερχόμενα από λιπώδη ιστό (Adipose Tissue Mesenchymal Stem Cells-AD- MSCs). Η όλη διαδικασία επιτυγχάνεται με τη χρήση βιοαντιδραστήρα.Τα μεσεγχυματικά κύτταρα είναι πολυδύναμα βλαστοκύτταρα που μπορούν να απομονωθούν από το μυελό των οστώνκαι το λιπώδη ιστό. Τα κύτταρα αυτά έχουν τη δυνατότητα να διαφοροποιούνται, υπό εργαστηριακές συνθήκες, σε οστεοκύτταρα, χονδροκύτταρα, και λιποκύτταρα. Στην παρούσα μελέτη ανθρώπινα μεσεγχυματικά κύτταρα απομονώθηκαν από λιπώδη ιστό και καλλιεργήθηκαν in vitro. Η έκφραση των αντιγόνων επιφανείας CD90, CD73, σε συνδυασμό με την απουσία του μάρτυρα CD45 επιβεβαιώνουν την επιτυχή απομόνωση μεσεγχυματικών βλαστικών κυττάρων, με χρήση κυτταρομετρίας ροής. Έπειτα από 21 ημέρες από την επαγωγή στοχευόμενης διαφοροποίησης τα βλαστοκύτταρα διαφοροποιήθηκαν σε χονδροκύτταρα και χαρακτηρίστηκαν ιστολογικά με χρώση κυανού της τολουιδίνης και μοριακά με RTPCR για δείκτες διαφοροποίησης όπως η αγκρεκάνη. Με τη χρήση του τρισδιάστατου εκτυπωτή δημιουργήθηκε υπό κλίμακα ικρίωμα ρινικού χόνδρου από PLA. Η διαδικασία θα ολοκληρωθεί με την εκτύπωση του υπό διερεύνηση υλικού χιτοζάνης/ζελατίνης σε 3D ικρίωμα και αφού εμποτιστεί με χονδροκύτταρα θα μεταφερθεί στον βιοαντιδραστήρα.Mesenchymal stem cells (MSCs) are multipotent cells isolated from various tissues, mainly from the bone marrow and adipose tissue. Their ability to differentiate into osteoblasts, chondrocytes or adipocytes renders them a promising clinical tool for injury repair and tissue regeneration. In the current study, MSCs were isolated from human adipose tissue (hAD-MSCs) and were triggered to differentiate into chondrocytes in vitro. Expression of mesenchymal stem cell markers, such as CD90 and CD73, in combination with the absence of hematopoietic markers, such as CD45, proves via flow cytometry the successful isolation of MSCs. Histologic staining with Toluidine blue and real time PCR analysis for the expression of the chondrogenic marker aggrecan (ACAN) verified the successful chondrogenic differentiation of AD-MSCs. Using Poly Lactic-Acid as scaffolding material, a three-dimensional scaffold with customized architecture, controlled porosity and interconnected porous structure was fabricated using 3D printing. The produced scaffold represents the morphology of the nasal septum cartilage. We aspire, to see this scaffold with the differentiated chondrocytes and culture the complex under the appropriate micoenvironmental conditions of a bioreactor system in order to regenerate a potential cartilage transplant. This in vitro study expands the potentials of human AD-MSCs to be used in clinic for alleviation of cartilage defects and tissue engineering in Greece and worldwide

Histological Alterations in Hashimoto’s Disease: A Case-Series Ultrastructural Study

Background: Hashimoto’s thyroiditis (HT) is an autoimmune disease exhibiting stromal fibrosis and follicular cell destruction due to lymphoplasmacytic infiltration. Besides deprecated analyses, histopathological approaches have not employed the use of electron microscopy adequately toward delineating subcellular-level interactions. Methods: Biopsies for ultrastructural investigations were obtained from the thyroids of five patients with HT after a thyroidectomy. Transmission electron microscopy (TEM) was utilized to study representative tissue specimens. Results: Examination indicated interstitial extravasated blood cells and a plethora of plasma cells, based on their subcellular identity landmarks. These antibody-secreting cells were profoundly spotted near follicular cells, fibroblasts, and cell debris entrenched in collagenous areas. Pathological changes persistently affected subcellular components of the thyrocytes, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and other intracellular vesicles. Interestingly, significant endothelial destruction was observed, specifically in the larger blood vessels, while the smaller vessels appeared comparatively unaffected. Conclusions: Our TEM findings highlight the immune-related alterations occurring within the thyroid stroma. The impaired vasculature component and remodeling have not been described ultrastructurally before; thus, further exploration is needed with regards to angiogenesis in HT in order to achieve successful prognostic, diagnostic, and treatment-monitoring strategies

Life in space: can we live and multiply?

Humanity has a perpetual presence in orbit outside the Earth, with an arising interest in human activities in space. To date, more than 550 astronauts have traveled beyond the one-hundred-kilometer distance that theoretically separates Earth from space. The International Space Station, as a unique research laboratory orbiting around Earth, is a fact, facilitating the investigation of factors affecting life in space. Experiments are set up to investigate the development of life under special conditions, such as earth’s magnetic fields, radiation, microgravity. Negative effects on human body have been registered but we still know little about the exact effects on male and female reproductive pathways, on the regulation of hypothalamus and pituitary function, on prenatal maturation of gametes and on future generations. For humanity to expand, it is crucial for humans to survive and develop in space. Small mammals and creatures have evolved in space, but we are still a long way from removing the barriers and easing the difficulties that come with this project. Although there has been a lot of research done up to this point, more data collection is still required in order to completely comprehend why it is so challenging for organisms to grow there

Addressing Spaceflight Biology through the Lens of a Histologist–Embryologist

Embryogenesis and fetal development are highly delicate and error-prone processes in their core physiology, let alone if stress-associated factors and conditions are involved. Space radiation and altered gravity are factors that could radically affect fertility and pregnancy and compromise a physiological organogenesis. Unfortunately, there is a dearth of information examining the effects of cosmic exposures on reproductive and proliferating outcomes with regard to mammalian embryonic development. However, explicit attention has been given to investigations exploring discrete structures and neural networks such as the vestibular system, an entity that is viewed as the sixth sense and organically controls gravity beginning with the prenatal period. The role of the gut microbiome, a newly acknowledged field of research in the space community, is also being challenged to be added in forthcoming experimental protocols. This review discusses the data that have surfaced from simulations or actual space expeditions and addresses developmental adaptations at the histological level induced by an extraterrestrial milieu