Dentil! Traumatology 2003: 19: 314-320Printed in Detwtark. All rights reserved

Copyright <Q Biackweil Munksgaard 2003

DENTAL TRAUMATOLOGY

Comparison of bioactive glass, mineral trioxideaggregate, ferric sulfate, and formocresol aspulpotomy agents in rat molarSalako N, Joseph B, Ritwik P, SaloncnJ, John P, JunaidTA.Comparison of bioactive glass., mineral trioxide aggregate, ferricsulfate and formocresol as pulpotomy agents in rat molar. DentTraumatol 2003; 19: 31^320. © BlackweU Munksgaard, 2003.

Abstract - Bioactive glass (BAG) is often used as a filler material forrepair of dental bone defects. Although there is evidence of osteogeniepotential ofthis material, it is not clear yet whether the materialexhibits potential for dentinogenesis. Hence, the aim ofthe presentstudy was to evaluate BAG as a pulpotomy agent and to compare itwith three commercially available pulpotomy agents such asformocresol (FC), ferric sulfate (FS), and mineral trioxide aggregate(MTA). Pulpotomies were performed in 80 maxillary first molars ofSprague Dawley rats, and pulp stumps were covered with BAG, FC,FS, and MTA. Histologic analysis was performed at 2 weeks and thenat 4 weeks after treatment. Experimental samples were compared withcontra-lateral normal maxillary first molars. At 2 weeks, BAG showedinflammatory changes in the pulp. After 4 weeks, some samplesshowed normal pulp histology, with evidence of vasodilation. At2 weeks, MTA samples showed some acute inflammatory cells aroundthe material with evidence of macrophages in the radicular pulp.Dentine bridge formation with normal pulp histology was a consistentfinding at 2 and 4 weeks with MTA. Ferric sulfate showed moderateinflammation of pulp with widespread necrosis in coronal pulp at 2and 4 weeks. Formocresol showed zones of atrophy, inflammation, andfibrosis. Fibrosis was more extensive at 4 weeks with evidence ofcalcification in certain samples. Among the materials tested, MTAperformed ideally as a pulpotomy agent causing dentine bridgeformation while simuhaneously maintaining normal pulpal histology.It appeared that BAG induced an inflammatory response at 2 weekswith resolution of inilammation at 4 weeks.

Nathanael Salako\ Bobby Josepb\

Priyansbi Ritwik^ Jukka Salonen ,̂

Preetli)Jobn\T.A.Junaid*

'Faculty of Dentistry. Kuwait University, Kuwait;^Louisiana State University School of Dentistry, USA;^urku Centre for Biomaterials, Finland; ""Faculty ofMedicine, Kuwait University, Kuwait

Key words: pulpotomy; MTA; BAG; pulp histology

Dr Bobby Joseph, Associate Professor, Department ofDiagnostic Sciences, Faculty of Dentistry, KuwaitUniversity, PO Box: 24923. Safat 13110. KuwaitTel:+965 266 4502 Ext. 7105Fax:+965 263 4247e-mail: bobby (((• hsc.kuniv.edu.kw

Accepted 11 March, 2003

Pulpotomy is a therapeutic procedure, which consistsofthe surgical amputation of coronally inflamed pulp.The wounded surface ofthe radicular pulp is treatedwith a medicament or dressing agent to promote heal-ing or to cause fixation of the underlying tissue (1).The objective is to maintain vitality ofthe radicularpulp. Pulpotomy is a common procedure in the treat-ment of acutely inflamed primary teeth. It is also usedin the management of young permanent teeth withopen apices. Various materials have been recom-

mended for pulptomy, and these are formocresol {FC),gluteraldehyde, ferric sulfate (FS), zinc oxide eugenol,polycarboxylate cement, and calcium hydroxide (1).Of these, formocresol and. more recently, ferric sulfateare the most commonly used agents in pulpotomy pro-cedure in primary molars, Bioactive glass (BAG)and mineral trioxide aggregate (MTA) are two rela-tively new materials in the field of dentistry (2,3).

