Poly(propylene glycol) and urethane dimethacrylates improve conversion of dental composites and reveal complexity of cytocompatibility testing

dental materials 3 2 ( 2 0 1 6 ) 264–277 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema Poly(propylene glycol) and urethane dimethacrylates improve conversion of dental composites and reveal complexity of cytocompatibility testing Nick J. Waltersa,1, Wendy Xiaa, Vehid Saliha,b, Paul F. Ashleyc, Anne M. Younga,∗ aDivision of Biomaterials & Tissue Engineering, UCL Eastman Dental Institute, 256 Gray’s Inn Road, London WC1X 8LD, UK bPlymouth University Peninsula Schools of Medicine and Dentistry, Portland Square, Drake Circus, Plymouth, Devon PL4 8AA, UK cPaediatric Dentistry, UCL Eastman Dental Institute, 256 Gray’s Inn Road, London WC1X 8LD, UK a r t i c l e i n f o Article history: Received 7 January 2015 Received in revised form 20 May 2015 Accepted 30 November 2015 Keywords: Dental composite Dental material Monomer Cytocompatibility Degree of conversion Polymerization shrinkage PPGDMA TEGDMA UDMA Bis-GMA a b s t r a c t Objectives. To determine the effects of various monomers on conversion and cytocompatibility of dental composites and to improve these properties without detrimentally affecting mechanical properties, depth of cure and shrinkage. Methods.Composites containing urethane dimethacrylate (UDMA) or bisphenol A glycidyl methacrylate (Bis-GMA) with poly(propylene glycol) dimethacrylate (PPGDMA) or triethylene glycol dimethacrylate (TEGDMA) were characterized using the following techniques: conversion (FTIR at 1 and 4 mm depths), depth of cure (BS EN ISO 4049:2009 and FTIR), shrinkage (BS EN ISO 17304:2013 and FTIR), strength and modulus (biaxial flexural test) and water sorption. Cytocompatibility of composites and their liquid phase components was assessed using three assays (resazurin, WST-8 and MTS). Results.UDMA significantly improved conversion, BFS and depth of cure compared to BisGMA, without increasing shrinkage. UDMA was cytotoxic at lower concentrations than Bis-GMA, but extracts of Bis-GMA-containing composites were less cytocompatible than of those containing UDMA. PPGDMA improved conversion and depth of cure compared to TEGDMA, without detrimentally affecting shrinkage. TEGDMA was shown by all assays to be highly toxic. Resazurin, but not WST-8 and MTS, suggested that PPGDMA exhibited improved cytocompatibility compared to TEGDMA. ∗Corresponding author. Tel.: +44 20 3456 2353. E-mail addresses: nick.walters@uta.fi (N.J. Walters), anne.y[email protected] (A.M. Young). 1Current address: Adult Stem Cell Group, BioMediTech (Institute of Biosciences & Medical Technology), FinnMedi 5, 33014 University of Tampere, Finland. http://dx.doi.org/10.1016/j.dental.2015.11.017 0109-5641/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 265 Significance. The use of UDMA and PPGDMA results in composites with excellent conversion, depth of cure and mechanical properties, without increasing shrinkage. Composites containing UDMA appear to be slightly more cytocompatible than those containing Bis-GMA. These monomers may therefore improve the material properties of dental restorations, particularly bulk fill materials. The effect of diluent monomer on cytocompatibility requires further investigation. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Dental composites are widely used as dental restorative materials for their high strength and excellent aesthetics. These consist primarily of: a liquid phase containing monomers and an initiator system (typically photo-activated); a filler phase (typically boro-aluminosilicate glass particles), which provides optimal mechanical and aesthetic properties; and silane, a coupling agent which enables bonding of the polymer to the filler. Bisphenol A glycidyl methacrylate (Bis-GMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) are some of the most commonly used monomers in dental composites. Upon photoinitiation, they form a crosslinked polymer network which hardens and entraps the fillers [1]. Composites are typically layered in increments and this is time-consuming for clinicians, since it requires curing each increment before