Immunolocalization of dentin matrix protein-1 in human primary teeth treated with different pulp capping materials dia C. T. Marques,1 Ana Paula Fernandes,1 Camila O. Rodini,2 Natalino Lourenc¸o Neto,1 Na 3 Vivien T. Sakai, Ruy Cesar C. Abdo,1 Maria Aparecida A. M. Machado,1 Carlos F. Santos,2 Thais M. Oliveira1 1
~o Paulo, Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of Sa ~o Paulo, Brazil Bauru, Sa 2 ~o Paulo, Bauru, Sa ~o Paulo, Brazil Department of Biological Sciences, Bauru School of Dentistry, University of Sa 3 Department of Clinics and Surgery, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil Received 1 July 2014; revised 21 November 2014; accepted 9 January 2015 Published online 10 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33379 Abstract: The aim of this study was to evaluate the immunolocalization of dentin matrix protein (DMP)-1 in human primary teeth treated with different pulp capping materials. Twenty-five primary molars were divided into the following groups: formocresol (FC), calcium hydroxide (CH), mineral trioxide aggregate (MTA), corticosteroid/antibiotic solution 1 CH (O 1 CH), and Portland cement (PC), and all received conventional pulpotomy treatment. The teeth at the regular exfoliation period were extracted for histological analysis and immunolocalization of DMP-1. Statistical analysis was performed using the v2 test (p < 0.05). Histological analysis revealed statistically significant differences in the comparison among the groups through the use of a score system regarding the presence of hard tissue barrier, odon-
toblastic layer, and internal resorption, but not regarding pulp calcification. Immunohistochemical analysis showed immunostaining for DMP-1 in groups CH, MTA, O 1 CH, and PC. Internal resorption was observed in the groups FC and CH. MTA and PC showed pulp repair without inflammation and with the presence of hard tissue barrier. DMP-1 immunostaining was higher for MTA and PC, confirming the repC 2015 arative and bioinductive capacity of these materials. V Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 165–169, 2016.
Key Words: immunohistochemistry, pulpotomy, deciduous tooth, biomaterials availability
How to cite this article: Lourenc¸o Neto N, Marques NCT, Fernandes AP, Rodini CO, Sakai VT, Abdo RCC, Machado MAAM, Santos CF, Oliveira TM. 2016. Immunolocalization of dentin matrix protein-1 in human primary teeth treated with different pulp capping materials. J Biomed Mater Res Part B 2016:104B:165–169.
INTRODUCTION
Dentin matrix protein (DMP)-1 is a highly phosphorylated acidic noncollagenous protein present in the mineralized dentin matrix localized in several soft tissues and is involved in the mineralization of hard dental tissues, but its role is still unclear.1,2 Some studies describe that DMP-1 regulates the mineralization of dentin through the nucleation of hydroxyapatite crystals and also induces the differentiation of dental pulp cells into odontoblast-like cells.3 Animal model studies show the possible role of DMP-1 in amputated pulps, which exhibited a newly formed dental bridge with positive DMP-1 immunostaining.4 Pulpotomy is the treatment to remove the infected and inflamed coronal pulp, preserving the vital pulp tissue and retaining the functional tooth until its exfoliation.5–7 In the studies on pulpotomy of primary teeth, the lack of clinical signs and symptoms of pulp degeneration and the absence of radiographic evidences of periapical or inter-radicular lesions
are reasonable clinical and radiographic parameters that have been evaluated to confirm the success of the capping materials.5,8,9 However, only the histological examination allows the assessment of the cellular organization and the structure of mineralized tissue that is produced in response to these materials. There are few histological and immunohistochemistry studies on human pulp tissue and its reaction after pulpotomy, some of which have shown re-establishment of normal pulp tissue and hard tissue barrier formation after pulpotomy using biocompatible materials.6,10,11 The aim of this study was to evaluate the immunolocalization of DMP-1 in human primary teeth treated with different pulp capping materials. MATERIAL AND METHODS
Participants The Institutional Review Board approved the protocol of the study (process #121/2009) regarding ethical aspects.
