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Copyright <Q Biackweil Munksgaard 2003

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

DENTAL TRAUMATOLOGY

Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar Salako N, Joseph B, Ritwik P, SaloncnJ, John P, JunaidTA. Comparison of bioactive glass., mineral trioxide aggregate, ferric sulfate and formocresol as pulpotomy agents in rat molar. Dent Traumatol 2003; 19: 31^320. Š BlackweU Munksgaard, 2003. Abstract - Bioactive glass (BAG) is often used as a filler material for repair of dental bone defects. Although there is evidence of osteogenie potential ofthis material, it is not clear yet whether the material exhibits potential for dentinogenesis. Hence, the aim ofthe present study was to evaluate BAG as a pulpotomy agent and to compare it with three commercially available pulpotomy agents such as formocresol (FC), ferric sulfate (FS), and mineral trioxide aggregate (MTA). Pulpotomies were performed in 80 maxillary first molars of Sprague Dawley rats, and pulp stumps were covered with BAG, FC, FS, and MTA. Histologic analysis was performed at 2 weeks and then at 4 weeks after treatment. Experimental samples were compared with contra-lateral normal maxillary first molars. At 2 weeks, BAG showed inflammatory changes in the pulp. After 4 weeks, some samples showed normal pulp histology, with evidence of vasodilation. At 2 weeks, MTA samples showed some acute inflammatory cells around the material with evidence of macrophages in the radicular pulp. Dentine bridge formation with normal pulp histology was a consistent finding at 2 and 4 weeks with MTA. Ferric sulfate showed moderate inflammation of pulp with widespread necrosis in coronal pulp at 2 and 4 weeks. Formocresol showed zones of atrophy, inflammation, and fibrosis. Fibrosis was more extensive at 4 weeks with evidence of calcification in certain samples. Among the materials tested, MTA performed ideally as a pulpotomy agent causing dentine bridge formation while simuhaneously maintaining normal pulpal histology. It appeared that BAG induced an inflammatory response at 2 weeks with resolution of inilammation at 4 weeks.

Pulpotomy is a therapeutic procedure, which consists ofthe surgical amputation of coronally inflamed pulp. The wounded surface ofthe radicular pulp is treated with a medicament or dressing agent to promote healing or to cause fixation of the underlying tissue (1). The objective is to maintain vitality ofthe radicular pulp. Pulpotomy is a common procedure in the treatment of acutely inflamed primary teeth. It is also used in the management of young permanent teeth with open apices. Various materials have been recom314

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 of Medicine, Kuwait University, Kuwait

Key words: pulpotomy; MTA; BAG; pulp histology Dr Bobby Joseph, Associate Professor, Department of Diagnostic Sciences, Faculty of Dentistry, Kuwait University, PO Box: 24923. Safat 13110. Kuwait Tel:+965 266 4502 Ext. 7105 Fax:+965 263 4247 e-mail: bobby (((â&#x20AC;˘ hsc.kuniv.edu.kw Accepted 11 March, 2003

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 sulfate are the most commonly used agents in pulpotomy procedure in primary molars, Bioactive glass (BAG) and mineral trioxide aggregate (MTA) are two relatively new materials in the field of dentistry (2,3). Bioactive glasses have been studied for more than 30 years as hone substitutes. They react with aqueous


Comparison of pulpotomy agents in rat molar

solutions and produce a carbonated apatite layer. Therefore, BAGs are biocompalible materials, which readily bind to the bone. Originally, BAG was considered as osteoconductive material. Today, however, there is an increasing amount of evidence showing that they also have the capacity to serve as inductive materials for hard tissue formation and mineralization (2). Because of their biocompatibility and antibacterial property, BAGs might be ideal materials for pulpotomies, pulp capping, and periapical bone heahng (4). As BAGs have been reported to stimulate osteoblasts and to promote healing of bone lesions (5), there are strong indications to examine whether BAGs stimulate odontoblasts and induce the formation of rcparative dentine in pulpal wounds. Mineral trioxide aggregate has attracted attention in the field of endodonties (3). Its principal components are tricalcium silicate, tricalcium aluminate, tricalcium oxide, and silicate oxide. It also contains oxides of iron and magnesium, and bismuth oxide is added for radiopacity. Many favorable features have been reported on the use of MTA, when used with other conventional endodontic materials. These are its excellent sealing ability (6), biocompatibility (7), ability to form dentine bridge (8), and cementum and periodontal ligament regeneration (9). Hence, MTA has been recommended as a retrograde filling material in the repair of perforations, pulp capping, and apexifieation (10). The purpose ofthis study was to compare the dental pulp 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 Central Animal Breeding House, Kuwait University. They were divided into four treatment groups, 20 each for BAG, MTA, ferric sulfate, and formocresol. Pulpotomy was performed on the maxillary first molar. The animals were anesthetized by intraperitoneal injection of pentobarbitone with a dose of approximately 0.6 ml per 250 g weight. The animals were pinned on their back to a surgical board. Visualization ofthe working area was aided with a surgical microscope. Every effort was made to ensure consistency ofthe size ofthe exposure ofthe pulp with the use of 330 tungsten carbide bur. Hemorrhage was controlled with sterile endodontic paper points.

