Tooth Discoloration after Regenerative Endodontic Procedures with Calcium Silicate-Based Cements—An Ex Vivo Study

applied sciences A icle Too h Discolo a ion a e Regene a i e Endodon ic P ocedu es wi h Calcium Silica e-Based Cemen s—An Ex Vi o S udy Paulo J. Palma 1,2,* , Joana A. Ma ques 1, Joana San os 3, Rui I. Falacho 4, Diana Sequei a 1, Pa ícia Diogo 1,2, F ancisco Ca amelo 5, João C. Ramos 2,6 and João Miguel San os 1,2 1Ins i u e o Endodon ics, Facul y o Medicine, Uni e si y o Coimb a, 3000-075 Coimb a, Po ugal; [email p o ec ed] (J.A.M.); [email p o ec ed] (D.S.); [email p o ec ed] (P.D.); [email p o ec ed] (J.M.S.) 2Cen e o Inno a ion and Resea ch in O al Sciences (CIROS), Facul y o Medicine, Uni e si y o Coimb a, 3000-075 Coimb a, Po ugal; [email p o ec ed] 3Den is y Depa men , Facul y o Medicine, Uni e si y o Coimb a, 3000-075 Coimb a, Po ugal; [email p o ec ed] 4Ins i u e o O al Implan ology and P os hodon ics, Facul y o Medicine, Uni e si y o Coimb a, 3000-075 Coimb a, Po ugal; [email p o ec ed] 5Coimb a Ins i u e o Clinical and Biomedical Resea ch (iCBR) and Labo a o y o Bios a is ics and Medical In o ma ics (LBIM), Facul y o Medicine, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] 6Ins i u e o Ope a i e Den is y, Facul y o Medicine, Uni e si y o Coimb a, 3000-075 Coimb a, Po ugal *Co espondence: [email p o ec ed]; Tel.: +351-239-249-151 Recei ed: 13 July 2020; Accep ed: 19 Augus 2020; Published: 21 Augus 2020   Abs ac : The aim o he p esen ex i o s udy was o assess and compa e co onal discolo a ion induced by ou endodon ic bioma e ials used in egene a i e endodon ic p ocedu es (REPs). Roo esec ion was execu ed ho izon ally, 2 mm apical o he cemen oenamel junc ion, in all i y- ou ee h. A e accessing he pulp chambe , specimens we e andomly di ided in g oups and illed wi h ei he saline solu ion o blood, ollowed by calcium silica e-based cemen s (CSCs) placemen : P oRoo mine al ioxide agg ega e (MTA) (Den sply Si ona), Bioden ine (Sep odon ), To alFill BC (FKG), o pulp capping ma e ial (PCM) (Col è ne). Colo change ( ∆ E) was assessed using he L* a* b* sys em a i e di e en imepoin s (be o e and immedia ely a e bioma e ial applica ion, 72 h, 7 days, and 6 mon hs). The signi icance le el o s a is ical analysis was se a p<0.05. The e a e s a is ically signi ican di e ences ega ding ∆ E o e ime (p<0.001). S a is ical di e ences a e ound conside ing ma e ial (p<0.001), ea men (p=0.007), o bo h (p=0.002). I solely he ma e ial o ea men is conside ed, ega dless o ime, s a is ically signi ican di e ences a e de ec ed (p<0.001). A e a six-mon h pe iod o e alua ion, blood exposu e migh be a c i ical ac o in bioma e ials’ colo a ia ion. Bioden ine p esen s he lowes discolo a ion po en ial, ollowed by To alFill and PCM, albei wi hou s a is ically signi ican di e ences. MTA exhibi ed he g ea es colo a ia ion. The selec ion o bioma e ial should conside he ma e ial’s discolo a ion po en ial. Keywo ds: calcium silica e-based cemen s; endodon ic bioma e ials; egene a i e endodon ic p ocedu es; oo h discolo a ion; i al pulp he apy 1. In oduc ion Regene a i e endodon ic p ocedu es (REPs) demons a e excellen success a es o he esolu ion o pe iapical pa hology and inc ease su i al o he imma u e oo h [ 1 – 3 ]. Howe e , oo h discolo a ion Appl. Sci. 2020,10, 5793; doi:10.3390/app10175793 www.mdpi.com/jou nal/applsci Appl. Sci. 2020,10, 5793 2 o 13 is a majo es he ic conce n epo ed in mul iple s udies as an undesi able esul o egene a i e endodon ic p ocedu es [ 4 – 6 ] o i al pulp he apy [ 7 ]. REPs a e de ined as “biologically based p ocedu es designed o eplace damaged oo h s uc u es, including den ine and oo s uc u es, as well as cells o he pulp-den ine complex” [ 8 ]. The e o e, REPs aim is o egene a e he pulp-den ine complex damaged in imma u e pe manen ee h wi h pulp nec osis [ 9 ]. The associa ion be ween colo a ia ion and he use o di e en endodon ic ma e ials in REPs, namely calcium silica e-based cemen s (CSCs) such as mine al ioxide agg ega e—MTA (P oRoo MTA, Den sply Si ona, Johnson Ci y, TN, USA)—and Bioden ine (Sep odon , Sain -Mau -des-Foss é s, F ance), has been desc ibed [ 10 , 11 ]. Thus, he selec ion o he bioma e ial mus conside unc ional, biological, and es he ic aspec s, including i s discolo a ion po en ial [12–14]. MTA was de eloped as a illing ma e ial, p esen ing a wide span o clinical applica ions including REPs, pulp capping p ocedu es, apexi ica ion and apexogenesis, as well as oo eso p ions, u ca ion de ec s, and pe o a ion epai s [ 15 , 16 ]. Al