Marginal Microleakage of Newly Synthesized Nanostructured Biomaterials Based on Active Calcium Silicate Systems and Hydroxyapatite

Abstract Introduction Calcium silicate cements can be successfully used for the treatment of root perforations due to their exceptional biological and sealing properties. The aim of this study was to test, using dye penetration method, marginal microleakage of newly synthesized nanostructured biomaterials based on calcium silicate system and hydroxyapatite after their application in interradicular perforation of extracted teeth. Material and Methods The study included 34 extracted human molars. Newly synthesized nanostructured materials: one based on calcium silicate system (CS), and the other one based on hydroxyapatite and active calcium silicate system (HA-CS) were tested. Mineral trioxide agreaggate (MTA; Angelus, Londrina, Brazil) was used as control. Marginal microleakage was evaluated using dye penetration test 6 months after the application of materials in experimentally prepared inter-radicular perforations in extracted human molars. Dye penetration was analyzed using light microscope at 30X magnification, a method of quantifying visual information in computer software for image processing (Adobe Photoshop CS5 Extended, version 12.0 x 32). The values were expressed in millimeters, and the results statistically analyzed using one-way ANOVA and Tukey post-hoc test (α=0.05). Results The shortest dye penetration was measured for calcium silicate system (0.44 mm), while slightly higher values were found for MTA (0.54 mm). Dye penetration for hydroxyapatite and active calcium silicate system (2.00 mm) was longer than for the other two materials (p<0.05). Conclusion The lowest marginal microleakage was observed in CS and was comparable to MTA. Microleakage in HA-CS was significantly higher than CS and MTA.


INTRODUCTION
Marginal seal or adequate marginal adaptation of material along the cavity walls should be able to prevent leakage of tissue fluid and consequently bacterial microleakage.Therefore, it is considered as significant factor for long term success of endodontic treatment [1].
Calcium silicate cements were introduced in endodon tic practice in the mid 90ies of the last century in order to be used as materials for closing root perforations and root apical closure after apical surgery.Studies have demon strated superior marginal seal achieved by calcium silicate cements compared to amalgam and cements based on zincoxideeugenol (ZOE; Super EBA and IRM cement), which have been commonly used in these indications [1].It has also been demonstrated that calcium silicate cements achieve good marginal seal in cavities contami nated with blood [1,2], they are biocompatible [1], bio active [3] and induce regeneration of hard dental [4,5] and periodontal tissue [6].Consequently, their indications have expanded significantly [7].
The main issue for clinical use of calcium silicate cements is their long setting time [8].Initial binding of several hours increases the risk of material dissolu tion and leaching from the site of application.Moisture is needed for hydration and setting of these materials, which is why additional external moisture is needed dur ing initial bonding.That inevitably delays completion of endodontic treatment [9].In previous years, numerous studies have been conducted in order to overcome this problem [1013].
Modern research has focused on synthesis and evalu ation of new nanostructured biomaterials in similar endodontic indications [14].Nanomaterials are charac terized among others, by pronounced activity of particles and consequently, faster setting in comparison with con ventional microstructural materials [15].The Institute of Nuclear Sciences in Vinča, according to the recipe of Jokanović et al. [16], synthesized two new biomaterials: one based on calcium silicate system (CS) and the other one based on hydroxyapatite and active calcium silicate system (HACS).These materials showed initial setting of 10 minutes (CS) and 15 minutes (HACS), respectively.Previous studies have shown that these materials have less genotoxic and cytotoxic effects, but higher osteogenic po tential than commercial calcium silicate cements [17,18].
The aim of this study was to test, using dye penetra tion method, marginal microleakage of newly synthesized nanostructured biomaterials based on calcium silicate system and hydroxyapatite (HA CS) after their applica tion in interradicular perforation of extracted teeth.

MATERIAL AND METHODS
Three materials in total were tested in this study: new material based on calcium silicate system (CS), new ma terial based on mixtures of hydroxyapatite and calcium silicate system (HACS) and control material MTA (MTA Angelus®, Londrina, Brazil).Experiments were performed on 34 human, extracted, maxillary and mandibular molars with fully developed and nonfused roots.Access cavities were prepared with high speed handpiece, which after hand Kfiles (Kfiles, VDW GmbH, Germany) were used to establish working length of root canals up to 1 mm shorter than the anatomy of the foramen.Root canals were instrumented with Crowndown technique using series of rotary files BioRace (FKG Dentaire, Swiss Dental Products, Switzerland).During instrumentation, canals were irrigated with 0.5% NaOCl.For the final rinse 5 mL of 10% citric acid solution (during 1 min) and 5 mL of 0.5% sodium hypochlorite solution were used.After dry ing the canals were obturated using monocone technique and sealer Acroseal (Septodont, France).
After sealer setting, in the center of the pulp chamber floor, using slow speed handpiece and round bur size # 4 interradicular perforations were prepared.The size of perforations was the same as diameter of round bur while the depth depended on the thickness of the pulp cham ber floor.After washing perforations with distilled water and airdrying, the teeth were randomly assigned into the three experimental groups according to the materials used: CS, HACS and MTA (n=10).Positive controls were teeth with perforations that were not sealed and negative controls were two teeth without perforations.
Prior to the application of the test material, the teeth were placed in a sponge soaked with artificial tissue fluid up to the level of enamelcement junction (Eng.Hank's balanced salt solution, HBSS).Materials were mixed with distilled water in the ratio of 3: 1 and placed in the prepared cavities using condensers.On the top of the material a moist cotton pellet was placed and the teeth were incubated at 37°C for 24 h.After complete setting of materials access cavities were permanently closed using composite.Teeth were incubated in sponges at 37°C for the next 6 months.
After 6 months marginal microleakage was evaluated using passive dye penetration method.The teeth were coated with two coats of varnish, except in the area of the material in the furcation (about 1 mm around material) and placed in 50% solution of silver nitrate (AgNO 3 ) for 2 h.After quick rinsing teeth were immersed in photo devel oper for 6 h.Teeth were cut longitudinally in the region of perforation using diamond disc of 0.7 mm thickness and linear speed saw with water cooling (Isomet testers 4000, Buehler, Lake Bluff, IL, USA).Dye penetration between the material and cavity walls was analyzed using light microscopy and photographed at 30X magnification.The depth of dye penetration was measured using the method of quantifying of visual information in computer software for image processing (Adobe Photoshop CS5 Extended, version 12.0 x 32).The obtained values were expressed in millimeters.The results were statistically analyzed using oneway ANOVA with Tukey posthoc test.The level of significance was set at α=0.05

