Elsevier

Acta Biomaterialia

Volume 5, Issue 5, June 2009, Pages 1399-1404
Acta Biomaterialia

Microbial biofilm growth vs. tissue integration: “The race for the surface” experimentally studied

https://doi.org/10.1016/j.actbio.2008.12.011Get rights and content

Abstract

Biomaterial-associated infections constitute a major clinical problem. Unfortunately, microorganisms are frequently introduced onto an implant surface during surgery and start the race for the surface before tissue integration can occur. So far, no method has been forwarded to study biofilm formation and tissue integration simultaneously. The aim of this study is to describe an in vitro method to investigate this “race for the surface”. First, a suitable growth medium was prepared that allowed both bacterial and tissue growth in a parallel plate flow chamber. Staphylococci were deposited on the glass bottom plate of the flow chamber in different surface densities, after which U2OS osteosarcoma cells were seeded. U2OS cells did not grow in the absence of flow, possibly due to poisoning by bacterial endotoxins, but under flow both staphylococci and U2OS cells grew. The number of adhering cells and area per spread cell were determined after 48 h in relation to the initial number of bacteria present. Both the number and spread area per cell decreased with increasing density of adhering staphylococci. This demonstrates that the outcome of the race for the surface between bacteria and tissue cells is dependent on the number of bacteria present prior to cell seeding.

Introduction

Biomaterials play a major role in modern medicine for the restoration of function, frequently used examples being prosthetic joints or heart valves. Biomaterial-associated infections (BAIs) pose a serious complication, which is of growing concern due to the increasing use of biomaterial implants and devices. On average, BAI occurs in approximately 0.5–6% [1], [2] of all cases, strongly depending on the implant site, and more often in cases of trauma or revision surgery [3], [4], [5]. BAIs are difficult to treat, as the biofilm mode of growth protects the infecting organisms against the host immune system and antibiotic treatment [6], [7]. In most cases, the final outcome of a BAI is removal of the implant. There are various routes along which a BAI can develop. The best-documented route is direct contamination of the implant during surgery (perioperative contamination) or contamination during hospitalization [8], [9], [10]. Since microorganisms can remain dormant for several years on a biomaterial surface [9], [11] inside the human body or in adjacent tissue [9], BAI can become clinically manifest years after insertion of an implant. Moreover, late BAI can develop by microbial spreading through blood from infections elsewhere in the human body, but evidence for haematogenous spreading is mainly anecdotal.

In 1987, the orthopedic surgeon Anthony G. Gristina coined the term “race for the surface” to describe the fate of biomaterial implants in relation with the development of BAI [9]. The fate of a biomaterial implant was pictured as a race between microbial adhesion and biofilm growth on an implant surface vs. tissue integration. If the race is won by tissue cells, then the surface is covered by tissue and less vulnerable to bacterial colonization. On the other hand, if the race is won by bacteria the implant surface will become rapidly covered by a biofilm and tissue cell functions will be hampered by bacterial virulence factors and toxins. To the aid of the implant, its surface may generate an inflammatory reaction at the tissue interface, resulting in the activation of the immune system, which may hamper bacterial colonization [9], [12]. Unfortunately, microorganisms are frequently introduced on an implant surface during surgery and in vivo, so microorganisms start the race for the surface before tissue integration can occur.

The concept of the race for the surface has been embraced by many researchers in the field, but hitherto there has been no in vitro experimental methodology forwarded to study the actual race. New biomaterials or functional coatings are evaluated either for their ability to resist bacterial adhesion and biofilm formation [13], [14], [15], [16] or for their ability to support tissue cell adhesion and proliferation [13], [16], [17], [18]. The aim of this study is to describe an in vitro experimental methodology to investigate the race for the surface between bacteria and tissue cells in a single experiment.

Section snippets

U2OS cell culturing and harvesting

U2OS osteosarcoma cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) low glucose supplemented with 10% fetal calf serum (FBS), 0.2 mM of ascorbic acid-2-phosphate, denoted in the paper as DMEM + FBS. U2OS cells were maintained at 37 °C in a humidified 5% CO2 atmosphere, and cells were passaged at 70–90% confluency using trypsin–EDTA.

Bacterial growth conditions and harvesting

S. epidermidis ATCC 35983, originally isolated from human blood of a patient with an infected intravascular catheter, was used throughout this

Development of a modified culture medium

U2OS cells were cultured in different ratios of DMEM + FBS and TSB media, but did not show any growth in media containing more than 30% TSB (see Fig. 1). No changes in cell morphology were observed when U2OS cells were cultured in media containing 2% or 4% TSB compared to cells grown in 100% DMEM + FBS. For higher percentages of TSB, a change in U2OS cell morphology and subsequent cell death were observed. Analogously, the growth of S. epidermidis ATCC 35983 dropped considerably when the TSB

Discussion

This paper presents the first experimental set-up to study the race between bacteria and tissue cells for a biomaterial surface in vitro. The need for such a system is enormous, as many of the coatings that are currently being propagated to attract low numbers of adhering bacteria, such as polymer brush coatings [15], [20], may also be expected to support poor adhesion and spreading of tissue cells. This is stimulating the development of bifunctional coatings that support cell spreading and

Conclusion

A novel in vitro methodology to study the race between bacteria and tissue cells for a biomaterial surface has been developed. Although due to the cell type chosen and use of a staphylococcal strain, the methodology described here may seem geared toward orthopedic applications, we emphasize that its principles are equally applicable to other implant systems, such as surgical meshes and vascular grafts. Both the absence and presence of flow, as well as the number of adhering bacteria, appear to

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