Regulating Blood Clot Fibrin Films to Manipulate Biomaterial-Mediated Foreign Body Responses

Abstract

The clinical efficacy of implanted biomaterials is often compromised by host immune recognition and subsequent foreign body responses (FBRs). During the implantation, biomaterials inevitably come into direct contact with the blood, absorbing blood protein and forming blood clot. Many studies have been carried out to regulate protein adsorption, thus manipulating FBR. However, the role of clot surface fibrin films formed by clotting shrinkage in host reactions and FBR is often ignored. Because of the principle of fibrin film formation being relevant to fibrinogen or clotting factor absorption, it is feasible to manipulate the fibrin film formation via tuning the absorption of fibrinogen and clotting factor. As biological hydroxyapatite reserved bone architecture and microporous structure, the smaller particle size may expose more microporous structures and adsorb more fibrinogen or clotting factor. Therefore, we set up 3 sizes (small, <0.2 mm; medium, 1 to 2 mm; large, 3 to 4 mm) of biological hydroxyapatite (porcine bone-derived hydroxyapatite) with different microporous structures to investigate the absorption of blood protein, the formation of clot surface fibrin films, and the subsequent FBR. We found that small group adsorbed more clotting factors because of more microporous structures and formed the thinnest and sparsest fibrin films. These thinnest and sparsest fibrin films increased inflammation and profibrosis of macrophages through a potential signaling pathway of cell adhesion-cytoskeleton-autophagy, leading to the stronger FBR. Large group adsorbed lesser clotting factors, forming the thickest and densest fibrin films, easing inflammation and profibrosis of macrophages, and finally mitigating FBR. Thus, this study deepens the understanding of the role of fibrin films in host recognition and FBR and demonstrates the feasibility of a strategy to regulate FBR by modulating fibrin films via tuning the absorption of blood proteins.

Fig. 1.

Experimental flow of the study. (A) The preparation of PHA particles with 3 different sizes. (B) Different numbers of microporous structures lead to different numbers of clotting factor absorption and then different absorptions leading to different clotting factors in the remaining blood, leading to different clot fibrins including surface fibrin film formation. (C) The thickness and density of fibrin films affect macrophages through a potential pathway of cell adhesion–cytoskeleton–autophagy. (D) Activated macrophages by fibrin films regulate the collagen secretion and collagen matrix of fibroblasts. (E) Thinner and sparser fibrin films induce obvious FBR, while thicker and denser fibrin films induce low FBR in vivo. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

The effects of 3 sizes of PHA on fibrin films. (A) Microporous structures of the prepared PHA detected by Brunauer–Emmett–Teller. (B) Residual plasma protein concentration after PHA adsorption. (C) Plasma fibrinogen concentration after PHA adsorption. (D) The APTT, PT, TT, and INR tests using PHA-adsorbed plasma. (E) Plasma clotting factors concentration (in international units per deciliter) after PHA adsorbed. (F and G) MSB staining and SEM observation of manipulated fibrin films. (H) Semiquantitative analysis of fibrin film’s thickness. (I) Semiquantitative analysis of fibrin films porosity. (J) Residual plasma Ca²⁺ concentration. ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

The adhesion behavior of macrophages is reduced on the thinner and sparser fibrin films. (A) Gene Ontology analysis shows the top 20 terms enriched in differentially genes for cellular component. (B and C) Gene Set Enrichment Analysis reveals the difference of adherens junction and focal adhesion. |Normalized enrichment score| (|NES|) > 1, nominal P < 0.05, and false discovery rate (FDR) q < 0.25 are considered significant. (D) Heatmap of cell adhesion related gene expressions. (E and F) RT-qPCR results of cell-adhesion-related gene expressions and representative immunofluorescence images of Itgb2 and Zyxin (classic adhesion marker) in macrophages cultured on fibrin films. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.

