Decoding biomaterial-associated molecular patterns (BAMPs): influential players in bone graft-related foreign body reactions

Protein adsorption phase of FBR

FBR begins with forming a provisional matrix as plasma proteins adsorb onto the surface of the implanted biomaterial (Davenport Huyer et al., 2020). After implantation, host blood proteins, including albumin, fibronectin, vitronectin, fibrinogen, immunoglobulins, coagulation and complement factors, rapidly adsorb to the biomaterial’s surface, primarily within the first four hours. The type and concentration of these adsorbed proteins influence subsequent cellular events and the inflammatory responses of FBR. For instance, fibrinogen adsorption increases macrophage production of the pro-inflammatory cytokine TNF-α, as its P2 domain interacts with integrin αX/β2 on M1 macrophages, triggering a pro-inflammatory response (Lee et al., 2019; Zhou & Groth, 2018).

In addition to protein deposition, blood-biomaterial interaction activates both the complement and coagulation cascades, forming a provisional matrix rich in fibrin that surrounds the biomaterial. The conversion of fibrinogen to fibrin during clotting generates fibrinopeptides that increase vascular permeability and promote leukocyte chemotaxis (Binder et al., 2017). Furthermore, the cleavage of complement factors C3 and C5 releases the anaphylatoxins C3a and C5a, which enhance vascular permeability, chemotaxis, and leukocyte extravasation. Complement activation also stimulates platelet activation and contributes to coagulation through platelet-related coagulation factor IV and the release of clotting factors and activators (Eriksson et al., 2019; Kizhakkedathu & Conway, 2022).

Protein adsorption on biomaterial surfaces triggers platelet activation, activates the complement system, and promotes coagulation, creating a fibrin-rich matrix around the implant. Furthermore, protein adsorption plays a vital role in modulating macrophage phenotypes at the implantation site.

Acute inflammation phase of FBR

Platelets within the fibrin mesh of the provisional matrix release cytokines and chemokines, aiding in the recruitment of immune cells. Transforming growth factor-beta (TGF-β), platelet factor 4 (CXCL4), and platelet-derived growth factor (PDGF) from platelets attract neutrophils to implanted bone grafts (Gleissner et al., 2010; Pitchford, Pan & Welch, 2017). Additionally, CXCL8 released from surrounding endothelial cells interacts with C-X-C chemokine receptor type 2 (CXCR2) on neutrophils, directing them to the bone graft site. Neutrophils are the first immune responders to biomaterial implantation (Abaricia et al., 2021a). Following this, circulating monocytes are recruited and differentiate into macrophages, mainly driven by monocyte chemoattractant protein-1 (MCP-1) binding to C–C motif chemokine receptor 2 (CCR2).

During the acute inflammatory stage, macrophages polarize into the M1 phenotype, secreting pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-12, IL-18, macrophage inflammatory protein 1α/β (MIP-1α/β), and MCP-1 (McKiel, Woodhouse & Fitzpatrick, 2020). Unlike short-lived neutrophils, macrophages can persist around the implanted bone graft for several months (McKiel, Woodhouse & Fitzpatrick, 2020). This acute inflammatory phase may lead to tissue restoration and osseointegration of bone grafts (restitutio ad integrum) or progress into chronic inflammation, resulting in fibrous encapsulation and graft failure (Ivanovski & Mark, 2022). The sustained presence of classically activated macrophages (caMac) or pro-inflammatory macrophages tends to drive the FBR process toward fibrotic encapsulation, while alternatively activated macrophages (aaMac) or anti-inflammatory macrophages promote osseointegration and graft success.

Macrophages also affect the adaptive immune response to bone graft implantation. M1 macrophages, through cytokines such as IL-12, CXCL9, and CXCL10, drive the recruitment and polarization of Th1 cells. In contrast, M2 macrophages release IL-10, CCL17, and CCL22, which promote Th2 responses. An enhanced Th1 response around the bone graft leads to fibrotic encapsulation and graft rejection, while increased Th2 activity supports osseointegration and graft success (Davenport Huyer et al., 2020).

