The Effect of Altered pH on Push-Out Bond Strength of Biodentin, Glass Ionomer Cement, Mineral Trioxide Aggregate and Theracal

Summary Introduction Throughout the history of dentistry, a wide variety of materials such as gold-foil, silver posts, amalgam, zinc oxide eugenol, glass ionomer cements, mineral trioxide aggregate have been used as retrograde fillings. Altered pH in periapical lesions can affect push-out bond strength of these materials. The aim of this study was to evaluate the effect of altered pH on push-out bond strength of Biodentin, Glass ionomer cement (GIC), Mineral trioxide aggregate (MTA) and Theracal. Material and Methods Forty-eight dentin slices of extracted single-rooted human teeth were sectioned and their canal portion instrumented to achieve a diameter of 1.4 mm. The specimens were then assigned into the four groups (one group for each material) with 12 samples in each group. All groups were further divided into 3 subgroups (with 4 specimens in each subgroup): acidic (butyric acid buffered at pH 6.4), neutral (phosphate buffer saline solution at pH 7.4) and alkaline (buffered potassium hydroxide at pH 8.4). Samples were incubated for 4 days at 37°C in acidic, neutral or alkaline medium. Push-out bond strength was measured using a Universal Testing Machine. The slices were examined under a stereomicroscope to determine the nature of bond failure. Results GIC showed the highest bond strength (33.33MPa) in neutral and acidic medium (26.75MPa) compared to other materials. Biodentin showed the best result in alkaline medium. Conclusion Altered pH level affected push-out bond strength of root end materials. GIC demonstrated good push-out bond strength that increased with decrease of pH whereas newer materials Biodentin and Theracal showed satisfying results in altered pH.


INTRODUCTION
Infected or inflamed tissue may have a normal pH of 7.4 or an acidic pH as low as 5.0. Acidic pH may inhibit setting reaction, affect adhesion, or increase solubility of materials. If infection or inflammation persists, erosion of filling materials can occur in acidic environment generated by bacteria or inflammation [1]. Also, exposure of root end filling material to an alkaline environment after pretreatment with calcium hydroxide might affect its properties [2]. Therefore, sealing ability of material may be directly or indirectly affected by environmental conditions and pH of medium [1]. Over the years various root end filling materials such as gold-foil, silver posts, amalgam (with and without bonding agent), zinc oxide eugenol, glass ionomer cements, mineral trioxide aggregate have been used as retrograde fillings [3].
Glass ionomer cements (GICs) are widely used for a variety of purposes such as intermediate restorations, permanent restoration of micro-cavities, fissure sealing of erupting molars and root end filling material [4]. GICs are formed by the reaction between calcium-aluminosilicate glass particles and aqueous solutions of polyacrylic acid.
The main advantages of GICs are their strong chemical bond with dentin and their ability to release fluoride ions. Due to this chemical bonding to dentin, GICs have shown higher bond strength even when used as root end filling material. It has been reported that these cements are easy to handle and they do not cause any adverse histological reaction in the periapical tissue [3]. However, push-out bond strength of GICs under altered pH has not been studied yet.
In the recent decades, mineral trioxide aggregate (MTA) has shown promising results when used as repair material of lateral root walls or furcation perforations, root-end filling, apical plug, and root canal filling [5,6]. It consists of a fine powder of tricalcium silicate, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite, and bismuth oxide. During clinical application as root-end and perforation filling material or as an apical plug in necrotic teeth with open apices, MTA may be exposed to an acidic environment because of the presence of periradicular inflammation [7,8]. pH change of host tissues because of the presence of pre-existing disease may affect physical and chemical properties of material [9,10]. It has been reported that hardness [11], diametric tensile strength [8], push-out bond strength [10], and sealing ability of MTA [12] decrease after placement in acidic environment. As MTA has been shown to be suitable material, other calcium silicate-based materials have been developed recently to improve MTA drawbacks such as prolonged setting time, difficult handling, high cost, and potential tooth discoloration.
