Immune Response to Extracellular Matrix Bioscaffolds: A Comprehensive Review

Abstract

Extracellular matrix (ECM) bioscaffolds have demonstrated therapeutic potential across a variety of clinical and preclinical applications for tissue repair and regeneration. In parallel, these scaffolds and their components have shown the capacity to modulate the immune response. Unlike synthetic implants, which are often associated with chronic inflammation or fibrotic encapsulation, ECM bioscaffolds interact dynamically with host cells, promoting constructive tissue remodeling. This effect is largely attributed to the preservation of structural and biochemical cues—such as degradation products and matrix-bound nanovesicles (MBV). These cues influence immune cell behavior and support the transition from inflammation to resolution and functional tissue regeneration. However, the immunomodulatory properties of ECM bioscaffolds are dependent on the source tissue and, critically, on the methods used for decellularization. Inadequate removal of cellular components or the presence of residual chemicals can shift the host response towards a pro-inflammatory, non-constructive phenotype, ultimately compromising therapeutic outcomes. This review synthesizes current basic concepts on the innate immune response to ECM bioscaffolds, with particular attention to the inflammatory, proliferative, and remodeling phases following implantation. We explore how specific ECM features shape these responses and distinguish between pro-remodeling and pro-inflammatory outcomes. Additionally, we examine the impact of manufacturing practices and quality control on the preservation of ECM bioactivity. These insights challenge the conventional classification of ECM bioscaffolds as medical devices and support their recognition as biologically active materials with distinct immunoregulatory potential. A deeper understanding of these properties is critical for optimizing clinical applications and guiding the development of updated regulatory frameworks in regenerative medicine.

1. Introduction

The extracellular matrix (ECM) plays a central role in tissue repair. In the context of regenerative medicine, biologic scaffolds derived from ECM have emerged as promising biomaterials capable of modulating the immune response and promoting tissue remodeling [1,2]. Unlike synthetic materials, which often elicit chronic inflammation or fibrotic encapsulation [3,4], ECM bioscaffolds engage with host cells in a dynamic manner, facilitating constructive healing [5]. These interactions are largely attributed to the structural and biochemical cues preserved in the ECM, including growth factors, matrix-bound nanovesicles (MBV), and degradation products that influence immune cell behavior [2]. However, the successful clinical translation of ECM bioscaffolds depends heavily on understanding their complex immunomodulatory effects—both those intrinsic to the ECM source and those introduced during scaffold manufacturing. This review explores the immune response elicited by ECM bioscaffolds and how this response is shaped by scaffold composition, processing methods, and structural properties. We begin by overviewing the immune response triggered upon biomaterial implantation, with emphasis on the inflammatory, proliferative, and remodeling phases. Subsequent sections examine specific ECM features that promote either pro-inflammatory or pro-remodeling immune responses. Finally, we discuss the implications of these findings for manufacturing practices, clinical applications, and regulatory classification. We emphasize that ECM bioscaffolds should be considered not as inert medical devices but as biologically active materials with therapeutic potential and immunoregulatory properties.

2. Overview of the Immune Response to Implanted Biomaterials

The immune response to an implanted biomaterial is initiated by the inflammation caused by surgery itself. The disruption of blood vessels, basement membranes, and tissues, in general, triggers coagulation cascade events, which result in the accumulation of platelets and leukocytes [6,7]. The contact of blood with the implanted biomaterial enables the adsorption of proteins such as albumin, immunoglobulins, and fibrinogen [3], which modulate the platelets’ activation and coagulation events [8], therefore influencing the progression of the healing. Among leukocytes accumulated from peripheral blood, antigen-presenting cells (APCs) of the innate immune system are the first to initiate the reaction to the biomaterial [9]. Neutrophils and circulating monocytes are generally acknowledged as the first responders [7,10]. In addition, tissue resident innate immune cells such as dendritic cells [11] or tissue resident macrophages, together with other lymphoid cells such as natural killers or innate lymphoid cells (ILC), are also attracted to the inflammation area. This recruitment is driven by chemokines released during the initial inflammation and coagulation [12]. These chemokines, together with cytokines derived from the neighboring cells signaling, clotting, and fibrinolytic events, activate further inflammatory molecular pathways in the recruited innate immune cells. To name some of these molecules, they include inflammatory cytokines such as TNFa, IL-1, IL-2, IL-6, IL-8, and IL-12 [12,13] and chemokines such as CCR1, CCR2, CCR6, CXCR2, and CXCL16 [7,10,13,14]. Whereas the initial inflammatory events are independent of the type of material implanted, the surgical technique and other factors can greatly impact the magnitude and durability of the inflammation [6]. However, from the inflammation onset onwards, the properties of the implanted material will critically influence the healing response, classically organized in three overlapping stages: inflammation, proliferative phase and remodeling phase (Figure 1).

After the inflammatory response onset, innate immune cells interact with the implanted material through transmembrane receptors called integrins [15] that activate metabolic pathways in response to mechanical [16,17] and biochemical stimuli [18]. These metabolic pathways include cascade factors such as NF-κB, MAPK, TGF-β, JAK/STAT, PI3K/Akt/mTOR [19], and control the release of cytokines and growth factors that communicate and amplify the immune response at the local level. Recruited neutrophils dominate the mononuclear cell population initially, initiating their phagocytic and proteolytic activity on adsorbed proteins and cellular debris [7]. Within days, as neutrophils clear the area, recruited circulating monocytes differentiate into macrophages in response to the initial inflammatory response. These monocyte-derived macrophages, together with tissue resident macrophages, are the main conductors of the orchestrated cellular response to biomaterials [20]. Most research on immune responses to biomaterials has focused on macrophages. However, contributions from the adaptive immune system and other hematopoietic innate immune cells are increasingly recognized in the context of long-term scaffold integration or rejection. For instance, eosinophils are also recruited during the initial coagulation steps and are key in fibrinolytic events [21], and their recruitment is promoted by ECM biomaterials in cardiac [22] and muscle regeneration [23]. Mast cells have been shown to be essential in the recruitment of phagocytic cells during biomaterial-related inflammation [24] and contribute to the regulation of the three phases of wound healing [24]. Basophils, less prominent granulocytes related to allergic reactions and response to IgE, similarly to mast cells, have also been linked to the progression of fibrosis in different tissues [25].

