Citations

ABSTRACT Objective To compare antigen removal processing with conventional sodium dodecyl sulfate decellularization for generating laryngotracheoesophageal scaffolds, focusing on preservation of extracellular matrix integrity and removal of immunogenic material. Methods Laryngotracheoesophageal complexes were harvested from C57BL/6 mice and processed using either antigen removal or sodium dodecyl sulfate‐based protocols. Scaffolds were analyzed for residual double‐stranded DNA content, collagen preservation, glycosaminoglycan retention, and ultrastructural integrity via histology, quantitative image analysis, and scanning electron microscopy. Results Both antigen removal and sodium dodecyl sulfate protocols achieved effective decellularization, with comparable dsDNA removal relative to native controls ( p = 0.001; p < 0.001). Antigen removal scaffolds demonstrated better collagen preservation compared to sodium dodecyl scaffolds, which exhibited marked collagen depletion (Muscle: Collagen I, p < 0.001; Collagen III, p < 0.01; Cartilage: Collagen I, p < 0.05; Collagen III, p < 0.01). Both antigen removal and sodium dodecyl sulfate groups showed significant glycosaminoglycan depletion compared with controls ( p < 0.001). Scanning electron microscopy confirmed superior ultrastructural preservation in antigen removal‐treated scaffolds, characterized by intact fibrillar architecture and lacunar morphology, while sodium dodecyl sulfate scaffolds demonstrated collapsed lacunae and irregular extracellular matrix organization. Conclusion Antigen removal processing generates laryngotracheoesophageal scaffolds with enhanced extracellular matrix preservation compared with conventional sodium dodecyl sulfate decellularization, while maintaining equivalent levels of antigen elimination. Retention of collagen and ultrastructure suggests antigen removal scaffolds may provide superior biomechanical and biologic performance for laryngeal tissue engineering. Although glycosaminoglycan depletion remains a limitation in both approaches, antigen removal scaffolds represent a promising pathway toward clinically translatable alternatives to immunosuppression‐dependent laryngotracheoesophageal repair. Level of Evidence Preclinical.

Abstract Heart failure (HF) is a major cause of hospitalization and mortality worldwide. Ventricular assist devices (VADs) represent an alternative to cardiac transplantation for advanced HF. The main FDA-approved VAD (HeartMate III, Abbott), while effective, requires major cardiac surgery to insert the inflow cannula into the cardiac apex, and is associated with numerous co-morbidities and re-hospitalizations related to continuous flow and an external drive line and power supply. Mi-VAD, Inc. aspires to improve outcomes and quality of life with a miniature, minimally invasive, pulsatile, transcutaneously-powered VAD that is implanted above the aortic valve and/or the pulmonary valve and offering a wide range of hemodynamic support suitable for both advanced-stage and less advanced HF. Through its unique placement, there is the potential for right ventricular and biventricular cardiac assist for patients with restrictive cardiomyopathy and HFpEF for whom current VAD therapy is ineffective. Mi-VAD, Inc. has achieved proof of concept with its prototype, providing pulsatile ECG gated cardiac assist up to 10L/minute. Following initial bench top performance testing and in vivo efficacy and safety studies future work will focus on the development of wireless charging capability and conducting GLP builds for verification, regulatory clearance and commercialization. Additional technical development will focus on AI-driven hemodynamic optimization using multimodal physiologic sensing towards patient-specific digital twin of the Mi-VAD platform.

Abstract Description Despite significant efforts to identify targetable, immunogenic antigens in vitro, variability in population wide immune responses, combined with our inability to rank the relative immunogenicity of individual antigens, has led to frequent failures in validating predicted candidates in vivo. Recently, in an animal model of biomaterial biocompatibility, we observed that only ∼3% of residual antigens induced a graft-specific IgG-response ∼30% as strong as native tissue. Leveraging an optimized, shotgun immunoproteomics pipeline, we identified and quantified the magnitude of response toward each, individual, xeno-antigen present in this in vivo model. Additionally, we quantify the abundance (i.e. dose) of each antigen, within native tissue vs. the antigen removed biomaterial. By combining data on the strength of the residual immune response with the dose of each antigen, we developed and validated the Ratio of Immunogenicity (ROI; % residual response/% residual abundance), a novel and quantitative metric which can rank the relative immunogenicity of all antigens in an in vivo system. The ROI metric reveals that inherent properties (i.e., independent of dose) of individual antigens modulate their immune stimulatory capacity, from highly (i.e. strong) to minimally (i.e. weak) antigenic. The ROI is a new tool, with broad utility, which can identify highly immunogenic target antigens, and offers insight into basic immune biology and guiding developments to enhance clinical practice. Funding Sources Supported by R01HL153098 & the Mayo Clinic Foundation for Medical Research Topic Categories Technological Innovations in Immunology (TECH)

