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

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

Body image, emotional intelligence and quality of life in peritoneal dialysis patients

Background: End-stage-renal-disease is one of the most common chronic diseases, and peritoneal dialysis constitutes one of the replacement therapies. The aim of this study was to investigate the views of patients on peritoneal dialysis regarding their body image, to assess their quality of life and level of emotional intelligence. Methods: A cross-sectional study was performed with structured questionnaires. The sample of the study was the patients undergoing peritoneal dialysis and monitored by the nephrology clinics of 7 public hospitals in Greece. Results: A total of 102 completed questionnaires were collected and analyzed (68% response rate). The participants showed moderate degree of body-image dysphoria (mean = 1.29, SD = 0.94), moderate levels of emotional intelligence and experienced moderate quality of life. According to the statistical analysis, women reported worse body image (p = 0.013) and university graduates showed higher levels of emotionality (p = 0.016). The correlations between the quality of life questionnaire subscales and demographic characteristics revealed statistically significant relationships between marital status and the Physical Functionality subscale, where unmarried people had a better quality of life in this subscale (p = 0.042) and between postgraduate/doctoral degree holders and the subscale Patient Satisfaction (p = 0.035). Also, statistically significant relationships were found between occupation and the Social Interaction subscale, where those engaged in household activities and were unemployed (p = 0.022) showed better quality of life. Participants living in semi-urban areas had better quality of life on the subscale Burden of Kidney Disease (p = 0.034). Conclusion: ESRD patients on peritoneal dialysis suffer significant limitations related to disease and treatment modality. According to our findings, these affect both their body image as well as their quality of life. Improvement in emotional intelligence is the factor which plays an important mediating role in improving both body image and quality of life in patients on peritoneal dialysis