hough his bioce amic shows excellen biocompa ibili y and bioac i i y, he o iginal o mula ion (g ay MTA) was associa ed wi h co onal discolo a ion [ 16 ]. This well-known d awback igge ed he de elopmen o whi e MTA, by modi ying he o iginally ma ke ed composi ion. Howe e , he la e o mula ion s ill esul s in den al s aining [ 11 , 14 , 17 , 18 ]. Conside ing MTA’s main disad an ages, including i s discolo a ion po en ial, a new gene a ion o CSCs was in oduced, such as Bioden ine (wi h zi conium oxide as adiopaci ie ), p e-mixed To alFill BC RRM Pu y (FKG, La Chaux-de-Fonds, Swi ze land) and an unde de elopmen expe imen al ma e ial (pulp capping ma e ial (PCM), Col ène/Whaleden , Al s ä en, Swi ze land). Nowadays, mul iple hypo heses ha e been ou lined ega ding he unde lying mechanisms o colo al e a ion ollowing REPs, wi h he p esence o bismu h oxide wi hin he ma e ial composi ion as adiopaci ie [ 19 – 21 ] and blood con amina ion [ 22 , 23 ] being sugges ed as key ac o s o igge and exace ba e discolo a ion, espec i ely. Cu en li e a u e s a es ha Bioden ine p esen s supe io colo s abili y o e MTA, which migh be associa ed wi h he as e se ing ime o he o me ly men ioned bioma e ial [ 11 , 18 , 24 ]. The e o e, bea ing his po en ial explana ion in mind, his s udy aims o analyze he discolo a ion po en ial o wo mo e ecen ly p esen ed calcium silica e-based cemen s—To alFill BC Pu y and a new expe imen al cemen (PCM)—which we e selec ed because o hei sho e se ing imes (app oxima ely 2 h and 3 min, espec i ely) compa ed o MTA. Mo eo e , he p esen s udy belongs o an ongoing esea ch line [ 3 , 11 ] ha aims o in es iga e and compa e he discolo a ion po en ial o se e al a ailable bioac i e cemen s used in egene a i e he apy. Since he las s udy [ 24 ] o he men ioned esea ch p ojec was conduc ed on ac ylic ee h and e alua ed he ole played by blood on colo s abili y o bo h Bioden ine and MTA, he p esen s udy in ends o con i m he possible in e ac ion be ween di e en bioma e ials and blood, when in con ac wi h he human oo h den al s uc u e. Thus, he aim o he p esen ex i o s udy was o assess and compa e co onal discolo a ion induced by ou endodon ic bioma e ials used in REPs—P oRoo MTA, Bioden ine, To alFill BC Pu y, and an expe imen al pulp capping ma e ial (PCM)—in he p esence o saline solu ion o blood. The null hypo hesis s a es ha he e a e no s a is ically signi ican di e ences be ween he expe imen al g oups. 2. Expe imen al Sec ion 2.1. Specimen P epa a ion Fi y- ou p emola s ex ac ed o o hodon ic pu poses o pe iodon al easons we e included in he p esen s udy. The numbe o samples included in he p esen s udy was based on a p e ious sample size calcula ion, pe o med in G*Powe 3.1 so wa e. Only ee h clinically and adiog aphically ee o ca ies, c acks, es o a ions, and pa hologic o ex insic discolo a ions we e selec ed o he expe imen al p ocedu es. Ex e nal su aces o each oo h we e isually inspec ed and cleaned wi h ul asonic scale and pe iodon al scale s, and polished wi h pumice and wa e in o de o emo e any o ganic ma e ial, calculus, o ex insic s aining. Appl. Sci. 2020,10, 5793 3 o 13 Roo esec ion was execu ed ho izon ally, 2 mm apical o he cemen oenamel junc ion, in all ee h. The access ca i y was p epa ed in all specimens h ough oo -end p epa a ion using a cylind ical diamond bu (2-mm diame e , D endel+Zweiling Diaman GmbH, Kalle al, Ge many) wi h high speed u bine, unde copious i iga ion. Ca i ies cen e ed on he pulp chambe wi h 4 mm dep h and 2 mm diame e we e ob ained, always ensu ing a pe iphe al minimum o 1 mm o enamel and 1 mm o den in. The access ca i ies we e hen i iga ed wi h sodium hypochlo i e (2.5% NaOCl) o emo e pulp issue emnan s, ollowed by a 17% EDTA (CanalP o EDTA, Col è ne/Whaleden , Al s ä en, Swi ze land) i iga ion o elimina e he smea laye and expose ubula den in, and hen a inal inse wi h saline solu ion (0.9% NaCl). Specimens we e s o ed in saline solu ion un il expe imen al p ocedu es s a ed. 2.2. Blood Collec ion In o med consen was ob ained om a pa icipan , acco ding o he app o al o he E hical Commi ee o IRB o he Facul y o Medicine—Uni e si y o Coimb a (no i ica ion CE001/2013), and a blood (6 mL) sample was collec ed by enipunc u e. The blood collec ion ubes we e s e ile and in e nally coa ed wi h sp ay-d ied ipo assium e hylenediamine e aace ic acid (K3EDTA) o p e en clo ing, hus allowing bo h hema ology analysis and handling du ing sample p epa a ion. Hema ologic pa ame e s included hema oc i and hemoglobin de e mina ions and an e y h ocy e coun . The ob ained alues we e 37.2% o hema oc i , 4.22 × 10 12 /L o e y h ocy es, and 11.9 g/dL o hemoglobin. Blood samples we e sealed and s o ed a 4 ◦C un il use. 2.3. Expe imen al Se up Samples we e andomly di ided in o 10 g oups by he s a i ied andom sampling me hod: one nega i e con ol g oup (n=3), one posi i e con ol g oup (n=3), and 8 expe imen al g oups (n=48), as shown in Figu e 1. Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 o 13 adiog aphically ee o ca ies, c acks, es o a ions, and pa hologic o ex insic discolo a ions we e selec ed o he expe imen al p ocedu es. Ex e nal su aces o each oo h we e isually inspec ed and cleaned wi h ul asonic scale and pe iodon al scale s, and polished wi h pumice and wa e in o de o emo e any o ganic ma e ial, calculus, o ex insic s aining. Roo esec ion was execu ed ho izon ally, 2 mm apical o he cemen oenamel junc ion, in all ee h. The access ca i y was p epa ed in all specimens h ough oo -end p epa a ion using a cylind ical diamond bu (2-mm diame e , D endel+Zweiling Diaman GmbH, Kalle al, Ge many) wi h high speed u bine, unde copious i iga ion. Ca i ies cen e ed on he pulp chambe wi h 4 mm dep h and 2 mm diame e we e ob ained, always ensu ing a pe iphe al minimum o 1 mm o enamel and 1 mm o den in. The access ca i ies we e hen i iga ed wi h sodium hypochlo i e (2.5% NaOCl) o emo e pulp issue emnan s, ollowed by a 17% EDTA (CanalP o EDTA, Col ène/Whaleden , Al s ä en, Swi ze land) i iga ion o elimina e he smea laye and expose ubula den in, and hen a inal inse wi h saline solu ion (0.9% NaCl). Specimens we e s o ed in saline solu ion un il expe imen al p ocedu es s a ed. 2.2. Blood Collec ion In o med consen was ob ained om a pa icipan , acco ding o he app o al o he E hical Commi ee o IRB o he Facul y o Medicine—Uni e si y o Coimb a (no i ica ion CE001/2013), and a blood (6 mL) sample was collec ed by enipunc u e. The blood collec ion ubes we e s e ile and in e nally coa ed wi h sp ay-d ied ipo assium e hylenediamine e aace ic acid (K3EDTA) o p e en clo ing, hus allowing bo h hema ology analysis and handling du ing sample p epa a ion. Hema ologic pa ame e s included hema oc i and hemoglobin de e mina ions and an e y h ocy e coun . The ob ained alues we e 37.2% o hema oc i , 4.22 × 10 12 /L o e y h ocy es, and 11.9 g/dL o hemoglobin. Blood samples we e sealed and s o ed a 4 °C un il use. 2.3. Expe imen al Se up Samples we e andomly di ided in o 10 g oups by he s a i ied andom sampling me hod: one nega i e con ol g oup (n = 3), one posi i e con ol g oup (n = 3), and 8 expe imen al g oups (n = 48), as shown in Figu e 1. Expe imen al g oups (N=54) Con ol Con ol / Saline (n=3) Con ol / Blood (n=3) MTA MTA / Saline (n=6) MTA / Blood (n=6) Bioden ine Bioden ine / Saline (n=6) Bioden ine / Blood (n=6) To alFill To alFill / Saline (n=6) To alFill / Blood (n=6) PCM PCM / Saline (n=6) PCM / Blood (n=6) Figu e 1. Expe imen al g oups desc ip ion (Saline: saline solu ion; MTA: P oRoo MTA; To alFill: To alFill BC Pu y; PCM: expe imen al pulp capping ma e ial). Appl. Sci. 2020,10, 5793 4 o 13 A e p eope a i e colo measu emen o each specimen, ca i ies in he con ol g oup we e illed wi h a s e ile co on pelle mois ened wi h saline solu ion (nega i e con ol–Con ol/Saline) and blood (posi i e con ol-Con ol/Blood), espec i ely. The access ca i ies o he emaining expe imen al g oups we e illed wi h di e en combina ions o liquid solu ion (saline solu ion o blood) and bioma e ial (P oRoo MTA, Bioden ine, To alFill BC, and PCM) as ollows: MTA/Saline; MTA/Blood; Bioden ine/Saline; Bioden ine/Blood; To alFill/Saline; To alFill/Blood; PCM/Saline and PCM/Blood. Ca i ies we e i s ly illed wi h ei he saline solu ion o blood, using a sy inge and needle wi h la e al exi (Kendall, Monojec , New Yo k, NY, USA). Subsequen ly, bioma e ials we e p epa ed acco ding o he manu ac u e s’ ins uc ions (Table 1) and inse ed in o he ca i ies di ec ly o e he liquid solu ions. All ca i ies we e hen sealed wi h a esin-based lowable composi e—SDR Flow+ Bulk Fill Flowable A2 (Den sply DeT ey GmbH, Kons anz, Ge many)—, and ligh -cu ed o 20 s wi h a polywa e LED cu ing ligh sou ce (Bluephase S yle, I ocla Vi aden AG, Schaan, Liech ens ein). Table 1. Ma e ials’ composi ions, manu ac u e s, p epa a ion p ocedu es, lo numbe s and expi a ion da es. Ma e ial Composi ion Manu ac u e P epa a ion P ocedu e Lo Numbe Expi a ion Da e P oRoo ®MTA T icalcium silica e, bismu h oxide, dicalcium silica e, icalcium alumina e, calcium sul a e dehyd a es o gypsum Den sply Si ona, Johnson Ci y, TN, USA Mix powde /liquid a io 1:3 177918 08/2020 Bioden ineTM Powde : icalcium silica e, dicalcium silica e, calcium ca bona e and oxide, i on oxide, zi conium oxide Liquid: calcium chlo ide, hyd osoluble polyme Sep odon , Sain -Mau -des-Fossés, F ance Pou 5 d ops o liquid in o he capsule Place he capsule on a mixing de ice Mix o 30 s B21190 11/2019 To alFill®BC RRMTM Pu y T icalcium silica e, an alum oxide, zi conium oxide FKG, La Chaux-de-Fonds, Swi ze land No mixing is equi ed Remo e he ma e ial and place i on glass slab. Place he ma e ial in o canal wi h an ins umen and comp ess i . 