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ORGINAL ARTICLE

SEM evaluation of pulp reaction to different pulp

capping materials in dog’s teeth

Saeed Asgary1*

, Masoud Parirokh2, Mohammad Jafar Eghbal

3, Jamileh Ghoddusi

4

1. Department of Endodontics, Iranian Center for Endodontic Research, Dental School, Shaheed Beheshti University of Medical Sciences,Tehran, Iran2. Department of Endodontics, Dental School, Kerman University of Medical Sciences, Kerman, Iran3. Department of Endodontics, Iran Center for Dental Research, Dental School, Shaheed Beheshti University of Medical Sciences, Tehran,Iran4. Department of Endodontic Department, Dental School, Mashad University of Medical Sciences, Mashad, Iran

AbstractIntroduction: This investigation evaluates the effects of mineral trioxide aggregate (MTA),calcium hydroxide (CH) and calcium enriched mixture (CEM) as pulp capping materials ondental pulp tissues.Materials and Methods: The experimental procedures were performed on eighteen intactdog canine teeth. The pulps were exposed. Cavities were randomly filled with CEM, MTA, orCH followed by glass ionomer filling. After 2 months, animals were sacrificed, each toothwas sectioned into halves, and the interface between each capping material and pulp tissuewas evaluated by scanning electron microscope (SEM) in profile view of the specimens.Results: Dentinal bridge formation as the most characteristic reaction was resulted fromSEM observation in all examined groups. Odontoblast-like cells were formed and create denscollagen network, which was calcified gradually by deposition of calcosphirit structures toform newly dentinal bridge.Conclusion: Based on the results of this in vivo study, it was concluded that these testmaterials are able to produce calcified tissue in underlying pulp in the case of being used as apulp capping agent. Additionally, it appears that CEM has the potential to be used as a directpulp capping material during vital pulp therapy.

Keywords: Calcium enriched mixture; Calcium hydroxide; MTA, Pulp capping agent; SEM

Received: 23 Jul 2006; Revised: 09 Oct 2006; Accepted: 02 Nov 2006

*Corresponding author at: Saeed Asgary, Department of Endodontics, Iranian Center for Endodontic Research,Dental School, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. Tel: +98-2122413897, Fax: +98-2122427753. E-mail; saasgary@yahoo.com

Introduction

Direct pulp capping (DPC) is defined as usageof a suitable dental material over an exposedpulp to protect it from additional injury and alsoinitiates the formation of irritation dentin at thesite of injury. This technique permitspreservation of pulp tissue to continue itsfunctions (1). Pulp capping is mainly indicatedfor reversible pulp tissue injury of developing ormature teeth. Inducing the dentinogenicpotential of pulpal undifferentiatedmesanchymal cells as the ultimate goal of DPCmaterial application is widely accepted (2).Various factors including pulp status, rootformation stage, periodontal condition, age, time

elapsed between pulp exposures and protection,pulp protecting materials, the nature and size ofexposure and bacterial microleakage caninfluence the ultimate result of pulp preservation(3). Although a wide variety of various dentalmaterials have been used for DPC procedures,but the most pertinent include differentformulations of calcium hydroxide (CH), dentin-bonding materials, and mineral trioxideaggregate (MTA).Hess reported a technique for pulpotomy usingCH in 1929 (4). CH has widespread use forprotecting the exposed dental pulp. As itpromoted reparative dentine bridge formationand maintained pulp vitality, this material wasthought to be more biologically acceptable (5).

