Engrailed-Positive Fibroblasts: The Primary Cell Type Present in Fibrotic Capsules During Foreign Body Response

Abstract

Introduction: We have recently shown that En1 positive fibroblasts (EPFs) predominate in wounds and are responsible for scar formation. Meanwhile, foreign body response (FBR) is a common surgical complication whereby a fibrotic capsule forms around an implant. Though believed to have some mechanistic overlap with normal wound healing, the specific cell populations responsible are unknown. Methods: En1Cre transgenic driver mice were crossed with R26mTmG reporter mice, generating En1Cre; Rosa26mTmG (En1+) mice. In these mice, cells that express En1 also express GFP, while all other cells express RFP. Silicone discs were surgically implanted below the subcutaneous layer of En1+ mice dorsi, and fibrotic capsules surrounding the implants were harvested at POD30. Immunohistochemistry and flow cytometry (FACS) analysis were used to evaluate the cellular characteristics of the En1+ cells. Results: Using immunohistochemistry, En1+ cells were found to be more prevalent in the fibrotic capsule relative to unwounded tissue (*P < 0.05, n = 6). Furthermore, immunofluorescence staining and FACS revealed that GFP+ cells within the fibrotic capsules expressed pro-fibrotic markers such as Col-I, a-SMA, and Col-III. These cells were also negative for CD41 and CD45, endothelial and immune cell markers, respectively. These data indicate that En1+ cells seen within fibrotic capsules were primarily of fibroblast origin. Conclusion: As with normal wound healing, where EPFs are prevalent within a healing scar, our findings strongly suggest that En1 plays a key role in fibrotic capsule formation. Further understanding of the mechanisms that drive En1 positive fibroblasts towards fibrosis may offer potential therapeutic targets to prevent fibrotic capsule formation.