Chronic inflammatory phase of FBR

FBR can progress into a chronic inflammatory stage due to a predominant M1 macrophage population or Th1 response, the release of toxic or degraded biomaterial byproducts, movement of the biomaterial at the implantation site, or inadequate mechanical compliance, including overloading or underloading conditions (Carnicer-Lombarte et al., 2021; Davenport Huyer et al., 2020). In the later stages of chronic inflammation, macrophages fuse to form FBGCs, which are a hallmark of FBR (Stewart et al., 2024). The formation of FBGCs on implanted biomaterials is primarily induced by IL-4 and IL-13 (Eslami-Kaliji et al., 2023). McNally & Anderson (2011) investigated the effects of adsorbed proteins on polystyrene substrates coated with complement factors C3bi, collagens, fibrinogen, plasma fibronectin, laminin, thrombospondin, vitronectin, and von Willebrand factor to assess monocyte adhesion, macrophage development, and IL-4-induced FBGC formation. While all adsorbed proteins facilitated monocyte adhesion, only vitronectin significantly promoted macrophage growth and FBGC formation. This indicates that surfaces favoring vitronectin adsorption may drive macrophage activation and FBGC formation (Eslami-Kaliji et al., 2023; McNally & Anderson, 2011; Sheikh et al., 2015a). FBGCs contribute to FBR-related fibrosis by releasing reactive oxygen species (ROS) and enzymes that degrade the biomaterial (Eslami-Kaliji et al., 2023). Additionally, profibrotic factors, such as PDGF, vascular endothelial growth factor (VEGF), and TGF-β, released from FBGCs, facilitate fibroblast recruitment, further promoting fibrosis (Zhou & Groth, 2018).

Fibrous encapsulation phase of FBR

FBGCs expressing PDGF and TGF-β stimulate fibroblast proliferation, collagen synthesis, and wound healing (Eslami-Kaliji et al., 2023; Zhou & Groth, 2018). Additionally, FBGCs promote the differentiation of fibroblasts into myofibroblasts by inducing α-smooth muscle actin (α-SMA) expression. During normal wound healing and biomaterial integration (or foreign body equilibrium), myofibroblasts in the surrounding tissue either undergo apoptosis or enter a quiescent state, halting collagen production (Lebonvallet et al., 2018). However, in chronic inflammation, myofibroblasts persist and continue to produce excessive collagen fibers, resulting in extensive fibrosis and scarring (McKiel, Woodhouse & Fitzpatrick, 2020; Noskovicova, Hinz & Pakshir, 2021). This fibrotic response hinders bone graft integration by restricting oxygen and nutrient transport to surrounding tissues, ultimately compromising graft function (Capuani et al., 2022).

In vitro studies

THP-1 monocytes exposed to titanium dioxide nanoparticles (TiO₂ NPs) and microparticles (TiO₂MPs), with sizes <100 nm and <5 µm, respectively, exhibited an enhanced inflammatory response during in vitro analysis. Elevated ROS levels confirmed the uptake of these particles, and the resulting inflammation was compared to controls, and the increase in ROS generation with TiO₂NPs was concentration-dependent (Kheder, Soumya & Samsudin, 2021). Additionally, research conducted by our group employed human peripheral blood monocyte-derived macrophages (PBMMs) to assess the immunological response of demineralized (DMB) and decellularized (DCC) bovine bone substitutes. The findings indicated that PBMMs treated with DMB demonstrated increased expression of inflammatory cytokine markers IL-1β and TNF-α, along with proinflammatory cell surface markers CD86 and CD14, while DCC substitutes exhibited immunoregulatory effects on PBMMs (Rani et al., 2024). In another in vitro study (Toledano-Serrabona et al., 2022), researchers utilized titanium metal particles released during implantoplasty of dental implants on macrophage cell cultures (THP-1). The results indicated an increased pro-inflammatory expression of TNF-α and a decreased expression of anti-inflammatory markers TGF-β and CD206. These findings suggest that titanium particles play a role in developing bone resorption or peri-implant tissue inflammatory response.

