Multiphoton Label-Free ex-vivo imaging using a custom-built dual-wavelength microscope with chromatic aberrations compensation

Label-Free Multiphoton Microscopy is a very powerful optical microscopy that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of a Multiphoton approach, like longer penetration depths and intrinsic optical sectioning, while opening the possibility of serial examinations with different kinds of techniques. Among the many variations of Label-Free MPM, Higher Harmonic Generation (HHG) is one of the most intriguing due to its generally low photo-toxicity, which enables the examination of specimens particularly susceptible to photo-damages. HHG and common Two-Photon Microscopy (TPM) are well-established techniques, routinely used in several research fields. However, they require a significant amount of fine-tuning in order to be fully exploited and, usually, the optimized conditions greatly differ, making them quite difficult to perform in parallel without any compromise on the extractable information. Here we present our custom-built Multiphoton microscope capable of performing simultaneously TPM and HHG without any kind of compromise on the results thanks to two, separate, individually optimized laser sources with full chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples in order to prove the significant advantages of our approach

The Rapidly Evolving Concept of Whole Heart Engineering

Whole heart engineering represents an incredible journey with as final destination the challenging aim to solve end-stage cardiac failure with a biocompatible and living organ equivalent. Its evolution started in 2008 with rodent organs and is nowadays moving closer to clinical application thanks to scaling-up strategies to human hearts. This review will offer a comprehensive examination on the important stages to be reached for the bioengineering of the whole heart, by describing the approaches of organ decellularization, repopulation, and maturation so far applied and the novel technologies of potential interest. In addition, it will carefully address important demands that still need to be satisfied in order to move to a real clinical translation of the whole bioengineering heart concept

The light and shadow of senescence and inflammation in cardiovascular pathology and regenerative medicine

Recent epidemiologic studies evidence a dramatic increase of cardiovascular diseases, especially associated with the aging of the world population. During aging, the progressive impairment of the cardiovascular functions results from the compromised tissue abilities to protect the heart against stress. At the molecular level, in fact, a gradual weakening of the cellular processes regulating cardiovascular homeostasis occurs in aging cells. Atherosclerosis and heart failure are particularly correlated with aging-related cardiovascular senescence, that is, the inability of cells to progress in the mitotic program until completion of cytokinesis. In this review, we explore the intrinsic and extrinsic causes of cellular senescence and their role in the onset of these cardiovascular pathologies. Additionally, we dissect the effects of aging on the cardiac endogenous and exogenous reservoirs of stem cells. Finally, we offer an overview on the strategies of regenerative medicine that have been advanced in the quest for heart rejuvenation

A Comprehensive Comparison of Bovine and Porcine Decellularized Pericardia: New Insights for Surgical Applications

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e. bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species' scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species' scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use

Towards an increased biocompatibility in total heart replacements: a new hybrid membrane for artificial blood pumps and the whole bioengineered heart

