Statement of Purpose: During wound healing, fibrin is one of the first scaffolds encountered by immune cells. Fibrinogen, a protein found in blood plasma, is cleaved by thrombin, a proteolytic enzyme, to produce fibrin monomers, initiating the polymerization process. The three dimensional network of branching fibrin fibers enables neutrophils and macrophages to attach upon arrival to the site of injury and later allows fibroblasts to anchor as tissue rebuilding takes place. Infiltrating fibroblasts lay down new extracellular matrix, resulting in collagen-rich granulation tissue. Although many cell types interact with both fibrin and collagen during this process, macrophages play an important role in regulating the inflammatory versus wound healing response. As plastic cells that can alter their behavior in response to cues in their microenvironment, macrophages can assume an inflammatory subtype as well as an alternatively activated, pro-healing phenotype. While soluble fibrinogen has been shown to stimulate inflammatory chemokine expression of macrophages, the effect of fibrin matrices on macrophage activation has not been previously investigated. Here, we created 3D fibrin/collagen composite hydrogels of varied compositions, and examined the effects of composition on fiber architecture and macrophage phenotype polarization. Methods: To form 3D gels, tissue culture plastic (TCP) were coated with ECM solutions and incubated overnight at 37°C. Pure fibrin gels were fabricated at 2.0 mg/ml protein content using bovine plasma Type IS fibrinogen (Sigma). The lyophilized protein was reconstituted in PBS containing calcium and magnesium. Pure collagen substrates at 2.0 mg/ml protein content were fabricated using rat tail Type Ⅰ collagen (Coming) according to the manufacturer's suggested protocol. Fibrin-collagen gels were made at various ratios to obtain a final protein content of 2.0 mg/ml. For fibrin containing gels, 0.2 U of bovine plasma thrombin (Sigma) was used per mg of total protein. Fiber architecture was characterized using back reflection microscopy by illuminating substrates with 488 nm laser light with a 40X objective; images were taken along the z-axis at sequential focal lengths. Macrophage phenotype behavior was examined by seeding murine bone-marrow derived macrophages on resulting thin 3D gel films. Supernatants were collected at 42 hours after seeding. ELISAs were performed to assess secretion of interiukin-10 (IL10, an anti-inflammatory cytokine) and tumor necrosis factor α (TNFα, a pro-inflammatory cytokine). Results: Back reflection microscopy reveals distinct fiber architectures in gels of different composition (Figure 1a). Pure fibrin gels display finer and shorter fibrils (top), whereas pure collagen gels have long, thick, and branching fibers (bottom). Gels containing a 1-1 mixture of fibrin and collagen display an interpenetrated network of both thin and thick fibers (middle). Hydrogel composition significantly influenced cytokine secretion of macrophages. The amount of secreted IL-10, an anti-inflammatory cytokine, is enhanced in macrophages seeded on gels with increasing amount of fibrin (Figure 1 b, left). The amount of secreted TNFa, a pro-inflammatory cytokine, is also elevated with increased fibrin content although at a more moderate level (right). These data suggest that fibrin may enhance the polarization of macrophages towards an anti-inflammatory, pro-healing phenotype. Conclusions: In this work, we find that fibrin and collagen gels display distinct fiber architectures, and gels containing both polymers exhibit a blend of the different fiber geometries. Macrophages seeded onto gels containing fibrin display an anti-inflammatory phenotype, with increasing fibrin content directly correlated with an increase in IL-10 secretion. Future work will include characterization of gel mechanical properties as well as examination of additional macrophage phenotype markers.
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