Bioactive glasses have been studied for more than30 years as hone substitutes. They react with aqueous

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Comparison of pulpotomy agents in rat molar

solutions and produce a carbonated apatite layer.Therefore, BAGs are biocompalible materials, whichreadily bind to the bone. Originally, BAG was consid-ered as osteoconductive material. Today, however,there is an increasing amount of evidence showingthat they also have the capacity to serve as inductivematerials for hard tissue formation and mineraliza-tion (2).

Because of their biocompatibility and antibacterialproperty, BAGs might be ideal materials for pulpo-tomies, pulp capping, and periapical bone heahng(4). As BAGs have been reported to stimulate osteo-blasts and to promote healing of bone lesions (5), thereare strong indications to examine whether BAGs sti-mulate odontoblasts and induce the formation ofrcparative dentine in pulpal wounds.

Mineral trioxide aggregate has attracted attentionin the field of endodonties (3). Its principal compo-nents are tricalcium silicate, tricalcium aluminate, tri-calcium oxide, and silicate oxide. It also containsoxides of iron and magnesium, and bismuth oxide isadded for radiopacity. Many favorable features havebeen reported on the use of MTA, when used withother conventional endodontic materials. These areits excellent sealing ability (6), biocompatibility (7),ability to form dentine bridge (8), and cementumand periodontal ligament regeneration (9). Hence,MTA has been recommended as a retrograde fillingmaterial in the repair of perforations, pulp capping,and apexifieation (10).

The purpose ofthis study was to compare the dentalpulp response in rat molars when BAG, MTA, FS,and FC were used as pulpotomy agents.

Materials and methods

Eighty male Sprague Dawley rats, 14 weeks old,weighing 250-300 g were obtained from the CentralAnimal Breeding House, Kuwait University. Theywere divided into four treatment groups, 20 each forBAG, MTA, ferric sulfate, and formocresol. Pulpot-omy was performed on the maxillary first molar.

The animals were anesthetized by intraperitonealinjection of pentobarbitone with a dose of approxi-mately 0.6 ml per 250 g weight. The animals werepinned on their back to a surgical board. Visualizationofthe working area was aided with a surgical micro-scope. Every effort was made to ensure consistencyofthe size ofthe exposure ofthe pulp with the use of330 tungsten carbide bur. Hemorrhage was controlledwith sterile endodontic paper points.

Pulpotomy agents

Four pulpotomy agents were investigated in this study.Two agents -formocresol and ferric sulfate - are themost commonly used agents for primary molar

pulpotomy procedure, and the other two -MTAand BAG - are new agents currently being investi-gated as potential agents for pulp therapies in both pri-mary and permanent dentitions.

Bioactive glass material used for the study wasS53P4 (AbminTechnologigies Ltd., Turku, Finland)with the composition (by weight): SiO^ (53.0%),CaO (20.0%), NaaO (23.0%), and P2O5 (4~.0%).Theglass was used in the form of fine powder with the par-ticle size of <20 |im. The powder was mixed with asmall amount of saline to make a creamy suspension.After obtaining hemorrhage control by paper pointpressure, BAG was placed directly on the pulpalstump, and the cavity was filled with amalgam.

Mineral trioxide aggregate used was ProRoot MTA(Tulsa Dentsply). The material was mixed as per themanufacturer's recommendations. One gram of pow-der was mixed with one vial of distilled water pro-vided. A smooth mix of MTA was obtained, whichwas applied on the pulp stump after hemorrhage con-trol with paper point. The cavity was then sealed withamalgam.

Paper points moistened with 15.5% ferrie sulfatesolution (Astringdent Ultra dent Product, Inc.) wereplaced in contact with the pulpal stumps until hemor-rhage control was obtained. The site was then coveredwith zinc oxide—eugenol cement and amalgam.

Paper point moistened with climcal grade formo-cresol was placed in contact with the pulpal tissuefor 5 min. The exposed site was covered with zincoxide-eugenol cement amalgam.

Tissue preparation

The animals were anesthetized by an intraperitonealinjection of pentobarbitone and perfused intracar-dially with Bouin's solution; 0.9% (v/v) picric acid,9% (v/v) formaldehyde, and 5% (v/v) acetic acid.The maxillae were dissected out and postfixed inBouin's solution. The maxillae were then decalcifiedwith Rapid Decalcifier (Apex Engineering ProductsCorporation, USA). Each maxilla was sectioned intotwo halves, each containing the first molars. The tissuewas processed and then embedded in paraffin by stan-dard histologic procedures (11). Serial sections(5)J.m) were cut for H&E staining and stored forfuture analysis. In each experimental group, 10 ratswere sacrificed at 2 weeks, and 10 rats were sacrificedat 4 weeks {Table 1).

