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The effect of titanium surface topography on macrophage behaviour in vitro Refai, Ali K.

Abstract

Macrophages are pivotal to the survival of endosseous implants. Researchers have shown that macrophage-like cells attached to rough SLA surfaces and such rough surfaces were associated with a greater percentage of bone formation than smooth surfaces. Therefore, it is the hypotheses of this study to investigate the effect of surface topography on macrophage morphology, activation, and secretion of selected cytokines and chemokines, and the effect of the secretions on early events that take place during bone formation. RAW 264.7 cells were cultured on replicas of four titanium topographies initially produced by polishing (PO), large-grit sandblasting (CB), acid-etching (AE), and sandblasting plus acid etching (SLA) with and without lipopolysaccharides (LPS). Morphology and motility were observed via scanning electron microscopy (SEM) and time-lapse video microscopy respectively. Cytokines and chemokines were measured using an enzyme-linked immunosorbent assay (ELISA) and cells counted by nuclear staining. Factorial design and two-way analysis of variance were employed to assess statistical significance and interaction. SEM demonstrated that macrophages increased in number and exhibited diverse morphologies on the four tested surfaces. Although no differences in motility were found macrophages exhibited either an oscillatory movement or remained stationary. Unstimulated macrophages on AE and SLA increased secretion of tumor necrosis factor-α compared to those cultured on the PO surface. Cells cultured with suboptimal doses of LPS on SLA produced higher levels of interleukin-1β, interleukin-6, and TNF-α at 24 and 48 hours. Relative to cells on PO surfaces unstimulated macrophages on SLA surfaces down-regulated their production of chemokines CCL2and CCL3, but increased their relative production when stimulated by LPS. Using a Transwell co-culture system to determine the effects of macrophage secretions on rat osteoblasts we found that ALP activity was increased significantly after 1 week of culture for co-cultures on SLA surfaces compared to co-cultures on PO surfaces or control surfaces. However, the number of bone-like nodules was unaffected by the presence or duration of macrophages in the co-culture model. This in vitro study demonstrated that surface topography modulates production of cytokines and chemokines in a time-dependent manner. The success of these approaches in examining cytokines typical of classically activated macrophages, suggests that other activation patterns such as immunosuppression and wound healing should be similarly studied.

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