Altered Inflammatory State and Mitochondrial Function Identified by Transcriptomics in Paediatric Congenital Heart Patients Prior to Surgical Repair.

Congenital heart disease (CHD) remains the most common birth defect, with surgical intervention required in complex cases. Right ventricle (RV) function is known to be a major predictor of sustained cardiac health in these patients; thus, by elucidating the divergent profiles between CHD and the control through tissue analysis, this study aims to identify new avenues of investigation into the mechanisms surrounding reduced RV function. Transcriptomic profiling, in-silico deconvolution and functional network analysis were conducted on RV biopsies, identifying an increase in the mitochondrial dysfunction genes RPPH1 and RMPR (padj = 4.67 × 10-132, 2.23 × 10-107), the cytotoxic T-cell markers CD8a, LAGE3 and CD49a (p = 0.0006, p < 0.0001, and p = 0.0118) and proinflammatory caspase-1 (p = 0.0055) in CHD. Gene-set enrichment identified mitochondrial dysfunctional pathways, predominately changes within oxidative phosphorylation processes. The negative regulation of mitochondrial functions and metabolism was identified in the network analysis, with dysregulation of the mitochondrial complex formation. A histological analysis confirmed an increase in cellular bodies in the CHD RV tissue and positive staining for both CD45 and CD8, which was absent in the control. The deconvolution of bulk RNAseq data suggests a reduction in CD4+ T cells (p = 0.0067) and an increase in CD8+ T cells (p = 0.0223). The network analysis identified positive regulation of the immune system and cytokine signalling clusters in the inflammation functional network, as there were lymphocyte activation and leukocyte differentiation. Utilising RV tissue from paediatric patients undergoing CHD cardiac surgery, this study identifies dysfunctional mitochondrial pathways and an increase in inflammatory T-cell presence prior to reparative surgery.

Growth capacity of a Wharton's Jelly derived mesenchymal stromal cells tissue engineered vascular graft used for main pulmonary artery reconstruction in piglets.

Background: Surgical treatment of congenital heart defects affecting the right ventricular outflow tract (RVOT) often requires complex reconstruction and multiple reoperations due to structural degeneration and lack of growth of currently available materials. Hence, alternative approaches for RVOT reconstruction, which meet the requirements of biocompatibility and long-term durability of an ideal scaffold, are needed. Through this full scale pre-clinical study, we demonstrated the growth capacity of a Wharton's Jelly derived mesenchymal stromal cells (WJ-MSC) tissue engineered vascular graft used in reconstructing the main pulmonary artery in piglets, providing proof of biocompatibility and efficacy. Methods: Sixteen four-week-old Landrace pigs were randomized to undergo supravalvar Main Pulmonary Artery (MPA) replacement with either unseeded or WJ-MSCs-seeded Small Intestinal Submucosa-derived grafts. Animals were followed up for 6 months by clinical examinations and cardiac imaging. At termination, sections of MPAs were assessed by macroscopic inspection, histology and fluorescent immunohistochemistry. Results: Data collected at 6 months follow up showed no sign of graft thrombosis or calcification. The explanted main pulmonary arteries demonstrated a significantly higher degree of cellular organization and elastin content in the WJ-MSCs seeded grafts compared to the acellular counterparts. Transthoracic echocardiography and cardiovascular magnetic resonance confirmed the superior growth and remodelling of the WJ-MSCs seeded conduit compared to the unseeded. Conclusion: Our findings indicate that the addition of WJ-MSCs to the acellular scaffold can upgrade the material, converting it into a biologically active tissue, with the potential to grow, repair and remodel the RVOT.

Protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber.

Surgical treatment of pediatric congenital heart disease with tissue grafts is a lifesaving intervention. Decellularization to reduce immunogenicity of tissue grafts is an increasingly popular alternative to glutaraldehyde fixation. Here, we present a protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber. We describe steps for 3D printing the flow rig, preparing porcine tissue, and using the flow rig to utilize shear forces for decellularization. We then detail procedures for characterizing the acellular scaffold. For complete details on the use and execution of this protocol, please refer to Vafaee et al.1.

Graft rejection in paediatric congenital heart disease.

Congenital heart disease (CHD) affects around 1.35 million neonates worldwide per annum, and surgical repair is necessary in approximately 25% of cases. Xenografts, usually of bovine or porcine origin, are often used for the surgical reconstruction. These xenografts elicit an immune response due to significant immunological incompatibilities between host and donor. Current techniques to dampen the initial hyperacute rejection response involve aldehyde fixation to crosslink xenoantigens, such as galactose-α1,3-galactose and N-glycolylneuraminic acid. While this temporarily masks the epitopes, aldehyde fixation is a suboptimal solution, degrading over time, resulting in cytotoxicity and rejection. The immune response to foreign tissue eventually leads to chronic inflammation and subsequent graft failure, necessitating reintervention to replace the defective bioprosthetic. Decellularisation to remove immunoincompatible material has been suggested as an alternative to fixation and may prove a superior solution. However, incomplete decellularisation poses a significant challenge, causing a substantial immune rejection response and subsequent graft rejection. This review discusses commercially available grafts used in surgical paediatric CHD intervention, looking specifically at bovine jugular vein conduits as a substitute to cryopreserved homografts, as well as decellularised alternatives to the aldehyde-fixed graft. Mechanisms of biological prosthesis rejection are explored, including the signalling cascades of the innate and adaptive immune response. Lastly, emerging strategies of intervention are examined, including the use of tissue from genetically modified pigs, enhanced crosslinking and decellularisation techniques, and augmentation of grafts through in vitro recellularisation or functionalisation with human surface proteins.

Biological Scaffolds for Congenital Heart Disease.

Congenital heart disease (CHD) is the most predominant birth defect and can require several invasive surgeries throughout childhood. The absence of materials with growth and remodelling potential is a limitation of currently used prosthetics in cardiovascular surgery, as well as their susceptibility to calcification. The field of tissue engineering has emerged as a regenerative medicine approach aiming to develop durable scaffolds possessing the ability to grow and remodel upon implantation into the defective hearts of babies and children with CHD. Though tissue engineering has produced several synthetic scaffolds, most of them failed to be successfully translated in this life-endangering clinical scenario, and currently, biological scaffolds are the most extensively used. This review aims to thoroughly summarise the existing biological scaffolds for the treatment of paediatric CHD, categorised as homografts and xenografts, and present the preclinical and clinical studies. Fixation as well as techniques of decellularisation will be reported, highlighting the importance of these approaches for the successful implantation of biological scaffolds that avoid prosthetic rejection. Additionally, cardiac scaffolds for paediatric CHD can be implanted as acellular prostheses, or recellularised before implantation, and cellularisation techniques will be extensively discussed.