The α-Gal KO Mouse Animal Model is a Reliable and Predictive Tool for the Immune-Mediated Calcification Assessment of Heart Valve Bioprostheses.

BACKGROUND: Recent studies highlighted the presence of anti-α-Gal antibodies in patients implanted with commercial bioprosthetic heart valves (BHVs). BHVs expose residual α-Gal xenoantigen and their recognition by the circulating anti-Gal antibodies leads to opsonization of the device's tissue component with the consequent triggering of a deterioration pathway that culminates with calcification. Small animal models such as mice and rats have been broadly involved in the in vivo testing of biomaterials by subcutaneous implantation, especially for the effectiveness of BHVs anti-calcific treatments. However, since models employed for this purpose express α-Gal antigen, the implantation of BHVs' leaflets does not elicit a proper immunological response, so the calcification propensity may be dramatically underestimated. METHODS: An α-Gal knockout (KO) mouse model has been created, using the CRISP/Cas9 approach, and adopted to assess the calcification potential of commercial BHVs leaflets through the surgical implantation in the back subcutis area. Calcium quantification was performed by inductively coupled plasma analysis; immune response against the BHVs leaflets and α-Gal silencing was evaluated through immunological assays. RESULTS: Two months after the implantation of commercial BHV leaflets, the anti-Gal antibody titers in KO mice doubled when compared with those found in wild-type (WT) ones. Leaflets explanted from KO mice, after one month, showed a four-time increased calcium deposition concerning the ones explanted from WT. The degree of silencing of α-Gal varied, depending on the specific organ that was assessed. In any case, the animal model was suitable for evaluating implanted tissue responses. CONCLUSIONS: Such mouse model proved to be an accurate tool for the study of the calcific propensity of commercial BHVs leaflets than those hitherto used. Given its reliability, it could also be successfully used to study even other diseases in which the possible involvement of α-Gal has been observed.

Correlations between the alpha-Gal antigen, antibody response and calcification of cardiac valve bioprostheses: experimental evidence obtained using an alpha-Gal knockout mouse animal model.

Introduction: Preformed antibodies against αGal in the human and the presence of αGal antigens on the tissue constituting the commercial bioprosthetic heart valves (BHVs, mainly bovine or porcine pericardium), lead to opsonization of the implanted BHV, leading to deterioration and calcification. Murine subcutaneous implantation of BHVs leaflets has been widely used for testing the efficacy of anti-calcification treatments. Unfortunately, commercial BHVs leaflets implanted into a murine model will not be able to elicit an αGal immune response because such antigen is expressed in the recipient and therefore immunologically tolerated. Methods: This study evaluates the calcium deposition on commercial BHV using a new humanized murine αGal knockout (KO) animal model. Furtherly, the anti-calcification efficacy of a polyphenol-based treatment was deeply investigated. By using CRISPR/Cas9 approach an αGal KO mouse was created and adopted for the evaluation of the calcific propensity of original and polyphenols treated BHV by subcutaneous implantation. The calcium quantification was carried out by plasma analysis; the immune response evaluation was performed by histology and immunological assays. Anti-αGal antibodies level in KO mice increases at least double after 2 months of implantation of original commercial BHV compared to WT mice, conversely, the polyphenols-based treatment seems to effectively mask the antigen to the KO mice's immune system. Results: Commercial leaflets explanted after 1 month from KO mice showed a four-time increased calcium deposition than what was observed on that explanted from WT. Polyphenol treatment prevents calcium deposition by over 99% in both KO and WT animals. The implantation of commercial BHV leaflets significantly stimulates the KO mouse immune system resulting in massive production of anti-Gal antibodies and the exacerbation of the αGal-related calcific effect if compared with the WT mouse. Discussion: The polyphenol-based treatment applied in this investigation showed an unexpected ability to inhibit the recognition of BHV xenoantigens by circulating antibodies almost completely preventing calcific depositions compared to the untreated counterpart.

Preventing extrinsic mechanisms of bioprosthetic degeneration using polyphenols.