In the case of the adaptive immune cells, the activation of CD8⁺ and CD4⁺ T cells by the recognition of major histocompatibility complex (MHC) class I and II molecules in innate cells leads to cytotoxic response, cytokine secretion, and modulation of macrophage polarization [26]. In parallel, damage-associated molecular patterns (DAMPs) can activate APCs, with special contribution of dendritic cells [11], initiating complement cascades and the release of pro-inflammatory molecules C3a and C5a, which promote Th1 and Th17 responses. B cells may further contribute by producing antibodies against residual antigens within bioscaffolds, thereby exacerbating scaffold degradation [27].

For adaptive immune cells, engagement proceeds via antigen presentation. As noted above, innate APCs are the first to sense implanted biomaterials [7,9,10,11]. ECM-bioscaffold degradation products function as damage-associated molecular patterns (DAMPs) that ligate pattern-recognition and scavenger receptors on dendritic cells and macrophages; in some contexts, they also signal via integrins [2,26,28,29]. This early activation drives effective T-cell priming: CD8⁺ T cells via MHC I cross-presentation and CD4⁺ T cells via MHC II, supported by co-stimulatory signals [26]. In vivo, ECM-conditioned APCs bias T-cell differentiation toward Th2 and Treg lineages, reinforcing type-2 macrophage polarization and promoting resolution [26,30,31,32]. By contrast, high DAMP/complement signaling (C3a/C5a) or certain synthetic implants can favor Th1/Th17-linked programs, underscoring material- and context-dependence [26,33]. B lymphocyte responses depend on material source and processing, yet are generally compatible with tolerance and remodeling when bioscaffolds are appropriately prepared (parameters for optimal ECM preparation are detailed in subsequent sections). Although B lymphocytes can generate antibodies to residual antigens—potentially accelerating scaffold degradation [27]—xenogeneic ECM is commonly associated with a Th2-restricted humoral profile consistent with constructive outcome [2,34]. For example, in volumetric muscle-loss models, biologic scaffolds promote early B lymphocyte recruitment in wounds, germinal center formation in draining lymph nodes, and transient antigen presentation; in contrast, synthetic polymers (e.g., PCL) sustain B lymphocyte presence and maintain more persistent antigen-presentation programs [35]. Collectively, the evidence indicates that degradation products from ECM-bioscaffolds condition antigen presentation and favor regulatory T lymphocyte induction and B lymphocyte tolerance, sustaining a pro-resolution set point that supports scaffold integration [2,26,34].

Taken together, non-macrophage innate cells and adaptive lymphocytes influence scaffold integration and remodeling. However, the implication of these cell types in biomaterial-induced responses remains underrepresented in the literature and requires further studies. Taken together, non-macrophage innate cells and adaptive lymphocytes also influence scaffold integration and remodeling. However, the implication of these cell types in biomaterial-induced responses is underrepresented in the literature and requires further studies.

As inflammation progresses, released pro-inflammatory cytokines, chemokines, and proliferative and angiogenic growth factors attract surrounding cells such as fibroblasts, mesenchymal stem cells, and endothelial cells in order to initiate the proliferation phase. In this phase, new ECM is rapidly produced to repair the damaged tissues, and is dominated by the deposition of fibronectin, collagen type III, and collagen type I [36,37]. In optimal healing conditions, the pro-inflammatory phenotypes of immune cells gradually shift to pro-remodeling phenotypes, expanding the change in phenotypes to other cells and enabling the resolution of the inflammation. Classically, these phenotypes were discretely classified as M1 (pro-inflammatory) and M2 (remodeling), mainly due to the shift observed in some macrophage markers [38,39,40]. However, advancement in research of biomaterial-induced phenotypes has shown that the rainbow of phenotypes that macrophages (and other immune cell types) can adopt during healing processes is far more numerous and complex [41,42,43]. Within pro-inflammatory and pro-remodeling phenotypes, several subclasses have been identified, where M1/M2 markers’ exclusivity is lost and their presence and proportion are highly dependent on several factors, which, in the case of biomaterial implantation, include the biochemical [34,44,45,46], physical, and mechanical properties of the biomaterial [47,48]. Notably, the conventional combination of histology, reporter genes, and flow cytometry is now augmented by advances in multiparametric profiling—including mass cytometry (CyTOF), spectral flow cytometry, lineage-tracing systems, and single-cell transcriptomics [41]. These methods detect rare and transitional subpopulations and, across diverse contexts including bioscaffolds, reveal that macrophage (and other immune cell types) phenotypes during healing form a rainbow or broad continuum [41,42,43]. CyTOF enables simultaneous measurement of more than 30 parameters per cell [49], and single-cell RNA-seq (scRNA-seq) profiles thousands of transcripts per cell, providing an unbiased view of macrophage states [50]. Coupled with modern dimensionality-reduction and clustering algorithms (e.g., t-SNE [51], FlowSOM [52]), these platforms resolve continuous phenotypic gradients and novel subpopulations [41]. Additionally, spatial proteomics and spatial transcriptomics can map these state scores across the implant–tissue interface, linking microanatomical niches to function [53,54]. Nonetheless, the capacity of the biomaterial to interact with the host’s cells and promote or allow a shift from M1-like to M2-like phenotypes is crucial to support a functional remodeling of the damaged tissue, whereas the incapacity to resolve the inflammation leads to a reaction also known as foreign body reaction (FBR) [3,41,42]. Synthetic materials are known to induce this reaction because they lack interactions with immune cells [3,4] and resistance to degradation by proteolytic enzymes. As a result, ECM is deposited uncontrollably around the biomaterial, forming a capsule composed of a single or double layer of cells. In this case, M1-like macrophages stay in the implantation area together with numerous giant cells, specialized phagocytic multinucleated cells resulting from the fusion of several macrophages [4], a common response to FBR. On the other hand, ECM biomaterials are recognized by surrounding cells, and, gradually degraded by proteolytic enzymes, mainly matrix metalloproteinases (i.e., MMP-1, MMP-8, MMP-3, MMP-9) [55,56]. This continuous degradation allows the penetration of surrounding cells and replacement of the implanted material by the host’s ECM during the proliferation phase. In addition, this pro-remodeling phenotype can be amplified in lymphoid cells, which have been reported to undergo a Th2 phenotype in the presence of ECM bioscaffolds [34,57,58] due to the indirect signaling from innate immune cells. Nonetheless, it is noteworthy to mention that degradable materials can still elicit a pro-inflammatory response, given other factors like cytotoxic byproducts of degradation [59,60], presence of processing chemicals [61], and pathogen contamination [10,62], as it will be further discussed in the following sections of the review.

