Methods and compositions for reducing foreign body response

Abstract

Methods of reducing foreign body response (FBR) in a subject are provided. Aspects of the methods include implanting a foreign body, e.g., an implantable device, in the subject in combination with an effective amount of an SPP1 active agent, e.g., a SPP1 polypeptide, to reduce FBR in the subject. Also provided are implantable devices coated with a SPP1 active agent composition, e.g., a controlled release composition, and kits including an implantable device and a SPP1 active agent composition.

Description

[0001] METHODS AND COMPOSITIONS FOR REDUCING FOREIGN BODY

[0002] RESPONSE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 535,205, filed August 29, 2023, the disclosure of which application is incorporated herein by reference in its entirety.

[0005] REFERENCE TO AN ELECTRO IC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (STAN-2132WO_S23-249_SEQLIST.xml; Size: 5,630 bytes; and Date of Creation: August 28, 2024) is herein incorporated by reference in its entirety.

[0007] INTRODUCTION

[0008] Despite the significant potential of implantable biomedical devices for improving health and quality of life, all biological implants are limited by foreign body response (FBR), an immune- mediated reaction involving chronic inflammation, foreign body giant cell formation, and ultimately fibrous encapsulation of foreign material. Pathologic FBR can lead to implant malfunction, superimposed infection, soft tissue disfigurement, and may ultimately necessitate reoperation procedures and material explant. While FBR is an expected inflammatory response to foreign material, the mechanisms underlying progressive chronic inflammation and capsule formation are poorly understood. Prior approaches to reducing FBR involve the use of acellular dermal matrix (ADM), a decellularized tissue with an intact extracellular matrix (ECM), that is thought to attenuate FBR by providing a biomimetic scaffold for native tissue incorporation. However, the use of ADM is limited by high cost and availability in addition to related risks such as necrosis or infection. There is a need for new methods and compositions to reduce FBR in a scalable and cost-effective manner.

[0009] SUMMARY

[0010] The present disclosure provides methods for reducing foreign body response (FBR) in a subject. Aspects of the methods of reducing FBR in a subject include implanting a foreign body in the subject in combination with an effective amount of an SPP1 active agent to reduce FBR in the subject. In some embodiments, the SPP1 active agent comprises an SPP1 polypeptide. In some cases, the SPP1 active agent is a recombinant SPP1 polypeptide. In some cases, the SPP1 active agent is present in an SPP1 active agent composition. In some embodiments, the SPP1 active agent composition comprises a controlled release composition. In some embodiments, the SPP1 active agent composition comprises a gel. In some cases, the gel is a hydrogel. In some embodiments, the hydrogel comprises a polymer. In some embodiments, the polymer comprises a polysaccharide. In some cases, the polysaccharide comprises hydroxypropyl methylcellulose. In some embodiments, the SPP1 active agent composition comprises a nanoparticle. In some embodiments, the nanoparticle comprises a diblock copolymer. In some cases, the diblock copolymer comprises polyethylene glycol) and poly(lactic acid). In some embodiments of the methods, the foreign body is coated with a SPP1 active agent composition of the present disclosure. In some cases, the foreign body is an implantable device.

[0011] In some embodiments, the methods of the present disclosure produce an implant with a reduced foreign body capsule thickness around the implant compared to a control. In some embodiments of the methods, the foreign body capsule has reduced collagen density compared to a control. In some embodiments of the methods, the foreign body capsule has increased elastin density compared to a control. In some embodiments of the methods, the foreign body capsule has increased elasticity compared to a control. In some embodiments of the methods, the subject is a mammal. In some cases, the subject is a human. In some cases, the human is an adult. In some cases, the subject is a mouse.

[0012] The present disclosure additionally provides implantable devices coated with a SPP1 active agent composition of the present disclosure. Also provided are kits including an implantable device and an SPP1 active agent composition of the present disclosure.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] Figures 1A-1 F: Acellular Dermal Matrix (ADM) alters extracellular matrix ultrastructure in human capsule specimens and leads to a reduction in fibrotic encapsulation. (A) Schematic of paired capsule specimen retrieval obtained from human patients. (B) Hematoxylin and eosin (H&E) staining of ADM (left) and Native (right) capsule. Black dotted regions indicate areas from which magnified images were captured. (C) Trichrome staining of ADM and Native capsule. Quantification of collagen density in Native and ADM capsule (right). (D) Picrosirius red histology (top) and uniform manifold approximation and projection (UMAP) displaying quantified extracellular matrix (ECM) ultrastructure parameters bottom; ADM and Native capsule data displayed in blue and red, respectively). (E) Elastin staining of ADM and Native capsule (left). Quantification of percentage of elastin+ staining in ADM and Native capsule (right). (F) Schematic of Luminex workflow (left). Luminex secretion assay comparing median fluorescent intensity (MFI) of Stromal cell-derived factor 1 A and B (SDF1A+B), C-X-C motif chemokine ligand 10 (CXCL10), and lnterleukin-1 alpha (IL-1 a) secretion, between Native and ADM capsule (right). Linked data points represent Native and ADM capsules retrieved from the same patient. (C, E) Data shown as mean ± standard deviation (S.D.). (F) Data shown as absolute values. *P< 0.05, **P< 0.01 , ****P < 0.0001 . Scale bars, (B, C, top row) 300 pm; (B, C, bottom row) 60 pm; (D) 50 pm; (E) 100 pm; n = 13 human specimens unless otherwise specified.

[0015] Figures 2A-2H: Single-cell transcriptomic analyses reveal changes in myeloid cell dynamics during foreign body capsule formation in humans with Acellular Dermal Matrix (ADM) application. (A) Schematic of paired capsule specimen retrieval obtained from human breast reconstruction patients followed by single cell RNA sequencing (scRNA-seq) analysis. (B) Uniform-manifold approximation and projection (UMAP) of scRNA-seq data from all human captured cells, colored by cell type. Black dotted region indicates cells transcriptomically classified as myeloid cells (i.e., in silica selection) that were used for downstream analysis. (C) UMAP of myeloid cells colored by Seurat cluster (0-3). (D) Relative representation of myeloid cells belonging to Seurat subclusters 0-3 from ADM and Native capsule. (E) Dot plot illustrating relative expression of the top 3 differentially expressed genes in each myeloid Seurat subcluster. (F) Violin plots showing expression of Osteopontin (SPP1) in myeloid cells by Seurat cluster (top) and experimental condition (bottom). (G) Inferred SPP1 signaling network in cells from ADM (above) and Native (below) capsule. (H) Immunofluorescence (IF) staining of SPP1 (green signal) and Integrin alpha X (CD11c) (red signal) in Native and ADM capsule. Bottom, quantification of SPP1 and CD11c co-expression from IF staining. DAPI (4',6-diamidino-2-phenylindole), nuclear counterstain (blue signal). * *P < 0.01. (H) Scale bars 100pm. n = 13 human specimens unless otherwise specified.

[0016] Figures 3A-3G: CODEX analysis of ADM and Native Human Capsules. (A) Schematic of the CO-Detection by indEXing (CODEX) experiment. (B) Example of Acellular Dermal Matrix (ADM) and Native capsules following CODEX conjugation. (C) Example of staining of CODEX markers in ADM (top) and Native (bottom) capsules [Integrin ocX (CD11C); White, Collagen Type 1 (COL1); Green, CD45; Blue, Yes-associated protein (YAP); Yellow and Osteopontin-1 (SPP1); Red]. (D) Uniform-manifold approximation and projection (UMAP) of CODEX defined clusters. (E) Bar graph quantifying Myeloid clusters in ADM and Native capsules. *P < 0.05 (F) Differential interaction maps in Native vs. ADM capsules. (G) Ridge plot showing expression of SPP1 in Myeloid clusters. Scale bars 100 pm. n = 3 unless otherwise specified. Figures 4A-4E: Murine Acellular Dermal Matrix (ADM) FBR model recapitulates decreased fibrosis as observed in human ADM capsule specimens. (A) Silicone implants either coated with ADM (mADM) or alone (mNative) were implanted subcutaneously in the dorsi of C57BL / 6j (wild-type) mice. Implant and peri-implant tissue were retrieved at postoperative day (POD) 28 for histological analyses. (B) Hematoxylin and eosin (H&E) staining of mNative and mADM capsule (left and middle columns). Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (top right). Quantification of capsule thickness in mNative and mADM capsule (bottom right). (C) Trichrome staining of mNative and mADM capsule (left and middle columns). Schematic representing skin layers and capsule (top right). Quantification of collagen density in mNative and mADM capsule (bottom right). (D) Representative picrosirius red histology from mADM capsule (top left, blue) and mNative capsule (top right, red). Uniform manifold approximation and projection (UMAP) displaying quantified extracellular matrix (ECM) ultrastructure parameters (bottom; mADM and mNative capsule data displayed in blue and red, respectively). (E) Immunofluorescence (IF) staining of alpha Smooth Muscle Actin (oc-SMA) (red signal) in mNative and mADM capsule (left). Bright red tissue is autofluorescence of panniculus carnosus. Quantification of oc-SMA coexpression from IF staining. DAPI (4',6-diamidino-2-phenylindole), nuclear counterstain (blue signal). (C, D) Data shown as mean ± standard deviation 0.0001 , Scale bars, (B,

[0017] C, top row) 100 pm; (B, C, bottom row) 30 pm; (D) 50 pm. (E) 50 pm. n = 3 unless otherwise specified.

[0018] Figures 5A-5H: Single-cell transcriptomic analyses comparing murine Native and Acellular Dermal Matrix (ADM) capsule mimics myeloid cell dynamics in human FBR. (A) Silicone implants either coated with ADM (mADM) or alone (mNative) were implanted subcutaneously in the dorsi of C57BL / 6J (wild-type) mice. Implant and peri-implant tissue were retrieved at postoperative day (POD) 28 for single-cell RNA sequencing (scRNA-seq). (B) Uniform-manifold approximation and projection (UMAP) of scRNA-seq data from all mouse capsule cells, colored by cell type. Black dotted region indicates cells transcriptomically classified as monocytes (i.e., in silica selection) that were used for downstream analysis. (C) UMAP of monocytes colored by Seurat subcluster (0-6). (D) Relative representation of monocytes belonging to Seurat subclusters 0-6 from mNative and mADM capsule. (E) Dot plot of relative expression of the top 2 differentially expressed genes in each monocyte Seurat subcluster. (F) Violin plots showing expression of Osteopontin (Spp1) in monocytes by Seurat subcluster (top) and experimental condition (right below). (G) Inferred SPP1 signaling network in cells from mADM (above) and mNative (below) capsule. (H) Immunofluorescence (IF) staining of SPP1 (green signal) and Integrin alpha X (CD11 c) (red signal) in mNative and mADM capsule (top). Bright green tissue is autofluorescence of panniculus carnosus. Quantification of SPP1 and CD11 c coexpression from IF staining. DAPI (4',6-diamidino-2-phenylindole), nuclear counterstain (blue signal). DAPI (4',6-diamidino-2-phenylindole), nuclear counterstain (blue signal). Scale bars 50 pm. n = 3 unless otherwise specified.

[0019] Figures 6A-6H: Osteopontin (SPP1) is both necessary and sufficient to decrease fibrotic encapsulation due to foreign body response. (A) Schematic of experimental flow of the murine FBR model used. Silicone implants either alone (KO mNative), coated with Acellular Dermal Matrix (KO mADM), or combined with an ipsilateral injection of recombinant SPP1 -loaded PNP hydrogel (KO +SPP1 ) were implanted subcutaneously in the dorsi of Spp1 Knock-Out (SPP1 KO) mice, Implant and peri-implant tissue retrieved at postoperative day (POD) 28 for histologic analyses. (B) Hematoxylin and eosin (H&E) staining of KO mNative (far left column), KO mADM (middle left column), and KO +SPP1 (middle right column) capsule. Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (far right column, top). Quantification of capsule thickness in KO mNative, KO mADM, and KO +SPP1 capsule (bottom). (C) Trichrome staining of KO mNative (far left column), KO mADM (middle left column), and KO +SPP1 (middle right column) capsules. Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (far right column, top). Quantification of collagen density in KO mNative, KO mADM, and KO +SPP1 capsule (bottom). (D) Immunofluorescence (IF) staining of alpha Smooth Muscle Actin (a-SMA) (red signal) in KO mNative, KO mADM, and KO +SPP1 capsule (top and bottom left). Quantification of a-SMA expression from IF staining (right). (E) Schematic of experimental flow of the murine FBR model used. Silicone implants either combined with an ipsilateral injection of empty PNP hydrogel (mNative -SPP1 ), coated with Acellular Dermal Matrix (mADM), or combined with an ipsilateral injection of recombinant SPP1 -loaded PNP hydrogel (mNative +SPP1 ) were implanted subcutaneously in the dorsi of C57BL / 6J (Wildtype) mice. Implant and peri-implant tissue retrieved at postoperative day (POD) 28 for histologic analyses. (F) H&E staining of mNative -SPP1 (far left column), mADM (middle left column), and mNative +SPP1 (middle right column) capsule. Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (far right column, top). Quantification of capsule thickness in mNative -SPP1 , mADM, and mNative +SPP1 capsule (bottom). (G) Trichrome staining of mNative-SPP1 (far left column), mADM (middle left column), and mNative +SPP1 (middle right column) capsule. Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (far right column, top). Quantification of collagen in mNative -SPP1 , mADM, and mNative+SPP1 capsule (bottom). (H) IF staining of a-SMA (red signal) in mNative -SPP1 , mADM, and mNative +SPP1 (top and bottom left). Quantification of a-SMA expression from IF staining (bottom right). Bright red tissue is autofluorescence of panniculus carnosus. (B, C, F, G) Data shown as mean ± standard deviation (S.D.). (D, H) DAPI (4',6-diamidino-2-phenylindole), nuclear counterstain (blue signal). **P< 0.01 , ***P< 0.001 , 0.0001. Scale bars, (B, C, F, G top row) 100 pm; (B, C,

[0020] F, G, bottom row) 30 pm. (D, H) 50 pm; n = 3 unless otherwise specified.

[0021] Figures 7A-7F: (A) Intraoperative photograph following breast implant removal showing acellular dermal matrix (ADM right) and Native capsule (left) retrieval (black arrows). (B) Hematoxylin and eosin (H&E) staining of ADM alone. (C) Trichrome staining of ADM alone. (D) Elastin staining of ADM alone. (E) Luminex secretion assay comparing median fluorescent intensity (MFI) of the following chemokine markers between implant alone (Native) and Acellular Dermal Matrix-coated (ADM) capsule: Eotaxin-2, Eotaxin-3, Interleukin 5 (IL-5), Chemokine ligand 2 (CCL2), Chemokine ligand 21 (CCL21 ), Chemokine ligand 22 (CCL22), and Chemokine ligand 27 (CCL27). Linked data points represent Native and ADM capsules retrieved from the same patient. (F) Tensile Young’s modulus of ADM and Native capsule. (E) Data shown as absolute values. (F) Data shown as mean modulus of elasticity (MPa) ± standard deviation (S.D.). *P < 0.05. (B-D) Scale bars 600 pm. n = 13 human specimens unless otherwise specified.

[0022] Figures 8A-8F: (A) Uniform-manifold approximation and projection (UMAP) of single-cell RNA-sequencing (scRNA-seq) data from all human capsule cells, colored by patient. (B) UMAP of all cells colored by experimental condition [implant alone (Native) or Acellular Dermal Matrix- coated (ADM) capsule]. (C) UMAP of fibroblast cells (i.e., in silico selection) colored by Seurat subcluster (0-6). (D) UMAP of fibroblasts colored by experimental condition (Native or ADM capsule). (E) UMAP of myeloid cells colored by experimental condition (Native or ADM capsule). (F) Heatmap displaying top differentially expressed genes for each myeloid cell Seurat subcluster. The red square emphasizes that SPP1 is a gene highly expressed in monocyte Seurat subcluster 2. Figures 9A-9B: (A) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic to cells in myeloid cell Seurat subclusters 1 and 2. (B) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic to cells in myeloid cell Seurat subclusters 0 and 3. n = 13 human specimens unless otherwise specified. (A, B) Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow).

[0023] Figures 10A-10C: (A) Cell-cell interaction maps among cells from ADM capsule (left) and Native capsule (right). (B) Relative information flow for each significant pathway identified via CellChat. X-axis indicates the percentage of interactions per pathway identified in the dataset, split by experimental condition. Top signaling pathways colored in red are more enriched in ADM capsule, while those in green are more enriched in Native capsule. Red arrows highlight specific pathways enriched in ADM capsule, while green arrows highlight specific pathways enriched in Native capsule. (C) Immunofluorescence staining of Osteopontin (SPP1 ) (green signal) and Collagen Type 1 (COL1A1 , green signal) in Native and ADM capsule. DAPI (4',6-diamidino-2- phenylindole), nuclear counterstain (blue signal). Scale bars 100 pm. n = 3 unless otherwise specified.

[0024] Figures 11A-11 E: (A) Uniform-manifold approximation and projection (UMAPs) of macrophage cells colored by Seurat subcluster (0-4). (B) UMAP of macrophages colored by experimental condition (Native or ADM capsule). (C) Relative representation of macrophages belonging to Seurat subclusters 0-4 from ADM and Native capsule. (D) Violin plot displaying Osteopontin (SPP1) expression in macrophages by experimental condition (ADM or Native capsule). (E) Heatmap displaying top differentially expressed genes for each macrophage Seurat subcluster. The red square emphasizes that SPP1 is a gene highly expressed in macrophage Seurat subcluster 1 .

[0025] Figures 12A-12B: (A) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic of cells in macrophage Seurat subclusters 0, 1 , and 4. (B) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic of cells in macrophage Seurat subclusters 2 and 3. (A, B) Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow), n = 13 human specimens unless otherwise specified. Figures 13A-13F: (A) Bar graphs of Osteopontin (SPP1 ), Integrin Alpha M (CD11 c), and Collagen Type 1 (COL1 ) protein expression in Acellular Dermal Matrix-coated (ADM) and implant alone (Native) capsules. (B) Bar graph quantifying Myeloid 1 and Myeloid 3 communication in ADM and Native capsules. (C) Differential interaction maps in ADM (top) and Native (bottom) capsules (D) Bar graph quantifying Smooth muscle cell and Myeloid cell communication in ADM and Native capsules (left, top and bottom). Bar graph quantifying cluster of differentiation 4 (CD4) cell and Myeloid cell communication in ADM and Native capsules (right, top and bottom). (E) Ridge plot showing Yes-associated protein (YAP) expression in all cells. (F) YAP expression in Myeloid clusters, n = 3 unless otherwise specified.

[0026] Figures 14A-14B: (A) Gross photograph of mNative and mADM capsule retrieval (left). Photos on the right show magnified images of mNative and mADM capsules. (B) Tensile Young’s modulus of PDMS and ADM.