Correspondence to: T. M. Oliveira; e-mail: [email protected] ~o Paulo Research Foundation (FAPESP grant); contract grant number: 2009/11284-4 (to T. M. O.) Contract grant sponsor: Sa
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During the pretreatment screening period, the parents or guardians of the children received detailed information concerning the procedures involved in the study, and signed informed consent forms. The criteria for selection of the teeth to be included in the study were as follows: children aged 7–9 years, mandibular primary molar first and second teeth compromised by deep caries cavities with vital pulp; absence of history of spontaneous pain; no clinical or radiographic evidence of pulp degeneration, such as excessive bleeding from the root canal, fistula or abscess, internal or external root resorption, inter-radicular and/or furcal bone destruction, and the possibility of proper restoration of the teeth. Exclusion criteria included the presence of systemic pathology and history of allergic reaction to latex (used in the rubber dam isolation), local anesthetics, or to the constituents of the tested pulp dressing agents. Sample size The sample size was calculated to detect a difference of 75% points, assuming that 100% of success would be obtained in the controls based in a previous study.12 Considering a significance level of 5% and power of 80%, five teeth per group would be necessary. Clinical procedures Twenty-five children participated in the study. Each child had one mandibular primary molar requiring pulpotomy. The teeth were assigned by a random number producing system (computerized random numbers) into the following groups: diluted formocresol (FC) group, calcium hydroxide (CH) group, mineral trioxide aggregate (MTA) group, CH preceded by corticosteroid/antibiotic solution (O 1 CH) group, and Portland cement (PC) group. In all groups, the coronal pulp amputation technique was performed in one single session. After local anesthesia with 4% articaine with 1:100,000 epinephrine and rubber dam isolation, caries removal was accomplished with the aid of a handpiece with a round bur. The opening of the pulp chambers was conducted with high speed and round bur (#1014, #1015, KG Sorensen, Sao Paulo, SP, Brazil) under water spray. Complete coronal pulp tissue was removed manually with an excavator, followed by irrigation with saline solution in order to clear off the debris. The wound surface was continuously irrigated with saline solution until bleeding ceased. Hemorrhage control was achieved by placing a dry sterile cotton pellet on the radicular pulp stumps under slight pressure and waiting 5 min for primary homeostasis. In FC group, a sterile cotton pellet dampened with diluted FC (1:5 Buckley’s solution; Biodin^amica Quımica e Farmac^eutica, Ibipor~a, PR, Brazil) was placed on the amputated pulp and removed after 5 min, and the pulp stumps were covered with zinc oxide–eugenol paste. In CH group, the canal orifices were dressed with dry powdered CH (Biodin^amica Quımica e Farmac^eutica) with the aid of a sterile amalgam carrier. In MTA group, the amputated pulp stumps and chamber floor ^ were covered with MTA (Angelus, Londrina, PR, Brazil). In O 1 CH group, a cotton pellet with two drops of the corticosteroid/antibiotic solution (Polymyxin B sulfate 10,000 IU,
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neomycin sulfate 5 mg, and hydrocortisone 10 mg; Otosporin, Farmoquımica S/A, Rio de Janeiro, RJ, Brazil) was placed on the amputated pulp and removed after 5 min, and then the powder of CH was placed into the pulp chamber as in the CH group. In PC group, the amputated pulp stumps and chamber floor were covered with PC (Votorantim-Cimentos, S~ao Paulo, SP, Brazil). All groups received a layer of reinforced zinc oxide-eugenol (IRM, Dentsply, Petr opolis, RJ, Brazil) prior to restoration with resin-modified glass ionomer cement (Vitremer, 3M ESPE, S~ao Paulo, SP, Brazil).6,13,14 Periodic follow-up examinations were carried out at 3month intervals after the end of the treatment until the teeth achieved the regular exfoliation period to be extracted for histological and pulp response analyses.15 Histological analysis Pulpotomized teeth were extracted at the regular exfoliation period.15 Mean tooth extraction time was 12.63 months. Following extraction, the teeth were immediately fixed in 10% neutral buffered formalin for 24 h, decalcified in 4% neutral EDTA for 45–60 days, and processed for histological analysis.16,17 Five-micrometer-thick serial sections were cut longitudinally, stained with hematoxylin and eosin, and assessed under a light microscope. The morphology of the dentin–pulp complex was analyzed from three-step serial sections and graded based on the criteria for methodology as previously described by Caicedo et al.18 with some modifications. The parameters were individually analyzed regarding the presence (score 1) or absence (score 0), quantity, and/or concentration of the observed structures. Immunohistochemical analysis Tissue sections were digested with Proteinase K (DAKO North America, Carpinteria, CA) for 25 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution in methanol (0.01M) (Easy Path (EP) 12-20522, S~ao Paulo, SP, Brazil) for 10 min. Sections were incubated with polyclonal rabbit anti-human DMP-1 antibody (HPA037465, SigmaAldrich, St. Louis, MO) at 1.3 mg/mL (1:150) dilution for 30 min. Sections were rinsed with phosphate-buffered saline and incubated with a horseradish peroxidase-conjugated secondary antibody (Advance, DAKO North America) for 30 min. Color was developed with 3,3?-diaminobenzidine tetrahydrochloride solution, and slides were counterstained with Lillie– Mayer’s hematoxylin for 60 s. Descriptive analysis of dentin– pulp complex was carried out under light microscope. Blinding Two blinded and previously calibrated investigators performed the histopathological analysis (Kappa test for interexaminer reproducibility was 0.94). Statistical methods Data were analyzed using the v2 test followed by a multiple comparison test in order to determine significant differences to nominal-qualitative variables (hard tissue barrier, odontoblastic layer, pulp calcification, and internal resorption). Statistical significance was established at 5%.
IMMUNOLOCALIZATION OF DMP-1 IN HUMAN PRIMARY MOLARS
ORIGINAL RESEARCH REPORT
TABLE I. Comparison of the Histologic Parameters Among the Teeth Treated With Different Capping Materials. Statistical analysis was performed using the v2 test followed by a multiple comparison test to nominal-qualitative variables.