OBJECTIVES: The purpose of this study was to evaluate the impact of a polyphenols-based treatment on the extrinsic mechanisms responsible for early bioprosthetic heart valve (BHV) degeneration. Structural degeneration can be driven by both extrinsic and intrinsic mechanisms. While intrinsic mechanisms have been associated with inherent biocompatibility characteristics of the BHV, the extrinsic ones have been reported to involve external causes, such as chemical, mechanical and hydrodynamic, responsible to facilitate graft damage. METHODS: The chemical interaction and the stability degree between polyphenols and pericardial tissue were carefully evaluated. The detoxification of glutaraldehyde in commercial BHVs models and the protective effect from in vivo calcification were taken into relevant consideration. Finally, the hydrodynamic and biomechanical features of the polyphenols-treated pericardial tissue were deeply investigated by pulse duplicator and stress-strain analysis. RESULTS: The study demonstrated the durability of the polyphenols-based treatment on pericardial tissue and the stability of the bound polyphenols. The treatment improves glutaraldehyde stabilization's current degree, demonstrating a surprising in vivo anti-calcific effect. It is able to make the pericardial tissue more pliable while maintaining the correct hydrodynamic characteristics. CONCLUSIONS: The polyphenols treatment has proved to be a promising approach capable of acting simultaneously on several factors related to the premature degeneration of cardiac valve substitutes by extrinsic mechanisms.

Polyphenols could be Effective in Exerting a Disinfectant-Like Action on Bioprosthetic Heart Valves, Counteracting Bacterial Adhesiveness.

Background: The incidence of infective endocarditis in patients with bioprosthetic heart valves is over 100 times that of the general population with S. aureus recognized as the causative organism in approximately 1/3 of cases. In this study, (1) the microbicidal and virucidal effect of a polyphenolic solution was carefully evaluated. The same solution was then adopted for the treatment of a commercial bioprosthetic heart valve model for (2) the assessment of inhibition of S. aureus adhesiveness. Methods: (1) the viability of 9 microorganisms strains (colony-forming units) and the infectivity degree of 3 viral strains (cellular infection capacity) were evaluated after suspension in the polyphenolic solution. (2) Leaflets from a treated and untreated commercial surgical valve model were incubated with a known concentration of S. aureus. After incubation, the leaflets were homogenized and placed in specific culture media to quantify the bacterial load. Results: (1) The polyphenolic solution proved to be effective in eliminating microorganisms strains guaranteeing the killing of at least 99.9%. The effectiveness is particularly relevant against M. chelonae (99.999%). (2) The polyphenol-based treatment resulted in the inhibition of the S. aureus adhesiveness by 96% concerning untreated samples. Conclusions: The data suggest an interesting protective effect against infections and bacterial adhesiveness by a polyphenolic-based solution. Further studies will plan to extend the panel of microorganisms for the evaluation of the anti-adhesive effect; however, the use of optimized polyphenolic blends could lead to the development of new treatments capable to make transcatheter-valve substitutes more resistant to infection.

Altered Blood Levels of Anti-Gal Antibodies in Alzheimer's Disease: A New Clue to Pathogenesis?

Alzheimer's disease is a neurodegenerative disorder whose pathological mechanisms, despite recent advances, are not fully understood. However, the deposition of beta amyloid -peptide and neuroinflammation, which is probably aggravated by dysbiotic microbiota, seem to play a key role. Anti-Gal are the most abundant xenoreactive natural antibodies. They are supposed to stem from immunization against the gut microbiota and have been implicated in the pathogenesis of several diseases, including multiple sclerosis. These antibodies target the alpha-Gal epitope, expressed on the terminal sugar units of glycoprotein or glycolipid of all mammals except apes, Old World monkeys and humans. The alpha-Gal is constitutively expressed in several bacteria constituting the brain microbiota, and alpha-Gal-like epitopes have been detected in gray matter, amyloid plaque, neurofibrillary tangles and corpora amylacea of the human brain, suggesting a potential link between anti-Gal and Alzheimer's disease etiopathogenesis. For the first time, our study searched for possible alterations of anti-Gal immunoglobulin levels in Alzheimer's disease patients. IgG and IgM blood levels were significantly lower, and IgA significantly higher in patients than in healthy subjects. These results suggest that such immunoglobulins might be implicated in Alzheimer's disease pathogenesis and open new scenarios in the research for new biomarkers and therapeutic strategies.

Different approaches to heart valve decellularization: A comprehensive overview of the past 30 years.

Xenogeneic decellularized heart valve scaffolds have the potential to overcome the limitations of existing bioprosthetic heart valves that have limited duration due to calcification and tissue degeneration phenomena. This article presents a review of 30 years of decellularization approaches adopted in cardiovascular tissue engineering, with a focus on the use, either individually or in combination, of different detergents. The safety and efficacy of cell-removal procedures are specifically reported and discussed, as well as the structure and biomechanics of the treated extracellular matrix (ECM). Detergent residues within the ECM, production of hyaluronan fragments, safe removal of cellular debris, and the persistence of the alpha-Gal epitope after the decellularization treatments are of particular interest as parameters for the identification of the best tissue for the manufacture of bioprostheses. Special attention has also been given to key factors that should be considered in the manufacture of the next generation of xenogeneic bioprostheses, where tissues must retain the ability to be remodeled and to grow in weight along with body reshaping.

