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

den al ma e ials 3 2 ( 2 0 1 6 ) 264–277 A ailable online a www.sciencedi ec .com ScienceDi ec jou nal homepage: www.in l.else ie heal h.com/jou nals/dema Poly(p opylene glycol) and u e hane dime hac yla es imp o e con e sion o den al composi es and e eal complexi y o cy ocompa ibili y es ing Nick J. Wal e sa,1, Wendy Xiaa, Vehid Saliha,b, Paul F. Ashleyc, Anne M. Younga,∗ aDi ision o Bioma e ials & Tissue Enginee ing, UCL Eas man Den al Ins i u e, 256 G ay’s Inn Road, London WC1X 8LD, UK bPlymou h Uni e si y Peninsula Schools o Medicine and Den is y, Po land Squa e, D ake Ci cus, Plymou h, De on PL4 8AA, UK cPaedia ic Den is y, UCL Eas man Den al Ins i u e, 256 G ay’s Inn Road, London WC1X 8LD, UK a i c l e i n o A icle his o y: Recei ed 7 Janua y 2015 Recei ed in e ised o m 20 May 2015 Accep ed 30 No embe 2015 Keywo ds: Den al composi e Den al ma e ial Monome Cy ocompa ibili y Deg ee o con e sion Polyme iza ion sh inkage PPGDMA TEGDMA UDMA Bis-GMA a b s a c Objec i es. To de e mine he e ec s o a ious monome s on con e sion and cy ocompa i- bili y o den al composi es and o imp o e hese p ope ies wi hou de imen ally a ec ing mechanical p ope ies, dep h o cu e and sh inkage. Me hods.Composi es con aining u e hane dime hac yla e (UDMA) o bisphenol A glycidyl me hac yla e (Bis-GMA) wi h poly(p opylene glycol) dime hac yla e (PPGDMA) o ie hylene glycol dime hac yla e (TEGDMA) we e cha ac e ized using he ollowing echniques: con e - sion (FTIR a 1 and 4 mm dep hs), dep h o cu e (BS EN ISO 4049:2009 and FTIR), sh inkage (BS EN ISO 17304:2013 and FTIR), s eng h and modulus (biaxial flexu al es ) and wa e so p ion. Cy ocompa ibili y o composi es and hei liquid phase componen s was assessed using h ee assays ( esazu in, WST-8 and MTS). Resul s.UDMA significan ly imp o ed con e sion, BFS and dep h o cu e compa ed o Bis- GMA, wi hou inc easing sh inkage. UDMA was cy o oxic a lowe concen a ions han Bis-GMA, bu ex ac s o Bis-GMA-con aining composi es we e less cy ocompa ible han o hose con aining UDMA. PPGDMA imp o ed con e sion and dep h o cu e compa ed o TEGDMA, wi hou de imen ally a ec ing sh inkage. TEGDMA was shown by all assays o be highly oxic. Resazu in, bu no WST-8 and MTS, sugges ed ha PPGDMA exhibi ed imp o ed cy ocompa ibili y compa ed o TEGDMA. ∗Co esponding au ho . Tel.: +44 20 3456 2353. E-mail add esses: nick.wal e s@u a.fi (N.J. Wal e s), anne.y[email p o ec ed] (A.M. Young). 1Cu en add ess: Adul S em Cell G oup, BioMediTech (Ins i u e o Biosciences & Medical Technology), FinnMedi 5, 33014 Uni e si y o Tampe e, Finland. h p://dx.doi.o g/10.1016/j.den al.2015.11.017 0109-5641/© 2015 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 265 Significance. The use o UDMA and PPGDMA esul s in composi es wi h excellen con e sion, dep h o cu e and mechanical p ope ies, wi hou inc easing sh inkage. Composi es con- aining UDMA appea o be sligh ly mo e cy ocompa ible han hose con aining Bis-GMA. These monome s may he e o e imp o e he ma e ial p ope ies o den al es o a ions, pa - icula ly bulk fill ma e ials. The e ec o diluen monome on cy ocompa ibili y equi es u he in es iga ion. © 2015 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). 1. In oduc ion Den al composi es a e widely used as den al es o a i e ma e- ials o hei high s eng h and excellen aes he ics. These consis p ima ily o : a liquid phase con aining monome s and an ini ia o sys em ( ypically pho o-ac i a ed); a fille phase ( ypically bo o-aluminosilica e glass pa icles), which p o- ides op imal mechanical and aes he ic p ope ies; and silane, a coupling agen which enables bonding o he polyme o he fille . Bisphenol A glycidyl me hac yla e (Bis-GMA), u e hane dime hac yla e (UDMA) and ie hylene glycol dime hac yla e (TEGDMA) a e some o he mos commonly used monome s in den al composi es. Upon pho oini ia ion, hey o m a c oss- linked polyme ne wo k which ha dens and en aps he fille s [1]. Composi es a e ypically laye ed in inc