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The antibacterial effect of CH on infected pulptissue is of its considerable importance (6).Although the beneficial effect of CH is mainlyattributed to the initial low-grade irritation ofthe traumatized underlying pulp tissue_ due toOH- release _ (7) but the exact mechanism bywhich CH forms a dentin bridge has not beenexplained yet (8).Franz et al. (9) studied the ultrastructure of thedentinal bridge formed under CH. They reportedthe presents of necrotic tissue in the layeradjacent to CH. Mentioned disadvantages forthis material were its degradation over the time,tunnel defects in dentinal bridges and its poorsealing properties (10). At the present time, DPCwith CH remains an unpredictable method fortreatment of mature teeth.MTA, which was introduced in 1993 byTorabinejad at Loma Linda University, has beentested since 1996 as potential DPC material ofchoice (11-12). Pitt Ford et al. have reported thatthe MTA, which was placed in mechanicallyexposed pulps of mandibular incisors inmonkeys, stimulates pulp healing with minimalinflammatory reactions and dentinal bridgeformation (11). MTA stimulates significantlymore hard-tissue formation, and results in lessinflammation demonstrated remarkable successcompared with CH (12-14). The authors havedocumented, in a SEM study, that the mostcharacteristic reaction of pulp cells was theintimate connection of cell processes andsecretion extra-cellular fibers with the crystals ofMTA. They also found the progressivemineralization of dentinal bridge formation fromthe periphery towards the central area (15).Although MTA has superior biocompatibility incomparison with CH, it has a delayed settingtime (16), poor handling characteristics (17),while it is an expensive material.The advantages and disadvantages of differentcommercial types of MTA have beendocumented (18-19). These preliminary datawere used in a project on production of a newdental material which benefits from superiorbiocompatibility of MTA and also appropriatesetting time (less than 1 hour), handlingcharacteristics, chemical properties, andreasonable price. This novel endodontic cement(CEM) was formulated using different calciumcompounds (20). The results of our other studies(unpublished data) revealed that the mixed CEM

comprises water-soluble calcium and phosphate,and immediately forms hydroxyapatite duringand after setting time. This cement form aneffective seal against entrance ofmicroorganisms, has antibacterial effect,resistant to washout, and also it is able to set inan aqueous environment.The aim of present in vivo study was toevaluate the dentinogenic activity of CEM onmature dog teeth and compare it with MTA andCH by using the scanning electron microscope.

Materials and Methods

Eighteen intact canine teeth with healthyperiodontium in five healthy 18- 24 months oldbeagle dogs were used. All experimentalprocedures were carried out according toprotocols approved by the Ethics Committee ofDental Research Center of the Shaheed BeheshtiMedical University (M/8512). Under generalanesthesia gained with intramuscular injection of20 mg/kg Ketamine hydrochloride (Alfasan,Woerden, The Nederlands) and 1 mg/kgXylazine (Bayer, Munich, Germany), dog teethwere polished and washed with 0.2%chlorhexidine gluconate. Intraoral anesthesia formandibular teeth was achieved by anestheticblock injection of 1.8 ml of mepivacaine 3%(ESPE, Dental AG, Seefeld, Germany), and inmaxilla by infiltration injection of 1.8 mlmepivacaine 3%. The teeth were then isolatedby rubber dam. Using a No. 1 round bur(Dentsply, Tulsa, Ok, USA) in an air turbinewith sterile saline spray, Class V cavities wereprepared at labial surfaces of the teeth, and pulpexposures (approximately 1 mm in diameter)were obtained. Bleeding was controlled byirrigation with sterile saline solution and thecavities were dried with sterile cotton pelletsbefore placing test materials. The cavities wererandomly divided in to three test groups.In group 1, the exposed pulps were capped withProRoot MTA (Tooth-colored formula,Dentsply, Tulsa, Ok, USA) which was mixedaccording to the manufacturer’s directions toprovide a grainy, sandy mixture. Using CH (LDCaulk, Milford, DE, USA) which was mixedaccording to the manufacturer’s directions, thepulp exposures were capped in group 2. In group3, the calcium enriched mixture (CEM) wasmixed with powder and liquid in a 3: 1 ratio to