Tablet Materials used and number of rats in each experimental group

Material

BAGMTAFerric sulateFormocresol

2 weeks

10101010

4 weeks

10101010

Totai

20202020

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Salako et al.

Table 2. Summary of histologic results

Observation 2 weeks

BAG

4 weeks 2 weeks

MA

Material

Ferric sulfate

4 weeks 2 weeks 4 weeks

Formocfesol

2 weeks 4 weeks

Inflammation, localizedInflammation, widespreadNecrosis, localizedNecrosis, widespreadLeucocytesMacrophagesFibrosisDentine bridge

+/-

+ : Present; + + : present in high number; - : not present; -\-l-: not consistent finding.

Results

The experimental animals tolerated the surgical proce-dure well. No apparent adverse events occurred dur-ing the observation period of 2 and 4 weeks. In eachrat, the maxillary first molar on the right side wasoperated on, and the contra-lateral first molar waskept as control. Results were obtained by comparingthe experimental side with the control side (Table 2).

BAG

Light microscopic examination of 2-weok-old BAGsamples showed localized areas of inflammation in

the pulp, especially in the mid-root portion. Areasof necrotic tissue were seen both in the coronaland in the radicular part of the pulp {Fig. 1). Onesample showed changes consistent with root re.sorp-tion with an hour glass-shaped root surface andpolymorphonuclear leucocytes in the radicular pulpas well as in the adjacent periodontal tissue (Fig.2).None of the samples had an odontoblastic layerintact.

Histologic examination of the 4-week-old samplesshowed comparatively belter results than the 2-week-old samples (Fig. 3). Most of the slides showed normal

Fig. 1. Pulpotomy with BAG at 2 weeks. Arrows show a pool ofariuc iniliimmatory cells (hematoxylin & eosin; original mag-nification X10).

Fig.2. Pulpotomy with BACl at 2 weeks showing polymorpho-nuclear leukocytes around an area of root resorption. A rootcanal (RC) is also visible [hematoxylin&eosin; original magni-fication x20).

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Comparison of pulpotomy agents in rat molar

Fig. 3. Pulpotomy with BAG at 4 weeks showing normal pulphistology. A hlood vessel (BV) is also seen (hematoxylin &fosin; original niagnitication x20).

pulp tissue with an odontoblastic layer. Some samplesshowed dilated blood vessels {Fig.4).

Results with FS in both the 2- and the 4-week groupswere similar. Both groups showed complete pulpaldestruction, with some areas of acute inflammatoryinfiltrate (Figs. 5 and 6).

In case of FC, two-week-old samples showednecrotie zone just beyond the pulpotomy site, fol-

Fig. 5. Pulpotoniy with ferric SLiiratcat4 weeks showing areas ofnecrosis (N), inflammation [Pj and calcification (C) (hematox-ylin & (.'(Lsin; x20).

lowed by an area of inflammatory infiltrate. Four-week-old samples showed an atrophic zone beyondthe pulptomy site, followed by areas of fibrous tissueformation (Fig.7). The fibrous tissue was also evidentin the radicular pulp. Some samples in this groupshowed some calcific deposition in the coronalregion of the pulp, close to the orifice of the rootcanal (Fig. 8).

Fig. 4. Pulpotomy with BAG at 4 weeks showing areas consis-tent wiili necrosis, and odontoblasts {arrows] are also seen (he-maloxylin & eosin; xlO).

Fig. 6. Ferric sulfate at 2 weeks showing apical third of root withexcess cementum (GMl and root remodeling {hematoxylin &eosin; xlO).

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Salako et al.

Fig. 7. Formocresol al 2 weeks showingareas of inflammation (P), calcification(C), and fibrosis (F) (hematoxylin & eo-sin; x20).