If inflammation is resolved, and the surrounding innate immune cells phenotype switches from pro-inflammatory to a dominantly pro-remodeling or M2-like state, the presence of giant cells, eosinophils, and neutrophils decreases [63], and the remodeling phase of healing takes place. During this phase orchestrated by M2-like macrophages through cytokines such as IL-4 or IL-10 [13,64,65], the ECM is reorganized to replace unorganized deposited granulation tissue. Over months, the implanted material is progressively replaced by host tissue in an effort to restore organ function. If inflammation persists or a foreign body response (FBR) blocks progression to the remodeling phase, inflammatory cytokines and degradative enzymes are continually released, and disorganized ECM is deposited. These processes lead to fibrosis and complications—pain, stiffness, swelling—and can ultimately result in loss of organ function [4,66].

The clinical literature indicates that frank “rejection” of ECM bioscaffolds is uncommon; most failures are organ- and context-dependent and more often reflect mechanical demands or processing quality than allogenic/xenogeneic immunity. In the shoulder, repairs reinforced with small intestinal submucosa (SIS) can re-tear as assessed by magnetic resonance imaging (MRI), and randomized trials in moderate-to-large tears show no clear benefit [67,68,69]. In abdominal wall reconstruction, systematic reviews underscore outcome dependence on mesh properties [70,71]. By contrast, a multicenter randomized controlled trial of paraesophageal hernia repair showed reduced recurrence with a biologic prosthesis [72]. Mechanistically, inadequate decellularization retains high amounts of damage-associated molecular patterns (DAMPs) or endotoxin, and excessive crosslinking biases macrophages toward pro-inflammatory phenotypes that drive fibrosis and poor remodeling [73,74]. As noted above, xenogeneic ECM typically elicits a Th2–skewed response [26,30,31,32] that preserves systemic protective immunity, arguing against pharmacologic immunosuppression. Moreover, matrix-bound nanovesicles (MBV), intrinsic to ECM, recapitulate pro-remodeling effects locally and systemically without compromising host defense [75,76,77]. Thus, in the absence of infection or pharmacologic immunosuppression, scaffold composition and processing govern the host response.

Overall, a key prerequisite to prevent a chronic inflammatory reaction or FBR by an implanted biomaterial is its ability to interact with the host’s cells without causing cytotoxic reactions while being gradually degraded and resorbed. Adequately processed ECM bioscaffolds, which are virtually depleted from the donor’s cell material and processing residuals, excel at this requirement. What is more, it is widely accepted that during the degradation of implanted ECM bioscaffolds, ECM components are released into the environment and promote the transition to a remodeling phase [18,44,74,78]. While the mechanism by which this occurs is not completely understood, it is accepted that the direct effect of such ECM degradation products on surrounding innate immune cells is the main driver [18,64,79]. Nonetheless, degradable materials can still elicit a pro-inflammatory response given other factors like cytotoxic byproducts of degradation [59,60], presence of processing chemicals [61], and pathogen contamination [10,62]. These factors are discussed further in the following sections of the review.

3. ECM Biomaterials

Scaffolds derived from ECM represent a unique class of materials that actively participate in the immune microenvironment and can modulate the host response toward resolution and constructive tissue remodeling [2,77]. As described in the previous section, ECM bioscaffolds deliver immunomodulatory signals through retained bioactive molecules and native-like architecture that influence immune cell activation, polarization, and effector function [48,80]. ECM biomaterials can be broadly categorized into two groups: (a) decellularized tissue-derived ECM, and (b) scaffolds fabricated from purified ECM components [2,81].

3.1. ECM Bioscaffolds

Decellularized scaffolds (also called ECM biologic scaffolds or ECM bioscaffolds) retain the complex biochemical and structural landscape of native tissues. They have demonstrated therapeutic potential across a variety of clinical and preclinical applications, including abdominal wall reconstruction [57,74,82], musculoskeletal repair [64,83,84], cardiac tissue regeneration [84,85,86], bladder augmentation [87,88], and esophageal repair [89,90,91], among others [2]. Their immunomodulatory capacity is attributed to the preserved ECM biophysical and biochemical properties, which provide instructive cues for immune cells, particularly macrophages, guiding the transition from inflammation to tissue regeneration. Another factor underlying limited xenogeneic rejection is high cross-species ECM conservation. Comparative proteomics defines a conserved core matrisome—collagens, glycoproteins, proteoglycans—across mammals, while matrisomes remain organ-specific (heart versus kidney) [92,93]. However, some inconsistent clinical outcomes and complications—such as seroma formation, scaffold degradation, and fibrotic encapsulation—have been reported [67,73,94,95,96]. These inconsistencies are often linked to differences in tissue source, decellularization efficiency, and post-processing methods. Thus, the immune compatibility of decellularized scaffolds is highly dependent on the preservation of ECM integrity and the efficient removal of immunogenic cellular remnants.

Decellularization is a critical process aimed at eliminating cells and nuclear content (e.g., DNA, phospholipids, intracellular proteins) while preserving the scaffold’s native architecture and functional components [2,97,98]. Incomplete decellularization may result in the persistence of DAMPs, triggering sustained inflammation and promoting a pro-inflammatory (M1-like) macrophage response. Conversely, over-processing can degrade essential ECM proteins, growth factors, and biomechanical properties, reducing the scaffold’s regenerative potential. Decellularization protocols typically combine physical, chemical, and enzymatic strategies (Figure 2A), tailored to the source tissue’s structure, density, and lipid content [1,97,99]. Physical methods, such as freeze–thaw cycles and sonication, disrupt cell membranes and facilitate the detachment of cellular remnants from the ECM. In multilayered tissues (e.g., urinary bladder, small intestine), mechanical delamination is often used to isolate specific ECM-rich layers [34,100,101,102]. Although chemical methods are generally more disruptive to ECM ultrastructure than physical approaches, they are essential for effective removal of cell debris. Ionic detergents such as SDS are highly effective for cell lysis but can damage collagen networks and basement membrane components [103]. Non-ionic or zwitterionic detergents are less aggressive and better preserve bioactivity, though they may be insufficient for decellularizing thicker tissues [44,86]. In lipid-rich tissues, solvents such as ethanol or acetone aid in delipidation [104,105,106]. The success of decellularized ECM scaffolds in releasing bioactive signals, promoting a pro-remodeling type 2 immune response, and recruiting endogenous stem/progenitor cells depends on a critical balance. Immunogenic cell remnants must be eliminated while immune-modulating ECM components are preserved. Once effective cellular removal has been verified, it is essential to assess how decellularization affects the mechanical properties of the remaining ECM scaffold, where the mechanical testing should be selected based on the demands of the intended clinical application [76]. The development of tissue-specific, standardized protocols remains essential to ensure reproducibility and enable successful clinical translation.