Recently, a new calcium silicate-based material Biodentin (Septodont, Saint-Maur-des-Fossés, France) has been introduced to the market. Biodentin is composed of tricalcium silicate, calcium carbonate, zirconium oxide, and a water-based liquid containing calcium chloride used as setting accelerator and water-reducing agent [13]. Biodentin is a fast-setting calcium silicate-based material that can be used as dentin restorative material as well as endodontic material with characteristics comparable to MTA [14].
The aim of periradicular surgery is to remove the cause of disease and provide favourable environment for surgical wound healing. Placement of a root-end filling is one of the key steps in managing root end [18]. However, periapical lesions affect pH with consequent alteration of bond strength of root end filling materials.
The aim of the current study was to evaluate the effect of altered pH on push-out bond strength of Biodentin, Glass ionomer cement, Mineral trioxide aggregates and Theracal.

MATERIAL AND METHODS
Forty-eight single-rooted human anterior teeth with straight canals, extracted for periodontal and orthodontic reasons, were collected and stored in phosphate-buffered saline solution until used. Midroot dentin was sectioned horizontally into slices 1 mm thick. A diamond disc was used to obtain 48 root dentin slices ( Figure 1). The canal portions of root dentin slices were instrumented to achieve a standardized diameter of 1.4 mm using round carbide bur ( Figure 2). All used materials were mixed according to their manufacturers' instructions and introduced incrementally with no pressure into the lumens of the root-dentin slices. The specimens were then divided into the four groups (n=12), i.e. Biodentin, Glass ionomer cement, MTA (Mineral trioxide aggregates) and Theracal. These groups were further divided into 3 subgroups (n s =4), i.e. acidic (butyric acid buffered at pH 6.4), neutral (phosphate buffer saline solution at pH 7.4) and alkaline (buffered potassium hydroxide at pH 8.4). They were then incubated for 4 days at 37°C.
Push-out bond strength was measured using a universal testing machine ( Figure 3). The samples were placed   on a metal slab with a central hole to allow free motion of the plunger. Compressive load was applied by exerting downward pressure on the surface of materials using 1 mm diameter cylindrical jig at a speed of 1 mm/min. The jig had a clearance of approximately 0.2 mm from the margin of dentinal wall to insure contact with material only. Maximum load applied to material at the time of dislodgement was recorded in newton's.
In order to express bond strength in MPa, recorded value was divided by the adhesion area of root canal filling calculated by the following formula: 2πr×h, where r is the root canal radius and h is the thickness of the root-dentin slice in millimeters. The slices were then examined under a stereomicroscope to determine the nature of bond failure. Each sample was categorized into one of three failure modes: adhesive failure at the material and dentin interface, cohesive failure within materials, or mixed failure. The data were analyzed using one-way analysis of variance followed by the Tukey's Post Hoc Test.

RESULTS
Results showed a statistically significant difference among groups (p<0.001) after 4 days, where GIC had the highest bond strength in acidic and neutral environment while Biodentin showed the highest bond strength in alkaline environment. MTA showed the lowest push-out bond strength in all mediums compared to other root end filling materials. Statistical analysis also showed that the type of cement and storage solution significantly affected micropush-out bond strength. Three modes of bond failure were found: adhesive failure at the material and dentin interface, cohesive failure within material, or mixed failure (Figure 4a-c; Graph 1; Table 1).

DISCUSSION
Russian biologist Metchnikoff reported first evidence of acidic pH inside phagocytes in 1893. Later, Jensen and Bainton (1973) demonstrated that pH of a phagosome was reduced to approximately 6.5 within 3-4 min after initiation of phagocytosis. Also pH of pus aspirated from periapical tissues has been confirmed as acidic (6.68±0.324) [9]. Under certain clinical applications calcium silicatebased materials are used for the repair of root and furcation perforations, root-end fillings, and apical plugs. They  are often placed in an environment where inflammation may be present and surface of unset material exposed to low pH. This altered pH may affect its physical and chemical properties [18,19].
