Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Non-physiologic Bioreactor Processing Conditions for Heart Valve Tissue Engineering.

PURPOSE: Conventional methods of seeding decellularized heart valves for heart valve tissue engineering have led to inconsistent results in interstitial cellular repopulation, particularly of the distal valve leaflet, and notably distinct from documented re-endothelialization. The use of bioreactor conditioning mimicking physiologic parameters has been well explored but cellular infiltration remains challenging. Non-characteristic, non-physiologic conditioning parameters within a bioreactor, such as hypoxia and cyclic chamber pressure, may be used to increase the cellular infiltration leading to increased recellularization. METHODS: To investigate the effects of novel and perhaps non-intuitive bioreactor conditioning parameters, ovine aortic heart valves were seeded with mesenchymal stem cells and cultured in one of four environments: hypoxia and high cyclic pressures (120 mmHg), normoxia and high cyclic pressures, hypoxia and negative cyclic pressures (- 20 mmHg), and normoxia and negative cyclic pressures. Analysis included measurements of cellular density, cell phenotype, and biochemical concentrations. RESULTS: The results revealed that the bioreactor conditioning parameters influenced the degree of recellularization. Groups that implemented hypoxic conditioning exhibited increased cellular infiltration into the valve leaflet tissue compared to normoxic conditioning, while pressure conditioning did not have a significant effect of recellularization. Protein expression across all groups was similar, exhibiting a stem cell and valve interstitial cell phenotype. Biochemical analysis of the extracellular matrix was similar between all groups. CONCLUSION: These results suggest the use of non-physiologic bioreactor conditioning parameters can increase in vitro recellularization of tissue engineered heart valve leaflets. Particularly, hypoxic culture was found to increase the cellular infiltration. Therefore, bioreactor conditioning of tissue engineered constructs need not always mimic physiologic conditions, and it is worth investigating novel or uncharacteristic culture conditions as they may benefit aspects of tissue culture.

Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture.

Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic "niche," a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

A novel extracellular matrix-based leukemia model supports leukemia cells with stem cell-like characteristics.

Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton's jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.

Wharton's Jelly Matrix Decellularization for Tissue Engineering Applications.

Scaffolds, both natural and synthetic, used in tissue engineering provide mechanical support to cells. Tissue decellularization has been used to provide natural extracellular matrix scaffolds for tissue engineering purposes. In this chapter we focus on describing the methodology used to decellularize Wharton's jelly matrix, the mucous connective tissue that surrounds umbilical cord vessels, to obtain decellularized Wharton's jelly matrix (DWJM); an extracellular matrix that can be used for tissue engineering purposes. We also, briefly, describe our experience with processing DWJM for cell seeding and recellularization.

Decellularized Wharton's Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 µg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

Design and efficacy of a single-use bioreactor for heart valve tissue engineering.

Heart valve tissue engineering offers the promise of improved treatments for congenital heart disorders; however, widespread clinical availability of a tissue engineered heart valve (TEHV) has been hindered by scientific and regulatory concerns, including the lack of a disposable, bioreactor system for nondestructive valve seeding and mechanical conditioning. Here we report the design for manufacture and the production of full scale, functional prototypes of such a system. To evaluate the efficacy of this bioreactor as a tool for seeding, ovine aortic valves were decellularized and subjected to seeding with human mesenchymal stem cells (hMSC). The effects of pulsatile conditioning using cyclic waveforms tuned to various negative and positive chamber pressures were evaluated, with respect to the seeding of cells on the decellularized leaflet and the infiltration of seeded cells into the interstitium of the leaflet. Infiltration of hMSCs into the aortic valve leaflet was observed following 72 h of conditioning under negative chamber pressure. Additional conditioning under positive pressure improved cellular infiltration, while retaining gene expression within the MSC-valve interstitial cell phenotype lineage. This protocol resulted in a subsurface pilot population of cells, not full tissue recellularization. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 249-259, 2017.

Performance of allogeneic bioengineered replacement pulmonary valves in rapidly growing young lambs.

