Leigh G. Griffiths

Immunogenicity in xenogeneic scaffold generation: Antigen removal vs. decellularization

Decades of research have been undertaken towards the goal of tissue engineering using xenogeneic scaffolds. The primary advantages associated with use of xenogeneic tissue-derived scaffolds for in vitro development of replacement tissues and organs stem from the inherent extracellular matrix (ECM) composition and architecture. Native ECM possesses appropriate mechanical properties for physiological function of the biomaterial and signals for cell binding, growth and differentiation. Additionally, xenogeneic tissue is readily available. However, translation of xenogeneic scaffold-derived engineered tissues or organs into clinical therapies requires xenoantigenicity of the material to be adequately addressed prior to implantation. Failure to achieve this goal will result in a graft-specific host immune rejection response, jeopardizing in vivo survival of the resultant scaffold, tissue or organ. This review explores (i) the appropriateness of scaffold acellularity as an outcome measure for assessing reduction of the immunological barriers to the use of xenogeneic scaffolds for tissue engineering applications and (ii) the need for tissue engineers to strive for antigen removal during xenogeneic scaffold generation.

Stepwise solubilization-based antigen removal for xenogeneic scaffold generation in tissue engineering

The ability of residual antigens on decellularized tissue to elicit the immune response upon implantation motivates development of a more rigorous antigen removal (AR) process for xenogeneic scaffold generation. Antigen removal strategies promoting solubilization of hydrophilic proteins (predominantly cytoplasmic) enhance the reduction of hydrophilic antigenicity in bovine pericardium (BP); however, the diversity of protein antigens within a tissue necessitates development of AR strategies capable of addressing a spectrum of protein antigen solubilities. In the present study, methods for promoting solubilization of lipophilic proteins (predominantly membrane) were investigated for their ability to reduce lipophilic antigenicity of BP when applied as a second AR step following our previously described hydrophilic AR method. Bovine pericardium following AR (BP-AR) was assessed for residual hydrophilic and lipophilic antigenicity, removal of known lipophilic xenoantigens, tensile properties, and extracellular matrix structure and composition. Facilitating hydrophile solubilization (using dithiothreitol and potassium chloride) followed by lipophile solubilization (using amidosulfobetaine-14 (ASB-14)), in a two-step sequential, differential AR strategy, significantly reduces residual hydrophilic and lipophilic antigenicity of BP-AR beyond that achieved with either one-step hydrophilic AR or decellularization using 1% (w/v) sodium dodecyl sulfate. Moreover, use of 1% (w/v) ASB-14 for lipophilic AR eliminates the two most critical known barriers to xenotransplantation (galactose-α(1,3)-galactose and major histocompatibility complex I)) from BP-AR without compromising the structure-function properties of the biomaterial. This study demonstrates the importance of a sequential, differential protein solubilization approach to reduce biomaterial antigenicity in the production of a xenogeneic scaffold for heart valve tissue engineering.

Companion animals: Translational scientist's new best friends.

Naturally occurring diseases in companion animals represent an underused resource that holds promise for providing predictive proof of efficacy in the evaluation of new therapeutics and devices. Knowledge and resources derived from veterinary medicine represent an underused resource that could serve as a bridge between data obtained from diseases models in laboratory animals and human clinical trials. Naturally occurring disease in companion animals that display the defining attributes of similar, if not identical, diseases in humans hold promise for providing predictive proof of concept in the evaluation of new therapeutics and devices. Here we outline comparative aspects of naturally occurring diseases in companion animals and discuss their current uses in translational medicine, benefits, and shortcomings. Last, we envision how these natural models of disease might ultimately decrease the failure rate in human clinical trials and accelerate the delivery of effective treatments to the human clinical market.

The role of protein solubilization in antigen removal from xenogeneic tissue for heart valve tissue engineering.

