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Original Article

Histopathologic Responses of Dog’s Dental Pulp to Mineral Trioxide Aggregate, Bio Active Glass, Formocresol, Hydroxyapatite Ebrahim Jabbarifar DDS, MSc*, Sayed Mohammad Razavi DDS, MSc**, Neda Ahmadi DDS, MSC***

ABSTRACT Introduction: Bio Active Glass (BAG) is often used as a filler material for regeneration of dental bone defects. Mineral trioxide aggregate (MTA) is used as retrofilling agent, repair of root resorption and pulpotomy agent in primary dentition. Formocresol (FC) is old and standard fixation agent in pulpotomy procedures. Hydroxyapatite (HA) is a biologic constitute. It is used in ridge augmentation, bony defect repair and pulpotomy agent in teeth. The purpose of this study was evaluation of histopathologic responses of dog’s dental pulp to MTA, BAG, FC and HA after three months follow up. Materials and Methods: For this experimental prospective study forty-eight teeth were randomly selected in four dogs. Animals were anesthetized and pulpotomized. Three months later, animals were anesthetized and sacrificed. Then, teeth were extracted and histologic sections were prepared. Histologic sections were observed by an oral pathologist. The sections were observed from the view of inflammation, hyperemia, necrosis, vitality, calcification and dentinal bridge. Relative frequency of histopathologic outcomes of dental pulp was analyzed and reported. Mean differences of four groups were analyzed by Kruskal-Wallis and Mann-Whitney tests. Results: Relative frequency of inflammation of FC, MTA, HA and BAG groups were 29.2, 25.9, 42.4 and 29.2 percent, respectively. Relative frequency of hyperemia of FC, MTA, HA and BAG groups were 75.1, 63.6, 59.2, 81.6 and 62.5 percent, respectively. Relative frequency of necrosis of FC, MTA, HA and BAG groups were 29.2, 11.1, 12.1, 0 percent, respectively. Relative frequency of calcification of FC, MTA, HA and BAG groups were 75, 51.9, 69.7 and 54.8 percent, respectively. Relative frequency of dentinal bridge of FC, MTA, HA and BAG groups were 29.2, 70.4, 30.3 and 29.2 percent, respectively. Conclusion: Histopathologic reactions of dental pulp to four materials (MTA, HA, BAG, FC) were different. Histopathologic outcomes of MTA was more proper than those of BAG, HA and FC. Considering these findings, use of mineral trioxide aggregate and bioactive glass in pulpotomy is more favorable than using Formocresol and Hydroxyapatite. Key words: Animal study, Bio Active Glass (BAG), Calcification, Dental pulp, Dentinal bridge, Formocresol (FC), Histopathologic Response, Hydroxyapatite, Necrosis, Mineral Trioxide Aggregate (MTA), Pulpotomy. Received: August 2006 Accepted: March 2007

Introduction

Primary tooth pulpotomy procedures are based on the rationale that radicular pulp tissue is healthy or is capable of healing after amputation of the af-

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fected pulp. Ideal pulpotomy procedure after extirpated coronal pulp should be bactericidal, harmless to pulp and surrounding structures, promote

* Associate Professor, Department of Pedodontics and Torabinejad Dental Research Center, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran. ** Assistant Professor, Department of Oral Pathologist and Torabinejad Dental Research Center, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran. *** Assistant Professor, Pedodontics, Hamadan Dental School, Hamadan, Iran. Correspondence to: E. Jabbarifar, Department of Pedodontics, School of Dentistry, Isfahan University of Medical sciences, Isfahan, Iran. E-mail: Jabarifar@dnt.mui.ac.ir

