Alessandro Gandaglia

The influence of heart valve leaflet matrix characteristics on the interaction between human mesenchymal stem cells and decellularized scaffolds

The potential for in vitro colonization of decellularized valves by human bone marrow mesenchymal stem cells (hBM-MSCs) towards the anisotropic layers ventricularis and fibrosa and in homo- vs. heterotypic cell-ECM interactions has never been investigated. hBM-MSCs were expanded and characterized by immunofluorescence and FACS analysis. Porcine and human pulmonary valve leaflets (p- and hPVLs, respectively) underwent decellularization with Triton X100-sodium cholate treatment (TRICOL), followed by nuclear fragment removal. hBM-MSCs (2x10(6) cells/cm(2)) were seeded onto fibrosa (FS) or ventricularis (VS) of decellularized PVLs, precoated with FBS and fibronectin, and statically cultured for 30 days. Bioengineered PVLs revealed no histopathological features but a reconstructed endothelium lining and the presence of fibroblasts, myofibroblasts and SMCs, as in the corresponding native leaflet. The two valve layers behaved differently as regards hBM-MSC repopulation potential, however, with a higher degree of 3D spreading and differentiation in VS than in FS samples, and with enhanced cell survival and colonization effects in the homotypic ventricularis matrix, suggesting that hBM-MSC phenotypic conversion is strongly influenced in vitro by the anisotropic valve microstructure and species-specific matching between extracellular matrix and donor cells. These findings are of particular relevance to in vivo future applications of valve tissue engineering.

First quantification of alpha-Gal epitope in current glutaraldehyde-fixed heart valve bioprostheses

Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti‐αGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out.

First quantitative assay of alpha-Gal in soft tissues: presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.

Decellularized 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.

Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

Cells, scaffolds and bioreactors for tissue-engineered heart valves: a journey from basic concepts to contemporary developmental innovations

The development of viable and functional tissue-engineered heart valves (TEHVs) is a challenge that, for almost two decades, the scientific community has been committed to face to create life-lasting prosthetic devices for treating heart valve diseases. One of the main drawbacks of tissue-based commercial substitutes, xenografts and homografts, is their lack of viability, and hence failure to grow, repair, and remodel. In adults, the average bioprostheses life span is around 13 years, followed by structural valve degeneration, such as calcification; in pediatric, mechanical valves are commonly used instead of biological substitutes, as in young patients, the mobilization of calcium, due to bone remodeling, accelerates the calcification process. Moreover, neither mechanical nor bioprostheses are able to follow childrens body growth. Cell seeding and repopulation of acellular heart valve scaffolds, biological and polymeric, appears as a promising way to create a living valve. Biomechanical stimuli have significant impact on cell behavior including in vitro differentiation, and physiological hemodynamic conditioning has been found to promote new tissue development. These concepts have led scientists to design bioreactors to mimic the in vivo environment of heart valves. Many different types of somatic and stem cells have been tested for colonizing both the surface and the core of the valve matrix but controversial results have been achieved so far.

The use of thermal techniques for the characterization and selection of natural biomaterials.

In this paper we explore the ability of thermal analysis to check elastin and collagen integrity in different biomaterial applications. Differential Scanning Calorimetry (DSC) has been used to analyze the first and second order transitions of the biological macromolecules in the hydrated and dehydrated state. First, we report the characterization of control cardiovascular tissues such as pericardium, aortic wall and valvular leaflet. Their thermal properties are compared to pure elastin and pure collagen. Second, we present results obtained on two collagen rich tissues: pericardia with different chemical treatments and collagen with physical treatments. Finally, more complex cardiovascular tissues composed of elastin and collagen are analyzed and the effect of detergent treatment on the physical structure of collagen and elastin is brought to the fore.

Alpha-Gal detectors in xenotransplantation research: a word of caution

Naso F, Gandaglia A, Iop L, Spina M, Gerosa G. Alpha‐Gal detectors in xenotransplantation research: a word of caution. Xenotransplantation 2012; 19: 215–220.

Fine structure of glycosaminoglycans from fresh and decellularized porcine cardiac valves and pericardium.

Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds.

Physiological Performance of a Detergent Decellularized Heart Valve Implanted for 15 Months in Vietnamese Pigs: Surgical Procedure, Follow‐up, and Explant Inspection

This study features the longest experimental follow-up for decellularized heart valves implanted in an animal model. Porcine aortic heart valves were decellularized according to a disclosed standardized method in which TRITON X-100 and sodium cholate (TRICOL) are used in succession, followed by a further treatment with the endonuclease Benzonase to completely remove the nucleic acid remnants. Experimental animals (n = 17), represented by Vietnamese pigs (VPs), received a decellularized aortic allograft as a substitute for the replacement of their right ventricular outflow tract. The surgical implantation of the TRICOL-treated aortic valve conduit was successful in 11 VPs, while perioperative or postoperative complications occurred in the remaining six animals. In the sham-operated group (n = 4), the native pulmonary root was excised and immediately reimplanted orthotopically in the same animal. Echocardiography demonstrated a satisfactory hemodynamic performance of the TRICOL-treated valves during follow-up as well as the absence of relevant leaflet alterations concerning thickness and motility or valve insufficiency. At explantation, macroscopic inspection of tissue-engineered heart valve conduits did not evidence calcifications and showed a decreased wall thickness, comparable to that of the reimplanted native pulmonary roots. Noteworthy, extended functional performance, recovery of DNA content, and active extracellular matrix precursor incorporation are apparently compatible with the properties of a living self-supporting substitute.

Differential distribution of structural components and hydration in aortic and pulmonary heart valve conduits: impact of detergent-based cell removal

Evaluation of the physiological performance of biological scaffolds for tissue engineering applications has been mostly based on biophysical and morphological methods, with limited attention paid to the quantitative contribution of the main structural components to native and/or treated valve assemblies. In the present study quantitation addressed the porcine leaflet, sinus and adjacent wall of aortic and pulmonary valved conduits before and after detergent-based cell removal. Collagen, elastin, glycosaminoglycan, lipid and water contents were expressed in terms of relative concentration and volume fraction in order to assess their effective contribution to the native tissue and to changes following decellularization procedures. The main findings were recognition of unexpectedly large water and underestimated collagen contents, differential distribution of elastin between the sectors and of glycosaminoglycan along the conduits and pulmonary scaffold destabilization upon cell removal, not found in the aortic case. Simultaneous investigations allowed consistent comparisons between native and decellularized tissues and added analytical knowledge crucial for designing realistic constitutive models. We have provided a quantitative structural foundation for earlier biomechanical findings in pulmonary leaflets and the basis for validation of theoretical assumptions still lacking the support of experimental evidence in both conduits. Future insights into the distribution of load-bearing components in human conduits are likely to provide indications important to optimize the surgical positioning of valvular grafts.