Macrophage responses to silk
Introduction
The in vivo use of synthetic and biologically derived polymers in biomedical applications such as tissue engineering and drug delivery introduces an interaction with the host immune system that can determine the efficacy of the particular application. Activation of the innate immune response, specifically the macrophage, is a useful determinant of the biocompatibility of biomaterials. This correlation has been especially well described with regard to orthopaedic implants, which sometimes require replacement after inflammation-mediated osteolysis and implant failure [1], [2], [3], [4], [5], [6], [7], [8], [9]. It is important, therefore, in any potential application for biomaterials that the innate immune response to the material in question be determined.
Polyethylene wear particulates are taken up by macrophages via phagocytosis and elicit proinflammatory cytokine production, contributing to the failure of the implant [10]. Wooley et al., demonstrated that ultra high molecular weight polyethylene and polymethylmethacrylate (PMMA) particles induced cellular infiltrate and cytokine release in a murine air pouch model [1]. The effects of size, shape, and morphology of PMMA particles was also examined in the air pouch model, indicating that particles less than 20 μm in diameter induced a greater inflammatory reaction than particles larger than 50 μm, suggesting that the ratio of total surface area to volume of the particles may be the major factor in determining the degree of the inflammatory reaction [11].
Native silkworm silk fibers from Bombyx mori consist of a core structural protein fibroin that is coated with sericin, a family of glue-like proteins that hold the fibroin core fibers together [12]. These silk fibers have been used for decades as sutures in biomedical applications [13] and have potential as scaffolds in tissue engineering [14], [15], [16]. Immune responses mounted against silk sutures have been largely attributable to the glue-like sericin proteins, not the core fibroin fibers [17], [18], [19], [20]. Soluble factors generated by sonication of native silkworm fibers induced pro-inflammatory cytokine production and increased phagocytosis [21]. Santin et al., recently examined the inflammatory potential of silk fibroin membranes and determined that the fibroin membranes elicited lower levels of macrophage activation than polystyrene and poly (2-hydroxyethyl) methacrylate [22].
The objective of the present study was to examine the direct activation of the innate immune response by silk fibers in more detail in order to gain a better understanding of the relationship between these fibers and biological responses. A broader goal was to gain a better understanding of the source of any immune response in relation to silk within the context of other natural degradable biomaterials such as collagen. To accomplish this goal native silk fibers from B. mori were prepared and analyzed in an in vitro macrophage assay to assess cytokine activation. Responses were compared to collagen and commercially available black braided silk suture.
Section snippets
Cell lines and materials
The murine macrophage cell line, RAW 264.7 (TIB-71, ATCC, Manassas, VA) was maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FCS) (GIBCO, Carlsbad, CA). All media contained 50 μg/ml of gentamycin (Cell-Gro, Herndon, VA). Lipopolysaccharide (LPS) (from E. coli 055:B5), used as a positive control in macrophage assays, and polymyxin B were purchased from Sigma (St. Louis, MO). Native B. mori silkworm cocoons were generously provided by M. Tsukada, Japan and
Raw 264.7 cell activation in response to silk fibers
In two separate experimental protocols designed to examine the effects of short- and long-term cultures (Figs. 1A, and B), silk fibers failed to stimulate significant TNF release from RAW 264.7 cells. Macrophages stimulated for 18 h released baseline levels of TNF in response to the two-collagen fibers, the extracted silk, and non-extracted (native) silk fibers, while responding strongly to LPS stimulation (Fig. 1A). Due to the chronic nature of exposure of the immune system in vivo, long-term
Discussion and conclusions
A significant issue to consider with the use of any biomaterial is the potential activation of the immune system. The innate immune system has an important role in determining the course of a potential immune response to any foreign substance in a host. This has been well documented in the case of orthopedic implants as described earlier. The studies presented here explored the response of the innate immune system, specifically macrophages, to silk fibers, as well as extracts from the silk.
Acknowledgments
We thank the NIH (RO1 DE13405-01), and Tissue Regeneration, Inc. for support of this research.
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