Biocompatibility and immunogenicity of decellularised allogeneic aorta in the orthotopic rat model

Background and aim of the study: Peripheral arterial disease causes blood vessel dysfunction that requires surgical intervention. Current surgical interventions employ synthetic or allogeneic vascular grafts, which offer biocompatible materials solutions that are not able to regenerate or grow with the patient. Decellularised scaffolds have gained significant momentum in the past few years, since they have the potential to regenerate in the patient. The aim of this study was to investigate the effects of modified decellularisation protocol on the biocompatibility and immunogenicity of allogeneic rat abdominal aorta in an orthotopic rat model. Methods: Native syngeneic Wistar (W) and allogeneic Dark Agouti (DA) aortas, together with decellularised allogeneic DA aortas, were assessed histologically, immunohistochemically and biomechanically. The immunogenicity of the untreated and decellularized syngeneic and allogeneic grafts was assessed in W rats, implanted orthotopically. Following implantation for 6 weeks, the grafts were explanted and assessed for the presence of T cells and macrophages by immunohistochemistry, and for their biomechanical integrity and histoarchitecture. Results: No obvious histoarchitectural differences were observed between the native W and DA aortas, with both presenting similar three-layered structures. Histological analysis of decellularized DA aortas did not reveal any remaining cells. Explanted native DA allografts showed media necrosis, partial elastic fibre degradation and adventitia thickening, as well as infiltration by lymphocytes (CD3+, CD4+) and macrophages (CD68+) in the adventitia. The explanted decellularized DA allografts indicated reduced immune injury compared to the explanted native DA allografts. The explanted native W syngeneic grafts showed a mild immune response, with an intact media and no lymphocyte infiltration. The explanted native DA allografts showed significantly lower collagen phase slope than the decellularized DA allografts prior implantation, and significantly higher thickness than the explanted decellularized DA allografts. Conclusions: The results indicated that the modified decellularization protocol did not affect significantly the mechanical and histological properties of the native DA rat aorta. Overall, the immune response was improved by decellularization. Native DA allografts induced an adverse immune response in W rats, whereas syngeneic W grafts showed good tissue integration

Scaffold creation from human umbilical cord for restoring disorders of the peripheral nervous system

