Structural assessments in decellularized extracellular matrix of porcine semilunar heart valves: Evaluation of cell niches.

Tissue-engineered heart valves aim to reproduce the biological properties of natural valves with anatomically correct structure and physiological performance. The closest alternative to creating an ideal heart valve substitute is to use decellularized porcine heart valves, due to their anatomy and availability. However, the immunological barrier and the structural maintenance limit the long-term physiological performance of decellularized porcine heart valves. This study investigated the extracellular matrix (ECM) structure of aortic and pulmonary porcine valves decellularized by a low concentration sodium dodecyl sulfate (SDS)-based method in order to determine the ECM scaffold (ECMS) conditions related to remodeling potential. To assess the structures of the leaflets and conduits of the heart valves, ECM components and their organization were evaluated by histology, biochemical analysis (BC), scanning electron microscopy, multiphoton microscopy, tensile test, immunofluorescence labeling (IF), and Raman microspectroscopy used to draw a profile of the cell niches. Histology and multiphoton imaging of decellularized aortic and pulmonary leaflets and conduits revealed a collagen and elastin histoarchitecture with rearrangement, loosening fibers, and glycosaminoglycan depletion confirmed by biochemistry quantification. The potential cytotoxicity of SDS residues was eliminated after 10 wash cycles. The mechanical properties of the structure of the valve indicated a functional resistance of decellularized ECM. The IF demonstrated the presence of basement membrane, suggesting a potential structure for host cell attachment. The RM analysis showed evidence of molecular interactions, suggesting conservation of the chemical composition, particularly among the protein molecular structures. The structural analyses performed in the semilunar porcine heart valves demonstrate that decellularized ECMS has structural properties that support physiological performance and potential host tissue integration. In fact, decellularized leaflet scaffolds were prone to cell interaction after human adipose-derived stromal cell seeding and culturing. Further analysis of biocompatibility, particularly the ECM-cell interaction, can elucidate the remodeling process, in preserved decellularized heart valve scaffold.

In vitro evaluation of bovine pericardium after a soft decellularization approach for use in tissue engineering.

Pericardial membrane derived from bovine heart tissues is a promising source of material for use in tissue-engineering applications. However, tissue processing is required for its use in humans due to the presence of animal antigens. Therefore, the purpose of this study was to evaluate the structural integrity and biocompatibility of the bovine pericardium (BP) after a soft decellularization process with a 0.1% sodium dodecyl sulfate (SDS) solution, with the aim to remove xenoantigens and preserve extracellular matrix (ECM) bioactivity. The decellularization process promoted a mean reduction of 77% of the amount of DNA in the samples in which cell nuclei staining was undetectable. The ECM content was maintained as mostly preserved after decellularization as well as its biomechanical properties. In addition, the decellularization protocol has proven to be efficient in removing the xenoantigen alpha-gal, which is responsible for immune rejection. The decellularized BP was noncytotoxic in vitro and allowed human adipose-derived stem cell (hASC) adhesion. Finally, after 7 days in culture, the tissue scaffold became repopulated by hASCs, and after 30 days, the ECM protein pro-collagen I was seen in the scaffold. Together, these characteristics indicated that soft BP decellularization with 0.1% SDS solution allows the acquirement of a bioactive scaffold suitable for cell repopulation and potentially useful for regenerative medicine.

Human Heart Explant-Derived Extracellular Vesicles: Characterization and Effects on the In Vitro Recellularization of Decellularized Heart Valves.

Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.

Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking.

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (104 to 10-1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤ 0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p < 0.05). The positivity for growth in the automated method was higher in concentrated inoculum (> 102 CFU/mL) (p < 0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 103 CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.

A Literature Review on the Use of Aortic Allografts in Modern Cardiac Surgery for the Treatment of Infective Endocarditis: Is There Clear Evidence or Is It Merely a Perception?