emen s and his is ime-consuming o clinicians, since i equi es cu ing each inc emen be o e p oceeding wi h he nex . This is pa ic- ula ly an issue in deepe pos e io ca i ies, in which many inc emen s may be equi ed. As a esul , bulk fill ma e ials aim o o e come his issue by u ilizing pho oini ia o s which a e e ec i e a dep hs o 4–5 mm, as well as monome s wi h low double bond concen a ion and, in some cases, monome s which a e clea ed du ing polyme iza ion. One o he majo limi ing ac o s o composi es is he close in e play be ween deg ee o con e sion and o he cha - ac e is ics, including mechanical p ope ies, polyme iza ion sh inkage, wa e so p ion and elu ion o oxic componen s. Since he le el o esidual monome in a composi e a ec s i s biocompa ibili y, mechanical p ope ies and aes he ics [2], high con e sion is ideal o op imiza ion o hese p ope ies. Con e sely, howe e , high con e sion is ypically associa ed wi h high olume ic sh inkage. In he pa ien , his can esul in mic obial mic oleakage (pene a ion o pa hogens be ween he composi e and oo h), ecu en ca ies and, ul ima ely, ailu e o he es o a ion. Monome s wi h low double bond concen a ion and subsequen low sh inkage a e he e o e op imal, pa icula ly in he case o bulk fill ma e ials, due o he la ge olume o each inc emen . The aim o he p esen esea ch was o imp o e he con- e sion, s eng h and cy ocompa ibili y o den al composi es wi hou de imen ally a ec ing polyme iza ion sh inkage o dep h o cu e by ully eplacing Bis-GMA wi h UDMA and TEGDMA wi h PPGDMA. This is due o he g ea e flexibili y and c oss-linking densi y o UDMA han Bis-GMA [3,4] and he g ea e flexibili y and significan ly lowe double bond concen- a ion o PPGDMA han TEGDMA. In o de o in es iga e he e ec o each monome on cy ocompa ibili y, human gingi al fib oblas s (HGF) we e cul u ed in solu ions o each indi idual liquid phase componen a a ying concen a ions, as well as in ex ac s o each composi e o mula ion. Due o he a iabil- i y be ween di e en cell iabili y assays, which a ises om he a ge ing o di e en enzymes wi hin he cell, and he ambigui y o he widely used e m ‘biocompa ibili y’ [5], h ee assays we e compa ed. The null hypo hesis was ha eplace- men o Bis-GMA wi h UDMA and TEGDMA wi h PPGDMA would ha e no e ec on hese p ope ies. 2. Ma e ials and me hods 2.1. Ma e ials 2.1.1. Composi e pas e p epa a ion Mic ohyb id den al composi es [1] we e p epa ed using 10 w % 40 nm umed silica (Ae osil OX-50, E onik Indus- ies AG, Essen, Ge many) and 90 w % silane- ea ed ba - ium bo o-aluminosilica e glass pa icles o a ious sizes (DMG Chemisch-Pha mazeu ische Fab ik GmbH, Hambu g, Ge many). These we e combined wi h ou dime hac yla e- based liquid phases. The liquid phases consis ed o bulk monome UDMA (DMG) o Bis-GMA (Polysciences Inc., Eppel- heim, Ge many) combined wi h diluen monome PPGDMA (Polysciences) o TEGDMA (DMG). The bulk o diluen mola a io was 3.5:1. The liquid phases also con ained 40 mM (0.58–0.61 w %) pho oini ia o campho quinone (CQ, DMG), 60 mM (0.82–0.86 w %) co-ini ia o N,N-dime hylaminoe hyl me hac yla e (DMAEMA, Sigma–Ald ich, Gillingham, UK) and 100 ppm inhibi o bu yla ed hyd oxy oluene (Sigma–Ald ich). The wo phases we e combined o o m composi e pas es using a cen i ugal plane a y mixe (SpeedMixe , Hauschild Enginee ing, Hamm, Ge many), in o de o minimize ai inco - po a ion and ensu e comple e we ing o fille pa icles. The powde o liquid a io (PLR) was kep cons an a 40 ol% liq- uid (19.3–20.3 w %, depending on liquid phase densi y). Den al composi es we e designa ed abb e ia ions based on hei bulk and diluen monome con en : UP, UT, BP and BT, whe e U, B, P and T ep esen UDMA, Bis-GMA, PPGDMA and TEGDMA, espec i ely. Comme cial composi e Fil ek Z250 (3M ESPE, S . Paul, MN, USA) was used o compa ison. 2.1.2. Disc specimen p oduc ion Excep whe e o he wise s a ed, disc-shaped specimens we e moulded by applying composi e pas es o me al ci clips (in e nal diame e 10.2 mm, hickness 1 mm) and p essing hem be ween wo shee s