Alpha-Gal Inactivated Heart Valve Bioprostheses Exhibit an Anti-Calcification Propensity Similar to Knockout Tissues.

BACKGROUND: Glutaraldehyde (GLA) has been used to crosslink bioprosthetic heart valve (BHVs) tissues to enhance their stability, besides ensuring a satisfactory degree of immunological tolerance. Unfortunately, GLA fixation does not guarantee a complete tissue biocompatibility of BHVs in currently used devices. The interaction between preformed human anti-alpha-Gal antibody and alpha-Gal antigens promotes the calcification of GLA-treated alpha-Gal-positive tissue. Recently, an alarming correlation between the presence of the alpha-Gal epitope and a premature BHVs degeneracy was reported. This article presents the results of a novel treatment called FACTA, for the inactivation of the alpha-Gal epitopes in porcine aortic valve tissue and commercial BHVs. METHODS: Evaluation of the alpha-Gal epitope inactivation was performed through a patented ELISA test, confirmed by western blot, immunofluorescence, and immunohistochemical analyses. Investigations were also conducted to assess the in vitro propensity to trigger thrombosis, calcification, and worsening of FACTA-treated tissue. To explain the mechanism of action through which the FACTA treatment acts, a specific experimental model, based on the mass spectroscopy approach, was performed. RESULTS: The study confirms that GLA is able to ensure the inactivation of approximately half alpha-Gal epitopes originally present in both porcine aortic valve tissue and marketed BHVs. By subjecting tissues to the FACTA procedure, it was possible to obtain an alpha-Gal inactivation degree of about 95% alongside to a reduced propensity from 72.6% to 85.4% to the in vitro calcification for porcine aortic valve tissue and 80.5% for commercial treated BHVs. FACTA was effective in decreasing oxidative tissue damage and protecting collagen from degradation. Finally, FACTA could further mitigate or even abrogate the need for early anticoagulation therapies after BHV implantation. CONCLUSION: A novel treatment, called FACTA, is effective to produce biological tissues that are less susceptible to enzymatic and oxidative stress and structural degradation, calcification, and thrombus formation. FACTA-treated tissues display a clear improvement of their biocompatibility that is characterized by an almost complete inactivation of the alpha-Gal epitope. FACTA prevents the xenogeneic tissue antigens from reacting with the host immune system, ensuring an effective shield effect that makes the tissue surface less reactive and more biocompatible.

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells.

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality.

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.

Tissue-specific expression and regulatory networks of pig microRNAome.

BACKGROUND: Despite the economic and medical importance of the pig, knowledge about its genome organization, gene expression regulation, and molecular mechanisms involved in physiological processes is far from that achieved for mouse and rat, the two most used model organisms in biomedical research. MicroRNAs (miRNAs) are a wide class of molecules that exert a recognized role in gene expression modulation, but only 280 miRNAs in pig have been characterized to date. RESULTS: We applied a novel computational approach to predict species-specific and conserved miRNAs in the pig genome, which were then subjected to experimental validation. We experimentally identified candidate miRNAs sequences grouped in high-confidence (424) and medium-confidence (353) miRNAs according to RNA-seq results. A group of miRNAs was also validated by PCR experiments. We established the subtle variability in expression of isomiRs and miRNA-miRNA star couples supporting a biological function for these molecules. Finally, miRNA and mRNA expression profiles produced from the same sample of 20 different tissue of the animal were combined, using a correlation threshold to filter miRNA-target predictions, to identify tissue-specific regulatory networks. CONCLUSIONS: Our data represent a significant progress in the current understanding of miRNAome in pig. The identification of miRNAs, their target mRNAs, and the construction of regulatory circuits will provide new insights into the complex biological networks in several tissues of this important animal model.

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

BACKGROUND: 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. METHODS: In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard αGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the αGal epitopes, was used as negative control. RESULTS: Epic™ valve was the only model among those tested, in which the αGal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive αGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an αGal amount not significantly different from that exhibited by porcine Mosaic(®) valve (5.2 ± 0.6 × 10(10) each 10 mg of tissue). CONCLUSIONS: For the first time, the quantitative evaluation of the αGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for αGal-free long-lasting substitutes.

Tissue-engineered heart valves: intra-operative protocol.