Infective valve endocarditis is caused by different pathogens and 60% of those involve the aortic valve with valve failure. Although S. aureus is recognized as the most frequently isolated causative bacterium associated with IE in high-income countries, Gram-positive cocci nevertheless play a crucial role in promoting infection in relation to their adhesive matrix molecules. The presence of pili on the surface of Gram-positive bacteria such as in different strains of Enterococcus faecalis and Streptococcus spp., grants these causative pathogens a great offensive capacity due to the formation of biofilms and resistance to antibiotics. The indications and timing of surgery in endocarditis are debated as well as the choice of the ideal valve substitute to replace the diseased valve(s) when repair is not possible. We reviewed the literature and elaborated a systematic approach to endocarditis management based on clinical, microbiological, and anatomopathological variables known to affect postoperative outcomes with the aim to stratify the patients and orient decision making. From this review emerges significant findings on the risk of infection in the allograft used in patients with endocarditis and no endocarditis etiology suggesting that the use of allografts has proved safety and effectiveness in patients with both pathologies.

Histological and Biomechanical Characteristics of Human Decellularized Allograft Heart Valves After Eighteen Months of Storage in Saline Solution.

Background: The best storage preservation method for maintaining the quality and safety of human decellularized allograft heart valves is yet to be established. Objective: The aim of the present study was to evaluate the stability in terms of extracellular matrix (ECM) integrity of human heart valve allografts decellularized using sodium dodecyl sulfate-ethylenediaminetetraacetic acid (SDS-EDTA) and stored for 6, 12, and 18 months. Methods: A total of 70 decellularized aortic and pulmonary valves were analyzed across different storage times (0, 6, 12, and 18 months) for solution pH measurements, histological findings, cytotoxicity assay results, biomechanical test results, and microbiological suitability test results. Continuous data were analyzed using one-way analysis of variance comparing the follow-up times. Results: The pH of the stock solution did not change during the different time points, and no microbial growth occurred up to 18 months. Histological analysis showed that the decellularized allografts did not present deleterious outcomes or signs of structural degeneration in the ECM up to 12 months. The biomechanical properties showed changes over time in different aspects. Allografts stored for 18 months presented lower tensile strength and elasticity than those stored for 12 months (p < 0.05). The microbiological suitability test suggested no residual antimicrobial effects. Conclusion: Changes in the structure and functionality of SDS-EDTA decellularized heart valve allografts occur after 12 months of storage.

Conventional culture method and qPCR using 16S rDNA for tissue bank: a comparison using a model of cardiac tissue contamination.

Real-time polymerase chain reaction (qPCR) using 16S rDNA is an alternative to conventional culture-based tests. The aim of this study was to compare the conventional culture method with qPCR using 16S rDNA in a model of cardiac tissue contamination. Samples of cardiac tissue for artificial contamination with Escherichia coli and control samples were submitted for DNA extraction, which was conducted by selective and alkaline lysis and purification steps. A standard curve for 16S rDNA was constructed to determine the efficiency and analytical sensitivity of the assay in concentrations from 106 to 102 c.f.u. ml-1 using TaqMan Master Mix. 16S rDNA was detected in all contaminated samples; however, it was not detected in the the final washing step solution of the sample with a bioburden of 102 c.f.u. ml-1. Using qPCR is a potential alternative to conventional culture for microbiological safety testing of allograft tissues for biobanking, reducing the time and labour input required.

Decellularized Allografts for Right Ventricular Outflow Tract Reconstruction in Children.

OBJECTIVE: Determine the midterm outcomes of decellularized allografts for right ventricular outflow tract (RVOT) reconstruction in children less than 12 years of age. METHODS: The study included all consecutive patients submitted to RVOT reconstruction with decellularized allografts between June 2006 and June 2016. Besides clinical and echocardiographic control, 20 patients with more than five years of follow-up were evaluated with computed tomography (CT) scans to determine allograft diameters and calcium scores. Structural valve deterioration was defined as any peak gradient above 40 mm Hg and/or insufficiency of moderate or severe degree. Conduit failure was defined as the need for allograft reintervention. RESULTS: There were 59 patients with a median age of six years (range = 0.01-12 years). The most common operation was the Ross procedure (34%). Mean clinical follow-up was 5.4 (2.8) years and was 94% complete. At eight years, only two patients needed a reintervention, with a 90.9% freedom from this event. Structural valve deterioration occurred in 13 patients, 5 due to stenosis and 8 due to insufficiency, with a freedom from structural valve deterioration due to any cause of 64.9% at eight years. Late CT scans demonstrated the absence or minimal calcification of the conduits. CONCLUSIONS: Decellularized allografts for RVOT reconstruction in children were associated with a low incidence of structural valve deterioration and conduit failure. Although these results still need to be confirmed in larger series and with longer follow-up, our data suggest favorable outcomes, at least in the first decade after the operation.

