Zinc-Calcium-Fluoride Bioglass-Based Innovative Multifunctional Dental Adhesive with Thick Adhesive Resin Film Thickness.

TLDR: An experimental three/two-step E&R/SE Exp_2UA combines high bonding potential and bond-degradation resistance with long-term ion release rendering the adhesive anti-enzymatic and anti-bacterial potential.

Abstract: Apart from producing high bond strength to tooth enamel and dentin, a dental adhesive with biotherapeutic potential is clinically desirable, aiming to further improve tooth restoration longevity. In this laboratory study, an experimental two-step universal adhesive, referred to as Exp_2UA, applicable in both the etch-and-rinse (E&R) and self-etch (SE) modes and combining a primer, containing 10-methacryloyloxydecyldihydrogen phosphate as a functional monomer with chemical binding potential to hydroxyapatite, with a bioglass-containing hydrophobic adhesive resin, was multifactorially investigated. In addition to primary property assessment, including measurement of bond strength, water sorption, solubility, and polymerization efficiency, the resultant adhesive-dentin interface was characterized by transmission electron microscopy (TEM), the filler composition was analyzed by energy-dispersive X-ray spectroscopy, and the bioactive potential of the adhesive was estimated by measuring the long-term ion release and assessing its antienzymatic and antibacterial potential. Four representative commercial adhesives were used as reference/controls. Application in both the E&R and SE modes resulted in a durable bonding performance to dentin, as evidenced by favorable 1 year aged bond strength data and a tight interfacial ultrastructure that, as examined by TEM, remained ultramorphologically unaltered upon 1 year of water storage aging. TEM revealed a 20 μm thick hydrophobic adhesive layer with a homogeneous bioglass filler distribution. Adequate polymerization conversion resulted in extremely low water sorption and solubility. In situ zymography revealed reduced endogenous proteolytic activity, while Streptococcus mutans biofilm formation was inhibited. In conclusion, the three-/two-step E&R/SE Exp_2UA combines the high bonding potential and bond degradation resistance with long-term ion release, rendering the adhesive antienzymatic and antibacterial potential.

Figures

Figure 2. Water sorption (A) and solubility (B) (both in μg/mm3) of the dental adhesives investigated after 7-day (7d), 6-month (6m) and 1-year (1y) water storage. The lowest water sorption and solubility were recorded for BZF-21 and the adhesive resin (‘Bond’) of CSE2 (Figure 2A,B). The same small or capital letters indicate absence of statistically significant difference (p>0.05). Columns distinguished with a hash key ‘#’ are significantly different in pairwise comparisons (p<0.05). (C) Insufficiently polymerized fragment of GPrB immersed in water, not allowing measurement of water sorption and solubility. Polymerization kinetics (D) with progression in degree of conversion (DC in %) upon application of the different adhesives to dentin in etch-and-rinse (E&R) and self-etch (SE) mode, as measured within the first 20min after application to achieve maximum DC, as was also graphically presented in the bar graph. In the latter graph, the same small or capital letters indicate absence of statistically significant difference (p>0.05).

Figure 6. Ultra-structural TEM photomicrographs of the adhesive-dentin interface produced by CSE2 when it was applied in E&R (a,b and e,f) and SE (c,d and g,h) modes upon 1w (a-d) and 1y (e-h) water storage. Overview of the adhesive-dentin interface when CSE2 was applied in E&R bonding mode upon 1w water storage (a). Higher magnification of CSE2’s E&R adhesive interface (b). A gradual transition with a solely partially demineralized zone was observed at the hybrid layer (Hy) bottom. Overview of the adhesive-dentin interface when CSE2 was applied in SE mode upon 1w water storage, revealing a tight adhesive-dentin interface, presenting with an adhesive-resin (Ar) layer of ca. 5-6 µm thickness (c). Higher magnification of CSE2’s SE adhesive interface (d). Overview of CSE2’s E&R adhesive interface upon 1y water storage, disclosing filler de-bonding within the adhesive resin (e). Higher magnification of CSE2’s E&R adhesive interface (f). Overview of CSE2’s SE adhesive interface upon 1y water storage, revealing filler de-bonding within the whole adhesive layer and hereby having left interfacial porosities (black handpointers) as indications of hydrolytic bond-degradation effects (g). Higher magnification of CSE2’s SE adhesive interface (h). Ar: adhesive resin; Fl: flowable composite; Hy: hybrid layer; Rt: resin tag; Ud: unaffected dentin.