3.2. Purified ECM-Based Scaffolds

In contrast to tissue-derived scaffolds, purified ECM-based materials offer a more controlled composition and engineering versatility [107]. These scaffolds are engineered from isolated ECM molecules—such as collagen, fibronectin, elastin, fibrin, and laminin—and can be formulated into scaffolds, gels, particles, and films to support specific biological responses [107]. Collagen, particularly type I, is the most widely used component due to its abundance, low immunogenicity, and ability to support cell adhesion, proliferation, and differentiation via integrin signaling [108]. Collagen-based scaffolds have been extensively used in wound healing, where they promote angiogenesis and epithelialization [109]. Fibronectin, another key ECM protein, facilitates early matrix organization, cell migration, and integrin-mediated signaling essential for tissue repair [110,111]. Other purified ECM molecules—including laminin, elastin, and hyaluronic acid—have been incorporated into scaffolds to provide mechanical flexibility, support basement membrane assembly, or modulate immune cell behavior [112,113,114,115,116]. These components can be isolated or produced recombinantly, enabling greater consistency and the ability to decouple and study specific matrix-derived signals.

Purified ECM scaffolds typically exhibit minimal immunogenicity, high biocompatibility, and reproducible fabrication, as their isolation protocols eliminate or minimize other molecules, including DAMPs [117]. However, they lack the full complexity of native ECM and may require additional engineering (e.g., bioactive ligand presentation, mechanical tuning) to emulate natural tissue cues, limiting their immunomodulatory potential when compared to ECM bioscaffolds. Despite their simplified architecture, these materials play a central role in designing immuno-instructive environments, particularly coatings, injectable matrices, or components of composite scaffolds [107,117]. The ability of ECM-derived scaffolds—whether obtained from decellularized tissues or engineered from purified components—to modulate the host immune response, particularly macrophage activity, in a context-dependent manner underscores their therapeutic potential [5,80,118].

Optimizing ECM materials for clinical use requires a deeper understanding of how scaffold composition and processing influence immune dynamics. Given their higher complexity and immunomodulatory potential, the following sections will focus on ECM-bioscaffolds (i.e., decellularized tissues) and the factors that influence their immunomodulatory properties. Nonetheless, many of the concepts discussed below can also be applied to purified ECM-based scaffolds to a certain extent, depending on their processing. In addition, the main focus of the present review is to understand the effect of ECM bioscaffolds’ biological and biochemical properties. Although other physical properties could also impact the immune response (

Table 1. Physical properties of ECM scaffolds with likely influence on the immune response.

Physical Properties of the ECM	Pro-Inflammatory Promotion	Pro-Remodeling Promotion	Limitations	Ref.
Surface charge and wettability	Anionic hydrophilic surfaces are related to inflammatory responses.	Cationic hydrophilic and hydrophobic surfaces are related to proremodeling responses and promote migration.	Few studies have been conducted in complex environments where the influence of the surface can be limited.	[119,120]
Surface topography	Higher roughness promotes migration and phagocytosis, but has no clear effect on phenotype.	Smoother surfaces can promote higher attachment of immune cells.	Very limited studies on synthetic materials that lack the biological component.	[121,122]
Stiffness	Higher stiffness (as compared to tissue in homeostasis) is usually related to inflammation.	Low stiffness can promote pro-remodeling but impair migration.	Few studies, mostly on 2D platforms. Complex interactions in 3D.	[48,123,124]

), very few studies on ECM materials have investigated these effects. How these properties evolve during the healing process and their hierarchical influence compared to biological properties is still poorly understood and must be a matter of future studies.

4. Factors Influencing Immune Response to ECM Bioscaffolds

In the absence of infection, metabolic disorder, or pharmacological immunosuppression, the immune response to an implanted ECM bioscaffold is primarily determined by the material’s biochemical composition, structural integrity, and associated molecular cues [1,99]. Early efforts in the biomaterials field emphasized the use of “inert” materials—such as medical-grade silicone—that would minimize immune activation [5]. However, as noted in Section 2, it is now widely accepted that no implanted material is entirely inert. All biomaterials interact with the host immune system, eliciting a response that can either promote tissue repair or lead to chronic inflammation and graft failure. Synthetic materials typically elicit a foreign body response (FBR), characterized by persistent inflammation and fibrous encapsulation. In contrast, ECM bioscaffolds modulate the host response in a more physiological manner, often supporting constructive and regenerative outcomes. However, the outcome of this immune interaction is not binary but rather shaped by a combination of factors related to source tissue, decellularization methods, post-processing techniques, and residual bioactive molecules. Below, we outline key factors that can drive the response toward either a pro-inflammatory (Section 4.1) or pro-remodeling (Section 2) response (Figure 2B). In addition,

Table 2. Summary of major ECM constituents (e.g., collagens, glycosaminoglycan, MBV) and typical processing residues (e.g., nucleic acids, detergents). For each entry, we indicate risks if retained and recommend handling/removal considerations to balance safety with preservation of bioactivity. NA: not applicable.