1702BPP 11/2019 PCM Silica es, polydime hylsiloxane, silicon oils, pla inum ca alys , zinc oxide, zi conium dioxide, bioac i e glass, pigmen Col ène/Whaleden , Al s ä en, Swi ze land Ready o apply using au o-mixing ips 2018120-P3-RR 08/2019 SDR TM Bulk ill lowable composi e Ba ium-alumino- luo o-bo osilica e glass, s on ium-alumino- luo o-silica e glass, modi ied u e hanedime hac yla e esin, EBPADMA, TEGMA, CQ, pho oaccele a o , BHT, UV s abilize , i anium dioxide, i on oxide pigmen s, luo escing agen Den sply DeT ey GmbH, Kons anz, Ge many Dispense SDRTM ma e ial Ligh -cu e o a leas 20 s 1803000656 02/2021 Too h illing and pho og aphic eco d we e pe o med in he labo a o y a 23.5 ◦ C in a 52% humidi y en i onmen . Immedia e pos ope a i e colo measu emen s we e eco ded, and specimens we e s o ed in a da k en i onmen , in an incuba o (Gallenkamp, London, UK) a 37 ◦ C and 100% humidi y un il subsequen colo measu emen e alua ion pe iods. 2.4. Pho og aphic Reco d Pho og aphic egis e was pe o med wi h a Canon EOS 5DsR came a using a Canon EF 100 mm /2.8 L Mac o IS USM Lens and a Canon Mac o Twin Li e MT-24EX wi h emi e s posi ioned a a 45 ◦ angle and c oss pola iza ion il e s (pola _eyes, Emula ion G oup). The ollowing se ings we e used: F22 ape u e, ISO 100, 1/125 shu e speed, Flash a Manual 1 2 powe and cus om whi e balance (wi h a 18% g ey ca d–eLAB–Emula ion G oup). The pho og aphs we e aken om he buccal wall/side o he ee h and we e sa ed in RAW ile o ma . In o de o assu e s anda d posi ioning o he samples, a silicone de ice (Vi ual Re ill Pu y Fas Se , I ocla Vi aden AG, Schaan, Liech ens ein) was p epa ed o hold he specimens and he g ey ca d scale was used as a e e ence o p ope ly cen e and place he samples, as seen in Figu e 2. Appl. Sci. 2020,10, 5793 5 o 13 Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 o 13 In pos -p oduc ion, be o e aking colo measu emen s, all pho og aphic egis e s we e calib a ed using he Whi e Balance Selec o and he Exposu e slide o Adobe Pho oshop Ligh oom (Adobe Sys ems, San Jose, CA, USA). Figu e 2. Posi ioning de ice o pho og aphic eco d. The blue dashed line delimi s he o al a ea conside ed o shade analysis wi hin he Commission In e na ional de l’Eclai age (CIE) L* a* b* colo space o all ime ames. 2.5. Too h Colo Measu emen In shade analysis he e a e i e e alua ion pe iods (Figu e 3): T0: baseline (a e ca i y p epa a ion, be o e bioma e ial placemen ); TPO: immedia ely a e bioma e ial placemen and p o isional es o a ion; T72H: a e 72 h o s o age; T7D: a e 7 days o s o age; and T6M: a e six mon hs o s o age. Colo assessmen was pe o med by a single ope a o using ImageJ (Na ional Ins i u es o Heal h, NIH) so wa e, conside ing a cen al ci cula a ea, ocusing on he heigh and wid h o he oo h c own, wi h he o al a ea o 256 × 256 pixels. The colo space was measu ed wi h he ules o he Commission In e na ional de l’Eclai age (CIE) L * a * b * sys em, wi h L * alues co esponding o he ligh ness o luminance ( anging om 0 [black] o 100 [whi e]), a * alues ma ching he ed-g een axis ( ed—posi i e a *; g een—nega i e a *) and he alues o b* exhibi ing he yellow-blue axis (yellow— posi i e b *; blue—nega i e b *). ∆E desc ibes he colo a ia ion be ween baseline and each o he subsequen colo measu emen pe iods o e alua ion (TPO, T72H, T7D, and T6M), de e mined by using he ollowing o mula: ∆E = [󰇛∆L󰇜+ 󰇛∆a󰇜+ 󰇛∆b󰇜] / (1) Pe cep ible ch oma ic al e a ions o he human eye we e de ined a he h eshold alue o ∆E ≥ 3.3 [25]. Figu e 2. Posi ioning de ice o pho og aphic eco d. The blue dashed line delimi s he o al a ea conside ed o shade analysis wi hin he Commission In e na ional de l’Eclai age (CIE) L* a* b* colo space o all ime ames. In pos -p oduc ion, be o e aking colo measu emen s, all pho og aphic egis e s we e calib a ed using he Whi e Balance Selec o and he Exposu e slide o Adobe Pho oshop Ligh oom (Adobe Sys ems, San Jose, CA, USA). 