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healing of the remaining pulp, not interfere with the physiologic root resorption in primary dentition, has no systemic distribution and hazard biologic and immunologic effects and has proper histologic and radiographic and clinical outcomes 1,2. The most commonly used pulpotomy agent is formaldehyde derivates 3. Calcium hydroxide with alkalinity exposed after one month leads to superficial necrosis of pulp zone, new area of coarse fibrous tissue likened to a primitive bone layer, odontoblastic like fibrous tissue zone, calcific bridge zone, vital and normal pulp tissue free of inflammatory cells zone 4. Glutaraldehyde dialdehyde (GA) has received attention as fixative and potential substitute in primary tooth pulpotomy. The superiority and advantages of GA vs. Formocresol as a pulpotomy agent is attributed to the irreversibility of histologic outcomes. Larger glutaraldehyde than formocresol molecules, less penetration to adjacent tissue and delayed distribution, require a longtime exposed for efficiency task than formocresol 5,6. Ferric sulfate more recently has been devoted to investigating its effectiveness as pulpotomy agent in primary dentition. Ferric sulfate (Astringedent 15.5 percent) agglutinates blood proteins and controls hemorrhage without clot formation 7. Tricalcium phosphate, pure calcium hydroxyl appatite, cholorhexidine gluconate (2%) and distilled water have been studied as pulp therapy procedure in animal and human case, but these materials have not been approved as satisfactory as calcium hydroxide and formocresol 7,8. Findings from pulpotomy experiments using frozen-dried bone, chlorhexidine 2%, feracrylum, calcium phosphate ceramic, bone morphogenic protein type 4, 7, platelet protein derivates, collagen, Portland cement, Argon CO2 laser, electrosurgery, powdered bovine dentin, Bio Active Glass and other biologic agents have been tested and the preliminary results were promising 9. More recently better histologic, radiographic and clinical outcomes have been obtained with white and gray mineral trioxide aggregate. Pulp canal obliteration has been detected in 91% of samples 10. Histologic, radiographic, clinical outcomes after various pulpotomy methods are attributed to a- previous pulpal health status, bsystemic condition of patient, c- biocompatibility and biophysical and immunologic properties of dressing material, d- different methodology and design studies, e- safety and innocency of

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Histopathologic Responses of Dog’s Dental Pulp to …

pulpotomy techniques and period of exposure. Common failure outcomes of pulpotomy procedures in primary dentitions are internal root resorption, dentoalveolar abscess, necrosis, granulation tissue substituted pulp of root canal, early exfoliation, over retained tooth, hypoplastic defect in succedaneous tooth, odontogenic tumor, narrow PDL space, delayed eruption and deflected eruptive route 11. The purpose of this study was comparison of histopathologic responses of dog’s dental pulp to mineral trioxide aggregate, formocresol, hydroxyapatite and Bio Active Glass.

Materials and Methods

Four male dogs of Iranian mixed generation were obtained with the weight of 15-20 kg from animal house of Torabinejad research center of Isfahan dental school. Forty-eight teeth were randomly divided into MTA, FC, HA and BAG groups by throwing toss. The animals were examined, vaccinated and isolated for ten days. General anesthesia with intramuscular injection of 20 mg/kg of ketamine HCL (Alfasan, Netherland) and 12 mg/kg of xylazine (Alfasan, Netherland) was done. An infiltration injection of xylocaine 2% (Darupakhsh, Iran) was used for local anesthesia. Using a number 1 round bur in high-speed handpiece with copious water spray, access cavities were prepared in occlusal and lingual surface and pulp was exposed. Access cavity was completed. Coronal pulp was extirpated and bleeding was stopped with sterile moistened cotton pellets. Then, pulp stumps were covered with purry mixture MTA (Pro-Root MTA Densply, Tulsa USA), Hydroxyapatite (HA, Switzerland), Bioactive glass (Nova Bone, Germany), Tricresolformalin (PD, Brazil), which were prepared according to the manufacture’s direction. Next, the cavities were restored with hard setting zonaline (PD, England) and Amalgam (SDI, Australia) Capsules. After 3 months, vital perfusion fixation was performed with 10% formalin solution (Merk, Germany). Then, animals’ teeth were separated and dissected and placed in formalin solution for 14 days. Following decalcification, specimens were prepared for pathologic processing. Sections were stained with Hematoxylin and Eosin (H & E). Sections were observed under a light microscope (Zeiss, Germany) by an oral pathologist. Assessment of G coronal pulpal responses was performed according to the following criteria 12:

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Histopathologic Responses of Dog’s Dental Pulp to ‌