proceeding with the next. This is particularly an issue in deeper posterior cavities, in which many increments may be required. As a result, bulk fill materials aim to overcome this issue by utilizing photoinitiators which are effective at depths of 4–5 mm, as well as monomers with low double bond concentration and, in some cases, monomers which are cleaved during polymerization. One of the major limiting factors of composites is the close interplay between degree of conversion and other characteristics, including mechanical properties, polymerization shrinkage, water sorption and elution of toxic components. Since the level of residual monomer in a composite affects its biocompatibility, mechanical properties and aesthetics [2], high conversion is ideal for optimization of these properties. Conversely, however, high conversion is typically associated with high volumetric shrinkage. In the patient, this can result in microbial microleakage (penetration of pathogens between the composite and tooth), recurrent caries and, ultimately, failure of the restoration. Monomers with low double bond concentration and subsequent low shrinkage are therefore optimal, particularly in the case of bulk fill materials, due to the larger volume of each increment. The aim of the present research was to improve the conversion, strength and cytocompatibility of dental composites without detrimentally affecting polymerization shrinkage or depth of cure by fully replacing Bis-GMA with UDMA and TEGDMA with PPGDMA. This is due to the greater flexibility and cross-linking density of UDMA than Bis-GMA [3,4] and the greater flexibility and significantly lower double bond concentration of PPGDMA than TEGDMA. In order to investigate the effect of each monomer on cytocompatibility, human gingival fibroblasts (HGF) were cultured in solutions of each individual liquid phase component at varying concentrations, as well as in extracts of each composite formulation. Due to the variability between different cell viability assays, which arises from the targeting of different enzymes within the cell, and the ambiguity of the widely used term ‘biocompatibility’ [5], three assays were compared. The null hypothesis was that replacement of Bis-GMA with UDMA and TEGDMA with PPGDMA would have no effect on these properties. 2. Materials and methods 2.1. Materials 2.1.1. Composite paste preparation Microhybrid dental composites [1] were prepared using 10 wt% 40 nm fumed silica (Aerosil OX-50, Evonik Industries AG, Essen, Germany) and 90 wt% silane-treated barium boro-aluminosilicate glass particles of various sizes (DMG Chemisch-Pharmazeutische Fabrik GmbH, Hamburg, Germany). These were combined with four dimethacrylatebased liquid phases. The liquid phases consisted of bulk monomer UDMA (DMG) or Bis-GMA (Polysciences Inc., Eppelheim, Germany) combined with diluent monomer PPGDMA (Polysciences) or TEGDMA (DMG). The bulk to diluent molar ratio was 3.5:1. The liquid phases also contained 40 mM (0.58–0.61 wt%) photoinitiator camphorquinone (CQ, DMG), 60 mM (0.82–0.86 wt%) co-initiator N,N-dimethylaminoethyl methacrylate (DMAEMA, Sigma–Aldrich, Gillingham, UK) and 100 ppm inhibitor butylated hydroxytoluene (Sigma–Aldrich). The two phases were combined to form composite pastes using a centrifugal planetary mixer (SpeedMixer, Hauschild Engineering, Hamm, Germany), in order to minimize air incorporation and ensure complete wetting of filler particles. The powder to liquid ratio (PLR) was kept constant at 40 vol% liquid (19.3–20.3 wt%, depending on liquid phase density). Dental composites were designated abbreviations based on their bulk and diluent monomer content: UP, UT, BP and BT, where U, B, P and T represent UDMA, Bis-GMA, PPGDMA and TEGDMA, respectively. Commercial composite Filtek Z250 (3M ESPE, St. Paul, MN, USA) was used for comparison. 