Allometrically scaling tissue forces drive pathological foreign-body responses to implants via Rac2-activated myeloid cells

Abstract

Small animals do not replicate the severity of the human foreign-body response (FBR) to implants. Here we show that the FBR can be driven by forces generated at the implant surface that, owing to allometric scaling, increase exponentially with body size. We found that the human FBR is mediated by immune-cell-specific RAC2 mechanotransduction signalling, independently of the chemistry and mechanical properties of the implant, and that a pathological FBR that is human-like at the molecular, cellular and tissue levels can be induced in mice via the application of human-tissue-scale forces through a vibrating silicone implant. FBRs to such elevated extrinsic forces in the mice were also mediated by the activation of Rac2 signalling in a subpopulation of mechanoresponsive myeloid cells, which could be substantially reduced via the pharmacological or genetic inhibition of Rac2. Our findings provide an explanation for the stark differences in FBRs observed in small animals and humans, and have implications for the design and safety of implantable devices.

Fig. 1. Pathological FBR in humans is mediated by RAC2 mechanotransduction signalling, regardless of implant properties, and is associated with increased mechanical signalling.

a, Trichrome staining of fibrotic capsules from the fibrous capsule formed around silicone-based breast implants, titanium-based pacemakers and stainless-steel-based orthopaedic implants are all similar to one another. Implant located at the bottom of each image. Scale bar, 200 µm. b,c, Quantification of collagen (b) and mature collagen (c) shows no significant differences between the different types of human implant. For breast implants, n = 6 independent capsules; cardiac pacemakers, n = 4 independent capsules; neurostimulators, n = 4 independent capsules; and orthopaedic hardware, n = 5 independent capsules. d, Heat map of the top-100 genes upregulated in Baker-IV versus Baker-I breast implants, organized in decreasing order of P value. e,f, Pathways significantly upregulated in Baker-IV (e) or Baker-I (f) samples analysed using DAVID. Selected pathways highlighted in red are mechanotransduction pathways and those highlighted in green are inflammatory pathways. g, Selected cell-activating and inflammatory genes upregulated in Baker-IV (red) (n = 10 independent capsules) versus Baker-I (grey) capsules (n = 10 independent capsules; RAC2, *P = 0.241; PLAUR, *P = 0.0388; CXCR4, *P = 0.0332; CD44, P = 0.052; PDGFRA, P = 0.0585; MIF, *P = 0.0211). h, STRING analysis showing that RAC2 is a central mechanotransduction mediator of both cell-activating and inflammatory signalling genes that were all upregulated in Baker-IV specimens. STRING analysed interactions between the different genes based on experimental evidence and predicted interactions. Statistical comparisons for b and c were made by using a one-way ANOVA with Tukey’s multiple comparisons tests; statistical comparisons for g were made using a two-tailed t-test comparing Baker-IV with Baker-I samples for each gene. Each data point represents an independent capsule from a different patient. All data represent mean ± s.e.m. Representative images are shown across all experiments. NS, not statistically significant. Source data

Fig. 2. Altering extrinsic tissue-scale forces using MSIs produces a human-like FBR capsule architecture in mice.

a,b, FEM of murine (a) and human (b) implants showing that human implants are subject to 100-fold higher mechanical stress than murine implants. c, Schematic and picture of the MSI model. FEM confirming that MSIs recreate human levels of mechanical stress in the mouse. Sx refers to the mechanical stress in the x direction. d, Trichrome staining of FBR capsules in the human implant capsules, SM model and the MSI murine model. Scale bar, 50 µm. e, Herovici staining showing mature (red) and immature (blue) collagen. Scale bar, 50 µm. f, Immunostaining for αSMA, a marker for myofibroblasts. Scale bar, 50 µm. Implant located at the bottom of each image. g, SEM imaging of the surface of the capsules. Scale bar, 10 μm. h, Top: quantification of percent area positive for collagen in each capsule (far right; n = 5 biological replicates for each group; *P = 0.0261, **P = 0.0012). Second from top: quantification of mature collagen deposition in the FBR tissue (far right; n = 5 independent capsules per group; **P = 0.0044). Third from top: quantification of αSMA normalized to cell density using image analyses in each capsule (n = 5 biological replicates for each group; **P = 0.0028). Bottom: quantification of surface collagen percent area associated with each capsule (n = 8 biological replicates for each group; ****P < 0.0001). Statistical comparisons were made by using a one-way ANOVA with Tukey’s multiple comparisons tests. Each data point represents an independent capsule from a different patient or mouse. All data represent mean ± s.e.m. Representative images are shown across all experiments. H, human. Source data

Fig. 3. MSIs generate a sustained inflammatory response at the implant–tissue interface.