1) Inflammation cell response scoring: Score 0: No scattered inflammatory cells in the pulp area corresponding to the pulp-exposed characteristic tissue. Score 1: Slight inflammatory cell infiltrate with PMNs or MNLs. Score 2: Moderate inflammatory cell infiltrate involving the coronal third of radicular pulp. Score 3: Severe inflammatory cell infiltrate involving the coronal third of radicular pulp. 2) Hard tissue formation scoring: Score 0: Absent Score 1: Modest hard tissue deposition beneath or lateral to the exposed area. Score 2: Moderate hard tissue deposition beneath or lateral to the exposed area. Score 3: Complete dentin bridge formation beneath to the exposed area. 3) Hyperemia scoring: Score 0: Absent. Score 1: Three blood vessels. Score 2: More than 5 blood vessels. Score 3: More than 8 blood vessels. 4) Necrosis scoring: Score 0: Absent. Score 1: Denaturation of proteins, autolysis. 5) Vitality scoring: Score 0: Abnormal cell, blood vessel, disintegration pulp tissue. Score 1: Natural dental pulp cell, blood vessel, intact pulp tissue. 6) Dentinal bridge scoring: Score 0: Absence of any dentinal structure. Score 1: Atubular and tubular dentinal structure. Frequency and relative frequency of histopathologic variables in MTA, FC, HA and BAG groups were analyzed by SPSS software version 11.5. Relative frequencies of difference in vitality, dentinal bridge, inflammation, calcification, hyperemia, necrosis between MTA, FC, HA and BAG groups were analyzed by Kruskal-Wallis and Man-Whitney tests included in SPSS software.

Results

Based on light microscopic observations, no inflammatory reaction was seen in 70.8 Percent of BAG sections, 57.6 percent of HA samples, 70.8 percent of FC group and 74.1 percent of MTA cases (Table 1). Differences of inflammation reactions in four groups were not significant (Kruskal-Wallis, P=0.32). Relative frequency of inflammation in HA group was more than that in BAG, MTA and FC groups (Mann-Whitney test). Hyperemia was observed in 62.5 percent of BAG sections, 81.6 percent of HA samples, 75.1 percent of FC group and 59.2 percent of MTA cases. Mean differences of hyperemia in MTA, FC, HA and BAG groups were not significant. Pulpal necrosis was observed in 12.1 percent of HA samples, 29.2 percent of FC cases and 11.1 percent of MTA sections. Necrosis among four groups was significantly different (Kruskal-Wallis test, P=0.03) and average of necrosis in FC group was more than that in MTA, HA and BAG samples (Mann-Whitney test). 62.5 percent of the BAG, 57.6 percent of HA, 45.8 percent of FC and 40.7 percent of MTA group were vital in the remaining dental pulp after 3 months follow up. Averages of differences of vitality in four groups were not significant (Kruskal-Wallis test, P>0.05). Calcification loci as diffuse and irregular were observed in 54.2 percent of BAG, 69.7 percent of HA, 75 percent of FC and 51.9 percent of MTA samples. The differences in calcification between four groups were not significant (Kruskal-Wallis test). The formation of dentinal bridge was observed in 29.2 percent of BAG, 30.3 percent of HA, 29.2 percent of FC and 70.4 percent of MTA group. The formation of dentinal bridge in MTA group was more than that in BAG, HA and FC samples (Figure 1, 2). Average difference in formation of dentinal bridge in four groups was significant (Kruskal-Wallis test, P=0.006).

Table 1. Frequency and Relative frequency of histopathologic responses in 1/3 coronal root pulp to MTA, BAG, HA and FC. Pulpotomy

Inflammation

Vitality

Calcification

Hyperemia

Necrosis

Dentinal bridge

MTA

25.9 (7)

40.7 (11)

51.9 (14)

59.2 (16)

11.1 (3)

70.4 (19)

BAG

29.2 (7)

62.5 (15)

54.2 (13)

62.5 (15)

0(0)

29.2 (7)

HA

42.4 (14)

57.6 (10)

69.7 (23)

81.6 (21)

12.1 (4)

30.3 (10)

FC

29.2 (7)

45.8 (11)

75 (18)

75.1 (18)

29.2 (7)

29.2 (7)

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Discussion

A

B Figure 1. Histopathologic responses of dental pulp to MTA (Dentinal bridge formation) D= Dentin, DB= Dentinal bridge, P= Pulp (H&E stain, original magnification, A X40, B X10).

Figure 2. Histopathologic responses of dental pulp to MTA (Calcification foci) Cal= Calcification foci, P= Pulp (H&E stain, original magnification, X10).