RESULTS
The shortest dye penetration was found for CS (0.44±0.54 mm) (Figure 1).Similar values were measured for MTA (0.54±0.76 mm) (Figure 2).The longest dye penetration was recorded for HACS (2.00±0.70 mm) (Graph 1, Fig ure 3).There was no statistically significant difference in marginal dye penetration between CS and MTA.HACS showed dye penetration that was significantly longer than for CS and MTA (p<0.05).
In addition to color penetration between restorations and dentin, staining of tested materials was also noticed (Figures 4, 5 and 6).

DISCUSSION
Dye or bacteria penetration or fluid filtration methods are most commonly used for the evaluation of marginal microleakage of different materials in vitro [1922].In the current study dye penetration method was used due to simplicity as described in numerous studies [19,20,23,24,25].One of the main objections to dye penetra tion method is related to the size of dye molecules that are commonly used which are actually smaller than the size of bacteria [19].Therefore, for materials that show penetration of small dye molecules, it can be expected that leakage of larger molecules (bacteria and their products) will be absent [1].
When choosing dye for the assessment of marginal leakage chemical nature of the tested material should be taken into account.Wu et al. [26] found that methylene blue is unstable in the presence of alkaline substances resulting in its decolorization.As calcium hydroxide is major ingredient of MTA, the use of methylene blue for Graph 1.Average microleakage of the tested materials Gra fi kon 1. Sred nje vred no sti pro do ra bo je is pi ti va nih ma te ri ja la   Sli ka 5.In ter ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom hi drok si apa ti tom i kal ci jum si li kat nim ce men tom.Uoča va se ap sorp ci ja bo je sa pre bo ja va njem ve ćeg de la ma te ri ja la (×30).Sli ka 6.In ter ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom MTA.Uočava ju se pro dor i ap sorp ci ja bo je ce lom de blji nom ma te ri ja la (×30).Sli ka 3.In ter ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom hi drok si apa ti tom i kal ci jum si li kat nim ce men tom.Uoča va ju se pro dor i apsorp ci ja bo je sa de li mič nim pre bo ja va njem ma te ri ja la (×30).Sli ka 4. In ter ra dik sna per fo ra ci ja is pu nje na kal ci jum si li kat nim mate ri ja lom.Uoča va se pro dor i ap sorp ci ja bo je sa de li mič nim pre boja va njem ma te ri ja la (×30).
the evaluation of MTA and other materials of similar composition would cause its discoloration and eventually unreliable results.A solution of silver nitrate was chosen as dye due to its stability in the presence of the high pH and high molecular weight of silver particles [25].
Marginal microleakage was assessed after the appli cation of the test material in experimentally prepared, interradicular perforations of extracted teeth.During the experimental period the teeth were stored in an incubator, with a sponge soaked in artificial tissue fluid to simulate clinical conditions [21].In the current study, the deepest dye penetration was measured after the teeth were longi tudinally cut.Therefore, it was not possible to determine precisely in which part of the cavity dye penetrated the most.Dye penetration method done in vitro cannot be directly related to the complex in vivo microleakage but it can be possible indicator of microleakage in clinical conditions [20].
The lowest dye penetration in the current study was observed for CS material.The values of dye penetration for MTA were slightly higher but with no statistically sig nificant difference compared to CS.Given that CS and MTA have similar composition and physical properties are likely similar.Good marginal seal of calcium silicate cements is associated with mild expansion of cement during setting [27,28], bioactivity and hydroxyapatite formation on the surface of the material in contact with phosphates from tissue fluids [29].
However, different microleakage evaluating methods have shown that MTA and similar calcium silicate ce ments do not have the ability of absolute hermetic sealing [21,22,25,30], which is consistent with the results of the current study.In the group of samples with CS, the mea sured values of dye penetration were lower compared to the MTA samples, however, larger number of samples in the CS group showed microleakage than in the MTA group.The measured values of dye penetration in the MTA group were very different, and the results are consistent with the findings of De Deus et al. [30].They evaluated the margin al microleakage of different calcium silicate cements (PC, MTA Angelus and MTA Bio) and reported very variable results within groups.They also detected microleakage in all samples but with no statistically significant difference between different calcium silicate cements [30].In contrast, Hashem et al. [19] observed differences in the permeability of various commercial calcium silicates, considering that different chemical composition and different setting time may play a role in the material attachment to the cavity walls.Also, studies that evaluated permeability of calcium silicate cements using bacteria penetration method have reported different results.Montelano et al. [2] recorded the most pronounced microleakage in the first days of ap plication of materials, while Parirokh et al. [21] detected significantly delayed bacterial penetration (between 39 and 73 days of experiment) highlighting the positive impact of phosphate solution on the adhesion of material.
Literature reviews describe inconsistency in results for the permeability of calcium silicate cements using differ ent testing methods.Many factors influence microleakage of MTA: dye, pH of dye, storage conditions of samples before experiment, and setting time of material prior to immersion of samples in dye solution [1].Also, differ ent results may be due to the difference in diameter and depth of perforations, number of samples, or duration of observational period [30].
For adequate understanding of results obtained in the current study, it is important to explain the way of dye penetration.Samples that showed dye penetration did not confirm strictly marginal microleakage.In fact, besides the dye detected at the junction of materials and dentin, staining of materials was noted likely due to dye absorp tion (Figures 4, 5 and 6).Similar results were reported by TobónArroyave et al. [20].They found that dye penetra tion in MTA was different in nature than in conventional cements (IRM and Super EBA).When IRM and Super EBA cements were used as apical plug, dye penetration was noted as a circle around the material, between ma terial and dentin.In contrast, in all MTA samples color change was noted in the entire thickness of the material [20].The reason for these results could be found in MTA structure made of numerous pores and capillaries that could cause more pronounced permeability.It is impor tant however to note that MTA samples in that study had setting time of just 30 minutes before their immersion in dye, therefore results could reflect incomplete setting of material.In the current study, samples were allowed 24 hours setting time before incubation and then after incubation in humid environment for 6 months.This pro tocol could allow more complete setting and consequently lower dye penetration and absorption.
HACS showed the deepest dye penetration and in the majority of samples material disintegration was observed.Since hydroxyapatite is well known for its porous structure and inadequate mechanical properties, the results were somewhat expected.During synthesis HACS, calcium silicate was added to hydroxyapatite in order to improve its mechanical properties.Based on the current results it can be noted that additional quantity of calcium silicate (HA:CS=2:1) under the experimental conditions of this study, did not prevent dissolution and leaching of material from the site of application.Literature findings also suggest that calcium phosphate cements do not provide adequate sealing due to their porosity and solubility and in that sense they are inferior to calcium silicate cements [24,25].
It is worth noting that in some MTA and CS samples surface disintegration or dissolution of the outer layer material was noted but still less than in HACS samples.In contact with water or phosphate solution, cement re leases calcium ions with partial decalcification of calcium silicate hydrate, whereas the expansion of cement after contact with liquid may result in microfractures.All these may constitute an obstacle to longterm stability of the material [13].However, when interpreting results of an in vitro study, it must be noted that tissue response was ab sent.Namely, one of the characteristics of the tested bioac tive materials in this study is formation of hydroxyapatite on their surface in contact with tissue fluids [3,29], as well as the formation of calcified tissue via calcium silicate or calcium phosphate cements.Therefore, one might expect that in clinical setting material disintegration could be selflimiting and hydroxyapatite formed over time could fill the pores within the material and provide chemical bond to dentin contributing to better marginal seal [31].

