Implant Fibrosis is Caused by a Unique Subgroup of Collagen Producing Macrophages
Britta A. Kuehlmann, MD, PhD1, Clark Andrew Bonham1, Lukas Prantl, MD, PhD2, Matthew Philip Murphey, MD1, Geoffrey C. Gurtner, MD, FACS1.
1Stanford University School of Medicine, Palo Alto, CA, USA, 2University Hospital Regensburg and Caritas Hospital St. Josef, Regensburg, Germany.
PURPOSE: All implanted medical devices stimulate collagen deposition resulting in a surrounding fibrotic capsule. Implants are frequently used for reconstruction, such as breast implants following mastectomy. The most common complication following breast implants is capsular fibrosis (CF), with its severity clinically measured by the Baker I-IV classification. Despite the clinical prevalence, the mechanisms underlying CF are still poorly understood and therapeutic interventions are limited. For the first time, we analyze human capsule samples to identify the pathways activated in human capsular fibrosis, and use animal models to clarify the mechanisms underlying the fibrotic process resulting in CF. METHODS: We have established the largest tissue bank worldwide for breast capsular tissues, including histological and clinical data. Consisting of over 600 breast tissues, this registry provides the opportunity to analyze mechanisms underlying CF. Using RNA analysis with a 2,559-gene probeset, the gene expression from a total of 40 patients was analyzed. 20 patients expressing the mildest form of capsular fibrosis (Baker I) were compared to 20 human samples of the most severe form of this disease (Baker IV). All breast capsule samples were obtained after implant-based revision surgery. To recreate CF within an animal model, miniature silicone implants were placed subcutaneously in Bl6-mice. Cells from the capsule were then isolated and characterized by fluorescence-activated cell sorting (FACS), single cell quantitative polymerase chain reaction (qPCR) and single cell RNA sequencing to determine the genetic profiles of those most responsible for fibrotic development. Further, en-bloc capsule was additionally resected for SEM, TEM, 3D-confocal imaging and several stains.
RESULTS: 1,543 genes were upregulated and 1,016 were downregulated in Baker IV vs Baker I. Interestingly, genes regulating macrophage activation, macrophage surface markers and extracellular matrix (ECM) production were among the most highly expressed in Baker IV specimens. Conversely, we did not observe significant increases in myofibroblast genes. To determine whether our findings in the human fibrotic tissues were similar to murine capsules we used vav-reporter-mice for lineage tracking. Here we observed the recruitment of mainly immune cells in fibrotic capsule development and identified the presence of a hematopoietic-derived cell with distinct characteristics of macrophages, confirming their existence within the chronology of CF. To confirm this, we placed silicone implants into Bl6-mice. Surprisingly, in the murine capsules we found the predominant cells across different time-points were myeloid cells and not fibroblasts or endothelial cells. We were able to confirm that these macrophages could produce ECM at the protein level by subjecting the cells to FACS analysis. We found over 80% of the macrophages expressing collagen 1. Our findings were confirmed by analysis of immunohistochemistry stains of the fibrotic sections showing that F4/80+ macrophages deposited collagen.
CONCLUSION: For the first time we demonstrate that collagen depositingmacrophages are responsible for CF by analyzing both patient samples and murine models. Taken together, our findings suggest that macrophages seem to be one of the predominant sources of ECM deposition in fibrotic capsules and may have promising therapeutic implications for the treatment of CF.
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