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provide a putty mixture and capped the pulpexposures. A light pressure was applied with awet cotton pellet in groups 1 and 3, resulting inan acceptable adaptation of material with thepulp tissue and cavity walls. Excess water wasremoved with sterile cotton pellets. Theremainders of the cavities in all groups wereimmediately restored with Fuji II glass ionomer(GC International Corp, Tokyo, Japan)permanent filling material. In order to minimizepain and discomfort, a nonsteroidal anti-inflammatory drug (Ibuprofen, Rose Daro, Iran)was prescribed immediately followingoperation.The controls were selected fromuntreated, two intact canines on which no pulpcapping were performed.After 2 months, the animals were sacrificed byan overdose of Pentothal, and vital perfusionfixation was performed with Karnovskysolution. Then, the teeth and their surroundingtissues were removed as block sections. Acrack was created on each sample by a vice. Inthe next step, the teeth were placed in 2.5%gluteraldehyde solution for 14 days. Followingcomplete fixation of the samples, each toothwas carefully notched (mark) in abuccolingual direction by a diamond disc toobtain a cross-crack of filled cavity andprobably underlying dentinal bridge. Afterwashing the tooth halves with normal saline,the samples were then processed for scanningelectron microscopic (SEM) analysis usingcritical point drying technique and then put inan oven and were allowed to dry at 50°C for24 h.All the samples were gold-coated using aDynavac CS300 (Dynavac, Sydney, Australia)coating unit. A Cambridge Instruments Stereo-scan 360 (Cambridge Instruments, Cambridge,UK) SEM was used for imaging and observingbasic morphology of dental pulp reaction anddentinal bridge formation in cross-sectionalmode. SEM operating conditions were used asfollows: 20-kV accelerating voltage, 6-mmworking distance and 71-pA probe current.

Results

The DPC procedures were well-tolerated by theexperimental animals. The postoperative periodwas uneventful, confirming the absence of anyapparent adverse effect caused by the

experimental protocol.In the control group, the pulps were healthy andshowed normal components. Odontoblast cellsappear as large, closely aligned, multilayered,sweet potato- shaped cells. They averaged 3-5μm in width and 6-10μm in length. A thin layer of unmineralized dentin (predentin) waspresent and odontoblastic processes passed thislayer and entered dentinal tubules (Figure 1).According to statistical analysis, there was nosignificant difference between test groups.All samples in group 1 showed vital pulp.Complete dentin bridge formation were foundin all cases of direct pulp capping with MTA.Profile view of the complete dentin barriershowed that the bridges consisted of threedifferent aspects. The outer aspect wascomposed of MTA in direct contact withnewly formed hard tissue. In the middleportion, a dentin like bridge with irregulardentinal tubules was identified. The pulpal orinner aspects exhibited predentin layer whichwas wider than normal condition and it wasfast mineralized. The vital pulp consists ofnormal elements. Young odontoblast like cellsdifferentiated and elaborated collagen matrixand predentin layer as shown in Figure 2. Inthis figure, the amorphous crystals ofhydroxyapatite are shown in the odontoblasticintercellular spaces as a sign of rapidmineralization process.In group 2, vital pulp was seen in all thesamples. Profile view of the barrier tissueshowed that the bridges consisted of threedifferent aspects. The outer aspect wascomposed of CH in direct contact with newlyformed tissue. In the middle portion, a dentinlike bridge in some areas was identified. Theinner aspects exhibited predentin layer whichwas wider than normal condition, and themineralization process was not fast.The predentin layer made of coarse collagenbundles which were prepared for calcification(Figure 3). The pulp consists of normalelements. Odontoblast-like cells were absent.All samples in group 3 showed vital pulp.Complete dentinal bridge formation was foundin all cases of direct pulp capping with CEM.Profile view of the complete dentin barriershowed that the bridges consisted of threedifferent aspects. The outer aspect wascomposed of CEM in direct contact with newly