CONCLUSION
The lowest marginal microleakage or the best marginal seal was observed in CS and MTA.Microlekage of HACS was significantly higher compared to the other two tested materials.

KRATAK SADRŽAJ
Uvod Pri me na ce me na ta od kal ci jum-si li ka ta u le če nju per fo ra ci ja ko re na zu ba za sni va se na nji ho vim iz u zet nim bi o lo škim svojstvi ma i od go va ra ju ćem rub nom zap ti va nju.Cilj ovog ra da bio je da se te stom pro do ra bo je is pi ta mar gi nal na mi kro pro pu stlji vost no vo sin te ti sa nih na no struk tur nih bi o ma te ri ja la na ba zi kal ci jum si li kat nih si ste ma i hi drok si a pa ti ta na kon pri me ne ma te ri ja la u in ter ra dik sne fo ra ci je tra ho va nih zu ba.Ma te ri jal i me to de ra da Is pi ti va nje je re a li zo va no na 34 eks tra ho va na hu ma na mo la ra.Te sti ra ni su no vo sin te ti sa ni na no struk tur ni ma te ri ja li: ma te ri jal na ba zi ak tiv nih kal ci jum si li kat nih si ste ma (CS) i ma te ri jal na ba zi hi drok si a pa ti ta i ak tiv nih kal ci jum si li kat nih si ste ma (HA-CS).Kao kon trol ni ma te ri jal ko ri šćen je ko mer ci jal ni kal ci jum si li kat ni ce ment (MTA An ge lus, Lon dri na, Bra zil).Mar ginal na mi kro pro pu stlji vost je is pi ti va na te stom pro do ra bo je šest me se ci na kon pri me ne ma te ri ja la u eks pe ri men tal no pre pa ri sa ne in ter ra dik sne per fo ra ci je na eks tra ho va nim hu ma nim mo la ri ma.Pro dor bo je je ana li zi ran sve tlo snim mi kro sko pom na uve li ča nju od 30 pu ta me to dom kvan ti fi ko va nja vi zu el nih in for ma ci ja kom pju ter skim pro gra mom za ob ra du sli ke (Ado be Pho tos hop CS5 Ex ten ded, ver zi ja 12.0x32).Do bi je ne vred no sti su iz ra že ne u mi li me tri ma, a do bi je ni re zul ta ti sta ti stič ki su ob ra đe ni pri me nom jedno fak tor skog te sta ANO VA sa Tu ki je vim (Tu key) post-hoc te stom (α=0,05).Re zul ta ti Naj ma nji pro dor bo je iz me ren je kod ma te ri ja la CS (0,44 mm), dok su ne što ve će vred no sti za be le že ne kod MTA (0,54 mm).Pro dor bo je kod ma te ri ja la HA-CS (2,00 mm) bio je zna čaj no ve ći u od no su na ma te ri ja le CS i MTA (p<0,05).Za klju čak Naj ma nja mar gi nal na mi kro pro pu stlji vost za be le že na je kod ma te ri ja la CS i bi la je kom pa ra bil na sa MTA.Mi kro pro pustlji vost ma te ri ja la HA-CS bi la je zna čaj no ve ća u od no su na ma te ri jal CS i kon trol ni ma te ri jal MTA.Ključ ne re či: mar gi nal na mi kro pro pu stlji vost; kal ci jum si li kat ni ce men ti; hi drok si a pa tit UVOD Kva li tet no rub no zap ti va nje, od no sno od go va ra ju ća mar gi nal na adap ta ci ja ma te ri ja la uz zi do ve ka vi te ta, tre ba da one mo gu ći pro tok tkiv nih teč no sti i po sle dič no bak te rij sko mi kro cu re nje, zbog če ga se sma tra zna čaj nim fak to rom za du go ro čan uspeh en do dont skog le če nja [1].
Ce men ti od kal ci jumsi li ka ta uve de ni su u en do dont sku prak su sre di nom de ve de se tih go di na pro šlog ve ka, naj pre kao ma te ri ja li na me nje ni le če nju ko ren skih per fo ra ci ja i za tva ra nju vr ha ko re na zu ba na kon apek sne hi rur gi je.Is tra ži va nja su uka za la na znat no bo lje rub no zap ti va nje kal ci jumsi li ka ta u od no su na amal gam i ko mer ci jal ne ce men te na ba zi ZOE (Su per EBA i IRM ce ment), ko ji su obič no ko ri šće ni u na ve de nim in di ka ci ja ma [1].Ka sni je je utvr đe no da kal ci jum si li kat ni ce men ti ostva ru ju kva li tet no rub no zap ti va nje i u ka vi te ti ma ko ji su kon ta mi ni ra ni kr vlju [1,2], da su bi o kom pa ti bil ni [1], bi o ak tiv ni [3] i da in du ku ju re ge ne ra ci ju čvr stih zub nih tki va [4,5] i pe ri o don ci ju ma [6], zbog če ga su nji ho ve in di ka ci je da nas zna čaj no pro ši re ne [7].
Glav ni pro blem ko ji ote ža va kli nič ku pri me nu kal ci jumsi li ka ta je du go vre me ve zi va nja [8].Ini ci jal no ve zi va nje od ne ko li ko sa ti no si ri zik od ras tva ra nja i is pi ra nja ma te ri ja la s me sta apli ka ci je.Za hi dra ta ci ju i očvr šća va nje ma te ri ja la neo p hod na je vla ga, pa se pre po ru ču je da se na kon pri me ne (u pe ri o du ini ci jal nog ve zi va nja) ma te ri ja lu obez be di eks ter na vla ga, što ne mi nov no od la že za vr še tak en do dont skog le če nja [9].Ra ni jih go di na ura đe na su broj na is tra ži va nja ra di pre va zi la že nja ovog pro ble ma [1013].
Sa vre me na is tra ži va nja su fo ku si ra na na sin te zu i is pi ti va nja mo gu će pri me ne na no struk tur nih bi o ma te ri ja la u slič nim en do dont skim in di ka ci ja ma [14].Na no ma te ri ja le, iz me đu osta log, od li ku je iz ra že ni ja ak tiv nost če sti ca, te po sle dič no br že ve zi va nje u od no su na kon ven ci o nal ne mi kro struk tur ne ma te ri ja le [15].U In sti tu tu za nu kle ar ne na u ke u Vin či, pre ma re cep tu ri Jo ka no vi ća i sa rad ni ka [16], sin te ti sa na su dva no va bi o ma te ri ja la: ma te ri jal na ba zi ak tiv nih kal ci jum si li kat nih si ste ma (CS) i ma te ri jal na ba zi hi drok si a pa ti ta i ak tiv nih kal ci jum si li kat nih si ste ma (HACS).Na no teh no lo gi jom do bi je ni su ma te ri ja li s ini ci jal nim ve zi va njem od 10 mi nu ta (CS), od no sno 15 mi nu ta (HACS).Do sa da šnja is tra ži va nja su po ka za la da do bi je ni ma te ri ja li ima ju sla bi ja ge no tok sič na i ci to tok sič na dej stva, kao i ve ći oste o ge ni po ten ci jal u od no su na ko mer ci jal ne kal ci jum si li kat ne ce men te [17,18].Cilj ovog is tra ži va nja bio je da se te stom pro do ra bo je is pi ta mar gi nal na mi kro pro pu stlji vost ma te ri ja la CS i HACS na kon pri me ne ma te ri ja la u in ter ra dik sne per fo ra ci je eks ta ho va nih hu ma nih zu ba.