Dentin bridge Odontoblastic layer Pulp calcification Internal resorption
FC
CH
A
B
MTA B
O 1 CH B
PC B
p
0 0A
1 2B
1 1.75B
1 1.75B
1 1.75B
0.4A
0.66A
0.5A
1A
1A
>0.05
0.8A
0.66A
0B
0B
0B
0.01585*
0.00079* 0.01333*
*Statistically significant difference (p < 0.05) Same letters indicate no statistically significant difference among the groups (horizontal line)
RESULTS
Histological results The groups did not demonstrate any type of clinical or radiographic pathological conditions during the observed period. The histopathological analysis showed that the remaining radicular pulp of all teeth remained vital, irrespective of the capping material used. The comparison among the groups through the use of a score system revealed statistically significant differences regarding the presence of hard tissue barrier, odontoblastic layer, and internal resorption, but not regarding pulp calcification (p > 0.05; Table I). In the FC group, most of the cases presented moderate inflammatory infiltrate without granulation tissue and regular vascularization in a loose connective tissue. A superficial necrotic layer surrounded by a band of inflammatory cells was observed in some cases. Internal resorption was a common finding at the entrance of the root canal in contact with the capping material. None of the cases showed either
dentin bridge formation or odontoblastic layer, which was statistically significantly different from the other groups studied. Pulp calcification was detected in two teeth [Figure 1(A,B)]. CH group showed variable inflammatory infiltrate according to the concentration of mononuclear cells observed in a dense connective tissue. In all cases, formation of hard tissue barrier underlined by an odontoblastic layer and a regular vascularization was observed. Pulp calcification and internal resorption occurred in some cases [Figure 1(C,D)]. Both FC and CH groups showed statistically significant differences from the other groups regarding the presence of internal resorption, because in MTA, PC, and O 1 CH groups this histological finding was not observed. MTA group exhibited absence of both pulp inflammation and internal resorption. Dense collagen fibers, pulp calcification, and hard tissue barrier formation underlined by an odontoblastic layer were common findings [Figure 1(E,F)]. Teeth of O 1 CH group presented either loose or dense connective tissue, hard tissue barrier formation with an organized odontoblastic layer, pulp calcification, and absence of inflammatory infiltrate and internal resorption [Figure 1(G,H)]. PC group showed absence of inflammation with loose connective tissue and great amount of blood vessels in most of the cases. Additionally, hard tissue barrier formation with an organized odontoblastic layer, pulp calcification, and absence of internal root resorption were verified [Figure 1(I,J)]. Immunohistochemistry results In FC group, DMP-1 immunostaining was confined eventually on the lateral walls of root canal. Cells stuck inside of the calcified matrix were also positive for DMP-1 [Figure 2(A,B)]. CH and MTA groups presented a marked immunostaining of the odontoblasts at different stages of organization of the odontoblastic layer and of the odontoblastic
FIGURE 1. (A, B) Loose connective tissue with moderate inflammatory infiltrate (ii), internal resorption (IR), and presence of superficial necrosis layer (SN) (310 and 340 magnifications). (C, E, G, I) Hard tissue barrier formation (HTB) (310 magnification). (D, F, H, J) Dense connective tissue and lack of inflammatory infiltrate (CT); presence of odontoblastic layer (OL) (340 magnification).
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FIGURE 2. (A) Lack of barrier; DMP-1 immunostaining restricted to the odontoblastic processes of the root wall (ii) (310 magnification). (B) DMP-1 immunostaining of cells within calcified matrix (iii) (340 magnification). (C, E, G, I) DMP-1 immunostaining of odontoblastic processes within the hard tissue barrier (ii) (310 magnification). (D, F, H, J) DMP-1 immunostaining of odontoblastic layer (OL) and odontoblastic processes within the hard tissue barrier (ii) (340 magnification).
processes toward the hard tissue barrier [Figure 2(C,D)]. The MTA group showed that the pattern of DMP-1 expression was similar in the tubules of both the radicular dentin and the hard tissue barrier formation [Figure 2(E,F)]. O 1 CH group showed DMP-1 immunostaining of the organized odontoblast layer just below and following the hard tissue barrier. At a greater magnification, a differential expression of DMP-1 could be noted at the hard tissue barrier and the radicular dentin [Figure 2(G,H)]. In contrast, the hard tissue barrier formation and the radicular dentin of all teeth from PC group displayed a similar DMP-1 expression in a well-organized tissue [Figure 2(I,J)].
DISCUSSION
Although many studies have reported high clinical and radiographic success rates of the pulp capping materials used in this study,7–9,11,13,14,19,20 few studies involving histological and immunohistochemistry evaluations have shown the state of the remaining pulp tissue after the contact with these capping materials. The efficacy of the pulp capping materials employed in pulpotomy is an important point to consider for the technique success, whereas histological features such as tissue vitality, inflammation, dentin resorption, and dentin bridge formation are desirable points for a biocompatible material.6,18,21 The presence of internal resorption in some of the samples suggests that a silent chronic inflammation may have developed and remained undiagnosed, thus resulting in the activity of the odontoclasts. The factors that have been related to this alteration are: inadequate control of hemorrhage during the procedure, contact of the material with the blood clot, improper restorations, lack of sealing, which enables bacterial leakage and pulp inflammation, and even the