In vivo animal models and human studies

The study by Ciobanu et al. (2024) investigated the treatment of critical-sized bone defects (CsBDs) in a rat model using four approaches: untreated defects, defects treated with Bio-Gen^®, Bio-Gen^® combined with platelet-rich fibrin (PRF), and autologous bone grafts (ABG). The ABG group achieved the most successful healing outcomes. In the Bio-Gen^® group, histological analysis revealed the formation of a fibrous callus with numerous capillaries, a giant cell reaction to the bone graft fragments, and sparse lymphocytes. Combining PRF with Bio-Gen^® enhanced healing compared to Bio-Gen^® alone, with improved tissue regeneration, reduced inflammation, and better vascularization. A study (Fernandes et al., 2024) on critical-size calvarial defects in 50 Wistar rats compared to blood (G1), autogenous bone (G2), bioglass (G3), hydroxyapatite (G4), and xenograft (G5) grafts, with or without expanded polytetrafluoroethylene (e-PTFE) barriers. Autogenous bone (G2) demonstrated the best bone formation and resorption outcomes, followed by G4, G5, and G3. Synthetic biomaterials (G3 and G4) yielded comparable results, while G5 resulted in 22% new bone formation after 45 days. Among the synthetic materials, G4 showed a superior degradation profile.

An in vivo study on mineralized collagen-polycaprolactone implants in a porcine ramus critical-size defect model found that only 2 out of 22 implants achieved effective bone regeneration, whereas the majority showed limited bone formation and fibrous encapsulation (Dewey et al., 2021). Another group (Tanneberger et al., 2021) explored the cellular response to porcine-derived resorbable collagen membranes in Wistar rats over a span of 30 days. The membrane induced mononuclear cell infiltration, forming multinucleated giant cells (MNGCs) by day 15. These cells increased in number and migrated centrally by day 30, expressing CD-68, calcitonin receptor, and MMP-9. The disintegration of the collagen membrane was linked to MNGC activity and significantly increased vascularization compared to the controls. Another group of researchers studied the foreign body response of PCL scaffolds implanted into the dorsal window chamber of 10–12-week-old C57BL/6 mice. Over two to four weeks, they observed the formation of an immature neurovascular network alongside the development of a dense fibrous capsule (Dondossola et al., 2016). An in vivo study on Beagle dogs found that hydroxyapatite-coated poly-l-lactic acid (PLLA) screws exhibited superior biocompatibility, reduced inflammation, and improved bone integration compared to uncoated PLLA screws, which resulted in significant foreign body reactions characterized by the formation of fibrous tissue and infiltration by histiocytes (Akagi et al., 2013).

Histological analysis of 14 tissue samples from patients who underwent sinus augmentation before tooth implantation revealed mild inflammatory responses, including increased immune cells and blood vessels around both xenogeneic (Bio-Oss^®) and synthetic (NanoBone^®) bone substitutes. Multinucleated giant cells were observed more frequently on the synthetic material, indicating a stronger immune response compared to the xenogeneic substitute (Barbeck et al., 2017). A randomized clinical trial (Koo et al., 2020) examined bone formation after grafting periodontally damaged extraction sockets using deproteinized bovine bone mineral (DBBM) or deproteinized porcine bone mineral (DPBM) with collagen membrane coverage. A total of 100 patients participated, and 81 biopsy samples (42 from the DBBM group and 39 from the DPBM group) were included in the final analysis. Both groups showed comparable histologic bone formation, although some specimens from both groups exhibited fibrous encapsulation of biomaterial particles in the coronal region.

Biomaterial proteins adsorption and their fundamental mechanism

A dynamic layer of adsorbed proteins forms when bone grafts are placed into the host because their surface characteristics allow both the adsorption and desorption of blood proteins. As a component of BAMPs, the adsorbed protein consists of either autologous proteins (found in blood or extracellular fluid) or allogenic proteins (derived from materials). Autologous host serum proteins begin to adsorb at the millisecond level, and a protein layer will have formed by the time immune cells are attracted to the biomaterial implantation site (Eslami-Kaliji et al., 2020; Wang et al., 2022).