Component Present in the ECM	Risks	Removal Trade-Off
Glycosaminoglycans (GAGs, incl. hyaluronan/HA)	Low-MW HA fragments act as DAMPs recognized by PRRs.	Beneficial if preserved: hydration, growth-factor sequestration, support for recellularization; pro-remodeling cues. Avoid over-aggressive steps that fragment HA; prefer less-aggressive (non-ionic/zwitterionic) detergents with thorough rinsing.
Degradation peptides (matrikines/matricryptins)	Some peptides are sensed as DAMPs/chemotactic signals; overall bias toward pro-remodeling responses.	Beneficial if preserved: ECM behaves as a sustained peptide-release matrix. Avoid over-crosslinking, which reduces bioactive peptide release and promotes pro-inflammatory bias; avoid excessive fragmentation that increases the DAMP burden.
Integrin-recognition motifs (RGD, GxOGER, PHSRN)	NA	Beneficial if preserved: integrin-mediated adhesion and repair signaling. Minimize SDS exposure; avoid crosslinking that masks ligands.
Matrix-bound nanovesicles (MBV)	NA	Beneficial if preserved: potent local immunomodulators, replicate ECM pro-remodeling effects. Present across decellularization methods—resilient and tightly ECM-associated.
Collagen and basement-membrane (BM) architecture	Architectural damage elevates inflammatory bias and impairs endothelial behavior.	Beneficial if preserved: adhesion, epithelialization, angiogenesis; proper presentation of recognition motifs. Limit ionic detergents (SDS) that disrupt BM/collagen; use mechanical delamination; avoid over-crosslinking that reduces biodegradability and shifts immune tone.
Residual cellular membranes (processing residual)	Reservoir of antigens/DAMPs.	Emphasize complete membrane removal using physical (e.g., freeze–thaw, sonication) plus mild chemical steps.
α-gal epitope (largely membrane-associated carbohydrate)	Relevance remains under investigation; anti-Gal recognition may activate complement, recruit macrophages, and cause matrix degradation.	Reduce membrane reservoirs during decellularization.
Residual DNA (processing residual)	Quality control concern.	DNase with rinses to meet quality control without harsher steps that damage the ECM.
Residual detergents (processing residual)	Cytotoxic; hinders recellularization; damages BM/collagen.	Minimize ionic-detergent dose/time; favor non-ionic/zwitterionic where feasible; validate long rinses to sub-toxic levels.
Endotoxin (LPS) (processing residual)	Among the most potent inducers of innate activation, trace amounts can activate TLRs; FDA endotoxin limits apply.	Low-endotoxin sourcing and asepsis; validated depyrogenation; lot-release endotoxin testing; avoid over-sterilization that denatures ECM cues.

summarizes the risks, benefits and removal trade-offs of ECM components.

4.1. Factors Promoting a Pro-Inflammatory Response

As outlined in Section 3, decellularization aims to remove cellular content while preserving the ECM’s three-dimensional structure and bioactivity. However, harsh processing methods or incomplete decellularization may result in the release or retention of DAMPs, including fragmented matrix components (e.g., decorin, low molecular weight hyaluronan, fibronectin), mitochondrial and nuclear DNA, and heat shock proteins, among others [125]. These molecules are recognized by pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), expressed on both immune and stromal cells [126]. DAMP-PRR interactions initiate inflammatory signaling cascades, leading to the production of cytokines (e.g., IL-1β, IL-6, TNF-α) and chemokines that recruit immune cells to the scaffold [105,127]. Macrophages play a central role in this early response. Excessive DAMP signaling can polarize macrophages toward a pro-inflammatory M1-like phenotype [27], resulting in sustained inflammation, impaired resolution, and suboptimal remodeling. Although innate immune activation is necessary for tissue repair, dysregulation of this response—particularly when the balance between pro-inflammatory and pro-regenerative macrophage subsets is disrupted—can lead to graft degradation and fibrosis.

4.1.1. Residual DNA

Residual DNA is a frequently assessed indicator of decellularization efficiency and scaffold immunogenicity. For its quantification, DNA is normally isolated from dry material using classic DNA isolation methods or affinity columns and kits, and then quantified employing absorbance (nanodrop) or fluorescence-based (double-stranded DNA-binding dyes) techniques. Although thresholds of <50 ng dsDNA per mg dry ECM and DNA fragments < 200 bp are widely used, the precise levels required to avoid immune activation remain context dependent [128,129]. Some studies have shown that insufficient DNA removal correlates with increased inflammatory infiltration and impaired remodeling [73]. Others suggest that DNA alone does not elicit significant immune responses unless presented alongside adjuvants or in pro-inflammatory environments [130]. In fact, recent studies have shown that xenogeneic DNA present in porcine biologic scaffolds is not linked to an inflammatory immune response [130], but to other factors such as endotoxins [131]. However, it is logical to assume that a higher presence of DNA is related to a higher presence of other DAMPs, such as cellular membranes or cytosolic components. Thus, while residual DNA may not be a crucial determinant of the immune response to biologic scaffolds, its quantification remains an important component of quality control. However, the safety threshold of residual DNA established in the literature remains arbitrary and should be revised on a case-by-case basis.

4.1.2. Residuals from Processing Agents

Detergents are effective for decellularization, but also well known to induce cytotoxicity, which can hinder scaffold recellularization [97,132,133,134]. Therefore, their quantification is a common quality control point in order to prevent cytotoxicity events, normally using chromatography and fluorescent dye-based techniques. For instance, SDS treatment may reduce soluble collagen or alter its structure, disrupting the basement membrane and leading human microvascular endothelial cells to poor confluence and abnormal cell morphology [103]. Crosslinking is another factor that alters the biochemical and mechanical properties of ECM biomaterials. While it helps reduce immunogenicity by restoring structural integrity after decellularization, excessive crosslinking—particularly with chemical agents—impairs biodegradability [135,136] and promotes a higher M1/M2 macrophage ratio, leading to increased inflammation and FBR compared to a non-crosslinked scaffold [137].

4.1.3. ECM Carbohydrates

Immunogenic carbohydrate epitopes, such as galactose-α (1,3)-galactose (α-gal), are commonly found in ECM derived from non-primate mammals. Humans naturally produce anti-Gal antibodies due to continuous microbial exposure, and recognition of residual α-gal in xenogeneic ECM bioscaffolds may activate the classical complement pathway, promoting inflammation through complement-mediated macrophage recruitment and matrix degradation. Although α-gal is primarily associated with hyperacute rejection in vascularized xenotransplantation, its relevance in the context of ECM bioscaffolds remains under investigation [5]. Strategies to mitigate the risk include enzymatic deglycosylation or the use of tissues from α-gal-knockout animals. For instance, subcutaneous implantation of decellularized lungs from wild-type and α-gal knockout pigs in non-human primates suggested that removal of the Gal epitope can reduce adverse inflammatory responses. These responses are linked to chronic exposure to xenogeneic tissues [138]. Notably, α-gal has been detected in porcine small intestinal submucosa (SIS); however, SIS did not induce complement activation in vitro [139], and its capacity to support tissue remodeling appears unaffected by naturally occurring anti-αgal antibodies in αgal^−/− mouse models [140]. Furthermore, SIS-derived ECM has been employed in both preclinical and clinical settings for decades without reports of significant adverse immunological events [72,141,142,143,144].