2.1.2. Disc specimen production Except where otherwise stated, disc-shaped specimens were moulded by applying composite pastes to metal circlips (internal diameter 10.2 mm, thickness 1 mm) and pressing them between two sheets of acetate. This prevents oxygen inhibition during polymerization and expels excess paste, 266 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 ensuring similar specimen thickness. Specimens were photopolymerized using a blue light emitting diode curing unit with a wavelength of 450–470 nm and power output with periodic level shifting of 1100–1330 mW/cm2(Demi Plus, Kerr Dental, Orange, CA, USA), in direct contact with the acetate. The curing duration for each testing method varied and is detailed in each corresponding section. 2.2. Methods 2.2.1. Handling properties and wet-point determination The wet-point of each liquid was determined by gradually adding a small quantity of liquid phase to a known mass of filler phase and mixing, until the filler was sufficiently wetted and a cohesive paste had been formed. The quantity of liquid phase added was recorded and notes were made regarding the handling properties of each formulation. The wet-point (vol%) was then calculated from the total mass of liquid and the density of each component. 2.2.2. Polymerization properties 2.2.2.1. Degree of conversion. The conversion of each composite was determined using Fourier transform infrared spectroscopy (FTIR, System 2000, PerkinElmer, Seer Green, UK). Composite paste was applied to either a single or four stacked circlips of the same dimensions. These were placed on the diamond of an attenuated total reflectance accessory (Golden Gate ATR, Specac Ltd., Orpington, UK) and covered with a sheet of acetate. After an initial spectrum of the uncured composite had been obtained, spectra were recorded continuously for 1000 s (n = 3). The specimens were photo-polymerized from the top for the first 20 s. Spectra were recorded at a wavelength range of 800–1800 cm−1and resolution of 8 cm−1. Absorbance profiles were obtained at 1319 ± 1 cm−1(C–O stretch bond) and 1334 ± 2 cm−1(baseline) and used to calculate conversion at 1 mm and 4 mm depths using Eq. (1): C =1 −Af Ao× 100 (1) where C is conversion. Afand A0are final and initial absorbance above baseline, respectively [6]. 2.2.2.2. Polymerization shrinkage. 2.2.2.2.1. Shrinkage based on conversion. The volumetric shrinkage of composites was calculated from the conversion data using Eq. (2) [7], which is based on the finding that methacrylate esters typically undergo volumetric shrinkage of s = 22.5 cm3/mol upon polymerization [8,9]: S = 100 ×smCinixi Wi (2) where S, m, C, , ˙, i, n, x and W are shrinkage (vol%), monomer mass fraction within composite, conversion, composite density, sum of all monomers in liquid phase, each monomer in liquid phase, number of C C bonds per molecule, mass fraction of monomer in liquid phase and molecular mass, respectively. 2.2.2.2.2. Shrinkage based on volume change. The shrinkage of composites was also obtained by measuring the density of polymerized and unpolymerized specimens according to BS EN ISO 17304:2013 [10]. This technique uses an analytical balance equipped with a density determination apparatus (AG 204 & MS-DNY-43, Mettler Toledo, Beaumont Leys, UK) and is based on Archimedes’ principle. Disc specimens were cured for 40 s from each side to ensure complete conversion. Specimen edges were polished to remove loose chips. The mass of three cured and three uncured specimens of each formulation were measured in air and under deionized water. Each value for mass under water was averaged from 10 readings. The shrinkage of composites and their SD were calculated using the equations provided in the standard. 2.2.2.3. Depth of cure. The depth of cure of composites (n = 3) was measured according to BS EN ISO 4049:2009 [11]. Briefly, composite paste was applied to a brass split-mould (internal diameter 4 mm, height 6 mm) and photo-polymerized for 20 s from the top. The specimen was removed from the mould and a plastic spatula was used to remove any uncured material from the bottom. The depth of cured material was measured using digital callipers to an accuracy of ±0.01 mm and the reading was halved, as required by the standard, in order to give a value for depth of cure. 2.2.2.4. Water sorption. In order to determine water sorption, disc specimens were cured for 40 s from each side to ensure maximum conversion. The mass of composites before and after immersion in 10 mL deionized water for one week was then determined using an analytical balance. 