Table 1 Qualitative analysis of green fluorescence-signal intensity at the adhesive-dentin interface for Exp_2UA and the reference adhesive CSE2.

Figure 7. In-situ zymography to evaluate the anti-enzymatic properties of Exp_2UA and CSE2. Representative CLSM photomicrographs of proteolytic activity within the hybrid layer and resin tags of adhesive-dentin interfaces produced by Exp_2UA (a1-a3, b1-b3) and CSE2 (c1-c3, d1-d3), when both adhesives were applied in E&R (a1-a3, c1-c3) and SE (b1-b3, d1-d3) bonding modes. Green fluorescence (ad1) indicates that the fluorescein-conjugated gelatin was hydrolyzed and endogenous proteolytic activity

Figure 1. STEM/EDS of zinc-calcium-fluoride (Zn-Ca-F) bioglass incorporated into the experimental multifunctional adhesive resin BZF-21 (a-c) and ion release from the experimental adhesive resin BZF-21 in function of time, as measured by ICP-OES and a F ion-specific electrode (d-f). Release kinetics of the ions Zn, Ca and F from the BZF-21 during a period of 1 year, with the highest cumulative ion release measured for Zn, then Ca and the least for F (Figure 1e). To avoid a saturation effect, the immersion solution Milli-Q water was changed every week (Figure 1f). Release kinetics of the ions Zn, Ca and F from the BZF-21 during a period of 28 days, with an initial high release of Zn, Ca and F over the first day (Figure 1f). Overview (S)TEMs illustrate the filler-matrix configuration of BZF-21 in the Figure S1.

Figure 8. Anti-bacterial properties of the experimental adhesive resin BZF-21. Collection of solutions extracted from experimental BZF-21 adhesive-resin disks (A). S. mutans growth measured spectrophotometrically (OD: optical density) in function of time (B) and after 24h incubation (C). The data are presented as mean ± standard deviation. The same letters indicate absence of statistically significant

Full text

!
!"#$%$&'$"()%*'(+,"-./0"+1'&22%0&2.-/"##+3&4"3./)('4"%*(#$4"+#&'/-.#4&'/
&-5.2"3./6"45/45"$7/&-5.2"3.%,.2"#/*"')/45"$7#.22/
!
Chenmin!Yao,
†,‡
!Mohammed!H.!Ahmed,
†,§
!Xin!Li,
†
!Ivana!Nedeljkovic,
†,//
!Jennifer!Vandooren,
#
!
Ben!Mercelis,
†
!Fei!Zhang,
†,&
!Kirsten!L.!Van!Landuyt,
†
!Cui!Huang,
‡
!Bart!Van!Meerbeek
†,*
/
!
†
KU! Leuven! (University! of! Leuven),! Department! of! Oral! Health! Sciences,! BIOMAT! &! UZ! Leuven!
(University!Ho sp itals!Le uve n),!D en tistry,!3000!Leuven,!Belgium;!
‡
Wuhan!University,!the!State!Key!Laboratory! Breeding! Base! of!Basic! Science! of!Stomatology! (Hubei-
MOST)!&!Key!Laboratory!of!Oral!Biomedicine!Ministry!of!Education!(KLOB M ),! Schoo l! &! Hospital!of!
Stomatology,!430079!Wuhan,!China;!
§
Tanta!University,!Faculty!of!Dentistry,!Department!of!Dental!Biomaterials,!31511!Tanta,!Egypt;! !
//
University!of!Amsterdam!and!Vrije!Universiteit!Amsterdam,!Department!of!Dental!Material!Sciences,!
Academic!Centre!for!Dentistry!Amsterdam!(ACTA),!1081!LA!Amsterdam,!The!Netherlands;!
#
KU!Leuven!(University!of!Leuven),!Laboratory!of!Immunobiology,!Rega!Institute!for!Medical!Research,!
3000!Leuven,!Belgium;!
&
KU!Leuven!(University!of!Leuven),!Department!of!Materials!Enginee ring,!3001!Leuven,!Belgiu m.!
*Corresponding!author.!
!
Chenmin' Yao,'Mohammed'H.'Ahmed,' Xin'Li,' Ivana'Nedeljkovic,'Jen nife r'V an do ore n,'Ben' Mercelis,' F e i'
Zhang,'Kirsten'L.' Van' Landuyt,'Cui'Huang,' B art' V a n ' Meer b e ek .
'
Zinc-calcium-fluoride ' bioglass-based'
innovat ive ' m u lti-functional' den tal' ad he sive ' with' thick ' adh esive -re sin' film ' thickne ss.' ACS ' App lied'
Materials'&'Interfaces.
'
/
89:;<8=;>/Apart!from!producing!high!bond!strength!to !tooth!enamel!and!dentin,!a!dental!adhesive!
with! bio-therapeutic! potential! is! clinically! desirable,! aiming! to ! further! imp rove ! too th-restoration!
longevity . In!this!laboratory!stu d y ,!an!experimental! two-step! universal!adhesive,!being!referred!to!as!
Exp_2UA,!ap plicable!in!both!etch-and-rinse!(E&R)!and !self-etch!(SE)!mode!and!com bining!a!primer,!
containing! 10-methacryloyloxydecyldihydrogen! phosphate! (10-MDP)! as! functional! monomer! with!

