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Modeling Cardiac Congenital Diseases: From Mathematic Tools to Human Induced Pluripotent Stem Cells
Full text linkToward the Effective Bioengineering of a Pathological Tissue for Cardiovascular Disease Modeling: Old Strategies and New Frontiers for Prevention, Diagnosis, and Therapy
Full text linkCardiovascular diseases (CVDs) still represent the primary cause of mortality worldwide. Preclinical modeling by recapitulating human pathophysiology is fundamental to advance the comprehension of these diseases and propose effective strategies for their prevention, diagnosis, and treatment. In silico, in vivo, and in vitro models have been applied to dissect many cardiovascular pathologies. Computational and bioinformatic simulations allow developing algorithmic disease models considering all known variables and severity degrees of disease. In vivo studies based on small or large animals have a long tradition and largely contribute to the current treatment and management of CVDs. In vitro investigation with two-dimensional cell culture demonstrates its suitability to analyze the behavior of single, diseased cellular types. The introduction of induced pluripotent stem cell technology and the application of bioengineering principles raised the bar toward in vitro three-dimensional modeling by enabling the development of pathological tissue equivalents. This review article intends to describe the advantages and disadvantages of past and present modeling approaches applied to provide insights on some of the most relevant congenital and acquired CVDs, such as rhythm disturbances, bicuspid aortic valve, cardiac infections and autoimmunity, cardiovascular fibrosis, atherosclerosis, and calcific aortic valve stenosis
From the anatomical study to the application of different bioengineering techniques for the creation of new vital heart valve substitutes
Full text linkThis doctoral thesis focuses on the approaches currently in use to develop new vital valve devices: heart valve tissue engineering and heart valve tissue guided regeneration. Indeed, it investigates some aspects able to modify the preservation of the implanted construct, i.e. the immunogenic properties of the extracellular matrix and the biological entity of stem cell populations in heart valve leaflets.
The porcine semilunar valves, as the bovine pericardial derived ones, find large clinical usage for the substitution of dysfunctional valves. The rational of their employ is the high morphological and functional analogies with the native human ones and hence they can provide a valid extracellular matrix (ECM) for bioengineered prosthesis. Once eliminated the xenogeneic cell component through a decellularizing
treatment, it is possible to benefit from the extracellular fiber mesh as a template for repopulation with human stem cells in order to develop autologous-like replacements. So far the influence on cellular attachment exerted by the anisotropic ECM distribution in fibrosa and ventricularis has never been
investigated. Porcine pulmonary leaflets were decellularized with a Triton X110/Sodium Cholate-based protocol. The absence of alpha-Gal epitopes, highly immunogenic for the human species, has been demonstrated through a double fluorescence technique using an isolectin and a specific monoclonal antibody. After treatment with fetal bovine serum and fibronectin to increase adhesion, human bone marrow mesenchymal stem cells have been seeded either onto the ventricularis or fibrosa and statically maintained in culture for 30 days. Not only ventricularis is able to consent a higher attachment, but also increased spreading and early cell differentiation in the cusp stroma. The interaction between the same cells and decellularized human leaflets has been further evaluated by ventricularis seeding. The homologous combination favors proliferation, with decrease of apoptotic events and enhanced cell maturation level, so that it is possible to appreciate the expression of typical mature smooth muscle markers in ventricularis. ECM performs consequently an essential role for cell integration by furnishing specific signals for the acquisition of the correct valvular phenotype.
A further hypothesis for the achievement of new vital substitutes is likely represented by tissue guided regeneration operated in vivo by ECM in respect to the recipient's cells. Decellularized aortic roots were used to mimic a reconstruction of the RVOT in minipigs and performances were followed echocardiographically for 12-14 months, revealing a progressive function improvement. A continuous engraftment is observed in the explanted specimens, even if the layers less exposed to blood circulation are still devoid of cells. The observations on primary cultures of the vessel and leaflet tissues lead to hypothesize the contribution of two main phenotypes to repopulation: pulmonary artery smooth muscle
cells and mobilized mesenchymal stem cells.
Particularly important is the preservation of the bioconstruct once the function has been rescued, but at the same time it is essential to understand the key cell effectors participating to valve dysfunction in order to prevent the phenomenon. Scarcely studied, possible stem cell populations in the valves could be involved in the homeostatic tissue remodeling or in adverse pathophysiological events. Aortic roots (n=27) with related mitral leaflets (n=27) have been classified in 5 groups depending on allograft donor's age (10- 60 years). In addition, aortic and mitral pathological leaflets (n=10) were harvested during valve replacements. Cusp cryosections have been analysed through classical histology, Mallory's trichrome, Von
Kossa, Oil Red O and immunohistochemistry for differentiated, inflammatory, calcifying, different lineagederived stem cell markers. With aging the typical observed feature is the accumulation of lipids, both as small droplets in the subventricularis layer or as cholesterol crystals in the fibrosa. Calcifications appear rare. Stem cell epitopes are highly expressed in the leaflet to reveal a specific spatial distribution. Primary cultures obtained from these specimens showed a phenotype and a differentiation potentiality, suggestive
of mesenchymal stem progenitors without apparent osteogenic induction. Further studies are needed to better understand the contribution of these cells in heart valve calcification.
