Nitrogen plasma surface modification enhances cellular compatibilityof aluminosilicate glass

Georgia Kaklamani a, Nazia Mehrban b, James Bowen b,n, Hanshan Dong a,Liam Grover b, Artemis Stamboulis a

a University of Birmingham, School of Metallurgy and Materials, Edgbaston, Birmingham B15 2TT, UKb University of Birmingham, School of Chemical Engineering, Edgbaston, Birmingham B15 2TT, UK

a r t i c l e i n f o

Article history:Received 29 July 2013Accepted 25 August 2013Available online 31 August 2013

Keywords:Active screen plasma nitridingFibroblastGlassIonomerSurface

a b s t r a c t

The effect of Active Screen Plasma Nitriding (ASPN) treatment on the surface-cellular compatibility of aninert aluminosilicate glass surface has been investigated. ASPN is a novel surface engineering technique,the main advantage of which is the capacity to treat homogeneously all kind of materials surfaces of anyshape. A conventional direct current nitriding unit has been used together with an active screenexperimental arrangement. The material that was treated was an ionomer glass of the composition4.5SiO2–3Al2O3–1.5P2O5–3CaO–2CaF2. The modified glass surface showed increased hardness and elasticmodulus, decreased surface roughness. The incorporation of nitrogen-containing groups was confirmedusing X-ray photoelectron spectroscopy. The modified surface favoured attachment and proliferation ofNIH 3T3 fibroblasts.

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1. Introduction

Surface modification is employed to alter the surface charac-teristics of materials while maintaining the bulk properties [1–3].Surface modification of biomaterials is often required to alterthose characteristics that play a role in their interaction with thebiological environment [4–7]. Plasma treatment modifies the sur-face properties of materials [8] resulting in changes extendingfrom a few nanometres to �10 μm without affecting the bulkproperties of the material [9], surface chemistry can be selectivelycontrolled and a variety of chemical structures can be produced[10]. Plasma treatment affords control over wettability, chemicalinertness, hardness and biocompatibility [11,12]. Plasma-modifiedmaterials exhibit amended response characteristics when in con-tact with the biological environment [13]. Active Screen PlasmaNitriding (ASPN) is a surface engineering method historically usedto modify metallic surfaces such as stainless steel, chromium andtitanium by introducing nitrogen into the surface improving thesurface microhardness, wear and corrosion resistance, microstruc-ture and morphology [14–17]. ASPN offers reduced gas and energyconsumption, minimisation of pollutant emission, and short treat-ment times. Despite the fact that conventional direct currentplasma nitriding has been proved efficient in the treatment ofsimple shapes and small loads, there exist many difficulties during

the treatment; these include (i) maintaining uniform chambertemperature, (ii) arcing, and (iii) non-uniform appearance at edges[18–22]. Conversely, ASPN is a relatively novel surface modifica-tion that allows the homogeneous treatment of surfaces of anymorphology [23].

Ionomer glasses based on the general composition SiO2–Al2O3–

P2O5–CaO–CaF2 are used glass ionomer cements in dentistry [24].Recent developments led to glass compositions that offer radio-pacity, translucency, controlled setting reaction, as well as release oftherapeutic ions such as fluorine and strontium [25–28]. The glasscomposition used in this study, 4.5SiO2–3Al2O3–1.5P2O5–3CaO–2CaF2, has been extensively characterised and both its structureand crystallisation mechanism are well understood [29–31]. Plasmasurface modification on a glass surface (without the addition ofcoatings) in order to study the cellular compatibility of the modifiedsurface has not previously been conducted. Therefore the aim of thiswork is to investigate the effect of ASPN on the surface properties ofan inert glass composition as well as the effect of the treatment onfibroblast/surface interactions.

2. Materials and methods

The glass composition 4.5SiO2–3Al2O3–1.5P2O5–3CaO–2CaF2was produced by a melt quench route that has been previouslyreported in detail [29]. Cylindrical samples (diameter 15 mm,length 20 mm) were cast and polished following this method. ASPNsurface modification has been described previously by Li et al. [32].

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n Corresponding author. Tel.: þ44 121 414 5080.E-mail address: j.bowen.1@bham.ac.uk (J. Bowen).