!
chemical! b inding!potential!to!hydroxyapatite,!with!a!bioglass-containing! hydrop hobic!adhesive!resin,!
was!multifactorially!investigate d .!In!additio n !to!prim ar y!pro p e rty!assessment,!including!measu re ment!
of! bond! strength,! water! sorption,! solubility! and! polymerization! efficiency,! the! resultant! adhesive -
dentin!interface!was!characterized!by!TEM ,!the!filler!composition!analyzed!by!EDS!and!th e! a dh es ive’s!
bioactive!potential!estimated!by!measuring!long-te rm !ion!release!and!assessing!its!anti-enzymatic!and!
anti-bacterial! potential.! Four! rep rese nta tive! commercial! adhesives! served! as! reference/controls.!
Application!in!both!E&R!and!SE!mode!resulted!in!durable!bonding!performance!to!dentin,!as!evidenced!
by! fa vora ble !1-year!aged!bond-stren gth!data!and!a!tight!inte rfa c ial!ultra-structure!that,!as!e x a mined!
by! TEM,!re m ain ed !u ltra-morphologically!unaltered! upon!1-year!water-storage! a ging.!TEM !revealed! a!
20-µm!thick!hydrophobic! adhesive!layer!with!a!homogeneous! bioglass!filler!distribution.! A d e q ua te !
polymerization!conversion!resulted!in!extremely!low!water!sorption!and!solubility.!In-situ!zymography!
revealed!reduced!endogenou s!proteolytic!activity,!while!Streptococcus'mutans!biofilm!formation!was!
inhibited .!In!conc lu s io n ,!th e !three/two-step!E&R/SE !Exp_2UA!com bines!high!bonding!potential!and!
bond-degradation!resistance!with!long-term!ion!release! rendering!the!a dh esiv e! anti-enzymatic!and!
anti-bacterial!potential./
!
?@ABC<D:>! bioactive;! io n ! release;! anti-enzymatic;! anti-bacterial;! bond! durability;! adhesive-dentin!
interface ; !methacryloxydecyl!dihydrogen!phosphate!
!
EF/GH;<CDI=;GCH/
The!use!of!dental!adh esives!to!bo nd !tooth-colored!resin -based!com posites!(RBC)!to!tooth!tissu e ,!so !
enabling! to! minimally! invasively ! restore! teeth,! has! revolutio nize d! today’s! dental! practice,! hereby!
closely!meeting!each!patient’s!major!dema nd!for!aesth etic!and!ne arly!invisible!tooth!restorations.!The!
RBC!resto ratio n’s!longe vity!dep en ds!high ly!on!the!clinica l!performance!of!dental!adhesives,!bonding!
the! restorative! comp osite ! to! tooth! enam e l! and! dentin .! In! a! challenging! humid! mouth,! tooth!
restorations! are! cyclically! sub jected/expo sed! to! mecha nica l! stress! d urin g! chewin g,! tempera ture !
changes!and!acid!attacks,!as!well!as!bacterial!cariogenic!effects!during!continuous!biofilm!activity.! !
The!fundam ental!principle!of!adhesion!is!based!on!a!biomaterial-tooth!exchange!process,!involving !
substitution!of!inorganic!to oth!material!by!synthetic!resin!m ono m ers!that!upon!in-situ'polymerization!
micro-mechanically! interlock! within! beforehand! created! surface! micro-retention.
1
! This! exchange!