2.2.3. Mechanical properties Composite disc specimens (n = 10) were cured for 40 s from each side. Specimens were stored dry for 24 h, before placement in 10 mL deionized water at 37 ◦C for one week. They were then tested using a biaxial flexural test (Autograph AGSX, Shimadzu, Milton Keynes, UK), with a 2 kN load cell and ball-on-ring jig at a cross-head speed of 1 mm/min, until specimen failure. Biaxial flexural strength (BFS, MPa) and modulus of tensile elasticity (E, GPa) were calculated using Eqs. (3) and (4), respectively, BFS =P t2(1 + v)0.485 ln a t+ 0.52+ 0.48(3) E =P wc×ˇca2 t3(4) where BFS, P, t,  and a are biaxial flexural strength, failure load (N), specimen thickness (mm), Poisson’s ratio (0.3) and jig support radius (4 mm), respectively, and E, (P/Wc) and ˇc are modulus, gradient of elastic region and center deflection function (0.5024) [12], respectively. 2.2.4. Cytocompatibility 2.2.4.1. Cell culture. Primary HGF were obtained from a commercial source (ScienCell Research Laboratories, Carlsbad, CA, USA). HGF were cultured under standard conditions (37 ◦C, 95% air, 5% CO2, 95% relative humidity) in Dulbecco’s d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 267 modified Eagle medium (DMEM, Gibco, Life Technologies, Paisley, UK) supplemented with 10% foetal bovine serum (Gibco) and 1% penicillin/streptomycin (PAA Laboratories, GE Healthcare, Chalfont St. Giles, UK). Passage numbers 4–8 were used for cytocompatibility studies. 2.2.4.2. Preparation of test solutions. Test solutions were prepared in serum-free DMEM. As is typical in biomaterials testing, serum-free medium was used in order to prevent adsorption of serum proteins to material components. Controls consisted of serum-free DMEM. 2.2.4.2.1. Liquid phase components. Five ten-fold serial dilutions of each component were prepared in serum-free DMEM, ranging from 0.01 to 100 mM for DMAEMA, UDMA, PPGDMA and TEGDMA, and 0.001 to 10 mM for CQ and BisGMA, due to their lower solubility. The solutions were stored for ∼30 min at 60 ◦C and then stirred using a sterile spatula, in order to aid dissolution of components with low solubility, particularly the bulk monomers. 2.2.4.2.2. Composite extracts. In order to prepare specimens for extract testing, a 1 mm thick circlip atop a sheet of acetate was filled with composite and covered with acetate. A further three circlips were stacked on top and filled with composite and covered with acetate. The resultant 4 mm deep stack was then photo-polymerized for 20 s from the top. The bottom 1 mm thick section was removed from the mould and incubated in 650 ␮L serum-free DMEM at 37 ◦C. This provided an extraction ratio of 1 mL/3 cm2surface area, as required by ISO 10993-12:2009 [13]. Specimens (n = 3) were agitated at 100 rpm during extraction (orbital shaker, Stuart Scientific, Stone, UK). After 24 h, specimens were transferred to fresh medium and incubated for a further 6 days, yielding extracts from 1 and 7-day time-points. 2.2.4.3. Cytocompatibility assays. HGF were seeded at a density of 30,000 cells/cm2in 96 well plates (n = 3 per specimen, time-point and assay). After an initial 24 h seeding period, cell culture medium was replaced with 100 ␮L of the specimen (component solution, composite extract or control). The solutions were thoroughly mixed using a vortex mixer (Stuart SA8, Bibby Scientific, Stone, UK) in order to ensure even dispersion of dissolved components. Cytocompatibility was assessed after a further 24 h of culture (48 h time-point), in accordance with ISO 10993-5:2012 [14]. The specimen was then replaced with serum-free DMEM and cultured for a further 24 h recovery period (72 h time-point). Three water-soluble cell metabolic activity assays were used to assess cytocompatibility: resazurin (alamarBlue Cell Proliferation Assay, AdB Serotech, Bio-Rad Laboratories Inc., Hemel Hempstead, UK); water-soluble tetrazolium salt-8 (WST-8, Cell Counting Kit-8, Sigma–Aldrich); and 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS, CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Southampton, UK). Medium was aspirated from the cells and replaced with 100 ␮L cell culture medium containing the corresponding substrate (Table 1). After incubation at 37 ◦C, fluorescence (FLx800, BioTek, Potton, UK) or absorbance (Infinite M200, Tecan, Männedorf, Switzerland) was measured. Standard curves were obtained by seeding a Table 1 – Cytocompatibility assay parameters. Substrate Incubation time (min) Measurement Resazurin, 10% 90 Fluorescence – excitation: 560 nm, emission: 590 nm WST-8, 10% 80 Absorbance – 460 nm (reference: 650 nm) MTS, 20% 60 Absorbance – 490 nm (reference: 630 nm) range of cell concentrations in 96 well plates, 2 h prior to each assay. 