Targeted plasma functionalization of titanium inhibits polymicrobial biofilm recolonization and stimulates cell function

Highlights

• Low temperature plasma simultaneously decontaminates and functionalizes titanium.

• Plasma exposure creates nanoscale features and superhydrophilic characteristics.

• Plasma exposure is highly effective in eliminating polymicrobial biofilms.

• Plasma treated surface inhibits microbial recolonization and biofilm formation.

• Biocompatibility improved through enhanced cell adhesion and proliferation.

Abstract

Biofilm contamination on an implanted medical device represents a particularity resilient reservoir of infection that inevitably leads to device failure. In this study, we demonstrate that an atmospheric pressure air plasma treatment can simultaneously eradicate biofilm contamination while beneficially functionalizing the underlying surface, creating long-lasting characteristics that inhibit microbial recolonization and promote fibroblast proliferation. By comparing two contrasting plasma treatments the interplay between plasma generated reactive species, biofilm contamination and the underlying surface was uncovered. The composition, wettability and topography of titanium surfaces were characterized using X-ray photoelectron spectroscopy, water contact angle measurements and atomic force microscopy. Exposure to plasma generated chemical species created nanoscale surface features and the introduction of oxygen and nitrogen containing functional groups, resulting in changes to the surface wettability. Using a polymicrobial biofilm model comprising of E. coli and S. epidermidis, it was shown that plasma can effectively eliminate biofilm contamination from the surface, while simultaneously functionalizing the surface to inhibit recolonization. To assess the biocompatibility of treated surfaces the adhesion and proliferation of murine fibroblasts was assessed using fluorescent microscopy, cell viability assays and flow cytometry. It was shown that plasma exposure led to surface characteristics that promote fibroblast adhesion and proliferation.

Introduction

In natural and manmade environments, bacterial biofilms are ubiquitous, yet the significance of biofilm contamination in a clinical setting is often underestimated [1]. Biofilms have a complex architecture, with physiologically organized bacterial microcolonies. It is the structure and organization of the biofilm that conveys numerous advantages over unprotected planktonic cells, providing protection against immune system defense and the diffusion of antibiotics. Several studies have posited that resistance against antimicrobial agents in a biofilm can be up to 1000 times greater compared to single cells [2]. Such contamination presents a particularly virulent form of infection and provides ideal conditions for the emergence of multidrug-resistant colonies. A plethora of life-threatening infections can arise as a result of biofilm formation on indwelling and implanted medical devices, such as urinary catheters, mechanical heart valves, prosthetic joints and endotracheal tubes [3]. In the field of orthopedics, infections related to biofilm colonization of prosthetic devices are one of the most serious and devastating complications. In such cases, complex and expensive revision procedures are typically required, often involving an attempt to clean the device in-vivo or its complete removal and replacement [4]. Despite the incidence of such cases being relatively low, estimated to be in the range of 0.5–5% for total joint replacements, the large population of patients with orthopedic implants means such infections have a major impact in terms of morbidity, mortality and medical costs [5].

One of the most common bacterial species linked to orthopedic implant-related infections are coagulase-negative Staphylococcus species. >40% of all infections are linked to species from this Gram-positive bacterial genus, with S. epidermidis and S. aureus being the most commonly diagnosed bacteria in implant related infections [6]. Infections often arise because the surface of the implanted device provides an ideal environment for bacterial adhesion and the formation of biofilms, which can reach a thickness of >100 mm in some cases [7]. Orthopedic implant infections can develop during different stages of a patient's medical treatment; pre-operatively, when infections are associated with fraction fixation, intra-operatively and during the post-operative period, mainly because of abnormal wound healing. The development of biofilms on an implant surface is a highly dynamic and competitive process, involving different constituents, such as extracellular matrix (ECM) proteins, host cells (endothelial cells, fibroblast, bone cells) and microorganisms. Initially, the adsorption of proteins and macromolecules of ECM occurs, and under normal physiological conditions, these components govern the adhesion, migration, proliferation and differentiation of tissue cells. In the case of implanted material, ECM bound on the surface can also act as a substrate for bacterial attachment thus a competitive process is initiated. A positive surgical outcome relies on the ability of fibroblast to adhere and proliferate on the surface of implant, rather than bacterial colonization and ultimately biofilm formation [8].

A number of different technologies are currently under investigation to inhibit biofilm formation on implanted devices. Certainly, the deposition of functional coatings on to devices prior to implantation has shown promise, with drug eluting coatings [[9], [10], [11]], bactericidal coatings [[12], [13], [14]], adhesion resistant chemistries [15,16], and nano-topologies all demonstrating the potential to prevent device colonization [17,18]. While these approaches are highly successful in vitro, it is not clear how effective they are in vivo or how they affect clinical outcomes. Alternative approaches focusing on the in-situ cleaning of a contaminated device are also under investigation, including the use of electrical stimulation on the orthopedic implant surface [19,20], applied pulsed electromagnetic fields [21], laser-generated shockwaves [22] and sonication [23]. Such methods typically rely on a mechanical mode of action to remove adhered bacteria from the implant surface; clearly, these methods are highly successful ex-vivo, but their application in-vivo is problematic meaning recurrent infections are unavoidable [7].

Low temperature plasma is an alternative technique that has shown great promise for both the surface functionalization of medical implants and the rapid decontamination of biofilms on clinical surfaces. Low and atmospheric pressure plasmas have long been used to functionalize biomaterials [24], including the creation of protein-resistant surfaces [25], the modification of surfaces to promote haemocompatibility and encourage cell adhesion [26,27], and the introduction of antimicrobial characteristics [28]. Beyond surface functionalization, low temperature plasma has been used to remove microbial contamination from a wide range of biomaterials [29], including the destruction of biofilms from both polymeric and metallic surfaces [[30], [31], [32]]. Developments within the field of atmospheric pressure plasma technology have enabled the creation of plasma devices capable of creating stable discharges in ambient air that produce controlled mixtures of highly Reactive Oxygen and Nitrogen Species (RONS) directly at the point of need. While the interplay between the plasma, biofilm contamination and the underlying surface is complex and not yet fully understood, there exists an exciting possibility that such plasmas could be used to simultaneously decontaminate and functionalize an implant surface in-vivo, thus removing the infection while imparting preferential surface characteristics to promote cell attachment and inhibit bacterial recolonization.

In this study, we investigated the surface treatment of medical grade titanium using two common atmospheric pressure air plasma systems. By comparing and contrasting the unique characteristics of each plasma treatment, their ability to inactivate polymicrobial biofilms while simultaneously imparting long-lasting antimicrobial and cell-promoting characteristics over timescales that are relevant for post-surgery wound healing were uncovered. It was demonstrated that a direct plasma treatment, where highly reactive RONS interact with the titanium surface, had a significant and beneficial impact on both the composition and morphology of the surface while being able to simultaneously eliminate biofilm contamination. The study unravels the complex interaction between biofilm contamination and the underpinning surface during and after plasma treatment; ultimately demonstrating that a targeted air plasma treatment can be successfully applied to decontaminate and functionalize a titanium surface in a single step. These results suggest that a targeted air plasma treatment could play a considerable role in the fight against implant-associated infections.