BACKGROUND: Cardiac allometric organ growth after pediatric valve replacement can lead to patient-prosthesis size mismatch and valve re-replacement, which could be mitigated with allogeneic decellularized pulmonary valves treated with collagen conditioning solutions to enhance biological and mechanical performance, termed "bioengineered valves." In this study, we evaluated functional, dimensional, and biological responses of these bioengineered valves compared with traditional cryopreserved valves implanted in lambs during rapid somatic growth. METHODS: From a consanguineous flock of 13 lambs, the pulmonary valves of 10 lambs (mean weight, 19.6 ± 1.4 kg) were replaced with 7 bioengineered valves or 3 classically cryopreserved valves. After 6 months, the 10 lambs with implanted valves and 3 untreated flock mates were compared by echocardiography, cardiac catheterization, and explant pathology. RESULTS: Increases in body mass, valve geometric dimensions, and effective orifice areas were similar in the 2 groups of lambs. The bioengineered valves had higher median cusp-to-cusp coaptation areas (34.6%; interquartile range, 21.00%-35.13%) and were more similar to native valves (43.4%; interquartile range, 42.59%-44.01%) compared with cryopreserved valves (13.2%; interquartile range, 7.07%-13.91%) (P = .043). Cryopreserved valves cusps, but not bioengineered valve cusps, were thicker than native valve cusps (P = .01). Histologically, cryopreserved valves demonstrated less than native cellularity, whereas bioengineered valves that were acellular at the time of surgery gained surface endothelium and subsurface myofibroblast interstitial cells in pulmonary artery, sinus wall, and cusp base regions. CONCLUSIONS: Biological valve conduits can enlarge via passive dilatation without matrix synthesis, but this would result in decreased cusp coaptational areas. Bioengineered valves demonstrated similar annulus enlargement as cryopreserved valves but usually retained larger areas of cuspal coaptation. Heat-shock protein 47-positive (collagen-synthesizing) cells were present in previously acellular bioengineered sinus walls and cusp bases, but rarely in more distal cusp matrices.

Potential mechanisms underlying ectodermal differentiation of Wharton's jelly mesenchymal stem cells.

Wharton's jelly mesenchymal stem cells (WJMSCs) are being increasingly recognized for their ectodermal differentiation potential. Previously, we demonstrated that when WJMSC were seeded onto an acellular matrix material derived from Wharton's jelly and cultured in osteogenic induction media, generated CK19 positive cells and hair-like structures indicative of ectodermal differentiation of WJMSCs. In this manuscript, we examine the underlying mechanism behind this observation using a variety of microscopy and molecular biology techniques such as western blotting and qPCR. We demonstrate that these hair-like structures are associated with live cells that are positive for epithelial and mesenchymal markers such as cytokeratin-19 and α-smooth muscle actin, respectively. We also show that up-regulation of ß-catenin and noggin, along with the expression of TGF-ß and SMAD and inhibition of BMP4 could be the mechanism behind this ectodermal differentiation and hair-like structure formation.

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-ß), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-ß in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Pulmonary arterioplasty with decellularized allogeneic patches.

BACKGROUND: Decellularized allogeneic nonvalved pulmonary artery patches for arterioplasty are a relatively new option compared with cryopreserved allogeneic, crosslinked xenogeneic bioprosthetic or synthetic materials. This study examines the midterm experience with a new decellularized allogeneic patch for congenital cardiac reconstructions. METHODS: For this prospective postmarket approval, nonrandomized, inclusive observational study, we collected data on a consecutive cohort of 108 patients with cardiovascular reconstructions using 120 decellularized allogeneic pulmonary artery patches (MatrACELL; LifeNet Health, Inc, Virginia Beach, VA) between September 2009 and December 2012. One hundred of the patches were used for pulmonary arterioplasties. Two patients were lost early to follow-up and excluded from subsequent survival and durability analyses. Data included demographics, surgical outcomes, subsequent reoperations, and catheter reinterventions. These variables were also collected for an immediately preceding retrospective consecutive cohort of 100 patients with 101 pulmonary arterioplasty patches who received classical cryopreserved pulmonary artery allografts (n=59 patches and patients) or synthetic materials (n=41 patients with 42 patches) for pulmonary arterioplasties between 2006 and 2009. RESULTS: In 106 patients with 118 decellularized patches, there were no device-related serious adverse events, no device failures, and no evidence of calcifications on chest roentgenograms. In contrast, the prior comparative pulmonary arterioplasty cohort of 100 patients experienced an overall 14.0% patch failure rate requiring device-related reoperations (p<0.0001) at mean duration of 194±104 days (range, 25 to 477 days). CONCLUSIONS: The intermediate-term data obtained in this study suggest favorable performance by decellularized pulmonary artery patches, with no material failures or reoperations provoked by device failure.

Generating CK19-positive cells with hair-like structures from Wharton's jelly mesenchymal stromal cells.

Wharton's jelly mesenchymal stromal cells (WJMSCs) are considered mesenchymal, multipotent, and capable of differentiating into cells of mesodermal origin. Ectodermal differentiation from mesenchymal cells has been recently reported. Herein, we show for the first time that we can generate cytokeratin 19-positive cells and hair-like structures from WJMSCs in vitro using 2 separate methodologies that utilize osteogenic media to induce WJMSCs to undergo osteogenic differentiation. In one method, WJMSCs were seeded on a matrix isolated from Wharton's jelly following decellularization. In the other method, WJMSCs were cultured to form spheroids. Our findings demonstrate that WJMSCs may have the capacity for ectodermal differentiation.

Bioengineered human and allogeneic pulmonary valve conduits chronically implanted orthotopically in baboons: hemodynamic performance and immunologic consequences.

OBJECTIVE: This study assesses in a baboon model the hemodynamics and human leukocyte antigen immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning treatments) human and baboon heart valve scaffolds. METHODS: Fourteen baboons underwent pulmonary valve replacement, 8 with decellularized and conditioned (bioengineered) pulmonary valves derived from allogeneic (N = 3) or xenogeneic (human) (N = 5) hearts; for comparison, 6 baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel-reactive serum antibodies (human leukocyte antigen class I and II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms measured valve function and geometry. Differences were analyzed with Kruskal-Wallis and Wilcoxon rank-sum tests. RESULTS: All animals survived and thrived, exhibiting excellent immediate implanted valve function by transesophageal echocardiograms. Over time, reference valves developed a smaller effective orifice area index (median, 0.84 cm(2)/m(2); range, 1.22 cm(2)/m(2)), whereas all bioengineered valves remained normal (effective orifice area index median, 2.45 cm(2)/m(2); range, 1.35 cm(2)/m(2); P = .005). None of the bioengineered valves developed elevated peak transvalvular gradients: 5.5 (6.0) mm Hg versus 12.5 (23.0) mm Hg (P = .003). Cryopreserved valves provoked the most intense antibody responses. Two of 5 human bioengineered and 2 of 3 baboon bioengineered valves did not provoke any class I antibodies. Bioengineered human (but not baboon) scaffolds provoked class II antibodies. C1q(+) antibodies developed in 4 recipients. CONCLUSIONS: Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves.

The specialized roles of immature and mature dendritic cells in antigen cross-presentation.

Exogenous antigen cross-presentation is integral to the stimulation of cytotoxic T-lymphocytes against viruses and tumors. Central to this process are dendritic cells (DCs), which specialize in cross-presentation. DCs may be considered to exist in two radically different states of activation, generally referred to as immature and mature. In each of these states, the cell has a series of separate and specialized abilities for the induction of T-cell immunity. In the immature state, the DC is adept in surveying the periphery, acquiring and storing antigen, but has a limited capacity for direct T-cell activation. During a brief and defined window of time following DC stimulation, nearly every aspect of antigen handling changes, as it transitions from an entity focused on protein preservation to one capable of efficient cross-presentation. It is this time period and the underlying molecular mechanisms active here, which form the core of our studies on cross-presentation.

Initial pediatric cardiac experience with decellularized allograft patches.

PURPOSE: This study examines use patterns, early outcomes, and technical surgical adaptability of a newly approved decellularized allograft pulmonary artery patch for right ventricular outflow tract reconstructions in neonates and infants, including primary operations and reoperations. DESCRIPTION: The study includes the 44 consecutive initial patients in which 46 patches were used for cardiovascular reconstruction between September 2009 and September 2010. Recorded variables include sex, age at operation, adverse outcome end points (eg, death, stenosis, aneurysm), congenital diagnoses, postoperative complications, and number, type, and location of patches. EVALUATION: Patients were a mean age of 290±343 days at operation, and 54.5% were boys. Three types of patch were used: 58.7% thin, 21.7% thick, and 19.6% hemipulmonary. No device-related deaths or adverse events occurred. A biopsy specimen of one patch at elective reoperation demonstrated active recellularization, with no inflammation. CONCLUSIONS: These patches have a wide range of applicability. Handling and technical flexibility is excellent and superior to standard options. There appears to be potential for autologous revitalization of the tissue.

Performance and morphology of decellularized pulmonary valves implanted in juvenile sheep.

BACKGROUND: Because of cryopreserved heart valve-mediated immune responses, decellularized allograft valves are an attractive option in children and young adults. The objective of this study was to investigate the performance and morphologic features of decellularized pulmonary valves implanted in the right ventricular outflow tract of juvenile sheep. METHODS: Right ventricular outflow tract reconstructions in juvenile sheep (160±9 days) using cryopreserved pulmonary allografts (n=6), porcine aortic root bioprostheses (n=4), or detergent/enzyme-decellularized pulmonary allografts (n=8) were performed. Valve performance (echocardiography) and morphologic features (gross, radiographic, and histologic examination) were evaluated 20 weeks after implantation. RESULTS: Decellularization reduced DNA in valve cusps by 99.3%. Bioprosthetic valves had the largest peak and mean gradients versus decellularized valves (p=0.03; p<0.001) and cryopreserved valves (p=0.01; p=0.001), which were similar (p=0.45; p=0.40). Regurgitation was minimal and similar for all groups (p=0.16). No cusp calcification was observed in any valve type. Arterial wall calcification was present in cryopreserved and bioprosthetic grafts but not in decellularized valves. No autologous recellularization or inflammation occurred in bioprostheses, whereas cellularity progressively decreased in cryopreserved grafts. Autologous recellularization was present in decellularized arterial walls and variably extending into the cusps. CONCLUSIONS: Cryopreserved and decellularized graft hemodynamic performance was comparable. Autologous recellularization of the decellularized pulmonary arterial wall was consistently observed, with variable cusp recellularization. As demonstrated in this study, decellularized allograft valves have the potential for autologous recellularization.

Decellularization reduces calcification while improving both durability and 1-year functional results of pulmonary homograft valves in juvenile sheep.

OBJECTIVE: The juvenile sheep functional valve chronic implant calcification model was used to compare long-term calcification rates, functional performance, and durability for 3 types of right ventricular outflow tract implants: classically cryopreserved homografts and 2 decellularized pulmonary valved conduits. METHODS: Fifteen juvenile sheep were randomly assigned to one of 3 study arms and underwent pulmonary valve replacement. The arms included the following: (1) cryopreserved ovine pulmonary valves; (2) cryopreserved, decellularized, saline (1 degrees C-10 degrees C)-stored ovine pulmonary valves; and (3) cryopreserved, decellularized, glycerolized (-80 degrees C) stored ovine pulmonary valves. Animal growth, serial echocardiographic results (with valve performance assessment), dimensions, and tissue-specific calcification measurements were compared with pre-explant angiographic analysis and right ventricular outflow tract pressure measurements, cardiac magnetic resonance imaging, specimen radiographic analysis, gross explant pathology, and histopathology. Parametric and nonparametric statistical analysis were performed. RESULTS: All but 2 study animals receiving implants thrived postoperatively, with similar growth rates, explant valve dimensions, ventricular functions, cardiac output, and indices during the study. As determined by means of echocardiographic analysis, 3 animals in arm 1 (and one in arm 2) had leaflet dysfunction. Valve regurgitation was recognized in 1 survivor each from both arms 1 and 2. Although 1 arm 1 animal died with calcified subacute bacterial endocarditis, and the other 4 had leaflet and conduit wall calcification by the time of death, no arm 2 or arm 3 animals demonstrated leaflet calcium, and no arm 3 and only 1 arm 2 animals had calcium in the conduit wall over the entire year, as determined with any measurement method. All cryopreserved conduit walls had calcium by 20 weeks, whereas only 1 of 10 decellularized conduits (arms 2 plus 3) had wall calcium. CONCLUSION: Cryopreserved-decellularized-glycerolized valves retained normal valve function, with absent leaflet and minimal wall calcifications 1 year postoperatively, as opposed to classically cryopreserved allografts. These results might be predictive of the prolonged durability and functionality of a cryopreserved-decellularized-glycerolized allograft valve.

Aortic annuloplasty with aortic root reconstruction to prevent patient-prosthesis mismatch.

Part of the ongoing argument concerning patient-prosthesis mismatch (PPM) following aortic valve replacement (AVR) is due to the perception that aortic annulus enlargement procedures increase the risk and technical difficulty of aortic valve surgery. Here, an aortic root reconstruction that involves enlargement of the annulus and tailoring of the aortic root to accommodate larger stented prostheses is presented that has been personally performed in 196 patients with no technique-related surgical deaths or complications, and thus can be carried out without additional risk. This aortic root enlargement aortoplasty and annuloplasty method can be calibrated to all AVRs involving stented manufactured prostheses when these are deemed the prosthesis of choice for the patient with a relatively small annulus and/or aortic root, severe left ventricular hypertrophy, compromised LV function or a very active lifestyle, to achieve predicted EOA values > or = 1.00 cm2/m2.