One of the biggest challenges in surgery is the restoration of the peripheral nerves. Despite the great advances in microsurgery in cases where there is a gap between the nerve stumps, complete recovery of nerve function has not been achieved. In such lesions, the use of nerve conduit is necessary for bridging the gap between the nerve stumps. A wide variety of biomaterials are being used to promote functional recovery of injured nerves, such as autologous nerve graft, synthetic materials that are non-biodegradable, or biodegradable, natural materials such as collagen, chitosan, and keratin. Furthermore, various tissues of non-neural origin such as vessels, muscles, and intestine submucosa, have been used as nerve conduits. The aim of this study is to accomplish a successful decellularization of the human umbilical artery (hUA) and then its use as a nervous guide.Initially, the hUAs were decellularized, with the use of hypertonic solutions and detergents. Subsequently, evaluation of the decellularization performed, with the use of histological stains, DNA quantification and cytotoxicity analysis. Furthermore, a segmental peripheral nerve injury created in 24 Sprague–Dawley rats. The animals were organized into three experimental groups with different forms of repair: autologous nerve graft (n = 12), decellularized hUA (n = 12), de-cellularized hUA in combination with PRP (n = 12).For functional recovery evaluation, sciatic faction index and gastrocnemius muscle weight ratio estimated. For the nerve fibre regeneration evaluation, nerve morphometry and immunohistochemistry analysis performed. According to the results of this study, the hUA can support the proliferation of regenerated nerve fibers in a 10 mm nerve gap. Additionally, the combination of hUA and PRP, have the same results using an autologous nerve graft.Μια από της μεγαλύτερες προκλήσεις της χειρουργικής είναι η αποκατάσταση διατμηθέντων περιφερικών νευρών. Παρά τη μεγάλη πρόοδο στη μικροχειρουργική στις περιπτώσεις που υπάρχει κενό μεταξύ των κολοβωμάτων του διατμηθέντος νεύρου, η πλήρης αποκατάσταση παραμένει ζητούμενο. Για την αποκατάσταση τέτοιων βλαβών, είναι απαραίτητη η εφαρμογή νευρικών οδηγών ώστε να γεφυρωθεί το κενό μεταξύ των κολοβωμάτων. Ως νευρικοί οδηγοί έχουν χρησιμοποιηθεί αυτόλογα νευρικά μοσχεύματα, συνθετικά υλικά μη βιοαποικοδο-μήσιμα ή βιοαποικοδομήσιμα, φυσικά υλικά όπως το κολλαγόνο, η χιτοζάνη και η κερατίνη. Ακόμα ως νευρικοί οδηγοί έχουν χρησιμοποιηθεί διάφοροι ιστοί μη νευ-ρικής προέλευσης όπως αγγεία, μύες και ο υποβλεννογόνος του εντέρου. Ο σκοπός της παρούσας μελέτης είναι η επιτυχής αποκυτταροποίηση της ανθρώπινης ομφάλιας αρτηρίας και στη συνέχεια η χρήση της ως νευρικός οδηγός. Αρχικά αποκυτταροποιήθηκαν n= 25 ομφάλιες αρτηρίες με τη χρήση υπέρτονων διαλυμάτων και απορρυπαντικών. Ακολούθησε η αξιολόγηση της αποκυτταροποίησης με τη χρήση ιστολογικών χρώσεων, ποσοτικοποίηση του DNA που παραμένει μετά την αποκυτταροποίηση και τέλος πραγματοποιήθηκε έλεγχος κυτταροτοξικότητας. Στη συνέχεια οι ΑΟΑ χρησιμοποιήθηκαν ως νευρικοί οδηγοί σε μοντέλο ισχιακού τραύματος σε επίμυ με έλλειμμα 10 mm. Οι επίμυες καταμε-ριστήκαν σε τρεις διαφορετικές ομάδες: ομάδα control n= 12 που ως νευρικός οδηγός χρησιμοποιήθηκε αυτόλογο ισχιακό νεύρο, η ομάδα ΑΟΑ n= 12 που χρη-σιμοποιήθηκαν ΑΟΑ, και η ομάδα ΑΟΑ+PRP n= 12 που χρησιμοποιήθηκαν ΑΟΑ που στο εσωτερικό τους εγχύθηκε ενεργοποιημένο PRP. Για την αξιολόγηση της νευρικής αποκατάστασης εφαρμόστηκε η δοκιμασία της ανάλυσης βαδίσματος και ο υπολογισμός του δείκτη λειτουργικότητας του ισχιακού νεύρου. Επιπλέον πραγματοποιήθηκε μορφομετρική και ανοσοϊστοχημική ανάλυση του ισχιακού νεύρου, καθώς επίσης υπολογίστηκε ο λόγος διατήρησης μυϊκής μάζας. Το συμπέρασμα της παρούσας διατριβής είναι ότι η ΑΟΑ μπορεί να υπο-στηρίξει την επιμήκυνση των αναγεννηθέντων νευρικών ινών σε νευρικό έλλειμμα 10 mm. Ωστόσο υστερεί σε σχέση με τη χρήση αυτόλογου νεύρου ως νευρικός οδηγός. Αντίθετα όταν η ΑΟΑ συνδυάστηκε με PRP είχε εξίσου καλά αποτελέσματα με τη χρήση αυτόλογου νεύρου

Δημιουργία ικριώματος από ανθρώπινο ομφάλιο λώρο για την αποκατάσταση δυσλειτουργιών του περιφερικού νευρικού συστήματος.