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irritating effect of the pulp capping materials. However, the exact mechanism accounting for this process remains unkown.13,18,19,22–25 In this study, the hard tissue barrier formation was radiographically and histologically detected in the CH, O 1 CH, MTA, and PC groups, denoting that the biocompatible materials may have induced pulp tissue repair.18,21,26 During the pulp repair after pulpotomy, hard tissue barrier formation may occur, which is suggestive of a successful procedure.6,13,14 The rationale for the aforementioned concept is that the pulp capping material was not irritating to the remaining tissue, decreasing the inflammation, collaborating with the repair process, and making viable the deposition of the mineralized material by the odontoblasts.6,12–14,20,21,27 This result is in agreement with other studies in the recent literature.6,10,12,15,20,21 The formation of the hard tissue barrier and the presence of an odontoblastic layer were not observed in the FC group, suggesting the lack of biocompatibility and inductive property of FC.20 DMP-1 expression was evaluated in order to assess the viability of the odontoblasts through the secretion and mineralization of a dentin matrix.28,29 The “dentin bridges” formed in teeth of CH, MTA, O 1 CH, and PC groups as well as the cells in close contact with them were positively stained with DMP-1 antibody, suggesting the odontoblast differentiation in the remaining pulp just below the hard tissue barrier.1,29,30 Interestingly, the immunostaining was more evident in the hard tissue barrier of MTA and PC groups than in that of the other groups. According to the studies on animal models,2,31 this feature would suggest that MTA and PC are less irritating to pulp tissues because little or no inflammation is described. Besides, these materials are capable of cell induction proved by DMP-1 immunostaining observed in the hard tissue barrier and in the
IMMUNOLOCALIZATION OF DMP-1 IN HUMAN PRIMARY MOLARS
ORIGINAL RESEARCH REPORT
cells just below it, confirming the secretion of reparative dentin by odontoblast-like cells. In addition, the similar DMP-1 expression in the tubules of the hard tissue barrier and in the radicular dentin suggests the uniformity of dentin formation in both areas and the clear bioinductive ability of MTA and PC, resulting in the differentiation of odontoblast-like and odontoblast cells. These findings are in agreement with other studies,28–30,32 which verified the relationship between DMP-1 expression and dentin secretion in cell culture models. This study showed that despite the clinical and radiographic outcomes, the histological and immunohistochemistry analyses were suitable tools to determine the pulp condition after pulpotomy with different capping materials. The assessment of DMP-1 expression provided important information on the odontoblastic differentiation and hard tissue barrier formation. CONCLUSION
MTA and PC showed pulp repair without inflammation and with the presence of hard tissue barrier. DMP-1 immunostaining was higher for MTA and PC, confirming the reparative and bioinductive capacity of these materials. ACKNOWLEDGMENTS
The authors acknowledge all the volunteers. They thank Daniele Santi Ceolin and Patrıcia De Sa Mortagua Gemino for excellent laboratorial assistance. REFERENCES 1. Almushayt A, Narayanan K, Zaki AE, George A. Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Therapy 2006; 13:611–620. 2. Aguiar MC, Arana-Chavez VE. Immunocytochemical detection of dentine matrix protein 1 in experimentally induced reactionary and reparative dentine in rat incisors. Arch Oral Biol 2010; 55:210– 214. 3. Abd-Elmeguid A, Yu DC, Kline LW, Moqbel R, Vliagoftis H. Dentin matrix protein-1 activates dental pulp fibroblasts. J Endod 2012; 38:75–80. 4. Ishimatsu H, Kitamura C, Morotomi T, Tabata Y, Nishihara T, Chen KK, Terashita M. Formation of dentinal bridge on surface of regenerated dental pulp in dentin defects by controlled release of fibroblast growth factor-2 from gelatin hydrogels. J Endod 2009; 35:858–865. 5. Cohenca N, Paranjipe A, Berg J. Vital pulp therapy. Dent Clin North Am 2013; 57: 59–73. 6. Oliveira TM, Moretti ABS, Sakai VT, Lourenc¸ o, Neto N, Santos CF, Machado MAAM, Abdo RCC. Clinical, radiographic and histologic analysis of the effects of pulp capping materials used in pulpotomies of human primary teeth. Eur Arch Paediatr Dent 2013; 14: 65–71. 7. Lin PY, Chen HS, Wang YH, Tu YK. Primary molar pulpotomy: A systematic review and network meta-analysis. J Dent 2014; 42: 1060–1077. 8. Huth KC, Hajek-Al-Khatar N, Wolf P, Ilie N, Hickel R, Paschos E. Long-term effectiveness of four pulpotomy techniques: 3-year randomised controlled trial. Clin Oral Investig 2012; 16:1243–1250. 9. Shirvani A, Hassanizadeh R, Asgary S. Mineral trioxide aggregate vs. calcium hydroxide in primary molar pulpotomy: A systematic review. IEJ 2014; 9:83–88. 10. Accorinte ML, Loguercio AD, Reis A, Carneiro E, Grande RH, Murata SS, Holland R. Response of human dental pulp capped with MTA and calcium hydroxide powder. Oper Dent 2008; 33: 488–495.
11. Haghgoo R, Abbasi F. A histopathological comparison of pulpotomy with sodium hypochlorite and formocresol. Iran Endod J 2012; 7:60–62. 12. Agamy HA, Bakry NS, Mounir MM. Comparison of mineral trioxide aggregate and formocresol as pulp-capping agents in pulpotomized primary teeth. Pediatr Dent 2004; 26:302–309. 13. Moretti AB, Sakai VT, Oliveira TM, Fornetti AP, Santos CF, Machado MA, Abdo RC. The effectiveness of MTA, calcium hydroxide and formocresol for pulpotomies in primary teeth. Int Endod J 2008; 41:547–555. 14. Sakai VT, Moretti AB, Oliveira TM, Fornetti AP, Santos CF, Machado MA, Abdo RC. Pulpotomy of human primary molars with MTA and Portland cement: A randomised controlled trial. Br Dent J 2009; 207:128–132. lu I, Sari S, Gu € nhan O. The effect of sodium hypo15. Tunc¸ ES, Sarog chlorite application on the success of calcium hydroxide pulpotomy in primary teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102: 22–26. 16. Freitas P, Novaretti CP, Rodini CO, Batista AC, Lara VS. Mast cells and lymphocyte subsets in pulps from health and carious teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 103:95–102. 17. Sanjai K, Kumarswamy J, Patil A, Papaiah L, Jayaram S, Krishnan L. Evaluation and comparison of decalcification agents on the human teeth. J Oral Maxillofac Pathol 2012; 16:222–227. 18. Caicedo R, Abbott P, Alongi D, Alarcon M. Clinical, radiographic and histological analysis of the effects of mineral trioxide aggregate used in direct pulp capping and pulpotomies of primary teeth. Aust Dent J 2006; 51:297–305. 19. Godhi B, Sood PB, Sharma A. Effects of mineral trioxide aggregate and formocresol on vital pulp after pulpotomy of primary molars: An in vivo study. Contemp Clin Dent 2011; 2:296–301. 