MATERIJAL I METODE RADA
U ovom is tra ži va nju te sti ra ni su ma te ri jal na ba zi kal ci jum si li kat nih si ste ma (CS), ma te ri jal na ba zi me ša vi ne hi drok si a pa ti ta i kal ci jum si li kat nih si ste ma (HACS) i kon trol ni ma te ri jal MTA (MTA An ge lus®, Lon dri na, Bra zil).Is tra ži va nje je ura đe no na 34 hu ma na eks tra ho va na mak si lar na i man di bu lar na mo la ra s pot pu no raz vi je nim i ne fu zi o ni sa nim ko re no vi ma.Pri stup ni ka vi te ti su pre pa ri sa ni vi so ko tu ra žnom bu ši li com, na kon če ga je ruč nim Ktur pi ja ma (K-fi les, VDW GmbH, Ne mač ka) utvr đe na rad na du ži na ka na la ko re na zu ba, na 1 mm kra će od ana tom skog fo ra me na.Ka na li ko re na su pre pa ri sa ni teh ni kom crown-down, se ri jom ma šin skih en do dont skih in stru men ta Bio Ra ce (FKG Den ta i re, Swiss Den tal Pro ducts, Švaj car ska).To kom in stru men ta ci je ka na li su is pi ra ni sa Na OCl u kon cen tra ci ji od 0,5%.Za fi nal no is pi ra nje je ko ri šće no 5 ml de se to pro cent ne li mun ske ki se li ne (to kom jed nog mi nu ta) i 5 ml 0,5% Na OCl.Na kon su še nja ka na li su op tu ri sa ni mo no ko nom teh ni kom uz si ler Ac ro seal (Sep to dont, Fran cu ska).
Po ve zi va nju si le ra, u cen tru po da pulp ne ko mo re, ko le nja kom i okru glim bo re rom ve li či ne #4, pre pa ri sa ne su in ter ra dik sne per fo ra ci je ta ko da je ši ri na per fo ra ci ja od go va la pro me bo re ra, dok je du bi na za vi si la od de blji ne po da pulp ne ko mo re.Po sle is pi ra nja pre pa ri sa nih ka vi te ta de sti lo va nom vo dom i su še nja, zu bi su me to dom slu čaj nog iz bo ra po de lje ni u tri eks pe ri men tal ne gru pe, a per fo ra ci je za tvo re ne test ma te ri ja li ma CS, HACS i MTA (n=10).Po zi tiv nu kon trol nu gru pu či ni la su dva zu ba kod ko jih per fo ra ci je ni su za tvo re ne ma te ri ja li ma, a ne ga tiv nu dva zu ba bez per fo ra ci ja.
Pre pri me ne is pi ta nih ma te ri ja la zu bi su do ni voa gleđ no ce ment ne gra ni ce po sta vlje ni u sun đer na to pljen ve štač kom tkiv nom teč no šću -Hen ko vim urav no te že nim sla nim ras tvo rom (engl.Hank's ba lan ced salt so lu tion -HBSS).Ma te ri ja li su za me ša ni sa de sti lo va nom vo dom u od no su 3:1 i u ka vi te te kon de nzo va ni na bi ja či ma.Pre ko ma te ri ja la je po sta vlje na vla žna va ti ca, a za tim su zu bi in ku bi ra ni na 37°C to kom 24 ča sa.Po ve zi va nju ma te ri ja la pri stup ni ka vi te ti su ko nač no za tvo re ni kom po zi tom, a zu bi u na to plje nom sun đe ru in ku bi ra ni na 37°C na red nih šest me se ci.
Po sle šest me se ci mar gi nal na mi kro pro pu stlji vost je is pi ti va na me to dom pa siv nog pro do ra bo je.Zu bi su pre ma za ni sa dva slo ja la ka, osim u pre de lu ma te ri ja la u fur ka ci ji (1 mm oko ma te ri ja la) i po to plje ni u pe de se to pro cent ni ras tvor sre broni tra ta (Ag NO 3 ) to kom dva ča sa.Na kon krat kog is pi ra nja zu bi su osta vlje ni u fo to graf skom raz vi ja ču na red nih šest sa ti.Za tim su lon gi tu di nal no se če ni u pre de lu in ter ra dik snih per fo ra ci ja di ja mant skim di skom de blji ne 0,7 mm, li ne ar nom pre ci znom te ste rom sa vo de nim hla đe njem (Iso met te ste ra 4000, Bu e hler, La ke Bluff, IL, SAD).Pro dor bo je iz me đu ma te ri ja la i zi do va ka vi te ta je ana li zi ran sve tlo snim mi kro sko pom i fo to gra fi san pri uve li ča nju od 30 pu ta.Du bi na pro do ra bo je je me re na me to dom kvan ti fi ko va nja vi zu el nih in for ma ci ja kom pju ter skim pro gra mom za ob ra du sli ke (Ado be Pho tos hop CS5 Ex ten ded, ver zi ja 12.0x32).Do bi je ne vred no sti su iz ra že ne u mi li me tri ma, a re zul ta ti sta ti stič ki ob ra đe ni pri me nom jed no fak tor skog te sta ANO VA sa Tu ki je vim (Tu key) post-hoc te stom.Ni vo zna čaj no sti utvr đen je na α=0,05.

REZULTATI
Naj ma nji pro dor bo je iz me ren je kod ma te ri ja la CS (0,44±0,54 mm) (Sli ka 1).Slič ne vred no sti iz me re ne su za ma te ri jal MTA (0,54±0,76 mm) (Sli ka 2).Naj ve ći pro dor bo je za be le žen je kod ma te ri ja la HACS (2,00±0.70 mm) (Gra fi kon 1, Sli ka 3).Ni su uoče ne sta ti stič ki zna čaj ne raz li ke u mar gi nal nom pro do ru bo je iz me đu ma te ri ja la CS i MTA.Kod ma te ri ja la HACS iz me re ne vred no sti pro do ra bo je bi le su sta ti stič ki zna čaj no ve će u od no su na pro dor bo je kod ma te ri ja la CS i MTA (p<0,05).
Osim pro do ra bo je, na spo ju ma te ri ja la i zub nih struk tu ra uoče no je pre bo ja va nje te sti ra nog ma te ri ja la pri me nje nog u ve štač ki for mi ra ne in ter ra dik sne per fo ra ci je (Sli ke 4, 5 i 6).