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

Role of the nuclear receptor HNF-4a in the hepatic gene expression

The Hepatocyte Nuclear Factor-4α (HNF-4α) is a liver enriched transcription factor that is important for hepatic differentiation and for the maintenance of the liver phenotype in the adult. In addition, HNF-4α is a crucial regulator of a large number of genes involved in glucose, fatty acid and cholesterol metabolism. Mutations in HNF-4α impair insulin secretion and cause type 2 diabetes (MODY-1). Like other members of the nuclear receptor superfamily, HNF-4α has a modular structure composed of six functional domains. Two well conserved domains to those of other nuclear receptors are the DNA-binding domain (DBD) and the ligand-binding domain (LBD), where it is located the ligand-dependent transactivation function AF- 2. HNF-4α was long considered an orphan receptor because it activates transcription in the absence of exogenously added ligand, but the recent solved crystal structure revealed the existence of constitutively bound fatty acids in the ligand binding pocket, thus defining fatty acids as the endogenous ligands. In order to map residues that are critical for HNF-4α constitutive activity, a number of site-directed mutagenesis studies was performed. Transcriptional analysis of point mutations of the residues in helices 3, 4 and 5, that have been shown to line the ligand-binding pocket and come in direct contact with the ligand, highlighted their importance in HNF-4α mediated transactivation. In particular mutations S181Y (H3), M182K (H3) and L219Q (H5) impair transactivation potential without affecting DNA binding and dimerization. Structural modelling reveals that the observed loss in transactivation can be attributed to local changes that the mutations produced in interactions with residues of their vicinity. Thus, it was demonstrated that these residues play an important role in maintaining the structural integrity of the HNF-4α ligand binding pocket. Interestingly, mutation R212G (Η4) has no dramatic effect on the transcriptional activity of HNF-4α. It was also studied the involvement of these amino acid residues in coactivation function, using the coactivators PGC-1 and SRC- 3. It was observed that mutations M182K and L219Q could not be enhanced by the action of the coactivators whereas S181Y that was inactive in the absence of coactivators was shown to regain transcriptional activation potential in their presence. These results reveal the existence of distinct residues with dual function in the LBD pocket of HNF-4α affecting both constitutive and coactivator-stimulated activity. On the other hand distinct residues may influence only ligand interaction. The functional data were confirmed with protein interaction assays. In addition, itΟ πυρηνικός παράγοντας των ηπατοκυττάρων (HNF-4α) είναι ένας μεταγραφικός παράγοντας που εκφράζεται κυρίως στο ήπαρ όπου ελέγχει λειτουργίες καθοριστικές για τη διαφοροποίηση των ηπατικών κυττάρων καθώς και για τη διατήρηση του ηπατικού φαινοτύπου. Επιπλέον ο HNF-4α ρυθμίζει την έκφραση ενός μεγάλου αριθμού γονιδίων που συμμετέχουν στο μεταβολισμό της γλυκόζης, των λιπαρών οξέων και της χοληστερόλης. Μεταλλάξεις του γονιδίου διαπιστώθηκε να εμποδίζουν την έκκριση ινσουλίνης οδηγώντας σε μια μορφή νεανικού διαβήτη τυπού ΙΙ (MODY-1). Ο HNF-4α ανήκει στην υπεροικογένεια των πυρηνικών υποδοχέων και όπως τα υπόλοιπα μέλη αποτελείται απο έξι διακριτές λειτουργικά περιοχές, με περισσότερο συντηρημένες μεταξύ των μελών, την περιοχή πρόσδεσης στο DNA (DNA Binding Domain, DBD) και την περιοχή πρόσδεσης του συνδέτη (Ligand Binding Domain, LBD) στην οποία και εντοπίζεται μια περιοχή μεταγραφικής ενεργοποίηση, η AF-2. Μέχρι πρόσφατα ο HNF-4α ανήκε στην κατηγορία των ορφανών υποδοχέων ενεργοποιώντας τη μεταγραφή απουσία εξωγενούς συνδέτη, όμως με τα πρόσφατα κρυσταλλογραφικά δεδομένα φάνηκε πως τα λιπαρά οξέα αποτελούν τους ενδογενείς συνδέτες του μορίου. Στην παρούσα εργασία μελετήθηκε η λειτουργική σημασία συγκεκριμένων αμινοξέων της LBD περιοχής και η επίδραση τους στη μεταγραφική ενεργότητα του HNF-4α. Η μεταγραφική ανάλυση πραγματοποιήθηκε με την εισαγωγή σημειακών μεταλλάξεων σε αμινοξέα στις έλικες 3, 4 και 5 της LBD περιοχής, τα οποία εντοπίζονται στην κοιλότητα πρόσδεσης του συνδέτη και έρχονται σε επαφή μαζί του. Πιο συγκεκριμένα οι μεταλλάξεις S181Y (H3), M182K (H3) και L219Q (H5) οδήγησαν σε μείωση της μεταγραφικής ενεργότητας του HNF-4α, χωρίς να επηρεάζουν τις ιδιότητες του μορίου να προσδένεται στο DNA και να διμερίζεται. Με τη βοήθεια δομικών μοντέλων διαπιστώθηκε πως οι μεταλλάξεις αυτές οδηγούν σε μείωση της ενεργότητας εξαιτίας των τοπικών αλλαγών που προκαλλούν στις αλληλεπιδράσεις με τα γειτονικά αμινοξέα, υποδηλώνοντας οτι τα αμινοξέα αυτά είναι σημαντικά για τη διατήρηση της δομικής ακεραιότητας της LBD περιοχής του HNF-4α. Αντίθετα, η μετάλλαξη R212G (Η4) δε φάνηκε να επηρεάζει τη μεταγραφική ενεργότητα του HNF-4α. Στη συνέχεια μελετήθηκε η επίδραση των μεταλλάξεων αυτών στη μεταγραφική ενεργότητα του μορίου παρουσία των συνενεργοποιητών της μεταγραφής, PGC-1 και SRC-3. Παρατηρήθηκε πως οι μεταλλάξεις M182K και L219Q παραμένουν ανενεργές ακόμη και παρουσία συνενεργοποιητή σε αντίθεση μ

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells

Background/Aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion

Evaluation of Cocaine Effect on Endogenous Metabolites of HepG2 Cells Using Targeted Metabolomics

Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties

Abstract Background Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. Methods DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. Results Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions. Conclusions These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications