Case-control association study of congenital heart disease from a tertiary paediatric cardiac centre from North India.

BACKGROUND: Congenital Heart diseases (CHDs) account for 1/3rd of all congenital birth defects. Etiopathogenesis of CHDs remain elusive despite extensive investigations globally. Phenotypic heterogeneity witnessed in this developmental disorder reiterate gene-environment interactions with periconceptional factors as risk conferring; and genetic analysis of both sporadic and familial forms of CHD suggest its multigenic basis. Significant association of de novo and inherited variants have been observed. Approximately 1/5th of CHDs are documented in the ethnically distinct Indian population but genetic insights have been very limited. This pilot case-control based association study was undertaken to investigate the status of Caucasian SNPs in a north Indian cohort. METHOD: A total of 306 CHD cases sub-classified into n = 198 acyanotic and n = 108 cyanotic types were recruited from a dedicated tertiary paediatric cardiac centre in Palwal, Haryana. 23 SNPs primarily prioritized from Genome-wide association studies (GWAS) on Caucasians were genotyped using Agena MassARRAY Technology and test of association was performed with adequately numbered controls. RESULTS: Fifty percent of the studied SNPs were substantially associated in either allelic, genotypic or sub-phenotype categories validating their strong correlation with disease manifestation. Of note, strongest allelic association was observed for rs73118372 in CRELD1 (p < 0.0001) on Chr3, rs28711516 in MYH6 (p = 0.00083) and rs735712 in MYH7 (p = 0.0009) both on Chr 14 and were also significantly associated with acyanotic, and cyanotic categories separately. rs28711516 (p = 0.003) and rs735712 (p = 0.002) also showed genotypic association. Strongest association was observed with rs735712(p = 0.003) in VSD and maximum association was observed for ASD sub-phenotypes. CONCLUSIONS: Caucasian findings were partly replicated in the north Indian population. The findings suggest the contribution of genetic, environmental and sociodemographic factors, warranting continued investigations in this study population.

The study of expression levels of DNA methylation regulators in patients affected with congenital heart defects (CHDs).

BACKGROUND: Congenial heart defects (CHDs) have multifactorial etiology with complex interplay of genetic and environmental factors. Environmental impact can have epigenetic mechanism of CHD development. Many studies have reported the causal association between CHD and distinct DNA methylation profile which is one of the key epigenetic events, which has vital role in normal embryonic development. The products of DNMT1, DNMT3A, DNMT3B, and MBD2 are important regulators of DNA methylation process. Changes in the expression of these genes are implicated in congenital structural cardiac defects. Hence, in this proof-of-concept study, we have compared the expression levels of these genes in the blood samples of healthy controls and CHD cases while investigating the etiology of CHD. METHODS: In this study with 48 CHD cases and 47 healthy controls, total RNA was isolated from the whole blood samples using TRI reagent. Quantitative RT PCR (qRT-PCR) was used to analyze the mRNA levels of DNMT1, DNMT3A, DNMT3B, and MBD2. The expression levels have been analyzed by relative quantification. RESULTS: We observed that DNMT3B (fold change = -2.563; p = .0018) and DNMT3A (fold change = -2.169; p = .05) were significantly downregulated in CHD patients, whereas the expression of DNMT1 and MBD2 was not significantly different between cases and controls. CONCLUSIONS: Lower expression of de novo methyltransferases, namely, DNMT3B and DNMT3A in CHD cases, may be an important contributor to the mechanism of CHD pathogenesis. Further studies with age-matched controls and analysis of global DNA methylation profile are required to investigate the proposed causal association.

A critical appraisal of humanized alternatives to fetal bovine serum for clinical applications of umbilical cord derived mesenchymal stromal cells.

OBJECTIVE: The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB+ plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations. METHODS: The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed. RESULTS: Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signature of unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44+, CD73+, CD105+, CD90+, and CD166+ in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1+ (~ 21-10%) and TNAP+ (~ 29-11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05). CONCLUSION: Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient.

Isolation, growth kinetics, and immunophenotypic characterization of adult human cardiac progenitor cells.

The discovery of cardiac progenitor cells (CPCs) has raised expectations for the development of cell-based therapy of the heart. Although cell therapy is emerging as a novel treatment for heart failure, several issues still exist concerning an unambiguous definition of the phenotype of CPC types. There is a need to define and validate the methods for the generation of quality CPC populations used in cell therapy applications. Considering the critical roles of cardiac cell progenitors in cellular therapy, we speculate that long term culture might modulate the immunophenotypes of CPCs. Hence, a strategy to validate the isolation and cell culture expansion of cardiac cell populations was devised. Isolation of three subpopulations of human CPCs was done from a single tissue sample using explant, enzymatic isolation, and c-kit+ immunomagnetic sorting methods. The study assessed the effects of ex vivo expansion on proliferation, immunophenotypes, and differentiation of CPCs. Additionally, we report that an explant culture can take over 2 months to achieve similar cell yields, and cell sorting requires a much larger starting population to match this expansion time frame. In comparison, an enzymatic method is expected to yield equivalent quantities of CPCs in 2-3 weeks, notably at a significantly lower cost, which may intensify their use in therapeutic approaches. We determined that ex vivo expansion caused changes in cellular characteristics, and hence propose validated molecular signatures should be established to evaluate the impact of ex vivo expansion for a safe cell therapy product.

Generation of clinical-grade red blood cells from human umbilical cord blood mononuclear cells.

A xeno-free method for ex vivo generation of red blood cells (RBCs) is attempted in order to replicate for large-scale production and clinical applications. An efficient milieu was formulated using injectable drugs substituting the animal-derived components in the culture medium. Unfractionated mononuclear cells isolated from human umbilical cord blood were used hypothesizing that the heterogeneous cell population could effectively contribute to erythroid cell generation. The strategy adopted includes a combination of erythropoietin and other injectable drugs under low oxygen levels, which resulted in an increase in the number of mature RBCs produced in vitro. The novelty in this study is the addition of supplements to the medium in a stage-specific manner for the differentiation of unfractionated umbilical cord blood mononuclear cells (MNCs) into erythropoietic lineage. The erythropoietic lineage was well established by day 21, wherein the mean cell count of RBCs was found to be 21.36 ± 0.9 × 108 and further confirmed by an upregulated expression of CD235a+ specific to RBCs. The rationale was to have a simple method to produce erythroid cells from umbilical cord blood isolates in vitro by mitigating the effects of multiple erythroid-activating agents and batch to batch variability.

A Patient-Inspired Ex Vivo Liver Tissue Engineering Approach with Autologous Mesenchymal Stem Cells and Hepatogenic Serum.

Design and development of ex vivo bioengineered liver tissue substitutes intended for subsequent in vivo implantation has been considered therapeutically relevant to treat many liver diseases that require whole-organ replacement on a long-term basis. The present study focus on patient-inspired ex vivo liver tissue engineering strategy to generate hepatocyte-scaffold composite by combining bone marrow mesenchymal stem cells (BMSCs) derived from cardiac failure patients with secondary hyperbilirubinemia as primers of hepatic differentiation and hepatocyte growth factor (HGF)-enriched sera from same individuals as hepatic inducer. A biodegradable and implantable electrospun fibrous mesh of poly-l-lactic acid (PLLA) and gelatin is used as supporting matrix (average fiber diameter = 285 ± 64 nm, porosity = 81 ± 4%, and average pore size = 1.65 ± 0.77 µm). The fibrous mesh supports adhesion, proliferation, and hepatic commitment of patient-derived BMSCs of adequate stemness using HGF-enriched sera generating metabolically competent hepatocyte-like cells, which is comparable to the hepatic induction with defined recombinant growth factor cocktail. The observed results confirm the combinatorial effects of nanofiber topography and biochemical cues in guiding hepatic specification of BMSCs. The fibrous mesh-hepatocyte construct developed in this study using natural growth factors and BMSCs of same individual is promising for future therapeutic applications in treating damaged livers.

Simulation of Cardiac Arrhythmias Using a 2D Heterogeneous Whole Heart Model.

Simulation studies of cardiac arrhythmias at the whole heart level with electrocardiogram (ECG) gives an understanding of how the underlying cell and tissue level changes manifest as rhythm disturbances in the ECG. We present a 2D whole heart model (WHM2D) which can accommodate variations at the cellular level and can generate the ECG waveform. It is shown that, by varying cellular-level parameters like the gap junction conductance (GJC), excitability, action potential duration (APD) and frequency of oscillations of the auto-rhythmic cell in WHM2D a large variety of cardiac arrhythmias can be generated including sinus tachycardia, sinus bradycardia, sinus arrhythmia, sinus pause, junctional rhythm, Wolf Parkinson White syndrome and all types of AV conduction blocks. WHM2D includes key components of the electrical conduction system of the heart like the SA (Sino atrial) node cells, fast conducting intranodal pathways, slow conducting atriovenctricular (AV) node, bundle of His cells, Purkinje network, atrial, and ventricular myocardial cells. SA nodal cells, AV nodal cells, bundle of His cells, and Purkinje cells are represented by the Fitzhugh-Nagumo (FN) model which is a reduced model of the Hodgkin-Huxley neuron model. The atrial and ventricular myocardial cells are modeled by the Aliev-Panfilov (AP) two-variable model proposed for cardiac excitation. WHM2D can prove to be a valuable clinical tool for understanding cardiac arrhythmias.

Low frequency magnetic force augments hepatic differentiation of mesenchymal stem cells on a biomagnetic nanofibrous scaffold.

Liver tissue engineering using polymeric nanofibrous scaffold and stem cells holds great promises for treating end-stage liver failures. The aim of this study was to evaluate hepatic trans-differentiation potential of human mesenchymal stem cells (hMSCs) on a biomagnetic electrospun nanofibrous scaffold fabricated from a blend of poly-L-lactide (PLLA), collagen and fibrin-rich blood clot, under the influence of a low frequency magnetic field. The scaffold was characterized for surface properties, biochemical and biomechanical parameters and bio-magnetic behaviour. Cell proliferation assay revealed that the scaffold was suitable for hMSCs adhesion and proliferation. Hepatic trans-differentiation potential of hMSCs was augmented on nanofibrous scaffold in magnetic field exposure group compared to control groups, as evident by strong expression of hepatocyte specific markers, albumin release, urea synthesis and presence of an inducible cytochrome P450 system. Our results conclude that biomagnetic scaffold of PLLA/collagen/blood clot augments hepatic trans-differentiation of hMSCs under magnetic field influence.

Nanofibers coated on acellular tissue-engineered bovine pericardium supports differentiation of mesenchymal stem cells into endothelial cells for tissue engineering.

AIM: This study aimed to develop biodegradable, polymer-based nanofibers coated on acellular tissue-engineered bovine pericardium (ATEBP) for cell interfaces, enabling more exquisite functionality, such as mesenchymal stem cell (MSC) adhesion, proliferation and differentiation into endothelial cells for tissue engineering. MATERIALS & METHODS: ATEBP coated with nanofibers of poly(L-lactic acid)-co-poly(ε-caprolactone) (PLACL) and a blend of PLACL and gelatin were analyzed for human bone marrow-derived MSC adhesion, proliferation and differentiation into endothelial cells. RESULTS: The cell culture-based approach showed an increase in human bone marrow-derived MSC adhesion, proliferation and differentiation into endothelial cells on ATEBP coated with PLACL/gelatin nanofibers compared with ATEBP and PLACL nanofibers coated on ATEBP. CONCLUSION: ATEBP coated with PLACL/gelatin nanofibrous scaffolds, along with human bone marrow-derived MSCs differentiated into endothelial cells, might improve the scaffolds' functionality for tissue engineering.

In vitro hepatic trans-differentiation of human mesenchymal stem cells using sera from congestive/ischemic liver during cardiac failure.

Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro.

Nanofiber-reinforced myocardial tissue-construct as ventricular assist device.

OBJECTIVES: This study aimed to create a myocardial tissue construct by tissue engineering to repair, replace, and regenerate damaged cardiac tissue. METHODS AND RESULTS: Human cardiac muscles harvested from a homograft heart retrieval system were decellularized followed by coating with electrospun nanofibers to make them amenable to scaffolding. These processed cardiac tissues were nourished in modified media having ischemic cardiac tissue conditioned media in 6 separate experimental variants, and cord blood mononuclear cells were injected into 4 of them. On the 17th day of culture, the nanofiber-coated scaffolds injected with mononuclear cells and/or reinforced by electrical and mechanical forces, started contracting spontaneously at varying rates, while the control remain noncontractile. Histological staining confirmed the pre-culture acellularity as well as post-culture stem cell viability, and revealed expression of troponin I and cardiac myosin. The acellular processed scaffold when implanted into sheep ischemic myocardial apex revealed transformation into sheep myocardium after 4 months of implantation. CONCLUSION: These results provide direct evidence for the re-cellularization of decellularized cardiac tissue grafts reinforced with a polymer nanofiber coating, by human mononuclear cells injection, leading to generation of a tissue-engineered myocardial construct.

Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering.

Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p>0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP-G-CA-ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP-G-CA-ET). DBP-G-CA-ET exhibited a significant (p>0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p<0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions.

Large-scale in-vitro expansion of RBCs from hematopoietic stem cells.

The quest for RBCs in transfusion medicine has prompted scientists to explore the large-scale expansion of human RBCs from various sources. The successful production of RBCs in the laboratory depends on the selection of potential cell source, optimized culture, bio-physiological parameters, clinically applicable culture media that yields a scalable, contamination-free, non-reactive, non-tumorogenic, stable and functional end product. The expansion protocol considering the in vivo factors involved in homeostasis can generate a cost-effective and readily available cell source for transfusion. This review paper discusses several approaches used to expand RBCs from various sources of stem cells.

Trans-differentiation of human mesenchymal stem cells generates functional hepatospheres on poly(l-lactic acid)-co-poly(ε-caprolactone)/collagen nanofibrous scaffolds.

Mesenchymal stem cell (MSC)-based liver tissue engineering on nanofibrous scaffold holds great promise for cell-based therapy in liver injuries and end-stage liver failure treatments. We investigated the hepatic trans-differentiation potential of human MSCs on a biocomposite poly(l-lactic acid)-co-poly (ε-caprolactone)/collagen (PLACL/collagen) nanofibrous scaffold. The nanofibrous scaffolds comprised of PLACL, collagen and a PLACL/collagen blend (2 : 1) were fabricated by electrospinning and also evaluated for fiber morphology, surface wettability, functional groups, porosity and tensile properties. Hepatic trans-differentiation of human bone marrow-derived MSCs (hMSCs) was carried out on these scaffolds over a period of 28 days using sequential induction with hepatogenic growth factors. Hepatogenesis was confirmed by scanning electron microscopy (SEM), cell phenotype tracking dye expression, quantitative expression of hepatic genes, immunofluorescence staining of hepatocyte-specific markers and albumin release. The results proved that the porous PLACL/collagen nanofibrous scaffold supported enhanced hMSC proliferation and hepatic trans-differentiation compared to individual PLACL and collagen scaffolds as well as a monolayer culture on tissue culture plate (TCP). Interestingly, hMSC-derived hepatocyte-like cells on PLACL/collagen nanofibrous scaffolds could aggregate to form functional 'hepatospheres' similar to normal hepatic spheroids. The present study concludes that PLACL/collagen nanofibrous scaffolds are potentially biomimetic and upon sequential induction with hepatogenic growth factors/cytokines, it augments trans-differentiation of hMSCs towards functional hepatosphere formation. Such bioengineered nanofibrous scaffold hepatic construct provides a promising approach for cellular therapy of damaged livers in end-stage liver failure treatments.

Crosslinked acellular saphenous vein for small-diameter vascular graft.

OBJECTIVE: Patients with congenital and acquired heart diseases or arteriopathy require small-diameter vascular grafts for arterial reconstruction. Autologous veins are the most suitable graft, but when absent, an alternative is necessary. This work addresses the issue. BACKGROUND: Tissue-engineering efforts to create such grafts by modifications of acellular natural scaffolds are considered a promising area. METHODS: Homologous saphenous veins harvested from cadavers and organ donors were processed by decellularization with detergent and enzymatic digestion, followed by crosslinking by dye-mediated photooxidation. They were validated for acellularity, mechanical strength, and crosslink stability. In-vitro and in-vivo cytotoxicity and hemocompatibility studies were conducted. Collagen conformity was studied by Fourier transform infrared spectroscopy, and heat stability by differential scanning calorimetry. A limited large animal study was performed. RESULTS: The processing method delivered biocompatible, hemocompatible, effectively crosslinked grafts, with high heat stability of 126 , an enthalpy value of 183.5 J·g(-1), and collagen conformity close to that of the native vein. The mechanical strength was 250% better than the native vein. The presence of extracellular matrix proteins allowed the acellular vein to become a triple-layered vascular structure in the sheep venous system. CONCLUSION: Crosslinking after decellularization by the dye-mediated photooxidation method could be reproduced in any human vein to obtain a small-diameter vascular grafts.

Ischemic cardiac tissue conditioned media induced differentiation of human mesenchymal stem cells into early stage cardiomyocytes.