Normal tissue pH is 7.4 but it can be affected by certain clinical conditions. Tronstad et al. [20] showed pH in the range of 6.4-7 in the pulp, dentin, cementum, and periodontal ligament of vital or necrotic pulp teeth. As calcium hydroxide is preferred intracanal medicament, after its placement pH values of the most inner part of circumpulpal dentin change to pH range 11.1-12.2. It might be beneficial to provide pretreatment with calcium hydroxide in necrotic open apices or root perforations before application of biomaterials [21,22]. There are conflicting results regarding the effect of calcium hydroxide dressing on sealing ability of various biomaterials [23,24]. It has been suggested that residual calcium hydroxide might interfere with material adaptation to the root canal walls or chemically interact with them.
In the present study, push-out bond strength of Biodentin, Glass ionomer cement, MTA and Theracal was evaluated and compared after exposure to acidic, neutral and alkaline pH. For acidic environment, butyric acid, a byproduct of anaerobic bacteria metabolism, buffered at pH 6.4 was used. For alkaline environment buffered potassium hydroxide at pH 8.4 was used while for neutral environment phosphate buffer saline solution at pH 7.4 was used.
The results of present study indicate that push-out bond strength of GIC (26.75 MPa) in acidic medium was significantly higher while MTA had the lowest (2.78 MPa) push-out bond strength compared to other materials. In alkaline medium, the highest push-out bond strength was shown by Biodentin (28.89 MPa) and the lowest by MTA (8.03 MPa). In neutral medium, the greatest pushout bond strength was shown by GIC (33.22 MPa) and the lowest by MTA (8.92 MPa). MTA showed inferior bond strength in all environmental conditions, which is in agreement with Shokouhinejad et al. [10]. It is possible that pH inhibits setting reaction, affects adhesion, or increases solubility of calcium silicate-based materials eventually affecting mechanical properties of material including surface microhardness.
In the current study, Biodentin showed greater bond strength in both acidic and alkaline medium than MTA. Bond strength of MTA was most likely affected by the alkaline pH of dentin [12]. Biodentin also showed superior bond strength in alkaline medium than in neutral conditions. GIC demonstrated the highest bond strength compared to other root end filling materials in acidic and neutral pH. This result is probably related to to its strong chemical bonding to dentin.
Theracal is pulp-capping material but due to its good sealing ability we used it as root end filling material. It showed higher bond strength than MTA (8.03), almost comparable to GIC (13.78) in alkaline environment and higher than Biodentin in acidic environment. Further in vivo and in vitro research should be done in order to assess Theracal as root end filling material.

CONCLUSION
Altered pH levels affect the properties of root end materials. MTA showed inferior push-out bond strength that was affected by pH of surrounding environment. GIC has good bond strength that increased with decrease in pH opposite to other materials. Further research is needed for Biodentin and Theracal as root end filling materials.

UVOD
In fi ci ra na i upa lje na tki va mo gu ima ti nor mal nu vred nost pH od 7,4, ali i ki se li pH od oko 5,0. Ki se li pH mo že spre či ti us posta vlja nje re ak ci je, uti ca ti na pri a nja nje ili po ve ća ti ras tvor lji vost ma te ri ja la. Ako in fek ci ja ili upa la tra ju du go, to mo že do ve sti i do ero zi je ma te ri ja la [1]. S dru ge stra ne, i iz lo že nost ba znoj sre di ni na sta loj na kon me di ka ci je ka na la kal ci jum-hi drok si dom mo že uti ca ti na svoj stva ma te ri ja la za pu nje nje apek sa ko re na zu ba [2]. Na taj na čin spo sob nost op tu ra ci je ma te ri ja la mo že bi ti di rekt no ili in di rekt no po re me će na [1]. Kroz isto ri ju sto mato lo gi je raz li či ti ma te ri ja li su ko ri šće ni za re tro grad no pu nje nje ka na la ko re na, kao što su zlat ne fo li je, sre br ni ko či ći, amal gam (sa ad he zi vom i bez nje ga), cink-ok sid ni euge nol, gla sjo no merce ment, mi ne ral ni tri ok sid ni agre gat i dr. [3].