Initial Surgical Experience with Aortic Valve Repair: Clinical and Echocardiographic Results.

INTRODUCTION: Due to late complications associated with the use of conventional prosthetic heart valves, several centers have advocated aortic valve repair and/or valve sparing aortic root replacement for patients with aortic valve insufficiency, in order to enhance late survival and minimize adverse postoperative events. METHODS: From March/2012 thru March 2015, 37 patients consecutively underwent conservative operations of the aortic valve and/or aortic root. Mean age was 48±16 years and 81% were males. The aortic valve was bicuspid in 54% and tricuspid in the remaining. All were operated with the aid of intraoperative transesophageal echocardiography. Surgical techniques consisted of replacing the aortic root with a Dacron graft whenever it was dilated or aneurysmatic, using either the remodeling or the reimplantation technique, besides correcting leaflet prolapse when present. Patients were sequentially evaluated with clinical and echocardiographic studies and mean follow-up time was 16±5 months. RESULTS: Thirty-day mortality was 2.7%. In addition there were two late deaths, with late survival being 85% (CI 95% - 68%-95%) at two years. Two patients were reoperated due to primary structural valve failure. Freedom from reoperation or from primary structural valve failure was 90% (CI 95% - 66%-97%) and 91% (CI 95% - 69%-97%) at 2 years, respectively. During clinical follow-up up to 3 years, there were no cases of thromboembolism, hemorrhage or endocarditis. CONCLUSIONS: Although this represents an initial series, these data demonstrates that aortic valve repair and/or valve sparing aortic root surgery can be performed with satisfactory immediate and short-term results.

Immunological and echocardiographic evaluation of decellularized versus cryopreserved allografts during the Ross operation.

OBJECTIVE: Compare the immunological and echocardiographic data of decellularized versus cryopreserved allografts used for RVOT reconstruction during Ross operation. METHODS: From 16/01/03 thru 07/10/03, 20 Ross operations were performed using decellularized (n=11) or cryopreserved (n=9) allografts. Echocardiography was done at discharge, 1, 3, 6 and 12 months and annually thereafter. Samples for determination of antibodies against HLA class I and II were obtained preoperatively and at days 5, 10, 30, 90 and 180 postoperatively. These samples were tested by the ELISA method in LAT-M dishes (unspecific) for identification of circulating antibodies and the results expressed as mean sample values (Is=DO/cutoff). If positive, LAT-E (specific) was performed and PRA levels determined. RESULTS: There was no mortality. Cryopreserved allografts showed marked Is values elevations for class I and II antibodies which started at the first month and remained elevated up to 6 months. In contrast, of the patients receiving decellularized allografts, seven remained negative, two patients had only marginal elevation of class I antibodies and two patients showed abnormal elevations of PRA levels. This response happened earlier than in the cryopreserved group, starting on the 5th postoperative day and has returned to baseline levels in one case. Echocardiography showed mild, but significant, elevation of gradients in cryopreserved valves but none in the decellularized. CONCLUSIONS: Decellularized allografts had normal function up to 18 months and showed important reduction of the immunogenic response when compared to cryopreserved valves.

Long-term results of the Ross operation: an 18-year single institutional experience.