In conclusion, both proposed modalities for the achievement of new valve substitutes appear valid instruments for the aim, because relying on fully decellularized alpha-Gal negative matrices permissive of cell-repopulation. Future efforts and application of new biomimetic strategies would surely implement the encouraging results here demonstrated. New insights on stem cell valvular biology have been proposed here for the first time with important confirmations on the continuous cell remodeling interesting the cusp tissue.Tale tesi di dottorato si focalizza sugli approcci correntemente in uso per lo sviluppo di nuovi sostituti valvolari con caratteristiche di vitalità: l’ingegneria tissutale valvolare e la rigenerazione valvolare tessuto-guidata.
Inoltre, mette in luce alcuni aspetti in grado di influenzare la preservazione dell’impianto, come le proprietà immunogeniche della matrice extracellulare e l’entità biologica delle popolazioni staminali nei lembi valvolari cardiaci.
Le valvole semilunari porcine, come quelle di pericardio bovino, trovano largo impiego per la sostituzione di valvole non più funzionanti. Il razionale per l’impiego di esse è l’alta analogia morfologica e funzionale con le valvole umane, che le rende fonte ottimale di matrice extracellulare (ECM) per protesi bioingegnerizzate. Una volta rimossa la componente cellulare xenogenica attraverso un trattamento decellularizzante, è possibile beneficiare della trama di fibre extracellulari come supporto tridimensionale per il ripopolamento con cellule staminali umane in modo da sviluppare dei sostituti simil-autologhi.
Finora, non è mai stata indagata l’influenza esercitata dalla distribuzione anisotropica delle fibre dell’ECM in ventricularis e fibrosa sull’adesione cellulare. Lembi polmonari porcini sono stati sottoposti a decellularizzazione mediante un trattamento a base di TritonX100/ Sodio Colato. L’assenza dell’epitopo alpha-Gal, altamente immunogeno per l’uomo, è stata dimostrata attraverso una doppia tecnica di fluorescenza con un’isolectina e uno specifico anticorpo monoclonale. Dopo condizionamento con siero bovino fetale e fibronectina per aumentare l’attaccamento, le cellule staminali mesenchimali del midollo osseo sono state seminate sia su ventricularis o fibrosa e mantenute staticamente in vitro per 30 giorni. Non
solo la ventricularis favorisce una maggiore adesione, ma anche un’aumentata diffusione all’interno dello stroma e un precoce differenziamento cellulare nei citotipi valvolari. Successivamente, è stata valutata l’interazione fra cellule umane e matrice decellularizzata umana, effettuando ancora una volta la semina su ventricularis. La combinazione omologa permette di ottenere una maggiore proliferazione , una
diminuzione degli eventi apoptotici e un maggiore grado di maturazione del costrutto, tanto da
apprezzare l’espressione di proteine tipiche del muscolo liscio proprio nella tonaca di pertinenza, la vetricularis. L’ECM effettua quindi un ruolo essenziale per l’integrazione cellulare fornendo segnali specifici per l’acquisizione del fenotipo valvolare corretto.
Un’ulteriore ipotesi per l’ottenimento di nuovi sostituti vitali può essere rappresentata dalla rigenerazione tessuto-guidata, favorita in vivo dall’ECM in grado di esercitare un ruolo attrattivo e di guida per le cellule
dell’ospite. Radici aortiche decellularizzate sono state impiantate in minipig per mimare la ricostruzione del tratto di efflusso del ventricolo destro e la funzionalità valvolare è stata monitorata ecocardiograficamente per 12-14 mesi, rivelando un progressivo miglioramento della funzione. Un ripopolamento continuo può essere osservato attraverso le analisi ex vivo negli espianti, anche se le regioni meno esposte al flusso sanguigno sono ancora prive di cellule. Le osservazioni sulle colture primarie dei tessuti parietale vascolare e del lembo portano a ipotizzare il contributo di due maggiori fenotipi al ripopolamento: cellule muscolari lisce dell’arteria polmonare e cellule staminali mesenchimali mobilizzate.