o ace a e. This p e en s oxygen inhibi ion du ing polyme iza ion and expels excess pas e, 266 d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 ensu ing simila specimen hickness. Specimens we e pho o- polyme ized using a blue ligh emi ing diode cu ing uni wi h a wa eleng h o 450–470 nm and powe ou pu wi h pe iodic le el shi ing o 1100–1330 mW/cm2(Demi Plus, Ke Den al, O ange, CA, USA), in di ec con ac wi h he ace a e. The cu - ing du a ion o each es ing me hod a ied and is de ailed in each co esponding sec ion. 2.2. Me hods 2.2.1. Handling p ope ies and we -poin de e mina ion The we -poin o each liquid was de e mined by g adually adding a small quan i y o liquid phase o a known mass o fille phase and mixing, un il he fille was su ficien ly we ed and a cohesi e pas e had been o med. The quan i y o liquid phase added was eco ded and no es we e made ega ding he handling p ope ies o each o mula ion. The we -poin ( ol%) was hen calcula ed om he o al mass o liquid and he densi y o each componen . 2.2.2. Polyme iza ion p ope ies 2.2.2.1. Deg ee o con e sion. The con e sion o each com- posi e was de e mined using Fou ie ans o m in a ed spec oscopy (FTIR, Sys em 2000, Pe kinElme , See G een, UK). Composi e pas e was applied o ei he a single o ou s acked ci clips o he same dimensions. These we e placed on he diamond o an a enua ed o al eflec ance acces- so y (Golden Ga e ATR, Specac L d., O ping on, UK) and co e ed wi h a shee o ace a e. A e an ini ial spec um o he uncu ed composi e had been ob ained, spec a we e eco ded con inuously o 1000 s (n = 3). The specimens we e pho o-polyme ized om he op o he fi s 20 s. Spec a we e eco ded a a wa eleng h ange o 800–1800 cm−1and esolu ion o 8 cm−1. Abso bance p ofiles we e ob ained a 1319 ± 1 cm−1(C–O s e ch bond) and 1334 ± 2 cm−1(baseline) and used o calcula e con e sion a 1 mm and 4 mm dep hs using Eq. (1): C =1 −A Ao× 100 (1) whe e C is con e sion. A and A0a e final and ini ial abso bance abo e baseline, espec i ely [6]. 2.2.2.2. Polyme iza ion sh inkage. 2.2.2.2.1. Sh inkage based on con e sion. The olume ic sh inkage o composi es was calcula ed om he con e sion da a using Eq. (2) [7], which is based on he finding ha me hac yla e es e s ypically unde go olume ic sh inkage o s = 22.5 cm3/mol upon polyme iza ion [8,9]: S = 100 ×smCinixi Wi (2) whe e S, m, C, , ˙, i, n, x and W a e sh inkage ( ol%), monome mass ac ion wi hin composi e, con e sion, composi e den- si y, sum o all monome s in liquid phase, each monome in liquid phase, numbe o C C bonds pe molecule, mass ac ion o monome in liquid phase and molecula mass, espec i ely. 2.2.2.2.2. Sh inkage based on olume change. The sh ink- age o composi es was also ob ained by measu ing he densi y o polyme ized and unpolyme ized specimens acco ding o BS EN ISO 17304:2013 [10]. This echnique uses an analy ical bal- ance equipped wi h a densi y de e mina ion appa a us (AG 204 & MS-DNY-43, Me le Toledo, Beaumon Leys, UK) and is based on A chimedes’ p inciple. Disc specimens we e cu ed o 40 s om each side o ensu e comple e con e sion. Speci- men edges we e polished o emo e loose chips. The mass o h ee cu ed and h ee uncu ed specimens o each o mula ion we e measu ed in ai and unde deionized wa e . Each alue o mass unde wa e was a e aged om 10 eadings. The sh inkage o composi es and hei SD we e calcula ed using he equa ions p o ided in he s anda d. 2.2.2.3. Dep h o cu e. The dep h o cu e o composi es (n = 3) was measu ed acco ding o BS EN ISO 4049:2009 [11]. B iefly, composi e pas e was applied o a b ass spli -mould (in e nal diame e 4 mm, heigh 6 mm) and pho o-polyme ized o 20 s om he op. The specimen was emo ed om he mould and a plas ic spa ula was used o emo e any uncu ed ma e ial om he bo om. The dep h o cu ed ma e ial was measu ed using digi al callipe s o an accu acy o ±0.01 mm and he eading was hal ed, as equi ed by he s anda d, in o de o gi e a alue o dep h o cu e. 2.2.2.4. Wa e so p ion. In o de o de e mine wa e so p ion, disc specimens we e cu ed o 40 s om each side o ensu e maximum con e sion. The mass o composi es be o e and a e imme sion in 10 mL deionized wa e o one week was hen de e mined using an analy ical balance. 