Sviluppo di scaffold ingegnerizzati con cellule staminali e valutazione del loro potenziale uso in cardiochirurgia attraverso un modello suino con circolazione extracorporea

Abstract

Congenital Heart Defects (CHDs) are the leading global cause of death in the first year of life. Truncus Arteriosus (TA) is a CHD characterized by a common aorta/pulmonary artery and a ventricular septal defect. When treating paediatric patients with CHD, corrective surgery often requires patches, conduits or valves to repair the defect. Right ventricular outflow tract (RVOT) reconstruction in patients with TA involves the use of a replacement patch. Synthetic and fixed grafts currently used in cardiac surgery are non-living, non-contractile and they lack growth potential, leading to increased risk of thrombosis, stenosis, calcification and reoperation. Several studies suggest that tissue-engineered grafts made from living, functional cells could grow and remodel in parallel with the patient, facilitate healing and help to recover cardiac function. To date, autologous stem cells represent the most promising cell source to engineer cardiovascular patches. The aim of this study was to develop engineered cardiac patches with the potential to be used for correction of CHD. Additionally, we established a cardiopulmonary bypass recovery piglet model for RVOT reconstruction to test tissue-engineered grafts. Mesenchymal stem cells (MSCs) were isolated from the thymus gland of both newborn piglets and children undergoing open-heart surgeries. Cell surface markers and functional characterization demonstrated the mesenchymal lineage of the thymus-derived stem cells. Three differentiation protocols were tested in order to identify the best strategy to generate cardiac committed cells from MSCs. Three scaffolds were developed and tested for their capability to allow cell engraftment and proliferation. Porcine pericardium and myocardium were decellularized and recellularized with undifferentiated and differentiated cells, which were cultured under static conditions. The biocompatibility of CorMatrix® was also investigated by culturing the seeded graft under either static or dynamic conditions. 20 - 25 kg piglets underwent general anaesthesia and the heart was accessed by sternotomy. Cardiopulmonary bypass was established by cannulating the ascending aorta and the inferior and superior vena cava. An incision over the RVOT and below the pulmonary valve was performed. The defect created was then patched using either an engineered-CorMatrix® or an unseeded control scaffold. The patches were stitched with the cell-seeded side facing the inner side of the heart. Two animals were terminated 4 months after surgery and one was sacrificed after 24 hours. Histological and immunofluorescent stainings were performed on the explanted grafts and right ventricles in order to evaluate the grafts degradation and integration within the surrounding tissue, cells migration and penetration into the scaffolds, possible immune-response generated and to characterize the cellular content of the explants. Human and porcine thymus-derived MSCs express the mesenchymal surface markers and efficiently differentiate into the three mesenchymal phenotypes. Cardiomyocyte-like cells were obtained from all the developed protocols as cardiac-specific markers were detected in the differentiated cells by immunocytochemistry and RT-PCR. No cellular nor nuclear materials were detected in the decellularized pericardium and myocardium, while the extracellular matrix structure and composition was well preserved, indicating that a successful decellularization was achieved. The recellularization of the decellularized materials and the CorMatrix® was efficient for both undifferentiated and differentiated cells. The optimal cell-scaffold-stimuli combination appeared to be CorMatrix® seeded with undifferentiated cells cultured under dynamic conditions. Two out of three piglets successfully recovered the surgical procedure. One animal was terminated prior the end point as the piglet was too anaemic after surgery. The histological examination of the 4 months-patches showed good integration of the scaffold within the surrounding tissue as the CorMatrix® was no longer detectable. New collagen-rich tissue built up from the implanted grafts and some cardiac-like areas were found in the graft-side of the explants. Immunofluorescent characterization of the samples confirmed that these areas expressed cardiac markers and that they were more abundant in the engineered-patch then in the unseeded scaffold. High degree of vascularization was observed in the newly formed tissue. The quantification of both the capillaries and the greater vessels demonstrated that there was more angiogenesis in the engineered graft then in the control. No major differences were observed in terms of endothelialisation as a mature layer of endothelium was observed in both samples. These data suggest that the presence of the seeded cells helped the remodelling of the graft and triggered the generation of new blood vessels. Evaluation of the engineered-graft that was explanted 24 hours post-surgery, showed the presence of a thick cell layer growing on the inner side of the scaffold. A strong inflammation process was on going, most likely due to the surgery performed on the day before. In conclusion, with this study we have validated a protocol for isolation and differentiation of thymus-derived MSCs and the use of these cells to engineer living grafts from decellularized xenogeneic materials and commercially available scaffolds. These results allow the generation of cardiac patches that have the potential to be used in corrective heart surgery. Furthermore, the in vivo study established a cardiopulmonary bypass juvenile porcine model for RVOT grafting.