[0027] Figures 15A-15F: (A) Uniform-manifold approximation and projection (UMAPs) of fibroblasts colored by Seurat subcluster (0-4) (B) UMAP of fibroblasts colored by experimental condition [Acellular Dermal Matrix-coated (mADM) or implant alone (mNative) capsule]. (C) UMAP of monocytes colored by experimental condition (mNative or mADM capsule). (D) Heatmap displaying top differentially expressed genes for each monocyte Seurat subcluster. (E) Enrich R analysis results for Pathways and Gene Ontology (GO) terms characteristic of cells in monocyte Seurat subclusters 2, 3, and 4. (F) Immunofluorescence staining of SPP1 (green signal) and Collagen Type I (COL1 A1 ) (red signal) in mNative and mADM capsule. Bright green tissue is autofluorescence of panniculus carnosus. (E) Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow). (F) DAPI (4',6- diamidino-2-phenylindole), nuclear counterstain (blue signal), n = 3 unless otherwise specified.

[0028] Figure 16: EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic of cells in monocyte Seurat subclusters 0, 1 , 5, and 6. n = 3 unless otherwise specified. Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow).

[0029] Figures 17A-17E: (A) Schematic of Osteopontin-1 (SPP1) release from polymer- nanoparticle (PNP) hydrogel wherein 100 pL of PNP hydrogel with 15 pL of Spp1 -AF647 is injected into the bottom of a capillary tube and PBS buffer added (left). Buffer is sampled over time to quantify Spp1 release. Quantification of release assay (right). (B) Picrosirius red histology (bottom) and uniform manifold approximation and projection (UMAP) displaying quantified extracellular matrix (ECM) ultrastructure parameters for KO mNative, KO mADM, and KO +SPP1 capsules (top), with capsule data displayed in red, blue, and yellow, respectively. (C) Immunofluorescence (IF) co-staining of Osteopontin-1 (SPP1 ) (green signal) and Integrin alpha X (CD11c) (red signal) (top) and co-staining of SPP1 (green signal) and Collagen Type I (COL1A1) (red signal) bottom in KO mNative, KO mADM, and KO +SPP1 capsules. (D) IF co- staining of Osteopontin-1 (SPP1 ) (green signal) and CD11c (red signal) (top) and co-staining of SPP1 (green signal) and COL1A1 (red signal) bottom in mNative -SPP1 , mADM, and mNative +SPP1 capsules. (E) Picrosirius red histology (bottom) and uniform manifold approximation and projection (UMAP) displaying quantified extracellular matrix (ECM) ultrastructure parameters for mNative -SPP1 , mADM, and mNative +SPP1 capsules (top), with capsule data displayed in red, blue, and yellow, respectively. (A) Data are shown as mean ± SD and fit with a one-phase decay in GraphPad Prism and half-life of 16.11 days calculated. (C, E) DAPI (4',6-diamidino-2- phenylindole), nuclear counterstain (blue signal). (B, D) Scale bars 50 pm. (C, E) Scale bars 100 pm. n = 3 unless otherwise specified.

[0030] Figures 18A-18B: (A) Relative representation of fibroblasts belonging to Seurat clusters 0-6 from ADM and Native capsules. (B) Enrichr analysis results for Pathways and Gene Ontology (GO) terms characteristic to cells in fibroblast Seurat clusters 0 and 3. Pathways: NCI- Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow).

[0031] Figures 19A-19B: (A) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic to cells in monocytes / macrophages cell Seurat clusters 1 and 2. (B) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic to cells in monocytes / macrophages Seurat clusters 0 and 3. (A, B) Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red) WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow).

[0032] Figures 20A-20B: (A) Violin plots showing expression of Osteopontin (SPP1) in monocytes / macrophages by Seurat cluster (left) and experimental condition (right) following downsampling of Native capsule cells (ADM monocytes / macrophages = 253, Native monocytes / macrophages = 253). (B) Violin plot showing expression of SPP1 in macrophages by condition following downsampling of Native capsule cells (ADM macrophages = 165, Native macrophages = 165).

[0033] Figures 21A-21 B: (A) All human cell single-cell RNA-sequencing Uniform-manifold approximation and projection (UMAP) colored by expression level for Integrin Alpha M (CD11b) (top) and Integrin Alpha X (CD11c) (bottom). (B) IF staining of SPP1 (green signal) and CDU b (red signal) in Native and ADM capsule. DAPI (4Z,6-diamidino-2-phenylindole), nuclear counterstain (blue signal). Scale bars 100 i m.

[0034] Figures 22A-22B: (A) Haematoxylin and eosin (H&E) staining of Native -ADM capsule. (B) Immunofluorescence (IF) staining of Osteopontin (SPP1 ) (green signal) and Integrin alpha X (CD11c) (red signal, left). Quantification of SPP1 and CD11 c co-expression from IF staining (right). (B) DAPI (4Z,6-diamidino-2-phenylindole), nuclear counterstain (blue signal). Data shown as mean ± standard deviation (S.D.). DAPI (4Z,6-diamidino-2-phenylindole), nuclear counterstain (blue signal). **** P< 0.0001 , ns = not significant. Scale bars, (A) 400 m; (B) 100 n m. n = 3 human specimens.

[0035] Figures 23A-23D: (A) Schematic of experimental flow of the murine FBR model applied using ADM on its own. Implant and peri-implant tissue retrieved at postoperative day (POD) 28 for histology analysis. (B) Hematoxylin and eosin (H&E) staining of ADM alone capsule. Quantification of capsule thickness in mADM and ADM alone capsule (right). (C) Trichrome staining of ADM alone capsule. Quantification of collagen density in mADM and ADM alone capsule (right). (D) Immunofluorescence (IF) staining of a-Smooth Muscle Actin (a-SMA) (red signal) in ADM alone (far left). Quantification of a-SMA expression from IF staining (second panel from left). (D) DAPI (4Z,6-diamidino-2-phenylindole), nuclear counterstain (blue signal). Data shown as mean ± standard deviation (S.D.). ns = not significant. Scale bars (B, C) 50 i m; (D) 100 fi m. n = 3 unless otherwise specified.

[0036] Figures 24A-24B: (A) Relative representation of fibroblasts belonging to fibroblast Seurat clusters 0-4 from mADM and mNative capsules. (B) EnrichR analysis results for Pathways and Gene Ontology (GO) terms characteristic to cells in fibroblast Seurat clusters 0 and 1 . Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow).

[0037] Figures 25A-25C: (A) Violin plot displaying Osteopontin (SPP1) expression in macrophages by experimental condition [Acellular Dermal Matrix-coated (mADM) or implant alone (mNative) capsule]. (B) All mouse single cell RNA-sequencing (scRNA-seq) UMAP colored by expression level for Integrin alpha M (Cd11b) (top) and Integrin alpha X (Cd11c) (bottom). (C) IF staining of SPP1 (green signal) and CD11 b (red signal) in mNative and mADM capsule. Bright green tissue is autofluorescence of panniculus carnosus. (C) DAPI, nuclear counterstain (blue signal). Pathways: NCI-Nature 2016 (blue), KEGG 2021 Human (red), WikiPathway 2021 Human (yellow). GO Terms: GO Cellular Component 2021 (blue), GO Molecular Function 2021 (red), GO Biological Process (yellow). Scale bars = 100 m.

[0038] Figures 26A-26B: (A) Summary of correlations between human monocyte / macrophage cluster 2 from Fig. 2 to mouse monocyte clusters 1 , 2, and 4 from Fig. 5. (B) Violin plot depicting the similarity of human monocyte / macrophage cluster 2 onto mouse monocyte derived Seurat clusters.

[0039] Figures 27A-27F: (A) Schematic of experimental flow of the murine FBR model used. Silicone implants coated with Acellular Dermal Matrix combined with an ipsilateral injection of empty PNP hydrogel (KO mADM -SPP1) and silicone implants coated with ADM combined with an ipsilateral injection of recombinant SPP1 -loaded PNP hydrogel (KO mADM +SPP1 ) were implanted subcutaneously in the dorsi of Spp1 Knock-Out (SPP1 KO) mice. Implant and periimplant tissue retrieved at postoperative day (POD) 28 for histologic analyses. (B) Hematoxylin and eosin (H&E) staining of KO mADM -SPP1 (left) and KO mADM +SPP1 capsule (middle). Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (right, top). Yellow lines indicate capsule area. Quantification of capsule thickness in KO mADM -SPP1 and KO mADM +SPP1 capsule (right, bottom). (C) Trichrome staining of KO mADM -SPP1 (left) and KO mADM +SPP1 capsules (middle). Black dotted regions indicate areas from which magnified images were captured. Schematic representing skin layers and capsule (right, top). Quantification of collagen density in KO mADM -SPP1 and KO mADM +SPP1 capsule (right, bottom). (D) Picrosirius red histology of KO mADM -SPP1 and KO mADM +SPP1 . (E) Immunofluorescence (IF) staining of alpha Smooth Muscle Actin (a-SMA) (red signal) in KO mADM -SPP1 and KO mADM +SPP1 (top left and top right). Quantification of a-SMA expression from IF staining (bottom). (F) IF costaining of Osteopontin-1 (SPP1 ) (green signal) and Integrin alpha M (CD11 b) (top) and costaining of SPP1 (green signal) and Collagen Type 1 (COL1) (red signal) (bottom) in KO mADM -SPP1 and KO mADM +SPP1 capsules. Quantification of COL1 expression from IF staining (bottom right). (B, C, E, F) Data shown as mean ± standard deviation (S.D.). (E, F) DAPI (4Z,6- diamidino-2-phenylindole), nuclear counterstain (blue signal). ** P< 0.01 , **** P< 0.0001. (B, C) Scale bars 50 n m; Scale bar insets 100 n m (D) Scale bars 50 ,« m. (E, F) Scale bars 100 n m. n = 3 unless otherwise specified.

[0040] Figures 28A-28E: (A) Hematoxylin and eosin (H&E) staining of mouse skin injected subcutaneously every other day with SPP1 resuspended in PBS. (B) Schematic of experimental flow of the murine FBR model used. Silicone implants combined with a subcutaneous injection of SPP1 in PBS (mNative + SPP1 PBS). Implant and peri-implant tissue retrieve at postoperative day (POD) 28 for histology analysis. (C) Hematoxylin and eosin (H&E) staining of mNative + SPP1 PBS capsule. Quantification of capsule thickness in mNative, mNative + SPP1 , and mNative + SPP1 PBS capsule (right). (D) Trichrome staining of mNative + SPP1 PBS. Quantification of collagen density in mNative, mNative + SPP1 , and mNative + SPP1 PBS capsule (right). (E) Immunofluorescence (IF) staining of a-Smooth Muscle Actin (a-SMA) (red signal) in mNative+SPP1 PBS (left). Quantification of a-SMA expression from IF staining (right). (E) DAPI (4Z,6-diamidino-2-phenylindole), nuclear counterstain (blue signal). Data shown as mean ± standard deviation (S.D.). *P<0.05, **P<0.01 , ****P<0.0001. Scale bars (B, C, D) 50pm; (E) 100|im. n = 3 unless otherwise specified.

[0041] Figures 29A-29E: (A) Violin plots displaying Integrin Subunit Alpha V (ITGAV) and Integrin Subunit Beta 3 (ITGB3) expression in human monocytes / macrophages by Seurat cluster (0-6). (B) Violin plot displaying Cluster of Differentiation 44 (CD44) expression in human monocytes / macrophages by Seurat cluster (0-6). (C) Immunofluorescence (IF) staining of CD44 (red signal) in ADM and Native capsule (left and right). (D) Violin plot displaying Matrix Metallopeptidase 9 (MMP9) in human monocytes / macrophages by experimental condition (ADM or Native capsule). (E) IF co-staining of MMP9 (green signal) and Integrin Alpha M (CD11 b) in ADM and Native capsules (top left and top right). Quantification of MMP9 expression from IF staining (bottom). (E) Data shown as mean ± standard deviation (S.D.). (C, E) DAPI (4Z,6- diamidino-2-phenylindole), nuclear counterstain (blue signal). * P< 0.05. (C, E) Scale bars 100 pm.

[0042] DEFINITIONS

[0043] As used herein in its conventional sense, a “foreign body” refers to any material, composition, or object that is foreign or exogenous to the tissue, and / or organism in which the foreign body is implanted. A “foreign body” encompasses implantable devices.

[0044] As used herein in its conventional sense, a “foreign body response” refers to an immune- mediated inflammatory and fibrotic response to a foreign body implanted in a tissue and / or organism. The “foreign body response” includes the encapsulation of a foreign body by a layer of fibrous tissue (i.e., a foreign body capsule).

[0045] As used herein in its conventional sense, a “myeloid cell” refers to cells derived from hematopoietic stem cells and encompasses granulocytes and monocytes. A “myeloid cell” includes a cell characterized by expression of the surface marker integrin ocX (i.e., CD11c).

[0046] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0047] The term “oligonucleotide” refers to a polynucleotide of between 3 and 100 nucleotides of single- or double-stranded nucleic acid (e.g., DNA, RNA, or a modified nucleic acid). However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and can be isolated from genes, transcribed (in vitro and / or in vivo), or chemically synthesized. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0048] The terms ‘’peptide,” ‘’polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term "fibrosis" as used herein in its conventional sense refers to the formation or development of excess fibrous connective tissue in an organ or tissue as a result of injury or inflammation of a part or interference with its blood supply. It can be a consequence of the normal healing response that leads to a scar, an abnormal reactive process or no known or understood cause.

[0049] As used herein, the term “modulating” means increasing, reducing or inhibiting an attribute of a biological cell, population of cells, component of a cell, or a tissue (e.g., a protein, nucleic acid, etc.). In some cases, the attribute includes, e.g., activation of a signaling pathway. In some cases, the attribute includes an amount and / or activity of one or more cells or a tissue. In some cases, the attribute includes a mechanical property of a cell, population of cells, or a tissue. In some cases, "modulate" or "modulating" or “modulation” may be measured using an appropriate in vitro assay, cellular assay or in vivo assay. In some cases, the increase or decrease is 10% or more relative to a reference, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, up to 100% relative to a reference. For example, the increase or decrease may be 2 or more times, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 50 times or more, or 100 times or more relative to a reference.

[0050] DETAILED DESCRIPTION

[0051] Methods for reducing foreign body response (FBR) in a subject are provided. Aspects of the methods include implanting a foreign body in the subject in combination with an effective amount of an SPP1 active agent to reduce FBR in the subject. In some embodiments, the SPP1 active agent is present in a SPP1 active agent composition. Implantable devices coated with a SPP1 active agent composition of the present disclosure are provided. Also provided are kits including an implantable device and a SPP1 active agent composition of the present disclosure.

[0052] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0053] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0055] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0056] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0057] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0058] In further describing various aspects of embodiments of the invention, aspects of the methods will be reviewed first in greater detail, followed by a review of various aspects of related devices and kits.

[0059] METHODS

[0060] As summarized above, the present disclosure provides methods of reducing foreign body response (FBR) in a subject, the methods comprising implanting a foreign body in the subject in combination with an effective amount of a SPP1 active agent to reduce FBR in the subject. By ‘‘in combination” it is meant that the SPP1 active agent, or a composition thereof, is implanted in the subject such that the SPP1 active agent, or a composition thereof, is adjacent to, or otherwise in contact with, at least some of the tissue surrounding, and / or disrupted by, the foreign body. In some embodiments, the SPP1 active agent, or a composition thereof, is first applied to the foreign body, after which the foreign body combined with the applied SPP1 active agent can be subsequently implanted into the subject.

[0061] The foreign body response (FBR) is an inflammatory and fibrotic process that is mounted in response to implantation of any foreign material that has consequently damaged surrounding tissue. A fibrotic aspect of the FBR includes eventual encapsulation of the foreign body in a layer of fibrous tissue that acts as a barrier (i.e., foreign body capsule) between the foreign body and surrounding tissue. The thickness and composition of the foreign body capsule can impact the function of an implantable device and the health of the surrounding tissue. Pathologic formation of a foreign body capsule can critically impede the function of the implantable device, lead to disfigurement of surrounding soft tissues or infection, and necessitate additional surgical procedures to remove and / or replace the implantable device.

[0062] SPP1 ACTIVE AGENTS

[0063] As summarized above, aspects of the methods include implanting a foreign body in the subject in combination with an effective amount of a SPP1 active agent. Secreted Phosphoprotein 1 (SPP1 ), also known as Osteopontin (OPN), is an integrin-binding glycophosphoprotein with established roles in tumorigenesis and bone mineralization. The SPP1 protein is secreted, can bind to hydroxyapatite with high affinity, and can function as a cytokine. In some instances, the SPP1 active agent is an SPP1 protein or a fragment thereof. The SPP1 protein is encoded by the Secreted Phosphoprotein-1 gene. Example gene, transcript, and protein sequences of SPP1 may be accessed at Genbank at NG_030362.1 , NM 000582.3, and NP 000573.1 , respectively. As used in the present disclosure, a SPP1 polypeptide encompasses any natural variants, alternative sequences, isoforms, or mutant proteins that can be found in any mammal (e.g., that can be found in a human). Examples of SPP1 protein isoforms found in humans include, without limitation, the protein sequences which may be accessed at Genbank at NP 001035147.1 , NP 001035149.1 , NP 001238758.1 , and NP 001238759.1 . Examples of SPP1 protein variants found in the human population include, without limitation, L10I, T73A, D107N, F136S, and T152R. Variants of SPP1 include SPP1 variants which are not glycosylated or substantially less glycosylated than endogenously secreted SPP1 proteins. Additional variants of SPP1 include SPP1 proteins that are not phosphorylated or substantially less phosphorylated than endogenously secreted SPP1 proteins.

[0064] The SPP1 protein used in the methods and compositions described herein may be endogenously derived (e.g., from demineralized bone) or may be a recombinant SPP1 protein. In some embodiments, the SPP1 polypeptide is a fusion protein comprising a heterologous polypeptide. In some cases, the heterologous polypeptide is an affinity tag polypeptide that may aid in purification and isolation of a recombinant SPP1 polypeptide (e.g., FLAG, hemagglutinin(HA), poly-histidine, glutathione-S-transferase (GST), or HALO tags). Methods of making and / or purifying endogenously secreted or recombinant SPP1 proteins are described, for example, in: (Prince, Charles W., et al. "Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone." Journal of Biological Chemistry 262.6 (1987): 2900- 2907), (Ashkar, S. A. M. Y., M. J. Glimcher, and R. A. Saavedra. "Mouse osteopontin expressed in E. coli exhibits autophosphorylating activity of tyrosine residues." Biochemical and biophysical research communications 194.1 (1993): 274-279), and (Ashkar, S., et al. "In vitro phosphorylation of mouse osteopontin expressed in E. coli." Biochemical and biophysical research communications 191.1 (1993): 126-133), the disclosures of which are herein incorporated by reference.

