Direct capping of human pulps with a dentin bonding system and calcium hydroxide: an immunohistochemical analysis
Alexandre M. Fernandes, DDS, MSc, PhD,a Gerluza A. B. Silva, DDS, MSc, PhD,a Nelson Lopes, Jr., DDS, MSc, PhD,a Marcelo H. Napimoga, DDS, MSc, PhD,b Bruno B. Benatti, DDS, MSc, PhD,b and José B. Alves, DDS, MSc, PhD,b Belo Horizonte and Uberaba, Minas Gerais, Brazil
FEDERAL UNIVERSITY OF MINAS GERAIS AND UNIVERSITY OF UBERABA
Pulp capping is a treatment where a protective agent is applied to an exposed pulp to allow the maintenance of its vitality and function. The present study analyzed the immunohistochemical expression of ﬁbronectin and type III collagen in human dental pulps submitted to direct pulp capping with calcium hydroxide [Ca(OH)2] or the Single Bond adhesive system (SBAS). The results demonstrated that both proteins were not expressed in the SBAS group, although in the group capped with Ca(OH)2 a diffuse labeling in the extracellular matrix was initially observed, followed by a late expression in the odontoblast-like layer and beneath the dentin bridge. It seems that application of adhesive systems in direct contact with healthy pulps will not lead to expression of proteins that are believed to be essential for pulpal repair. Moreover, Ca(OH)2 showed good biocompatibility properties with the dental pulp tissue, inducing the expression of reparative molecules, and therefore remains the material of choice for the treatment of accidental pulp exposures. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:385-90)
In many clinical situations during tooth preparation or traumatic injuries, it is possible to accidentally expose the dental pulp without the involvement of microorganisms. In these cases, direct pulp capping may be indicated for maintaining pulp vitality and function. Complete healing of the pulp-dentinal complex, which involves maintenance of pulp vitality and function and the restoration of dentin continuity beneath the injury, is a prerequisite for the long-term success of direct pulp capping treatment.1,2 Materials able to stimulate pulp tissue repair and tertiary dentine formation have been investigated extensively in pulp capping situations.3-5 Traditionally, various formulations of Ca(OH)2-containing materials represent the more usual treatment modalities in capping treatment. Experimental and clinical investigations6-8 have demonstrated that Ca(OH)2-based materials have the ability to stimulate reparative dentine formation. High rates (more than 80% of treated teeth) of pulpal survival after capping treatment with Ca(OH)2 have been reported in long-term follow-up
Supported in part by FAPEMIG (Fundo de Amparo à Pesquisa de Minas Gerais) and PAPE (Programa de Apoio à Pesquisa da Universidade de Uberaba). a Institute of Biological Science, Federal University of Minas Gerais. b Laboratory of Molecular Biology, University of Uberaba. Received for publication May 21, 2007; returned for revision Aug 27, 2007; accepted for publication Aug 28, 2007. 1079-2104/$ - see front matter © 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2007.08.031
studies.8,9 However, physical limitations of Ca(OH)2based materials, such as nonadherence to dentine, dissolution in tissue ﬂuids or other dental materials, and degradation upon tooth ﬂexure, has led scientists to seek new approaches.7,8 Recognized because of their qualities in restorative procedures, dentin adhesives have been suggested as a possible alternative material to Ca(OH)2 during direct capping. In addition to the conﬁrmed adhesive properties of these compounds, this new pulp therapy was based on the possible “inherent healing potential” of the exposed pulp which would react favorably to the exposure irrespective of the material used, as long as bacteria are excluded.10-17 However, results of studies evaluating the viability of this new proposal have been extremely controversial. In general, nonhuman experimental models and clinical and radiographic parameters have yielded positive results, but histologic studies in humans have led to discouraging conclusions.8 The ability of the pulp to respond depends on the integration of numerous cellular and extracellular factors. Among the extracellular matrix components identiﬁed in the pulp tissue and involved in a series of interactions with cellular components during regeneration processes are ﬁbronectin and type III collagen. Fibronectin is a glycoprotein involved in a variety of cellular functions, such as adhesion, migration, growth, and differentiation.18,19 Fibronectin has also been suggested as a marker for reparative dentin formation.20 Type III collagen accounts for 43% of total pulp collagen, shows a strong afﬁnity for ﬁbronectin,21 and is 385