It is anticipated that the “big twelve” blood-derived host proteins, which are typically present in human plasma at concentrations of one mg/ml or higher, will compete for the first interaction on the biomaterial’s surface. These proteins include albumin, α-macroglobulin, haptoglobin, low-density lipoprotein (LDL), high-density lipoprotein (HDL), fibrinogen, transferrin, α-antitrypsin, complement factor C3, IgG, IgA, and IgM (McKiel, Woodhouse & Fitzpatrick, 2020). High molecular weight proteins and those present in higher concentrations, such as albumin, immunoglobulins, fibrinogen, factor XII (Hageman factor), and high molecular weight kininogen (HMWK), are the first to arrive and initially adhere to the surface of bone grafts. Eventually, these proteins will be replaced by medium- and low-molecular-weight proteins with strong surface affinity. This phenomenon of protein adsorption and desorption on the biomaterial surface is called the Vroman effect (Wei et al., 2021).

A variety of factors influence the adsorption of proteins on bone graft surfaces. These include the concentrations and chemical properties of blood proteins, their protein-protein interactions, and the physicochemical characteristics of biomaterials (such as surface area, hydrophobicity, charge, roughness, thickness, porosity, and chemical composition) (Adams et al., 2019; Barberi & Spriano, 2021; Ping et al., 2021; Stanciu & Diaz-Amaya, 2021). The rate of protein adsorption is directly related to protein concentration and inversely related to protein molecular weight. Van der Waals forces, electrostatic interactions, hydrophobic interactions, and hydrogen bonds play a role in protein adsorption. Some proteins are reversibly adsorbed on the surfaces of biomaterials and tend to desorb over time. However, others are unlikely to desorb as they bond permanently to the surface. The interactions between proteins and the biomaterial’s surface and between proteins determine the final profile of the adsorbed protein layer (Stanciu & Diaz-Amaya, 2021; Talha et al., 2019).

In an actual scenario, protein adsorption occurs when multiple serum proteins interact with one another simultaneously, as seen in the case of blood plasma. Protein-protein interactions influence protein adsorption on the surface of biomaterials (Zheng, Kapp & Boccaccini, 2019). For instance, these interactions between proteins can either cooperative or competitive protein adsorption on the surface of biomaterials. Adsorption, where deposited proteins influence the adsorption of “new” proteins (those that are not adsorbed), is known as cooperative adsorption (Liu, 2015). In contrast, competitive protein adsorption entails different proteins having varying affinities for various solid surfaces. In these cases, a protein with a higher affinity for the surface adsorbs at a greater concentration than a protein with a lower affinity (Lundqvist, 2013).

The amino acid sequence forming the fundamental structure of proteins is one of the most crucial aspects of protein adsorption. Proteins are polypeptides [-NH-CHR-CO-] featuring functional groups and a main backbone structure that consists of the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). The unique characteristics of the protein structure are derived from the functional group ‘R’. Based on the chemical structure of their functional groups, proteins can be hydrophilic (polar), hydrophobic (nonpolar), or carry anionic/cationic charges. The functional group determines the active sites of proteins available for surface interaction, which influences protein adsorption on biomaterials (Sanvictores & Farci, 2022; Stanciu & Diaz-Amaya, 2021). Proteins generally fold in a way that exposes hydrophilic and charged groups to the external environment while positioning hydrophobic groups deep within the protein. During protein adsorption, the adsorbed proteins spread out to expose their core, forming a monolayer of adsorbed proteins and releasing water molecules complexed with the native protein state (Stanciu & Diaz-Amaya, 2021).

Furthermore, the primary site for cell attachment in all proteins is the bioactive motif or domain Arg-Gly-Asp (RGD) (Ryu et al., 2013; Wang et al., 2022). Numerous ECM proteins, including collagen, laminin, vitronectin, fibronectin, fibrinogen, and others, have been found to contain the RGD sequence (Love & Jones, 2013; Rowley et al., 2019). Fibronectin, von Willebrand factor, and vitronectin proteins containing the RGD sequence interact with β1 integrin receptors on macrophages. Additionally, complement C3 fragments, fibrinogen, factor X, and high-molecular-weight kininogen bind to β2 integrins on macrophages to initiate initial monocyte attachment (Kizhakkedathu & Conway, 2022; Piatnitskaia et al., 2024; Sheikh et al., 2015a). The RGD sequence influences cell adhesion on biomaterials and the immunogenic cellular phenotype. Studies have shown that the RGD sequence of proteins affects the immunogenic cellular phenotype and function without altering the composition of the adsorbed protein layer (Acharya et al., 2010; Acharya et al., 2011; Swartzlander et al., 2015).