pulpotomy procedure, and the other two -MTA and BAG - are new agents currently being investigated as potential agents for pulp therapies in both primary and permanent dentitions. Bioactive glass material used for the study was S53P4 (AbminTechnologigies Ltd., Turku, Finland) with the composition (by weight): SiO^ (53.0%), CaO (20.0%), NaaO (23.0%), and P2O5 (4~.0%).The glass was used in the form offinepowder with the particle size of <20 |im. The powder was mixed with a small amount of saline to make a creamy suspension. After obtaining hemorrhage control by paper point pressure, BAG was placed directly on the pulpal stump, and the cavity was filled with amalgam. Mineral trioxide aggregate used was ProRoot MTA (Tulsa Dentsply). The material was mixed as per the manufacturer's recommendations. One gram of powder was mixed with one vial of distilled water provided. A smooth mix of MTA was obtained, which was applied on the pulp stump after hemorrhage control with paper point. The cavity was then sealed with amalgam. Paper points moistened with 15.5% ferrie sulfate solution (Astringdent Ultra dent Product, Inc.) were placed in contact with the pulpal stumps until hemorrhage control was obtained. The site was then covered with zinc oxideâ&#x20AC;&#x201D;eugenol cement and amalgam. Paper point moistened with climcal grade formocresol was placed in contact with the pulpal tissue for 5 min. The exposed site was covered with zinc oxide-eugenol cement amalgam. Tissue preparation

The animals were anesthetized by an intraperitoneal injection of pentobarbitone and perfused intracardially 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 in Bouin's solution. The maxillae were then decalcified with Rapid Decalcifier (Apex Engineering Products Corporation, USA). Each maxilla was sectioned into two halves, each containing thefirstmolars. The tissue was processed and then embedded in paraffin by standard histologic procedures (11). Serial sections (5)J.m) were cut for H&E staining and stored for future analysis. In each experimental group, 10 rats were sacrificed at 2 weeks, and 10 rats were sacrificed at 4 weeks {Table 1). Tablet Materials used and number of rats in each experimental group Material

Pulpotomy agents

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

BAG MTA Ferric sulate Formocresol

2 weeks

4 weeks

Totai

10 10 10 10

10 10 10 10

20 20 20 20

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Salako et al. Table 2. Summary of histologic results Material MA

BAG Observation Inflammation, localized Inflammation, widespread Necrosis, localized Necrosis, widespread Leucocytes Macrophages Fibrosis Dentine bridge

2 weeks

4 weeks

2 weeks

Ferric sulfate 4 weeks

2 weeks

4 weeks

Formocfesol 2 weeks

4 weeks

+/-

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

Results The experimental animals tolerated the surgical procedure well. No apparent adverse events occurred during the observation period of 2 and 4 weeks. In each rat, the maxillary first molar on the right side was operated on, and the contra-lateral first molar was kept as control. Results were obtained by comparing the experimental side with the control side (Table 2). BAG

the pulp, especially in the mid-root portion. Areas of necrotic tissue were seen both in the coronal and in the radicular part of the pulp {Fig. 1). One sample showed changes consistent with root re.sorption with an hour glass-shaped root surface and polymorphonuclear leucocytes in the radicular pulp as well as in the adjacent periodontal tissue (Fig.2). None of the samples had an odontoblastic layer intact. Histologic examination of the 4-week-old samples showed comparatively belter results than the 2-weekold samples (Fig. 3). Most of the slides showed normal

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

Fig. 1. Pulpotomy with BAG at 2 weeks. Arrows show a pool of ariuc iniliimmatory cells (hematoxylin & eosin; original magnification X10).

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Fig.2. Pulpotomy with BACl at 2 weeks showing polymorphonuclear leukocytes around an area of root resorption. A root canal (RC) is also visible [hematoxylin&eosin; original magnification x20).