Petrópolis. Louis. Control of hemorrhage and drying of the exposed pulp were performed with a sterilized cotton pellet soaked in 0. Brazil). 90%. The sections were then immersed in 0. 1012 bur (KGS). and the underlying molecular mechanisms are still unclear. n 20): pulps were exposed and capped with Single Bond®adhesive system (3M Dental Products. Brazil).9% physiologic saline solution. Immediately after tooth extraction.386
Fernandes et al. Dentsply. a ﬁrst layer was gently applied over the perforation site. Burlingame. São Paulo. Immunohistochemical procedure The slices were deparafﬁnized in xylene and rehydrated in 100%. Wehrheim. group I (SBAS. Paul. Brieﬂy. such as ﬁbronectin and type III collagen.23 Therefore. Signed consent was given by patients and their parents after they had received a thorough explanation related to the study. CA) for 60 minutes at room temperature and rinsed with TBS for 3 minutes 3 times. Olympus.3) at room temperature for a period ranging from 120 to 180 days. Teeth from the experimental groups were extracted after 7 or 30 days (n 10) for immunohistochemical analysis. The streptavidin-biotin-peroxidase complex (Vector) was then applied to the slides. the purpose of the present study was to evaluate the expression of extracellular matrix elements. Brazil) under water irrigation. Staining speciﬁcity was ascertained by omission of primary antibodies. 3M) was poured over the Ca(OH)2 cement and photopolymerized for 20 seconds to prevent Ca(OH)2 disintegration during the step of acid conditioning. and 70% alcohol. CA) was then applied to the sections. Tokyo. and group II (Ca(OH)2. A second layer was then applied in the same way and light-cured for 20 seconds at 500-600 mW/cm2. Schaan. St. Japan). In group I (SBAS). applied with light pressure for 30 seconds. MATERIALS AND METHODS Subjects Forty-six healthy premolars showing no clinical or radiographic impairment. The secondary biotinylated antibody (Vector Laboratories. In group II. and distilled water and washed in Tris-buffered saline (TBS). immersed in xylene. a layer of Ca(OH)2 powder PA was packed in the exposure site. the teeth were sectioned transversally and ﬁxed in 10% buffered neutral formalin for 48 h. and submitted to immunohistochemical analysis. At least 10 representative sections of each specimen were analyzed under light microscope (BX50. St.® Vivadent. followed by a base of Ca(OH)2 cement (Dycal. Petrópolis. and embedded in parafﬁn using conventional procedures. The slides were then incubated with monoclonal antibodies for ﬁbronectin or type III collagen (Santa Cruz Biotechnology. MN). rinsed in TBS and counterstained with Mayer hematoxylin. Clinical and histologic procedures After local anesthesia. dental
enamel (30 seconds) and dentin and pulp (15 seconds) were conditioned with 37% phosphoric acid gel (Email Gel Blue. ETIC 010/98). Dentsply. All clinical procedures were executed by a single professional. Demineralization was carried in 10% ethylene diaminotetraacetic acid (Sigma. The tissue was dehydrated in ascending series of ethanol. All teeth (groups I and II) were restored with Charisma® compound resin (Heraeus Kulzer. 3099 bur (KGS. 1996). Immunohistochemical analysis was performed individually in a blind fashion by 2 cali-
. the teeth were isolated with a rubber dam and polished with a rubber cup and lowabrasion prophylactic paste (Odahcam.3% hydrogen peroxide in TBS to block the endogenous peroxidase activity for 1 hour. incubated for 30 minutes and rinsed again with TBS. Liechtenstein). Teeth were randomly divided into 3 groups according to the capping materials used: control group (n 6): no pulp exposition. the remaining dentin wall was delicately ruptured with a sterile spherical diamond no. Sagittal sections of 6 m were mounted on glass slides pretreated with 3-aminopropyltriethoxysilane (Sigma). The cavity was then gently dried with absorbent paper and sterilized cotton pellets. n 20): pulps were exposed and capped with calcium hydroxide (Biodinâmica.