The only definitive treatment currently used for end-stage heart failure (HF) is cardiac transplantation. However, the procedure is limited by organ shortage and the side-effects of life-long immunosuppression. The need for new therapeutic strategies paved the way for the development of mechanical circulatory supports (e.g., total artificial hearts, TAHs) and the whole bioengineered heart. Although the two approaches may appear to be in conflict, TAHs are intended for short- to middle-term applications and can be developed and clinically applied relatively faster; the bioengineered heart approaches a definitive solution but, although current results are promising, a full organ equivalent has not yet been achieved. The creation of a novel TAH internal lining and the generation of a decellularized matrix for total heart bioengineering were the aim of this doctoral thesis, in order to improve the common goal of biocompatibility in formulating whole heart replacements. The TAH approach was investigated by coupling a TriCol-decellularized bovine pericardial scaffold (DBP) with a medical-grade polycarbonate urethane. This combination gave rise to a hybrid membrane (HM) capable of overcoming materials’ reciprocal limitations. The DBP was assessed by histology, two-photon microscopy (TPM)-combined immunofluorescence and TPM-mediated morphometric quantifications. Following HM assembly, surface analyses showed that the polymer penetrated the DBP scaffold. In vitro cytocompatibility was performed according to ISO 10993-5 with human umbilical vein endothelial cells (HUVECs) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs). All tests confirmed unaltered morphology, proliferation, viability, and absence of cytotoxicity. No activation of the complement system, part of ISO 10993-4, was reported for HM. The poor endothelialization and occurrence of thromboembolism in TAH may be prevented by accelerating endothelial adhesion through immobilization of short synthetic peptides. TriCol DBP scaffolds were selectively and covalently linked by Arg-Glu-Asp-Val (REDV) and rhodamine-conjugated REDV (RhodREDV) at different concentrations. After functionalization, the amount of bound RhodREDV was quantified with a TPM. The bioactivity of REDV was evaluated by in vitro static seeding of HUVECs. Live/dead staining and MTS reduction quantification demonstrated improved early adhesion, viability and proliferation of HUVECs on 10-5 M REDV-functionalized DBP at 24 hours. The LDH assay reported negligible levels of cytotoxicity. Histological evaluations revealed a near-continuous type of cell lining. The first step towards whole heart bioengineering was performed by developing a preservative decellularization protocol for cardiac organs. Rat hearts were decellularized by a combination of myorelaxant, protease inhibitors, sodium dodecyl sulfate (SDS) and Triton X-100. All solutions were administered by retrograde coronary perfusion. The effectiveness of the protocol was evaluated by histology, TPM-combined immunofluorescence, DNA quantification, and proteomic analysis. Cytocompatibility was assessed according to ISO 10993-5 by static seeding of hBM-MSCs on isolated ventricles. The results confirmed unaltered morphology, maintained proliferation and the absence of cytotoxicity. Histology showed continuous monolayer of hBM-MSCs at 24 hours, whereas cell penetration was visible at days 7 and 14. Concluding, this doctoral thesis describes the progresses towards the biocompatibility of heart replacements. A first generation of HM, based on DBP and polyurethane, was created and demonstrated its suitability for TAH internal lining. The covalent and selective functionalization of 10-5 M REDV peptide on DBP proved its efficacy in accelerating endothelial adhesion and promoting cell proliferation. A more preservative and cytocompatible protocol for whole heart decellularization was developed and achieved complete cell removal well-maintaining cardiac matrix.Il trapianto di cuore è l’unico trattamento dell’insufficienza cardiaca terminale, ma la sua applicazione è limitata dalla carenza di organi e dagli effetti collaterali delle terapie immunosoppressive. La necessità di nuove strategie terapeutiche ha portato allo sviluppo delle assistenze meccaniche ed al cuore bioingegnerizzato. Sebbene possano sembrare in contrasto, i cuori artificiali totali (TAH) sono impiantati a breve/medio termine e possono essere sviluppati ed introdotti nella clinica più rapidamente, mentre il cuore bioingegnerizzato si avvicina ad una soluzione definitiva, ma nonostante i risultati promettenti non è ancora stato prodotto un organo equivalente completo. La creazione di un nuovo rivestimento interno per i TAH e la generazione di una matrice decellularizzata per la bioingegnerizzazione del cuore sono stati l’obiettivo di questa tesi di dottorato al fine di migliorare la biocompatibilità dei sostituti cardiaci. Il rivestimento per i TAH è stato realizzato accoppiando pericardio bovino decellularizzato TriCol (DBP) ad un poliuretano, producendo una membrana ibrida (HM). Il DBP è stato valutato con istologie, immunofluorescenza combinata con microscopia a due fotoni (TPM) e quantificazione morfometrica. Le analisi superficiali della HM hanno mostrato la penetrazione del polimero. La citocompatibilità in vitro (ISO 10993-5) è stata valutata con cellule endoteliali di vena ombelicale umana (HUVEC) e mesenchimali staminali derivate da midollo osseo umano (hBM-MSC). I test hanno confermato inalterate morfologia, proliferazione, vitalità ed assenza di citotossicità. Nessuna attivazione del sistema del complemento (ISO 10993-4) è stata segnalata per HM. La scarsa endotelizzazione ed il verificarsi di tromboembolia nei TAH possono essere prevenuti accelerando l’adesione endoteliale con brevi peptidi sintetici. Gli scaffold TriCol DBP sono stati selettivamente e covalentemente legati ad Arg-Glu-Asp-Val (REDV) e REDV coniugato con rodamina (RhodREDV). Dopo la funzionalizzazione, la quantità di RhodREDV legato è stata quantificata con il TPM. La bioattività di REDV è stata valutata mediante semina statica di HUVEC in vitro. Il live/dead staining e la quantificazione della riduzione di MTS hanno dimostrato adesione precoce e migliore vitalità e proliferazione delle cellule nei DBP funzionalizzati con 10-5 M di REDV a 24 ore. Il saggio LDH ha riportato livelli trascurabili di citotossicità. Le valutazioni istologiche hanno rivelato un rivestimento cellulare quasi continuo. Il primo passo verso la bioingegnerizzazione del cuore è stato eseguito sviluppando un protocollo di decellularizzazione più conservativo. I cuori di ratto sono stati decellularizzati con miorilassante, inibitori delle proteasi, sodio dodecil solfato e Triton X-100. Le soluzioni sono state somministrate mediante perfusione coronarica retrograda. L’efficienza del protocollo è stata valutata con istologie, immunofluorescenza combinata con TPM, quantificazione del DNA e analisi proteomica. La citocompatibilità è stata eseguita mediante semina statica di hBM-MSC in ventricoli isolati (ISO 10993-5). I risultati hanno confermato morfologia inalterata, mantenimento della proliferazione e assenza di citotossicità. Le hBM-MSC hanno formato un monolayer continuo a 24 ore, mentre a 7 e 14 giorni era visibile la penetrazione. Concludendo, questa tesi di dottorato descrive i progressi verso la biocompatibilità dei sostituti cardiaci. La prima generazione di HM, basata su DBP e poliuretano, ha dimostrato la sua idoneità per il rivestimento interno dei TAH. La funzionalizzazione covalente e selettiva di DBP con 10-5 M di REDV ha provato la sua efficacia nell’accelerare l’adesione e proliferazione endoteliale. Un protocollo più conservativo e citocompatibile per la decellularizzazione del cuore totale è stato sviluppato e ha determinato la completa rimozione delle cellule endogene, preservando la matrice cardiaca

Il modello di Hodgkin-Huxley: simulazioni MATLAB dell'andamento del potenziale d'azione. The Hodgkin-Huxley model: MATLAB simulations of the action potential trend

Il lavoro sviluppato in questo elaborato riguarda lo studio del modello di Hodgkin-Huxley per la membrana neuronale e, in particolare, la simulazione dell’andamento del potenziale d’azione e l’analisi qualitativa di altre grandezze a esso connesse, quali le conduttanze tempo-varianti e le correnti ioniche. Tali simulazioni sono state svolte al fine di verificare come il modello considerato riesca a ricreare, in maniera adeguata e puntuale, i meccanismi di generazione del potenziale d’azione, ovvero le modalità con cui la cellula nervosa trasmette e riceve le informazion

Il cuore bioingegnerizzato: valutazione di differenti metodi di decellularizzazione totale d'organo dal punto di vista biologico e biomeccanico

Nel presente lavoro, è stata applicata la tecnica della decellularizza- zione totale d’organo per perfusione retrograda e, sulla matrice risultante, sono state eseguite analisi per la caratterizzazione bio- logica e biomeccanica. I risultati hanno messo in evidenza che è stato possibile ottenere, mediante due dei tre protocolli adottati, scaffold matriciali efficacemente decellularizzati e dotati di geometria tridimensionale e vascolarizzazione intatt

The Biocompatibility Challenges in the Total Artificial Heart Evolution

There are limited therapeutic options for final treatment of end-stage heart failure. Among them, implantation of a total artificial heart (TAH) is an acceptable strategy when suitable donors are not available. TAH development began in the 1930s, followed by a dramatic evolution of the actuation mechanisms operating the mechanical pumps. Nevertheless, the performance of TAHs has not yet been optimized, mainly because of the low biocompatibility of the blood-contacting surfaces. Low hemocompatibility, calcification, and sensitivity to infections seriously affect the success of TAHs. These outstanding issues have led to the withdrawal of many prototypes during preclinical phases of testing. This review offers a comprehensive analysis of the pathophysiological events that may occur in the materials that make up TAHs developed to date. In addition, this review illustrates bioengineering strategies to prevent these events and describes the most significant steps toward the achievement of a fully biocompatible TAH. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 21 is June 4, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates

RegenHeart: A Time-Effective, Low-Concentration, Detergent-Based Method Aiming for Conservative Decellularization of the Whole Heart Organ

Heart failure is the worst outcome of all cardiovascular diseases and still represents nowadays the leading cause of mortality with no effective clinical treatments, apart from organ transplantation with allogeneic or artificial substitutes. Although applied as the gold standard, allogeneic heart transplantation cannot be considered a permanent clinical answer because of several drawbacks, as the side effects of administered immunosuppressive therapies. For the increasing number of heart failure patients, a biological cardiac substitute based on a decellularized organ and autologous cells might be the lifelong, biocompatible solution free from the need for immunosuppression regimen. A novel decellularization method is here proposed and tested on rat hearts in order to reduce the concentration and incubation time with cytotoxic detergents needed to render acellular these organs. By protease inhibition, antioxidation, and excitation–contraction uncoupling in simultaneous perfusion/submersion modality, a strongly limited exposure to detergents was sufficient to generate very well-preserved acellular hearts with unaltered extracellular matrix macro- and microarchitecture, as well as bioactivity