[0065] In some cases, the SPP1 active agent comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater amino acid sequence identity to the following SPP1 amino acid sequence: MRIAVICFCLLGITCAIPVKQADSGSSEEKQLYNKYPDAVATWLNPDPSQKQNLLAPQTLPSKSNESHDH MDDMDDEDDDDHVDSQDS IDSNDSDDVDDTDDSHQSDESHHSDESDELVTDFPTDLPATEVFTPVVPTVD TYDGRGDSVVYGLRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQDLNAPSDWDSRGKDS YETSQLDDQSAETHSHKQSRLYKRKANDESNEHSDVIDSQELSKVSREFHSHEFHSHEDMLVVDPKSKEE DKHLKFRI SHELDSASSEVN ( SEQ ID NO : 1 )

[0066] In some cases, the SPP1 active agent comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater amino acid sequence identity to the following SPP1 amino acid sequence: IPVKQADSGSSEEKQLYNKYPDAVATWLNPDPSQKQNLLAPQTLPSKSNESHDHMDDMDDEDDDDHVDSQ DSIDSNDSDDVDDTDDSHQSDESHHSDESDELVTDFPTDLPATEVFTPVVPTVDTYDGRGDSVVYGLRSK SKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQDLNAPSDWDSRGKDSYETSQLDDQSAETHSH KQSRLYKRKANDESNEHSDVIDSQELSKVSREFHSHEFHSHEDMLVVDPKSKEEDKHLKFRI SHELDSAS SEVN ( SEQ ID NO : 2 )

[0067] In some cases, the SPP1 active agent comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater amino acid sequence identity to the following SPP1 amino acid sequence: MRLAVICFCLFGIASSLPVKVTDSGSSEEKLYSLHPDP IATWLVPDPSQKQNLLAPQNAVSSEEKDDFKQ ETLPSNSNESHDHMDDDDDDDDDDGDHAESEDSVDSDESDESHHSDESDETVTASTQADTFTPIVPTVDV PNGRGDSLAYGLRSKSRSFQVSDEQYPDATDEDLTSHMKSGESKESLDVIPVAQLLSMPSDQDNNGKGSH ESSQLDEPSLETIIRLEnSKESQESADQSDVIDSQASSKASLEnQSnKFIISIIKDKLVLDPKSKEDDRYLKF RISHELESSSSEVN ( SEQ ID NO : 3 )

[0068] In some cases, the SPP1 active agent comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater amino acid sequence identity to the following SPP1 amino acid sequence: LPVKVTDSGSSEEKLYSLHPDP IATWLVPDPSQKQNLLAPQNAVSSEEKDDFKQETLPSNSNESHDHMDD DDDDDDDDGDHAESEDSVDSDESDESHHSDESDETVTASTQADTFTPIVPTVDVPNGRGDSLAYGLRSKS RSFQVSDEQYPDATDEDLTSHMKSGESKESLDVIPVAQLLSMPSDQDNNGKGSHESSQLDEPSLETHRLE HSKESQESADQSDVIDSQASSKASLEHQSHKFHSHKDKLVLDPKSKEDDRYLKFRISHELESSSSEVN ( SEQ ID NO : 4 )

[0069] In some instances, the SPP1 active agent is a peptidomimetic that exhibits the desired activity. Peptidomimetics of interest include, without limitation: peptidomimetics incorporating D- amino acids, that may, for example, improve enzymatic stability; peptidomimetics incorporating unnatural amino acids that may, for example, support bio-orthogonal chemistries (e.g., to enable selective cross-linking); -turn mimetics; cyclic peptides that may, for example, improve biological activity; or any combination thereof.

[0070] In some embodiments, the SPP1 active agent is a nucleic acid encoding a SPP1 polypeptide. Depending on the desired polypeptide, the nucleic acid sequence may vary. Nucleic acids of interest include those encoding a SPP1 polypeptide provided above. A nucleic acid of the present disclosure may be a sequence of DNA or RNA having an open reading frame that encodes a polypeptide of interest, i.e., a polypeptide coding sequence, and is capable, under appropriate conditions, of being expressed as a polypeptide of interest, e.g., a SPP1 protein or active fragment thereof. Also encompassed in this term are nucleic acids that are homologous, substantially similar or identical to the specific nucleic acids described above. In addition to the above described specific nucleic acid compositions, also of interest are homologues of the above sequences. In certain embodiments, sequence similarity between homologues is 20% or higher, such as 25 % or higher, and including 30 %, 35%, 40%, 50%, 60%, 70% or higher, including 75%, 80%, 85%, 90% and 95% or higher. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence may be 18 nt long or longer, such as 30 nt long or longer, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using default settings, i.e., parameters w=4 and T=17). Of particular interest in certain embodiments are nucleic acids of substantially the same length as nucleic acids mentioned above, where by substantially the same length is meant that any difference in length does not exceed about 20 number %, usually does not exceed about 10 number % and more usually does not exceed about 5 number %; and have sequence identity to any of these sequences of at 90% or greater, such as 95% or greater and including 99% or greater over the entire length of the nucleic acid. In some embodiments, the nucleic acids have a sequence that is substantially similar or identical to the above specific sequences. By substantially similar is meant that sequence identity is 60% or greater, such as 75% or greater and including 80, 85, 90, or even 95% or greater. Nucleic acids of interest also include nucleic acids that encode the proteins encoded by the above described nucleic acids, but differ in sequence from the above described nucleic acids due to the degeneracy of the genetic code.

[0071] In particular embodiments wherein the SPP1 active agent is a nucleic acid encoding a SPP1 polypeptide, the nucleic acid may be incorporated into an expression vector (e.g., a recombinant expression vector) to facilitate delivery of the nucleic acid to the tissue surrounding, and / or disrupted by, the foreign body. In some cases, the expression vector may be a viral vector or a non-viral vector.

[0072] In some cases, the nucleic acid encoding the SPP1 polypeptide in an expression vector may be operably linked to a suitable promoter sequence that enables expression of the SPP1 polypeptide in the target tissue. Suitable promoters for use in an expression vector described herein include any eukaryotic promoter (i.e., a promoter functional in a eukaryotic cell, e.g., a mammalian cell). The promoter can be a constitutively active promoter (i.e., a promoter always in an active or “ON” state), and inducible promoter (i.e., a promoter whose activity is controlled by an external stimulus, e.g., a specific temperature, small molecule, or protein). Suitable eukaryotic promoters are well known in the art and include, without limitation, cytomegalovirus (CMV) immediate early promoters, herpes simplex virus (HSV) thymidine kinase promoters, SV40 early and late promoters, Rous-Sarcoma virus (RSV) promoters, p-actin promoters, tubulin promoters, and EF1 a promoters. In some cases, the eukaryotic promoter may be a cell or tissue-specific promoter. Cell and tissue-specific promoters are well known in the art and described, e.g., in “Tissue-specific Promoters” (www[.]invivogen[.]com / tissue-specific-promoters). In some embodiments, the nucleic acid encoding the SPP1 polypeptide may further comprise a ribosome binding site for initiation of translation (e.g., an internal ribosome entry site; IRES) and / or a transcription terminator.

[0073] In some cases, the expression vector may be a viral vector. Viral vectors are known in the art and described, for example, in Warnock, James N., Claire Daigre, and Mohamed Al-Rubeai. "Introduction to viral vectors." Viral vectors for gene therapy: methods and protocols (2011 ): 1-25, the entirety of which is incorporated herein by reference. Suitable viral vectors for use in the present methods and compositions include, without limitation, adenovirus vectors (see, e.g., WO 94 / 12649, WO 93 / 03769; WO 93 / 19191 ; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655), adeno-associated virus (AAV) vectors (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et aL, PNAS 94:6916 6921 , 1997), lentiviral and retroviral vectors (e.g., lentivirus, Rous- Sarcoma virus, Murine Leukemia virus, and human immunodeficiency virus related vectors).

[0074] In some cases, the expression vector may be a non-viral vector. Non-viral vectors are known in the art and described, for example, in Yin, Hao, et al. "Non-viral vectors for gene-based therapy." Nature Reviews Genetics 15.8 (2014): 541 -555, the entirety of which is incorporated herein by reference. Suitable non-viral vectors for use in the present methods and compositions include, without limitation, lipid-based vectors (such as a liposome or lipid nanoparticle, see, e.g., Liposomes: Methods and Protocols, Volume 1 : Pharmaceutical Nanocarriers: Methods and Protocols, (ed. Weissig). Humana Press, 2009, ISBN 160327359X. and Liposome Technology, volumes I, II & III, (ed. Gregoriadis). Informa Healthcare, 2006.), polymeric vectors (e.g., polymeric poly(L-lysine), poly(lactic acid), and poly(D,L-lactide-co-glycolide) vectors, see, e.g., Functional Polymer Colloids and Microparticles volume 4 Microspheres, microcapsules & liposomes), (eds. Arshady & Guyot). Citus Books, 2002 and Polymers in Drug Delivery, (eds. Uchegbu & Schatzlein). ORC Press, 2006.)

[0075] In certain embodiments, the SPP1 active agent is a small molecule that can increase expression of SPP1 or activate SPP1 signaling. Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing screening protocols. Methods of screening for small molecules modulators of signaling are well known in the art and described, for example, in W02003038442A2, the entirety of which is incorporated herein by reference. As used herein, “an effective amount of a SPP1 active agent” refers to an amount of an a SPP1 active agent suitable to reducing foreign body response (FBR) in a subject. In some cases, an effective amount of a SPP1 active agent comprises a SPP1 active agent ranging from, e.g., 1.5 pg / ml to 5 pg / ml, 1 .5 pg / ml to 10 pg / ml, 1 .5 pg / ml to 15 pg / ml, 1 .5 pg / ml to 50 pg / ml, 1 .5 pg / ml to 100 pg / ml, 1.5 pg / ml to 150 pg / ml, 15 pg / ml to 50 pg / ml, 15 pg / ml to 100 pg / ml, 15 pg / ml to 150 pg / ml, 50 pg / ml to 75 pg / ml, 50 pg / ml to 100 pg / ml, 50 pg / ml to 150 pg / ml, or 150 pg / ml to 300 pg / ml. SPP1 ACTIVE AGENT COMPOSITIONS

[0076] A SPP1 active agent of the present disclosure, e.g., an effective amount of a SPP1 active agent, may be present in a SPP1 active agent composition. In more general terms, the active agent composition includes the active agent and a vehicle. The vehicle may comprise one or more known pharmaceutically acceptable excipients. In some cases, the vehicle may comprise a pharmaceutically acceptable salt. Suitable salts for use in a present composition include, without limitation, sodium chloride, sodium citrate, sodium benzoate, sodium phosphates (e.g., phosphate buffered saline), potassium phosphate, potassium citrate, calcium carbonate, and calcium phosphate, or combinations thereof. In some cases, the vehicle may include a pharmaceutically acceptable sugar or polyol. Suitable sugars or polyols for use in a present composition include, without limitation, sucrose, trehalose, mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, iso-maltose, maltitol, palatinit, stachyose, melezitose, dextran, or combinations thereof. In some cases, the vehicle may include an amino acid. Suitable amino acids for use in a present composition include, without limitation, glycine, arginine, lysine, and glutamine. In some embodiments, the vehicle may include a pharmaceutically acceptable thickening agent. Suitable thickening agents for use in a present composition include, without limitation, polysaccharides (e.g., hyaluronic acid, alginate, chitosan, dextran, cellulose), silicones (e.g., polysiloxane, polydimethylsiloxane), and other suitable polymers (e.g., polyethylene glycol).

[0077] In some embodiments, the vehicle is a controlled release vehicle that thus provides for a controlled release composition. By controlled release composition is meant any composition comprising an active agent which is formulated to provide a longer duration of exposure to an active agent after administration compared to a corresponding immediate release composition comprising an equivalent amount of an active agent. Controlled release compositions may alternatively be described, without limitation, as “extended release”, “sustained release”, “prolonged release”, “programmed release”, “time release”, or “rate controlled” compositions. In some embodiments, a controlled release composition may release an effective amount of a SPP1 active agent over any suitable period of time including e.g., one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 28 days or more, or 30 days or more. In some embodiments, a controlled release composition may retain a given amount of SPP1 active agent with a half-life from about 8 days to about 32 days, e.g., from about 8 to 9 days, 10 to 11 days, 12 to 13 days, 14 to 16 days, 17 to 18 days, 19 to 20 days, 21 to 22 days, 23 to 24 days, 25 to 26 days, 27 to 28 days, 29 to 30 days, or 31 to 32 days.

[0078] A controlled release vehicle may include one or more known pharmaceutically acceptable excipients, including any combination the pharmaceutically acceptable excipients discussed herein. In some embodiments, the controlled release vehicle comprises a gel. A gel of the present disclosure may be formed from cross-linking of one or more precursors. The gel precursors may be polymeric or non-polymeric. Crosslinks may be formed by covalent bonds or non-covalent interactions. Examples of non-covalent bonds include ionic bonds or hydrophobic interactions. In some cases, the gel is a hydrogel (i.e., the interstitial fluid comprising the gel is water). In some embodiments, a hydrogel of the present disclosure comprises a polymer. In some cases, the polymer may be synthetic. In some cases, the polymer may be naturally occurring. In some embodiments, the polymer comprises a polysaccharide. Examples of suitable polysaccharides include, without limitation, hyaluronic acid (HA), alginate, chitosan, dextran, cellulose, and natural or synthetic derivatives thereof. In some cases, the polysaccharide is hydroxypropyl methylcellulose. In some embodiments, a hydrogel of the present disclosure comprises a nanoparticle. In some embodiments, the nanoparticles comprise a polymer. In some cases, the polymer may be synthetic. In some cases, the polymer may be naturally occurring. Examples of suitable nanoparticle polymers include, without limitation, poly(amidoamine), PEGylated poly(amidoamine), polyethylene glycol) methyl ether methacrylate, polypeptides (e.g., albumin), polysaccharides (e.g., chitosan), poly(lactic acid), poly(lactic-co-glycolic acid), and polyethylene glycol) (i.e., PEG). In some embodiments, the polymer is a block copolymer. A block copolymer is a polymer comprised of two or more homopolymer subunits (i.e., “blocks”). In some embodiments, the block copolymer is a diblock copolymer (i.e., comprised of two distinct “blocks”). In some cases, the diblock copolymer comprises polyethylene glycol) and poly(lactic acid).

[0079] FOREIGN BODIES

[0080] As summarized above, aspects of the methods include implanting a foreign body in a subject in combination with an effective amount of an SPP1 active agent to reduce FBR in a subject. In some embodiments of the present invention, the foreign body is coated with a SPP1 active agent composition of the present disclosure. In some cases, the foreign body is an implantable device. Suitable implantable devices for use in the methods of the present disclosure include, without limitation, implants for reconstructive and / or cosmetic surgery (e.g., breast implants, calf implants, gastric implants, gluteal implants, penile implants, testicular implants), prostheses, tissue-expanders, surgical mesh implants, electro-stimulatory implants (e.g., pacemakers and neuromodulator implants), biosensors, drug-delivery ports, catheters, orthopedic implants, and vascular and non-vascular stents, among others.

[0081] In some cases, the foreign body may include a suitable amount of the SPP1 active agent composition, such that an amount of an SPP1 active agent suitable for reducing the foreign body response (FBR) in the subject (i.e., an effective amount of a SPP1 active agent). In some embodiments, the SPP1 active agent composition may comprise a SPP1 active agent ranging from, e.g., 1.5 pg / ml to 5 pg / ml, 1.5 pg / ml to 10 pg / ml, 1.5 pg / ml to 15 pg / ml, 1.5 pg / ml to 50 pg / ml, 1.5 pg / ml to 100 pg / ml, 1.5 pg / ml to 150 pg / ml, 15 pg / ml to 50 pg / ml, 15 pg / ml to 100 pg / ml, 15 pg / ml to 150 pg / ml, 50 pg / ml to 75 pg / ml, 50 pg / ml to 100 pg / ml, 50 pg / ml to 150 pg / ml, or 150 pg / ml to 300 pg / ml.

[0082] In certain embodiments, the SPP1 active agent composition may be applied to the foreign body. In some embodiments, the foreign body may be coated with the composition. All or a part of the foreign body may be coated with the SPP1 active agent composition, e.g., a controlled release composition. In particular embodiments, all or a part of the surface of the foreign body may be coated with the SPP1 active agent composition. By “surface” it is meant any part or area of the foreign body that, without a coating, would otherwise be in contact with the surrounding tissue when the foreign body is implanted in a subject. In some cases, the percent of the surface of the foreign body coated with a SPP1 active agent composition can range from, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the surface area of the foreign body coated with a SPP1 active agent composition. In some cases, the thickness (e.g., the average thickness) of the SPP1 active agent composition coating the foreign body may range from about 1 pm to about 100 pm, e.g., from about 5 pm to about 95 pm, from about 10 pm to about 90 pm, from about 15 pm to about 85 pm, from about 20 pm to about 80 pm, from about 25 pm to about 75 pm from about 30 pm to about 70 pm, from about 35 pm to about 65 pm, from about 40 pm to about 60 pm, or from about 45 pm to 55 pm across the surface of the foreign body. In some cases, the thickness (e.g., the average thickness) of the SPP1 active agent composition coating the foreign body may range from about 0.1 mm to about 1 mm, e.g., from about 0.2 mm to 0.9mm, from about 0.3 mm to about 0.8 mm, and from about 0.4mm to about 0.6 mm. In some cases, the thickness of the coating across the surface of the foreign body may vary from about 50% to about 150% of the average thickness of the coating, e.g., from about 75% to about 125%, from about 80% to about 120%, from about 90% to 110%, or from about 95% to about 105%. The percent area of the foreign body to coated and the thickness of the coating may vary depending on the size and nature of the foreign body and can be readily determined by the person of skill using the common knowledge in the art.

[0083] FOREIGN BODY RESPONSE

[0084] As summarized above, aspects of the methods include reducing foreign body response (FBR) in a subject. A fibrotic aspect of the FBR includes eventual encapsulation of the implant in a layer of the fibrous tissue that acts as a barrier (i.e., foreign body capsule) between the implant and surrounding tissue. The thickness and composition of the foreign body capsule can impact the function of an implantable device and the health of the surrounding tissue. Fibrous encapsulation of an implanted foreign body (i.e., formation of a foreign body capsule) is characterized by increased collagen density, decreased elastin density, and decreased elasticity compared to the surrounding tissue.

[0085] In some embodiments, the methods of the present disclosure result in a reduced foreign body capsule thickness compared to a control (e.g., a foreign body implanted without a SPP1 active agent). A suitable control may include a substantially identical foreign body implanted in combination with a vehicle (e.g., one or more pharmaceutically acceptable excipients) substantially identical to the vehicle in the corresponding SPP1 active agent composition, but lacking the SPP1 active agent. As a particular example, a suitable control can include a substantially identical foreign body implanted in combination with a controlled release vehicle comprising a hydrogel and a phosphate buffered saline (PBS) excipient, the controlled release vehicle and PBS together being equivalent in volume to a corresponding SPP1 active agent composition, substantially identical to the control vehicle composition with the exception that the active agent composition comprises an amount of SPP1 active agent. In some cases, the capsule thickness is decreased by 10% or more compared to a control, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, as comparted to a suitable control.

[0086] The methods of the present disclosure may result in decreased expression of known markers of fibrosis, e.g., alpha smooth muscle actin (a-SMA), in the foreign body capsule. In some embodiments, the methods of the present disclosure result in reduced a-SMA expression in the foreign body capsule compared to a control. A suitable control may include a substantially identical foreign body implanted in combination with a vehicle (e.g., one or more pharmaceutically acceptable excipients) substantially identical to the vehicle in the corresponding SPP1 active agent composition, but lacking the SPP1 active agent. In some cases, a-SMA expression is decreased by 10% or more compared to a control, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0087] In some embodiments, the methods of the present disclosure result in increased SPP1 expression in myeloid cells of the foreign body capsule compared to a control. A suitable control may include a substantially identical foreign body implanted in combination with a vehicle (e.g., one or more pharmaceutically acceptable excipients) substantially identical to the vehicle in the corresponding SPP1 active agent composition, but lacking the SPP1 active agent. In some cases, SPP1 expression is increased by 10% or more compared to a control, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.