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the type of collagen synthesized during the initial healing phase of aggressed tissues. with completely formed roots. incubated for 30 minutes. Class I occlusal cavities were prepared using a high-rotation sterile cylindric diamond no. Brazil) in the occlusal cavity ﬂoor. The adhesive system was used according to the manufacturer’s instruction. Then a thin layer of glass ionomer cement (Vitrebond.22 Human studies aimed at understanding the healing characteristics of pulp tissue submitted to direct capping have not been conclusive. This in vivo study was deemed to be ethical according to the Brazilian Guidelines (Resolution 196 of the National Health Council. Ibiporá. When transparency was detected in the most prominent pulpal horn. on human pulps submitted to direct capping with calcium hydroxide or Single Bond® adhesive system. and the protocol was approved by the Research Ethics Committee of the Federal University of Minas Gerais (process no. Germany) according to the manufacture’s instructions. MO) solution (pH 7. and indicated for tooth extraction for orthodontic reasons were selected for this study. Santa Cruz.
b Letter represent intragroup analysis (Kruskal-Wallis analysis of variance on ranks). D. No positive staining was observed 30 days after direct capping. No effective expression of type III collagen (Fig.
Fig. Staining levels of ﬁbronectin and type III collagen in the investigated areas
Adhesive system Control Fibronectin Odontoblast layer Predentin layer Pulp tissue Type III collagen Odontoblast layer Pre-dentin layer Pulp tissue 2.40)a 2. In addition. Pulp. Immunohistochemical photomicrographs of repairassociated markers type III collagen (A) and ﬁbronectin (B) from pulps capped with adhesive system. Tissue staining speciﬁcity was conﬁrmed by lack of immunostaining for all experimental groups (data not shown).50 (0.10 (0. tissue staining for type III collagen and ﬁbronectin was conﬁrmed by positive identiﬁcation mainly observed in the odontoblast and predentin layers. Immunohistochemical photomicrographs of repairassociated markers type III collagen (A) and ﬁbronectin (B) from unexposed human dental pulps. V. A) and ﬁbronectin (Fig.31)b 0. and pulp tissue) the treatment with the adhesive system signiﬁcantlly decreased the expression of type III collagen and ﬁbronectin (P .31)b 0.05).78)a 30 days 2.67)a 0.20 (0.31)a 1. Bar 10 m. In the control group (without pulp exposition). predentin.00)b 0. predentin layer.63)a 1.47)a 1.42)a 2. after 7 days. 1.80 (0. 2.
Ca(OH)2-treated group showed a diffuse labeling of type III collagen and ﬁbronectin expression throughout the pulp tissue.91). 1). In a similar pattern.42)a 2.42)a 1.40 (0. RESULTS Negative control staining was done by omitting exposure to the primary antibodies. Conversely. Relative staining intensity was assessed for each molecule at the odontoblast layer. a. 2 moderate staining intensity.20 (0.63)b 0. 4. and 3 strong staining intensity.83)a 0. Note immunostaining in odontoblasts (arrow) and predentin layers as well as at odontoblast elongations (arrowhead) penetrating the predentin layers. similar to the control samples (Fig.73)a .67)b Calcium hydroxide 7 days 2. A) and ﬁbronectin (Fig. Note the presence of connective tissue matrix hyalinization (*) indicating a possible response to tissue aggression.00 (0.48)b 30 days 0. 2.63)b 0.30 (0. B) was observed in group I (SBAS) in both periods.63)a 1.69)a 2. 4.20 (0. vessel.70 (0. Statistical analysis Mean values and standard deviation were statistically analyzed comparing ﬁbronectin and type III collagen staining independently.10 (0.50 (0. a uniform labeling pattern expression of type III collagen (Fig. Data analysis demonstrated that at all 3 evaluated areas (odontoblast layer.48)b 0.00 (0.67)a 0.05) at both periods (Table I).80 (0. 1 weak but visible staining intensity. P.63)b 0.83 (0.30 (0.63)b 0.54)a 2. Different letters differ statistically (P
brated examiners (kappa index 0. Number 3
Fernandes et al.83 (0. and pulp tissue.70 (0.20 (0.40)a 1.00 (0.80 (0.05) compared with the control and Ca(OH)2-treated groups at both periods. expression of type III collagen and ﬁbronectin was similar in teeth treated with Ca(OH)2 and in those of the control group (P . Bar 50 m. being diffuse in the pulp tissue and with low expression in the dentin area (Fig.00 (0.63)b 0. After 30 days. Samples were scored as follows: 0 no immunoreactivity. an evident expression of both molecules was observed in the subodontoblastic
.20 (0. PD.10 (0.90 (0. followed by the Dunn method for multiple comparisons to test for differences among the groups in the same area over time. 3).