4.1.4. Endotoxins

Residual microbial components introduced or not properly removed during processing, particularly endotoxins such as lipopolysaccharides (LPS), are among the most potent inducers of innate immune activation [145]. Even trace amounts of LPS can activate TLR4 signaling in APCs, leading to robust secretion of pro-inflammatory cytokines and skewing macrophage polarization toward an M1-like phenotype. The resulting inflammatory environment can inhibit scaffold remodeling, promote fibrotic encapsulation, or even contribute to graft rejection [131]. The U.S. Food and Drug Administration (FDA) recommends specific endotoxin limits depending on the intended use of the device [146,147], but studies have shown that some commercial and investigational ECM products exceed these thresholds [148]. Endotoxin contamination is highly context dependent; even low levels may be problematic in immunologically sensitive environments [27]. For this reason, endotoxin quantification (e.g., via Limulus Amebocyte Lysate assays) and validated sterilization methods—such as ethanol washes, or supercritical CO₂—are essential components of manufacturing. Reducing endotoxin burden enhances the immunomodulatory potential of ECM bioscaffolds and helps preserve their regenerative properties.

Overall, residual DAMPs, antigens (e.g., MHC, α-gal), chemicals, and endotoxins can each contribute to pro-inflammatory responses that compromise scaffold performance. Strategies aimed at minimizing these immunogenic cues—through tissue-specific decellularization and rigorous sterilization—are critical for achieving predictable and favorable host outcomes. Understanding and controlling these variables is essential for the development of next-generation ECM scaffolds with optimized immunomodulatory properties.

4.2. Factors Promoting a Pro-Remodeling Response

4.2.1. Degradation Peptides and Recognition Motifs

There is extensive work demonstrating the beneficial effects of ECM degradation peptides on cell proliferation [79,149,150], migration [79,149], stem cell differentiation [150,151], angiogenesis [152], and, as previously stated, promotion of M2-like phenotypes in vitro [40,44,153] and in vivo [152,154,155]. Upon degradation and release, these peptides interact with cell membrane receptors such as TLR and integrins that activate specific molecular pathways related to a pro-remodeling response [18]. Therefore, ECM bioscaffolds can conceptually be perceived as a sustained peptide release biomaterial. In this release, peptides that were inaccessible to cells become available and acquire biological activity. These newly released biologically active peptides are also known as matricryptins [156] or matrikines [157], and include PGP from collagen, VGVAPG from elastin, or PHSRN from fibronectin, for instance. These peptides contain three to hundreds of amino acids and have specific functions in regulating the immune response. Interestingly, some of these peptides are recognized as DAMPs and chemotactic signals by innate immune cells during inflammation. Therefore, degradation of ECM bioscaffolds does not prevent inflammation but promotes its resolution and the transition to pro-remodeling phenotypes in later stages. In addition, before degradation, some peptides or recognition motifs are also available for cells to attach and migrate into the material network and activate metabolic pathways, including motifs such as RGD in fibronectin or GxOGER in collagen [158]. Therefore, the plethora of components in ECM bioscaffolds includes different motifs that are vital for cell recognition and cytocompatibility. Preserving these recognition motifs and the complexity of the ECM should contribute to a pro-remodeling response if factors that promote inflammation are balanced. In addition, the complex nature of the ECM and its tissue specificity are also believed to influence the cell response. For instance, the basement membrane site of porcine peritoneum has been shown to elicit significantly different responses in the same cell type in vitro when compared to the connective tissue side [152,153], while esophageal matrix hydrogel has shown tissue-specific remodeling response in a esophageal cancer dog model, as opposed to an urinary bladder matrix hydrogel [154].

4.2.2. ECM Carbohydrates: Friend or Fool?

As discussed previously, ECM carbohydrates (GAGs) are recognized as inflammatory components in ECM bioscaffolds given the presence of the a-gal epitope. However, carbohydrates fulfill critical roles in the ECM. They act as water retention structures that modulate the hydration and compressive mechanical properties of the tissue [159], while also being important growth factor reservoirs [160]. In fact, they are believed to play a crucial role in scaffold recellularization [161]. Therefore, GAGs are a pivotal component of the ECM that should be preserved after ECM bioscaffolds processing. While some carbohydrates such as the α-gal epitope are well-known to activate pro-inflammatory responses, these immunogenic carbohydrates are mainly found in cell membranes [162], although some presence can be observed in ECM proteins in function of the tissue. Therefore, the development of decellularization processes that eliminate cellular membrane material and that promote other factors associated with the pro-remodeling immune response is crucial. In this respect, physical means that eliminate cells and their combination with chemical agents that solubilize membrane lipids (hydrophobic) while minimizing denaturation are promising strategies to promote GAGs (hydrophilic) preservation.

4.2.3. Matrix-Bound Nanovesicles

Matrix-bound nanovesicles (MBV) were recently identified [163] in ECM bioscaffolds as an additional factor that contributes to a pro-remodeling response in macrophages in vitro [75] and in vivo [164,165]. These small extracellular vesicles are able to replicate the ECM pro-remodeling response that the source ECM bioscaffold induces, indicating that MBVs are at least partially responsible for ECM bioscaffolds’ immune modulation. Moreover, to be isolated, MBV requires tissue decellularization and ECM digestion with enzymatic methods [166], which resembles the process that the host’s immune cells carry out in order to trigger the regenerative potential of ECM bioscaffolds. MBV independence at promoting a pro-remodeling response [163,166] further proves the necessity for the degradation of ECM biomaterials upon implantation to promote their pro-regenerative effect. If it was stated before that ECM bioscaffolds can be conceived as a peptide delivery system, MBVs are included in this delivery. Interestingly, independently of the methodology used to decellularize the ECM, all decellularized tissue has shown MBV to date, proving the resilience of the MBV and the tight ECM-MBV relationship [163,166,167].