2.2.5. Statistical analyses One-way analysis of variance (ANOVA) and post-hoc Tukey’s tests were used to determine significance (p ≤ 0.05) between composite formulations (UP, UT, BP, BT and Z250). Two-way ANOVA and post-hoc Tukey’s tests were also used in order to determine the significance of the effects of multiple factors, e.g. UDMA vs. Bis-GMA and PPGDMA vs. TEGDMA. For all techniques, the standard deviation (SD) of each formulation was displayed on graphs, except in the case of polymerization shrinkage, where the SD was averaged for each technique, due to the high variability of the volume change method. Statistical significance is presented on graphs (*p < 0.05, **p < 0.01, ***p < 0.005 or ****p < 0.001). 3. Results 3.1. Handling properties and wet-point All experimental formulations had wet-points of 33.3 ± 0.4 vol% liquid. At this level, formulation pastes containing Bis-GMA felt considerably less malleable during specimen moulding than those produced with UDMA. At 40 vol% liquid, all formulations were slightly more malleable, but formulations containing Bis-GMA still exhibited lower malleability than those containing UDMA. Z250 had handling properties between those of Bis-GMAand UDMA-containing composites. No discernible difference in handling properties was observed between PPGDMA and TEGDMA. 3.2. Polymerization properties 3.2.1. Conversion Experimental composites containing UDMA had, on average, 1.2 and 1.3 times higher conversion at 1 and 4 mm depth, respectively, than those containing Bis-GMA (average p-value of both depths = 0.001) (Fig. 1a). Replacement of TEGDMA with PPGDMA further increased conversion by 1.1 and 1.2 times at 1 and 4 mm, respectively (average p-value of both depths < 0.01). UP had the highest conversion (68% and 65% at 1 and 4 mm). At both depths, UP’s high conversion was highly statistically significant compared to BP, BT and Z250 (p < 0.001) and at 1 mm, it was significantly higher than that of UT (p < 0.05). Z250 had the lowest (50% and 39% at 1 and 4 mm) and was comparable to BT. Composites containing Bis-GMA (BP, BT and Z250), showed a significant reduction in conversion upon increasing depth from 1 to 4 mm. 268 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 Fig. 1 – (a) Conversion of composites at 1 and 4 mm depth. Columns represent mean, error bars represent SD. (b) Shrinkage of 1 mm thick composite discs, calculated from conversion or volume change. Columns represent mean, error bars represent mean SD of the corresponding technique (±0.05 and ±0.23 vol% for conversion and volume change, respectively). (c) Depth of cure of composites. (d) Water sorption of composites after immersion in deionized water for one week. (c, d) Crosses represent mean, boxes represent 25–75 percentiles, error bars represent SD. 3.2.2. Shrinkage Based on conversion at 1 mm depth, the shrinkage of UDMAcontaining composites was predicted to be 1.2 times higher, on average, than those containing Bis-GMA (p < 2 × 10−5) (Fig. 1b). Composites containing TEGDMA were estimated to have 1.1 times higher shrinkage than those containing PPGDMA (p < 0.04). When shrinkage was determined by volume change, however, no statistically significant effect of monomer could be observed. This was due to the high standard deviation of the technique. The shrinkage of Z250 could not be calculated using conversion data, since its exact composition was unknown. The measured shrinkage, however, of Z250 was comparable to all experimental formulations. 