Decellularization techniques have been developed in an attempt to reduce the antigenicity of xenogeneic biomaterials, a critical barrier in their use as tissue engineering scaffolds. However, numerous studies have demonstrated inadequate removal and subsequent persistence of antigens in the biomaterial following decellularization, resulting in an immune response upon implantation. Thus, methods to enhance antigen removal (AR) are critical for the use of xenogeneic biomaterials in tissue engineering and regenerative medicine. In the present study, AR methods incorporating protein solubilization principles were investigated for their ability to reduce antigenicity of bovine pericardium (BP) for heart valve tissue engineering. Bovine pericardium following AR (BP-AR) was assessed for residual antigenicity, tensile properties, and extracellular matrix composition. Increasing protein solubility during AR significantly decreased the residual antigenicity of BP-AR-by an additional 80% compared to hypotonic solution or 60% compared to 0.1% (w/v) SDS decellularization methods. Moreover, solubilizing agents have a dominant effect on reducing the level of residual antigenicity of BP-AR beyond that achieved by AR additives alone. Tested AR methods did not compromise the tensile properties of BP-AR compared to native BP. Furthermore, residual cell nuclei did not correlate to residual antigenicity, demonstrating that residual nuclei counts may not be an appropriate indicator of successful AR. In conclusion, AR strategies promoting protein solubilization significantly reduced residual antigens compared to decellularization methods without compromising biomaterial functional properties. This study demonstrates the importance of solubilizing protein antigens for their removal in the generation of xenogeneic scaffolds.

In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation.

The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potential to overcome limitations of current clinically utilized glutaraldehyde-fixed biomaterials. The objective of this work was to determine how residual antigenicity and ECM architecture preservation modulate recipient immune and regenerative responses towards unfixed bovine pericardium (BP) ECM scaffolds. Disruption of ECM architecture during scaffold generation, with either SDS-decellularization or glutaraldehyde-fixation, stimulated recipient foreign body response and resultant fibrotic encapsulation following leporine subpannicular implantation. Conversely, BP scaffolds subjected to stepwise removal of hydrophilic and lipophilic antigens using amidosulfobetaine-14 (ASB-14) maintained native ECM architecture and thereby avoided fibrotic encapsulation. Removal of hydrophilic and lipophilic antigens significantly decreased local and systemic graft-specific, adaptive immune responses and subsequent calcification of BP scaffolds compared to scaffolds undergoing hydrophile removal only. Critically, removal of antigenic components and preservation of ECM architecture with ASB-14 promoted full-thickness recipient non-immune cellular repopulation of the BP scaffold. Further, unlike clinically utilized fixed BP, ASB-14-treated scaffolds fostered rapid intimal and medial vessel wall regeneration in a porcine carotid patch angioplasty model. This work highlights the importance of residual antigenicity and ECM architecture preservation in modulating recipient immune and regenerative responses towards xenogeneic biomaterial generation.

Antigen removal for the production of biomechanically functional, xenogeneic tissue grafts

Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed.

Mesenchymal Stem Cell Seeding of Porcine Small Intestinal Submucosal Extracellular Matrix for Cardiovascular Applications

In this study, we investigate the translational potential of a novel combined construct using an FDA-approved decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) seeded with human or porcine mesenchymal stem cells (MSCs) for cardiovascular indications. With the emerging success of individual component in various clinical applications, the combination of SIS-ECM with MSCs could provide additional therapeutic potential compared to individual components alone for cardiovascular repair. We tested the in vitro effects of MSC-seeding on SIS-ECM on resultant construct structure/function properties and MSC phenotypes. Additionally, we evaluated the ability of porcine MSCs to modulate recipient graft-specific response towards SIS-ECM in a porcine cardiac patch in vivo model. Specifically, we determined: 1) in vitro loading-capacity of human MSCs on SIS-ECM, 2) effect of cell seeding on SIS-ECM structure, compositions and mechanical properties, 3) effect of SIS-ECM seeding on human MSC phenotypes and differentiation potential, and 4) optimal orientation and dose of porcine MSCs seeded SIS-ECM for an in vivo cardiac application. In this study, histological structure, biochemical compositions and mechanical properties of the FDA-approved SIS-ECM biomaterial were retained following MSCs repopulation in vitro. Similarly, the cellular phenotypes and differentiation potential of MSCs were preserved following seeding on SIS-ECM. In a porcine in vivo patch study, the presence of porcine MSCs on SIS-ECM significantly reduced adaptive T cell response regardless of cell dose and orientation compared to SIS-ECM alone. These findings substantiate the clinical translational potential of combined SIS-ECM seeded with MSCs as a promising therapeutic candidate for cardiac applications.