20. Srinivasan D, Jayanthi M. Comparative evaluation of formocresol and mineral trioxide aggregate as pulpotomy agents in deciduous teeth. Indian J Dent Res 2011; 22:385–390. 21. Waterhouse PJ, Nunn JH, Whitworth JM, Soames JV. Primary molars pulp therapy: Histological evaluation of failure. Int J Paediatr Dent 2000; 10:313–321. 22. Sonmez D, Duruturk L. Ca(OH)2 pulpotomy in primary teeth. Part I: Internal resorption as complication following pulpotomy. Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106:94–98. 23. Sonmez D, Sari S, Cetinbas¸ T. A Comparison of four pulpotomy techniques in primary molars: A long-term follow-up. J Endod 2008; 34:950–955. lu H, Baygın O. Clinical 24. Alac¸am A, Odabas ME, T€ uz€ uner T, Silleliog and radiographic outcomes of calcium hydroxide and formocresol pulpotomies performed by dental students. Med Oral Pathol Oral Radiol Endod 2009; 108:127–133. lu O, Is¸ik B. A new haemostatic 25. Odabas¸ ME, Cinar C, Tulunog agent’s effect on the success of calcium hydroxide pulpotomy in primary molars. Pediatr Dent 2011; 33:529–534. lu H, Deveci C. Clinical and radio26. Odabas¸ ME, Alac¸am A, Silleliog graphic success rates of mineral trioxide aggregate and ferric sulphate pulpotomies performed by dental students. Eur J Paeditr Dent 2012; 13:118–122. 27. Peng L, Ye L, Guo X, Tan H, Zhou X, Wang C, Li R. Evaluation of formocresol versus ferric sulphate primary molar pulpotomy: A systematic review and meta-analysis. Int Endod J 2007; 40:751–757. 28. Martinez EF, da Silva LAH, Furuse C, Ara ujo NS, de Ara ujo VC. Dentin matrix protein 1 (DMP-1) expression in developing human teeth. Braz Dent J 2009; 20:365–369. 29. Suzuki S, Haruyama N, Nishimura F, Kulkarni AB. Dentin sialoprotein and dentin matrix protein-1: Two highly phosphorylated proteins in mineralized tissues. Arch Oral Biol 2012; 57:1165–1175 30. Beniah E, Deshpande AS, Fang PA, Lieb NS, Zhang X, Sfeir CS. Possible role of DMP-1 in dentin mineralization. J Struct Biol 2011; 174:100–106. 31. Fang P, Wang X, Zhang L, Yuan G, Chen Z, Zhang Q. Immunohistochemical localization of LIM mineralization protein 1 during mouse molar development. J Mol Histol 2010; 41:199–203. 32. Deshpande AS, Fang PA, Zhang X, Jayaraman T, Sfeir C, Beniash E. Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization. Biomacromolecules 2011; 12:2933– 2945.
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~o Paulo, Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of Sa ~o Paulo, Brazil Bauru, Sa 2 ~o Paulo, Bauru, Sa ~o Paulo, Brazil Department of Biological Sciences, Bauru School of Dentistry, University of Sa 3 Department of Clinics and Surgery, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil Received 1 July 2014; revised 21 November 2014; accepted 9 January 2015 Published online 10 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33379 Abstract: The aim of this study was to evaluate the immunolocalization of dentin matrix protein (DMP)-1 in human primary teeth treated with different pulp capping materials. Twenty-five primary molars were divided into the following groups: formocresol (FC), calcium hydroxide (CH), mineral trioxide aggregate (MTA), corticosteroid/antibiotic solution 1 CH (O 1 CH), and Portland cement (PC), and all received conventional pulpotomy treatment. The teeth at the regular exfoliation period were extracted for histological analysis and immunolocalization of DMP-1. Statistical analysis was performed using the v2 test (p < 0.05). Histological analysis revealed statistically significant differences in the comparison among the groups through the use of a score system regarding the presence of hard tissue barrier, odon-
toblastic layer, and internal resorption, but not regarding pulp calcification. Immunohistochemical analysis showed immunostaining for DMP-1 in groups CH, MTA, O 1 CH, and PC. Internal resorption was observed in the groups FC and CH. MTA and PC showed pulp repair without inflammation and with the presence of hard tissue barrier. DMP-1 immunostaining was higher for MTA and PC, confirming the repC 2015 arative and bioinductive capacity of these materials. V Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 165–169, 2016.
Key Words: immunohistochemistry, pulpotomy, deciduous tooth, biomaterials availability
How to cite this article: Lourenc¸o Neto N, Marques NCT, Fernandes AP, Rodini CO, Sakai VT, Abdo RCC, Machado MAAM, Santos CF, Oliveira TM. 2016. Immunolocalization of dentin matrix protein-1 in human primary teeth treated with different pulp capping materials. J Biomed Mater Res Part B 2016:104B:165–169.
INTRODUCTION
Dentin matrix protein (DMP)-1 is a highly phosphorylated acidic noncollagenous protein present in the mineralized dentin matrix localized in several soft tissues and is involved in the mineralization of hard dental tissues, but its role is still unclear.1,2 Some studies describe that DMP-1 regulates the mineralization of dentin through the nucleation of hydroxyapatite crystals and also induces the differentiation of dental pulp cells into odontoblast-like cells.3 Animal model studies show the possible role of DMP-1 in amputated pulps, which exhibited a newly formed dental bridge with positive DMP-1 immunostaining.4 Pulpotomy is the treatment to remove the infected and inflamed coronal pulp, preserving the vital pulp tissue and retaining the functional tooth until its exfoliation.5–7 In the studies on pulpotomy of primary teeth, the lack of clinical signs and symptoms of pulp degeneration and the absence of radiographic evidences of periapical or inter-radicular lesions
are reasonable clinical and radiographic parameters that have been evaluated to confirm the success of the capping materials.5,8,9 However, only the histological examination allows the assessment of the cellular organization and the structure of mineralized tissue that is produced in response to these materials. There are few histological and immunohistochemistry studies on human pulp tissue and its reaction after pulpotomy, some of which have shown re-establishment of normal pulp tissue and hard tissue barrier formation after pulpotomy using biocompatible materials.6,10,11 The aim of this study was to evaluate the immunolocalization of DMP-1 in human primary teeth treated with different pulp capping materials. MATERIAL AND METHODS
Participants The Institutional Review Board approved the protocol of the study (process #121/2009) regarding ethical aspects.