3.2.3. Depth of cure The depth of cure (Fig. 1c) was highly comparable for all composites (2.7 ± 0.25 mm), regardless of monomer composition (F = 0.96). d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 269 3.2.4. Water sorption The water sorption of formulations containing TEGDMA was, on average, 1.2 times higher than that of those containing PPGDMA (p < 0.02) (Fig. 1d). On average, UDMA induced ∼1.1 times higher water sorption than Bis-GMA, although this was not statistically significant. Z250 had the lowest water sorption, comparable to that of BP. UT had the highest water sorption, significantly higher than that of BP and Z250 (p < 0.05). 3.3. Mechanical properties Experimental composites containing UDMA had significantly higher strength (153–158 MPa) than those containing Bis-GMA Fig. 2 – (a) Biaxial flexural strength and (b) modulus of tensile elasticity of composites after storage in deionized water at 37 ◦C for one week. Crosses represent mean, boxes represent 25–75 percentiles, error bars represent SD. (p = 1 × 10−7). The strength of BP was significantly higher than that of BT (127 and 99 MPa, respectively; p < 0.05) (Fig. 2a). The modulus of experimental formulations containing PPGDMA was, on average, slightly lower than that of formulations containing TEGDMA (6.15 and 6.75 GPa, respectively; p = 0.009) (Fig. 2b). Z250 had the highest strength (173 MPa) and lowest modulus (5.3 GPa). 3.4. Cytocompatibility 3.4.1. Comparison of resazurin, WST-8 and MTS assays The apparent cell density of HGF after 48 and 72 h culture in serum-free DMEM is shown in Fig. 3. Resazurin and WST-8 assays reported a similar increase in apparent cell density from 30,000 to ∼49,000 cells/cm2between 0 and 48 h. MTS reported a lesser increase in apparent cell density (from 30,000 to 37,000 cells/cm2after 48 h). After 72 h in culture, the resazurin assay reported cell density to have increased to 113,000 cells/cm2, whereas the WST-8 assay reported cell density to be 74,000 cells/cm2. MTS again reported only a small increase in apparent cell density (45,000 cells/cm2). Since the assays reported different cell densities in the controls at different time-points, subsequent data are normalized to the control for the corresponding assay and time-point and are reported in terms of relative metabolic activity. 3.4.2. Composite component cytocompatibility The relative metabolic activity of surviving HGF following exposure to serial dilutions of liquid phase components, as well as following a subsequent recovery period, is presented in Fig. 4. The dashed lines represent the mean value obtained for the DMEM control for each corresponding time-point and assay. All components caused a typical, concentrationdependent reduction in cell number as concentration was Fig. 3 – Initial seeding density of HGF determined by cell counting (0 h, cross), and apparent density after culture in DMEM for 48 h (unfilled symbols) and 72 h (filled symbols), as assessed by resazurin (circle), WST-8 (triangle) and MTS (square) assays. Error bars represent SD. 270 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 Fig. 4 – Metabolic activity (relative to the corresponding control) of surviving HGF after culture in aqueous solutions of composite liquid phase components (a) CQ, (b) DMAEMA, (c) UDMA, (d) Bis-GMA, (e) PPGDMA or (f) TEGDMA. Resazurin (circle), WST-8 (triangle) and MTS (square) assays were performed after 24 h culture in the solutions (48 h time-point, unfilled symbols), and after a subsequent 24 h recovery period in DMEM (72 h time-point, filled symbols). Component concentrations ranged from 0.001 to 10 mM (a and d) or 0.01 to 100 mM (b, c, e and f). Dashed lines represent the mean value of the control, error bars represent SD. d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 271 increased, though the extent of this relationship depended on the assay. In the case of all components, a concentration of 10 mM was sufficient to cause close to 100% reduction in metabolic activity. After 48 h, WST-8 indicated a direct correlation between CQ concentration (Fig. 4a) and metabolic activity, with 0.001 and 0.01 mM having no significant effect on metabolic activity. As concentration was increased to 0.1, 1 and 10 mM, metabolic activity was reduced by 17, 33 and 