The adhesion-mediated cytoskeleton of macrophages is reduced on the thinner and sparser fibrin films. (A) Interaction network analysis showed close interaction between focal adhesion and regulation of actin cytoskeleton. (B) Gene Set Enrichment Analysis reveals the difference of regulation of actin cytoskeleton. |NES| > 1, nominal P < 0.05, and FDR q < 0.25 are considered significant. (C) Heatmap of cytoskeleton related gene expressions. (D and E) RT-qPCR results of cytoskeleton related gene expressions and representative immunofluorescence images of F-actin and Src (classic cytoskeleton marker) in macrophages cultured on fibrin films. (F and G) Heatmap and RT-qPCR results of cytoskeleton-accompanied autophagy-related gene expressions. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

The cytoskeleton-accompanied autophagy of macrophages is increased on the thinner and sparser fibrin films. (A) Interaction network analysis showed close interaction between regulation of actin cytoskeleton and autophagy. (B) The Rac-mTOR pathway affected autophagy-related genes (Atgs) with negative regulation. (C) Autophagic-related pathways were enriched. (D) Heatmap of autophagy related gene expressions. (E and F) RT-qPCR results of autophagy-related gene expressions and representative immunofluorescence images of LC3 (classic autophagy marker) in macrophages cultured on fibrin films. FC, fold change. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6.

The autophagy-mediated inflammation and profibrosis of macrophages are increased on the thinner and sparser fibrin films. (A) Interaction network analysis showed close interaction between autophagy-related genes and inflammatory-related genes. (B and C) Heatmap and RT-qPCR results of inflammatory related gene expressions. (D) Representative immunofluorescence images of CCR7 (classic inflammation marker) in macrophages cultured on fibrin films. (E) Interaction network analysis showed close interaction between inflammatory-related genes and profibrosis-related genes. (F and G) Heatmap and RT-qPCR results of profibrosis-related gene expressions. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 7.

Activated macrophage regulates the collagen secretion and collagen matrix formation of fibroblasts. (A and B) Representative immunofluorescence images of α-SMA (activated fibroblast marker) and COL-I (collagen I marker) in fibroblasts cultured in the conditional medium from activated macrophages on fibrin films. (C) Representative images of collagen matrix formation by fibroblasts. (D) RT-qPCR results of collagen secretion and collagen-matrix-formation-related gene expressions. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8.

Fibrin films observed in vivo. (A and B) Hematoxylin and eosin staining showed a films (blue dashed line) covering the surface of PHA. (C) Sirius red staining showed this films (golden yellow) was not collagen fiber (red). (D) MSB staining showed this films was fibrin films (purple red), not collagen fiber (dark blue). M, materials.

Fig. 9.

The functions of generated fibrin film on the FBR in vivo. (A) After 2 d, MSB staining showed cells (yellow asterisk) adhered to the fibrin films (blue dashed line). (B) After 2 d, LC3 (classic autophagy marker, green arrow) staining showed increased autophagy on the thinner and sparser fibrin films. (C) After 2 d, hematoxylin and eosin (H&E) and INOS (inflammatory macrophage marker, green arrow) staining showed increased inflammation on the thinner and sparser fibrin films. (D) After 4 weeks, Sirius Red and Goldner staining showed the increased fibrous capsule (gray dashed line) and collagen deposition on the thinner and sparser fibrin films. FM, fibrin films; CF, collagen fiber.

Fig. 10.

Schematic figure of the underlying mechanisms of PHA microporous structures regulating blood clot fibrin films to manipulate biomaterial-mediated FBR. (A) PHA with different numbers of micropore structures, resulting in different clotting factor absorptions and Ca²⁺ release. (B) The residual clotting factor and Ca²⁺ in the blood cause different thicknesses, densities, and Ca²⁺ binding of fibrin films. (C) Fibrin films of different thicknesses, densities, and Ca²⁺ binding affect adhesion-related protein quantity and conformation to further regulate intracellular signaling. (D) Macrophages regulated by fibrin films produce different levels of inflammatory and profibrotic factors to regulate the collagen secretion and collagen matrix of fibroblasts, leading to FBR.