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Results of this study showed mild to moderate inflammatory changes in 32.3 percent samples of MTA, FC, HA and BAG groups 13,14. Average inflammatory changes in MTA group were less than that in BAG and equal to that in FC, less than that in HA samples. This may be an expected protective response from pulp to any foreign materials, manipulation and immunohistochemical events. Mild to moderate hyperemia in 27.8 percent samples of MTA, HA, FC and BAG groups was observed. This sign may be a transient outcome of dental pulp to these materials and pulpotomy procedures. 86.1 percent of MTA, FC, HA and BAG groups did not show any tissue necrosis and in 23.9 percent of four groups necrosis occurred. The reasons of necrosis may be attributed to hazardous effect of these materials, immunohistochemical events and reactions of dental pulp due to MTA, FC, HA or BAG. Diffuse irregular calcified loci were observed in 62.3 percent of MTA, FC, HA and BAG groups. This sign may be originated from biohistochemical properties of these materials and their reactions, chemical properties of these materials and reactions of dental pulp to theirs 15,16. In 70.4 percent of MTA samples dentinal bridge in the entrance of root canal pulp were observed. This may be from histochemicophysiological characteristics of dental pulp to these materials. Average dentinal bridge formation in MTA samples was more than those in HA and BAG but, was equal to that in FC groups. Histologic evaluation of pulp specimens after pulp capping is very important. Although it is important to have a calcific bridge under capping materials, making a calcification and dentinal bridge could be a sign of either healing or irritation 17. Authors have shown that healing pulpal reactions are more dependent on the capacity of capping materials to prevent bacterial microleakage rather than the specific properties of the materials themselves. MTA and BAG are insoluble materials and can provide tight seals, which could prevent bacterial access to pulp tissues. MTA and BAG do not appear to deteriorate and disintegrate with time, thus space for microleakage won't develop. The pulp responds favorably to the protection provided by a MTA layer. Nova bone won’t be resorbed, so it remains on the pulp for long time and assists in reliability of under tissues 17,18. Inflammation and hyperemia can be harmless or helpful (depending on severity, acuteness and conDental Research Journal (Vol. 4, No. 2, Autumn-Winter 2007)


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tinuity of reactions). All Specimens have shown mild to severe rates of inflammation. Necrosis, which is the improper outcomes of connective tissues to pulp capping agents, was observed in some samples of four groups.

Conclusion

Histologic reactions of dental pulp to MTA were more favorable than those in HA, FC and BAG groups. Findings of this study showed that MTA and BAG are more proper than Formocresol and hydroxyapatite as pulpotomy agents. Histopathologic reactions of dental pulp to BAG was more favorable than those of FC and HA. Considering the feasibility, cost, time, biocompatibility, long period of study, high success rate, conservative interpretation of the results of animal studies by other researchers, MTA and BAG can be used as alternatives in vital pulpotomy procedures in primary and permanent dentition.

References

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Through Adolescence. 4th ed. Philadelphia: Mosby; 2005: 341-53. 7. Smith NL, Seale NS, Nunn ME. Ferric sulfate pulpotomy in primary molars: a retrospective study. Pediatr Dent 2000; 22(3):192-99. 8. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new rootend filling material. J Endod 1995; 21(7):349-53. 9. Ford TR, Torabinejad M, Abedi HR, Bakland LK, Kariyawasam SP. Using mineral trioxide aggregate as a pulp-capping material. J Am Dent Assoc 1996; 127(10):1491-494. 10. Srinivasan V, Patchett CL, Waterhouse PJ. Is there life after Buckley's Formocresol? Part I -- a narrative review of alternative interventions and materials. Int J Paediatr Dent 2006; 16(2):117-27. 11. Patchett CL, Srinivasan V, Waterhouse PJ. Is there life after Buckley's formocresol? Part II - Development of a protocol for the management of extensive caries in the primary molar. Int J Paediatr Dent 2006; 16(3):199-206. 12. Salako N, Joseph B, Ritwik P, Salonen J, John P, Junaid TA. Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar. Dent Traumatol 2003; 19(6):314-20. 13. Jabbarifar E, Khademi AA, Ghasemi D. Clinical and radiographic evaluation ferricsulfate pulpotomy with Formocresol in primary molar teeth. J Res Med Sci 2001; 9:60-3. 14. Eskandarzadeh A, Parirokh M, Eslami S, Asgary S. A comparative study between MTA and Calcium hydroxide as pulp capping agents in dog's teeth. Dent Res J 2005; 2:88-97. 15. Norton MR, Wilson J. Dental implants placed in extraction sites implanted with bioactive glass: human histology and clinical outcome. Int J Oral Maxillofac Implants 2002; 17(2):249-257. 16. Stoor P, Soderling E, Salonen JI. Antibacterial effects of a bioactive glass paste on oral microorganisms. Acta Odontol Scand 1998; 56(3):161-165. 17. Shoji S, Nakamura M, Horiuchi H. Histopathological changes in dental pulps irradiated by CO2 laser: a preliminary report on laser pulpotomy. J Endod 1985; 11(9):379-384. 18. American Academy of Pediatric Dentistry. Guidelines. Pediatr Dent 1997; 19(7):28-85.

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