OBJECTIVES: The purpose of the study was to assess the 18-year outcome of the Ross operation (RO), with emphasis on survival, reoperations, and late function of the pulmonary autografts (PAs) and the right-sided pulmonary allografts. METHODS: Between May 1995 to July 2013, 414 patients with a mean age (mean ± standard deviation) of 30.8 ± 13.1 years were submitted to an RO with the root replacement (n = 356) or the inclusion (n = 58) technique. The most prevalent aetiology was bicuspid valve (n = 206, 49.8%). Patients were divided in four groups depending on the type of allograft used on the right side. The mean follow-up was 8.2 ± 5.2 years and was 97.7% complete. In addition to longitudinal outcomes determined by means of the Kaplan-Meier analysis, log-rank test and Cox regression analysis were used to identify predictors of valve failure. RESULTS: The early mortality rate was 2.7% and the late survival rate was 89.3% at 15 years, similar to an age- and sex-matched population. There were 22 reoperations on the PA (90.7% freedom at 15 years) and 15 on the pulmonary allografts (92.5% freedom at 15 years). The freedom rate from more than mild aortic insufficiency (AI) was 73.1% at 15 years. Thirty-three patients presented with a late root diameter >45 mm, corresponding to a freedom rate of 72.4% at 15 years. Patients with AI and a dilated annulus, especially males, are at greater risk for these complications. Among the right-sided allografts, fresh decellularized allografts showed significantly superior freedom from structural valve dysfunction. CONCLUSIONS: The RO was associated with excellent long-term survival and low incidence of reoperations up to 15 years. Male patients with AI and dilated annulus are at increased risk for late insufficiency and root dilatation. Fresh decellularized allografts presented the best results for reconstruction of the right ventricular outflow tract.

Aortic valve replacement with the Cardioprotese Premium bovine pericardium bioprosthesis: four-year clinical results.

OBJECTIVES: This study reports the initial clinical and echocardiographic results of the Premium bioprosthetic aortic valve up to 4 years of follow-up. METHODS: Between October 2007 and July 2011, 121 consecutive patients were submitted for aortic valve replacement with the Premium bioprosthetic valve. The mean age was 68 ± 9 years and 64 patients were males. The patients were periodically evaluated by clinical and echocardiographic examinations. The mean follow-up was 21 months (min = 2, max = 48), yielding 217 patients/year for the analysis. RESULTS: The hospital mortality was 8%. Late survival at 3 years was 89% (95% CI: 81.9-93.3%), and 80% of the patients were in NYHA functional class I/II. The rates of valve-related complications were low, with a linearized incidence of 0.9%/100 patients/year for thromboembolic complications, 0% for haemorrhagic events and 0.9%/100 patients/year of bacterial endocarditis. There was no case of primary structural valve dysfunction. The mean effective orifice area was 1.61 ± 0.45 cm(2); mean gradient 13 ± 5 mmHg and peak gradient 22 ± 9 mmHg. Significant patient-prosthesis mismatch was found in only 11% of the cases. CONCLUSIONS: The Premium bioprosthetic aortic valve demonstrated very satisfactory clinical and echocardiographic results up to 4 years, similar to other commercially available, third-generation bioprosthetic valves.

Human cardiac explant-conditioned medium: soluble factors and cardiomyogenic effect on mesenchymal stem cells.

The use of conditioned medium (CM) from human cardiac explants (HCEs) as a potential source of paracrine factors for adult stem cell signaling has never been evaluated. We hypothesized that HCEs might provide a source of soluble factors triggering the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte-like cells. By using two-dimensional electrophoresis (2-DE) gels/mass spectrometry and antibody macroarray assays, we found that HCEs release macromolecules, including cytokines, growth factors and myocardial and metabolism-related proteins into the culture medium. We identified a total of 20 proteins in the HCE-CM. However, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-DE, these 20 proteins account for only a fraction of the total number of proteins present in the HCE-CM. We also found that CM increased the proliferation of bone marrow-derived-MSCs (BM-MSCs) in vitro. Unlike the other effects, this effect was most evident after 48 h of culture. Moreover, we examined the effect of HCE-CM on levels of mRNA and protein for specific cardiac markers. We showed that a surprisingly big fraction of BM-MSCs (3.4-5.0%) treated in vitro with HCE-CM became elongated and began to express cardiac markers, consistent with their possible differentiation into cardiomyocyte-like cells. Our in vitro model may be useful not only per se, but also for studies of the mechanisms of action of soluble factors involved in cell differentiation, paving the way for possible new protein-based treatments in the future.