Μια από της μεγαλύτερες προκλήσεις της χειρουργικής είναι η αποκατάσταση διατμηθέντων περιφερικών νευρών. Παρά τη μεγάλη πρόοδο στη μικρο-χειρουργική στις περιπτώσεις που υπάρχει κενό μεταξύ των κολοβωμάτων του διατμηθέντος νεύρου, η πλήρης αποκατάσταση παραμένει ζητούμενο. Για την αποκατάσταση τέτοιων βλαβών, είναι απαραίτητη η εφαρμογή νευρικών οδηγών ώστε να γεφυρωθεί το κενό μεταξύ των κολοβωμάτων. Ως νευρικοί οδηγοί έχουν χρησιμοποιηθεί αυτόλογα νευρικά μοσχεύματα, συνθετικά υλικά μη βιοαποικοδο-μήσιμα ή βιοαποικοδομήσιμα, φυσικά υλικά όπως το κολλαγόνο, η χιτοζάνη και η κερατίνη. Ακόμα ως νευρικοί οδηγοί έχουν χρησιμοποιηθεί διάφοροι ιστοί μη νευρικής προέλευσης όπως αγγεία, μύες και ο υποβλεννογόνος του εντέρου. Ο σκοπός της παρούσας μελέτης είναι η επιτυχής αποκυτταροποίηση της ανθρώπινης ομφάλιας αρτηρίας και στη συνέχεια η χρήση της ως νευρικός οδηγός. Αρχικά αποκυτταροποιήθηκαν n= 25 ομφάλιες αρτηρίες με τη χρήση υπέρ-τονων διαλυμάτων και απορρυπαντικών. Ακολούθησε η αξιολόγηση της αποκυτταροποίησης με τη χρήση ιστολογικών χρώσεων, ποσοτικοποίηση του DNA που παραμένει μετά την αποκυτταροποίηση και τέλος πραγματοποιήθηκε έλεγχος κυτταροτοξικότητας. Στη συνέχεια οι ΑΟΑ χρησιμοποιήθηκαν ως νευρικοί οδηγοί σε μοντέλο ισχιακού τραύματος σε επίμυ με έλλειμμα 10 mm. Οι επίμυες καταμεριστήκαν σε τρεις διαφορετικές ομάδες: ομάδα control n= 12 που ως νευρικός οδηγός χρησιμοποιήθηκε αυτόλογο ισχιακό νεύρο, η ομάδα ΑΟΑ n= 12 που χρη-σιμοποιήθηκαν ΑΟΑ, και η ομάδα ΑΟΑ+PRP n= 12 που χρησιμοποιήθηκαν ΑΟΑ που στο εσωτερικό τους εγχύθηκε ενεργοποιημένο PRP. Για την αξιολόγηση της νευρικής αποκατάστασης εφαρμόστηκε η δοκιμασία της ανάλυσης βαδίσματος και ο υπολογισμός του δείκτη λειτουργικότητας του ισχιακού νεύρου. Επιπλέον πραγματοποιήθηκε μορφομετρική και ανοσοϊστοχημική ανάλυση του ισχιακού νεύρου, καθώς επίσης υπολογίστηκε ο λόγος διατή-ρησης μυϊκής μάζας. Το συμπέρασμα της παρούσας διατριβής είναι ότι η ΑΟΑ μπορεί να υποστηρίξει την επιμήκυνση των αναγεννηθέντων νευρικών ινών σε νευρικό έλλειμμα 10 mm. Ωστόσο υστερεί σε σχέση με τη χρήση αυτόλογου νεύρου ως νευρικός οδηγός. Αντίθετα όταν η ΑΟΑ συνδυάστηκε με PRP είχε εξίσου καλά αποτελέσματα με τη χρήση αυτόλογου νεύρου.One of the biggest challenges in surgery is the restoration of the peripheral nerves. Despite the great advances in microsurgery in cases where there is a gap between the nerve stumps, complete recovery of nerve function has not been achieved. In such lesions, the use of nerve conduit is necessary for bridging the gap between the nerve stumps. A wide variety of biomaterials are being used to promote functional recovery of injured nerves, such as autologous nerve graft, synthetic materials that are non-biodegradable, or biodegradable, natural materi-als such as collagen, chitosan, and keratin. Furthermore, various tissues of non-neural origin such as vessels, muscles, and intestine submucosa, have been used as nerve conduits. The aim of this study is to accomplish a successful decellularization of the human umbilical artery (hUA) and then its use as a nervous guide. Initially, the hUAs were decellularized, with the use of hypertonic solutions and detergents. Subsequently, evaluation of the decellularization performed, with the use of histological stains, DNA quantification and cytotoxicity analysis. Fur-thermore, a segmental peripheral nerve injury created in 24 Sprague–Dawley rats. The animals were organized into three experimental groups with different forms of repair: autologous nerve graft (n = 12), decellularized hUA (n = 12), de-cellularized hUA in combination with PRP (n = 12). For functional recovery evaluation, sciatic faction index and gastrocnemius muscle weight ratio estimated. For the nerve fibre regeneration evaluation, nerve morphometry and immunohistochemistry analysis performed. According to the results of this study, the hUA can support the proliferation of regenerated nerve fibers in a 10 mm nerve gap. Additionally, the combination of hUA and PRP, have the same results using an autologous nerve graft

