18. Tissue Microenvironment Affects Fibrotic Capsules Formed during Foreign Body Response

Abstract

PURPOSE: Foreign body response (FBR) is a common surgical complication, wherein a fibrotic capsule forms around an implant. Though it is posited that FBR has mechanistic overlap with normal wound healing, the specific cell populations responsible for this phenomenon remain elusive. Our group previously demonstrated that En1 positive fibroblasts (EPFs) predominate in murine dorsal wounds, and showed that these cells are responsible for scar formation within the wound bed. This observation led us to explore the role of EPFs and other fibroblast subpopulations during FBR. METHODS: En-1Cre transgenic driver mice were crossed with R26mTmG reporter mice, generating En-1Cre; Rosa26mTmG (En1+) mice. In this mouse line, cells expressing En1 also express GFP, while all others express RFP. Silicone discs were surgically implanted below the subcutaneous layer of En1+ mice dorsi, and fibrotic capsules surrounding the implants were harvested at POD28. Immunohistochemistry and flow cytometry (FACS) analysis were utilized to evaluate the cellular characteristics of the En1+ cells. A subset of fibrotic capsules placed in C57BL/6J for 28 days was also prepared for single-cell RNA sequencing. Software packages in R and Python were used to identify and define subtypes of fibroblasts present within fibrotic capsules. Crosstalk and signaling pathways were further identified via network analysis platforms. RESULTS: Immunohistochemistry and FACS analysis confirmed that En1+ cells predominate in the fibrotic capsule, and that GFP+ cells also expressed pro-fibrotic markers, including Col-1 and α-SMA. As these cells were also negative for the endothelial and immune cell markers CD41 and CD45, respectively, these data indicate that En+ cells within capsules were primarily of fibroblast origin. Interestingly, basic histology highlighted that capsule thickness (p < 0.0001; n = 4) and collagen deposition (p = 0.0016; n = 4) were higher in the capsule adjacent to the subcutaneous tissue (subcutaneous capsule) in comparison to that adjacent to muscle tissue (muscle capsule). In parallel, our transcriptional dataset revealed that the distribution of fibroblast subpopulations present in subcutaneous capsule to that in muscle capsule differed. Notably, cell clustering, cell-cell interaction, and gene ontology analyses underscored that fibroblasts in the subcutaneous capsules were more fibrotic in nature relative to those present in muscle capsule. CONCLUSION: These data strongly suggest that, as with normal wound healing in which EPFs are diffusely present within a healing scar, En1 appears to play a key role in fibrotic capsule formation. Further, the presence of multiple subpopulations of fibroblasts revealed by our transcriptional analysis underscores the significant function fibroblasts may have in FBR, and that the tissue microenvironment may play a role in the fibrotic response. Deeper understanding of the mechanism behind fibroblasts’ involvement and the effect the tissue microenvironment may have on fibrotic capsule formation may offer potential therapeutic targets to reduce FBR post surgical implantation in the future.