a,b, UMAP plots of all cells from murine FBR capsules classified by sample type (a) and time points (b). A total of 36,827 cells were analysed. c, Relative proportion of myeloid, lymphoid fibroblast, and endothelial cells in 2-week and 4-week capsules. d, UMAP plots coloured by cell type. e, Relative proportion of myeloid, lymphoid, fibroblast, and endothelial cells in SM implants and MSI capsules. Myeloid cells were the most abundant cell type in both capsules and were particularly enriched with mechanical stimulation. f,g, Gene expression of fibroblast-defining genes (f) and immune cell-defining genes (g) projected onto UMAP embeddings. Grey, no gene expression; light orange, low gene expression; bright orange, high gene expression. h, Violin plots of MSI capsules compared with SM implant capsules. i,j, CODEX immunofluorescence staining of Rac2 (i) and (j) F4/80. The implant is located at the bottom of each image (n = 3 independent capsules per group; Rac2, *P = 0.0212; F4/80, *P = 0.0154). Scale bar, 50 μm. Statistical comparisons for i and j were made by using a two-tailed t-test. Data are presented as mean ± s.e.m. Endo, endothelial. Source data

Fig. 4. Increased extrinsic tissue-scale forces activate Rac2 signalling in myeloid cells, which drives a Baker-IV fibrotic phenotype in mice.

a, UMAP plot of myeloid cells in SM and MSI implant capsules. Clusters 1, 4 and 7 (red dotted line) are highly enriched in MSI capsules. b,c, FeaturePlot of top averaged Baker-IV markers (Fig. 1d), including key mechanotransduction and inflammatory chemokine signalling pathways (b) and Baker-I markers (c). Color bar denotes the amount of gene expression. d, Violin plots of Baker-IV markers differentially upregulated in the MSI clusters (arbitrary units). e, CODEX immunofluorescence staining of co-localized pixels of Rac2 and F4/80 (n = 3 independent capsules per group, *P = 0.0254). White box denotes high magnification (HM) image area. Scale bar for SM and MSI, 50 μm. Scale bar for HM images, 10 μm. Statistical comparisons for e were made by using a two-tailed t-test. Data are presented as mean ± s.e.m. f, Selected pathways significantly upregulated in murine MSI samples analysed using DAVID. Pathways highlighted in red are also upregulated in Baker-IV human specimens. Source data

Fig. 5. Increasing tissue-scale forces activates fusogenic macrophages, MHC II lymphocytes and myofibroblasts—all classic features of a pathologic FBR.

a, Relative proportions of cell types in cluster 1, 4 and 7 cells (primarily MSI macrophage clusters). b, Violin plots of gene expression (arbitrary units) by cell cluster. Cluster 4 cells upregulate markers for fusogenic macrophages. c,d, UMAP (c) and violin plots (arb. units) (d) of lymphocytes from murine FBR capsules. MSI lymphocytes show upregulation of MHC class II signalling. e, CODEX immunofluorescence staining of co-localized pixels of MHC2 and CD3 protein (n = 3 independent capsules per group, ***P = 0.0003). White box denotes high magnification (HM) image area. Scale bar for SM and MSI, 50 μm. Scale bar for HM images, 10 μm. Statistical comparisons were made by using a two-tailed t-test. Data are presented as mean ± s.e.m. f,g, UMAP (f) and violin plots (arb. units) (g) of fibroblasts from murine FBR capsules. h, Violin plots (arb. units) of mechanotransduction markers in SM and MSI fibroblasts. NK, natural killer. Source data

Fig. 6. Blocking Rac2 signalling effectively reverses the human-like FBR induced by increased tissue-scale forces in mice.

Comparative analysis of histology sections of FBR capsules from the MSI mouse model either pharmacologically blocked with Rac inhibitor (RI) or genetically blocked in a Rac2^−/− global KO mouse. a, Immunostaining for αSMA signalling in FBR capsules. Quantification of percent area positive for αSMA in each capsule (n = 5 biological replicates for each group; ***P = 0.0003). b, Trichrome staining of FBR capsules. Quantification of percent area positive for collagen in each capsule (n = 5 biological replicates for each group; *P = 0.045, **P = 0.0051). c, Haematoxylin and eosin (H&E) staining of FBR capsules. Quantification of average capsule thickness (n = 4 biological replicates for each group; *P = 0.0153). Scale bar in a–c, 50 µm. d, In SM implants, there is a modest activation of inflammatory pathways at the early time point, which subsides at the late time point, resulting in minimal FBR. In contrast, in MSI capsules, increased tissue-scale forces lead to the activation of Rac2 mechanical signalling, which promotes a robust activation of inflammatory markers that is sustained over time, resulting in a human-like pathological FBR. Statistical comparisons were made by using a one-way ANOVA with Tukey’s multiple comparisons tests. Each data point represents an independent capsule from a different mouse. All data represent mean ± s.e.m. Representative images are shown across all experiments. S, saline. Source data

Extended Data Fig. 1. Severe pathological FBR in humans is characterized by similar fibrotic encapsulation, regardless of implant properties.