!
process!can! be! conducted!following!an! etch-and-rinse! (E&R)! or! self-etch! (SE)! bonding!approach.
2-3
!
Diffusion-based! micro-mechanical! interlocking! within! a! fully! demineralized! collagen-rich! and!
hydroxyapatite-free!3-!to!5-µm!hybrid!layer!constitutes!the!primary!bonding!m echanism !of!ad hesives!
employed!in!an!E&R!bonding!mode.
4
!Adhesives!employed!in!a!mild!SE!bonding!m ode!make!additionally!
use! of! primary! ionic! interaction! of! dedicated! functional! monomers! with! hydroxyapatite! within! a!
partially!demineralized!submicron!hybrid!layer.!Additional!chemical!binding!is!t ho u g ht !to!contribute !in!
particular!to!bond!durability.
4
!Both!the!E&R!and!SE!bonding!modes!have!potential,!especially!w hen!
applied!in!a!multi-step!application!procedure,!but!they!also!have!limitations.
2-4
!
So-called!universal!adhesives!(UAs)!represe nt!th e!ne w est!gen era tion!of!de ntal!adh es ives.!They!are!
rapidly!becoming!popular!in!today’s! clinical! practice! because!of!their!ease!of!use!and!two!bonding!
mode!options.
5
!Based!on!the!primary!bonding!m echanisms!mentioned!above,!UAs!most!effectively!
bond!to!too th!tissue!by!combining!se lective!phosphoric-acid!etching!of!enamel!with!a!successive!SE!
bonding!routine!app lied!both!on!the!beforehan d!etched!enamel!and!non- etched!dentin.
4
!Nevertheless,!
UAs!sho uld!b e!co nsidered!as!trade-off!adhesives!by!combining !the!primer!a nd !adhesive!resin!into!a!
single! solution.! Major! drawbacks! of! UAs! are! their! rela tively! high! hydrophilic! nature! and! thin! film!
thickness,! reduc ing! th e ir! inte rfa cia l! stress-absorbing! potential! an d! potentially! compromising! t h eir!
long-te rm !clinical!b on din g!performance,!which !due!to!lack!of!long-term!clinical!da ta!is!n o t !yet!known.
4
! !
Simplified! ad hesive!procedures!should!however!not!compromise!clinical!bonding!performance.
6-7
!
Previous!generation!one-step!a dhe sives!as!w ell!as!the!n ew est!genera tion!of!UAs!have!repeatedly!in '
vitro!been!proven!to!benefit!from!an!extra!hydrophobic!adhesive!layer,!basically !transf o rming!their!
two-step!E&R !bond ing!mo de!into!a!three-step !E&R!bo nding!m ode !or!their!one - step !SE!bond ing!mo de!
into!a!two-step!SE!bonding!mode.
8-10
!In!fact,!these!extended!application! procedures!resemble!those!
of! the! respective! gold-standa rd! th ree-step! E&R! and! two - step ! SE! ad hesives.
4
! Besides! thicker! film!
thickness!to!better!stabilize!the!adhesive!interface!with!enhanced!interfacial!stress- ab sorbing!potential,!
the!im p ro ve d !bonding!performance!of!th e!multi-step!application!procedure!should!be!attributed!to!
the! higher! interfa cial! hyd rop h ob icity! achieved! and! impro v e d ! sealing! of! the! adhesive! interface,!
protecting! it!better!against!water!uptake!through!osmosis!from!the!underlying!d entin!as!w ell!as!the!
outer!oral!en viro nment.!Water!sorption! resulting!in!hydrolysis!should!most!likely!be! regarded!as!the!
main!bond-degradation!mechanism!of!adhesive-dentin!interfaces.
11
!