Data are presented as median and standard deviation of the average staining of all sections analyzed per area. 387
Table I. predentin layer.43)a 1. Data were analyzed using the Kruskal-Wallis one-way analysis of variance on ranks.80 (0.20 (0.70 (0. B) was clearly concentrated at the odontoblast-like cells layer and in the lower zone of the dentin bridge (predentin zone). Dentin.OOOOE Volume 105. the
Fig.42)b 0. 2.63)a 7 days 0.20 (0.30 (0.
Finally. leading us to hypothesize that growth factors released from dentin in the pulp tissue may potentially inﬂuence ﬁbronectin and type III collagen expression. In fact. With this notion.28 Type III collagen is the predominant
collagen synthesized during the early phases of tissue repair concomitantly with ﬁbronectin.22 A ﬁbronectin-rich matrix may serve as a reservoir of growth factors. which are essential to promote tissue repair. the odontoblasts are stimulated to form tertiary dentin. Pulp. and although suggestions have been made for its mechanisms of action they still remain largely elusive. 4. noncollagenous proteins. as well as beneath the dentin bridge (DB). 3. it has been shown that reparative dentinogenesis is associated with ﬁbronectin-mediated differentiation of pulp cells into odontoblast-like cells during reparative dentinogenesis after pulp capping with Ca(OH)2. collagens are a family of ﬁbrous proteins found in all multicellular animals. such as transforming growth factor . Based on this evidence. constituting 25% of their total protein mass.24. including collagenous proteins. which mediate changes in cell behavior observed during regeneration.32 Studies by Davidson and Guo (2000)33 are consistent with a putative role of calcium ions during reparative dentin formation. permeating both the cells and the extracellular matrix DISCUSSION Calcium hydroxide has found wide usage as a pulpcapping agent for over 60 years. Bar 50 m. types I and III collagen constitute the bulk of the tissue collagen.
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Fig. In human dental pulp. (2006)34 showed that during pulp capping with Ca(OH)2 the cellular activities involved in dentin bridge formation may be mediated through the release of growth factors.388
Fernandes et al. ﬁbronectin immunoreactivity under the dentin barrier could be related to the functions of these molecules. with type III collagen constituting a large proportion (43%) of the total collagen amount. it has been postulated that the alkaline pH maintained at the injured/treated region creates favorable conditions for dentin formation31 and that the localized elevated calcium ion concentration increases expression of mineralization-promoting genes (osteopontin and bone morphogenic protein 2) in pulp cells.23 When pulpal injury occurs. In addition. giving rise to dentine bridge formation. which have been considered to be signaling molecules for differentiation of odontoblasts. Immunohistochemical photomicrographs of repairassociated markers type III collagen (A) and ﬁbronectin (B) from pulps capped with calcium hydroxide after 30 days.
layer of the pulp tissue. the synthesis of these 2 components decreases simultaneously with an increase in the secretion of type I collagen.29 Furthermore. in the present investigation
. whereas the adhesive system did not.26. With advanced recovery. The extracellular matrix of pulp tissue comprises a variety of macromolecules. such as the release of hydroxyl ions30 raising the pH of the exposed pulpal tissue. Immunohistochemical photomicrographs of repairassociated markers type III collagen (A) and ﬁbronectin (B) from pulps capped with calcium hydroxide after 7 days. and phospholipids.27 Based on the present ﬁndings. P. Also.21 Diverse hypotheses about the successful treatment with Ca(OH)2 have been made. Immunostaining for type III collagen and ﬁbronectin presented a diffuse pattern throughout the central and peripheral areas of the pulp tissue (*).25 Understanding the material mode of action may allow the optimization of its clinical application. Bar 10 m.35 In contrast. because variation in the quality of dentin bridges induced after its application is well recognized. this study showed that pulp capping using Ca(OH)2 induces expression of reparative molecules such as type III collagen and ﬁbronectin.
