Macrophage responses to silk

Abstract

Silk fibers have potential biomedical applications beyond their traditional use as sutures. The physical properties of silk fibers and films make it a promising candidate for tissue engineering scaffold applications, particularly where high mechanical loads or tensile forces are applied or in cases where low rates of degradation are desirable. A critical issue for biomaterial scaffolds is biocompatibility. The direct inflammatory potential of intact silk fibers as well as extracts was studied in an in vitro system. The results indicate that silk fibers are largely immunologically inert in short- and long-term culture with RAW 264.7 murine macrophage cells while insoluble fibroin particles induced significant TNF release. Soluble sericin proteins extracted from native silk fibers did not induce significant macrophage activation. While sericin did not activate macrophages by itself, it demonstrated a synergistic effect with bacterial lipopolysaccharide. The low level of inflammatory potential of silk fibers makes them promising candidates in future biomedical applications.

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.