!
In!addition,!scientific!literatu re !ha s !rich ly!d o cu mented! th e!sen sitivity!of!adh es ive-dentin!interfaces!
to! enzymatic! bio-degradation! processes.
12-13
! Acidic! dental! bonding! p rocedu res! have! been !
demonstrated! to! not! only! expose! but! also! activate! dentinal! enzymes,
14-15
! with! a! higher!
exposure/activity! recorded! following! an! E&R ! tha n! (mild)! SE! bon din g! mode.
4,14-15
! Previous! research!
identified ! the! presence! o f! several! MMPs! within! dentin,! among! w hich! the! gelatinases! M M P-2! and!
MMP-9,!and!the!collagenase!MMP-8!have!been!described!to!be!involved!most!in!bond!degradation!at!
human!dentin.
14,16-17
!In !respo n se!to!these!findings,!severa l!exogenou s !inh ib it o rs !of!MMPs!have!been!
investiga te d !on!their!MMP-inh ibit in g!co ntribution!to!bond!stability,!among!which!chlorhexidine!w as!
studied!mo st!frequently.
18
!Subsequent!research!has!also!prom oted!the!use!of!collagen!cross-linkers !
that! bes ides! anti-enzymatic! effects! make! exposed! collagen! more! resistant! against! degradation.
19
!
Although! zinc! at! physiological! concentrations! is! a! necessary! elemen t! for! collagen! h ydrolysis! by!
MMPs,
20
!zinc!w as!also!reported!to!act!as!a!potential!MMP!inhibitor!at!high!concentrations.
21
!Especially!
MMP-2!and!MMP-9!are!inhibite d!b y !zin c!a n d !ot h er !m e ta ls.
22-23
!Loading!an!adhesive!with!zinc!that!can!
gradually! be!released! to! the! immediate! d entin! environment! may! help! to! retard! biodegradation!of!
adhesive!interfaces.!
Secondary!caries!is!the!principal! cause!of!dental!restoration!failure!and!a!m ajor!clinical!cha llenge!
for! dental! RBC’s.
24
! Prevalence! of! secondary! caries! was! found! to! be! higher! with! composite! than!
amalgam!restorations,!rega rdle ss!of! the!patient's!caries-risk!status.
24
!In!this!light,!there!is!currently!a!
research! trend! to w ards! developing!dental! adhesives!(and! resto rative ! m ate rials)! with! anti-bacterial!
potential.!The!com mercial!adhesive scientifically!d ocum en ted!most!with!la b o ra to ry !research!revealing!
anti-bacterial!effects!is!Clearfil!SE!Protect
!
(Kuraray!Noritake ,!Tokyo ,!Japan ).!Th is!ad he siv e!co ntains!the!
anti-bacterial!monomer!12-methacryloyloxy!dodecypyridinium!bromide!(MDPB),!which!late r!ap p e a r e d !
also!to!possess!anti-MMP!activity.
25
!Clinical!evidence!if!such!additional!therapeutic!effects!prolong!the!
lifetime!of!adhesive !t o ot h !re s to ra tio n s!is!u n fo rt un a te ly !s till!in s u ffic ien tly !c o n vin c in g.
26
!Other!studies!
ascribed!anti-bacterial!effects!of!restorative!(and!luting)!materials!to!a!gradual!and!continuous!release!
of!ions!like!zinc
27
!and!fluoride
28
.!This!approach!has!stimulated!dental!materials!scientists!to !load!resin-
based! materials! w ith! active! bioglass! filler! particles;! such ! m aterials! co uld! slowly! and! continuously!
release!anti-bacterial!ions!to!the!restoration’s!imm ed iate!environment!with!potential!to!inhibit!biofilm!
development!and!grow th.!Gen erally,!the!main!function!o f!filler! p articles!is!to!enhance!the!material’s!
mechanical! properties.! Additional!bio-fun ctio na l,! anti-bacterial! and! anti-MMP! performance!of! ion-