[0088] In some embodiments, the methods of the present disclosure result in reduced foreign body capsule collagen density compared to a control. A suitable control may include a substantially identical foreign body implanted in combination with a vehicle (e.g., one or more pharmaceutically acceptable excipients) substantially identical to the vehicle in the corresponding SPP1 active agent composition, but lacking the SPP1 active agent. In some cases, the capsule collagen density is decreased by 10% or more compared to a control, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. In some embodiments, the methods of the present disclosure result in an increased foreign body capsule elastin density compared to a control. In some cases, the capsule elastin density is increased by 10% or more compared to a control, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% or more.

[0089] In some embodiments, the methods of the present disclosure result in increased foreign body capsule elasticity compared to a control. A suitable control may include a substantially identical foreign body implanted in combination with a vehicle (e.g., one or more pharmaceutically acceptable excipients) substantially identical to the vehicle in the corresponding SPP1 active agent composition, but lacking the SPP1 active agent. The elasticity of the foreign body capsule may be quantified by the tensile Young’s elastic modulus of the foreign body capsule. In some cases, the Young’s elastic modulus is decreased by 5% or more compared to a control, e.g., 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more. In particular embodiments, the methods of the present disclosure may result in all or any sub-combination of the effects described above, compared to a control. For example, the methods of the present disclosure may result in a reduced foreign body capsule thickness, a reduced foreign body capsule collagen density, and an increased foreign body capsule elasticity compared to a control. As another example, the methods of the present disclosure may result in reduced a- SMA expression in the foreign body capsule and increased SPP1 expression in myeloid cells of the foreign body capsule compared to a control. As yet another example, the methods of the present disclosure may result in a reduced foreign body capsule thickness, a reduced foreign body capsule collagen density, an increased foreign body capsule elasticity, reduced a-SMA expression in the foreign body capsule, and increased SPP1 expression in myeloid cells of the foreign body capsule compared to a control.

[0090] SUBJECTS

[0091] Embodiments of the methods of the present invention can be practiced on any suitable subject. A subject of the present invention may be a “mammal” or “mammalian”, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subjects are humans. The methods may be applied to human subjects of both genders and at any stage of development ( / .e., neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent or adult.

[0092] DEVICES

[0093] The present disclosure provides foreign bodies coated with a SPP1 active agent composition. In some cases, the foreign body is an implantable device coated with a SPP1 active agent composition. Implantable devices include, without limitation, implants for reconstructive and / or cosmetic surgery (e.g., breast implants, calf implants, gastric implants, gluteal implants, penile implants, testicular implants), prostheses, tissue-expanders, surgical mesh implants, electro-stimulatory implants (e.g., pacemakers and neuromodulator implants), biosensors, drugdelivery ports, catheters, orthopedic implants, and vascular and non-vascular stents, among others.

[0094] The SPP1 active agent composition may be any SPP1 active agent composition of the present disclosure. In some embodiments, the SPP1 active agent composition is a controlled release composition. In some embodiments, the controlled release composition may release an effective amount of a SPP1 active agent over any suitable period of time including e.g., one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 28 days or more, or 30 days or more. In some embodiments, the controlled release composition may retain an amount of SPP1 active agent with a half-life from about 8 days to about 32 days, e.g., from about 8 to 9 days, 10 to 11 days, 12 to 13 days, 14 to 16 days, 17 to 18 days, 19 to 20 days, 21 to 22 days, 23 to 24 days, 25 to 26 days, 27 to 28 days, 29 to 30 days, or 31 to 32 days.

[0095] In some embodiments, the SPP1 active agent composition comprises a gel. A gel of the present disclosure may be formed from cross-linking of one or more precursors. The gel precursors may be polymeric or non-polymeric. Crosslinks may be formed by covalent bonds or non-covalent interactions. Examples of non-covalent bonds include ionic bonds or hydrophobic interactions. In some cases, the gel is a hydrogel (i.e., the interstitial fluid comprising the gel is water). In some embodiments, a hydrogel of the present disclosure comprises a polymer. In some cases, the polymer may be synthetic. In some cases, the polymer may be naturally occurring. In some embodiments, the polymer comprises a polysaccharide. Examples of suitable polysaccharides include, without limitation, hyaluronic acid (HA), alginate, chitosan, dextran, cellulose, and natural or synthetic derivatives thereof. In some cases, the polysaccharide is hydroxypropyl methylcellulose. In some embodiments, a hydrogel of the present disclosure comprises a nanoparticle. In some embodiments, the nanoparticles comprise a polymer. Examples of suitable polymeric nanoparticles include, without limitation, poly(amidoamine), PEGylated poly(amidoamine), polyethylene glycol) methyl ether methacrylate, polypeptides (e.g., albumin), polysaccharides (e.g., chitosan), poly(lactic acid), poly(lactic-co-glycolic acid), and poly(ethylene glycol) (i.e., PEG). In some embodiments, the polymer is a block copolymer. A block copolymer is a polymer comprised of two or more homopolymer subunits (i.e., “blocks”). In some embodiments, the block copolymer is a diblock copolymer (i.e., comprised of two distinct “blocks”). In some cases, the diblock copolymer comprises polyethylene glycol) and poly (lactic acid).

[0096] All or a part of the foreign body (e.g., an implantable device) may be coated with the SPP1 active agent composition, e.g., a controlled release composition. In particular embodiments, all or a part of the surface of the foreign body may be coated with the SPP1 active agent composition. By “surface” it is meant any part or area of the foreign body that, without a coating, would otherwise be in contact with the surrounding tissue when the foreign body is implanted in a subject. In some cases, the percent of the surface of the foreign body coated with a SPP1 active agent composition can range from, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the surface area of the foreign body coated with a SPP1 active agent composition. In some cases, the thickness (e.g., the average thickness) of the SPP1 active agent composition coating the foreign body may range from about 1 pm to about 100 pm, e.g., from about 5 pm to about 95 pm, from about 10 pm to about 90 pm, from about 15 pm to about 85 pm, from about 20 pm to about 80 pm, from about 25 pm to about 75 pm from about 30 pm to about 70 pm, from about 35 pm to about 65 pm, from about 40 pm to about 60 pm, or from about 45 pm to 55 pm across the surface of the foreign body. In some cases, the thickness (e.g., the average thickness) of the SPP1 active agent composition coating the foreign body may range from about 0.1 mm to about 1 mm, e.g., from about 0.2 mm to 0.9mm, from about 0.3 mm to about 0.8 mm, and from about 0.4mm to about 0.6 mm. In some cases, the thickness of the coating across the surface of the foreign body may vary from about 50% to about 150% of the average thickness of the coating, e.g., from about 75% to about 125%, from about 80% to about 120%, from about 90% to 110%, or from about 95% to about 105%. The percent area of the foreign body to coated and the thickness of the coating may vary depending on the size and nature of the foreign body and can be readily determined by the person of skill using the common knowledge in the art. The percent area of the foreign body to coated and the thickness of the coating may vary depending on the size and nature of the foreign body and can be readily determined by the person of skill using the common knowledge in the art.

[0097] A foreign body of the present disclosure may be coated with an SPP1 active agent composition, or a precursor thereof, using any suitable method known in the art. A precursor of an SPP1 active agent composition includes compositions wherein the SPP1 active agent carried by a precursor vehicle that may undergo a physical or chemical transformation to arrive at the final vehicle compound (e.g., a gel precursor which can polymerize into a gel upon mixing with one or more additional gel precursors and / or addition of a physical or chemical stimulus). The method for coating a foreign body with an SPP1 active agent may vary depending on the size and shape of the foreign body. In some cases, the SPP1 active agent composition may be applied using drop-coating methods. In drop coating methods, a defined volume of the SPP1 active agent composition is applied to the surface of the foreign body (e.g., with a pipette) and the composition is allowed to adhere, dry, and / or polymerize on the surface (e.g., in the cases where the SPP1 active agent composition comprises a hydrogel). Drop-coating methods may be used to, e.g., coat defined areas of a foreign body. In some cases, the SPP1 active agent composition may be applied using knife-coating methods, also known as spread-coating, bar-coating, or blade-coating methods. In some knife-coating methods, a fixed knife (or blade, or other spreader) is fixed and the substrate to be coated is moved underneath at a defined distance. In other cases, the substrate to be coated is fixed and the knife (or blade, or other spreader) is moved over the substrate to be coated at a defined distance. In either case, the knife (or blade, or other spreader) can spread the SPP1 active agent composition over the substrate to be coated with a defined thickness set by the distance between the substrate and the knife. The SPP1 active agent composition is then allowed to adhere, dry, and / or polymerize on the surface (e.g., in the cases where the SPP1 active agent composition comprises a hydrogel). Knife-coating may be used, e.g., when the foreign body has a flexible surface without a defined surface pattern. In some cases, the SPP1 active agent composition may be applied using spray-coating methods. In spraycoating methods, the SPP1 active agent composition, or precursor thereof, is aerosolized or atomized through a suitable spray nozzle by pressurized air or gas (e.g., nitrogen or argon) and then transferred via the atomized spray onto the object to be coated. Once transferred to the surface of the object, the SPP1 active agent composition is allowed to adhere, dry, and / or polymerize on the surface (e.g., in the cases where the SPP1 active agent composition comprises a hydrogel). Spray-coating methods are contactless and may be useful, e.g., for sensitive substrate surfaces and / or substrate surfaces with complex shapes. In some cases, the SPP1 active agent composition may be applied using dip-coating methods. In dip-coating methods, the object to be coated is immersed, partially or completely, in the SPP1 active agent composition, or precursor thereof, for a set amount of time and then withdrawn to allow the SPP1 active agent composition to adhere, dry, and / or polymerize on the surface (e.g., in the cases where the SPP1 active agent composition comprises a hydrogel). Dip-coating methods may be used, e.g., for depositing layers 1 pm to 100 pm thick and / or for substrate surfaces with complex shapes. In some cases, the SPP1 active agent composition may be applied using spin-coating methods. In spin-coating methods, uniform thin layers of a SPP1 active agent composition, or precursor thereof, are spread across a substrate using centrifugal forces, after which the SPP1 active agent composition is adhere, dry, and / or polymerize on the surface (e.g., in the cases where the SPP1 active agent composition comprises a hydrogel). Spin coating may be useful, e.g., for applying coatings to flat surfaces.

[0098] The various suitable deposition methods are known in the art and described, e.g., in Bauer, Meike, Axel Duerkop, and Antje J. Baeumner. "Critical review of polymer and hydrogel deposition methods for optical and electrochemical bioanalytical sensors correlated to the sensor’s applicability in real samples." Analytical and Bioanalytical Chemistry 415.1 (2023): 83- 95; Fu Y, Dudley EG. Antimicrobial-coated films as food packaging: a review. Compr Rev Food Sci Food Saf. 2021 doi: 10.11 11 / 1541 -4337.12769; Park J, Shin K, Lee C. Roll-to-roll coating technology and its applications: a review. Int J Precis Eng Manuf. 2016 doi: 10.1007 / s12541 -016- 0067-z; Mavukkandy MO, McBride SA, Warsinger DM, Dizge N, Hasan SW, Arafat HA. Thin film deposition techniques for polymeric membranes- a review. J Membr Sci. 2020 doi: 10.1016 / j.memsci.2020.118258; and Ceratti DR, Louis B, Paquez X, Faustini M, Grosso D. A new dip coating method to obtain large-surface coatings with a minimum of solution. Adv Mater. 2015 doi: 10.1002 / adma.201502518, of which the entirety of each is incorporated by reference herein.

[0099] KITS

[0100] The present disclosure provides kits comprising an implantable device and a SPP1 active agent composition, e.g., as described above. Implantable devices include, without limitation, implants for reconstructive and / or cosmetic surgery (e.g., breast implants, calf implants, gastric implants, gluteal implants, penile implants, testicular implants), prostheses, tissue-expanders, surgical mesh implants, electro-stimulatory implants (e.g., pacemakers and neuromodulator implants), biosensors, drug-delivery ports, catheters, orthopedic implants, and vascular and non- vascular stents, among others. The SPP1 active agent composition may be any SPP1 active agent composition of the present disclosure.

[0101] In some embodiments, the kit may include an implantable device pre-coated with an SPP1 active agent composition. In some embodiments, the kit may include the SPP1 active agent composition separate from the implantable device. In some cases, the kit may include one or more SPP1 active agent composition precursor(s), in individual containers, each separate from the implantable device. For example, the kit may include an SPP1 active agent with an associated pharmaceutically acceptable vehicle or carrier in a first container, and one or more additional vehicle precursors (e.g., gel precursors which can polymerize into a gel upon mixing with one or more addition gel precursors) in one or more additional containers. Each of these SPP1 active agent composition precursors may then be combined to arrive at an SPP1 active agent composition. In certain embodiments, the SPP1 active agent composition, or precursors thereof, may be in liquid form or may be lyophilized. In some cases, some of the SPP1 active agent composition precursors may be in liquid form while others are lyophilized.

[0102] In some cases, the size of the container may depend on the volume of the SPP1 active agent composition, or composition precursor, to be held in the container. In certain embodiments, the container may be configured to hold an amount of an SPP1 active agent composition, or precursor thereof, ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to 1 mg, or 0.1 mg to 0.5 mg. In some cases, the container is configured to hold an amount of an SPP1 active agent composition, or precursor thereof, ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In some instances, the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml. In some cases, the container is configured to hold a volume (e.g., a volume of a liquid SPP1 active agent composition, or precursor thereof) ranging from 0.1 ml to 200 ml.

[0103] Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of suitable materials, including glass or plastic. For example, the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX™), fused quartz glass, fused silica glass, and the like. Other examples of suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.

[0104] In some embodiments, the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container. The seal of the container may also substantially prevent other substances from entering the container. For example, the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container. In some instances, the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container. In some instances, the seal is made of a resilient material to provide a barrier (e.g., a water-tight and / or air-tight seal) for retaining a sample in the container. Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container. Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). For example, in certain embodiments, the seal is a septum pierceable by a needle, syringe, or cannula. The seal may also provide convenient access to a sample in the container, as well as a protective barrier that overlies the opening of the container. In some instances, the seal is a removable seal, such as a threaded or snap-on cap or other suitable sealing element that can be applied to the opening of the container. For instance, a threaded cap can be screwed over the opening before or after a sample has been added to the container.

[0105] In addition to the above components, the subject kits may further include, in some embodiments, instructions for practicing one or more of the subject methods described herein. For example, a subject kit may include instructions for preparing a SPP1 active agent composition (e.g., a SPP1 active agent composition comprising a SPP1 active agent and a hydrogel) from one or more composition precursors (e.g., a SPP1 active agent and one or more gel precursors). Alternatively, or in addition to instructions for preparing an a SPP1 active agent composition, the subject kit may include instructions for applying the SPP1 active agent composition to the implantable device of the kit (e.g., instructions for coating all or a part of the device with the SPP1 active agent composition). One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site. The following examples are provided by way of illustrations and not by way of limitation.

[0106] EXAMPLES

[0107] A Summary

[0108] Implantation of biomaterials is limited by foreign body response (FBR). Mechanisms governing FBR are poorly understood, though acellular dermal matrix (ADM) has been observed to attenuate this chronic fibrosis. Using implant-based breast reconstruction as a model, paired patient foreign body capsule specimens treated with ADM were subjected to single-cell RNA sequencing (scRNA-seq) and spatial proteomics. Osteopontin-1 (SPP1) was identified as atarget given high levels of SPP1 and attenuated FBR associated with ADM use in patient-derived histologic and scRNA-seq data. Further investigation of ADM in a mouse model of FBR validated these findings. It was shown that Spp1 expression is necessary to decrease FBR by using Spp1- knockout mice to establish that Spp1 loss prevented ADM’s beneficial effects on FBR. Finally, it was demonstrated that sustained release of recombinant SPP1 using a novel hydrogel system is sufficient to mimic attenuated FBR seen with ADM, facilitating therapeutic translation for a globally applicable biological problem.

[0109] B Introduction

[0110] The use of implantable biomedical devices increases exponentially each year, propelled by a combination of ever-advancing technological innovation and the clinical demands of an expanding senior population. The implantable devices market was valued at approximately $120 billion dollars worldwide in 2021 , and is estimated to rise to $168.3 billion dollars within the next four years. Despite their significant potential for improving health and quality of life, all biological implants are limited by foreign body response (FBR), an immune-mediated reaction involving chronic inflammation, foreign body giant cell formation, and ultimately fibrous encapsulation of foreign material. Pathologic FBR can lead to implant malfunction, superimposed infection, soft tissue disfigurement, and may ultimately necessitate reoperation procedures and material explant. While FBR is an expected inflammatory response to foreign material, the mechanisms underlying progressive chronic inflammation and capsule formation were poorly understood. Acellular dermal matrix (ADM), a decellularized tissue with an intact extracellular matrix (ECM), is thought to attenuate FBR by providing a biomimetic scaffold for native tissue incorporation. Rigorous molecular characterization of this phenomenon presents an opportunity to not only revolutionize prosthetic-based medicine, but also highjack a conventionally inevitable biologic process.

[0111] In this study, a mechanistic approach to investigating FBR was undertaken in the context of ADM use. Implant-based breast reconstruction is used as a model for understanding how ADM modulates FBR. Through analysis of paired patient-derived capsule specimens harvested during breast reconstruction, ADM was identified as biologically active with significant differences seen in capsule architecture, microenvironment protein signaling, and transcriptional activity in capsule specimens exposed to ADM ("ADM capsule”) versus Native capsule not exposed to ADM (“Native capsule”). Using multi-omic studies, it was demonstrated that ADM promotes an attenuated FBR associated with activation of SPP1+ myeloid cells. To further interrogate these findings, an analogous mouse model of FBR was developed, treated with and without ADM. Transcriptional analysis of mouse capsules validated findings of attenuated FBR in the presence of ADM, associated with Spp1+ overexpression in monocytes. Building on these findings, genetic manipulation of Spp1 using transgenic Spp1 knockout mice showed that Spp1 expression was necessary for FBR attenuation in the presence of ADM. Finally, to translate these findings, a novel hydrogel packaged for sustained release of recombinant SPP1 was applied to the mouse model of FBR. Treatment as such was sufficient for rescue of the ADM capsule phenotype in the absence of ADM, demonstrating that Spp1+ overexpression, activated by the presence of ADM, is a key mechanism for attenuating FBR.