98%, respectively. Resazurin reported similar levels (±5%) of metabolic activity at most concentrations, whereas MTS values were up to 15% higher. After 72 h, resazurin reported similar levels of relative metabolic activity to after 48 h. WST-8, however, indicated similar levels of metabolic activity to the control (±7%) at all concentrations except ≥10 mM, at which cells did not recover. MTS showed elevated metabolic activity (10–38% greater than that of the control) at all concentrations after 72 h, except ≥10 mM. By contrast, at 48 h, 0.01 mM DMAEMA (Fig. 4b) caused an 86–95% reduction in metabolic activity in all assays, with no recovery after 72 h. All assays indicated that concentrations ≥0.1 mM caused total inhibition of metabolic activity. Similarly, at a critical concentration of 0.01 mM or higher, UDMA (Fig. 4c) and TEGDMA (Fig. 4f) both inhibited metabolic activity in all assays. Bis-GMA (Fig. 4d) appeared to be more cytocompatible than DMAEMA, UDMA and TEGDMA. After 48 h, WST-8 indicated that concentrations of 0.001–0.01 mM had no effect on metabolic activity. It fell, however, by 7 and 66% upon increasing concentration to 0.1 and 1 mM, respectively. The other assays showed a similar trend, but at 0.001–0.1 mM the values reported by resazurin and MTS were ∼10–15% and ∼23–28% lower, respectively. After 72 h, values obtained by WST-8 assay were elevated by between 7 and 20% compared to the previous time-point, except at 10 mM. At 0.001–0.1 mM, metabolic activity appeared higher than the controls. Resazurin reported similar values (±10%) after 72–48 h. HGF exposed to PPGDMA (Fig. 4e) exhibited more complex trends. The three assays gave different results for concentration-dependent effects on relative metabolic activity. According to all assays, at ≥10 mM, PPGDMA completely inhibited metabolic activity after 48 h and this did not recover after 72 h. Upon lowering concentration, there was a small increase in activity according to the WST-8 and MTS assays, but it remained well below control values and was not enhanced by providing additional recovery time. Conversely, at 48 h and PPGDMA level below 0.1 mM, the cell activity according to the Resazurin test tended to higher values than with the control. By 72 h, however, this “excess” metabolic activity was down to control levels. Concentrations of 0.01, 0.1 and 1 mM, PPGDMA caused a reduction in WST-8 activity of 76, 85 and 92%, respectively, after 48 h. These levels remained unchanged after 72 h. MTS showed similar values to WST-8. By contrast, after 48 h, resazurin activity was elevated by 63 and 35% above the control at 0.01 and 0.1 mM, respectively, and at 1 mM was similar to the control. After 72 h, these values fell, with resazurin activity remaining 12% above the control at 0.01 mM, and being 7 and 80% lower than the control at 0.1 and 1 mM, respectively. 3.4.3. Composite extract cytocompatibility The metabolic activity of HGF after culture in composite extracts is presented in Fig. 5. Significant differences were again observed between different assays. In order to facilitate elucidation of trends, data are expressed as a percentage of the control for each corresponding extract (1 or 7 days) and assay time-point (48 or 72 h). After 24 h exposure to 1 day composite extracts (48 h timepoint), a general downward trend in resazurin metabolism was observed as follows: UP > UT ≥ BP > BT ≥ Z250. Although WST-8 activity differed slightly (UP ≈ UT > BP ≈ BT > Z250), both assays confirmed that the extracts from the lower quadrant of 4 mm deep composite samples containing UDMA had less effect on the 48 h metabolic activity than those containing BisGMA. In the case of resazurin, this was statistically significant (p = 0.001). They also showed that Z250 extracts were less cytocompatible than all of the experimental formulations. MTS showed no significant differences between experimental formulations but confirmed the low cytocompatibility of Z250. Metabolic activity in all assays varied from being similar to the control for UP, down to ∼50% of control for Z250. Composites containing TEGDMA caused a slight but significant reduction in resazurin activity compared to PPGDMA (p < 