Correspondence to: T. M. Oliveira; e-mail: [email protected] ~o Paulo Research Foundation (FAPESP grant); contract grant number: 2009/11284-4 (to T. M. O.) Contract grant sponsor: Sa
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During the pretreatment screening period, the parents or guardians of the children received detailed information concerning the procedures involved in the study, and signed informed consent forms. The criteria for selection of the teeth to be included in the study were as follows: children aged 7–9 years, mandibular primary molar first and second teeth compromised by deep caries cavities with vital pulp; absence of history of spontaneous pain; no clinical or radiographic evidence of pulp degeneration, such as excessive bleeding from the root canal, fistula or abscess, internal or external root resorption, inter-radicular and/or furcal bone destruction, and the possibility of proper restoration of the teeth. Exclusion criteria included the presence of systemic pathology and history of allergic reaction to latex (used in the rubber dam isolation), local anesthetics, or to the constituents of the tested pulp dressing agents. Sample size The sample size was calculated to detect a difference of 75% points, assuming that 100% of success would be obtained in the controls based in a previous study.12 Considering a significance level of 5% and power of 80%, five teeth per group would be necessary. Clinical procedures Twenty-five children participated in the study. Each child had one mandibular primary molar requiring pulpotomy. The teeth were assigned by a random number producing system (computerized random numbers) into the following groups: diluted formocresol (FC) group, calcium hydroxide (CH) group, mineral trioxide aggregate (MTA) group, CH preceded by corticosteroid/antibiotic solution (O 1 CH) group, and Portland cement (PC) group. In all groups, the coronal pulp amputation technique was performed in one single session. After local anesthesia with 4% articaine with 1:100,000 epinephrine and rubber dam isolation, caries removal was accomplished with the aid of a handpiece with a round bur. The opening of the pulp chambers was conducted with high speed and round bur (#1014, #1015, KG Sorensen, Sao Paulo, SP, Brazil) under water spray. Complete coronal pulp tissue was removed manually with an excavator, followed by irrigation with saline solution in order to clear off the debris. The wound surface was continuously irrigated with saline solution until bleeding ceased. Hemorrhage control was achieved by placing a dry sterile cotton pellet on the radicular pulp stumps under slight pressure and waiting 5 min for primary homeostasis. In FC group, a sterile cotton pellet dampened with diluted FC (1:5 Buckley’s solution; Biodin^amica Quımica e Farmac^eutica, Ibipor~a, PR, Brazil) was placed on the amputated pulp and removed after 5 min, and the pulp stumps were covered with zinc oxide–eugenol paste. In CH group, the canal orifices were dressed with dry powdered CH (Biodin^amica Quımica e Farmac^eutica) with the aid of a sterile amalgam carrier. In MTA group, the amputated pulp stumps and chamber floor ^ were covered with MTA (Angelus, Londrina, PR, Brazil). In O 1 CH group, a cotton pellet with two drops of the corticosteroid/antibiotic solution (Polymyxin B sulfate 10,000 IU,
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neomycin sulfate 5 mg, and hydrocortisone 10 mg; Otosporin, Farmoquımica S/A, Rio de Janeiro, RJ, Brazil) was placed on the amputated pulp and removed after 5 min, and then the powder of CH was placed into the pulp chamber as in the CH group. In PC group, the amputated pulp stumps and chamber floor were covered with PC (Votorantim-Cimentos, S~ao Paulo, SP, Brazil). All groups received a layer of reinforced zinc oxide-eugenol (IRM, Dentsply, Petr opolis, RJ, Brazil) prior to restoration with resin-modified glass ionomer cement (Vitremer, 3M ESPE, S~ao Paulo, SP, Brazil).6,13,14 Periodic follow-up examinations were carried out at 3month intervals after the end of the treatment until the teeth achieved the regular exfoliation period to be extracted for histological and pulp response analyses.15 Histological analysis Pulpotomized teeth were extracted at the regular exfoliation period.15 Mean tooth extraction time was 12.63 months. Following extraction, the teeth were immediately fixed in 10% neutral buffered formalin for 24 h, decalcified in 4% neutral EDTA for 45–60 days, and processed for histological analysis.16,17 Five-micrometer-thick serial sections were cut longitudinally, stained with hematoxylin and eosin, and assessed under a light microscope. The morphology of the dentin–pulp complex was analyzed from three-step serial sections and graded based on the criteria for methodology as previously described by Caicedo et al.18 with some modifications. The parameters were individually analyzed regarding the presence (score 1) or absence (score 0), quantity, and/or concentration of the observed structures. Immunohistochemical analysis Tissue sections were digested with Proteinase K (DAKO North America, Carpinteria, CA) for 25 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution in methanol (0.01M) (Easy Path (EP) 12-20522, S~ao Paulo, SP, Brazil) for 10 min. Sections were incubated with polyclonal rabbit anti-human DMP-1 antibody (HPA037465, SigmaAldrich, St. Louis, MO) at 1.3 mg/mL (1:150) dilution for 30 min. Sections were rinsed with phosphate-buffered saline and incubated with a horseradish peroxidase-conjugated secondary antibody (Advance, DAKO North America) for 30 min. Color was developed with 3,3?-diaminobenzidine tetrahydrochloride solution, and slides were counterstained with Lillie– Mayer’s hematoxylin for 60 s. Descriptive analysis of dentin– pulp complex was carried out under light microscope. Blinding Two blinded and previously calibrated investigators performed the histopathological analysis (Kappa test for interexaminer reproducibility was 0.94). Statistical methods Data were analyzed using the v2 test followed by a multiple comparison test in order to determine significant differences to nominal-qualitative variables (hard tissue barrier, odontoblastic layer, pulp calcification, and internal resorption). Statistical significance was established at 5%.