[0112] C Results

[0113] ADM alters foreign body capsule phenotype and decreases FBR

[0114] Capsule specimens were collected from patients undergoing expander-based breast reconstruction. During this two-stage procedure, patients first receive a tissue expander and then underwent exchange of the expander with a permanent implant following tissue expansion. Expanders were partially wrapped in ADM at the time of placement. As a result, at the time of expander-implant exchange, patients had both capsule that developed adjacent to the expander (“Native capsule”) and capsule adjacent to ADM (“ADM capsule”). These capsule specimens were retrieved from the same expander, allowing for patient-matched capsule specimens of both native and ADM capsule. Paired capsule specimens (Native capsule and ADM capsule) were obtained from 13 patients, with a mean age of 51 .5 years (SD 11 .2, range 35-68) and mean time to implant exchange of 7 months (SD 3.9, range 4-15, Table 1). Capsule specimens were sampled at a minimum distance of 2 cm distal from Native and ADM capsule overlap to avoid Native and ADM capsule overlap, and minimize the possibility of introducing tissue overlap as a confounding variable (Fig. 1A), and subsequently processed for histology and a multiplexed immunoassay (Luminex). Importantly, capsules were taken consistently from the posterior side of the expander pocket to minimize differences in FBR capsule due to variability in overlying tissue composition and resultant mechanical force disparities. This region was also chosen to protect against any confounders from mastectomy technique. By always collecting capsule consistently against the pectoralis major muscle, it was ensured that perfusion remained consistent. Thus, these patient- matched capsules represent a rigorous comparative model for intraindividual, intraimplant FBR. Clinically, the use of ADM for the purposes of a decreased FBR is rarely applied on its own without an implantable device. This model therefore reflects what is observed in patients. Collected samples were processed for histology, a multiplexed immunoassay (Luminex), and scRNA-seq. Gross photos from specimen harvest are shown in Fig. 7A. At baseline, ADM is composed of elastin and collagen following the decellularization protocol. Hence, histological comparisons between ADM and Native capsule were normalized to ADM alone, as ADM was not dissected off the underlying tissue. On hematoxylin and eosin (H&E) histological analysis, Native capsule demonstrated denser fibrosis compared to ADM capsules (Fig. 1 B). These observations were most pronounced at the most superficial aspect (top) of the capsule specimen (Fig. 1 B). In comparison, ADM capsule showed a looser, more porous fibrosis phenotype at the most superficial aspect (top), with connective tissue integrated into the ADM layer (Fig. 1 B). H&E staining of ADM alone is shown in (Fig. 7B). This finding was supported by Masson’s trichrome staining of specimens, which confirmed significantly increased mean normalized collagen density in Native compared to ADM capsules (Fig. 1 C, 1.4 vs. 1.8, *P < 0.05). Normalized collagen density represents a ratio that was obtained by staining ADM alone with Masson’s trichrome stain (Fig. 7C), calculating the mean percentage area of collagen density for ADM alone, and dividing each individual value obtained for ADM capsule and Native capsule percentage area of collagen density by this mean. Additionally, though collagen density among ADM and Native capsules was significantly different (unpaired T-test, *P < 0.05), these data did not appear sufficient to explain the previously reported clinical findings. As such, additional features of fibrosis were explored, including elastin staining and ECM ultrastructure. Elastin staining of the Native and ADM capsules was performed to interrogate the presence of additional ECM proteins. Interestingly, there was a significant increase in normalized mean elastin density found in ADM capsules relative to Native capsules (Fig. 1 D, 2.0 vs. 0.10, **** P < 0.0001 ). Quantitative analysis of ECM ultrastructure revealed that the ECM of Native and ADM capsule exhibit notable differences. Uniform manifold approximation and projection (UMAP) mapping of 294 quantified ECM ultrastructure parameters illustrated considerable divergence between ADM capsule and Native capsule ultrastructures (Fig. 1 D). It has previously been observed that elastin participates in wound healing by providing mechanical elasticity, decreasing wound contracture, and ultimately assisting in improved regeneration of the dermis. Notably, elastin is known to confer elasticity to ECM, thus indicating that ADM capsules are more elastic than Native capsules. This increased elastin expression may provide insights into the reduced rates of pathologic capsular contracture with ADM application. These data further support the conclusion that application of ADM results in an altered fibrotic phenotype of the foreign body response. Quantitative analysis of ECM ultrastructure revealed that the ECM of Native and ADM capsule exhibit notable differences. Uniform manifold approximation and projection (UMAP) mapping of 294 quantified ECM ultrastructure parameters illustrated considerable divergence between ADM capsule and Native capsule ultrastructures (Fig. 1 E). Additionally, elastin staining of the Native and ADM capsules supported differences in the ECM ultrastructure with ADM use, with increased normalized mean elastin density found in ADM capsules (Fig. 1 E, 2.0 vs. 0.10, **** P < 0.0001). Normalized elastin density via van Gieson’s staining, similarly to normalized collagen density, represents a ratio obtained by staining ADM alone with van Gieson’s stain (Fig. 7D), calculating the mean pixel density of elastin staining for ADM alone, and dividing each individual value obtained for ADM capsule and Native capsule pixel density by this mean. Thus, ECM deposition and ultrastructure analyses demonstrate that ADM alters multiple features of FBR fibrotic capsules. Given previous work using ultrastructure analysis to evaluate fibrosis in human specimens, these data demonstrate that ECM analysis could be used to predict and quantify the extent of human capsular fibrosis.

[0115] Table 1. Patient Summary Demographics In order to interrogate protein mediators of altered FBR with ADM use, protein isolation was performed from freshly harvested human capsule specimens to assess tissue cytokine content using the Luminex multiplex immunoassay (Fig. 1 F). Multiple cytokines were significantly elevated in ADM capsule (Fig. 7E), with the greatest differences noted in stromal cell-derived factor-1 (SDF-1 ) a and b (mean difference 137.5, ** P< 0.01 ), C-X-C motif chemokine ligand 10 (CXCL10) (mean difference 3114, ** P < 0.01 ), and interleukin 1 -alpha (IL1 -a) (mean difference 15.31 , * P < 0.05), proteins implicated in inflammation, immune cell recruitment, and angiogenesis, respectively. These changes in multiple cellular processes demonstrate that ADM is not only altering the fibrosis seen with capsules, but rather is changing the implant environment on a broader cellular level. Finally, as fibrotic tissues tend to be stiffer, functional mechanical testing of Native vs. ADM capsule was performed. This revealed Native capsules to have a higher Young’s Elastic Modulus, although it was not statistically significant (Fig. 7F). Collectively, these data suggest that the divergence in cellular signaling may lead to the phenotypic differences in capsule formation in ADM and Native capsules.

[0116] Single-Cell Analysis of ADM Capsules Reveals Upregulation of Osteopontin-1

[0117] Given the dissimilarities observed at a histologic and chemokine level in ADM and Native capsules, it was sought to explore the molecular cell signaling mechanisms underlying the phenotypic differences between Native and ADM capsules. Patient-matched human (paired) Native and ADM-adjacent capsule tissue specimens were thus subjected to scRNA-seq. (Fig. 2A). The scRNA-seq analysis demonstrated significant heterogeneity across all cells (Fig. 2B). Sub-analysis confirmed gene expression from 13 unique individuals and relative overlap between the two experimental conditions (Fig. 8A-B). Further analysis centered on fibroblast and myeloid cells (macrophages and monocytes), as these cell types predominate in FBR.

[0118] Louvain-based (Seurat) clustering identified 7 transcriptionally distinct fibroblast Seurat subclusters, with differences in contribution between Native and ADM capsules (Fig. 8C-D). Exploring fibroblast subpopulations, two clusters with the greatest differential prevalence between ADM and Native capsules were cluster 0 (28.91% ADM vs 17.80% Native) and cluster 3 (7.03% ADM vs 19.36% Native) (Fig. 18A). However, deeper analyses via Enrichr illustrated that both subclusters exhibited overlapping pathways and terms, including ‘collagen organization’, ‘ECM- receptor interactions’, and ‘collagen-containing ECM’ (Fig. 18B). Given that few changes were observed among fibroblast subpopulations, attention was shifted to other cell types involved in FBR: macrophages and monocytes. As it has been previously demonstrated that myeloid subpopulations play unique roles in fibrosis, it was sought to interrogate the subpopulations of monocytes and macrophages within Native and ADM capsule. For this analysis of this scRNA- seq dataset, Seurat-identified macrophage and monocyte subclusters were combined into 4 unique myeloid cell subclusters (Fig. 2C). These monocyte / macrophage (myeloid) cell subpopulations revealed differential subclustering between ADM and Native capsules (Fig. 8E). Comparison of monocyte / macrophage (myeloid) Seurat subclusters between ADM and Native capsules was notable for a decreased proportion of cluster 1 (5.50% ADM vs 46.60% Native) and increased proportion of cluster 2 in ADM capsules (43.10% ADM vs 6.80% Native) (Fig. 2D). Investigation of the top differentially expressed genes for each Seurat myeloid subcluster revealed Osteopontin-1 (Spp1) as the most differentially expressed gene in cluster 2 (fold change of 4.19, adjusted p-value 2.72 x 1065) [Fig. 2E, Fig. 2F (top) and Fig. 8F], Spp1 was also the highest differentially expressed gene between ADM and Native capsule monocytes / macrophages (fold change of 3.70, adjusted p-value 6.83 x 1033) (Fig. 2F, bottom) Further interrogation using pathway and Gene Ontology (GO) analysis revealed enrichment for ‘collagen-containing ECM formation’, ‘autophagy regulation’, and ‘foam cell differentiation’ in cluster 2 (Fig. 9A). In comparison, cluster 1 was enriched in Native capsule, and notable for gene expression terms for ‘prostaglandin signaling’ and ‘VEGF signaling’. Cluster 0 and cluster 3 showed upregulation of more inflammatory terms including ‘IL-1 signaling’ and ‘antigen presentation’ (Fig. 9B). Further examination using pathway analysis revealed enrichment for ‘HIF-1 Signaling’ in cluster 2 cells, a pathway that is known to be associated with tissue protection and adaptation. Interestingly, this cluster also showed enrichment for ‘Lipoprotein particle binding.’ Lipoproteins have previously been shown to have a modulating effect on inflammatory and fibrotic disease, and the presence of this term for cluster 2 is thus demonstrative of a decreased fibrotic signature in ADM capsules (Fig. 19A, right top and bottom rows). In contrast, cluster 1 was enriched in Native capsule, and notable for ‘complement and coagulation cascades,’ and ‘phagosome’ pathways, as well as terms including ‘complement’ and ‘neutrophil degranulation’, all demonstrative of increased inflammatory signature (Fig. 19A, left top and bottom rows). Cluster 0 and cluster 3 showed upregulation of more inflammatory terms including ‘IL-1 signaling’ and ‘antigen presentation’ (Fig. 19B). Further subcluster analysis revealed similar changes to SPP1 expression (Figs. 20A-20B) CellChat, a computational tool to investigate cell-cell communication, showed elevated interactions between diverse cell types in ADM capsules compared to Native capsules (Fig. 10A). Cell-cell signaling pathways also differed in ADM versus Native capsules (Fig. 10A). Interestingly, Wingless and lnt-1 (WNT), neural cell adhesion molecule 1 (NCAM), and bone morphogenetic protein (BMP) signaling communication were enriched in ADM compared to non-canonical WNT (ncWNT) and receptor activator of nuclear factor kappa beta ligand (RANKL) in Native capsules (Fig. 10B). CellChat also suggested enhanced Spp1 signaling in ADM compared to Native capsules, especially between monocytes / macrophages (myeloid cell types) and fibroblasts and endothelial cells (Fig. 2G). Increased SPP1 signaling between macrophages / monocytes, fibroblasts, and endothelial cells is illustrated by the increased thickness in the brown, blue, and indigo vectors in the plot. Immunostaining further revealed significantly greater expression of SPP1 in ADM specimens compared to Native capsules in the Integrin Alpha X (CD1 1C+)- expressing cells (Fig. 2H) (** P < 0.01 ), and decreased expression of collagen 1 (COL1 ) associated with increased SPP1 expression in ADM capsules (Fig.lOC). CD1 1c was selected as a marker for monocytes and macrophages as its expression was specific to those two cell populations in comparison to the pan-myeloid marker CD11b, which was also expressed in some T cells, and fibroblasts (Figs. 21A-21 B). SPP1 is an integrin-binding glycophosphoprotein with multiple roles in bone mineralization and tumorigenesis. In wound healing, loss of function of SPP1 in mice has been shown to delay wound closure and cause defective macrophage infiltration. Given the parallels between fibrotic wound healing and FBR, it was chosen to investigate this gene further.

[0119] During the acute inflammatory phase of FBR, monocytes infiltrate the implant site and, over time, differentiate into macrophages. As the mean time to implant exchange was 7 months, and thus beyond the acute inflammatory phase of FBR, it was hypothesized that the predominant myeloid cells at this stage of FBR and chronic inflammation would be macrophages. Macrophage subset analysis reinforced findings from the myeloid cell analysis, demonstrating 5 Seurat subclusters, with distinct proportional distribution of macrophage Seurat subclusters between Native and ADM capsules (Figs. 11A-11 B). Comparison of macrophage clusters based on experimental condition revealed relatively decreased proportions of Cluster 0 (1.2% ADM vs 47.30% Native) and Cluster 4 (6.10% ADM vs 12.40% from Native) and increased proportions of Cluster 1 in ADM capsule (52.10% ADM vs 7.30% Native) (Fig. 11C). As with monocyte / macrophage (myeloid) analysis findings, SPP1 was relatively overexpressed in ADM capsule vs. Native capsule (Fig. 11 D), and also emerged as a top differentially expressed gene for Cluster 1 (Fig. 11 E). Furthermore, Cluster 1 was enriched in ‘phagosome’, ‘fibroblast growth factor (FGF) signaling’ pathways and ‘glycerophospholipid metabolism’ GO terms (Fig. 12A). Notably, recent literature has shown that lipid deposition profiles on the surface of biomaterials can influence FBR, with anti-fibrotic materials modifying lipid deposition and facilitating evasion of the immune response. In contrast, clusters 0 and 4, more prevalent by population in Native capsules, were enriched for ECM processes including ‘contraction’ and ‘actin cytoskeleton’, as well as immune signaling, including ‘interleukin 17 (IL-17) signaling’ and ‘nuclear factor kappa beta (NF-kappaB)’ (Fig. 12A). Pathways and GO terms for clusters 2 and 3, which were found to be in similar proportions between ADM and Native capsule conditions, are illustrated in Fig. 12B. Collectively, these data suggest that distinct SPP1 + myeloid subpopulations may exist in ADM to reduce FBR.

[0120] Due to this work’s study design, all patients received an ADM wrap; however, as there is a possibility that cells and fluids can move around an implant, which may alter signal diffusion across an implant, pathology capsule specimens from patients undergoing implant-based breast reconstruction without any ADM (“Native -ADM”) were compared to Native capsule specimens (Fig. 22A). Immunostaining demonstrated no significant difference in CD11c and SPP1 coexpression (P = 0.7830) (Fig. 22B). These data provide support in the validity of this work’s study design.

[0121] Spatial phenotyping by CO-Detection by indexing (CODEX) demonstrates that cell-cell crosstalk differs in ADM and Native Capsules

[0122] Though CellChat alongside scRNA-seq analysis and immunostaining support findings that SPP1 upregulation was observed in ADM capsule, cell-cell signaling analyses via CellChat are inference based. Thus, building upon the scRNA-seq analyses described above, the spatial cellcell profiles of ADM and Native capsules were examined. CO-Detection by indEXing (CODEX) was utilized, an assay in which a panel of at least 37 individual protein markers are sequentially labeled and iteratively imaged via cyclic additions and washouts of dye-labeled oligonucleotide- conjugated antibodies (Figs. 3A-3C). This technique allows for high-throughput, multiplexed, single-cell imaging to visualize the in situ expression of cellular markers and associated cellular populations, while maintaining spatial tissue architecture. Thus, CODEX permits building upon the scRNA-seq analyses described above, to also explore changes to the spatial organization of proteins and examine the spatial cell-cell profiles of ADM and Native capsules. Across all treatment groups, 14 cell clusters were based on CODEX protein expression signatures including cluster of differentiation antigen 4 (CD4) T cells, cluster of differentiation antigen 8 (CD8) T cells, two endothelial clusters, five fibroblast clusters, three myeloid clusters, and two smooth muscle cell clusters (Figs. 3B, 3D). Myeloid cells were defined based on marker expression of CD45, CD68, and CD1 1c. SPP1 expression was greater in ADM compared to Native capsules, while COL1 expression was greater in Native compared to ADM capsules (Fig. 13A). These results parallel the histologic and scRNA-seq data, wherein increased collagen was associated with increased fibrosis in Native capsule, while increased SPP1 was demonstrative of the less fibrotic phenotype in ADM capsule. Given the observations made in monocytes and macrophage subclusters in the scRNA-seq dataset, differential protein expression among myeloid subpopulations in ADM and Native capsule was homed in on, which was spatially resolved at a single-cell level using CODEX. The proportions of three myeloid CODEX-defined clusters differed in ADM and Native capsules. ADM capsules had greater numbers of Myeloid 1 and 3 (Fig. 3E, top and bottom row), while Native had greater numbers of Myeloid 2 clusters (Fig. 3E, middle row, * P < 0.05). The cross talk between Myeloid 1 and 3 was also significantly greater in ADM versus Native capsules (* P < 0.05) (Fig. 13B). Thus, paralleling what was observed at an RNA level, myeloid cells from ADM and Native capsules differed in their protein expression as well.

[0123] Differential interaction maps were then used to visualize the strength of spatial cell-cell interactions based on K-nearest-neighbor localization, which can be uniquely assessed using CODEX and other spatial phenotyping technologies. Cell segmentation was performed so that cell-level analyses were conducted based on protein expression within the boundaries of cells. These spatial cell-cell communications were found to differ in ADM and Native capsules (Fig. 3F and Fig. 13C). Interestingly, CODEX-defined Myeloid 2 showed strong interactions with smooth muscle cells in Native capsules (Fig. 3F and Fig. 13D, top left and bottom left, * P < 0.05). As smooth muscle cells are associated with contracture, additional crosstalk between immune cells, the acute stage of FBR, and these myeloid cells, is demonstrative of the increased contractile nature of fibrotic tissue from native capsule and increased fibrosis. In contrast, CODEX-defined Myeloid 1 and 3 showed strong interactions with CD4 cells and fibroblasts clusters (Fig. 3F and Fig. 13D, top right and bottom right, * P < 0.05).

[0124] Given the importance of SPP1 in the scRNA-seq data, the protein expression of SPP1 in the three CD11 C+-expressing myeloid clusters was analyzed. Myeloid 1 and 3 showed higher levels of SPP1 expression compared to Myeloid cluster 2 (Fig. 3G). Yes-associated protein-1 (YAP) is known to be associated with fibrosis in several organs. For example, YAP signaling has been shown to play a key role in pro-fibrotic fibroblasts during wound healing. YAP-mediated mechanotransduction signaling has also been implicated with inflammation and stiffness sensing in macrophages during FBR. Interestingly Myeloid cluster 2 was found to spatially express high levels of YAP. Given the established role of YAP in increased inflammation and fibrosis, elevated YAP expression in Myeloid cluster 2 may be responsible for the fibrotic phenotype in Native capsules. (Fig. 13E-F). Furthermore, smooth muscle cell clusters also demonstrated high levels of YAP1 , which were found to display high cross talk with Myeloid cluster 2 in Native capsules compared to ADM capsules (Fig. 13E). Collectively, these data indicated that the spatial cell proteomic expression of ADM and Native capsules is distinct and that spatially SPP1 - expressing myeloid cells could be identified.