DISKUSIJA
Za pro ce nu mar gi nal ne mi kro pro pu stlji vo sti ma te ri ja la u stu di ja ma in vi tro obič no se ko ri ste me to de pro do ra bo je [19,20], pro do ra bak te ri ja [21] i fil tra ci je teč no sti [22].U ovom is tra ži va nju za pro ce nu mi kro pro pu stlji vo sti je pri me nje na me to da pro do ra bo je, ko ja je zbog svo je jed no stav no sti ko ri šće na u ve li kom bro ju is tra ži va nja [19,20,23,24,25].Glav ni ne do sta tak ove me to de je, me đu tim, u to me što je ve li či na mo le ku la bo ja ko je se obič no ko ri ste ma nja od ve li či ne bak te ri ja [19].Kod ma te ri ja la kod ko jih se be le ži pro dor ma lih mo le ku la bo je mo že se oče ki va ti iz o sta nak mi kro cu re nja ve ćih mo le ku la, po put bak te ri ja i nji ho vih pro iz vo da [1].
Pri li kom oda bi ra bo je za pro ce nu mar gi nal ne pro pu stlji vo sti uze ta je u ob zir he mij ska pri ro da ma te ri ja la ko ji se is pi tu je.Na i me, Vu (Wu) i sa rad ni ci [26] su usta no vi li da je me ti len pla vo ne sta bil no u pri su stvu al kal nih sup stan ci, što do vo di do nje go vog obez bo ja va nja.Ka ko je kal ci jumhi drok sid glav na he mij ska sme sa ko ja se oslo ba đa iz MTA, upo tre ba me ti len pla vog pri li kom is pi ti va nja MTA i dru gih ma te ri ja la slič nog he mij skog sa sta va bi, usled dis ko lo ra ci je bo je, mo gla do ve sti do ne po u zda nih re zul ta ta.Ras tvor sre broni tra ta je sta bi lan u pri su stvu ma te ri ja la vi so ke vred no sti pH, a če sti ce sre bra ima ju ve li ku mo le ku lar nu ma su, što su i bi li raz lo zi za nje go vu pri me nu u ovom is tra ži va nju [25].
Mar gi nal na mi kro pro pu stlji vost je is pi ti va na na kon pri me ne te sti ra nih ma te ri ja la u eks pe ri men tal no pre pa ri sa ne in ter ra dik sne per fo ra ci je na eks tra ho va nim zu bi ma.To kom eks pe ri men tal nog pe ri o da zu bi su ču va ni u in ku ba to ru, u sun đe ru na to plje nom ve štač kom tkiv nom teč no šću, ka ko bi se što bli že si mu li ra li kli nič ki uslo vi [21].U ovom is tra ži va nju me re na je naj du blja tač ka pro do ra bo je uz na po me nu da lon gi tu di nal nim se če njem zu ba, ko je se obič no pri me nju je, ni je mo gu će pre ci zno utvr di ti u ko jem de lu ka vi te ta je bo ja naj du blje pro dr la.Zbog to ga se pro dor bo je pri od re đi va nju mi kro pro pu stlji vo sti u uslo vi ma in vi tro ne mo že di rekt no upo re di ti s kom plek snim mi kro cu re njem u uslo vi ma in vi vo, ali se mo že sma tra ti po ka za te ljem mo gu će pro pu stlji vo sti ma te ri ja la u kli nič kim uslo vi ma [20].
Naj ma nji pro dor bo je u ovom is tra ži va nju uočen je kod ma te ri ja la CS.Vred no sti pro do ra bo je kod MTA bi le su ne što ve će, ali bez sta ti stič ki zna čaj ne raz li ke u od no su na CS. S ob zi rom na to da su CS i MTA ma te ri ja li slič nog he mij skog sa sta va, i nji ho ve fi zič ke oso bi ne mo gu bi ti slič ne.Do bro rub no zap ti va nje kal ci jum si li kat nih ce men ta se po ve zu je sa bla gom eks pan zi jom ce men ta pri li kom ve zi va nja [27,28], od no sno bi o ak tiv nom pri ro dom i stva ra njem hi drok si a pa ti ta na po vr ši ni ma te ri ja la u kon tak tu s fos fa ti ma iz tkiv nih teč no sti [29].