Fig. we may hypothesize that matrix factors are stimulated by the action of Ca(OH)2 in pulp cells. The extracellular matrix is likely to inﬂuence this process. proteoglycans. owing to the presence of ﬁbronectin. The reason behind these results may be that bonding materials are toxic to the pulp tissue and do not work as a barrier against late infection from the oral ﬂora. suggesting that they may contribute separately or synergistically with the local elevated pH. Note that staining for type III collagen and ﬁbronectin was concentrated at the odontoblast-like layer (Od) as indicated by arrowheads. and other bioactive molecules (type I collagen and nestin) from the dentin. direct pulp capping using bonding materials has demonstrated only few hard tissue bridges and hyalinization areas and more pronounced inﬂammation compared with Ca(OH)2-based materials. Graham et al.
Lopes-Júnior N. 14. J Dent 1999.26:708-11. and differentiation. Histologic evaluation of direct pulp capping with a self-etching adhesive and calcium hydroxide in beagles. Lemmens IG. A histopathological study of direct pulp capping with adhesive resins. Tonino GJM. Immunohistochemical localization of tenascin.16:66-76. ﬁbronectin. Immunohistochemical localization of types I and III collagen and ﬁbronectin in the dentine of carious human teeth. Direct capping of human pulps with a dentin bonding system or with calcium hydroxide cement. Histomorphometric analysis of dentinal bridge formation and pulpal inﬂammation. 389
6. Horsted-Bindslev P. Direct Pulp capping with dentin bonding system in human teeth: a clinical and histological evaluation. In line with this evidence.
no effective expression of ﬁbronectin or type III collagen was observed in the group submitted to direct capping with dentin adhesives (group I) in the evaluated periods. Van Amerogen JP. Suzuki S. (2000)19 demonstrated high expression of ﬁbronectin.21. In addition. whereas treatment with Ca(OH)2 led to a reparative process. Ozawa H. Alves JB. Inokoshi S. J Dent Res 1985. Formation of a hard tissue barrier after pulp cappings in humans.
. 10. 20. Heitmann T. 21.22. 8. In contrast.27:257-63. Smith AJ. Hartmann DJ. J Endod 2000. Arch Oral Biol 1986. 19. Quintessence Int 2002. Tziafas D. Endod Dent Traumatol 1985. Petersson K. Liu T. Designing new treatment strategies in vital pulp therapy. 125:823-31. The concentration. Cox CF.26: 765-70. 22. 15. Thylstrup A. Effects of direct resin pulp capping techniques on short-term response of mechanically exposed pulps. A retrospective study of direct pulp capping with calcium hydroxide compounds. 7. Re-evaluating pulp protection: calcium hydroxide liners vs. Cavalcanti de Araujo V. Kitasako Y. extractability and characterization of collagen in human dental pulp. Pesqui Odontol Bras 2006.102:e78-84. Localization and synthesis of type III collagen and ﬁbronectin in human reparative dentine.39:429-42. in group II [Ca(OH)2]. 4. Tagami J. Sondergaard B. Tagami J. as indicated by their simultaneous expression especially at the pulp periphery (odontoblastic layer) and predentin. cohesive hybridization.
1.68:628-39. 5. Tziafas D.37 The presence of a persistent inﬂammatory process throughout the study period and the lack of expression of type III collagen and ﬁbronectin observed in group I (SBAS). Nakamura H.18:87-99. Inokoshi S. Lesot H.20:219-25. Am J Dent 2003. Kawamata-Kido H. Fejerskov O. Silva GAB.64:541-8. The marked afﬁnity between these 2 molecules. Direct pulp capping with dentinal adhesive resin system: a pilot study. Tagami J. Tsuneda Y. Olsson H. Stanley HR. Yoshiba N. Int J Periodontics Restorative Dent 1998. Sasaki T.33:639-47. Hafez AA.20:223-9. Archs Oral Biol 1995. Pi G. Providing an environment for reparative dentine induction in amputated rat molar pulp by high molecular-weight hyluronic acid.39:395-400. 18. Martinez et al. Int Endod J 2006.OOOOE Volume 105. Unterbrink G. Magloire H. Dezan Junior E. Rohlin M.18. 2. 11. Hayakawa T.28:77-92. and type III collagen in human dental pulp. Rahal V. Goldberg M. Number 3
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