2.2.3. Mechanical p ope ies Composi e disc specimens (n = 10) we e cu ed o 40 s om each side. Specimens we e s o ed d y o 24 h, be o e place- men in 10 mL deionized wa e a 37 ◦C o one week. They we e hen es ed using a biaxial flexu al es (Au og aph AGS- X, Shimadzu, Mil on Keynes, UK), wi h a 2 kN load cell and ball-on- ing jig a a c oss-head speed o 1 mm/min, un il spec- imen ailu e. Biaxial flexu al s eng h (BFS, MPa) and modulus o ensile elas ici y (E, GPa) we e calcula ed using Eqs. (3) and (4), espec i ely, BFS =P 2(1 + )0.485 ln a + 0.52+ 0.48(3) E =P wc×ˇca2 3(4) whe e BFS, P, ,  and a a e biaxial flexu al s eng h, ailu e load (N), specimen hickness (mm), Poisson’s a io (0.3) and jig suppo adius (4 mm), espec i ely, and E, (P/Wc) and ˇc a e modulus, g adien o elas ic egion and cen e deflec ion unc ion (0.5024) [12], espec i ely. 2.2.4. Cy ocompa ibili y 2.2.4.1. Cell cul u e. P ima y HGF we e ob ained om a com- me cial sou ce (ScienCell Resea ch Labo a o ies, Ca lsbad, CA, USA). HGF we e cul u ed unde s anda d condi ions (37 ◦C, 95% ai , 5% CO2, 95% ela i e humidi y) in Dulbecco’s d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 267 modified Eagle medium (DMEM, Gibco, Li e Technologies, Pais- ley, UK) supplemen ed wi h 10% oe al bo ine se um (Gibco) and 1% penicillin/s ep omycin (PAA Labo a o ies, GE Heal h- ca e, Chal on S . Giles, UK). Passage numbe s 4–8 we e used o cy ocompa ibili y s udies. 2.2.4.2. P epa a ion o es solu ions. Tes solu ions we e p e- pa ed in se um- ee DMEM. As is ypical in bioma e ials es ing, se um- ee medium was used in o de o p e en adso p ion o se um p o eins o ma e ial componen s. Con- ols consis ed o se um- ee DMEM. 2.2.4.2.1. Liquid phase componen s. Fi e en- old se ial dilu ions o each componen we e p epa ed in se um- ee DMEM, anging om 0.01 o 100 mM o DMAEMA, UDMA, PPGDMA and TEGDMA, and 0.001 o 10 mM o CQ and Bis- GMA, due o hei lowe solubili y. The solu ions we e s o ed o ∼30 min a 60 ◦C and hen s i ed using a s e ile spa ula, in o de o aid dissolu ion o componen s wi h low solubili y, pa icula ly he bulk monome s. 2.2.4.2.2. Composi e ex ac s. In o de o p epa e speci- mens o ex ac es ing, a 1 mm hick ci clip a op a shee o ace a e was filled wi h composi e and co e ed wi h ace a e. A u he h ee ci clips we e s acked on op and filled wi h composi e and co e ed wi h ace a e. The esul an 4 mm deep s ack was hen pho o-polyme ized o 20 s om he op. The bo om 1 mm hick sec ion was emo ed om he mould and incuba ed in 650 ␮L se um- ee DMEM a 37 ◦C. This p o ided an ex ac ion a io o 1 mL/3 cm2su ace a ea, as equi ed by ISO 10993-12:2009 [13]. Specimens (n = 3) we e agi a ed a 100 pm du ing ex ac ion (o bi al shake , S ua Scien ific, S one, UK). A e 24 h, specimens we e ans e ed o esh medium and incuba ed o a u he 6 days, yielding ex ac s om 1 and 7-day ime-poin s. 2.2.4.3. Cy ocompa ibili y assays. HGF we e seeded a a den- si y o 30,000 cells/cm2in 96 well pla es (n = 3 pe specimen, ime-poin and assay). A e an ini ial 24 h seeding pe iod, cell cul u e medium was eplaced wi h 100 ␮L o he specimen (componen solu ion, composi e ex ac o con ol). The solu- ions we e ho oughly mixed using a o ex mixe (S ua SA8, Bibby Scien ific, S one, UK) in o de o ensu e e en dispe sion o dissol ed componen s. Cy ocompa ibili y was assessed a e a u he 24 h o cul u e (48 h ime-poin ), in acco - dance wi h ISO 10993-5:2012 [14]. The specimen was hen eplaced wi h se um- ee DMEM and cul u ed o a u he 24 h eco e y pe iod (72 h ime-poin ). Th ee wa e -soluble cell me abolic ac i i y assays we e used o assess cy ocom- pa ibili y: esazu in (alama Blue Cell P oli e a ion Assay, AdB Se o ech, Bio-Rad Labo a o ies Inc., Hemel Hemps ead, UK); wa e -soluble e azolium sal -8 (WST-8, Cell Coun ing Ki -8, Sigma–Ald ich); and 3-(4,5-dime hyl hiazol-2-yl)-5-(3- ca