Materials Letters 111 (2013) 225–229

Prior to treatment all samples were cleaned with distilled water andethanol. Samples were ASPN treated at 400 1C in a gas mixture of25% N2 and 75% H2 for 1 h; the plasma chamber pressure was2.5 mbar, the voltage was 250 V and the current was 1A. Aftertreatment all samples were stored under vacuum (10�2 mbar). Thehardness and elastic modulus of the surfaces were measured beforeand after treatment by nanoindentation. The hardness and elasticmodulus were calculated from the mean of six measurements. Theequipment used was a Nano Test 600 machine (Micro Materials,UK). Surface topography was measured before and after treatmentusing a MicroXAM interferometer (Omniscan, UK) operating awhite light source. Scanning Probe Image Processor software (ImageMetrology, Denmark) was employed for the analysis of the acquiredimages, yielding average roughness (Sa) and root-mean-squareroughness (Sq) values, which were the mean of five measurementsat separate locations. Scan areas sufficiently large as to be repre-sentative of the overall surface of the sample were identified. X-rayphotoelectron spectroscopy (XPS) was performed to examine anychemical changes introduced on the surface of the treated glass.A bespoke XPS was used for the analysis of both treated anduntreated samples. The software used was produced by PSP Ltd, UK.The pass energy was 50 eV and the X-ray gun operated at 10 keV.Spectra were acquired over the binding energy range �10 to1200 eV. The vacuum pressure in the analysis chamber was lessthan 10�8 mbar.

All chemicals for the cell culture studies were purchased fromSigma-Aldrich UK. NIH 3T3 fibroblasts were used to study thecellular compatibility of the treated and untreated glass surfaces.Prior to cell seeding, the treated and untreated glass surfaces wereautoclaved for 15 min at 120 1C. 3T3 fibroblasts were cultured for aweek using the standard 3T3 protocol. Cells were grown inS-DMEM (Supplemented Dulbecco’s Modified Eagle's Medium)supplemented with 10% FBS, 2.4% L-glutamine, 2.4% HEPES bufferand 1% penicillin/streptomycin, and fed every second day duringthe cell culture. All sterilised glass samples were placed in 12 well-plates, seeded at a cell density of 1.2�106 cells/sample andincubated at 37 1C at 5% CO2 and 100% relative humidity for 4 days.The cell seeded surfaces were examined using scanning electronmicroscopy (SEM, JSM 6060 LV, JEOL, Oxford Instruments Inca,UK). The operating voltage was 10 kV whereas the workingdistance was 10 mm and the spot size was 3 nm. Prior to testing,the cell seeded samples were chemically fixed following a stan-dard protocol described previously in detail [33]. The fixed cellseeded samples were Pt coated by sputtering at 25 mA and 1.5 kV.The thickness of the Pt coating was 10–12 nm.

3. Results and discussion

Table 1 shows the nanoindentation results of the plasmatreated and untreated glass. The hardness and elastic moduluswere clearly increased after the treatment. According to theliterature, plasma treatment has been used in the past in orderto improve the mechanical properties of glasses, ceramics andpolymers [34–37]. Schrimph et al. reported that the incorporationof nitrogen into silicate melts increased the hardness, refractiveindex, chemical durability and glass transition temperature (Tg)proportionally to increasing nitrogen content [38]. Grande et al.

also reported that quenching of fused mixtures of nitrides andoxides or reacting NH3 with molten oxides on the surface ofsilicate and phosphate glasses resulted in an increase of thesurface hardness. This was explained based on the assumptionthat only one of the four divalent oxygen atoms surrounding theglass formers, in this case silicon and phosphorus, could bereplaced by trivalent nitrogen resulting in a more tightly linkedglass network [39]. In our case there is a significant increase in thehardness and elastic modulus after the ASPN treatment; theseresults are shown in Table 1. As the treatment temperature waswell below the glass transition temperature of the glass (around670 1C) as reported previously [40], at 400 1C there is a possibilitythat annealing effects may have resulted in the incorporation oftrivalent nitrogen in the glass network, having the effect ofcrosslinking further the glass network at the surface and thereforeincreasing hardness and elastic modulus. Further investigationhowever should be conducted in order to establish the hardeningmechanism.