Gla sjo no mer-ce ment (GJC) se ko ri sti u raz li či te svr he, kao što su pri vre me ni is pu ni, stal ni is pu ni ma lih ka vi te ta, za li va nje fi su ra i pu nje nje apek sa ko re na [4]. GJC se for mi ra re ak ci jom če sti ca kal ci jum-alu mi no si li kat nog sta kla i vo de nog ras tvo ra po li a kril ne ki se li ne. Glav na pred nost ovog ce men ta je ja ka hemij ska ve za sa den ti nom i spo sob nost oslo ba đa nja jo na flu o ri da. Za hva lju ju ći do broj he mij skoj ve zi sa den ti nom, GJC po ka zu je do bru ve zu ka da se ko ri sti kao ma te ri jal za pu nje nje apek sa ko re na. Ovaj ce ment je jed no sta van za upo tre bu i ne do vo di do lo ših re ak ci ja na hi sto lo škom ni vou u pe ri a pi kal nom tki vu [3]. Do sa da ni je ob ja vlje na ni jed na stu di ja ko ja me ri ot por nost na smi ca nje GJC u uslo vi ma iz me nje ne vred no sti pH.
U po sled njih ne ko li ko de ce ni ja mi ne ral ni tri ok sid ni agre gat (MTA) se po ka zao vr lo uspe šnim ka da se ko ri sti kao ma te ri jal za za tva ra nje per fo ra ci ja (boč nih zi do va ili fur ka ci ja), kao apikal ni čep i kao ma te ri jal za pu nje nje ka na la ko re na [5,6]. Sa sto ji se od fi nog pra ha tri kal ci jum-si li ka ta, di kal ci jum-si li ka ta, trikal ci jum-alu mi na ta, te tra kal ci ujm-alu mi no fe ri ta i bi zmut-oksi da. S ob zi rom na nje go vu kli nič ku upo tre bu, MTA mo že bi ti iz lo žen ki se loj sre di ni zbog po sto ja nja pe ri a pi kal nog za pa lje nja [7,8]. Pro me na vred no sti pH zbog već po sto je ćeg sta nja mo že uti ca ti na fi zič ke i he mij ske oso bi ne ma te ri ja la [9,10]. Ta ko je po ka za no da se tvr do ća [11], du žin ska za te zna čvr sto ća [8], ot por nost na smi ca nje [10] i spo sob nost zap ti va nja MTA [12] sma nju ju na kon po sta vlja nja ma te ri ja la u ki se loj sre di ni. No vi ma te ri ja li na ba zi kal ci jum-si li ka ta uve de ni su da bi se pre va zi šli ne do sta ci MTA po put du žeg vre me na ve zi va nja, te škog ru ko vanja, vi so ke ce ne i po ten ci jal nog pre bo ja va nja zu ba.
Naj no vi ji na tr ži štu je kal ci jum-si li kat ni ma te ri jal bi o den tin (Sep to dont, Sa int-Ma ur-des-Fossés, Fran cu ska), ko ji se sa sto ji od tri kal ci jum-si li ka ta, kal ci jum-kar bo na ta, cir ko ni jum-ok si da i teč no sti na ba zi vo de ko ja sa dr ži kal ci jum-hlo rid kao ak ce le rator i agens za od u zi ma nje vo de [13]. Bi o den tin je br zo ve zu ju ći kal ci jum-si li kat ni ma te ri jal ko ji se mo že ko ri sti ti kao re sto rativ ni ma te ri jal za den tin, ali i kao en do dont ski ma te ri jal sa osobi na ma slič nim MTA [14].
Te ra kal (Bi sco Inc, Scham burg, IL, SAD) je no vi sve tlo snopo li me ri zu ju ći kal ci jum-si li kat ni ce ment mo di fi ko van smo lom (ba za/laj ner) di zaj ni ran za di rekt no i in di rekt no pre kri va nje pulpe. On sa dr ži oko 45% mi ne ral nih ma te ri ja la po te ži ni (tip III Por tland ce men ta), 10% rend gen kon trast ne kom po nen te, 5% hidro fil nog agen sa za zgu šnja va nje (fu med si li ka) i oko 45% smole [15]. Po ka zu je do bru fi zič ko he mij sku ve zu za den tin, do bro zap ti va nje i do bru to le ran ci ju od stra ne odon to bla sta [16,17].