boxyme hoxyphenyl)-2-(4-sul ophenyl)-2H- e azolium sal (MTS, CellTi e 96 AQueous One Solu ion Cell P oli e a ion Assay, P omega, Sou hamp on, UK). Medium was aspi a ed om he cells and eplaced wi h 100 ␮L cell cul u e medium con aining he co esponding subs a e (Table 1). A e incu- ba ion a 37 ◦C, fluo escence (FLx800, BioTek, Po on, UK) o abso bance (Infini e M200, Tecan, Männedo , Swi ze land) was measu ed. S anda d cu es we e ob ained by seeding a Table 1 – Cy ocompa ibili y assay pa ame e s. Subs a e Incuba ion ime (min) Measu emen Resazu in, 10% 90 Fluo escence – exci a ion: 560 nm, emission: 590 nm WST-8, 10% 80 Abso bance – 460 nm ( e e ence: 650 nm) MTS, 20% 60 Abso bance – 490 nm ( e e ence: 630 nm) ange o cell concen a ions in 96 well pla es, 2 h p io o each assay. 2.2.5. S a is ical analyses One-way analysis o a iance (ANOVA) and pos -hoc Tukey’s es s we e used o de e mine significance (p ≤ 0.05) be ween composi e o mula ions (UP, UT, BP, BT and Z250). Two-way ANOVA and pos -hoc Tukey’s es s we e also used in o de o de e mine he significance o he e ec s o mul iple ac- o s, e.g. UDMA s. Bis-GMA and PPGDMA s. TEGDMA. Fo all echniques, he s anda d de ia ion (SD) o each o mula ion was displayed on g aphs, excep in he case o polyme iza ion sh inkage, whe e he SD was a e aged o each echnique, due o he high a iabili y o he olume change me hod. S a is- ical significance is p esen ed on g aphs (*p < 0.05, **p < 0.01, ***p < 0.005 o ****p < 0.001). 3. Resul s 3.1. Handling p ope ies and we -poin All expe imen al o mula ions had we -poin s o 33.3 ± 0.4 ol% liquid. A his le el, o mula ion pas es con aining Bis-GMA el conside ably less malleable du ing specimen moulding han hose p oduced wi h UDMA. A 40 ol% liquid, all o mula ions we e sligh ly mo e malleable, bu o mula ions con aining Bis-GMA s ill exhibi ed lowe malleabili y han hose con aining UDMA. Z250 had handling p ope ies be ween hose o Bis-GMA- and UDMA-con aining composi es. No disce nible di e ence in handling p ope ies was obse ed be ween PPGDMA and TEGDMA. 3.2. Polyme iza ion p ope ies 3.2.1. Con e sion Expe imen al composi es con aining UDMA had, on a e age, 1.2 and 1.3 imes highe con e sion a 1 and 4 mm dep h, espec i ely, han hose con aining Bis-GMA (a e age p- alue o bo h dep hs = 0.001) (Fig. 1a). Replacemen o TEGDMA wi h PPGDMA u he inc eased con e sion by 1.1 and 1.2 imes a 1 and 4 mm, espec i ely (a e age p- alue o bo h dep hs < 0.01). UP had he highes con e sion (68% and 65% a 1 and 4 mm). A bo h dep hs, UP’s high con e sion was highly s a is ically sig- nifican compa ed o BP, BT and Z250 (p < 0.001) and a 1 mm, i was significan ly highe han ha o UT (p < 0.05). Z250 had he lowes (50% and 39% a 1 and 4 mm) and was compa a- ble o BT. Composi es con aining Bis-GMA (BP, BT and Z250), showed a significan educ ion in con e sion upon inc easing dep h om 1 o 4 mm. 268 d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 Fig. 1 – (a) Con e sion o composi es a 1 and 4 mm dep h. Columns ep esen mean, e o ba s ep esen SD. (b) Sh inkage o 1 mm hick composi e discs, calcula ed om con e sion o olume change. Columns ep esen mean, e o ba s ep esen mean SD o he co esponding echnique (±0.05 and ±0.23 ol% o con e sion and olume change, espec i ely). (c) Dep h o cu e o composi es. (d) Wa e so p ion o composi es a e imme sion in deionized wa e o one week. (c, d) C osses ep esen mean, boxes ep esen 25–75 pe cen iles, e o ba s ep esen SD. 3.2.2. Sh inkage Based on con e sion a 1 mm dep h, he sh inkage o UDMA- con aining composi es was p edic ed o be 1.2 imes highe , on a e age, han hose con aining Bis-GMA (p < 2 × 10−5) (Fig. 1b). Composi es con aining TEGDMA we e es ima ed o ha e 1.1 imes highe sh inkage han hose con aining PPGDMA (p < 0.04). When sh inkage was de e mined by olume change, howe e , no s a is ically significan e ec o monome could be obse ed. This was due o he high s anda d de ia ion o he echnique. The sh inkage o Z250 could no be calcula ed using con e sion da a, since i s exac