2.5. Too h Colo Measu emen In shade analysis he e a e i e e alua ion pe iods (Figu e 3): T 0 : baseline (a e ca i y p epa a ion, be o e bioma e ial placemen ); T PO : immedia ely a e bioma e ial placemen and p o isional es o a ion; T 72H : a e 72 h o s o age; T 7D : a e 7 days o s o age; and T 6M : a e six mon hs o s o age. Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 o 13 Figu e 3. Colo assessmen o andomly selec ed ep esen a i e samples om each expe imen al g oup a he i e e alua ed ime ames (T0, TPO, T72H, T7D, and T6M). 2.6. S a is ical Analysis S a is ical analysis was pe o med using IBM SPSS S a is ics e sion 24 so wa e and he signi icance le el was se a α = 0.05. The desc ip ion o he esul s ega ding each ma e ial (MTA, Bioden ine, To alFill, and PCM) and each ea men op ion (saline solu ion o blood) was a ained using he mean and s anda d de ia ion. To e alua e colo a ia ion o e ime, conside ing bo h he bioma e ial and he ea men , a epea ed measu es ANOVA wi h wo independen ac o s was applied. Fo each ma e ial and condi ion, a g aph was c ea ed depic ing he ch oma ic coo dina es (L*, a*, b*) and he a ia ion o ΔE o e ime. A F iedman es allowed assessmen o he di e ences o e ime. K uskal–Wallis es ing was execu ed o e alua e he di e ences be ween he g oups a T6M. K uskal–Wallis es ing was epea ed o each condi ion. 3. Resul s Table 2 shows he desc ip i e s a is ics ob ained o L* a* b* ch oma ic coo dina es mean alues wi hin he di e en s udy g oups o e ime. The ini ial momen is ep esen ed by T0, which co esponds o he baseline. Also, colo a ia ion (ΔE) be ween baseline and each o he subsequen colo measu emen pe iods o e alua ion is shown in Table 2. Figu e 3. Colo assessmen o andomly selec ed ep esen a i e samples om each expe imen al g oup a he i e e alua ed ime ames (T0, TPO, T72H, T7D, and T6M). Appl. Sci. 2020,10, 5793 6 o 13 Colo assessmen was pe o med by a single ope a o using ImageJ (Na ional Ins i u es o Heal h, NIH) so wa e, conside ing a cen al ci cula a ea, ocusing on he heigh and wid h o he oo h c own, wi h he o al a ea o 256 × 256 pixels. The colo space was measu ed wi h he ules o he Commission In e na ional de l’Eclai age (CIE) L* a* b* sys em, wi h L* alues co esponding o he ligh ness o luminance ( anging om 0 [black] o 100 [whi e]), a* alues ma ching he ed-g een axis ( ed—posi i e a*; g een—nega i e a*) and he alues o b* exhibi ing he yellow-blue axis (yellow—posi i e b*; blue—nega i e b*). ∆E desc ibes he colo a ia ion be ween baseline and each o he subsequen colo measu emen pe iods o e alua ion (T PO , T 72H , T 7D , and T 6M ), de e mined by using he ollowing o mula: ∆E=h(∆L)2+(∆a)2+(∆b)2i1/2(1) Pe cep ible ch oma ic al e a ions o he human eye we e de ined a he h eshold alue o ∆ E ≥ 3.3 [ 25 ]. 2.6. S a is ical Analysis S a is ical analysis was pe o med using IBM SPSS S a is ics e sion 24 so wa e and he signi icance le el was se a α =0.05. The desc ip ion o he esul s ega ding each ma e ial (MTA, Bioden ine, To alFill, and PCM) and each ea men op ion (saline solu ion o blood) was a ained using he mean and s anda d de ia ion. To e alua e colo a ia ion o e ime, conside ing bo h he bioma e ial and he ea men , a epea ed measu es ANOVA wi h wo independen ac o s was applied. Fo each ma e ial and condi ion, a g aph was c ea ed depic ing he ch oma ic coo dina es (L*, a*, b*) and he a ia ion o ∆ E o e ime. A F iedman es allowed assessmen o he di e ences o e ime. K uskal–Wallis es ing was execu ed o e alua e he di e ences be ween he g oups a T 6M . K uskal–Wallis es ing was epea ed o each condi ion. 3. Resul s Table 2shows he desc ip i e s a is ics ob ained o L* a* b* ch oma ic coo dina es mean alues wi hin he di e en s udy g oups o e ime. The ini ial momen is ep esen ed by T 0 , which co esponds o he baseline. Also, colo a ia ion ( ∆ E) be ween baseline and each o he subsequen colo measu emen pe iods o e alua ion is shown in Table 2. Table 2. Mean and s anda d de ia ion alues o each o he L* a* b* coo dina es o each expe imen al g oup o all pe iods o e alua ion. ∆ E alues exp ess colo a ia ion om baseline (T 0 ) o each e alua ion imepoin (TPO, T72H, T7D, and T6M). G oups Coo dina es T0TPO T72H T7D T6M p Con ol/Saline L* 58.6 ±6.6 61.6 ±6.0 58.6 ±6.5 60 ±6.6 59.5 ±7.1 =0.139 a* 1 ±5.2 2.8 ±5.2 3.5 ±6.5 3.2 ±5.9 3.4 ±5.3 =0.615 b* 18.1 ±12.6 17.6 ±11.7 19.1 ±12.5 19.3 ±12.3 19 ±12.3 =0.162 ∆E 3.8 ±2.1 2.0 ±0.7 2.2 ±0.9 1.6 ±0.6 =0.615 MTA/Saline L* 59.2 ±5.6 60.4 ±5.6 55.6 ±5.2 52.3 ±5.1 52.2 ±7.3 <0.001 † a* 2.0 ±2.4 2.0 ±2.5 1.1 ±1.6 0.8 ±1.5 0.7 ±1.1 =0.070 b* 15.6 ±6.5 14.7 ±6.0 12.0 ±5.3 11.1 ±4.9 11.3 ±5<0.001 † ∆E 1.8 ±0.8 5.5 ±1.1 8.6 ±1.2 8.5 ±2.6 =0.001 † Bioden ine/Saline L* 55.6 ±4.9 56.0 ±4.0 55.6 ±4.4 52.5 ±5.0 55.5 ±4=0.012 † a* 2.8 ±3 3.2 ±2.8 3.7 ±2.6 3.3 ±2.7 4.8 ±2.6 <0.001 † b* 16.4 ±4.7 16.6 ±4.1 17.1 ±4.3 16.2 ±3.8 19 ±4.5 =0.003 † ∆E 1.5 ±0.6 1.4 ±0.7 3.4 ±0.9 3.4 ±0.9 =0.006 † To alFill/Saline L* 60.1 ±5.4 61.9 ±4.7 61.2 ±5.2 60.3 ±5.4 60.1 ±4.7 =0.162 a* 0.8 ±2.7 0.9 ±3.1 1.1 ±2.9 1.2 ±3.0 1.9 ±3.0 =0.011 † b* 13.0 ±6.8 14.2 ±6.8 14.8 ±6.4 14.6 ±6.6 17.7 ±5.5 =0.001 † ∆E 2.3 ±0.9 2.4 ±0.7 2.2 ±0.5 4.9 ±2.4 =0.102 PCM/Saline L* 59.2 ±3.4 60.8 ±3.6 60.7 ±3.6 56.8 ±3.6 61.6 ±1.3 =0.003 † a* 1.4 ±3.1 1.5 ±2.7 1.2 ±2.9 1.2 ±2.7 0.1 ±2.5 =0.171 b* 15.1 ±6.5 15.4 ±5.1 15.7 ±6.3 14.6 ±6.0 14.4 ±5.3 =0.139 ∆E 2.5 ±0.5 1.7 ±0.8 2.6 ±1.5 5.1 ±3.9 =0.284 Appl. Sci. 2020,10, 5793 7 o 13 Table 2. Con . G oups Coo dina es T0TPO T72H T7D T6M p Con ol/Blood L* 58.4 ±9.2 58.0 ±8.9 53.5 ±12.0 53.6 ±10.3 51.3 ±7.9 =0.053 a* 1.0 ±3.2 4.9±3.7 3.9 ±4.0 3.4 ±3.6 3.3 ±2.8 =0.034 † b* 12.5 ±5.9 10.6 ±5.3 7.5 ±4.8 8.7 ±4.9 10.8 ±5.1 =0.022 † ∆E 4.4 ±1.4 7.7 ±4 6.7 ±2.8 8.0 ±1.7 =0.122 MTA/Blood L* 58.4 ±5.1 58.6 ±4.3 54.5 ±4.1 51.3 ±4.0 52.1 ±5.1 <0.001 † a* 1.0 ±2.0 1.5 ±1.6 0.0 ±2.0 -0.4 ±2.0 0.6 ±1.9 <0.001 † b* 16.0 ±3.2 13.5 ±4.3 11.2 ±4 9.7 ±3.8 12.2 ±3.1 <0.001 † ∆E 3.4 ±2.7 6.4 ±3.1 9.8 ±2.6 7.6 ±2.4 =0.004 † Bioden ine/Blood L* 60.6 ±4.2 60.6 ±4.0 60.9 ±4.1 57.3 ±3.9 61.0 ±4.2 =0.013 † a* 0.8 ±2.4 0.9 ±2.2 1.4 ±2.5 1.0 ±1.9 1.9 ±2.3 =0.010 † b* 12.8 ±5.5 12.2 ±4.8 14.0 ±4.5 12.4 ±3.8 16.7 ±4.4 =0.004 † ∆E 1.6 ±1.4 2.1 ±1.0 3.8 ±1.2 4.6 ±1.6 =0.009 † To alFill/Blood L* 59.1 ±7.8 59.4 ±7.0 57.5 ±7.1 54.6 ±6.5 56.7 ±6.7 =0.001 † a* 0.5 ±2.5 2.7 ±3.2 1.7 ±3.0 1.5 ±2.9 1.8 ±2.9 =0.015 † b* 11.0 ±4.3 10.3 ±4.5 11.4 ±3.5 10.2 ±3.3 15.5 ±3.0 =0.006 † ∆E 3.1 ±2.1 2.5 ±0.9 5.1 ±1.3 5.5 ±2.3 =0.086 PCM/Blood L* 58.6 ±6.9 58.6 ±4.8 55.0 ±5.9 51.0 ±5.6 55.9 ±5.6 =0.001 † a* 2.0 ±2.2 3.9 ±2.0 4.8 ±3.7 4.0 ±3.4 3.2 ±2.6 =0.004 † b* 15.9 ±6.8 14.3 ±7.1 15.8 ±4.9 14.5 ±4.6 18.6 ±5.6 =0.003 † ∆E 3.3 ±1.2 5.5 ±2.9 8.4 ±3.0 4.5 ±2.5 =0.006 † T 0 : baseline (a e ca i y p epa a ion, be o e bioma e ial placemen ); T PO : immedia ely a e bioma e ial placemen and p o isional es o a ion; T 72H : a e 72 h o s o age; T 7D : a e 7 days o s o age; and T 6M : a e six mon hs o s o age. Mean ±s anda d de ia ion. †S a is ically signi ican di e ence (p<0.05). The e a e s a is ically signi ican di e ences (F(2.11 92.90) =33.58; p<0.001) ega ding colo a ia ion ( ∆ E) o e ime. All CSC g oups showed a s a is ically signi ican a ia ion (mainly dec easing) in L* alues o e ime, excep To alFill/Saline g oup. MTA p esen ed a no iceable educ ion in L* alues om T PO o T 7D , ega dless o he ea men (saline o blood), as well as PCM con amina ed by blood. Howe e , a T 6M , g oup compa ison ega ding ligh ness e ealed no s a is ical di e ences be ween he es ed bioma e ials ei he when in con ac wi h saline solu ion (p=0.065) o blood (p=0.102), which also applies o a* and b* alues in bo h condi ions (saline: a* p=0.072; b* p=0.355; blood: a* p=0.148; b* p=0.060). When in con ac wi h saline solu ion, bo h MTA and Bioden ine exhibi ed s a is ically signi ican colo al e a ion o e ime (p=0.001 and p=0.012, espec i ely). In he case o blood con amina ion, MTA kep p esen ing s a is ically signi ican colo a ia ion (p=0.001) alongside Bioden ine (p=0.009), and PCM (p=0.006). No s a is ically signi ican di e ences we e de ec ed ega ding ∆ E o To alFill g oups o e ime, ei he in con ac wi h saline solu ion (p=0.102) o blood (p=0.086). A 6 mon hs all CSC g oups p esen ed pe cep ible colo changes (∆E>3.3). S a is ical di e ences a e ound conside ing he ac o ma e ial (F(8.45, 92.90) =5.635; p<0.001), he ac o ea men (F(2.11, 92.90) =5.04; p=0.007) o bo h ma e ial and ea men (F(8.45, 92.90) =3.31; p=0.002). The g aphs on Figu e 4show he mean ∆ E o e ime, conside ing ma e ials (Figu e 4a) and ea men (Figu e 4b) a ia ion. Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 o 13 S a is ical di e ences a e ound conside ing he ac o ma e ial (F(8.45, 92.90) = 5.635; p < 0.001), he ac o ea men (F(2.11, 92.90) = 5.04; p = 0.007) o bo h ma e ial and ea men (F(8.45, 92.90) = 3.31; p = 0.002). The g aphs on Figu e 4 show he mean ΔE o e ime, conside ing ma e ials (Figu e 4a) and ea men (Figu e 4b) a ia ion. Figu e 4. Colo a ia ion (ΔE) o e ime conside ing only he (a) ma e ials (mine al ioxide agg ega e (MTA), Bioden ine, To alFill, and PCM) and (b) ea men (Saline and Blood) a ia ion. I solely he ma e ial is conside ed ega dless he iming o e alua ion, s a is ically signi ican di e ences a e ound (F(4, 44) = 13.05; p <0.001), which also applies o ea men (F(1, 44) = 23.11; p < 0.001). MTA is he ma e ial showing lowes colo s abili y, wi h s a is ically signi ican di e ences (p < 0.001) being de ec ed in ega d o all o he es ed CSCs (Bioden ine, To alFill, and PCM). Fu he mo e, no s a is ically signi ican di e ences a e ound be ween Bioden ine, To alFill, and PCM. Addi ionally, he e a e s a is ically signi ican di e ences (F(4.44) = 2.71; p = 0.042) conce ning he in e ac ion be ween ac o ma e ial and ac o ea men . F om all he CSC g oups, MTA/Saline and MTA/Blood showed he highes colo a ia ion, wi h s a is ically signi ican di e ences. The o al ∆E om T0 o each o he ollowing assessmen pe iods is p esen ed in Figu e 5. Figu e 4. Colo a ia ion ( ∆ E) o e ime conside ing only he ( a ) ma e ials (mine al ioxide agg ega e (MTA), Bioden ine, To alFill, and PCM) and (b) ea men (Saline and Blood) a ia ion. Appl. Sci. 2020,10, 5793 8 o 13 I solely he ma e ial is conside ed ega dless he iming o e alua ion, s a is ically signi ican di e ences a e ound (F(4, 44) =13.05; p<0.001), which also applies o ea men (F(1, 44) =23.11; p<0.001). MTA is he ma e ial showing lowes colo s abili y, wi h s a is ically signi ican di e ences (p<0.001) being de ec ed in ega d o all o he es ed CSCs (Bioden ine, To alFill, and PCM). Fu he mo e, no s a is ically signi ican di e ences a e ound be ween Bioden ine, To alFill, and PCM. Addi ionally, he e a e s a is ically signi ican di e ences (F(4.44) =2.71; p=0.042) conce ning he in e ac ion be ween ac o ma e ial and ac o ea men . F om all he CSC g oups, MTA/Saline and MTA/Blood showed he highes colo a ia ion, wi h s a is ically signi ican di e ences. The o al ∆ E om T0 o each o he ollowing assessmen pe iods is p esen ed in Figu e 5. Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 o 13 S a is ical di e ences a e ound conside ing he ac o ma e ial (F(8.45, 92.90) = 5.635; p < 0.001), he ac o ea men (F(2.11, 92.90) = 5.04; p = 0.007) o bo h ma e ial and ea men (F(8.45, 92.90) = 3.31; p = 0.002). The g aphs on Figu e 4 show he mean ΔE o e ime, conside ing ma e ials (Figu e 4a) and ea men (Figu e 4b) a ia ion. Figu e 4. Colo a ia ion (ΔE) o e ime conside ing only he (a) ma e ials (mine al ioxide agg ega e (MTA), Bioden ine, To alFill, and PCM) and (b) ea men (Saline and Blood) a ia ion. I solely he ma e ial is conside ed ega dless he iming o e alua ion, s a is ically signi ican di e ences a e ound (F(4, 44) = 13.05; p <0.001), which also applies o ea men (F(1, 44) = 23.11; p < 0.001). MTA is he ma e ial showing lowes colo s abili y, wi h s a is ically signi ican di e ences (p < 0.001) being de ec ed in ega d o all o he es ed CSCs (Bioden ine, To alFill, and PCM). Fu he mo e, no s a is ically signi ican di e ences a e ound be ween Bioden ine, To alFill, and PCM. Addi ionally, he e a e s a is ically signi ican di e ences (F(4.44) = 2.71; p = 0.042) conce ning he in e ac ion be ween ac o ma e ial and ac o ea men . F om all he CSC g oups, MTA/Saline and MTA/Blood showed he highes colo a ia ion, wi h s a is ically signi ican di e ences. The o al ∆E om T0 o each o he ollowing assessmen pe iods is p esen ed in Figu e 5. Figu e 5. ∆E o all expe imen al g oups o e ime. Table 3is gene a ed by pe o ming g oup compa ison acco ding o indi idual e alua ion o ma e ial/ ea men in e ac ion. PCM con amina ed by blood led o a s a is ically highe colo change when compa ed o Bioden ine/Saline (p=0.012). Table 3. p- alues ob ained om g oup compa ison ega ding he in e ac ion o ac o ma e ial and ac o ea men . MTA/Saline Bioden ine/Saline To alFill/Saline PCM/Saline Blood MTA/Blood Bioden ine/Blood To alFill/Blood PCM/Blood Saline 0.010 * 1.000 1.000 1.000 0.010 * 0.001 * 1.000 0.767 0.069 MTA/Saline 0.001 * 0.006 * 0.007 * 1.000 0.996 0.008 * 0.218 0.996 Bioden ine/Saline 1.000 0.999 0.002 * <0.001 * 0.999 0.544 0.012 * To alFill/Saline 1.000 0.010 * <0.001 * 1.000 0.918 0.069 PCM/Saline 0.010 * <0.001 * 1.000 0.921 0.071 Blood 1.000 0.012 * 0.174 0.943 MTA/Blood 0.001 * 0.028 * 0.750 Bioden ine/Blood 0.939 0.082 To alFill/Blood 0.739 * S a is ically signi ican di e ence (p<0.05). 