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Figure 1- Scanning electron micrograph of dentin andpulp border of coronal pulp in control group (boldarrow: predentin layer consist of collagen matrix,normal arrows: odontoblast cell bodies in multilayerand intercellular spaces contain a fine network ofreticular fibers, arrow heads: branched odontoblasticprocesses when entered to dentinal tubules, x900).

formed hard tissue. In the middle portion, adentin-like bridge with irregular dentinaltubules was identified. The pulpal or inneraspects exhibited predentin layer which wassimilar to normal condition. The vital pulpconsisted of normal elements. Youngodontoblast like cells were differentiated andthey elaborated collagen matrix and predentinlayer as shown in Figure 4. The tubular natureof dentinal bridge is shown in this figure.

Discussion

Mature dental pulp cells possess the ability ofdifferentiation into odontoblast-like cell lineageforming tubular dentine in the absence ofnormal developmental conditions, i.e. dentalepithelium and basement membrane (21).Although DPC procedures are taught in generalpractice as a temporary treatment for cariousand mechanically exposed teeth, someresearchers have suggested that vital pulptherapies can be permanent (13, 22). It has beenwell recognized that dentinal bridge formationcan be observed in exposed pulps without anyexogenous application (23-24). The superficialzone of extracellular matrix at the woundsurface of the repairing connective tissues isphysiologically followed in exposed dentalpulp by osteodentin or fibrodentin hard tissuedeposition. A layer of odontoblast-like cells,producing reparative dentine, is finally formedas a sign of normalized pulp function. In

Figure 2- Scanning electron micrograph of dentin andpulp border in MTA pulp capped tooth (bold arrow:predentin layer consist of collagen matrix, normalarrows: odontoblast-like cell bodies longer thannormal ones, arrow heads: hydroxyappatite crystals inthe intercellular space, x1500).

morphological terms, these cells are recognizedas the elongated pulpal cells with increasedcytoplasm/nucleus ratio and polarized nuclei,which form tubular matrix in a polar predentinlike pattern (21).Because the pulp has enough vital tissue, it wasadvocated that DPC procedures could beperformed successfully on an asymptomaticcarious exposure (22). A clinical study showedthat asymptomatic carious exposures couldsurvive for an average of 12 yr after pulpcapping (25). One of the newest DPC materialsthat have appeared in 1996 is MTA. Theproperties of MTA are well established. It has agood sealing ability, little cytotoxicity,antibacterial effect, a pH of 12.5, andbiocompatibility, and also it is not affected bytissue fluid or blood contamination, (16, 26).In the present study the pulp capped teeth withMTA showed vitality, complete dentinal bridgeformation, differentiated odontoblast-like cells,collagen matrix elaboration, and predentinlayer creation. Various studies have presentedexcellent results for MTA as DPC material(12,13,27,28). The initial effect of MTA on thesurface of mechanically exposed pulp is theformation of a superficial layer of crystallinestructures over the pulpal surface of thecapping material. Columnar cells undergoingnuclear and cytoplasmic polarization andshowing a well developed cytoplasmicorganization are further arranged along thecrystalline structures. Initiation of

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Figure 3- Scanning electron micrograph (SEM) of

collagenous fibers of predentin of CH pulp capping

(x1500).

dentinogenesis can be identified only inhistomorphological studies by the palisadeappearance of elongated and polarizedodontoblast like cell layer able to secretetubular matrix in a polar predentin-like pattern(27). The favorable results obtained in thisresearch on MTA are similar to the findings ofprevious DPC studies.CH was used in this study, because it wasshown that healing process following DPC ofhuman teeth with CH is successful. It wasreported that healing process initially consistsof proliferation and migration of cells under thewound surface and elaboration of new collagenalong the superficial necrotic zone or the pulpalsurface of capping agent. The necrotic zone andthe new collagen layer attract mineral salts,becoming calcified matrices. Then a layer ofOdontoblast-like cells is formed in associationwith the fibrodentin, and reparative dentine issecreted (29).In this study, the observed results of CH wereas well as MTA and CEM groups. All casesshowed an incomplete fibrodentin bridge andthe predentin layer made of coarse collagenbundles which were not calcified in some areas.Many data from capping studies suggest thatinitiation of reparative dentine formation mightnot be attributed to any specific dentinogeniceffect of CH, although its effect in controllingthe infection and stimulating the wound healingprocess might not be excluded. It waspreviously reported that CH dressing of hard-setting Dycal does not provide a long-termprotection against microleakage, as were