Physicochemical property of biomaterial regulating protein adsorption and macrophage phenotype

The variation in protein adsorption according to the surface characteristics of bone grafts is briefly described in these sections. Additionally, this segment highlights the phenotypic differentiation of macrophages based on the physicochemical traits of bone grafts. Table 1 summarizes the pro-inflammatory and anti-inflammatory responses of macrophages in relation to the different physicochemical properties of bone grafts found in existing literature. Figure 3 illustrates macrophages’ pro- and anti-inflammatory responses according to the surface attributes of bone grafts and their respective phenotypic differentiation markers.

Danger signals

Danger-associated molecular patterns (DAMPs) are molecules found in the intracellular space or hidden within the ECM that provoke an inflammatory response when released into the extracellular space (McKiel, Woodhouse & Fitzpatrick, 2020) during tissue injury (Vénéreau, Ceriotti & Bianchi, 2015). DAMPs include nuclear proteins such as HMGB1, heat shock proteins (HSPs), and elements of the extracellular matrix like fibronectin extra domain A (Fn EDA) and hyaluronan (McKiel, Woodhouse & Fitzpatrick, 2020; McKiel & Fitzpatrick, 2018). These danger-signaling molecules are typically absent in significant concentrations under physiological conditions. However, when they are present at substantial concentrations, they pose a danger to the microenvironment, leading to the activation of immune cells that work to eliminate the source of cellular distress or damage. These danger signals can activate pattern recognition receptors (PRRs) and toll-like receptors (TLRs) (Ma, Jiang & Zhou, 2024; McKiel & Fitzpatrick, 2018).

At the time of biomaterial implantation, the surrounding tissues are injured, and the damaged tissue releases DAMPs. These molecules bind to TLRs, initiating intracellular signaling events that lead to the production of pro-inflammatory cytokines. Upon binding to TLRs, they activate cytoplasmic adapter molecules that trigger cellular pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factors associated with mitogen-activated protein kinase (MAPK) pathways. Activating these pathways produces inflammatory cytokines like TNF-α, IL-1, and IL-6 and chemokines through transcriptional and post-transcriptional mechanisms (Tu et al., 2022). The prolonged presence of DAMPs guides the wound toward a chronic inflammatory response, disrupting the balance between pro- and anti-inflammatory reactions during biomaterial implantation (McKiel, Woodhouse & Fitzpatrick, 2020).

HSPs are stress response proteins cells produce when exposed to chemical and physical stimuli. Examples of HSPs include HSP 70A, 70B, 60, 90, and 47. Biomaterial-associated cell stress or necrosis has been associated with the release of HSPs, which activate TLR 2 and 4, thereby inducing the inflammatory response at implantation sites (Fang et al., 2011; Nonhoff et al., 2024; Wang et al., 2022). When adsorbed on poly (methyl methacrylate) (PMMA) and polydimethylsiloxane, HSP 60 has been shown to activate NF-κB/AP-1-dependent SEAP (secreted alkaline phosphatase) and induce the expression of inflammatory cytokines (McKiel & Fitzpatrick, 2018).

FBR occurs in the absence of pathogens, and the inflammatory response is induced in the presence of DAMPs and an adsorbed protein layer. This type of immune activation that occurs in the absence of pathogens is referred to as sterile inflammation (McKiel & Fitzpatrick, 2018). The characteristics of sterile inflammation include the infiltration of neutrophils and macrophages and the expression of pro-inflammatory mediators such as IL-1β, TNF-α, and ROS (Krysko et al., 2011).

Surface coating

Extensive research has focused on creating hydrophilic surfaces for biomaterials to minimize FBR. A key method involves modifying surfaces with zwitterionic polymers (Shao & Jiang, 2015; Sun et al., 2014). These polymers feature both anionic and cationic groups within a single molecular unit, leading to an overall neutral charge that effectively resists nonspecific protein adsorption in complex biological environments (Blackman et al., 2019; Shao & Jiang, 2015). This neutral charge facilitates the creation of a dense hydration layer on the biomaterial’s surface, which inhibits protein adsorption through electrostatic repulsion. Notable examples of zwitterionic polymers, such as those containing carboxybetaine (CB) and sulfobetaine (SB) groups, demonstrate significant potential in reducing protein adsorption (Blackman et al., 2019).