Myeloid-Derived Suppressor Cells (MDSC) in the Umbilical Cord Blood: Biological Significance and Possible Therapeutic Applications

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells that suppress immune responses in cancer, infection, and trauma. They mainly act by inhibiting T-cells, natural-killer cells, and dendritic cells, and also by inducing T-regulatory cells, and modulating macrophages. Although they are mostly associated with adverse prognosis of the underlying disease entity, they may display positive effects in specific situations, such as in allogeneic hematopoietic stem cell transplantation (HSCT), where they suppress graft-versus-host disease (GVHD). They also contribute to the feto-maternal tolerance, and in the fetus growth process, whereas several pregnancy complications have been associated with their defects. Human umbilical cord blood (UCB) is a source rich in MDSCs and their myeloid progenitor cells. Recently, a number of studies have investigated the generation, isolation, and expansion of UCB-MDSCs for potential clinical application associated with their immunosuppressive properties, such as GVHD, and autoimmune and inflammatory diseases. Given that a significant proportion of UCB units in cord blood banks are not suitable for clinical use in HSCT, they might be used as a significant source of MDSCs for research and clinical purposes. The current review summarizes the roles of MDSCs in the UCB, as well as their promising applications

Administration of Adipose Derived Mesenchymal Stem Cells and Platelet Lysate in Erectile Dysfunction: A Single Center Pilot Study

Erectile dysfunction (ED) affects more than 30 million men; endothelial dysfunction plays a significant role in EDs pathogenesis. The aim of this study was to administer mesenchymal stem cells (MSC) derived from adipose tissue and platelet lysate (PL) into patients with erectile dysfunction. This pilot study enrolled eight patients with diagnosed ED. Patients enrolled were suffering from organic ED due to diabetes melitus, hypertension, hypercholesterolaemia, and Peyronie disease. The patients were distributed in 2 groups. Patients in group A received adipose derived mesenchymal stem cells (ADMSC) resuspended in PL while patients in group B received only PL. ADMSCs were isolated from patients’ adipose tissue and expanded. In addition, blood sampling was obtained from the patients in order to isolate platelet lysate. After the application of the above treatments, patients were evaluated with an International Index of Erectile Function (IIEF-5) questionnaire, penile triplex, and reported morning erections. After MSCs and PL administration, patients presented improved erectile function after 1 and 3 months of follow-up. A statistically significant difference was observed in the IIEF-5 score before and after administration of both treatments after the first month (p < 0.05) and the third month (p < 0.05). No statistically significant difference was observed in the IIEF-5 score between group A and B patients. All patients were characterized by improved penile triplex and increased morning erections. No severe adverse reactions were observed in any patient except a minor pain at the site of injection, which was in the limits of tolerability. The results of this study indicated the satisfactory use of MSCs and PL in ED. MSCs in combination with PL or PL alone seems to be very promising, especially without having the negative effects of the current therapeutic treatment