Cardiac extracellular matrix proteomics: Challenges, techniques, and clinical implications

Extracellular matrix (ECM) has emerged as a dynamic tissue component, providing not only structural support, but also functionally participating in a wide range of signaling events during development, injury, and disease remodeling. Investigation of dynamic changes in cardiac ECM proteome is challenging due to the relative insolubility of ECM proteins, which results from their macromolecular nature, extensive post‐translational modification (PTM), and tendency to form protein complexes. Finally, the relative abundance of cellular and mitochondrial proteins in cardiac tissue further complicates cardiac ECM proteomic approaches. Recent developments of various techniques to enrich and analyze ECM proteins are playing a major role in overcoming these challenges. Application of cardiac ECM proteomics in disease tissues can further provide spatial and temporal information relevant to disease diagnosis, prognosis, treatment, and engineering of therapeutic candidates for cardiac repair and regeneration.

Effect of bovine pericardial extracellular matrix scaffold niche on seeded human mesenchymal stem cell function

Numerous studies have focused on generation of unfixed bovine pericardium (BP) extracellular matrix (ECM) for clinical application. However, the extent to which maintenance of native ECM niche is capable of directing behavior of repopulating cells remains relatively unexplored. By exploiting the sidedness of BP scaffolds (i.e., serous or fibrous surface), this study aims to determine the effect of ECM niche preservation on cellular repopulation using different scaffold generation methods. BP underwent either sodium dodecyl sulfate (SDS) decellularization or stepwise, solubilization-based antigen removal using amidosulfobetaine-14 (ASB-14). SDS scaffolds were toxic to repopulating human mesenchymal stem cells (hMSC). Scanning electron microscopy revealed distinct surface ultrastructure of ASB-14 scaffolds based on native BP sidedness. Basement membrane structures on the serous side stimulated hMSC cell monolayer formation, whereas fibrous side facilitated cell penetration into scaffold. Additionally, serous side seeding significantly increased hMSC adhesion and proliferation rate compared to the fibrous side. Furthermore, scaffold ECM niche stimulated sidedness dependent differential hMSC human leukocyte antigen expression, angiogenic and inflammatory cytokine secretion. This work demonstrates that ECM scaffold preparation method and preservation of BP side-based niches critically affects in vitro cell growth patterns and behavior, which has implications for use of such ECM biomaterials in clinical practice.

Xenogeneic cardiac extracellular matrix scaffolds with or without seeded mesenchymal stem cells exhibit distinct in vivo immunosuppressive and regenerative properties.

Cardiac extracellular matrix (cECM) scaffolds are promising biomaterials for reconstructive surgery applications since they possess the structure/function properties of native tissue. Production of cECM scaffolds has been achieved using decellularization approaches, which commonly employ denaturing detergents, such as sodium dodecyl sulfate (SDS). Our antigen removal (AR) method has been shown to remove cellular and nonmyocyte components, while preserving cECM scaffold structure/function relationships. Here, we demonstrate that more human mesenchymal stem cells (MSCs) invaded AR scaffolds compared to SDS controls. Additionally, AR scaffolds stimulated a constructive remodeling response similar to allograft controls, and were transformed to adipose tissue in a xenogeneic rat to mouse subpannicular in vivo model. Conversely, SDS scaffolds showed a chronic inflammatory response that worsened throughout the 12-wk time course preventing constructive remodeling and mirroring the response seen towards xenogeneic tissue. AR scaffolds and xenogeneic controls recellularized with murine MSCs (mMSCs) were also implanted to assess whether mMSCs would offer any additive benefit in overcoming residual scaffold-specific immune responses. Paradoxically, recellularization resulted in chronic inflammatory response in AR-recellularized scaffolds. We conclude that AR cECM scaffolds represent a promising biomaterial, which is accepted by the recipient as self in origin and fosters implantation site appropriate regenerative responses. STATEMENT OF SIGNIFICANCE We demonstrated that an antigen-removal (AR) approach utilizing principles of differential solubility for production of a xenogeneic rat cardiac extracellular matrix scaffold results in improved recellularization efficiency with human and mouse mesenchymal stem cells (MSCs) in vitro. Furthermore, we tested the immune response to AR scaffolds versus allograft and xenograft controls with or without MSC recellularization using a rat to mouse subcutaneous model. We showed that AR scaffolds and allograft controls resulted in significant adipose tissue transformation after 12weeks. Paradoxically, MSCs had a positive impact in the immune response to xenografts, but had the opposite effect in AR scaffolds, resulting in chronic inflammatory response, which might be attributed to a change of their phenotype following recellularization into scaffolds.