Ipak, raz li či tim me to da ma is pi ti va nja mi kro pro pu stlji vo sti uoče no je da MTA i slič ni kal ci jum si li kat ni ce men ti ne ma ju spo sob nost ap so lut nog her me tič kog zap ti va nja [21,22,25,30], što je u skla du s re zul ta ti ma ovog is tra ži va nja.U gru pi uzo ra ka is pu nje nih ma te ri ja lom CS iz me re ne su ne što ma nje vred no sti pro do ra bo je u od no su na uzor ke is pu nje ne sa MTA, ali je pro dor bo je uočen u ve ćem bro ju uzo ra ka ne go u MTA gru pi.Iz me re ne vred no sti pro do ra bo je u MTA gru pi bi le su vr lo raz li či te, a do bi je ni re zul ta ti su u skla du s na la zi ma De De u sa (De De us) i sa rad ni ka [30], ko ji su, is pi tu ju ći mar gi nal nu mi kro pro pu stlji vost raz li či tih kal ci jum si li kat nih ce me na ta (PC, MTA An ge lus i MTA Bio), pi sa li o vr lo va ri ja bil nim re zul ta ti ma unu tar gru pa.Isti auto ri su mi kro pro pu stlji vost ot kri li u svim uzor ci ma, ali bez sta ti stič ki zna čaj nih raz li ka u po gle du pro pu stlji vo sti raz li či tih kal ci jum si li kat nih ce me na ta [30].Su prot no to me, Ha šem (Hashem) i sa rad ni ci [19] su uoči li raz li ke u pro pu stlji vo sti raz li či tih ko mer ci jal nih kal ci jumsi li ka ta, sma tra ju ći da raz li čit he mij ski sa stav i raz li či ta br zi na ve zi va nja ce me na ta mo že ima ti ulo gu u adap ta ci ji ma te ri ja la za zi do ve ka vi te ta.I stu di je u ko ji ma je pro pu stlji vost kal ci jumsi li kat nih ce me na ta is pi ta na me to dom pro do ra bak te ri ja ta ko đe su uka za le na raz li či te re zul ta te.Ta ko su Mon te la no (Mon te la no) i sa rad ni ci [2] naj zra že je mi kro cu re nje za be le ži li u pr vim da ni ma pri me ne ma te ri ja la, dok su Pa ri roh (Pa ri rokh) i sa rad ni ci [21] ot kri li pro dor bak te ri ja zna čaj no ka sni je (iz me đu 39 i 73 da na eks pe ri men ta), is ti ču ći po zi ti van uti caj fos fat nog ras tvo ra na pri a nja nje ma te ri ja la.
Uop šte no po sma tra ju ći, pre gle dom li te ra tu re uoča va se ne kon zi stent nost re zul ta ta u po gle du do bi je nih vred no sti pro pu stlji vo sti kal ci jum si li kat nih ce me na ta u okvi ru raz li či tih me to da is pi ti va nja.Iz do sa da šnjih is tra ži va nja mo že se za klju či ti da broj ni fak to ri uti ču na mi kro pro pu stlji vost MTA: od vr ste bo je, pH vred no sti bo je, uslo va ču va nja uzo ra ka pre eks pe ri men ta, od no sno vre me na ve zi va nja uzo ra ka ma te ri ja la pre nji ho vog po ta pa nja u bo ju [1].Ta ko đe, raz li či ti re zul ta ti mo gu bi ti po sle di ca i raz li ka u preč ni ku i du bi ni per fo ra ci ja, bro ja uzo ra ka, od no sno raz li ka u tra ja nju op ser va ci o nog pe ri o da [30].
Za raz u me va nje do bi je nih re zul ta ta u ovom is tra ži va nju zna čaj no je na po me nu ti i na čin pro do ra bo je.U uzor ci ma u ko ji ma je uočen pro dor bo je ne mo že se go vo ri ti o strikt no mar gi nal noj mi kro pro pu stlji vo sti.Na i me, osim bo je usta no vlje ne na spo ju ma te ri ja la i zub nih struk tu ra, uoče no je pre bo ja va nje sa mog ma te ri ja la ko je je ve ro vat no po sle di ca ap sorp ci je bo je (Sli ke 4, 5 i 6).Do slič nih re zul ta ta do šli su i To bonAro ja ve (Tobón-Arroyave) i sa rad ni ci [20], ko ji su utvr di li da se na čin pro do ra bo je kod MTA raz li ku je od pro do ra bo je kod kon ven ci o nal nih ce mena ta (IRM i Su per EBA).Is pi tu ju ći mi kro pro pu stlji vost ma te ri ja la na kon nji ho ve pri me ne u for mi apek snog če pa, pro dor bo je kod IRM i Su per EBA ce men ta uočen je kru žno oko ma te ri ja la, od no sno iz me đu ma