Current clinically used electronic implants, including cardiac pacing leads for epicardial monitoring and stimulation of the heart, rely on surgical suturing or direct insertion of electrodes to the heart tissue. These approaches can cause tissue trauma during the implantation and retrieval of the pacing leads, with the potential for bleeding, tissue damage, and device failure. Here, we report a bioadhesive pacing lead that can directly interface with cardiac tissue through physical and covalent interactions to support minimally invasive adhesive implantation and gentle on-demand removal of the device with a detachment solution. We developed 3D-printable bioadhesive materials for customized fabrication of the device by graft-polymerizing polyacrylic acid on hydrophilic polyurethane and mixing with poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) to obtain electrical conductivity. The bioadhesive construct exhibited mechanical properties similar to cardiac tissue and strong tissue adhesion, supporting stable electrical interfacing. Infusion of a detachment solution to cleave physical and covalent cross-links between the adhesive interface and the tissue allowed retrieval of the bioadhesive pacing leads in rat and porcine models without apparent tissue damage. Continuous and reliable cardiac monitoring and pacing of rodent and porcine hearts were demonstrated for 2 weeks with consistent capture threshold and sensing amplitude, in contrast to a commercially available alternative. Pacing and continuous telemetric monitoring were achieved in a porcine model. These findings may offer a promising platform for adhesive bioelectronic devices for cardiac monitoring and treatment.

American Heart Association estimates the prevalence of valvular heart disease to be 2.5% in the United States. Biological heart valve replacements, such as glutaraldehyde‐fixed bovine pericardium (BP), are considered the current standard of care. The NHLBI xenotransplantation working group identified xenoantigenicity as the major barrier to expanding use of such xenogeneic tissues. Previous research has utilized an immunoproteomic approach based on two‐dimensional western blots to identify a small number of BP xenoantigens, which is time consuming and technically challenging. These limitations have resulted in an ever increasing use of shotgun proteomic approaches due to their superior throughput and reproducibility. However, traditional Liquid Chromatography‐Mass Spectrometry (LC‐MS) fails to separate proteins based on their antigenicity, and therefore it is impossible to determine which identified proteins are antigenic versus not. We hypothesized that an affinity chromatography method utilizing poly‐polyclonal serum would bridge this gap and represent an innovative shotgun proteomic antigen identification approach. Using affinity chromatography rabbit IgG antibodies were isolated from whole serum with Protein A HP SpinTrap (GE Healthcare). IgG capture was confirmed by serum protein electrophoresis and ELISA. Two types of antibody affinity columns were generated, with naive serum (D0) or serum from rabbits immunized with bovine pericardium (D84). Bovine pericardial protein extracts were run through both affinity columns, allowing for the specific capture of antigens, which were later eluted and collected. To confirm specificity of antigen capture, eluants were subjected to western blot, and probed with D84 serum. Affinity enriched antigenic proteins were identified by LC‐MS/MS, with statistical comparisons made between D0 and D84. This approach identified 133 putative antigenic proteins that were enriched on D84 compared to D0 columns. Ten of these antigens corresponded to previously published BP xenoantigens. Of the remaining 123 previously unidentified antigens, 30 have been indicated to be immunogenic in other fields such as cancer and autoimmunity. This method was then applied to three humans who had been implanted with bovine pericardial heart valves. Surprisingly, 300 to 500 proteins were identified from patient columns. Between 57 to 79% of the identified proteins were shared among the three individuals, as well as almost all of the proteins identified in the rabbit model were also present in the human. This work therefore represents the first high throughput method of antigen identification that can now be used to monitor currently implanted patients as well as inform the development of BP‐based biomaterials in the future. Support or Funding Information Research supported by grant number R01HL115205 from the National Heart Lung and Blood Institute (NHLBI) at the National Institutes of Health (NIH) and grant number 5T32OD010931‐10 from the NIH.