Tissue engineering of heart valves investigates the possibility to create a fully compatible and biomimetic graft able to provide host cell repopulation like the native living valve. Decellularized aortic and pulmonary valves and synthetic polymers have been used to promote the creation of a native-like scaffold suitable to be colonized by cells either in vitro, in dynamic bioreactors or in vivo using different animal models. The herein presented research provides the intra-operative protocol and details of surgical technique. Porcine aortic valve conduits were decellularized and implanted in the right ventricular outflow tract of Vietnamese pigs.

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

Xenogeneic tissues are currently employed in clinical practice to create biological substitutes (bioprosthetic heart valves) and in the repair of various damaged tissues (pericardium, gastric-mucosa, nerves, cartilage). Many studies have shown that xenogeneic tissues express superficial epitopes as alpha-Gal, capable of triggering hyperacute and acute vascular rejection phenomena. Currently, no tissue treatment has proven able to completely mask or inactivate such epitopes. In fact, neither glutaraldehyde fixation nor decellularisation procedures ensure a definitive solution because of the persistence of reactive xenoantigen residues. The ability to ascertain alpha-Gal epitope removal from a xenogeneic tissue is closely related to the possibility of its quantitative determination. In the past, detection of the alpha-Gal epitope relied on the use of alpha-Gal reactive isolectin molecules and was limited to isolated cells. Recently, the quantitative evaluation of this antigen has been carried out in whole connective tissue through the use of the monoclonal antibody M86. This article provides an overview of the implications of the alpha-Gal epitope in the current clinical scenario and a definitive comparison between the reliability and specificity of isolectines vs. M86 in alpha-Gal determination.

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.

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.

Cardiomyocytes in vitro adhesion is actively influenced by biomimetic synthetic peptides for cardiac tissue engineering.

Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding.

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.

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 children's 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 changing hydrodynamic performance of the decellularized intact porcine aortic root: considerations on in-vitro testing.

BACKGROUND AND AIM OF THE STUDY: The most effective method for decellularization of the intact porcine aortic root remains controversial. Additionally, the hydrodynamic effect that such treatment may have on aortic roots has never been previously investigated. The study aim was to compare the in-vitro hydrodynamic performances of intact porcine aortic roots, both before and after decellularization treatment. METHODS: Fifteen fresh porcine aortic roots were tested in the aortic chamber of the Sheffield pulse duplicator (SPD). For study purposes, the roots were first sutured to a silicone aortic root and then hydrodynamically tested. After in-vitro testing, the fresh porcine aortic roots, while still fixed within the silicone root, were decellularized according to various protocols (TRI-COL, TRI-DOC, sodium dodecyl sulfate (SDS) 0.03%, and SDS 0.1%). After decellularization, the valve roots were re-tested, adopting identical testing conditions. Forward flow pressure drop, closing leakage volumes, effective orifice area (EOA), and stroke work loss were each monitored. Three roots, used as a control group, were tested in identical fashion before and after storage (without decellularization) for comparative purposes. RESULTS: The TRI-COL- and TRI-DOC-treated porcine aortic roots showed significantly lower transvalvular gradients, lower stroke work loss, lower valve resistance, and higher EOA than fresh intact porcine roots. In contrast, SDS 0.1%-treated porcine aortic roots showed opposing results, with the transvalvular gradients, stroke work loss and valve resistance each higher, and the EOA lower, than pre-treatment values. SDS 0.03% treatment had no significant effect on the hydrodynamic performance. After decellularization in all treatment groups, the diastolic parameters, total regurgitant volume and valve closing volume were each non-significantly increased. The aortic roots used as a control group showed similar results before and after storage. CONCLUSION: Based on these results using the SPD, all treatments except for SDS 0.03% modified the systolic and diastolic functions of intact porcine aortic roots.

Characterization of aneurysmal aortas by biochemical, thermal, and dielectric techniques.

Abdominal aortic aneurysms (AAA) are characterized by structural alterations of the aortic wall resulting from the degradation of elastic fibres and an increase of collagen/elastin ratio. In this study we investigated the chain dynamics of AAA tissues by two techniques generally used for the characterization of polymers, Differential scanning calorimetry (DSC) and thermally stimulated currents (TSC), and we correlated the obtained data with biochemical analyses. The thermal denaturation of collagen observed by DSC allowed us to evaluate the thermal stability of the triple helix domain: notable modifications were evidenced between collagen from control tissue and collagen from AAA, particularly concerning the thermal denaturation. The dielectric analysis of pathologic aortic walls by TSC revealed a relevant change of collagen mobility in AAA, with the occurrence of a specific mode of relaxation between -60 and -40°C. Biochemical, thermal, and dielectric results are compatible with increase of new collagen deposition and/or impairment of the collagen phase stability in the extracellular matrix of AAAs.