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ORIGINAL RESEARCH REPORT
TABLE I. Comparison of the Histologic Parameters Among the Teeth Treated With Different Capping Materials. Statistical analysis was performed using the v2 test followed by a multiple comparison test to nominal-qualitative variables.
Dentin bridge Odontoblastic layer Pulp calcification Internal resorption
FC
CH
A
B
MTA B
O 1 CH B
PC B
p
0 0A
1 2B
1 1.75B
1 1.75B
1 1.75B
0.4A
0.66A
0.5A
1A
1A
>0.05
0.8A
0.66A
0B
0B
0B
0.01585*
0.00079* 0.01333*
*Statistically significant difference (p < 0.05) Same letters indicate no statistically significant difference among the groups (horizontal line)
RESULTS
Histological results The groups did not demonstrate any type of clinical or radiographic pathological conditions during the observed period. The histopathological analysis showed that the remaining radicular pulp of all teeth remained vital, irrespective of the capping material used. The comparison among the groups through the use of a score system revealed statistically significant differences regarding the presence of hard tissue barrier, odontoblastic layer, and internal resorption, but not regarding pulp calcification (p > 0.05; Table I). In the FC group, most of the cases presented moderate inflammatory infiltrate without granulation tissue and regular vascularization in a loose connective tissue. A superficial necrotic layer surrounded by a band of inflammatory cells was observed in some cases. Internal resorption was a common finding at the entrance of the root canal in contact with the capping material. None of the cases showed either
dentin bridge formation or odontoblastic layer, which was statistically significantly different from the other groups studied. Pulp calcification was detected in two teeth [Figure 1(A,B)]. CH group showed variable inflammatory infiltrate according to the concentration of mononuclear cells observed in a dense connective tissue. In all cases, formation of hard tissue barrier underlined by an odontoblastic layer and a regular vascularization was observed. Pulp calcification and internal resorption occurred in some cases [Figure 1(C,D)]. Both FC and CH groups showed statistically significant differences from the other groups regarding the presence of internal resorption, because in MTA, PC, and O 1 CH groups this histological finding was not observed. MTA group exhibited absence of both pulp inflammation and internal resorption. Dense collagen fibers, pulp calcification, and hard tissue barrier formation underlined by an odontoblastic layer were common findings [Figure 1(E,F)]. Teeth of O 1 CH group presented either loose or dense connective tissue, hard tissue barrier formation with an organized odontoblastic layer, pulp calcification, and absence of inflammatory infiltrate and internal resorption [Figure 1(G,H)]. PC group showed absence of inflammation with loose connective tissue and great amount of blood vessels in most of the cases. Additionally, hard tissue barrier formation with an organized odontoblastic layer, pulp calcification, and absence of internal root resorption were verified [Figure 1(I,J)]. Immunohistochemistry results In FC group, DMP-1 immunostaining was confined eventually on the lateral walls of root canal. Cells stuck inside of the calcified matrix were also positive for DMP-1 [Figure 2(A,B)]. CH and MTA groups presented a marked immunostaining of the odontoblasts at different stages of organization of the odontoblastic layer and of the odontoblastic
FIGURE 1. (A, B) Loose connective tissue with moderate inflammatory infiltrate (ii), internal resorption (IR), and presence of superficial necrosis layer (SN) (310 and 340 magnifications). (C, E, G, I) Hard tissue barrier formation (HTB) (310 magnification). (D, F, H, J) Dense connective tissue and lack of inflammatory infiltrate (CT); presence of odontoblastic layer (OL) (340 magnification).
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FIGURE 2. (A) Lack of barrier; DMP-1 immunostaining restricted to the odontoblastic processes of the root wall (ii) (310 magnification). (B) DMP-1 immunostaining of cells within calcified matrix (iii) (340 magnification). (C, E, G, I) DMP-1 immunostaining of odontoblastic processes within the hard tissue barrier (ii) (310 magnification). (D, F, H, J) DMP-1 immunostaining of odontoblastic layer (OL) and odontoblastic processes within the hard tissue barrier (ii) (340 magnification).
processes toward the hard tissue barrier [Figure 2(C,D)]. The MTA group showed that the pattern of DMP-1 expression was similar in the tubules of both the radicular dentin and the hard tissue barrier formation [Figure 2(E,F)]. O 1 CH group showed DMP-1 immunostaining of the organized odontoblast layer just below and following the hard tissue barrier. At a greater magnification, a differential expression of DMP-1 could be noted at the hard tissue barrier and the radicular dentin [Figure 2(G,H)]. In contrast, the hard tissue barrier formation and the radicular dentin of all teeth from PC group displayed a similar DMP-1 expression in a well-organized tissue [Figure 2(I,J)].