te ri ja la i den ti na ka na la ko re na.Na su prot to me, u svim uzor ci ma sa MTA uoče na je ap sorp ci ja bo je či ta vom de blji nom ma te ri ja [20].Raz log ova kvih re zul ta ta mo že bi ti sa ma struk tu ra ce men ta, ko ji su iz gra đe ni od broj nih po ra i ka pi la ra ko ji su mo gli do ve sti do iz ra že ni je pro pu stlji vo sti.Va žno je, me đu tim, na po me nu ti da su se u po me nu tom is tra ži va nju uzor ci MTA ve zi va li sa mo 30 mi nu ta pre nji ho vog po ta pa nja u bo ju, te do bi je ni re zul ta ti mo gu bi ti i po sle di ca ne pot pu nog ve zi va nja ma te ri ja la.U ovom is tra ži va nju uzor ci ma te ri ja la su se ve zi va li 24 ča sa i na kon to ga su in ku bi ra ni u vla žnoj sre di ni još šest me se ci.Ova kav pro to kol je mo gao do ve sti do pot pu ni jeg ve zi va nja i po sle dič no ma njeg pro do ra, od no sno ma njeg upi ja nja bo je.
Kod ma te ri ja la HACS iz me ren je naj ve ći pro dor bo je, a u ve ći ni uzo ra ka je uoče na i iz ra že na dez in te gra ci ja ma te ri ja la.S ob zi rom na to da je hi drok si a pa tit do bro po znat po po ro znoj struk tu ri i neo d go va ra ju ćim me ha nič kim svoj st vi ma, do bi je ni re zul ta ti su do ne kle oče ki va ni.Pri li kom sin te ze ma te ri ja la HA CS, kal ci jum si li kat ni si stem je do dat hi drok si a pa ti tu upra vo ra di po bolj ša nja nje go vih me ha nič kih svoj sta va.Na osno vu do bi je nih re zul ta ta mo že se za klju či ti da do da ta ko li či na kal ci jum si li ka ta (HA:CS, 2:1) u eks pe ri men tal nim uslo vi ma ove stu di je ni je spre či la ras tva ra nje i is pi ra nje ma te ri ja la s me sta apli ka ci je.U li te ra tu ri je po tvr đe no da su kal ci jum fos fat ni ce men ti, zbog svo je po ro zno sti i po sle dič ne ras tvor lji vo sti, ma te ri ja li sa neo d go va ra ju ćim rub nim zap ti va njem, te u tom po gle du zna čaj no ma nje efi ka sni od ce me na ta na ba zi kal ci jumsi li ka ta [24,25].
Va žno je na po me nu ti da je i u po je di nim uzor ci ma sa MTA i ma te ri ja lom CS uoče na po vr šin ska dez in te gra ci ja, od no sno ras tva ra nje spo lja šnjeg slo ja ma te ri ja la, ali u znat no ma njoj me ri u od no su na ma te ri jal HACS.U kon tak tu s vo dom ili fos fat nim ras tvo rom ce ment ot pu šta kal ci ju mo ve jo ne sa de li mič nom de kal ci fi ka ci jom kal ci jum si li kat nog hi dra ta, a eks pan zi ja ce men ta po sle kon tak ta s teč no šću mo že iza zva ti mi kro pu ko ti ne, što sve za jed no mo že pred sta vlja ti pre pre ku du go roč noj sta bil no sti ma te ri ja la [13].Ipak, pri li kom tu ma če nja re zul ta ta is tra ži va nja u uslo vi ma in vi tro mo ra se ima ti u vi du da to kom ovih is pi ti va nja iz o sta je kom plek san od go vor tki va.Na i me, jed na od od li ka bi o ak tiv nih ma te ri ja la is pi ta nih u ovom is tra ži va nju je ste stva ra nje hi drok si a pa ti ta na nji ho voj po vr ši ni u kon tak tu s tkiv nim teč no sti ma [3,29], kao i stva ra nje kal ci fi ko va nog tki va pre ko kal ci jum si li kat nih, od no sno kal ci jum fos fat nih ce me na ta.Sto ga bi se mo glo oče ki va ti da bi u kli nič kim uslo vi ma dez in te gra ci ja ma te ri ja la bi la sa mo o gra ni ča va ju ća i da bi hi drok si a pa tit, for mi ran u funk ci ji vre me na, mo gao is pu ni ti po re unu tar ma te ri ja la i obez be di ti he mij sku ve zu sa den ti nom i ti me do pri ne ti kva li tet ni jem rub nom zap ti va nju [31].