Fig. 1(a) and (b) shows the surface topography of both treatedand untreated glass samples. The Sa and Sq for the untreated glassare 96.8 nm and 121.0 nm respectively, while for the treated glasssurface the Sa and Sq were 66.2 nm and 89.5 nm respectively; thesevalues demonstrate that ASPN treatment significantly decreased thesurface roughness. Furthermore, some surface porosity wasobserved on the surface of the ASPN treated glass exhibiting depths4200 nm below the sample surface, as shown in Fig. 1(c). XPSmeasurements of the treated and untreated glass samples revealedthe presence of nitrogen containing species on the plasma modifiedglass surface indicated by the presence of a broad peak at a bindingenergy of 403 eV, associated with N 1 s photoelectrons, as shown inFig. 1(d). The untreated glass surface did not exhibit a peak at thisbinding energy. The nitrogen content of the treated glass surface wasfound to be 1.5% of the total elemental composition. The nitrogen-containing functional groups likely consist of amine (–NH2) moieties,given that the plasma composition is 25% N2 and 75% H2. The aminegroups are likely to be attached to the surface via Si–N bonds. Thetreated and untreated ionomer glass samples were also subjected tocell culture studies. The purpose of this preliminary study was toobserve the cellular response to ASPN treatment. SEM imagesshowing the absence of 3T3 fibroblasts after their seeding on thesurface of untreated glass, following a 4 day incubation period, areshown in Fig. 2(a)–(c). In contrast Fig. 2(d)–(f) shows the presence of3T3 fibroblasts seeded on the ASPN treated glass, following a 4 dayincubation period. This cellular response is in good agreement withprevious work by Freeman et al. [41] wherein an animal study of thesame glass composition did not integrate well with bone. Scar tissueformation was observed at the interface of the glass with the nativetissue, showing that the glass surface was not favourable to cellularcompatibility. On the surface of the ASPN treated glass studied in ourwork, it is clear that the fibroblasts are attached to the samplesurface, indicated by the presence of cytoplasmic projections. Thecells were not only interconnected but also exhibited the appro-priate stellate shape. In addition, the cytoplasmic extensions and thefilopodia of the adjacent cells were clearly conjoined. In vitro cellattachment on material surfaces is usually mediated by glycopro-teins such as fibronectin (Fn) and vitronectin (Vn).

The attachment of cells to surfaces depends on the substratemechanical properties, surface topography, and specifically on thesubstrate chemistry that controls the nature of the protein layer [42].It has been reported that plasma treatments enhance cell attach-ment and proliferation due to the presence of new functional groupson the surface that results in enhanced activity of glycoproteins [43–48]. For example, it has been reported that nitrogen plasma treatedpolymeric surfaces can develop such adsorptive characteristics toattract both Fn and Vn glycoproteins, suggesting that the presence ofamine groups can result in cell attachment enhancement [49,50].

Table 1Hardness and elastic modulus of untreated and ASPN treated glasses.

Material Hardness (GPa) Elastic modulus (GPa)

Untreated glass 8.3 (70.3) 106.5 (72.0)ASPN treated glass 16.5 (72.4) 177.8 (75.9)

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Previous work by Kaklamani et al. showed that a similar ASPNtreatment of ultra-high molecular weight polyethylene (UHMWPE)surfaces promoted fibroblasts attachment and proliferation, while

cells did not attach on the untreated UHMWPE surfaces [33]. Cellsexhibit great sensitivity to substrates with specific nano- and micro-scale topographies [51,52]. Cells are also able to react to the surface

Fig. 1. 2D topography of (a) untreated and (b) ASPN treated glass surfaces, (c) line section of pores on the ASPN treated glass surface, (d) XPS spectra for the N 1sphotoelectron binding energy range.

Fig. 2. SEM images of the untreated glass surfaces 4 days after seeding with 3T3 fibroblasts at magnifications (a) �500, (b) �1000 and (c) �2000; SEM images of ASPNtreated glass surfaces seeded with 3T3 fibroblasts at magnifications (d) �500, (e) �1000 and (f) �2000.

G. Kaklamani et al. / Materials Letters 111 (2013) 225–229 227

mechanical properties. A mechanical balance should be maintainedbetween the cells and the environment they grow in, for tissueformation, cohesion, homeostasis and signalling between the cellsand the substrate [53]. Hence, cellular behaviour depends on themechanical properties of the tissue substrate they are attached to[54]. In the work presented here, the plasma treatment has littleeffect on the surface topography. The surface exhibits many orders ofmagnitude greater modulus than cells, although the hardnessand Young's modulus of the surface are increased further by plasmatreatment. However it is not anticipated that this will affect cellattachment. It is likely that the change in surface chemistry,specifically the incorporation of nitrogen-containing groups suchas amine moieties, which enhances the adhesion of fibroblasts.

Generally, a study on the attachment of cells on a plasma modifiedsurface has not been reported and there is minimal literature availablein this area. Considering the simplicity of the plasma treatment, aswell as the advantage of a homogeneous surface treatment, makesthis technique extremely attractive and further study in the area ofcellular activity could lead to the technique reaching its full potentialin this area of research.

4. Conclusions

The surface of an ionomer glass composition was subjected toASPN treatment for 1 h at 400 1C in a gas mixture of 25% N2 and75% H2. The treatment resulted in:

(1) Increased hardness and elastic modulus of the glass surface.(2) Decreased surface roughness.(3) Incorporation of nitrogen containing chemical species on the

glass surface.(4) Greatly enhanced surface biocompatibility and adhesion of

3T3 fibroblasts.

Acknowledgements

The Interferometer and Nanoindenter used in this research wasobtained, through Birmingham Science City: Innovative Uses forAdvanced Materials in the Modern World (West Midlands Centrefor Advanced Materials Project 2), with support from AdvantageWest Midlands (AWM) and part funded by the European RegionalDevelopment Fund (ERDF).

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