MTA

Light microscopic examination of 2-week-old sam-ples showed calcified material being laid down, distalto the pulpotomy site. This can be inferred asattempted bridge formation (Figs. 9 and 10). Theodontoblast layer was found to be intact. Some macro-phages were seen in the radicular pulp in one sample.Acute inflammatory cells were seen around MTAitself in one sample. Increased cementum formationwas seen in the apical regions of the root. However,

Fig.S. Enlarged view of calcified area in Fig.7 with arrowspointing to lacunae (hemaluxylin & eosin; x40).

in one sample, an isolated small area of cementumresorption was seen.

The 4-wcek samples showed complete dentin bridgeformation with normal pulpal histology.

Discussion

Bioactive glasses have been in use since 1984 in ortho-pedic and plastic surgery applications (4, 5). Theyare osteoconductive biomaterials, and hence havebeen tried for various applications in dentistry. Theyhave been tested for infrabony periodontal defectsand furcation defects, and may be an effective adjunctin their management (12). BAGs have been shown toimprove osteointcgration with implants (13,14).Tlieyare successful in ridge augmentation procedures.Fiber-reinforced BAGs are being tested as rootimplants (15). BAGs also have definitive antibacterialeffect on oral microorganisms, both supragingivaland subgingival (16). Based on the various propertiesof BAG, the present study was designed to test BAGas a pulpotomy agent in rat molars. Resuits showedan acute inflammatory response from the pulp at2 weeks. This may be an expected protective responsefrom the body to any foreign material. However, wefound one sample with periapical abscess formation,and one with root resorption, implying that the initialinflammatory response with BAG may be widespreadand severe enough to be irreversible. Most of the 4-week-old BAG samples showed regular pulpal histo-logy, with some dilated blood vessels. However, therewere specimens where there was complete pulpalnecrosis. It can be inferred that BAG causes a phaseof acute inflammation, following which there is ahealing/recovery period during which restorationof pulpal morphology is attempted. If the initial

318

Comparison of pulpotomy ageuts in rat moiar

Fig. 9. MTA at 2 weeks with arrows point-ing lo the dentine bridge distal to the ptiNpotomy site and normal pulpal histologybeyond the dentine bridge (hematoxylin& eosin; xlO).

inflammatory response is overwhelming, the resultantnecrosis eliminates any chances of recovery. In thepresent study, we were unable to ascertain factors,which could indicate the reasons for necrosis.

The 2-week-old MTA samples showed dentinbridge formation and some inflammatory responseto t he material. With time, the dentin bridge formationcontinues and inflammation resolves, leaving ahealthy pulp walled off from the pulpotomy site by adentin bridge. Previous studies that have used MTA

Fig. 10. Enlarged view oi' Fig. 9. Arrows pointing to intact odon-toblast layer. DB, dentine bridge (hematoxylin & eosin; x40}.

for pulpotomy have shown continuation of dentin for-mation to the point of complete root obliteration.Some MTA-treated teeth showed excessive cemen-tum formation. Schwartz et al. (1999) have reportedthat MTA causes cementogenesis (17). The excesscementum in the MTA-treated teeth in the presentstudy may be attributed to MTA. However, itshould be kept in mind that the study was conduc-ted in developing rats, and the finding may also becaused by the normal processes of root formationand remodeling.

Histologic reaction of vital pulp after the applica-tion of formocresol depends on the application timeand the concentration used. The usual finding is a zoneof necrosis followed by a zone of Bxation. Beyond this,there is an inflammatory infiltrate, which graduallyleads to normal pulp. Perfect healing without inflam-mation is not seen with formocresol. Replacement ofinflammation and necrosis with granulation tissueand bone or osteodentin has also been reported. Inthe present study, the zones of necrosis, flxation, andinflammation were seen with formocresol. Galcifledareas were also seen in the pulp chamber in somesamples.

In the present study, ferric sulfate caused progres-sive replacement of pulp tissue with clot formation.There were some areas of inflammation seen in someslides. Some calcific changes were observed in the cor-onal as well as in the radicular portions ofthe pulp.These findings are consistent with Smith et al. (18).

Among the materials tested, MTA performs ideallyas a pulpotomy agent, causing dentin bridge forma-tion and simultaneously maintaining normal pulpalhistology. It appears that BAG induces an inflamma-tory response at 2 weeks with the resolution of inflam-mation at 4 weeks.

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