4. Discussion The aim o he p esen ex i o s udy was o e alua e and compa e he discolo a ion po en ial o ou di e en CSCs used in egene a i e endodon ic p ocedu es, as well as o assess he ole played by blood con amina ion on colo a ia ion se e i y when CSCs a e applied. Theo e ically, ecen ly in oduced bioma e ials (Bioden ine, To alFill, and PCM) wi h a as e se ing ime when Appl. Sci. 2020,10, 5793 9 o 13 compa ed o he gold s anda d MTA migh p esen supe io colo s abili y. Mo eo e , i is c ucial o un eil he possible ole played by blood when in con ac wi h CSCs since blood exposu e is likely o occu in bo h REPs and i al pulp he apy. An insigh in o hese ma e s would allow adequa e selec ion o he bioma e ial o use based on he clinical condi ions, ul ima ely allowing he accomplishmen o be e es he ic esul s. In ac , i has been ex ensi ely epo ed ha REPs lead o den al discolo a ion. This undesi able consequence o egene a i e he apy is a majo conce n o bo h clinicians and pa ien s, especially when an es he ic a ea is in ol ed [ 4 – 7 ]. REPs p o ocol in ol es he c ea ion o a ce ical plug by placing a 3–4 mm CSC laye in he co onal po ion o he oo canal. Endodon ic ma e ials should p esen ch oma ic s abili y and op ic p ope ies simila o den al s uc u es and no cause oo h discolo a ion o e ime [ 14 ]. Howe e , as p e iously men ioned, he associa ion o CSCs wi h colo a ia ion o e ime is well-desc ibed wi hin he li e a u e. The ob ained esul s show ha he e a e s a is ically signi ican di e ences in ∆ E o e ime (p<0.001). In ac , s a is ical di e ences we e ound conside ing he ac o ma e ial (p<0.001), he ac o ea men (p=0.007) o he in e ac ion o bo h ac o s (p=0.001), o e ime. The e o e, he null hypo hesis, s a ing ha he e a e no s a is ically signi ican di e ences be ween he expe imen al g oups, has been ejec ed. Se e al possible mechanisms ha e been ou lined o explain colo al e a ion ollowing CSC placemen , one being ha he composi ion o he bioma e ial is a majo con ibu ing ac o o CSCs’ discolo a ion po en ial [ 26 ]. MTA o mula con ains bismu h oxide as a adiopaci ie (Bi 2 O 3 ), which has been p o en o lead o oo h discolo a ion h ough i s (1) educ ion o (2) oxida ion when in con ac wi h s ong oxidizing agen s, such as den in collagen and sodium hypochlo i e, used as an i iga ion solu ion [ 26 ]. In ac , p e ious s udies consis en ly demons a e MTA o exhibi lowe colo s abili y when compa ed o al e na i e calcium silica e-based cemen s [ 10 , 27 ], hypo hesizing ha he ob ained esul s could a ise om he inco po a ion o di e en adiopaci ie s such as zi conium oxide and an alum oxide, ins ead o bismu h oxide, in he mo e ecen ly in oduced CSCs [26]. Conce ning blood con amina ion, he o e all a ailable scien i ic e idence shows blood exposu e o be a ac o ha signi ican ly exace ba es CSC colo al e a ion [ 2 ]. This discolo a ion se e i y inc ease by blood migh be ela ed o ma e ial po osi y and p esence o absence o smea laye , which can educe o inc ease den in pe meabili y, espec i ely. MTA has a longe se ing ime (2 h and 45 min) compa ed wi h all he emaining h ee es ed ma e ials (Bioden ine, 12 min; To alFill, 2 h; PCM, 3 min). The e o e, i seems MTA emains po ous o longe which esul s in inc eased blood abso p ion and subsequen hemolysis, wi h consequen ly g ea e discolo a ion. On he o he hand, a ecen in i o s udy ound ha con ac wi h blood a e a ollow-up pe iod o 6 mon hs does no modi y colo al e a ions su e ed by he bioma e ials [ 24 ]. Ou indings do no line up wi h he esul s o he p e iously men ioned s udy since, a e a 6-mon h pe iod o e alua ion, he p esen s udy ound s a is ically signi ican di e ences ega ding he ac o ma e ial, as well as ega ding he ea men (blood/saline solu ion), meaning ha blood exposu e migh be a c i ical ac o in bioma e ials’ colo a ia ion. The con as ing esul s in espec o he ole played by blood on colo s abili y o CSCs, among s udies wi h simila expe imen al p o ocols, migh be ela ed o he use o di e en expe imen al models. The s udy o Palma e al. [ 24 ] was conduc ed in an ac ylic model, while he p esen s udy was made unde ex i o condi ions. The use o na u al human ee h may ha e led o a discolo a ion enhancemen in he case o blood con amina ion due o he pene a ion o blood in o he den inal ubules [ 11 ]. Fo ha eason he p esence o blood has become a ac o ha signi ican ly in luences he ch oma ic esul in he p esen s udy, which is in ag eemen wi h he o e all li e a u e [2]. I is, he e o e, c ucial o ensu e comple e hemos asis [ 26 ] and pe o m adequa e blood clo s abiliza ion, as well as o clean he blood p esen on den in walls as me iculously as possible, o p e en blood in lux in o he oo h s uc u e p io o CSC placemen [ 24 ]. Mo eo e , he use o na u al ee h as expe imen al model ep oduces he clinical con ex mo e ai h ully, hus allowing a be e o ecas o he impac o blood con amina ion on he es he ic ou come o he ea men .