(a) Hematoxylin and Eosin, (b) Trichrome, and (c) Herovici staining of fibrotic capsules from the fibrous capsule formed around silicone-based breast implants, titanium-based pacemakers, and stainless steel-based orthopedic implants are all similar to each other on the tissue architectural level. Scale bar = 200 µm. (d) Immunostaining for CD45 signaling in human implant FBR capsules. Quantification of percent area positive for CD45 in each capsule. Scale bar = 50 µm. Representative images are shown from similar images across n = 5 independent capsules per group. *p < 0.05. Statistical comparisons were made by using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests. Data is presented as mean ± SEM. B = breast implants, C = cardiac pacemakers, O = orthopedic hardware. (e) Schematic showing the experimental methodology followed; FBR capsules from Baker I and Baker IV breast implants were subject to molecular analyses using a commercially available biomarker panel (HTG Molecular). A total of 10 Baker I specimen and 10 Baker IV specimen were used in this study. Source data

Extended Data Fig. 2. All human implants experience about 100-fold increased mechanical stress compared to standard murine implants.

(a) Schematic of standard murine implants and and three commonly used human implants: breast implants, pacemakers, and neurostimulator batteries. (b) FE modeling of standard murine models of FBR reveals minimal mechanical stress at the implant-tissue interface. FE modeling reveals high mechanical stress ( ~ 100-fold higher) around human implants including breast implants, pacemakers and neurostimulator batteries.

Extended Data Fig. 3. Increased tissue-scale forces result in increased fibrosis around implants in mice, independently of implant chemistry or mechanical properties.

(a,b) Trichrome staining and Herovici staining of FBR capsules formed around standard murine implants with low stiffness, standard implants with high stiffness, and MSIs reveals that MSI-model produces a more robust scar tissue, with increased collagen and mature collagen. Representative images are shown from similar images across n = 5 independent capsules per group. Scale bar = 500 μm.

Extended Data Fig. 4. Lower levels of mechanical stress at the implant-tissue interface result in a diminished FBR.

(a) Trichrome staining of low-dose MSI stimulated FBR capsules. Scale bar = 50 µm. Quantification of percent area positive for collagen in each capsule (n = 4 for each group. *p = 0.0161). (b) H&E staining of FBR capsules. Quantification of average capsule thickness (n = 4 for each group. *p = 0.0286). (c) Immunostaining for aSMA signaling in FBR capsules (n = 4 independent capsules per group. ***p = 0.0004). Scale bar = 50 µm. All statistical comparisons were made by using two-tailed t tests. All data is presented as mean ± SEM. Source data

Extended Data Fig. 5. scRNA-seq of cells from SM and MSI implant capsules.

(a) Violin plots showing expression of inflammatory genes in standard murine (SM) implants and MSI capsules. (b) UMAP of myeloid cells colored by cluster. (c) Heatmap of differentially regulated genes in myeloid cells. MSI myeloid cells upregulated inflammatory markers. (d) FeaturePlot of Rac2 marker. (e) Violin plots of Rac2 expression in myeloid clusters.

Extended Data Fig. 6. Differentially upregulated fibroblast and lymphoid cell genes.

(a) Differentially upregulated genes between lymphocytes cells from the standard murine implants and MSIs. (b) Differentially upregulated genes between fibroblast cells from the standard murine implants and MSIs. (c) UMAP of fibroblasts colored by cluster. (d) Feature Plot of Acta2 marker in fibroblast population. (e) Violin plot of Acta2 expression in SM and MSI myofibroblast cluster.

Extended Data Fig. 7. Immunostaining for upregulated genes.

(a) Immunostaining for RAC2 signaling in FBR capsules (*p = 0.0155). Scale bar = 50 µm. Quantification of percent area positive for RAC2 in each capsule. (b-d) CODEX immunofluorescent staining for cellular populations of (b) F4/80 (macrophages, *p = 0.0499), (c) CD45 (all immune cells, *p = 0.0231), and (d) aSMA (fibroblasts, *p = 0.0433). (e-h) CODEX immunofluorescent staining for top Baker IV human genes (e) CCL4 (*p = 0.0338), (f) CXCL2 (*p = 0.0231), (g) CD44 (*p = 0.0478), and (h) PDGFRA (*p = 0.0411). (i) CODEX immunofluorescent co-staining for F4/80 (macrophage) and αSMA (fibroblast) co-localization. Quantification of how many aSMA expressing cells also express F4/80. (j) CODEX immunofluorescent staining for collagen marker COL1A1 (**p = .0027). Scale bar = 100 µm). n = 3 independent capsules per group for all groups. All statistical comparisons were made by using two-tailed t tests. All data is presented as mean ± SEM. MSI = mechanically stimulated implant. RI = Rac inhibitor. Source data