Comparison of pulpotomy agents in rat molar

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

Fig. 5. Pulpotoniy with ferric SLiiratcat4 weeks showing areas of necrosis (N), inflammation [Pj and calcification (C) (hematoxylin & (.'(Lsin; x20).

pulp tissue with an odontoblastic layer. Some samples showed dilated blood vessels {Fig.4). Results with FS in both the 2- and the 4-week groups were similar. Both groups showed complete pulpal destruction, with some areas of acute inflammatory infiltrate (Figs. 5 and 6). In case of FC, two-week-old samples showed necrotie zone just beyond the pulpotomy site, fol-

lowed by an area of inflammatory infiltrate. Fourweek-old samples showed an atrophic zone beyond the pulptomy site, followed by areas of fibrous tissue formation (Fig.7). The fibrous tissue was also evident in the radicular pulp. Some samples in this group showed some calcific deposition in the coronal region of the pulp, close to the orifice of the root canal (Fig. 8).

Fig. 4. Pulpotomy with BAG at 4 weeks showing areas consistent wiili necrosis, and odontoblasts {arrows] are also seen (hemaloxylin & eosin; xlO).

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

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Salako et al.

Fig. 7. Formocresol al 2 weeks showing areas of inflammation (P), calcification (C), and fibrosis (F) (hematoxylin & eosin; x20).

MTA Light microscopic examination of 2-week-old samples showed calcified material being laid down, distal to the pulpotomy site. This can be inferred as attempted bridge formation (Figs. 9 and 10). The odontoblast layer was found to be intact. Some macrophages were seen in the radicular pulp in one sample. Acute inflammatory cells were seen around MTA itself in one sample. Increased cementum formation was seen in the apical regions of the root. However,

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

318

in one sample, an isolated small area of cementum resorption was seen. The 4-wcek samples showed complete dentin bridge formation with normal pulpal histology. Discussion Bioactive glasses have been in use since 1984 in orthopedic and plastic surgery applications (4, 5). They are osteoconductive biomaterials, and hence have been tried for various applications in dentistry. They have been tested for infrabony periodontal defects and furcation defects, and may be an effective adjunct in their management (12). BAGs have been shown to improve osteointcgration with implants (13,14).Tliey are successful in ridge augmentation procedures. Fiber-reinforced BAGs are being tested as root implants (15). BAGs also have definitive antibacterial effect on oral microorganisms, both supragingival and subgingival (16). Based on the various properties of BAG, the present study was designed to test BAG as a pulpotomy agent in rat molars. Resuits showed an acute inflammatory response from the pulp at 2 weeks. This may be an expected protective response from the body to any foreign material. However, we found one sample with periapical abscess formation, and one with root resorption, implying that the initial inflammatory response with BAG may be widespread and severe enough to be irreversible. Most of the 4week-old BAG samples showed regular pulpal histology, with some dilated blood vessels. However, there were specimens where there was complete pulpal necrosis. It can be inferred that BAG causes a phase of acute inflammation, following which there is a healing/recovery period during which restoration of pulpal morphology is attempted. If the initial


Comparison of pulpotomy ageuts in rat moiar

Fig. 9. MTA at 2 weeks with arrows pointing lo the dentine bridge distal to the ptiN potomy site and normal pulpal histology beyond the dentine bridge (hematoxylin & eosin; xlO).

inflammatory response is overwhelming, the resultant necrosis eliminates any chances of recovery. In the present study, we were unable to ascertain factors, which could indicate the reasons for necrosis. The 2-week-old MTA samples showed dentin bridge formation and some inflammatory response to t he material. With time, the dentin bridge formation continues and inflammation resolves, leaving a healthy pulp walled off from the pulpotomy site by a dentin bridge. Previous studies that have used MTA

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

for pulpotomy have shown continuation of dentin formation to the point of complete root obliteration. Some MTA-treated teeth showed excessive cementum formation. Schwartz et al. (1999) have reported that MTA causes cementogenesis (17). The excess cementum in the MTA-treated teeth in the present study may be attributed to MTA. However, it should be kept in mind that the study was conducted in developing rats, and the finding may also be caused by the normal processes of root formation and remodeling. Histologic reaction of vital pulp after the application of formocresol depends on the application time and the concentration used. The usual finding is a zone of necrosis followed by a zone of Bxation. Beyond this, there is an inflammatory infiltrate, which gradually leads to normal pulp. Perfect healing without inflammation is not seen with formocresol. Replacement of inflammation and necrosis with granulation tissue and bone or osteodentin has also been reported. In the present study, the zones of necrosis, flxation, and inflammation were seen with formocresol. Galcifled areas were also seen in the pulp chamber in some samples. In the present study, ferric sulfate caused progressive replacement of pulp tissue with clot formation. There were some areas of inflammation seen in some slides. Some calcific changes were observed in the coronal as well as in the radicular portions ofthe pulp. These findings are consistent with Smith et al. (18). Among the materials tested, MTA performs ideally as a pulpotomy agent, causing dentin bridge formation and simultaneously maintaining normal pulpal histology. It appears that BAG induces an inflammatory response at 2 weeks with the resolution of inflammation at 4 weeks.