[0125] ADM decreases foreign body response in a mouse model

[0126] To validate these novel findings from the patient-derived samples, an in vivo model that allowed modulation of SPP1 in ADM and FBR was sought. A mouse dorsal implant model was adapted in order to interrogate the role of Spp1 in FBR in vivo. C57BL / 6J mice underwent dorsal implantation with sterilized 0.2 mL PDMS implants, prepared as previously detailed. These implants were either placed subcutaneously without ADM on the mouse dorsum, referred to as “mNative capsule” (n=6), or covered with ADM, referred to as “mADM capsule” (n=6). Mice were divided into different experimental groups, each receiving one implant. As previously reported, a foreign body capsule forms, encasing the implant by 21 days after surgery. It has previously been shown that implant stiffness can affect the extent of fibrosis observed following implantation, where greater difference in stiffness between the implant and interface tissue causes a more extensive fibrotic response. T o establish whether there were differences in stiffness between ADM and the PDMS implants, tensile testing was conducted. No significant difference in material elastic modulus was observed between ADM and PDMS (P = 0.2455) (Fig. 14B). For these studies, it was sought to characterize foreign body capsule formation 1 month after surgery (POD 28). On POD 28, capsules were harvested and processed for histology (Fig. 4A). Gross images of implant placement and dissected capsule are shown in Fig. 14A. Histologic analyses revealed analogous findings to human Native and ADM capsules by H&E and Masson’s trichrome staining (Fig. 4B- C). On H&E, mNative capsule was significantly thicker than mADM capsule, with mean thickness of mNative capsules calculated to be 42.1 pm (SD 10.3) compared to a mean thickness of 25.9 pm (SD 3.5) in mADM capsules (**** P <0.0001 , Fig. 4B). Additionally, Masson’s trichrome staining revealed significantly increased collagen density measured by mean area of collagen staining in mNative capsules (84.7%, SD 7.2), compared to mADM capsules (65.2%, SD 6.3, **** P <0.0001 , Fig. 4C). In order to quantify fibrotic patterns between capsule conditions, 294 ECM ultrastructure parameters were measured from Picrosirius red-stained images of mNative and mADM capsules, again demonstrating divergent fibrotic phenotypes between experimental conditions (Fig. 4D), as observed in the human data. Importantly, immunostaining of capsule specimens demonstrated significantly increased alpha smooth muscle actin (a-SMA), a well- known marker of fibrosis with an established presence in FBR capsule, in mNative capsules compared to mADM capsules, mirroring patient-derived capsule phenotypes (Fig. 4E, ** P < 0.01). To control for potential crosstalk between PDMS and ADM, a separate cohort of mice underwent dorsal implantation with ADM alone (“ADM alone”). Histologic analyses confirmed no significant difference in capsule thickness or collagen deposition when compared to the mADM capsule condition (P = 0.8434) (Figs. 23A-23C). Immunostaining also revealed no significant different in aSMA expression in ADM alone relative to mADM (P = 0.320) (Fig. 23D).

[0127] Altogether, basic histology and immunostaining data collectively support that the mouse implant FBR model recapitulated the phenotypic characteristics of the patient-derived Native and ADM foreign body capsules, with attenuated fibrosis observed in the presence of ADM.

[0128] Increased expression ofSPPI (Osteopontin-1) in monocyte subclusters of mADM capsule

[0129] To explore whether application of ADM in the mouse FBR model would lead to similar transcriptional changes to those observed in the human capsules, mNative and mADM capsules were subjected to scRNA-seq at POD 28 (Fig. 5A). Similar to human samples, there was cell heterogeneity across all cells in mouse capsules (Fig. 5B), with fibroblasts and myeloid cells encompassing a large proportion of all cells. Examination of fibroblasts revealed 5 transcriptionally unique Seurat-based subclusters, with relative overlap between mNative and mADM conditions, analogous to the human dataset (Figs. 15A-15B). Two clusters with the greatest differential prevalence between mADM and mNative capsules were clusters 0 (42.98% mADM vs 34.19% mNative) and 1 (16.44% mADM vs 25.59% mNative) (Fig. 24A); however, as with the human fibroblast clusters, Enrichr analysis highlighted that both of these clusters exhibited overlapping pathways and terms, including complement and coagulation cascades, ECM organization, and collagen deposition (Fig. 24B). Thus, as with the human dataset, attention was shifted to macrophages and monocytes. As high cell counts were in both monocytes and macrophages, these cell populations separately were analyzed separately. On downstream analysis, although macrophages from mADM capsules demonstrated a slight increase in SPP1 expression compared to mNative macrophages (24C), more striking differences in subcluster analysis were observed among monocytes, and thus focused the analysis was focused on the latter cell type. Once monocyte populations were isolated, 7 transcriptionally unique Seurat subclusters were identified (Fig. 5C) with partial overlap when compared by condition (Fig. 15C).

[0130] Monocytes derived from mADM specimens showed relatively decreased proportions of subclusters 2 (12.26% mADM vs 22.85% mNative) and 3 (1 1.00% mADM vs 17.61% mNative) and an increased proportion of subcluster 4 compared to monocytes derived from mNative specimens (21.56% mADM vs 4.19% mNative) (Fig. 5D). The top differentially expressed genes from each subcluster are illustrated in Fig. 5E and Fig. 15D, with subclusters 2 and 3 enriched for genes involved in inflammation, including ‘apoptosis’, ‘oxidative stress-related signaling’ pathways, and ‘chemokine / cytokine’ response GO terms. Inflammation is a key trigger for fibrosis, and plays a crucial role in the acute and chronic stages of FBR. These clusters were also elevated in genes associated with mechanotransduction signaling pathways, such as focal adhesion kinase (Fig. 15E). Mechanotransduction signaling is known to be a driver of fibrosis and FBR. Focal adhesion kinase signaling has specifically been implicated in macrophage integrin binding to material surfaces. Formation of focal adhesions promotes fibroblast activation and encourages the development of contractile forces. Contraction and stiff ECM deposition can further activate integrins, ultimately leading to activation of TGF-beta and therefore driving pro-fibrotic programs. These pathways and terms are therefore evidence of pro-inflammatory, fibrotic function. These observations are in keeping with the inflammatory signature of human monocyte / macrophage cluster 1 (Fig. 19A), which was enriched in Native capsule. Cluster 4 from mouse monocytes was enriched for genes involved in ECM-related GO terms, as well as with ‘HIF-1 Signaling’ and ‘Vascular Endothelial Growth Factor (VEGF) A-VEGFR2 Signaling’. As stated earlier, HIF-1 signaling is known to be associated with tissue protection and adaptation. Meanwhile, VEGF signaling is known to play a role in angiogenesis and tissue regeneration. Notably, fibrosis often leads to dysfunctional and decreased vasculature, thus upregulation of this transcriptional profile is evidence of a pro-regenerative phenotype for this cluster. These data mirror those from human monocyte / macrophage cluster 2, which was enriched in ADM capsules and were evidence of a decreased fibrotic signature in ADM. The remaining subclusters showed relatively similar proportions between mADM and mNative conditions, with respective pathways and GO terms illustrated in Fig. 16. Interrogation of Spp1 expression revealed subcluster 4 to be one of 3 subclusters expressing this gene (Fig. 5F, left). Spp1 was found to be associated with ‘IL-18 signaling’, ‘toll-like receptor regulation’, ‘FGF signaling’, ‘osteopontin signaling’, and ‘focal adhesion signaling’ pathways upon EnrichR analysis. Spp1 expression exhibited increased expression in mADM specimens relative to mNative specimens, reflecting findings from the human data (Fig. 5F, right). Increased Spp1 signaling was also noted between fibroblasts and myeloid cells on CellChat in ADM specimens (Fig. 5G), paralleling cell-cell interaction inference observed in the human scRNA-seq dataset. Finally, immunostaining showed significantly enhanced SPP1 and CD11 C expression (Fig. 5H, *** P <0.001 ), and relatively decreased COL1 expression (Fig. 15F) in mADM capsules compared to mNative capsules, supporting the data from patient-derived capsules. Analogous to the approach with the human dataset, CD11c was selected as a marker for monocytes as its expression was specific to this cell population in comparison to the pan-myeloid marker CD11 b, which also showed more elevated expression in granulocytes, T cells and macrophages (Figs. 25B-25C).

[0131] To determine the similarity between the human and mouse datasets, an anchor-based label transfer approach was applied in order to project the mouse monocytes (Fig. 5) onto the human monocytes / macrophages (Fig. 2). Analysis supported a strong correlation between human monocyte / macrophage cluster 2 with high SPP1 expression, and mouse monocyte clusters 2, 3, and 4, which also had elevated SPP1 expression and pro-fibrotic gene ontology analyses (Figs. 26A-26B). These results indicate that these mouse monocyte subclusters share a similar transcriptional program to human monocyte / macrophage cluster 2. Further, they provide support that the mouse model recapitulates human foreign body capsule formation at a transcriptional level. These results in conjunction with similarities between human and mouse histologic data provide further evidence of the validity of the model in providing empirical support for the earlier clinical data.

[0132] Spp1 expression is necessary for attenuation of FBR

[0133] To determine if Spp1 expression is necessary to attenuate FBR, the murine FBR model was applied to Spp1 knockout (KO) mice. Spp1 KO mice exhibit disorganized remodeling of the ECM and defective macrophage infiltration in the context of wound healing, compared to wounds in wild-type mice. Histologically, the KO mice exhibit more cell debris and relatively homogeneous and smaller collagen fibrils at the site of wound healing, but this has been shown to have no effect on the tensile strength of healing incisional wounds. These observations were intriguing, as these attributes closely mirror features seen in FBR progression, and supported the hypothesis that ADM is modulating capsule formation via SPP1. As such, capsule formation would be expected to occur as it would in wild-type mice, though it may appear histologically distinct. FBR capsule formation was compared under 3 conditions in Spp1 KO mice: (1 ) implant only (KO mNative); (2) ADM covering the implant (KO mADM); and (3) implant together with recombinant SPP1 (R&D Systems, Inc., Minneapolis, MN) formulated in a sustained-release hydrogel (KO +SPP1 ); (4) ADM covering implant with hydrogel-delivered recombinant SPP1 (KO mADM +SPP1); and (5) ADM covering implant with a non-drug-loaded empty hydrogel (KO mADM -SPP1 ) (Fig. 6A and 27 A). Given the observations made on human and mouse scRNA-seq datasets, it was aimed to establish how capsule composition changed with ADM in the absence of Spp1, hypothesizing that the capsule would revert to an mNative state. To further confirm that the absence of Spp1 caused the change to capsule composition in this experimental group, the implant together with recombinant SPP1 as well as the implant coated with ADM together with recombinant SPP1 were evaluated in Spp1 KO mice. Finally, the implant coated with ADM together with the non-drug- loaded empty hydrogel acted as a negative control to confirm the gel itself did not alter FBR.

[0134] This polymer-nanoparticle (PNP) hydrogel, developed by Appel and colleagues, is easily injectable and rapidly self-heals, limiting cargo burst release, and has excellent biocompatibility in several species (rodents, sheep, and rabbits). The PNP hydrogel has previously been shown to facilitate slow release of a variety of cargos, including cells, small molecules, and proteins such as the receptor-binding domain of the SARS-CoV-2 spike protein, which is of comparable molecular size to SPP1. In vitro studies of SPP1 -formulated PNP hydrogel confirmed sustained release of SPP1 over the course of a month at physiologic temperature, with a half-life of 16 days (Fig. 17A). As softer materials have been shown to elicit less severe FBR, to avoid introducing a confounding variable with application of the gel, it was specifically injected as a sustained- released drug depot adjacent to the implant.

[0135] On histological analysis, no significant differences in capsule thickness or collagen density comparing KO mNative specimens and KO mADM specimens were observed (Figs. 6B-6C). KO +SPP1 capsules, however, measured a mean of 20.4 pm (SD 4.3), and were significantly thinner compared to KO mNative capsules (mean 47.5 pm, SD 7.9; **** P <0.0001 ) or KO mADM capsules (mean 48.9 SD 12.9; **** P <0.0001 ) (Fig. 6B). Similarly, collagen density, measured as percent area, was significantly lower in KO +SPP1 capsules (mean 54.2%, SD 28.5) compared to KO mNative capsules (mean 98.0%, SD 1.4; *** P < 0.001) or KO mADM capsules (mean 96.8%, SD3.3; *** P< 0.001) (Fig. 6C). llltrastructural analysis of 294 unique ECM features using picrosirius-red stained capsules revealed overlap of KO mNative and KO mADM capsules. Meanwhile, KO +SPP1 capsules were distinct in UMAP space relative to remaining experimental groups (Fig. 17B). Immunostaining showed increased SPP1 expression in CD11 c co-expressing cells (identified as myeloid cells) and decreased fibrotic marker expression (COL1A1 and aSMA) in KO +SPP1 capsules relative to KO mNative and KO mADM capsules (Fig. 6D and 17C, * P < 0.05, P < 0.001).

[0136] Comparing capsule thickness, KO mADM +SPP1 (mean 19.3 pm, SD 3.567) capsules were significantly thinner than KO mADM -SPP1 capsules (mean 47.84 pm, SD 11 .95; **** P < 0.0001 ) (Fig. 27B). Similarly, collagen density was significantly lower in KO mADM +SPP1 capsules (mean 44.85%, SD 19.09) relative to KO mADM -SPP1 capsules (mean 93.92%, SD 3.971 ; **** P < 0.0001 ) (Fig. 27C). KO mADM +SPP1 capsule thickness and collagen density paralleled those of KO +SPP1 capsules (Figs. 6B-6C), while KO +mADM -SPP1 capsule thickness and collagen density paralleled those of KO -SPP1 and KO mADM capsules (Figs. 6B- 6C). Upon Picrosirius red staining, KO mADM +SPP1 displayed similar architectural properties to KO +SPP1 capsule specimens (Fig. 17B, 27D). Immunostaining showed decreased fibrotic marker expression (aSMA and COL1) in KO mADM +SPP1 capsules (Figs. 27E-27F, ** P< 0.01 ), as well as increased SPP1 expression in CD11c co-expressing cells (Fig. 27F).

[0137] T aken together, these data demonstrate that SPP1 expression was necessary for inducing the attenuated fibrotic capsule phenotype normally seen in ADM and mADM capsules in wild-type mice.

[0138] Sustained release of recombinant SPP1 is sufficient to decrease FBR in mice

[0139] Having established that SPP1 was necessary to decrease fibrotic capsule formation in the murine FBR model, it was next investigated whether sustained release of recombinant SPP1 in a wildtype mouse would be sufficient to recapitulate the observed phenotypic change upon ADM application, as genetic knockouts are not clinically translatable. To this end, wild-type C57BL / 6J mice were treated with: (1 ) implant together with empty PNP hydrogel without recombinant SPP1 (mNative -SPP1 ); (2) ADM-covered implant (mADM) as the positive control; or (3) implant together with recombinant SPP1 -loaded PNP hydrogel (mNative +SPP1 ) (Fig. 6E). As before, gel was injected as a sustained-released drug depot adjacent to the implant to avoid changing the stiffness of the implant at the tissue interface.

[0140] Histology revealed significantly decreased capsule thickness and collagen density in mNative +SPP1 capsules relative to mNative -SPP1 capsules. No significant difference in capsule thickness was observed between mNative and mNative -SPP1 groups, indicating that the depot gel alone did not impact FBR (P = 0.2745). Though capsule thickness was greater in mNative +SPP1 capsules compared to mADM capsules, collagen density measurements were comparable between these two groups (Figs. 6F-6G). Mean mNative +SPP1 capsule thickness was 30.9 pm (SD 4.9), compared to 50.0 pm (SD 6.7; **** P< 0.0001 ) in mNative -SPP1 capsules and 26.2 pm (SD 2.7; ** P < 0.01 ) in mADM capsules (Fig. 6F). Mean percent area of collagen density was similarly significantly lower in mADM (29.5%, SD 15.3) and mNative +SPP1 (30.3%, SD 9.9) capsules compared to mNative -SPP1 capsules (75.7%, SD 16.8; **** P <0.0001 and *** P < 0.001 , respectively), and there was no difference between mADM and mNative +SPP1 with respect to collagen density (P = 0.99, Fig. 6G). ECM ultrastructural analysis of 294 unique parameters on picrosirius-red stained capsule specimens revealed overlap of capsule ultrastructure of mADM and mNative +SPP1 capsules, and divergence from mNative -SPP1 capsule (Fig. 17E). Immunostaining confirmed decreased a-SMA and COL1 expression in mNative +SPP1 compared to mADM and mNative -SPP1 capsules (Fig. 6H and Fig. 17D). Immunostaining also showed increased SPP1 expression in CD11c-expressing cells (identified as myeloid cells) in mNative +SPP1 and mADM relative to mNative -SPP1 capsules (Fig. 17D and Fig. 27E). Finally, ECM ultrastructural analysis of 294 unique parameters on picrosirius-red stained capsule specimens revealed overlap of capsule ultrastructure of mADM and mNative +SPP1 capsules, and divergence from mNative -SPP1 capsule (Fig. 17E). Collectively, these data support that SPP1 release could substitute for ADM and is sufficient to restore the fibrotic capsule phenotype in the murine FBR model.

[0141] In order to evaluate whether SPP1 recombinant protein caused any toxicity, SPP1 resuspended in PBS was injected subcutaneously every other day for 28 days in an otherwise healthy, wildtype mouse. As observed on H&E, no visible toxicity was observed with all expected skin architecture and associated appendages present (Fig. 28A). To ensure that the hydrogel on its own did not influence capsule formation, an additional group of wild-type C57BL / 6 mice received implants and injections of SPP1 resuspended in PBS every other day for 28 days (mNative +SPP1 PBS) (Fig. 28B). Relative to the implant alone control, capsule thickness and collagen density in mNative +SPP1 PBS were significantly reduced (mean 33.68pm, SD 5.496; * P < 0.05 and mean 47.78%, SD 12.52; P < 0.0001 , respectively) (Figs. 28C-28D). Immunostaining showed significantly reduced oSMA expression in mNative +SPP1 PBS relative to control group as well (* P < 0.05) (Fig. 28E). These data demonstrate reduced fibrosis as a result of SPP1 , in keeping with the results observed with the sustained released SPP1 -loaded gel.

[0142] SPP1 is well known to interact via two separate pathways. Firstly, it has been shown that SPP1 can bind to integrin av 3 and activate integrin-mediated signaling. Secondly, SPP1 can activate downstream gene expression via CD44. Investigation of expression of Integrin Subunit Alpha V(JTGV5) and Integrin Subunit Beta 3 (ITB3), genes encoding for subunits of integrin av[33, suggested expression of only one of the two subunits and only in Seurat cluster 2 of human monocytes / macrophages (29A). In contrast, CD44 showed expression across all subclusters of human monocytes / macrophages (29B), and was confirmed at a protein level via immunostaining (29C). Interestingly, MMP9, a known direct downstream target of SPP1-CD44 signaling, was expressed at significantly higher levels in ADM capsule monocytes / macrophages relative to Native capsule monocytes / macrophages (log fold change 1 .57, adjusted P-value 6 x 10'10) (29D). Staining via IF confirmed increased protein expression of MMP9 (* P < 0.05) (29E). Thus, these data may suggest a potential mechanism by which SPP1 acts, by binding CD44 and activating its downstream target, MMP9.