Despite a staggering 100,000 heart valve replacements performed in the United States per year, current glutaraldehyde‐fixed xenogeneic bioprosthetic replacements are far from ideal. Although glutaraldehyde‐fixation effectively masks tissue antigenicity in the short term, chronic graft‐specific immune responses still commonly occur, limiting valve life expectancy. Additionally, glutaraldehyde‐fixation crosslinks extracellular matrix proteins preventing remodeling and growth in juvenile patients. A heart valve bioprosthesis formed from an unfixed xenogeneic ECM scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current heart valve replacements and improve patient outcomes. Based on protein chemistry principles of sequential, differential solubilization, we hypothesized that amidosulfobetaines would improve lipophilic antigen solubilization and subsequent removal from bovine pericardium following antigen removal (BP‐AR). Antigen removal (AR) was achieved by subjecting native BP samples to hydrophilic AR (48 h), followed by lipophilic AR (hydrophilic solution plus ASB‐14 or ASB‐16 at 0, 1, 2, 5, 8 and 0.1, 2, 3, 4, 5% respectively) (48 h), nuclease digestion (24 h) and washout (48 h). Residual protein was extracted from minced BP‐AR samples using 0.1% sodium dodecyl sulfate (SDS) (hydrophile extract) followed by 1% SDS (lipophile extract). Hydrophile and lipophile extracts ( n = 6 per group) were assessed for residual antigenicity using 1‐DE western blots assessed for IgG positivity using anti‐BP immune serum. Uniaxial tensile testing was performed on BP‐AR scaffolds to determine Young's modulus and Ultimate Tensile Strength. No significant change in scaffold residual hydrophile antigenicity was found between groups, with an average reduction in hydrophile antigenicity of 93.59% compared to untreated control tissue. Both ASB‐14 and ASB‐16 demonstrated statistically significant concentration dependent decreases in residual lipophile antigenicity of BP‐AR, which plateaued at 5% for ASB‐14 and 3% for ASB‐16 (79% and 92% lipophilic antigen removal respectively) ( p < 0.0001). Compared to native BP, there were no significant changes in Young's modulus and Ultimate Tensile Strength between BP‐AR scaffolds generated in the presence of ASB‐14 at 5% or ASB‐16 at 3% ( p < 0.6198) This study demonstrates that increasing amidosulfobetaine concentration increases removal of lipophilic antigens from candidate xenogeneic heart valve biomaterial. The extent to which the achieved reduction in biomaterial antigenicity equates to avoidance of in vivo recipient graft‐specific adaptive immune responses remains to be determined. Additionally, future studies are necessary to quantify ECM protein composition and histology of the resultant xenogeneic BP‐AR scaffolds. However, BP‐AR scaffolds generated using 5% ASB‐14 or 3% ASB‐16 represent promising xenogeneic heart valve prosthesis biomaterials. Support or Funding Information National Institutes of Health (NIH) R01HL115205

Abstract Whole organ tissue engineering encompasses a variety of approaches, including 3D printed tissues, cell-based self-assembly, and cellular incorporation into synthetic or xenogeneic extracellular matrix (ECM) scaffolds. This review article addresses the importance of whole organ tissue engineering for various solid organ applications, focusing on the use of extracellular (ECM) matrix scaffolds in such engineering endeavors. In this work, we focus on the emerging barriers to translation of ECM scaffold-based tissue-engineered organs and highlight potential solutions to overcome the primary challenges in the field. The 3 main factors that are essential for developing ECM scaffold-based whole organs are (1) recapitulation of a functional vascular tree, (2) delivery and orientation of cells into parenchymal void spaces left vacant in the scaffold during the antigen elimination and associated cellular removal processes, and (3) driving differentiation of delivered cells toward the appropriate site-specific lineage. The insights discussed in this review will allow the potential of allogeneic or xenogeneic ECM scaffolds to be fully maximized for future whole organ tissue-engineering efforts.