!
releasing! filler! particles! added! to! dental! adhesives! (an d ! restorative! RBCs)! on! dentin! bonding! has!
insufficien t ly!been!a d d res se d!scientifically.!Whereas!the!design!and! development!of!bioactive!dental!
adhesives! that! relea se! ions! may! not!be! difficult,! combinin g ! ‘bioactivity ’! with! ‘mecha n ic a l! stability’!
presents!a!considerable!challenge.
4
! !
In!th is!study,!we!in ve s ti ga t e d !a!new!experimentally!des ig n ed !and!d ev elo p e d !multi-functional!UA!
that!was!intended! to!combine!better!resistance! against!hydrolytic! bond-degradation!processes!with!
additional! bioactive! potential! in! light! of! the! clinically! highly! desired! anti-bacterial! and! anti-MMP!
properties.! The! null! hypotheses! tested! were! that! (1)! water! sorption! and! solubility,! and! (2)!
polymerization! efficiency!of!the!new!m ulti-functional!a dh esiv e!was!at!least!statistically!equivalent!to!
those!of!the!reference!adhesives,!(3)!the!‘immediate’!and!‘aged’!bonding!efficacy!to!dentin!of!the!new!
multi- fu n ction al! ad he sive !d id!n o t!sig nifica ntly!underscore!those!obtained!by!the!reference!adhesives,!
and!that!the!n ew !multi-fu n ction al!adhe sive!poss ess ed!anti-MMP!(4)!and!anti-bacterial!(5)!bioactive!
potential.!
!
!
JF/@KL@<GM@H;8N/:@=;GCH/
JFEF/@OP.,").#4&'/46+%24.P/I8/&#-/$+)).,$"&'/,.*.,.#$./&-5.2"3.2F/The!experimental!two-step!UA,!
further!being!referred!to!as!‘Exp _ 2 U A ’ ,!comb i n es!an!experimental!primer!based! on! technology!of!the!
commercial!UA!G-Premio!Bond!(‘G-PrBp’,!GC),!used!as!primer!w ithout!being!separately!light-cured,!
with!an!experimental!particle-filled!adhe sive !resin!synthesized!by!GC!under!the!code !name!‘BZF-21’!
(Tokyo,! Jap an ).!The! basic!com position! is! detailed! in! Table! S 1.!Noteworthy!is!that!both!the!primer!and!
adhesive! resin!do!not!contain! the!highly!hydrophilic! m onom er!2-hydroxyethyl! methacrylate! (HEMA),!
while!the!adhesive!resin!contains!zinc-calcium-fluoride!bioglass!and!fumed!silica!filler./
Representing!the!adhesive!generation!of!UAs,!the!com m ercial!products!G-Premio!Bond!(‘G-PrB’,!
GC),! Prime&Bond! Active! (‘P&Ba’;! Dentsply! Sirona,! Konstanz,! Germany)! and! Scotchbond! Universal!
(‘SBU’;!3M!Oral!Care,!Seefe ld,!Germany),!and!the!go ld -standard!two-step!self-etch!adhesive!Clearfil!SE!
Bond!2!(‘CSE2’;!Kuraray!Noritake)!served!as!reference!(control)!adhesives.!
/
JFJF/ :$&##"#1/ 4,&#2)"22"+#/ .'.$4, + # / )"$,+2$+ P Q/ 6 "45 / .#.,1Q%-"2P.,2"3./ K%,&Q/ 2P.$4,+2$+PQ/
R:;@MS @D : T/+*/45./.OP.,").#4&'/&-5.2"3./,.2"#/9!U%JEF/To!reveal!the!adhesive’s!ultra-structure,!TEM!