Figure 4- SEM of dentin and pulp (left) border in

CEM pulp capped tooth. Dentinal bridge was broken

obliquely and odontoblastic process exit from broken

part and are seen naked (bold arrow: predentin layer

consist of collagen matrix, normal arrows: cross

section of dentinal tubules, arrow heads: odontoblastic

processes when entered to dentinal tubules, x1000)

dissolved within 1–2 years, and tunnel defectsin the majority of the dentin bridges whichallow pulp inflammation, infection or necrosis(30). This could explain the unfavorable resultsof the pulp capping with CH in other studiesMTA is commonly used for various clinicalapplications, but the material is expensivecauses it's restrict usage. The development ofnew materials that are biocompatible,bactericidal, inductive of hard tissue formation,and have similar or better sealing propertiescould solve this problem. Regarding favorableresults of CEM we suggest this cement as oneof these materials. On a comparative basis,CEM resulted in similar response to pulpcapping with MTA and better than CH.Complete dentinal bridge with more thicknessthan CH was formed by CEM and the presenceof Odontoblast-like cells was the major finding.This new cement contains calcium compoundssuch as tricalcium phosphate (TCP), calciumsulfate, calcium silicate, calcium hydroxide,calcium oxide and others to improve thebiocompatibility, chemical and physicalcharacteristics. Good biocompatibility of CEMis hypothesized to be due to its chemicalproperties. Different calcium compounds inCEM provide high calcium and phosphorousconcentration within the mixture. Theseelements can produce hydroxyapatite which isa natural product of dental pulp cells (31).

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However, it is unclear which components leachfrom CEM, but the cement contains CH andcalcium oxide (which produces CH through thehydration). CH, if leached out in sufficientquantities due to a direct effect on themicrovasculature resulting in less plasmaoutflow which, in turn, favors a calcificresponse in the capped tissue (32). Holland etal. observed granulations nearest to the openingof dentin tubes filled with MTA and implantedin an animal study. They reported that thesestructures are similar to the calcite crystalsobserved with CH (33). According to Seux etal. calcite crystals attract fibronectin, which isresponsible for cellular adhesion and differen-tiation (34). Therefore, we hypothesized thatCEM, MTA and CH have a similar mechanismfor hard tissue formation. In addition, somecompounds such as calcium sulfate andcalcium silicate may cause a slight expansionof CEM through continuous hydration afterinitial setting of the material and furthercrystalline maturation. This may be indirectlyresponsible for CEM biocompatibility via goodsealing ability.Thus, the property of CEM may be due to itstissue reactions in similarity with MTA and CHbut, in addition, providing an excellent tightseal (20).It should be emphasized that this experimentwas carried out in ideal conditions andintentionally exposed pulps of healthy teeth inanimals have exclusively been studied. Carefulconsideration is essential for correlating thesefavorable results to clinical situation.

Conclusion

In conclusion, according to the results of this invivo study in dogs, CEM and MTA are effectivepulp capping materials, able to stimulate hardtissue bridge formation and superior to CHcement for pulp capping procedures. Also, CEMappears to be an alternative to MTA accordingto their biological effects are identical.

Acknowledgement

This study was supported, in part, by DentalResearch Center of Shaheed Beheshti MedicalUniversity. The authors wish to thank the staffmembers of the Electron Microscopy Unit

(EMU) of the Australian National University(ANU) for their assistance with scanningelectron microscopy.

Conflict of Interest: ‘None declared’.

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