Short Term Results of Fibrin Gel Obtained from Cord Blood Units: A Preliminary in Vitro Study

BACKGROUND: Recent findings have shown that the fibrin gel derived from cord blood units (CBUs) play a significant role in wound healing and tissue regeneration. The aim of this study was to standardize the fibrin gel production process in order to allow for its regular use. METHODS: CBUs (n = 200) were assigned to 4 groups according to their initial volume. Then, a two-stage centrifugation protocol was applied in order to obtain platelet rich plasma (PRP). The concentration of platelets (PLTs), white blood cells (WBCs) and red blood cells (RBCs) were determined prior to and after the production process. In addition, targeted proteomic analysis using multiple reaction monitoring was performed. Finally, an appropriate volume of calcium gluconate was used in PRP for the production of fibrin gel. RESULTS: The results of this study showed that high volume CBUs were characterized by greater recovery rates, concentration and number of PLTs compared to the low volume CBUs. Proteomic analysis revealed the presence of key proteins for regenerative medicine. Fibrin gel was successfully produced from CBUs of all groups. CONCLUSION: In this study, low volume CBUs could be an alternative source for the production of fibrin gel, which can be used in multiple regenerative medicine approaches

Investigation of the biomechanical integrity of decellularized rat abdominal aorta

Objectives. The loss or damage of an organ or tissue is one of the most common and devastating problems in healthcare today. Tissue engineering applies the principles of engineering and biology toward the development of functional biological replacements that are able to maintain, improve, or restore the function of pathological tissues. The aim of the overall project is to study an already existing method for the decellularization of homograft vascular grafts for use in vascular surgery. Materials and Methods. The biomechanical integrity of native and decellularized rat aortas was assessed under uniaxial tension tests. For this purpose, 36 male rats (12 Wistar and 24 Dark Agouti [DA]) were used to excise their abdominal aortas. Twelve of the aortas were tested fresh (Wistar and DA rats), within 24 hours from euthanasia, and the rest were decellularized using a modified protocol (DA rats only). Fresh and decellularized samples (n ¼ 12) were subjected to uniaxial tensile loading to failure, and the recorded stress-strain behaviour of each specimen was assessed in terms of 6 biomechanical parameters. Results. No statistically significant differences were found in any of the biomechanical parameters studied between the decellularized DA rat aorta group and both the native DA and Wistar rat aorta groups (P > .05). Also, no significant difference was shown between the native DA and native Wistar rat aorta groups. Conclusions. The results from this study have shown that the decellularization protocol did not affect the mechanical properties of the native rat aorta. In addition to this, both native Wistar and native/decellularized DA rat aorta groups shared similar mechanical properties

Perspectives for the Use of Umbilical Cord Blood in Transplantation and Beyond: Initiatives for an Advanced and Sustainable Public Banking Program in Greece

The umbilical cord blood (UCB) donated in public UCB banks is a source of hematopoietic stem cells (HSC) alternative to bone marrow for allogeneic HSC transplantation (HSCT). However, the high rejection rate of the donated units due to the strict acceptance criteria and the wide application of the haploidentical HSCT have resulted in significant limitation of the use of UCB and difficulties in the economic sustainability of the public UCB banks. There is an ongoing effort within the UCB community to optimize the use of UCB in the field of HSCT and a parallel interest in exploring the use of UCB for applications beyond HSCT i.e., in the fields of cell therapy, regenerative medicine and specialized transfusion medicine. In this report, we describe the mode of operation of the three public UCB banks in Greece as an example of an orchestrated effort to develop a viable UCB banking system by (a) prioritizing the enrichment of the national inventory by high-quality UCB units from populations with rare human leukocyte antigens (HLA), and (b) deploying novel sustainable applications of UCB beyond HSCT, through national and international collaborations. The Greek paradigm of the public UCB network may become an example for countries, particularly with high HLA heterogeneity, with public UCB banks facing sustainability difficulties and adds value to the international efforts aiming to sustainably expand the public UCB banking system