composi ion was unknown. The measu ed sh inkage, howe e , o Z250 was compa able o all expe imen al o mula ions. 3.2.3. Dep h o cu e The dep h o cu e (Fig. 1c) was highly compa able o all com- posi es (2.7 ± 0.25 mm), ega dless o monome composi ion (F = 0.96). d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 269 3.2.4. Wa e so p ion The wa e so p ion o o mula ions con aining TEGDMA was, on a e age, 1.2 imes highe han ha o hose con aining PPGDMA (p < 0.02) (Fig. 1d). On a e age, UDMA induced ∼1.1 imes highe wa e so p ion han Bis-GMA, al hough his was no s a is ically significan . Z250 had he lowes wa e so p- ion, compa able o ha o BP. UT had he highes wa e so p- ion, significan ly highe han ha o BP and Z250 (p < 0.05). 3.3. Mechanical p ope ies Expe imen al composi es con aining UDMA had significan ly highe s eng h (153–158 MPa) han hose con aining Bis-GMA Fig. 2 – (a) Biaxial flexu al s eng h and (b) modulus o ensile elas ici y o composi es a e s o age in deionized wa e a 37 ◦C o one week. C osses ep esen mean, boxes ep esen 25–75 pe cen iles, e o ba s ep esen SD. (p = 1 × 10−7). The s eng h o BP was significan ly highe han ha o BT (127 and 99 MPa, espec i ely; p < 0.05) (Fig. 2a). The modulus o expe imen al o mula ions con aining PPGDMA was, on a e age, sligh ly lowe han ha o o mula ions con- aining TEGDMA (6.15 and 6.75 GPa, espec i ely; p = 0.009) (Fig. 2b). Z250 had he highes s eng h (173 MPa) and lowes modulus (5.3 GPa). 3.4. Cy ocompa ibili y 3.4.1. Compa ison o esazu in, WST-8 and MTS assays The appa en cell densi y o HGF a e 48 and 72 h cul u e in se um- ee DMEM is shown in Fig. 3. Resazu in and WST-8 assays epo ed a simila inc ease in appa en cell densi y om 30,000 o ∼49,000 cells/cm2be ween 0 and 48 h. MTS epo ed a lesse inc ease in appa en cell densi y ( om 30,000 o 37,000 cells/cm2a e 48 h). A e 72 h in cul u e, he esazu in assay epo ed cell densi y o ha e inc eased o 113,000 cells/cm2, whe eas he WST-8 assay epo ed cell densi y o be 74,000 cells/cm2. MTS again epo ed only a small inc ease in appa en cell densi y (45,000 cells/cm2). Since he assays epo ed di e en cell densi ies in he con ols a di e en ime-poin s, subsequen da a a e no - malized o he con ol o he co esponding assay and ime-poin and a e epo ed in e ms o ela i e me abolic ac i i y. 3.4.2. Composi e componen cy ocompa ibili y The ela i e me abolic ac i i y o su i ing HGF ollowing exposu e o se ial dilu ions o liquid phase componen s, as well as ollowing a subsequen eco e y pe iod, is p esen ed in Fig. 4. The dashed lines ep esen he mean alue ob ained o he DMEM con ol o each co esponding ime-poin and assay. All componen s caused a ypical, concen a ion- dependen educ ion in cell numbe as concen a ion was Fig. 3 – Ini ial seeding densi y o HGF de e mined by cell coun ing (0 h, c oss), and appa en densi y a e cul u e in DMEM o 48 h (unfilled symbols) and 72 h (filled symbols), as assessed by esazu in (ci cle), WST-8 ( iangle) and MTS (squa e) assays. E o ba s ep esen SD. 270 d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 Fig. 4 – Me abolic ac i i y ( ela i e o he co esponding con ol) o su i ing HGF a e cul u e in aqueous solu ions o composi e liquid phase componen s (a) CQ, (b) DMAEMA, (c) UDMA, (d) Bis-GMA, (e) PPGDMA o ( ) TEGDMA. Resazu in (ci cle), WST-8 ( iangle) and MTS (squa e) assays we e pe o med a e 24 h cul u e in he solu ions (48 h ime-poin , unfilled symbols), and a e a subsequen 24 h eco e y pe iod in DMEM (72 h ime-poin , filled symbols). Componen concen a ions anged om 0.001 o 10 mM (a and d) o 0.01 o 100 mM (b, c, e and ). Dashed lines ep esen he mean alue o he con ol, e o ba s ep esen SD. d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 271 inc eased, hough he ex en o his ela ionship depended on he assay. In he case o all componen s, a concen a ion o 10 mM was su ficien o cause close o 100% educ ion in me abolic ac i i y. A e 48 h, WST-8 indica ed a di ec co ela ion be ween CQ concen a ion (Fig. 4a) and me abolic ac i i y, wi h 