Recent studies have shown that the injection of MBV can cause immune modulation locally [164,165] and systemically [76,168]. This suggests that MBV can be used to harness the regenerative potential of the ECM bioscaffolds, concentrate it, and deliver it systemically without the necessity of a surgical implantation. What is more, the effects of the immune modulation after MBV injection seem to have a persistent effect on the immune system [76,77], suggesting an epigenetic effect. Such findings are a paradigm shift in our understanding of ECM bioscaffolds’ regenerative and immune regulation effects, representing a promising avenue to develop new therapies.

4.3. Patient Factors Affecting the Immune Response

Beyond intrinsic scaffold properties, patient factors such as age, gender, or even blood type can condition the immune response to ECM bioscaffolds, and therefore, they must not be overlooked. Age might be the most critical factor, given the well-known effects of aging on the immune system, also known as immunosenescence. With aging, the counts, proliferation capacity, migration, and differentiation of both innate and adaptive immune systems are considerably impaired. This results in a delayed immune response to biomaterials and in chronic inflammation [169]. While a transcriptomic study in vitro studying leukocyte response to different biomaterials showed no age nor sex-related bias in collagen type I [170], a more recent study has shown distinct responses to a porcine urinary bladder bioscaffold when comparing young and aged mice [171]. In this study by Kilkarni et al., an implanted synthetic mesh showed a continuous increase in the infiltration of immune cells. However, the ECM bioscaffold promoted a drop in cell infiltration after inflammation, although this decrease was significantly delayed in aged mice. Interestingly, the response in young and aged mice to the ECM bioscaffold was governed by different metabolic pathways. This and a previous study [172] point towards the more prominent activation of the Th17 response in aged individuals as a main driver of such differences. The immune system also presents notorious differences between sexes, where the female immune system tends to higher acute reactions and therefore cases of immune hypersensitivity and fibrosis [173]. This could imply that female patients may be more suitable for ECM bioscaffolds treatments. However, very few studies in the literature investigate such differences, marking a gap in the field.

ABO/Rh blood-group can also modulate host responses to biomaterials. The ABO system (A, B, AB, O; defined by A/B carbohydrate antigens) and the Rhesus (Rh) system have been variably linked to outcomes after biologic implantations and warrant consideration in study design and clinical selection. In xenogeneic cardiac bioprostheses, single-center data associated recipient group A with longer bioprosthesis longevity, and subsequent work suggested ABO-specific structural valve degeneration (SVD) phenotypes—hypothesized to reflect cross-reactivity to xenogeneic glycans such as α-Gal [174,175]. However, a population-based registry did not demonstrate an association between ABO phenotype and SVD, pointing to underlying population/device heterogeneity [176]. For human homograft valves, residual A/B antigens persist on cryopreserved conduits—most prominently within vasa vasorum endothelium—creating a substrate for humoral recognition [177]. Pediatric series link ABO mismatch to accelerated failure, whereas adult series show no difference, underscoring context dependence and the role of retained endothelial elements [178,179]. Overall, ABO/Rh effects appear context-specific, most evident when scaffolds retain carbohydrate antigens/endothelium. Future work should include prospective, scaffold-specific studies that quantify residual antigens/endothelium and stratify by patient ABO/Rh, integrating serology, complement, and macrophage readouts.

5. Implications in Clinical Translation

From what has been discussed about ECM bioscaffolds, it can be resolved that their immune response is linked to their adequate processing. However, the presence of residual cellular membrane material, chemicals, or pathogenic signaling is a likely formula that will generate an unwanted immune reaction. This concept has implications for how ECM bioscaffolds should be manufactured and which quality control measures should be established, which logically will have an effect on their clinical performance. In addition, the intimate relationship between ECM bioscaffolds and their effect on the immune system also poses some challenges at the regulatory level. In the section below, the implications of the immunomodulatory properties of ECM bioscaffolds at different stages of clinical translation are discussed.

5.1. Implications in Manufacturing

The goal of processing ECM bioscaffolds in current commercial decellularization is to eradicate the presence of DNA from the ECM to a certain threshold. However, DNA is a stable polymer with a negative charge that has affinity for positively charged molecules such as side chains of collagen fibrils [180]. Therefore, harsh treatments are required to solubilize DNA from ECM structures, which can collaterally affect ECM components that are a source of immune-modulation signaling. In addition, given that no specific threshold of DNA can be correlated to an adverse response in ECM bioscaffolds, regulatory bodies cannot provide a limit from a regulatory standpoint. From a manufacturing, quality, and research and development perspective, there is a need for an evolution in reagents, processes, and quality controls in the processing of ECM bioscaffolds. Implementation of processes that specifically focus on cellular membrane (reservoir of most markers) removal could be an avenue for improved processing of ECM bioscaffolds. In this matter, the use of enzymes is gaining traction [181,182,183,184,185]; however, they increase the already high cost of ECM materials and have immune reaction risks [185].

Cytotoxicity caused by residuals such as detergents and salts is also a source of immune reactions during the degradation of implanted ECM bioscaffolds, [133] as previously discussed. Similarly to DNA content, there is no established limit on the amount of residual detergent that an ECM bioscaffold can contain. Normally, these limits are established internally in quality controls and referred to as appropriate when required tests under regulatory guidelines and international standards are positive. Whereas some detergents with higher biocompatibility and lower risks for the environment are being investigated, it is difficult to outperform classically used ionic and non-ionic detergents since their cytotoxicity mechanism is what makes them effective at decellularization: the solubilization of cellular membranes and breakage of protein—lipid interaction [186]. Alternatively, the use of mechanical means to delaminate epitheliums or remove keratinized structures could reduce the exposure time and concentration of these reagents since these compacted structures prevent the penetration and diffusion during chemical washes [185,187]. Implementation of scalable systems of perfusion decellularization is also an alternative, especially in highly vascularized tissues [188,189,190]. In addition, for ECM bioscaffolds that do not require structural integrity (i.e., hydrogel or micronized forms of ECM bioscaffolds [191]), tissue can be homogenized on an industrial scale in cold conditions. This approach could increase the efficiency of decellularization processes and preserve important ECM biological cues.