DISCUSSION
Although many studies have reported high clinical and radiographic success rates of the pulp capping materials used in this study,7–9,11,13,14,19,20 few studies involving histological and immunohistochemistry evaluations have shown the state of the remaining pulp tissue after the contact with these capping materials. The efficacy of the pulp capping materials employed in pulpotomy is an important point to consider for the technique success, whereas histological features such as tissue vitality, inflammation, dentin resorption, and dentin bridge formation are desirable points for a biocompatible material.6,18,21 The presence of internal resorption in some of the samples suggests that a silent chronic inflammation may have developed and remained undiagnosed, thus resulting in the activity of the odontoclasts. The factors that have been related to this alteration are: inadequate control of hemorrhage during the procedure, contact of the material with the blood clot, improper restorations, lack of sealing, which enables bacterial leakage and pulp inflammation, and even the
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irritating effect of the pulp capping materials. However, the exact mechanism accounting for this process remains unkown.13,18,19,22–25 In this study, the hard tissue barrier formation was radiographically and histologically detected in the CH, O 1 CH, MTA, and PC groups, denoting that the biocompatible materials may have induced pulp tissue repair.18,21,26 During the pulp repair after pulpotomy, hard tissue barrier formation may occur, which is suggestive of a successful procedure.6,13,14 The rationale for the aforementioned concept is that the pulp capping material was not irritating to the remaining tissue, decreasing the inflammation, collaborating with the repair process, and making viable the deposition of the mineralized material by the odontoblasts.6,12–14,20,21,27 This result is in agreement with other studies in the recent literature.6,10,12,15,20,21 The formation of the hard tissue barrier and the presence of an odontoblastic layer were not observed in the FC group, suggesting the lack of biocompatibility and inductive property of FC.20 DMP-1 expression was evaluated in order to assess the viability of the odontoblasts through the secretion and mineralization of a dentin matrix.28,29 The “dentin bridges” formed in teeth of CH, MTA, O 1 CH, and PC groups as well as the cells in close contact with them were positively stained with DMP-1 antibody, suggesting the odontoblast differentiation in the remaining pulp just below the hard tissue barrier.1,29,30 Interestingly, the immunostaining was more evident in the hard tissue barrier of MTA and PC groups than in that of the other groups. According to the studies on animal models,2,31 this feature would suggest that MTA and PC are less irritating to pulp tissues because little or no inflammation is described. Besides, these materials are capable of cell induction proved by DMP-1 immunostaining observed in the hard tissue barrier and in the
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ORIGINAL RESEARCH REPORT
cells just below it, confirming the secretion of reparative dentin by odontoblast-like cells. In addition, the similar DMP-1 expression in the tubules of the hard tissue barrier and in the radicular dentin suggests the uniformity of dentin formation in both areas and the clear bioinductive ability of MTA and PC, resulting in the differentiation of odontoblast-like and odontoblast cells. These findings are in agreement with other studies,28–30,32 which verified the relationship between DMP-1 expression and dentin secretion in cell culture models. This study showed that despite the clinical and radiographic outcomes, the histological and immunohistochemistry analyses were suitable tools to determine the pulp condition after pulpotomy with different capping materials. The assessment of DMP-1 expression provided important information on the odontoblastic differentiation and hard tissue barrier formation. CONCLUSION
MTA and PC showed pulp repair without inflammation and with the presence of hard tissue barrier. DMP-1 immunostaining was higher for MTA and PC, confirming the reparative and bioinductive capacity of these materials. ACKNOWLEDGMENTS
The authors acknowledge all the volunteers. They thank Daniele Santi Ceolin and Patrıcia De Sa Mortagua Gemino for excellent laboratorial assistance. REFERENCES 1. Almushayt A, Narayanan K, Zaki AE, George A. Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Therapy 2006; 13:611–620. 2. Aguiar MC, Arana-Chavez VE. Immunocytochemical detection of dentine matrix protein 1 in experimentally induced reactionary and reparative dentine in rat incisors. Arch Oral Biol 2010; 55:210– 214. 3. Abd-Elmeguid A, Yu DC, Kline LW, Moqbel R, Vliagoftis H. Dentin matrix protein-1 activates dental pulp fibroblasts. J Endod 2012; 38:75–80. 4. Ishimatsu H, Kitamura C, Morotomi T, Tabata Y, Nishihara T, Chen KK, Terashita M. Formation of dentinal bridge on surface of regenerated dental pulp in dentin defects by controlled release of fibroblast growth factor-2 from gelatin hydrogels. J Endod 2009; 35:858–865. 5. Cohenca N, Paranjipe A, Berg J. Vital pulp therapy. Dent Clin North Am 2013; 57: 59–73. 6. Oliveira TM, Moretti ABS, Sakai VT, Lourenc¸ o, Neto N, Santos CF, Machado MAAM, Abdo RCC. Clinical, radiographic and histologic analysis of the effects of pulp capping materials used in pulpotomies of human primary teeth. Eur Arch Paediatr Dent 2013; 14: 65–71. 7. Lin PY, Chen HS, Wang YH, Tu YK. Primary molar pulpotomy: A systematic review and network meta-analysis. J Dent 2014; 42: 1060–1077. 8. Huth KC, Hajek-Al-Khatar N, Wolf P, Ilie N, Hickel R, Paschos E. Long-term effectiveness of four pulpotomy techniques: 3-year randomised controlled trial. Clin Oral Investig 2012; 16:1243–1250. 9. Shirvani A, Hassanizadeh R, Asgary S. Mineral trioxide aggregate vs. calcium hydroxide in primary molar pulpotomy: A systematic review. IEJ 2014; 9:83–88. 10. Accorinte ML, Loguercio AD, Reis A, Carneiro E, Grande RH, Murata SS, Holland R. Response of human dental pulp capped with MTA and calcium hydroxide powder. Oper Dent 2008; 33: 488–495.
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