[0143] D. Discussion

[0144] Since the biomaterial’s initial use in the early 2000s, ADM’s applications have grown in breadth and frequency, with clinical studies correlating its application with decreased capsular contracture and improved native tissue integration; however, the molecular mechanisms responsible for this biomaterial’s beneficial properties remains elusive. Applying ADM as a case study to understand the mechanisms underlying fibrotic encapsulation, this work evaluated patient-matched specimens from thirteen human patients alongside supportive investigations in murine FBR models. Using a molecular approach, it has been shown via histologic, transcriptomic, and proteomic studies that ADM use leads to an upregulation of SPP1 signaling in monocytes and macrophages (myeloid lineage cells), ultimately decreasing fibrotic encapsulation as a result of FBR. These data were supported with a loss of function experiment wherein ADM-coated implants placed in Spp1 constitutive knock-out mice mimicked Native capsule, supporting that Spp1 expression is necessary for this ADM-associated phenotype. Finally, when a a slow-release SPP1 -loaded hydrogel was added to the wild-type mouse model of FBR, it resulted in reduced fibrotic capsule formation. This reduction was similar to the effects observed with ADM capsules, demonstrating that overexpression of SPP1 alone is enough to mimic the effects of ADM on FBR.

[0145] SPP1 is an integrin-binding glycophosphoprotein that has been shown to play significant roles in cancer, bone homeostasis, and metabolism. In addition, SPP1 has been shown to also bind and mediate signals via CD44 in hair follicle stem cells. Associated with tumor progression, invasion, and metastasis, elevated expression levels of SPP1 have been connected with poor prognosis; yet, how it relates to gene mutation and immune cell infiltration is not well understood.

[0146] A key finding from these investigations show that upregulation of SPP1 leads to a reduction in fibrotic encapsulation following FBR.

[0147] In summary, these findings utilize clinical and mouse models to establish molecular pathways that provide therapeutic targets to overcome FBR. Through a histologic and transcriptomic approach, evidence is provided for a mechanism by which ADM may be altering the implant microenvironment and changing the resulting fibrotic capsule.

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[0261] F Materials and Methods

[0262] Mice:

[0263] Transgenic mouse strains (acquired from Jackson Laboratories): B6 (C57BL / 6J, Stock: 000664), Spp1 (B6.129S6(Cg)-Sppftmf Blh / f, Stock: 004936). Mice were housed at the Stanford University Comparative Medicine Pavilion per Stanford APLAC guidelines, under the supervision of the Veterinary Service Center (VSC). Patient Sample Collection:

[0264] All patients undergoing tissue expander-based breast reconstruction implant exchange by a single plastic surgeon (Dr. Arash Momeni) were informed of the IRB-approved study (n = 10). For those patients who wished to consent for involvement in the study, patient demographics and medical history were recorded in a secure encrypted database. The senior surgeon performed an incomplete ADM wrap of tissue expanders at the time of first stage tissue expander pre-pectoral reconstruction. Thus, capsule specimen can be obtained from (i) the peri-prosthetic capsule that develops adjacent to the expander (hereafter referred to as “native capsule”) but distinct from ADM capsule, and (ii) from the capsule that develops adjacent to the ADM (hereafter referred to as “ADM capsule”). At the time of tissue expander-implant exchange, a 3 x 1 cm area of native and ADM capsules were obtained and divided into three smaller portions for (i) histological analysis, (ii) Fluorescence activated cell sorting (FACS) and transcriptomic analysis, and (iii) proteomic analysis. Intra-operatively, capsule specimens were obtained from the native capsule and the ADM capsule. ADM capsule was removed with ADM attached, as it was well integrated into the surrounding tissues. If the ADM was not incorporated, the patient’s samples were excluded from analysis. An additional three capsule specimens were collected who underwent tissue expander implant exchange without ADM-wrap. Patient demographic information is presented in Table 3.

[0265] Table 3. Patient summary demographics of additional capsule specimens

[0266] Mouse Dorsal Implant Model:

[0267] Acellular dermal matrix (ADM) Preparation

[0268] ADM (Alloderm™ RTM (LifeCell Corporation, Branchburg, NJ) was donated from leftover operating room materials. The ADM was stored in PBS at 4°C for up to 4 weeks in a sterile environment. Prior to implantation in the mouse, the ADM underwent 3 phosphate buffered saline (PBS) washes to remove the associated preservative. Following washing, the ADM was cut into 1 cm x 1 cm squares with sterile surgical scissors.

[0269] Implant Fabrication

[0270] Implants were fabricated in a modified method as previously reported by Lin et. al. Briefly, hemispherical 0.2 mL implants were created by casting Sylgard® 184 (Sigma-Aldrich) polydimethylsiloxane (PDMS) in a 10:1 ratio of base-agent to curing agent in a prefabricated mold. The PDMS solution was then temperature treated at 65 °C for 15 minutes and the reaction was allowed to continue for an additional 15 minutes at room temperature before removing the implants from the mold. After fabrication, the molds were placed in 100% Ethanol to allow unreacted materials to leach out and sterilize the implants before storing them in sterile containers.

[0271] Implant Preparation:

[0272] The implants were sterilized in Betadine Surgical Scrub Veterinary (Avrio Health L.P.™, Stamford, CT) for 12 hours at 4°C prior to surgery. For ADM coverage of the implant, a 1 cm x 1 cm square of ADM was placed covering the converse surface of the implant. 3 equally spaced simple interrupted Monocryl monofilament 4-0 sutures (Ethicon Inc, Raritan, NJ) were used to anchor the ADM to the implant at the implant-ADM interface.

[0273] Hydrogel Preparation:

[0274] The hydrogel was formulated as previously described. Briefly, the hydrogel was formulated with final concentrations of 2 wt% (percentage by weight) hydroxypropylmethylcellulose (HPMC)-Ci2 and 10 wt% polyethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles (NPs). HPMC-Ci2 was synthesized as previously described, dissolved in PBS at 6 wt%, and loaded into a 1 mL luer-lock syringe. A 20 wt% solution of NPs in PBS was diluted with additional PBS, or PBS containing recombinant Osteopontin protein (SPP1 ) (R&D Systems, Inc., Minneapolis, MN) for a final dose of 15 pg per 100 pL and loaded into a separate 1 mL luer-lock syringe. The two syringes were connected with a female-female luer-lock elbow with care to avoid air at the interface of HPMC-C12 and the NP solution. The two solutions were mixed for 1 min or until a homogenous hydrogel was formed. After mixing, the elbow was removed and a luer lock cap was placed and the material was stored at 4°C for up to 7 days prior to surgery.

[0275] In vitro release: Labeled recombinant Osteopontin protein (SPP1) (R&D Systems, Inc., Minneapolis, MN) was made by reacting Alexa Fluor 647 NHS ester (Lumiprobe) at 8x molar excess with SPP1. Briefly, 200 pg SPP1 was dissolved in 200 pL PBS and 22 pL of AF647 NHS ester at 2.44 mg / mL was added dropwise with stirring. After four hours at room temperature, 750 pL PBS was added to quench the reaction and the solution was spin-filtered (Amicon 0.5mL, 10K MWCO) with PBS rinses until flowthrough had no more visible dye. SPP1 -AF647 was concentrated to 1 mg / mL and incorporated into hydrogel at 15 pg per 100 pL for the in vitro release assay. Glass capillary tubes were cut to 4 inches, sealed on one end with epoxy, and allowed to cure for at least 24 hours. 100 pL of hydrogel was injected into the bottom of each tube (n = 3), 400 pL PBS was injected on top carefully to not disrupt the gel surface, and tubes were sealed with parafilm and stored upright at 37 °C. At each time point, all 400 pL PBS was carefully removed from the tube and replaced with fresh PBS, avoiding disturbance of the gel surface. Samples were taken at: 6, 14, 24 hrs and 2, 3, 5, 8, 12, 16, 19, 28 days, and fluorescence was measured using a Synergy H1 Plate Reader (BioTek). At the end of the study, gel was collected from the tubes, diluted with PBS, and the remaining AF647-SPP1 was quantified. Data is presented as AF647-SPP1 remaining in gel, calculated as 1 - Mt / M~, where Mtis the accumulative amount released at each time point and M~ is the total amount loaded in the gel at the beginning of the assay. Data were fit with a one phase-decay in GraphPad Prism and the half-life of release was determined.

[0276] Implant Placement:

[0277] For dorsal implant insertion, anesthesia was induced and maintained with 1-3% isoflurane at a flow rate of 2L / min. Adequate anesthesia was confirmed with loss of hind-limb reflex to nociceptive stimuli. The dorsal skin was shaved and sterilized with Betadine Surgical Scrub Veterinary (Avrio Health L.P.™, Stamford, CT) followed by sterile alcohol prep pads (FisherScientific™, Pittsburgh, PA). Next, a transverse incision was made through the panniculus carnosus on the medial aspect of the left mid-back with sterile scissors and forceps. A 2 cm x 2 cm subcutaneous pocket was raised caudally and laterally, large enough to fit the implant. A PDMS implant alone or PDMS implant covered with ADM was then placed in the subcutaneous pocket. Mice were divided into different experimental groups, each receiving one implant. For mice that received the ADM alone, a 1 x 1 cm square of ADM was placed in the subcutaneous pocket. The wound was closed without tension using 6 interrupted nylon monofilament 4-0 sutures (Dynarex™, Orangeburg, NY). The incision was dressed using Krazyglue™ (Elmer’s Products, Inc., Westerville, OH). Incisions were inspected daily. For mice that received the hydrogel with recombinant SPP1 (Osteopontin), 100 pL of the gel suspension was injected adjacent to the implant once the incision was closed and glued. Mice were hydrated with 1 mL of PBS at the end of the procedure.

[0278] Mouse FBR Model Harvest:

[0279] On post-operative day (POD) 28, the capsule surrounding the implant was harvested with dissecting scissors under 2.5X Loupe magnification and processed for subsequent analysis. Mice were euthanized by carbon dioxide (CO2) narcosis and cervical dislocation. The superior surface of the capsule was meticulously dissected following the edge of the implant, with overlying skin intact. Harvested capsule was sent for histology. Additionally, capsule used for fluorescence- activated cell sorting (FACS) was meticulously removed from the overlying skin using 2.5X Loupe magnification and mechanically digested using dissecting scissors to finely mince each specimen. Harvested capsule for use in histology and immunofluorescent (IF) staining was placed in tissue embedding cassettes.

[0280] Tensile strength testing

[0281] Human Samples:

[0282] Capsule specimens obtained from patients (“native capsule” and “ADM capsule”) were tested using an Instron 5565 using a 100 N load cell (n = 6). The capsule was excised with sharp surgical scissors as above and carefully cut into tapered 4 mm by 15 mm pieces. Tissue pieces were subsequently anchored between grips. The tissue was slowly separated (1% increase per second) until failure (defined by a clear drop in measured stress as tension increased). Young's Modulus was determined by taking the slope of the linear portion of the stress-strain curve. ADM and PDMS:

[0283] ADM and PDMS samples were tested using an Instron 5565 using a 100 N load cell. Materials were cut into tapered 5mm by 10mm pieces. Tissue pieces were subsequently anchored between grips. The tissue was slowly separated (1% increase per second) until failure (defined by a clear drop in measured stress as tension increased). Young’s Modulus was determined by taking the slope of the linear portion of the stress-strain curve.

[0284] Histology and Immunofluorescent Staining (Human and Mouse Samples):

[0285] Fixation: Mouse capsule specimens were fixed in 4% paraformaldehyde (PFA) solution in PBS for 24h at 4°C (n = 3 per condition). All human samples were fixed in 10% neutral buffered formalin (NBF; ThermoFisher Scientific™, Waltham, MA) for 24 hours at room temperature (n = 10). Cryosectioning:

[0286] Fixed samples were soaked in 30% sucrose dissolved in PBS at 4°C. After one week, samples were removed from the sucrose solution and embedded as tissue blocks using Tissue Tek O.C.T. (Sakura Finetek, Torrance, CA) over dry ice and 100% ethanol to achieve rapid freezing. Frozen blocks were mounted on a Thermo Scientific CryoStar NX70 cryostat, and 8 pm- thick sections were transferred to Superfrost / Plus adhesive slides (ThermoFisher Scientific™, Waltham, MA).

[0287] Paraffin Sectioning:

[0288] Automated Tissue Processor (ThermoFisher Scientific™, Waltham, MA) was used to dehydrate samples in a gradient of alcohols. Tissue was then embedded using ThermoFisher Histostar Tissue Embedding station. Paraffin blocks were trimmed as necessary and cut into 8 pm-thick sections. Paraffin ribbons were placed in a water bath at 40°C and mounted onto Superfrost / Plus adhesive slides (ThermoFisher Scientific™, Waltham, MA). Sections were baked at 50°C overnight.

[0289] Staining:

[0290] Hematoxylin and eosin (Cat:H-3502; Vector Laboratories, Burlingame, California), Masson’s Trichrome (ab150686; Abeam®, Waltham, MA), Picro-sirius Red (ab150681 ; Abeam®, Waltham, MA) and Modified Verhoeff Van Gieson stain (Abeam®, Waltham, MA) with standard protocols were used. Cryosection samples were first dehydrated using a slide rack, submerged into 1% PBS for 10 minutes, followed by 30% ethanol (EtOH), 50% EtOH, 70% EtOH, 95% EtOH, and 100% EtOH for 15 minutes each. Paraffin sections were hydrated prior to staining by placement in xylene for 20 minutes, followed by 10 minutes each of 100% EtOH, 95% EtOH, 70% EtOH, 50% EtOH, and 30% EtOH. Slides were then submerged in running tap water for 10 minutes.

[0291] Immunofluorescent staining:

[0292] For immunofluorescent staining, slides were washed twice in Tween 20 (Sigma-Aldrich™, St. Louis, MO) followed by one wash in PBS. Slides were then blocked for 1 hour with Power Block (Biogenex™, Fremont, CA) prior to addition of the following primary antibodies: Abeam ab216028 (anti-lntegrin Alpha X), Abeam ab5694 (anti-a-SMA), ThermoFisher Scientific MA1 - 26771 (anti-collagen type I), Abeam ab214050 (anti-SPP1), ThermoFisher Scientific 14-0196-82 (anti-lntegrin Alpha M), ThermoFisher Scientific 14-0112-82 (anti-lntegrin Alpha M), ThermoFisher Scientific MA5-13890 (anti-Cluster of Differentiation 44), ThermoFisher Scientific MA5-32705 (anti-Matrix Metallopeptidase 9). Slides were then incubated for 1 h with Alexa Fluor 488, 594, or 647-conjugated anti-rabbit, anti-rat, or anti-mouse antibodies (Invitrogen, Waltham, MA). Finally, slides were mounted in Fluoromount-G mounting solution with or without DAPI (ThermoFisher Scientific™, Waltham, MA). Brightfield images were acquired with a Leica CTR4000 microscope, while fluorescent images were acquired with a LSM880 inverted confocal, Airyscan, AiryscanFAST, GaAsP detector upright confocal microscope.

[0293] Hematoxylin and Eosin Staining:

[0294] Hematoxylin and eosin staining started with submerging slides into Hematoxylin for 10 minutes. Slides were then submerged into tap water for 5 minutes, then dipped into eosin 12 times. DI water baths were prepared and slides were submerged until water was clear of eosin. Slide racks were then dipped into 70% EtOH 10 times, followed by 1 minute in 95% EtOH then 100% EtOH. Finally slides were dipped into Xylene 8 times and then mounted on Superfrost / Plus adhesive slides (ThermoFisher Scientific™, Waltham, MA) with Permount Mounting Medium (Electron Microscopy Sciences™, Hatfield, PA). Insets for hematoxylin and eosin staining were established by measuring capsule thickness across a given sample, identifying the average thickness measurement, and selecting the region to magnify for the inset, (n = 6).

[0295] Elastin stain:

[0296] Following cryosectioning (8 .m thickness, Leica2265, Germany), a modified Verhoeff Van Gieson stain was performed (Abeam™, Cambridge, UK). Here, elastic fibers stain black, collagen stain red and muscle stain yellow. Slides were dipped in Elastic Stain Solution for 15 minutes and then rinsed in running tap water until water was clear of excess stain. Slides were then placed in Differentiating solution 15 times and rinsed with tap water until water was clear of excess stain. These were then placed in Sodium Thiosulfate Solution for 1 minute and rinsed in running water before staining the slides using Van Gieson’s Solution for 2 minutes. Slides were submerged in two changes of 95% alcohol for 1 minute each, and then dehydrated in 100% ethanol for another 1 minute. Finally, slides were dipped into Xylene 8 times and mounted with Permount Mounting Medium. Normalized elastin density via van Gieson’s staining was determined by staining ADM alone with van Gieson’s stain, calculating the mean pixel density of elastin staining for ADM alone, and dividing each individual value obtained for ADM capsule and Native capsule pixel density by this mean. Masson’s Trichrome Stain:

[0297] For Trichrome staining, Bouin’s solution was added to the samples for 60 minutes in a humidity chamber. To create humidity chambers, slide boxes were lined with wet paper towels. Next, slides were submerged into running tap water for 5 minutes. Working Weigert’s Iron Hematoxylin was then added to samples for 5 minutes, followed by submersion into running tap water for 5 minutes. Biebrich Scarlet / Acid Fuchsin Solution was then added to samples for 4 minutes. After slides were submerged in running tap water for 5 minutes, Phosphomolybdic / Phosphotungstic Acid was added to the sample for 45 minutes. Phosphomolybdic / Phosphotungstic Acid was removed from slides and Aniline Blue Solution was added for 4 minutes without a washing step. After Aniline Blue staining, slides were submerged into running tap water for 5 minutes. Correct staining was confirmed using Leica CTR4000 microscope. Finally, slides were dipped into 1% Glacial Acetic Acid Solution then running water 12 times each, followed by 15 dips each in 95% EtOH then 100% EtOH. Slides were submerged into Xylene 8 times prior to mounting with Permount. Insets for Masson’s Trichrome figures were established by measuring collagen density across a given sample, identifying the average collagen measurements, and selecting that region to magnify for the inset (n = 6).

[0298] Picrosirius Red Staining:

[0299] Dehydration / rehydration steps were not completed for Picrosirius red staining. Slides were first washed 3 times with PBS and then submerged into running tap water for 1 minute. Picrosirius red was added to slides for 60 minutes in a humidity chamber, previously described. After the completion of the 1 -hour stain, slides were dipped into 2 different changes of 0.5% Glacial Acetic Acid 10 times (20 dips total), followed by 10 times into 2 different changes of 100% ETOH (20 dips total), and 8 times into Xylene. Slides were then mounted with Permount.

[0300] Picrosirius Red Stained Histologic Analysis:

[0301] Analysis of picrosirius red stained tissue sections took place using an image-processing algorithm. The algorithm profiles 26 ultrastructural features to provide a quantitative comparison of extracellular matrices. Each group (n > 3) was randomly imaged at 100 separate locations at 40x. Color deconvolution following previously described methods was performed to characterize each stain by absorbance in red, green and blue (RGB) channels. Ortho-normal transformation was then used to determine each color’s contribution to the captured image. Red and green images were produced, representing mature and immature extracellular matrix (ECM) fibers, and analyzed as separate groups. A Matlab script was used to achieve analysis, including noise reduction, preferential selection for smooth regions with low variance, and “skeletonization” of images to characterize the fiber networks. The algorithm also allowed for measurement of fiber length, width, persistence, alignment, and overall dimensionality.