Examples of zwitterionic polymers that effectively reduce protein adsorption on biomaterial surfaces include poly (2-hydroxyethyl methacrylate) (PHEMA) and poly (carboxybetaine methacrylate) (PCBMA) (Zhou et al., 2024c). Recently, a new class of zwitterionic polymers known as zwitterionic polypeptides (ZIPs) has been introduced (Zhou et al. 2024a). These hydrogels, which are characterized by alternating sequences of glutamic acid (E) and lysine (K), are specifically designed to minimize FBR. Zwitterionic polypeptides display anti-inflammatory properties and demonstrate strong resistance to FBR, enhancing the functional performance of implanted biomaterials (Zhou et al. 2024a; Zhou et al., 2024c).

Additionally, zwitterionic polymers, such as poly (sulfobetaine methacrylate) (PSB) (Dong et al., 2021) and poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) (Park et al., 2014), have been effectively utilized to create superhydrophilic antifouling coatings on hydrophobic substrates, leading to a significant reduction in FBR in vivo. Another promising antifouling biomaterial includes intrinsically disordered proteins (IDPs), derived from fused in sarcoma (FUS) proteins, which are rich in hydrophilic residues (Chang et al., 2022). When applied to biomaterial surfaces, these hydrophilic residues effectively prevent protein adsorption. The outstanding antifouling properties of zwitterionic polymers position them as highly promising candidates for minimizing foreign body reactions.

Modification of material property

The physical properties of biomaterials have been extensively studied, highlighting their essential role in modulating the FBR to implanted materials. Intrinsic characteristics such as size, geometry, porosity, surface topography, and stiffness significantly influence cellular behavior and the overall FBR at both the molecular and cellular levels. For instance, nanoscale surface roughness has been shown to enhance protein adsorption, emphasizing the significance of surface topography in shaping biomaterial-host interactions (Mariani et al., 2019; Zhou et al., 2024c).

Material stiffness is a particularly influential property that governs macrophage adhesion and activation, both of which are central to FBR. Research on poly (ethylene glycol)-arginine-glycine-aspartic acid (PEG-RGD) hydrogels has demonstrated that softer hydrogels reduce macrophage activation, leading to a diminished FBR (Scott, Kiick & Akins, 2021). Further insights were provided by Noskovicova, Hinz & Pakshir (2021), who investigated the effects of coating stiff silicone implants with a soft silicone layer. The study found that softer materials significantly reduced fibrosis formation and fibroblast activation, which are key contributors to developing a fibrous capsule around implants. These findings suggest that decreased material stiffness can lead to less fibrous encapsulation. However, this creates a challenge for applications such as bone regeneration, where biomaterials intended for load-bearing regions must maintain adequate mechanical strength to ensure structural stability and functional performance.

Surface topography is crucial in influencing the FBR, especially through its effect on surface properties. Studies have identified an optimal average surface roughness of about 4 µm for minimizing FBR in both in vivo models and human tissue samples (Doloff et al., 2021). This evidence underscores the importance of optimizing surface characteristics to decrease adverse immune responses and improve the biocompatibility of implanted materials.

Incorporation of immunomodulatory agents

The reduction of FBR can be effectively achieved by incorporating immunomodulatory agents into the design of biomaterials. Applying functional groups to polymer coatings significantly decreases FBR. For example, a polymer derived from Z2 Y12, called poly (tetrahydropyran phenyl triazole) (PTHPT), has been used as a surface coating to combat FBR (Zhou et al., 2024c). In vivo, research shows that PTHPT coatings notably reduce capsule formation and fibrous tissue development in implants placed in the peritoneal cavity. Variants of the Z2-Y12 moiety, such as Met-Z2-Y12, have demonstrated greater effectiveness in reducing FBR responses, particularly as coatings for subcutaneous implants in mouse studies (Wright et al., 2023). Another promising category of immunosuppressive coatings includes phospholipids like phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylinositol. Research shows that applying phospholipids to biomaterial surfaces increases the transcription of anti-inflammatory genes in murine models (Zhou et al., 2024c).