ZAKLJUČAK
Naj ma nja mar gi nal na mi kro pro pu stlji vost, od no sno naj bo lje rub no zap ti va nje za be le že no je kod ma te ri ja la CS i bi lo je slično sa MTA.Mi kro pro pu stlji vost ma te ri ja la HACS bi la je zna čaj no ve ća u od no su na ma te ri jal CS i kon trol ni ma te ri jal MTA.

Figure 1 .
Figure 1.Inter-radicular perforation filled with calcium silicate material.There is no dye marginal leakage (×30).Sli ka 1.In ter ra dik sna per fo ra ci ja is pu nje na kal ci jum si li kat nim mate ri ja lom.Ne uoča va se mar gi nal ni pro dor bo je (×30).

Figure 2 .
Figure 2. Inter-radicular perforation filled with MTA.There is no dye marginal leakage (×30).Sli ka 2. In tre ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom MTA.Ne uoča va se mar gi nal ni pro dor bo je (×30).

Figure 5 .
Figure 5.Inter-radicular perforation filled with hydroxyapatite-calcium silicate material.There is dye absorption with most of material having changed color (×30).Sli ka 5.In ter ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom hi drok si apa ti tom i kal ci jum si li kat nim ce men tom.Uoča va se ap sorp ci ja bo je sa pre bo ja va njem ve ćeg de la ma te ri ja la (×30).

Figure 6 .
Figure 6.Inter-radicular perforation filled with MTA.There is dye absorption and color change throughout the whole thickness of material (×30).Sli ka 6.In ter ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom MTA.Uočava ju se pro dor i ap sorp ci ja bo je ce lom de blji nom ma te ri ja la (×30).

Figure 3 .
Figure 3. Inter-radicular perforation filled with hydroxyapatite-calcium silicate material.There is dye marginal leakage and absorption with material color change (×30).Sli ka 3.In ter ra dik sna per fo ra ci ja is pu nje na ma te ri ja lom hi drok si apa ti tom i kal ci jum si li kat nim ce men tom.Uoča va ju se pro dor i apsorp ci ja bo je sa de li mič nim pre bo ja va njem ma te ri ja la (×30).

Figure 4 .
Figure 4. Inter-radicular perforation filled with calcium silicate material.There is dye marginal leakage and absorption with material color change (×30).Sli ka 4. In ter ra dik sna per fo ra ci ja is pu nje na kal ci jum si li kat nim mate ri ja lom.Uoča va se pro dor i ap sorp ci ja bo je sa de li mič nim pre boja va njem ma te ri ja la (×30).