[0302] Luminex Multiple Immunoassay Native and ADM Capsule Cytokine Analysis (Human Samples)

[0303] Freshly harvested human capsule specimens were collected from the operating room and placed on ice. The tissue was then minced and placed in liquid nitrogen. Protein isolation was then performed using per the Tissue Homogenate Protocol (MAN0017834 ThermoFisher), using the recommended Cell Lysis Buffer (ThermoFisher, EPX-99999-000). Following repeated freezethaw cycles and sonication, samples were centrifuged and supernatant collected for protein quantification and western blot analysis. Protein concentration was measured by the bicinchoninic acid (BCA) method (ThermoFisher Scientific™, Waltham, MA). The 62-plex Luminex assay (custom-built by eBioscience) to assess tissue cytokine content was conducted per the manufacturer’s protocol. Two biological replicates were analyzed per patient specimen.

[0304] Human Single-Cell RNA Sequencing

[0305] Tissue Preparation and Dissociation of Native and ADM Capsules

[0306] Capsule tissue specimens from human specimens were minced on ice (n = 6 per condition). The tissue was then digested for 60 min in a 37 °C water bath agitator in 2 mg / mL collagenase type IV (ThermoFisher Scientific™, Waltham, MA) digestion buffer in Medium 199 (HyClone, South Logan, UT) consisting of 5% fetal bovine serum (ThermoFisher Scientific™, Waltham, MA), DNase I (Fisher Scientific, Hampton, NH), Poloxamer 188 (Cat. P5556-100ML, Sigma-Aldrich, St. Louis, MO), HEPES, and CaCk. The digest was quenched with FACS buffer (ThermoFisher Scientific™, Waltham, MA) containing 2X FBS, 1X Penicillin-Streptomycin, 1 X Poloxamer 188), then centrifuged at 1250rpm for 5 min at 4 °C, resuspended in FACS buffer, and filtered through 100, 70, and 40 pm cell strainers (Falcon cell strainer, ThermoFisher Scientific™, Waltham, MA). Histopaque was performed using Histopaque-11 19 (Sigma-Aldrich, St. Louis, MO), per the manufacturer’s protocol. Cells were counted and resuspended in FACS buffer. Primary antibodies were applied, and cells were stained in the dark with gentle agitation for 30 min. Cells were then washed thoroughly in FACS buffer.

[0307] Single-cell Barcoding, Library Preparation, and Sequencing (Human Samples): Individual human native and ADM capsule dissociated tissue cellular suspensions were tagged with hashtag oligos (HTOs) per the manufacturer’s protocol and then pooled. Cells were counted and filtered just prior to loading into the 10X machine. Single cells were barcoded using the 10x Chromium Single Cell platform, and cDNA libraries were prepared according to the manufacturer’s protocol (Single Cell 3’ v3, 10x Genomics, USA). In brief, cell suspensions, reverse transcription master mix and partitioning oil were loaded on a single-cell chip, then run on the Chromium Controller. Reverse Transcription was performed within the droplets at 53 °C for 45 min. cDNA was amplified for a 12 cycles total on a BioRad C1000 Touch thermocycler. cDNA size selection was performed using SpriSelect beads (Beckman Coulter, USA) and a ratio of SpriSelect reagent volume to sample volume of 0.6. cDNA was analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA was fragmented using the proprietary fragmentation enzyme blend for 5 min at 32 °C, followed by end repair and A-tailing at 65 °C for 30 min. cDNA were double-sided size selected using SpriSelect beads. Sequencing adaptors were ligated to the cDNA at 20 °C for 15 min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection using SpriSelect beads. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a NextSeq 500 Illumina platform aiming for 50,000 reads per cell.

[0308] Data Processing, fastq Generation, and Read Mapping (Human Samples):

[0309] Base calls were converted to reads with the software Cell Ranger (10x Genomics; version 3.1 )‘s implementation mkfastq. These were then aligned against the GRCh38 v3.0.0 (for human) genome using Cell Ranger’s count function (an implementation of STAR v2.7.0) with SC3Pv3 chemistry and 5000 expected cells per sample. Cell barcodes representative of quality cells were delineated from barcodes of apoptotic cells or background RNA based on a threshold of having at least 200 transcripts profiled and less than 10% of their transcriptome of mitochondrial origin. Data Normalization, Hashtag Oligo Demultiplexing, and Cell Subpopulation Identification (Human Samples):

[0310] Unique molecular identifiers (UM Is) from each cell barcode were retained for all downstream_analysis. Raw UMI counts were normalized with a scale factor of 10,000 UMIs per cell and subsequently natural log transformed with a pseudocount of 1 using the R package Seurat (version 4.0.5). HTOs for human samples were_demultiplexed using Seurat’s implementation HTODemux. Briefly, k-medoid_clustering is performed on the normalized HTO values, after which a ‘negative’ HTO distribution is calculated. For each HTO, the cluster with the lowest average value is treated as the negative group and a negative binomial distribution is fit to this cluster. Using the 0.99 quantile of this distribution as a threshold, each cell is classified as positive or negative for each HTO. Cells that are positive for more than one HTOs are annotated as doublets and removed. Cells that are not positive for any HTO are also removed. Aggregated data were then evaluated using uniform manifold approximation and projection (UMAP) analysis over the first 15 principal components. Louvain-based clustering analysis was chosen for the scRNA-seq data as it is the most used graph-based clustering method for single-cell RNA-seq data and is used to assist users in identifying different cell types or cell subpopulations within a given dataset. It is commonly used in part due to its scalability for large single-cell RNA-seq analysis datasets. Further, Seurat was made use of for the scRNA-seq analyses, which applies the Louvain algorithm as the default clustering method. Cell annotations were ascribed using SingleR (version 3.11 ) against the Blueprint + ENCODE reference database for human cells.

[0311] Generation of Characteristic Subpopulation Markers and Enrichment Analysis (Human Samples):

[0312] Cell-type marker lists were generated with two separate approaches. In the first approach, Seurat’s native FindMarkers function was employed with a log fold change threshold of 0.25 using the ROC test to assign predictive power to each gene. However, in order to better account for the mutual information contained within highly correlated predictive genes, a characteristic direction analysis was also employed. The 100 most highly ranked genes from this analysis for each cluster were used to perform gene set enrichment analysis against the BROAD Institute databases (http: / / software(.)broadinstitute(.)org / gsea / index(.)jsp) in a programmatic fashion using EnrichR (version 2.1 ). To identify differences in gene expression between ADM and Native capsule monocytes / macrophages, a Wilcoxon rank-sum test was implemented in Seurat.

[0313] Mouse Single-Cell RNA Sequencing:

[0314] Tissue Preparation and Dissociation of Mouse Native and ADM Capsules

[0315] Mouse capsules were harvested at POD 28. Capsules were mechanically digested with sharp dissecting scissors (n = 4 per condition). Tissue was then added to an enzymatic digestion consisting of Collagenase II (ThermoFisher, Cat:17101015) and IV (ThermoFisher, Cat:17104019) in DMEM-F12 (GIBCOTM, Fischer Scientific, Hampton, NH). Samples were added to an orbital shaker at 150 rpm for 90 minutes at 37eC. FACS buffer was added to quench the digest and samples were filtered through 70 pm cell strainers. Samples were then centrifuged at 1500 for 5 minutes at 4°C.

[0316] Single-cell Barcoding, Library Preparation, and Sequencing

[0317] Mouse cell suspensions were centrifuged and resuspended with 0.04% UltraPure BSA (Thermo Fisher, Waltham, MA); cell counts were completed. A second filtration step took place using 40 pm cell strainers. Quality control and single cell RNAseq were performed on unsorted cells using the 10x Chromium Single Cell platform (Single Cell 3’ v3, 10x Genomics, USA) at the Stanford Functional Genomics Facility (SFGF), Stanford University, Palo Alto.

[0318] Data Processing, fastq Generation, and Read Mapping (Mouse Samples):

[0319] Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1 ) implementation mkfastq and then aligned against the Cell Ranger mml 0 reference genome, using Cell Ranger’s count function with SC3Pv3 chemistry and 5,000 expected cells per sample, as previously described. For mouse datasets, a maximum percent mitochondrial RNA cutoff of 15% and 7,500 maximum unique genes and 85,000 maximum RNA counts were used.

[0320] Data Normalization and Cell Subpopulation Identification (Mouse Samples):

[0321] Unique molecular identifiers (UM Is) from each cell barcode were retained for all downstream analysis, normalized with a scale factor of 10,000 UMIs per cell, and subsequently natural log transformed with a pseudocount of 1 using the R package Seurat (version 4.0.5). The first 15 principal components of the aggregated data were then used for uniform manifold approximation and projection (UMAP) analysis. Louvain-based clustering analysis was chosen for the scRNA-seq data as it is the most used graph-based clustering method for single-cell RNAseq data and is used to assist users in identifying different cell types or cell subpopulations within a given dataset. It is commonly used in part due to its scalability for large single-cell RNA-seq analysis datasets. Further, Seurat was made use of for the scRNA-seq analyses, which applies the Louvain algorithm as the default clustering method. Cell annotations were ascribed using SingleR (version 3.11 ) against the Mouse-RNAseq reference dataset, available at https: / / rdrr(.)io / qithub / dviraran / SinqleR / man / mouse(.)rnaseq(.)html. Cell-type marker lists were generated using Seurat’s native FindMarkers function with a log fold change threshold of 0.25 using the ROC test to assign predictive power to each gene. The 200 most highly ranked genes from this analysis for each cluster were used to perform gene set enrichment analysis in a programmatic fashion using EnrichR (version 2.1). For integration of human and mouse scRNA- seq datasets, integrated cross-species analyses was performed using ortholog mapping via Seurat’s label transfer approach, as previously described.

[0322] The scRNA sequencing data generated during this study has been sent to the NCBI’s Gene expression Omnibus for deposition and will be accessible on request.

[0323] CellChat Receptor-Ligand Analysis:

[0324] To evaluate the potential for interactions between different cell types in the dataset, the recently developed CellChat platform was applied. This was implemented using the scRNA-seq Seurat object in R, in conjunction with the standalone CellChat Shiny App for its Cell-Cell Communication Atlas Explorer. Cells were binned according to the SingleR-defined cell type classifications. Default parameterizations were used throughout, and Secreted Signaling, ECM- Receptor, and Cell-Cell Contact relationships were considered.

[0325] CODEX spatial analysis:

[0326] To spatially phenotype the human specimens, Co-Detection by Indexing (CODEX) was used, a novel assay in which markers are labeled with oligonucleotide-conjugated antibodies and iteratively imaged between cyclic additions and washouts of dye-labeled oligonucleotides. A custom CODEX panel was designed to assess capsule cells within the tissue see Table 2 and Table 4). In brief, primary antibodies were individually barcoded and validated using the commercial supplier's protocols. Paraffin blocks for human specimens (n = 3 per group) were sectioned at 8 pm thickness onto coverslips for CODEX antibody staining. Antigens were retrieved by standard citrate-EDTA processing prior to addition of CODEX antibodies. Using a CODEX- integrated Keyence BZ-X instrument (Akoya Biosciences) image acquisition was then performed. Using software from Akoya Biosciences the raw images were process, with cell segmentation, and rendering. Using a CODEX-integrated Keyence BZ-X instrument (Akoya Biosciences) image acquisition was then performed. Using software from Akoya Biosciences the raw images were process, with cell segmentation, and rendering. Cell segmentation (Voronoi-based) was performed using default platform settings, so that all downstream cell-level analyses were conducted based on protein expression within the boundaries of individual cells.

[0327] The CODEX was visualized using Akoya Biosciences Multiplex Analysis Viewer (MAV) in Imaged. The resulting .fcs files were then concatenated in FlowJo and imported into the Monocle3 and STvEA R packages for further analysis. After debris removal, the processed UMAP manifold was analyzed through Monocle3 with a post-manifold threshold of >10,000 cells per cluster. Analysis of the protein staining patterns was then used to assign cell types. The cell interactions were then inferred using STvEA for cell types at >2.5% of total abundance at k=20 nearest neighbors to quantify cell spatial interactions, and differential interaction maps were generated using graph scores.

[0328] Table 2: List of protein markers with their associated barcodes for the CODEX experiment performed in this study.

[0329] Table 4: List of protein markers with their associated barcodes for the CODEX experiment performed in this study and additional information.

[0330] Yes-Associated Protein; CDH1: E-Cadherin; CD68: Cluster of Differentiation 68; IL6: Interleukin

[0331] 6; MGP: Matrix Gia Protein; CD45: Protein Tyrosine Phosphatase Receptor Type C; CD11c: Integrin Subunit Alpha X; CD8: Cluster of Differentiation 8; IL1 B: Interleukin 1 Beta; CD20: Membrane Spanning 4-Domain A1 ; HLA-DR: Major Histocompatibility Complex Class II; S100A4: S100 Calcium Binding Protein A4; FN1 : Fibronectin 1 ; CD26: Dipeptidylpeptiidase IV; PDGFRa: Platelet Derived Growth Factor Receptor Alpha; COLIV: Collagen Type IV; aSMA: Alpha Smooth Muscle Actin; Ki67: Marker of Proliferation Ki-67; VIM: Vimentin; PANCK: Pan-Cytokeratin; FAK: Focal Adhesion Kinase; MYH11 : Myosin Heavy Chain 11 ; ADIPOQ: Adiponectin, C1Q and Collagen Domain Containing; PLIN1 : Perilipin 1 ; CK19: Cytokeratin 19; CALD1 : Caldesmon 1;

[0332] TAGLN: Transgelin; PDGFRB: Platelet Dervied Growth Factor Receptor Beta; DES: Desmin; PIEZO1 : Piezo Type Mechanosensitive Ion Channel Component 1 ; PIEZO2: Piezo Type Mechanosensitive Ion Channel Component 2; PDPN: Podoplanin; COL1 : Collagen Type I; SPP1 : Osteopontin. Statistical Methods:

[0333] Statistical testing was performed in GraphPad Prism v9 unless otherwise stated. For two- group comparisons, unpaired t-tests were used. For multi-group analysis, one-way ANOVAs were used with Tukey’s post hoc corrections to compare groups; p<0.05 conferred statistical significance for all tests.

[0334] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.)- It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0335] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0336] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[0337] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0338] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0339] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §1 12(6) is not invoked.

Claims

WHAT IS CLAIMED IS:1 . A method of reducing foreign body response (FBR) in a subject, the method comprising: implanting a foreign body in the subject in combination with an effective amount of an SPP1 active agent to reduce FBR in the subject.

2. The method according to Claim 1 , wherein the SPP1 active agent comprises a SPP1 polypeptide.

3. The method according to Claim 2, wherein the SPP1 polypeptide is a recombinant SPP1 polypeptide.

4. The method according to Claim 1 , wherein the SPP1 active agent comprises a nucleic acid encoding a SPP1 polypeptide.

5. The method according to any of the preceding claims, wherein the SPP1 active agent is present in an SPP1 active agent composition.

6. The method according to Claim 5, wherein the SPP1 active agent composition comprises a controlled release composition.

7. The method according to any of Claims 5-6, wherein the SPP1 active agent composition comprises a gel.

8. The method according to Claim 7, wherein the gel is a hydrogel.

9. The method according to Claim 8, wherein the hydrogel comprises a polymer.

10. The method according to Claim 9, wherein the hydrogel further comprises a nanoparticle11 . The method according to any of Claims 9-10, wherein the polymer comprises a polysaccharide.

12. The method according to Claim 11 , wherein the polysaccharide comprises hydroxypropyl methylcellulose.

13. The method according to any of Claims 10-12, wherein the nanoparticle comprises a diblock copolymer.

14. The method according to Claim 13, wherein the diblock copolymer comprises polyethylene glycol) and poly(lactic acid).

15. The method according to any of Claims 5-14, wherein the foreign body is coated with the SPP1 active agent composition.

16. The method according to any of the preceding claims, wherein the foreign body is an implantable device.

17. The method according to any of the preceding claims, wherein the method results in a reduced foreign body capsule thickness around the implant compared to a control.

18. The method according to any of the preceding claims, wherein the method results in a reduced foreign body capsule collagen density compared to a control.

19. The method according to any of the preceding claims, wherein the method results in an increased foreign body capsule elastin density compared to a control.

20. The method according to any of the preceding claims, wherein the method results in an increased foreign body capsule elasticity compared to a control.21 . The method according to any of the preceding claims, wherein the subject is a mammal.

22. The method according to Claim 21 , wherein the subject is a human.

23. The method according to Claim 22, wherein the subject is an adult.

24. The method according to Claim 21 , wherein the subject is a mouse.

25. An implantable device coated with a SPP1 active agent composition.

26. The device according to Claim 25, wherein the SPP1 active agent composition comprises a SPP1 active agent.

27. The device according to Claim 26, wherein the SPP1 active agent comprises a SPP1 polypeptide.

28. The device according to Claim 27, wherein the SPP1 polypeptide is a recombinant SPP1 polypeptide.

29. The device according to Claim 28, wherein the SPP1 active agent comprises a nucleic acid encoding a SPP1 polypeptide.

30. The device according to any of Claims 25-29, wherein the SPP1 active agent composition comprises a controlled release composition.31 . The device according to any of Claims 25-30, wherein the SPP1 active agent composition comprises a gel.

32. The device according to Claim 31 , wherein the gel is a hydrogel.

33. The device according to Claim 32, wherein the hydrogel comprises a polymer.

34. The device according to Claim 33, wherein the hydrogel further comprises a nanoparticle.

35. The device according to any of Claims 33-34, wherein the polymer comprises a polysaccharide.

36. The device according to Claim 35, wherein the polysaccharide comprises hydroxypropyl methylcellulose.

37. The device according to any of Claims 34-36, wherein the nanoparticle comprises a diblock copolymer.

38. The device according to Claim 37, wherein the diblock copolymer comprises polyethylene glycol) and poly(lactic acid).

39. A kit comprising: a) an implantable device; and b) a SPP1 active agent composition.

40. The kit according to Claim 39, wherein the SPP1 active agent composition comprises a SPP1 active agent.41 . The kit according to Claim 40, wherein the SPP1 active agent comprises a SPP1 polypeptide.

42. The kit according to Claim 41 , wherein the SPP1 polypeptide is a recombinant SPP1 polypeptide.

43. The kit according to Claim 40, wherein the SPP1 active agent comprises a nucleic acid encoding a SPP1 polypeptide.

44. The kit according to any of Claims 39-43, wherein the SPP1 active agent composition comprises a controlled release composition.

45. The kit according to any of Claims 39-44, wherein the SPP1 active agent composition comprises a gel.

46. The kit according to Claim 45, wherein the gel is a hydrogel.

47. The kit according to Claim 46, wherein the hydrogel comprises a polymer.

48. The kit according to Claim 47, wherein the hydrogel further comprises a nanoparticle.

49. The kit according to any of Claims 47-48, wherein the polymer comprises a polysaccharide.

50. The kit according to Claim 49, wherein the polysaccharide comprises hydroxypropyl methylcellulose.51 . The kit according to any of Claims 48-50, wherein the nanoparticle comprises a diblock copolymer.

52. The kit according to Claim 51 , wherein the diblock copolymer comprises polyethylene glycol) and poly(lactic acid).

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