0.01), but WST8 and MTS assays showed no clear trends with regards to the effect of diluent monomer. After 72 h, similar trends were observed in all three assays, with metabolic activity falling to approximately 50% of that of the control for all experimental materials. The only differences between assays were observed with Z250, where resazurin activity had remained similar to the previous timepoint at ∼50%, but WST-8 activity had fallen from 53 to 37% and MTS activity had fallen from 53 to 33%. These differences, however, were not statistically significant. Resazurin and WST-8 suggested that 7-day extracts were more cytocompatible than 1 day extracts, since metabolic activity returned to control levels after 72 h. The trend of the 48 h resazurin assay after 7 days extraction was similar to the corresponding 1 day extract, but with slightly higher values. For example, 48 h assays showed that BT – the least cytocompatible experimental composite – had 75% resazurin activity relative to the control after 7 days extraction, compared to 56% after 1 day. In the case of all experimental formulations, WST-8 activity was similar to the control or slightly elevated by up to 11% after 48 h. The 7-day extract of Z250, however, still caused a significant reduction in WST-8 activity. After 72 h, the resazurin and WST-8 activity of HGF exposed to 7-day extracts had, in general, recovered. Resazurin activity of BT and Z250 remained slightly reduced at 89 and 82%, respectively. After 72 h, WST-8 activity of HGF exposed to all composite extracts was elevated above that of the control by between 10 and 31%. The MTS assay gave significantly different values compared to the other two assays for the 7-day extracts. After 48 h, MTS activity of HGF exposed to all experimental formulations was 60–75% higher than the control and remained similar after 72 h. Z250 values were similar to those reported by WST-8. UDMA was also shown to be more cytocompatible than Bis-GMA in 7-day extracts (48 h resazurin assay, p = 0.03; 72 h WST-8 assay, p = 0.0008; 72 h MTS assay, p = 0.007). WST-8 activity was also significantly higher in cells exposed to 7-day 272 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 264–277 Fig. 5 – Metabolic activity (relative to the corresponding control) of HGF after 48 (a and c) or 72 (b and d) h culture in 1 (a and b) or 7 (c and d) day composite extracts, assessed by resazurin (dark gray), WST-8 (black) and MTS (light gray) assays. Dashed lines represent the mean value of the control, error bars represent SD. extracts of PPGDMA-containing composites than TEGDMAcontaining composites after 48 h (p = 0.04). 4. Discussion As hypothesized, dental composites containing UDMA had significantly higher monomer conversion and slightly better cytocompatibility than those containing Bis-GMA. Similarly, composites containing PPGDMA achieved more complete conversion than those containing TEGDMA. Importantly, the use of UDMA and/or PPGDMA as the sole bulk/diluent monomer had no detrimental effect on the shrinkage or depth of cure of the materials. In addition, composites containing UDMA had improved handling properties compared to those containing Bis-GMA. The variation in the conversion of the composites is attributed to differences in the chemical structures (Fig. 6) and physical characteristics (Table 2) of their constituent monomers. Although UDMA is a slightly smaller molecule than Bis-GMA and, as a result, has slightly higher double bond concentration (ratio of double bonds per molecule to molecular mass), its lower glass transition temperature (Tg) and greater flexibility enable more complete cross-linking [15]. The bulky aromatic groups of Bis-GMA cause steric hindrance, reflected in its very high viscosity value, which is ∼80 times that of UDMA. This reduced flexibility limits the likelihood of methacrylate groups coming into contact with each other and binding. PPGDMA has more than twice the molecular mass of TEGDMA and as a result, its greater flexibility and lower double bond concentration result in improved conversion. The similar depth of cure of the composites is likely to be due to the similar refractive indices of the monomers used