Mesenchymal stem cells (MSCs) are multipotent, can be easily expanded in culture and hence are an attractive therapeutic tool for cardiac repair. MSCs have tremendous potential to transdifferentiate to cardiac lineage both in vitro and in vivo. The present study examined the differentiation capacity of conditioned media derived from ischemic cardiac tissue on human MSCs. Human Bone marrow-derived MSCs after due characterization by immunocytochemistry and flow cytometry for MSC specific markers were induced by culture media derived from ischemic (n = 13) and non-ischemic (n = 18) human cardiac tissue. Parallel cultures were treated with 5-azacytidine (5-azaC), a potent cardiomyogen. MSCs induced with ischemic conditioned media formed myotube like structures, expressed sarcomeric Troponin I, alpha myosin heavy chain proteins and were positive for cardiac specific markers (Nkx2.5, human atrial natriuretic peptide, myosin light chain-2a, GATA-4) as was observed in 5-azaC treated cells. However, uninduced MSCs as well as those induced with non-ischemic cardiac conditioned media still maintained the fibroblast morphology even after 3 weeks post-induction. Transmission electron microscopic studies of cardiomyocyte-like cells derived from MSCs revealed presence of sarcomeric bands but failed to show gap junctions and intercalated discs as of adult cardiomyocytes. These findings demonstrate that ischemic cardiac conditioned media induces morphological and molecular changes in MSCs with cardiac features, but at a primitive stage. Proteomics analysis of the ischemic conditioned media revealed differential expression of three relevant proteins (C-type lectin superfamily member 13, Testis-specific chromodomain protein Y2 and ADP/ATP translocase 1), whose exact role in cardiac regeneration needs further analysis.

Nanofiber-reinforced biological conduit in cardiac surgery: preliminary report.

Several options are available for right ventricular outflow tract reconstruction, including commercially available bovine jugular vein and cryo-preserved homografts. Homograft non-availability and the problems of commercially available conduits led us to develop indigenously processed bovine jugular vein conduits with competent valves. They were made completely acellular and strengthened by non-conventional cross-linking without disturbing the extracellular matrix, which improved the luminal surface characteristics for hemocompatibility. Biocompatibility in vitro and in vivo, along with thermal stability, matrix stability, and mechanical strength have been evaluated. Sixty-nine patients received these conduits for right ventricular outflow tract reconstruction. Seven conduits dilated and 4 required replacement. To counteract dilatation, biodegradable polymeric nanofibers in various combinations and in isolation (collagen, polycaprolactone, polylactic acid) were characterized and used to reinforce the conduit circumferentially. Physical validation by mechanical testing, scanning electron microscopy, and in-vitro cytotoxicity was conducted. Thermal stability, spectroscopy studies of the polymer, and preclinical studies of the coated bovine jugular vein in animals are in progress. The feasibility studies have been completed, and the final polymer selection depends on evaluation of the functional superiority of the coated bovine jugular vein.

WITHDRAWN: Humanised substitutes for animal sera in human mesenchymal stem cell culture and differentiation.

This epub Ahead of Print has been withdrawn by the Publisher.

Inflammatory responses of tissue-engineered xenografts in a clinical scenario.

Acellular tissue-engineered (ATE) xenografts and homografts are used in clinical cardiovascular surgery. The present study examined the specific role of carbohydrate antigen (α-Gal and T-antigen) in immune response after decellularisation in tissue-engineered xenografts (porcine pulmonary artery and bovine jugular vein). An enzyme-linked immunosorbent assay (ELISA) was used to ascertain whether implantation of bioprostheses, ATE xenografts and mechanical valve replacement result in augmentation of anti-α-Gal IgM antibodies within eight days of surgery (each group, n=6). Kinetics of host inflammatory response on surgically explanted ATE xenografts was also studied. Immunostaining for α-Gal and T-antigen detected the presence of them in the native tissue but they were absent in processed ATE xenografts from the same tissue. A significant increase in the concentration of anti-α-Gal IgM antibodies was observed in the serum of bioprosthetic valve recipients as compared to ATE xenograft recipients (P<0.05). Organised collagen, and decreased inflammatory response with increase in endothelisation and vascularisation was evident beyond one year of surgery as compared to early periods in ATE xenografts. This study demonstrates that decellularisation of xenografts and further processing of these tissues enabled reduction of inflammatory stimulus with autologous recellularisation with no calcification.