Cilj pe ri a pi kal ne hi rur gi je je da uklo ni uzrok bo le sti i omogu ći po volj no okru že nje za za ra sta nje. Api kal no pu nje nje je jedno od ključ nih ko ra ka [18]. Me đu tim, po sto ja nje pe ri a pi kal ne le zi je uti če na pro me nu vred no sti pH, što do vo di do sma nje nja kva li te ta ve ze s ma te ri ja lom za pu nje nje apek sa ka na la ko re na.
Cilj ovog ra da je bio da se pro ce ni efe kat iz me nje ne vred no sti pH na ot por nost na smi ca nje bi o den ti na, GJC, MTA i te ra ka la.

MATERIJAL I METODE RADA
U stu di ju je bi lo uklju če no 48 jed no ko re nih zu ba s pra vim kana li ma ko ji su bi li eks tra ho va ni iz pa ro don to lo ških ili or to dont-skih raz lo ga. Pre eks pe ri men ta zu bi su ču va ni u pu fe ri zo va nom fi zi o lo škom ras tvo ru. Sva ki ko ren zu ba je pre se čen ho ri zon tal no di ja mant skim di skom, da bi se do bi li den tin ski di sko vi de blji ne 1 mm -ukup no 48 di sko va (Sli ka 1). Deo ka na la sva kog di ska je ob ra đen okru glim kar bid nim svr dlom, da bi se do bio preč nik od 1,4 mm (Sli ka 2). Te sti ra ni ma te ri ja li su za me ša ni pre ma uput stvu pro iz vo đa ča i u in kre men ti ma une se ni u lu men ka na la sva kog den tin skog di ska. Uzor ci su za tim svr sta ni u če ti ri gru pe od po 12 uzo ra ka: bi o den tin, GJC, MTA i te ra kal. Po tom je svaka gru pa po de lje na na tri pod gru pe od po če ti ri uzor ka: ki se la sre di na (bu ti rič na ki se li na pu fe ro va na na vred no sti pH od 6,4), ne u tral na sre di na (fi zi o lo ški ras tvor pu fe ri zo van fos fat nim pufe rom na pH 7,4) i ba zna sre di na (pu fe ri zo va na ka li jum-hi droksi dom na pH 8,4). Svi uzor ci su in ku bi ra ni če ti ri da na na 37°C.
Ot por nost na smi ca nje (tzv. push-out čvr sto ća) sva kog ma teri ja la me re na je po mo ću uni ver zal ne ma šine za me re nje (Sli ka 3). Uzor ci su po sta vlje ni na me tal nu plo ču s otvo rom u cen tru, da bi se klip mo gao slo bod no kre ta ti. Pri ti sno op te re će nje je apli ko va no pri ti skom na do le na po vr ši nu ma te ri ja la po mo ću ci lin dra preč ni ka 1 mm br zi nom od 1 mm u mi nu ti. Pri ti sni ci lin dar je bio bar 0,2 mm da le ko od den tin skog zi da, ka ko bi se osi gu rao kon takt sa mo s ma te ri ja lom. Mak si mal no op te reće nje ko je je pod neo ma te ri jal u tre nut ku iz ba ci va nja iz ra žen je u njut ni ma (N).
Ka ko bi iz ra zi li čvr sto ću u MPa, za be le že na vred nost je pode lje na po vr ši nom ma te ri ja la u ka na lu ko re na pre ma sle de ćoj for mu li: 2πr×h; gdje je r preč nik ka na la, a h de blji na den tin skog di ska iz ra že na u mm. Sva ki den tin ski disk je po tom is pi tan pomo ću ste re o mi kro sko pa, da bi se utvr di la pri ro da pre ki da kontak ta iz me đu ma te ri ja la i den ti na, i to kao: na ru ša va nje ad heziv ne ve ze iz me đu ma te ri ja la i den ti na, na ru ša va nje ko he ziv ne ve ze unu tar ma te ri ja la ili me šo vi ti pre kid kon tak ta. Pri ku plje ni po da ci su ana li zi ra ni po mo ću jed no smer ne ana li ze va ri jan se i post hoc Ta ki je vim (Tu key) te stom.