0.001 and 0.01 mM ha ing no significan e ec on me abolic ac i i y. As concen a ion was inc eased o 0.1, 1 and 10 mM, me abolic ac i i y was educed by 17, 33 and 98%, espec i ely. Resazu in epo ed simila le els (±5%) o me abolic ac i i y a mos con- cen a ions, whe eas MTS alues we e up o 15% highe . A e 72 h, esazu in epo ed simila le els o ela i e me abolic ac i i y o a e 48 h. WST-8, howe e , indica ed simila le els o me abolic ac i i y o he con ol (±7%) a all concen a ions excep ≥10 mM, a which cells did no eco e . MTS showed ele a ed me abolic ac i i y (10–38% g ea e han ha o he con ol) a all concen a ions a e 72 h, excep ≥10 mM. By con as , a 48 h, 0.01 mM DMAEMA (Fig. 4b) caused an 86–95% educ ion in me abolic ac i i y in all assays, wi h no eco e y a e 72 h. All assays indica ed ha concen a ions ≥0.1 mM caused o al inhibi ion o me abolic ac i i y. Simi- la ly, a a c i ical concen a ion o 0.01 mM o highe , UDMA (Fig. 4c) and TEGDMA (Fig. 4 ) bo h inhibi ed me abolic ac i i y in all assays. Bis-GMA (Fig. 4d) appea ed o be mo e cy ocompa ible han DMAEMA, UDMA and TEGDMA. A e 48 h, WST-8 indi- ca ed ha concen a ions o 0.001–0.01 mM had no e ec on me abolic ac i i y. I ell, howe e , by 7 and 66% upon inc eas- ing concen a ion o 0.1 and 1 mM, espec i ely. The o he assays showed a simila end, bu a 0.001–0.1 mM he alues epo ed by esazu in and MTS we e ∼10–15% and ∼23–28% lowe , espec i ely. A e 72 h, alues ob ained by WST-8 assay we e ele a ed by be ween 7 and 20% compa ed o he p e i- ous ime-poin , excep a 10 mM. A 0.001–0.1 mM, me abolic ac i i y appea ed highe han he con ols. Resazu in epo ed simila alues (±10%) a e 72–48 h. HGF exposed o PPGDMA (Fig. 4e) exhibi ed mo e com- plex ends. The h ee assays ga e di e en esul s o concen a ion-dependen e ec s on ela i e me abolic ac i - i y. Acco ding o all assays, a ≥10 mM, PPGDMA comple ely inhibi ed me abolic ac i i y a e 48 h and his did no eco e a e 72 h. Upon lowe ing concen a ion, he e was a small inc ease in ac i i y acco ding o he WST-8 and MTS assays, bu i emained well below con ol alues and was no enhanced by p o iding addi ional eco e y ime. Con- e sely, a 48 h and PPGDMA le el below 0.1 mM, he cell ac i i y acco ding o he Resazu in es ended o highe al- ues han wi h he con ol. By 72 h, howe e , his “excess” me abolic ac i i y was down o con ol le els. Concen a- ions o 0.01, 0.1 and 1 mM, PPGDMA caused a educ ion in WST-8 ac i i y o 76, 85 and 92%, espec i ely, a e 48 h. These le els emained unchanged a e 72 h. MTS showed simila alues o WST-8. By con as , a e 48 h, esazu in ac i i y was ele a ed by 63 and 35% abo e he con ol a 0.01 and 0.1 mM, espec i ely, and a 1 mM was simila o he con ol. A e 72 h, hese alues ell, wi h esazu in ac i i y emaining 12% abo e he con ol a 0.01 mM, and being 7 and 80% lowe han he con ol a 0.1 and 1 mM, espec i ely. 3.4.3. Composi e ex ac cy ocompa ibili y The me abolic ac i i y o HGF a e cul u e in composi e ex ac s is p esen ed in Fig. 5. Significan di e ences we e again obse ed be ween di e en assays. In o de o acili a e elucida ion o ends, da a a e exp essed as a pe cen age o he con ol o each co esponding ex ac (1 o 7 days) and assay ime-poin (48 o 72 h). A e 24 h exposu e o 1 day composi e ex ac s (48 h ime- poin ), a gene al downwa d end in esazu in me abolism was obse ed as ollows: UP > UT ≥ BP > BT ≥ Z250. Al hough WST-8 ac i i y di e ed sligh ly (UP ≈ UT > BP ≈ BT > Z250), bo h assays confi med ha he ex ac s om he lowe quad an o 4 mm deep composi e samples con aining UDMA had less e ec on he 48 h me abolic ac i i y han hose con aining Bis- GMA. In he case o esazu in, his was s a is ically significan (p = 0.001). They also showed ha Z250 ex ac s we e less cy o- compa ible han all o he expe imen al o mula ions. MTS showed no significan di e ences be ween expe imen al o - mula ions bu confi med he low cy ocompa ibili y o Z250. Me abolic ac i i y in all assays a ied om being simila o he con ol o UP, down o ∼50% o con ol o Z250. Composi es con aining