Abstract Valve replacement is an important therapeutic approach for management of valvular heart disease, accounting for over 200,000 surgeries annually worldwide. Deficiencies of current heart valve prostheses led the NHLBI cardiac surgery working group to highlight the need for improved heart valve leaflet biomaterials. Recently, our group has engineered a novel method to eliminate antigens from bovine pericardium (BP) extracellular matrix (ECM) scaffolds, while retaining biomaterial structure-function properties and regenerative capacity. However, despite reducing BP-ECM scaffold antigen burden by &gt;92%, graft-specific humoral IgG response persisted at ~30% of the level associated with native BP in a rabbit model. In this study, we sought to determine the identities of residual antigens present in antigen removed BP-ECM scaffolds. New Zealand White Rabbits were subpannicularly implanted with either native, glutaraldehyde fixed, or antigen-removed BP-ECM scaffolds (n = 6 per group). Rabbit serum, collected pre- and at 56 d post-implantation, was used to generate IgG affinity chromatography columns. Native BP protein extracts were loaded onto columns, and captured antigens were identified by LC-MS/MS. A total of 259 antigens were identified in native BP rabbits. The glutaraldehyde fixed and antigen-removed rabbits responded to ~4% and ~12% of these same antigens, respectively. A small subset of antigens were uniquely identified in each of the treatment groups, not identified in native BP. These results inform ongoing efforts to eliminate antigens from xenogeneic tissues in development of ECM scaffolds, and may provide insight into the relative immunogenicity of individual xenoantigens in the context of tissue engineering.

Abstract Although immunosuppressive drugs improve short term survival, rejection incidence and long term survival remain largely unchanged in heart transplant patients. While Human Leukocyte Antigen (HLA) is a common initiator of graft-specific immune responses, non-HLA antibody mediated rejection (AMR) also commonly occurs. Furthermore, non-HLA antibodies have been implicated in cell mediated rejection and cardiac allograft vasculopathy. Therefore, there is a need to identify the role of non-HLA antigens in heart transplant rejection. Serum from heart transplant patients lacking donor specific anti-HLA antibodies was collected longitudinally; pre-transplantation, acute rejection, and post-rejection for patients with AMR and matched controls. Serum IgG was used to form anti-human heart affinity columns and antigenic proteins from donor heart extracts captured. Antigens were eluted from the columns and subjected to LC-MS/MS. Longitudinal graft-specific changes in immune response were calculated. Response toward non-HLA antigens increased longitudinally, with 41 high prevalence antigens found in ≥50% of the cohort. Co-culturing cardiomyocyte and endothelial cells with patient IgG indicated cell function changes correlated with specific antigens (e.g., Prohibitin). Immunofluorescence staining of patient biopsies revealed that longitudinal increase in antigen expression correlated with non-HLA antibody response. The non-HLA antigens identified in this study elicit acute and chronic graft-specific adaptive immune responses in heart transplant recipients. Antibodies against these non-HLA antigens induced cellular dysfunction by changing expression of their target protein in the donor heart.

Chronic vascular diseases affect over 25 million patients in the U.S, alone. While non-invasive therapies are available, approximately 4.5 million individuals are estimated to require a vascular bypass annually, worldwide. Autologous vascular grafts remain the standard of care; yet the absence of a suitable donor vessel results in approximately one third of patients being ineligible for autologous grafting. “Off-the-shelf” alternatives have been proposed, but have had limited pre-clinical success, primarily due to graft failure via thrombosis. Thrombotic failure risk has been shown to be dramatically reduced by the formation of a luminal quiescent endothelial cell monolayer. Recently, our group engineered an unfixed, antigen-removed (AR) extracellular matrix (ECM) scaffold from bovine saphenous vein (SV) which is minimally immunogenic and avoids in vivo thrombosis. However, the mechanism by which AR-ECM SV scaffolds prevent thrombosis remains unknown. In this study, we utilized cell culture, immunofluorescence, and RNA-seq to assess the hypothesis that hAECs seeded on the basement membrane (BM) surface of AR-ECM SV scaffolds adopt a quiescent phenotype. Cells seeded on the BM surface proliferated and underwent significantly greater XY migration than those seeded on the non-BM surface. Additionally, unlike non-BM seeded cells, BM seeding resulted in quiescent hAEC phenotype (i.e., apical polarization of podocalyxin and adherence junction protein (i.e., cadherin and β-catenin) colocalization). Finally, the transcriptional phenotype of hAECs seeded on the BM surface was similar to cells treated with simvastatin (i.e. “quiescent”) and significantly different from those treated with TNFα (i.e. “activated”) (n=6/group), as assessed by gene set enrichment and t-SNE analysis. We conclude that seeding hAECs on the BM surface of AR-SV ECM scaffolds induces a favorable “quiescent” phenotype, while an unfavorable “activated” phenotype is avoided. Ultimately, these results show that the BM surface of AR-SV-ECM scaffolds possesses inherent characteristics which promote quiescent hAEC behavior and resultant vascular homeostasis and will help inform future pre-clinical studies of this novel small diameter vascular grafting biomaterial.