Elsevier

Biomaterials

Volume 32, Issue 4, February 2011, Pages 979-984
Biomaterials

Competitive time- and density-dependent adhesion of staphylococci and osteoblasts on crosslinked poly(ethylene glycol)-based polymer coatings in co-culture flow chambers

https://doi.org/10.1016/j.biomaterials.2010.10.011Get rights and content

Abstract

Biomaterial-associated infections (BAI) remain a serious clinical complication, often arising from an inability of host tissue-implant integration to out-compete bacterial adhesion and growth. A commercial polymer coating based on polyethylene glycol (PEG), available in both chemically inert and NHS-activated forms (OptiChem®), was compared for simultaneous growth of staphylococci and osteoblasts. In the absence of staphylococci, osteoblasts adhered and proliferated well on glass controls and on the NHS-reactive PEG-based coating over 48 h, but not on the inert PEG coating. Staphylococcal growth was low on both PEG-based coatings. When staphylococci were pre-adhered on surfaces for 1.5 h to mimic peri-operative contamination, osteoblast growth and spreading was reduced on glass but virtually absent on both reactive and inert PEG-based coatings. Thus although NHS-reactive, PEG-based coatings stimulated tissue–cell interactions in the absence of contaminating staphylococci, the presence of adhering staphylococci eliminated osteoblast adhesion advantages on the PEG surface. This study demonstrates the importance of using bacterial and cellular co-cultures compared to monocultures when assessing functionalized biomaterials coatings for infectious potential.

Introduction

Biomaterial-associated infections (BAI) remain a serious complication in modern medicine with devastating clinical consequences ranging from complete implant failure to lethal sepsis of the patient [1]. Economic consequences of BAI are also noteworthy, as the magnitude of BAI and requirements for resulting treatments are costly and significant [1]. As the use of implanted biomaterials continues to arise, BAI incidence, costs and morbidity will also increase. For this reason, the design of improved biomaterials or functional coatings capable of withstanding biofilm formation while simultaneously providing a strong interface with surrounding host tissue cells is essential to ensure the long-term success of many implanted biomedical devices.

Despite careful sterile and hygienic surgical suite conditions during implantation of medical devices, viable pathogens from ubiquitous human skin flora, such as Staphylococcus epidermidis, can enter the surgical site and contaminate the implanted device. Alternatively, airborne micro-organisms may contaminate device surfaces prior to implantation [2], [3]. Consequently, after device placement, host cells and bacteria will simultaneously compete for colonization of the biomaterial surface [4]. BAI incidence will decrease if host cells rapidly adhere and readily proliferate on the biomaterial surface to out-compete bacteria at the implant site – a scenario previously called “the race for the surface” [4]. As cell-surface adhesion motifs and strategies are often too general to select only mammalian cells over micro-organisms, deliberate designs to increase affinity of a biomaterial surface or coatings for tissue cells is frequently accompanied by increased bacterial adhesion.

Many strategies have sought to reduce microbial adhesion and subsequent biofilm formation on implant surfaces, including use of hydrophobic coatings and application of quaternary ammonium (cationic) compounds [5], [6], [7], [8]. However, these approaches may stimulate adsorption of host proteins, providing a conditioning film for bacterial attachment. Due to their intrinsically low protein adsorption and hundred-fold reductions in bacterial adhesion with respect to common biomaterials, poly(ethylene) glycol (PEG) coatings have become the “first choice” strategy for reducing bacterial adhesion [9]. Hydrated, sufficiently dynamic PEG-polymer chains (e.g. brushes) are also proposed to reduce bacterial adhesion through the steric repulsion between the hydrated PEG chains [10]. Although polymer brushes are designed to rapidly hydrate and suppress non-specific adhesion of biomolecules to surfaces, these coatings can also be chemically modified to promote specific immobilization of tissue cells [11], [12], [13]. If resulting tissue cell-surface interactions are weak, non-specific, or insufficient to enable rapid mammalian cell attachment, bacterial adhesion and biofilm formation may remain insignificant and clinically unaffected [12].

OptiChem® is a commercially available, crosslinked PEG-based coating with an amine-reactive (NHS active ester) chain-terminal chemical functionality in its reactive form to facilitate specific immobilization of biomolecules. The NHS-functionality is deactivated with methoxyethylamine to provide a non-reactive or ‘chemically inert’ PEG surface with very low, non-specific binding of biological molecules from its physiological milieu [14], [15], [16]. Our previous studies have shown that deactivated inert OptiChem® coatings reduce adhesion of a variety of clinical bacterial isolates in different physiological fluids [17] and delay formation of mature biofilms [18]. However, little is known about how bacteria interact with NHS-reactive PEG-based coatings, and the simultaneous growth of both bacteria and mammalian cells on inert and NHS-reactive OptiChem®.

Recently, a methodology has been forwarded to evaluate the simultaneous growth of tissue cells and bacteria in a single co-culture experiment under the presence of controlled, variable fluid shear and in different media [19]. The aim of this study was to compare the simultaneous growth of S. epidermidis and U2OS osteoblast co-cultures on both deactivated ‘inert’ and NHS-reactive crosslinked PEG-based (OptiChem®) in vitro.

Section snippets

Substrata

Simultaneous bacterial and osteoblast cell growth in co-cultures were studied on OptiChem®-coated glass slides (Accelr8 Technology, USA, commercially available as Schott-NexterionTM Slide H). The chemical formulation of the coating, its full characterization and bio-immobilization properties have been described previously [14], [15], [16]. OptiChem® was applied on glass slides by spin-coating and curing to crosslink the PEG matrix [14]. Coated-slides were stored continuously at −20 °C prior to

Results

Images of U2OS cells seeded on glass, inert OptiChem® and reactive OptiChem® at 1.5 h, in the presence of pre-adhering staphylococci on each substratum are shown in Fig. 1. Mammalian cells seeded on glass were well distributed over the surface, whereas cells seeded on both inert and reactive OptiChem® coatings tended to aggregate, irrespective of the presence of staphylococci. Cells seeded on glass and reactive OptiChem® attached and started spreading after 1.5 h. By contrast, cells seeded on

Discussion

Competing cell-surface interactions on biomaterial surfaces between opportunistic pathogens and host tissue cells is a critical determinant for the development of biomaterial-associated infections (BAI) and therefore an important design parameter for improving implanted devices. PEG-based coatings are recognized to be very effective in reducing in vitro bacterial adhesion and biofilm formation [18], [21]. Validation of this benefit in vivo has also been reported [18], [22]. Therefore, PEG-based

Conclusions

A crosslinked PEG-based NHS-reactive coating, previously characterized in cell and bacterial monocultures, was used in a co-culture assay for competitive time- and density-dependent bacterial versus cellular adhesion. Results demonstrate both reduced susceptibility to staphylococcal biofilm formation while ensuring adhesive interactions required to facilitate mammalian osteoblast cell culture. However, bacterial biofilm forms to a limited extent on these PEG-based coatings, and staphylococcal

Funding

This research was funded by the University of Groningen-University Medical Center Groningen, Groningen, The Netherlands.

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