Preadsorption of Serum Proteins Regulates Bacterial Infections and Subsequent Macrophage Phagocytosis on Biomaterial Surfaces

Abstract

Bacterial infection has been one of the main obstacles for extensive biomedical applications of biomaterial films. Understanding the interactions among macromolecules, cells, and bacteria in the microenvironment located on the film surface at the molecular level is essential for developing antibacterial films. Here we report the distinct influence of several key serum proteins adsorbed on diamond-like carbon (DLC) and traditional Ti films on initial bacterial adhesion, biofilm formation, and corresponding immune responses. Type I collagen, Fn, and IgG were selected as the typical serum proteins. Gram-positive bacterium Staphylococcus epidermidis and Gram-negative bacterium Escherichia coli were used as the model bacteria. Macrophage phagocytosis tests were carried out to examine the impact of adsorbed proteins on the ability of macrophages to clear the adhered pathogens. Results show that it was the specific molecular recognition between adsorbed proteins and bacteria, not the surface physiochemical properties such as surface wettability, surface roughness, and surfaces charge, that decisively affected bacterial adhesion and following biofilm formation. Collagen resisted bacterial adhesion on both DLC and Ti films, even though the molecules exhibited distinct conformations on the two surfaces, whereas for Fn and IgG, the specific molecular recognition was closely related to protein conformations. Fn molecules formed globular aggregates on Ti surfaces that greatly enhanced bacterial adhesion but exhibited a fibril conformation on DLC surfaces that inhibited bacterial adhesion. IgG showed an end-on orientation with free F(ab) 2 domains on Ti surfaces, facilitating bacterial adhesion and biofilm formation, while the flattened orientation on DLC films showed little effect on bacterial behaviors. Furthermore, preadsorption of Fn and IgG significantly promoted the phagocytosis ability of macrophages against S. epidermidis and affected the corresponding secretion of inflammatory cytokine. These results would give insights into understanding protein−surface interactions for developing appropriate surface modification techniques for biomaterials with desired anti-inflammatory functions.

In vitro co-culture models for the assessment of orthopedic antibacterial biomaterials

“…SEM imaging to see lysed cells or bacteria on the surface or on top of immune cells was a commonly used method to assess the survival of immune and bacterial cells ( Svensson et al, 2014 ; Mendoza et al, 2017 ; Hou et al, 2019 ; Reigada et al, 2020 ; Yang et al, 2021 ). The staining of both macrophages and bacteria, followed by confocal microscopy ( Zwicker et al, 2022 ) or, in some cases, pre-staining of bacteria ( Wagner and Bryers, 2004 ; Chen et al, 2017 ) or flow cytometry ( Chen et al, 2017 ; Mendoza et al, 2017 ), have been used to assess the number of internalized bacteria inside macrophages.…”

“…An early in vitro infection model should, therefore, include bacteria and inflammatory cells ( im-bac models) with the possible addition of blood components (to represent the early hemostasis phase), followed by the addition of stromal/progenitor cells at a later time point. Until now, only a few studies have focused on the hemostasis phase and the effects of serum or blood on the antibacterial efficiency of a biomaterial, even though the addition of blood provides a clinically relevant microenvironment, which is beneficial for reducing the discrepancy between in vitro and in vivo outcomes ( Wagner and Bryers, 2004 ; Guo et al, 2017 ; Li et al, 2017 ; Hou et al, 2019 ).…”