Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine

Abstract

Tissue-engineered decellularized extracellular matrix (ECM) scaffolds hold great potential to address the donor shortage as well as immunologic rejection attributed to cells in conventional tissue/organ transplantation. Decellularization, as the key process in manufacturing ECM scaffolds, removes immunogen cell materials and significantly alleviates the immunogenicity and biocompatibility of derived scaffolds. However, the application of these bioscaffolds still confronts major immunologic challenges. This review discusses the interplay between damage-associated molecular patterns (DAMPs) and antigens as the main inducers of innate and adaptive immunity to aid in manufacturing biocompatible grafts with desirable immunogenicity. It also appraises the impact of various decellularization methodologies (i.e., apoptosis-assisted techniques) on provoking immune responses that participate in rejecting allogenic and xenogeneic decellularized scaffolds. In addition, the key research findings regarding the contribution of ECM alterations, cytotoxicity issues, graft sourcing, and implantation site to the immunogenicity of decellularized tissues/organs are comprehensively considered. Finally, it discusses practical solutions to overcome immunogenicity, including antigen masking by crosslinking, sterilization optimization, and antigen removal techniques such as selective antigen removal and sequential antigen solubilization.

Keywords: Biologic scaffold; Decellularization; Extracellular matrix; Immune response; Recellularization; Tissue engineering; Translational medicine.

Fig. 1

Innate and adaptive immune response against dECM scaffolds. (1) Decellularization strategies inevitably produce various amounts of damage-associated molecular patterns (DAMPs), which trigger pattern recognition receptors (PRRs) on innate immune cells. (2) Stimulation of PRRs in antigen-presenting cells provides prerequisites of T cell activation, including pro-inflammatory cytokine and major histocompatibility complex and co-stimulatory molecules, as well as triggering complement cascade to produce C3a, C3b, and C5a. T cell activation is the essential trigger for B cells, which mediate antibody production against remaining antigens within dECM scaffolds. Antigen-antibody interactions further trigger complement cascades and implement a vicious cycle. (3) C3a and C5a recruit immune cells and induce T helper type 1 (TH1) and TH17 polarization in helper CD4⁺ T cells, increasing the production of pro-inflammatory cytokines. (4) Elevation of pro-inflammatory cytokine content in the implantation site leads to further recruitment of immune cells and induces M1 polarization in naïve macrophages. (5) C3b complements enhance matrix metalloproteinases (MMP) production in M1 macrophages, accelerating the decellularized extracellular matrix (dECM) scaffold degradation and failure

Fig. 2

ECM alterations and immune response. Decellularization-derived damages to the extracellular matrix affect its immunogenicity via exposing/producing immunogenic particles or depleting immunomodulatory molecules. (1) Decellularization may destroy immunosuppressive traits of high molecular weight hyaluronic acids (HMWHAs) by breaking them into low molecular weight hyaluronic acids (LMWHAs). HMWHAs induce a regulatory phenotype in T cells and macrophages while suppressing humoral immunity and dendritic cell maturation. (2) LMWHAs are demonstrated to interact with CD44 and toll-like receptors of leukocytes and invoke inflammatory responses such as enhancing leukocyte chemotaxis, dendritic cell maturation and polarization of naïve macrophages toward M1 phenotype. (3) Collagen denaturation during decellularization may expose some cryptic antigenic sites and trigger antibody production, or (4) upregulate immune system activity through the depletion of RGD motives, which impede leucocytes chemotaxis and take part in the M2 polarization of macrophages. (5) Elastin particles are released upon elastin damage and provoke monocyte chemotaxis, antibody-mediated immune response, and T helper cell differentiation toward inflammatory phenotypes

Fig. 3

Apoptosis and necrosis as the core mechanisms of cell death in decellularization. Necrosis or apoptosis is the ultimate fate of cells upon the decellularization process. A Cell necrosis resulting from applying conventional decellularization techniques drives the vast leakage of immunogenic cellular materials, which invokes inflammatory responses following the implantation of derived scaffolds. B Apoptosis-assisted decellularization techniques exploit environmental stresses (extrinsic pathway) or death ligands to induce apoptosis. Following apoptosis, immunogenic cellular contents are degraded and confined to membranous vesicles that exert an immunomodulatory response

Fig. 4

Common post-decellularization modifications to address the immunogenicity of dECM scaffolds. A Selective antigen removal techniques rely on enzymatic treatment to specifically remove antigens (i.e., α-gal, Neu5Gc, and Sda epitopes). B Crosslinking may reduce the immunogenicity of dECM by masking exposed antigenic sites. C Solubilization removes various protein antigens (hydrophobic and hydrophilic antigens) according to their common physiochemical properties. D Terminal sterilization eliminates pathogen-related immunogenicity of viruses and bacteria in the dECM scaffolds