Particolarmente importante non è solo la creazione, ma anche il mantenimento del biocostrutto: una volta che si è ripristinata la funzione, perciò, è essenziale comprendere i reali effettori cellulari coinvolti nella disfunzione valvolare per prevenire il fenomeno già nelle prime fasi. Scarsamente studiate, eventuali popolazioni cellulari staminali a livello valvolare potrebbero partecipare al rimodellamento tissutale
omeostatico, ma anche a eventi patologici avversi. Homograft valvolari aortici (n=27) con annesso lembo anteriore della mitrale (n=27) sono stati classificati in 5 gruppi a seconda della fascia d’età di appartenenza del donatore (10-60 aa). In aggiunta, sono stati esaminati anche lembi aortici e mitrali (n=10), rimossi in sede di sostituzione valvolare. Criosezioni di tessuto cuspidale sono state sottoposte all’analisi istologica
classica, istochimica (von Kossa, Oil Red O) e immunoistochimica per marker di cellule differenziate,
citotipi calcificanti precoci, flogosi e cellule staminali di vari lineages. All’aumentare dell’età, il tipico aspetto osservato é l’accumulo di lipidi, sottoforma di piccole gocce nella tonaca sottoventricolare o depositi di colesterolo nella fibrosa. Il riscontro di calcificazioni è raro. Gli epitopi delle cellule staminali sono espressi nel lembo valvolare a rivelare una precisa distribuzione spaziale. Le colture primarie ottenute
da questi campioni mostrano un fenotipo e proprietà di transdifferenziazione, caratteristiche di un possibile progenitore mesenchimale, senza tuttavia apparente induzione all’osteogenesi. Ulteriori studi sono necessari per meglio comprendere il coinvolgimento di tali cellule nella patologia valvolare.
Concludendo, entrambe le modalità considerate per la realizzazione di un nuovo sostituto vitale sembrano validi strumenti a raggiungere lo scopo, basandosi su matrici decellularizzate prive di alpha-gal e prontamente colonizzabili. Esperimenti futuri e l’applicazione di nuove strategie biomimetiche potranno sicuramente migliorare i risultati incoraggianti finora ottenuti. Sono state inoltre proposte per la prima volta nuove informazioni sulla biologia cellulare staminale della valvola cardiaca con importanti conferme sul processo di continuo rimodellamento cellulare a carico del tessuto cuspidale
Guided Tissue Regeneration in Heart Valve Replacement: From Preclinical Research to First-in-Human Trials
Full text linkMechanical Circulatory Support and Stem Cell-Based Heart Treatment in Europe—2018 Clinical Update
No full textCutting-Edge Regenerative Medicine Technologies for the Treatment of Heart Valve Calcification (Chapter 9)
Full text linkhttp://Laura Iop and Gino Gerosa (2013). Cutting-Edge Regenerative Medicine Technologies for the Treatment of Heart Valve Calcification, Calcific Aortic Valve Disease, Dr. Elena Aikawa (Ed.), ISBN: 978-953-51-1150-4, InTech, DOI: 10.5772/55327. Available from: http://www.intechopen.com/books/calcific-aortic-valve-disease/cutting-edge-regenerative-medicine-technologies-for-the-treatment-of-heart-valve-calcificatio
Biocompatibility Evaluation Criteria for Novel Xenograft Materials: Distribution and Quantification of Remnant Nucleic Acid and Alpha-Gal Epitope
No full textNEW ALPHA-GAL REMOVAL ASSAY: PRESENCE AND DISTRIBUTION OF THE EPITOPE BEFORE AND AFTER CELL REMOVAL FROM XENOGENEIC HEART VALVES
No full textFirst quantitative assay of alpha-Gal in soft tissues: presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.
No full textDecellularized xenograft heart valves might be the ideal scaffolds for tissue engineered heart valves as the alternative to the currently used biological and mechanical prostheses. However, removal of the alpha-Gal epitope is a prerequisite to avoid hyperacute rejection of untreated xenograft material. The aim of this study was to develop an ELISA soft-tissue assay for alpha-Gal quantification in xenograft heart valves before and after a detergent-based (TriCol) or equivalent cell removal procedure. Leaflets from porcine valves were enzymatically digested to expose the epitope and reacted with the alpha-Gal monoclonal antibody M86 for its recognition. Rabbit erythrocytes were used as a reference for the quantification of alpha-Gal. Native aortic and pulmonary leaflets exhibited different epitope concentration: 4.33×10(11) vs. 7.12×10(11)/10 mg wet tissue (p<0.0001). Sampling of selected zones in native valves revealed a different alpha-Gal distribution within and among different leaflets. The pattern was consistent with immunofluorescence analysis and was unrelated to microvessel density distribution. After TriCol treatment alpha-Gal was no longer detectable in both pulmonary and aortic decellularized valves, confirming the ability of this method to remove both cells and alpha-Gal antigen. These results hold promise for a reliable quantitative evaluation of alpha-Gal in decellularized valves obtained from xenograft material for tissues engineering purposes. Additionally, this method is applicable to further evaluate currently used xenograft bioprostheses
Are FDA and CE sacrificing safety for a faster commercialization of xenogeneic tissue devices? Unavoidable need for legislation in decellularized tissue manufacturing
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