TEGDMA caused a sligh bu significan educ ion in esazu in ac i i y compa ed o PPGDMA (p < 0.01), bu WST- 8 and MTS assays showed no clea ends wi h ega ds o he e ec o diluen monome . A e 72 h, simila ends we e obse ed in all h ee assays, wi h me abolic ac i i y alling o app oxima ely 50% o ha o he con ol o all expe imen al ma e ials. The only di - e ences be ween assays we e obse ed wi h Z250, whe e esazu in ac i i y had emained simila o he p e ious ime- poin a ∼50%, bu WST-8 ac i i y had allen om 53 o 37% and MTS ac i i y had allen om 53 o 33%. These di e ences, howe e , we e no s a is ically significan . Resazu in and WST-8 sugges ed ha 7-day ex ac s we e mo e cy ocompa ible han 1 day ex ac s, since me abolic ac i i y e u ned o con ol le els a e 72 h. The end o he 48 h esazu in assay a e 7 days ex ac ion was simila o he co esponding 1 day ex ac , bu wi h sligh ly highe alues. Fo example, 48 h assays showed ha BT – he leas cy ocom- pa ible expe imen al composi e – had 75% esazu in ac i i y ela i e o he con ol a e 7 days ex ac ion, compa ed o 56% a e 1 day. In he case o all expe imen al o mula ions, WST-8 ac i i y was simila o he con ol o sligh ly ele a ed by up o 11% a e 48 h. The 7-day ex ac o Z250, howe e , s ill caused a significan educ ion in WST-8 ac i i y. A e 72 h, he esazu in and WST-8 ac i i y o HGF exposed o 7-day ex ac s had, in gene al, eco e ed. Resazu in ac i i y o BT and Z250 emained sligh ly educed a 89 and 82%, espec i ely. A e 72 h, WST-8 ac i i y o HGF exposed o all composi e ex ac s was ele a ed abo e ha o he con ol by be ween 10 and 31%. The MTS assay ga e significan ly di e en alues com- pa ed o he o he wo assays o he 7-day ex ac s. A e 48 h, MTS ac i i y o HGF exposed o all expe imen al o - mula ions was 60–75% highe han he con ol and emained simila a e 72 h. Z250 alues we e simila o hose epo ed by WST-8. UDMA was also shown o be mo e cy ocompa ible han Bis-GMA in 7-day ex ac s (48 h esazu in assay, p = 0.03; 72 h WST-8 assay, p = 0.0008; 72 h MTS assay, p = 0.007). WST-8 ac i i y was also significan ly highe in cells exposed o 7-day 272 d e n a l m a e i a l s 3 2 ( 2 0 1 6 ) 264–277 Fig. 5 – Me abolic ac i i y ( ela i e o he co esponding con ol) o HGF a e 48 (a and c) o 72 (b and d) h cul u e in 1 (a and b) o 7 (c and d) day composi e ex ac s, assessed by esazu in (da k g ay), WST-8 (black) and MTS (ligh g ay) assays. Dashed lines ep esen he mean alue o he con ol, e o ba s ep esen SD. ex ac s o PPGDMA-con aining composi es han TEGDMA- con aining composi es a e 48 h (p = 0.04). 4. Discussion As hypo hesized, den al composi es con aining UDMA had significan ly highe monome con e sion and sligh ly be e cy ocompa ibili y han hose con aining Bis-GMA. Simila ly, composi es con aining PPGDMA achie ed mo e comple e con- e sion han hose con aining TEGDMA. Impo an ly, he use o UDMA and/o PPGDMA as he sole bulk/diluen monome had no de imen al e ec on he sh inkage o dep h o cu e o he ma e ials. In addi ion, composi es con aining UDMA had imp o ed handling p ope ies compa ed o hose con aining Bis-GMA. The a ia ion in he con e sion o he composi es is a ibu ed o di e ences in he chemical s uc u es (Fig. 6) and physical cha ac e is ics (Table 2) o hei cons i uen monome s. Al hough UDMA is a sligh ly smalle molecule han Bis-GMA and, as a esul , has sligh ly highe double bond concen a ion ( a io o double bonds pe molecule o molecula mass), i s lowe glass ansi ion empe a u e (Tg) and g ea e flexibili y enable mo e comple e c oss-linking [15]. The bulky a oma ic g oups o Bis-GMA cause s e ic hind ance, eflec ed in i s e y high iscosi y alue, which is ∼80 imes ha o UDMA. This educed flexibili y limi s he likelihood o me hac yla e g oups coming in o con ac wi h each o he and binding. PPGDMA has mo e han wice he molecula mass o TEGDMA and as a esul , i s g ea e flexibili y and lowe double bond concen a ion esul in imp o ed con e sion. The simila dep h o cu e o he composi es is likely o be due o he simila e ac i e indices o he monome s used