Crosslinkers are another source of controversy in ECM biomaterials. Whereas they effectively improve the mechanical properties of ECM [60,192,193], this is at the cost of lower degradability, lower cytocompatibility, and higher inflammation [185,194,195]. By creating intermolecular and intramolecular bonds, crosslinkers prevent the accessibility to recognition motifs in ECM and its degradation, and therefore the release of immunomodulatory peptides and MBVs, which could be considered as turning the ECM into a synthetic material. Nonetheless, the poor mechanical properties of ECM biomaterials are an intrinsic limitation, particularly in load-bearing or challenging clinical scenarios [185]. Alternative formulas, such as combination with synthetic materials [191], should be further explored to address such drawbacks.

Tissue sourcing, harvesting techniques, and storage of raw tissues should also be carefully controlled in the quality systems for ECM bioscaffolds manufacturing. In particular, appropriate handling of tissues and certain preprocessing can drastically reduce the spread and proliferation of pathogens, and therefore endotoxins, which are a challenging contaminant in ECM matrices [196]. This could be crucial in tissues with mucosa and, therefore, prominent bacterial flora, such as the intestine.

5.2. Implications for Clinical Applications

ECM bioscaffolds’ effect on the immune system seems to be a positive trait in any clinical application: prevention of chronic inflammation, remodeling of tissue, and integration with the host’s ECM. However, while we are still far from fully understanding these immunological effects, research indicates that this immune regulation is not only at the local level, but also systemic, as suggested in studies with decellularized tissues [57,58], or MBV [76,168,197], which have even shown potential epigenetic effects on macrophages [77]. Therefore, the extent of these immune effects must not be overlooked, especially when combined with other therapeutics. Nonetheless, preclinical research indicates that neither ECM bioscaffolds implantation [57] nor MBV injection [77] compromises processes where inflammation is crucial, such as vaccination-induced adaptive immunity.

Additionally, the use of ECM bioscaffolds in high-risk of infection scenarios (ventral hernias, field wounds, diabetic ulcers, etc.) and cancer progression (soft tissue repair after tumor resection) also poses some controversy given their immunomodulatory properties. M2-like and remodeling phenotypes in the immune system are believed to promote infection and tumor progression, given that a pro-inflammatory reaction from APCs and T-effector cells is required to neutralize these processes. However, one must consider that ECM scaffolds do not prevent inflammation, but aid in its resolution in the last stage (Figure 1). In addition, as previously discussed, the rainbow of phenotypes (including within M2-like) that the innate immune system expresses upon biomaterials implantation makes it challenging to associate it with the complex cancer-inducing phenotypes. To date, no investigation links the use of ECM bioscaffolds with tumor recurrence [198], while it has been shown that a synergy in the use of ECM biomaterials and immunotherapy in cancer could exist [58]. On the other hand, the use of ECM scaffolds in risk of infection scenarios is still an unclear option [62,96,185,199], since some studies point to the antimicrobial effect of ECM degradation peptides [200,201,202], while others do not prove improvement in infection [203]. One must note that pathogens have evolved to degrade and attach to mammalian ECM (including those in ECM bioscaffolds). On the other hand, as previously discussed, DAMPs from ECM bioscaffolds degradation also contribute to innate cell recruitment and initial inflammatory phases, which should be detrimental to pathogen proliferation. Therefore, further studies are required to fully understand the implications of the use of ECM bioscaffolds in clinical scenarios with a risk of infection.

What makes ECM scaffolds attractive from an immune modulation perspective can also make them inappropriate for certain clinical applications. The relatively fast degradation and remodeling of ECM bioscaffolds is a trade-off for mechanical support and integrity. As a result, uncrosslinked ECM scaffolds are a risky option in scenarios where a challenging mechanical load is required, such as tendon augmentation [68,204,205,206] or ventral hernia repair [207,208], leading to bulging or mechanical failure [191]. Strategies such as a combination of synthetic materials with a controlled resorption rate could be an alternative to these limitations. On the other hand, ECM bioscaffolds provide an alternative to congenital infant defects such as those related to heart valves [209,210], given their prominent integration with the host’s tissue and prevention of inflammation.

5.3. Implications in Regulation as Medical Devices

Current research demonstrates the immune effect at local and systemic levels elicited by ECM bioscaffolds. While no biomaterial is completely inert after implantation, the specific cellular processes that ECM bioscaffolds trigger after implantation make them a kind of their own. This is further supported by the fact that all ECM bioscaffolds contain their own kind of extracellular vesicles, the MBV [163]. However, most healthcare regulatory bodies, such as the FDA and EMA, still neglect their immunological effects and still consider ECM bioscaffolds as medical devices (FDA) or tissue-engineered products (EMA) [191,211] when they clearly have a biologic nature, given their regulation on metabolic and immune processes. Moreover, industry manufacturers are not interested in recognizing this characteristic in their ECM bioscaffold products, given the constraining and expensive regulatory pathways that biologic therapies undergo [191,211]. For instance, medical devices in FDA regulation can benefit from the 510(k) pathway if a similar predicate (with similarity demonstrated in preclinical validations) is already regulated in a concrete clinical application. Biologics do not benefit from this circumstance and require clinical data showing biosafety, which largely increases the costs associated with bringing a product to market. In addition, biologics require more stringent (and costly studies and Investigational New Drug (IND) review when compared to novel ECM bioscaffolds. While long-term outcomes confirm low safety concerns given the extended use of ECM scaffolds in regenerative medicine, it could limit the potential of ECM bioscaffolds in clinics. This is especially important for clinicians who are using these materials in the surgical theater, so they fully understand their potential and limitations in the long and short term. Therefore, a further effort by regulatory agencies and manufacturers in recognizing the immune modulation properties of ECM bioscaffolds is required to fully exploit their potential while minimizing risks derived from their use.

6. Conclusions and Future Directions

ECM bioscaffolds are widely used in regenerative medicine and research. Their high cytocompatibility, degradation upon implantation, and release of biologically active signals grant them immunomodulatory properties associated with a pro-remodeling or constructive healing upon implantation. This immunomodulation is not limited to innate cells around the implantation site but also has effects at the systemic level. However, the processing of ECM bioscaffolds is crucial to this immune response and depends on the elimination of cellular material, residual chemicals, and pathogen traces. Therefore, processing techniques and quality controls in industry, as well as in regulatory bodies, must evolve, taking into account the immunoregulatory properties of the ECM bioscaffolds.