319


Saiako et ai. References 1. Alacam A. Ptilpal tissne changes following ptilpotomies wilh rnrmocreso!, gluiaraldeliyde-calciLim hydroxide, gltitaraldehyde-zinc oxide eugenol pastes in primary teeth. J Pedo 1989;13:i23-32. 2. Stanley HR, Clark AE, Pameijer CH, Lotiw NH Pulp capping with a modified bioglass formula. Am J Dent

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3. Eidelman E, Holan G, Fuks AB. Mineral trioxide aggregate vs. formocresol in pulpotomized primary molars. Pediatr Dent 2001;23:13-8. 4. Schepers Ii, Clercq M, Ducheyne P, Kempeneers R. Bioactive glass particulate material as a filler for bone lesions. J Oral Rehabil ]99l;18:439-52. 5. Carry A, David C, Arthur E. Clinical use of a bioactive glass particulute in the treatment of human osseous defects. Compend Cnntin Educ Deiil t997;18:3ri2-63. 6. Nakata TT, B^if KG, Baumgarther JCI Perforation repair comparing mineral trioxide aggregate and amalgam using an anaerobic bacterial leakage mode!. J Endod 1998;2'1-: 184-6. 7. Pitt Ford TR, Torabinejad M, McKendry DJ, Hong GU Kariyawasam SP. Use of mineral trioxide aggregate for repair ol'iurcal perforations. Oral Surg 1995;79:756-62. 8. Torabinejad M, Hong CU. McDonald F, Pitt Ford TR. Physiological and chemical properties of a new root-end material'J Endod 1995;21:349-53. 9. Pitt FordTR/lbrabinejad M, Abedi HR, Backland LK, Kariyawasam SP, Using mineral trioxide aggregate as a pulp-capping material. J Am Dent Assoc i99(i;I27:491-4. 10. Junn D), M< Millian P. Backland LK,lorabinejad M. Qiiantitative assessment of dentine bridge formation Ibllowing

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pulp capping with mineral trioxide aggregate. J Endod 1998;24:27B( Abstract). 11. Joscpb BK, Symons AL, Matkay CA, Hume DA, Waters MJ, Marks SC, Jr. Insulin-like growth factor-1 and insulinlike growl ll factor-I immunoreactivity in normal and oslcopetrotic (toothless, tl/tl) rat tibia. Growth Factors i999;16i279-91. 12. Yukrta RA, Evans GH. Aichelmann-Reidy MB, Mayer ET. Clinical comparison of bioactive glass hone replacement graft material and expanded polytetrafluoroethylene barrier membrane in treating human mandibular molar class 11 furcations. J Periodontoi 2001:72:125-33. 13. Nevins ML, Camelo M, Nevins M, King CJ, Oringer RJ, Sciienk RK, Fiorellini JP. Human histologic evaluation of bioactive ceramic in the treatment of periodontai osseous delects. Int J Periodontics Restorative Dent 2000;20:4.58-65. 14. Sculean A. Barbe G, Chiantella GC, Arwdler NB, Berakdar M, Brecx M. Clinical evaluation of an enamel matrix pmtein derivative combined with a bioactive glass for the treatment ol" intrabony periodontal defects in humans. J Periodontoi 2002;73:40i-8. 1.5. Norton MR, Wilson J. Dental implants placed in extraetion sites implanted with bioactive glass: hutnati histology and clinical outcome. Int J Oral Maxillofac Impiants 2002; 17:249-57. Hi. Stoor P. Soderling E, Salonenjl. Antibacterial effects of bioactive glass on oral micro-organisms. Acta Odontol Can 1998;56:I61 -5. 17. Schwarz RS, Manger M. Clement DJ, Walker WA, IU. Mitieral trioxide aggregate: a new material for endodonties. J A m Dent Assoc 1999:130:967-75. l!i. Smith NL, Seale NS, Nunn ME. Ferric sulfate pulpotomy in pritnary molars. Pediatr Dent 2000:22:192-9.


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Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar