Direct Pulp Capping with Mineral Trioxide Aggregate: An Immunohistologic Comparison with Calcium Hydroxide in Rodents

Direct Pulp Capping with Mineral Trioxide Aggregate: An Immunohistologic Comparison with Calcium Hydroxide in Rodents

Basic Research—Biology Direct Pulp Capping with Mineral Trioxide Aggregate: An Immunohistologic Comparison with Calcium Hydroxide in Rodents Till Dam...

589KB Sizes 0 Downloads 15 Views

Basic Research—Biology

Direct Pulp Capping with Mineral Trioxide Aggregate: An Immunohistologic Comparison with Calcium Hydroxide in Rodents Till Dammaschke, Priv.-Doz. Dr. med. dent.,* Udo Stratmann, Prof. Dr. med. dent.,† ¨ fer, Prof. Dr. med. Philipp Wolff, Dr. med. dent.,‡ Darius Sagheri, Dr. med. dent.,§ and Edgar Scha dent.k Abstract Introduction: The aim was to evaluate the proliferation of pulp cells 1, 3, and 7 days after direct pulp capping with ProRoot MTA (MTA) and to compare the results with calcium hydroxide (Ca(OH)2). Methods: An occlusal cavity was prepared in 36 molar teeth of 18 Wistar rats. Then MTA or Ca(OH)2 was placed on the exposed pulp. All cavities were restored with composite. After 1, 3, and 7 days the animals were killed. One hour before scarification 5-bromo-2’-deoxyuridine (BrdU) was injected into the intraperitoneal cavity for immunohistologic analysis. BrdU was incorporated into the cell nucleus during the S phase of the cell cycle. Proliferating cells were tagged and counted by using alkaline phosphatase and anti–alkaline phosphatase antibody staining. Three animals (6 molar teeth) served as controls and were not further treated. The number of the tagged cells was statistically analyzed by comparing the results of the 3 groups. A Bonferroni correction was performed, because the data of the Ca(OH)2– group was used 3 times for pairwise comparison. Results: The marked cells were identified as fibroblasts, endothelial cells (after 1, 3, and 7 days), and Ho¨hl cells (after 7 days). The MTA group showed a similar amount of Ho¨hl cells when compared with the Ca(OH)2 group (P > .05). One day and 7 days after capping, no significant differences were observed between the 2 tested groups and the controls (P > .05). After 3 days, significantly more cells were stained in the MTA and Ca(OH)2 groups than in the control group (P < .016). Conclusions: Immunohistologic analysis demonstrated that MTA showed similar results when compared with Ca(OH)2 within the first week after direct pulp capping. (J Endod 2010;36:814–819)

Key Words Calcium hydroxide, direct pulp capping, immunohistology, mineral trioxide aggregate, rat molar teeth

M

ineral trioxide aggregate (MTA) can be used as endodontic reparation cement for root-end fillings, apexification, closure of radicular perforations, and for direct pulp capping according to the manufacturer’s product information (Dentsply Tulsa Dental, Tulsa, OK). MTA is a cement powder that contains different oxide compounds (sodium and potassium oxides, calcium oxide, silicon oxide, ferric oxide, aluminum oxide, and magnesium oxide) (1). To set, it must first be mixed with water. If set MTA gets in contact with tissue fluids, its calcium oxide converts into calcium hydroxide (Ca(OH)2). The Ca(OH)2– molecule dissociates into calcium and hydroxyl ions, which will increase the pH value to approximately 12.5 and results in the release of calcium ions (2–4). Hence, MTA and Ca(OH)2 have similar features (5); therefore, MTA might well be used as an alternative to Ca(OH)2 in dentistry (6). The advantages of MTA in direct pulp capping, when compared with Ca(OH)2, are its lower solubility, improved mechanical strength, better marginal adaptation, and better sealing ability. Furthermore, using MTA for direct pulp capping eliminates some of the disadvantages of Ca(OH)2, eg, resorption of the capping material, mechanical instability, and subsequent inadequate long-term sealing ability as a result of leakage (7). Several histologic studies already compared the reaction of vital pulp cells when directly pulp capped with MTA or Ca(OH)2 (3, 7–19). Most of these studies found MTA to be superior to Ca(OH)2 (3, 8–10, 13–16). However, some authors found no significant differences in pulp healing between the 2 substances (7, 11, 12, 17–19) (Table 1). Nevertheless, all these studies demonstrated that MTA is well suitable for direct pulp capping (7). Immunohistologic analysis revealed that the turnover rate of cells in healthy pulp tissue is low (20, 21) because odontoblasts do not undergo mitosis (22, 23). Cavity preparation alone leads to an increase of the mitosis rate, which results in a proliferation of cells in the direction toward the damaged pulp tissue (22). If rat molar teeth are ground occlusally, the mitosis activity of pulp cells will increase within 24 hours, with a maximum after 48 hours (24). Hence, the hypothesis of this animal study was that direct pulp capping with MTA will lead to an increase of the cell division rate in vivo as a result of the biocompatibility of MTA to pulp cells (25, 26). For this reason MTA was compared with Ca(OH)2, the gold standard for direct pulp capping (27), by examining the proliferation of pulp cells 1 day, 3 days, and 7 days after direct pulp capping of rat molar teeth. To quantify the ratio of cells undergoing mitosis by enumeration and calculation of a mitosis index (MI), an immunohistologic analysis was performed by the use of vital staining with 5-bromo-20 -deoxyuridine (BrdU). Thymidine is a nucleoside and a typical component of the DNA in the cell nucleolus. Therefore, it can

From the *Department of Operative Dentistry and †Institute of Anatomy, Westphalian Wilhelms-University, Mu¨nster, Germany; ‡Department of Oral and Maxillofacial Surgery, Knappschafts-Hospital, Recklinghausen, Germany; §Department of Orthodontics, University of Cologne, Ko¨ln, Germany; and kCentral Interdisciplinary Ambulance in the School of Dentistry, Westphalian Wilhelms-University, Mu¨nster, Germany. Address requests for reprints to PD Dr. Till Dammaschke, Poliklinik fu¨r Zahnerhaltung, Waldeyerstr. 30, 48149 Mu¨nster, Germany. E-mail address: [email protected] uni-muenster.de. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.02.001

814

Dammaschke et al.

JOE — Volume 36, Number 5, May 2010

Basic Research—Biology TABLE 1. Histologic Results of Direct Pulp Capping with MTA in Comparison to Ca(OH)2 Given in Literature Authors

Type of Ca(OH)2

Species

Observation period

Result

Pitt Ford et al, 1996 (8) Faraco Junior and Holland, 2001 (3) Aeinehchi et al, 2003 (9) Dominguez et al, 2003 (10) Queiroz et al, 2005 (11) Iwamoto et al, 2006 (12) Accorinte et al, 2008 (13) Asgary et al, 2008 (14) Min et al, 2008 (15) Nair et al, 2008 (16) Costa et al, 2008 (17) Sawicki et al, 2008 (18) Shayegan et al, 2009 (19) Dammaschke et al, 2010 (7)

Hard setting cement Hard setting cement Hard setting cement Light curing Aqueous paste Hard setting cement Hard setting cement Hard setting cement Hard setting cement Hard setting cement Aqueous paste Hard setting cement Hard setting cement Aqueous paste

Monkey Dog Human Dog Dog Human Human Dog Human Human Dog Human Pig Rat

5 months 2 months 1 week – 6 months 50d + 150d 90 d 136  24 d 30 d + 60 d 8 weeks 2 months 1 week + 1 month + 3 mon 60 d 47d – 609 d 3 weeks 1 d, 3 d, 7 d, 70 d

MTA significantly superior MTA significantly superior MTA significantly superior MTA significantly superior No significant difference No significant difference MTA significantly superior MTA significantly superior MTA significantly superior MTA significantly superior No significant difference No significant difference No significant difference No significant difference

be assumed that thymidine is specific for DNA. BrdU is an analogue to thymidine, ie, thymidine can be replaced by BrdU through incorporating it into the cell nucleus during the S phase of the cell cycle. By using a specific antibody staining with alkaline phosphatase and anti–alkaline phosphatase antibody (APAAP), the BrdU can be tagged and counted in proliferating cells (28). A cell that contains BrdU underwent cell division at the moment when BrdU was applied exogenously, because BrdU has only a short bioavailability (29). These tagged cells are cells of the S, G2, and M phases of the cell cycle. The MI is based on the amount of incorporated BrdU into the cell DNA, which provides information on cell activity. The MI is a quotient of the amount of tagged cell nuclei divided by the amount of all cell nuclei examined (28).

Materials and Methods Operative Procedure Eighteen male and female Wistar rats (36 molar teeth) with an age of 3 months and a body weight between 250 and 300 g were used in this study. This animal research project was reviewed and licensed by the regional government administration (Mu¨nster, Germany) with the registration no. G39/99 and was carried out in accordance with the European Communities Council Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes. Adequate measures were taken to minimize pain or discomfort for the rats. For anesthesia, a combination of 1 mL ketamine 10% (CEVA, Du¨sseldorf, Germany) and 0.2 mL xylazine 2 % (CEVA) was added to 3.8 mL isotonic saline solution. The rats were anesthetized with an intramuscular injection (0.1 mL per 50 g body weight). Because of the small size of rat molar teeth the use of rubber dam was not possible. Before cavity preparation, the teeth were cleaned mechanically with a small brush, chemically with NaOCl (5%), and disinfected with chlorhexidine digluconate (0.1%, ChlorhexamedFluid; GlaxoSmithKline, Bu¨hl, Germany). Aided with magnifying glasses (magnification 4.5; Zeiss, Aalen, Germany), occlusal cavities were prepared in the first upper, caries-free right and left molar teeth with a micromotor handpiece and a cylindrical diamond bur (ISO 008; NTI, Kahla, Germany) running at maximum 3000 min1 up to the vicinity of the pulp chamber. The cavities were prepared under permanent cooling with water spray. Subsequently, the roofs of the pulp chambers were perforated with a sterile sharp probe (EXD2H; Hu-Friedy, Chicago, IL) in a slot-like shape. Pulpal blood was removed from the cavities with sterile paper points (Roeko, Langenau, Germany). To remove all blood remnants from the cavity walls, the cavities were rinsed carefully with saline solution and then air-dried. The pulp tissue of the upper right molar teeth was directly capped with ProRoot MTA cement (Dentsply Tulsa Dental; lot 03081235). The application followed JOE — Volume 36, Number 5, May 2010

exactly the manufacturer’s specifications (1 g MTA mixed with 0.35 g water). The pulps of upper left molar teeth were capped with Ca(OH)2 paste. The Ca(OH)2 paste was freshly prepared from Calciumhydroxid pro analysi (Merck, Darmstadt, Germany) and isotonic saline solution. All cavities were then restored with a flowable composite restoration material (Tetric flow; Vivadent, Ellwangen, Germany) in combination with a self-etching dentin adhesive (Resulcin AquaPrime + MonoBond; Merz Dental GmbH, Lu¨tjenburg, Germany).

Immunohistology After defined postoperative time intervals of 1, 3, and 7 days, an intravital injection of 1 mL of a BrdU solution (Sigma-Aldrich, Munich, Germany) was applied intraperitoneally to quantify the rate of pulp cells capable of mitosis after direct pulp capping by enumeration and calculation of an MI. Six animals (12 molar teeth) were treated in each time interval. For this purpose, 50 mg BrdU powder was solubilized in 5 mL saline solution (the applied quantity was 50 mg per kg body weight and 10 mg BrdU for an average body weight of 200 g/animal; this resulted in 1 mL BrdU solution). The animals were killed by the use of CO2 inhalation after an exposure period to BrdU solution for 1 hour. A group of 3 animals (6 molar teeth) served as controls and were not treated, but they solely received an intravital injection with BrdU. The maxillary molar teeth and the surrounding bone were dissected, fixed in formalin (2.5%), demineralized for 8 weeks with ethylenediaminetetraacetic acid, and embedded in paraffin. All specimens were divided into 13 sections with a layer thickness of 8 mm by using a microtome (Ultracut; Reichert-Jung, Vienna, Austria) and positioned on glass slides (Superfrost; Menzel-Gla¨ser, Braunschweig, Germany). To expose the antigen, the paraffin sections must be completely deparaffinized and rehydrated. For this reason the sections were incubated twice in xylol for 10 minutes. The sections were subsequently incubated in descending ethanol series (100%, 96%, 70%) and washed with distilled water. To improve immunoreactivity, the sections were proteolytically digested with 0.05% protease for 6 minutes. This resulted in a demasking of the antigen and an exposure of hidden determinants. The specimens were subsequently rinsed with Tris-Tween buffer (Tween 20; Sigma-Aldrich). Then the sections were incubated for 60 minutes with monoclonal mouse antibromodeoxyuridine (M 0744; DakoCytomation, Hamburg, Germany), the so-called primary antibody, at a concentration of 1:20. The sections were then rinsed and incubated for 30 minutes with polyclonal rabbit anti-mouse immunoglobulin (Z 0259; DakoCytomation), the so-called bridge antibody (40 mL antibody + 200 mL human serum + 760 mL Tris). After another irrigation with Tris-Tween, the sections were incubated with an APAAP antibody immune complex (D 0651; MTA for Direct Pulp Capping

815

Basic Research—Biology TABLE 2. Results of the Statistical Evaluation of the Immunohistology: Mean Values and Standard Deviation of the MI of Cells in the S Phase Marked with BrdU

Interval (days) 0 1 3 7

Without operation, mean, ± SD

Calcium hydroxide, mean ± SD

MTA, mean ± SD

0.52  0.46 1.64  0.81* 0.96  0.64

0.55  0.42 1.71  0.64* 1.12  0.56

0.53  0.18

MI, mitosis index; MTA, mineral trioxide aggregate; SD, standard deviation. *Significant difference compared with the control group without treatment (P < .016).

DakoCytomation) for 30 minutes (20 mL + 980 mL Tris). The 2 last steps (incubation with bridge antibody and immunoenzyme complex) were then repeated with an incubation time of 10 minutes. During the second incubation with the rabbit anti-mouse antibody the human serum was omitted, but instead 960 mL Tris was added. The glass slides were subsequently rinsed with Tris-Tween. The sections were covered with a liquid permanent red solution (LPR, K0640; DakoCytomation), prepared according to manufacturer’s specification (20 mL LPR chromogen to 3 mL substrate buffer), incubated for 9 minutes, and then rinsed with distilled water. Then staining with hematoxylin was performed to present the cell nuclei. This effectuates a contrast to the antigen staining. Subsequently, the specimens were washed with water, dried, and incubated with xylol. All sections were then covered with a resin (DPX; BDH Ltd, Pool, UK) and a coverslip. Three sections with completely visible crown pulp were selected from each treated molar tooth. For digital image evaluation, ie, count of red tagged cells capable of mitosis with a picture analysis program, stained specimens were photographed with a photo microscope (Phomi III; Zeiss, Oberkochen, Germany) and a camera (XP 900; Sony, Tokyo, Japan) and processed with a picture imaging program (Lucia Net; Nikon, Kingston Surrey, UK) at a 250-fold magnification. To cover the complete extension of the crown pulp, approximately 15–20 photos were taken for each section. All pictures that cover the complete crown were combined together. The image analysis was conducted with the computer program UTHSCSA ImageTool (University of Texas Health Science Center, San Antonio, TX). The tagged and untagged cells were counted with this program, and the quantities were recorded. The counting process and identification of cells were performed by an experienced oral anatomist in a blind scoring method and without knowing how the specimens were treated. The numbers of red tagged dividable cells per tooth were summated and, to generate an MI, were divided by the number of all cells and then multiplied with 100: MI = Tagged cells/Number of all cells  100.

A statistical evaluation of the MI of the 2 used materials, MTA and Ca(OH)2, was performed with the univariable, nonparametric MannWhitney U test and a multivariate variance analysis. Furthermore, the tagged cells of the S phase were determined with regard to their type and quantitative distribution for each section. Additional staining was omitted because these cell types (fibroblasts, endothelial cells, and Ho¨hl cells) can be easily distinguished even in light micrographs as a result of their specific morphology and location. The statistical evaluation of the counts and distribution of the different cell types was performed with the univariable, nonparametric MannWhitney U test as well. Because the data of the Ca(OH)2– group were used 3 times for pairwise comparison, a Bonferroni correction was performed for the time period ‘‘3 days’’. Normally, a is set at 0.05. In this case, a had to be divided by the number of performed tests (3). Therefore, a had to be set at 0.016.

Results Marking Index of All Tagged Pulp Cells One day after direct pulp capping, no significant differences could be observed between the 2 groups. The MI had its maximum 3 days after direct pulp capping in both capping materials (Ca(OH)2 and MTA). Significantly more cells were tagged in both the MTA and the Ca(OH)2 groups, when compared with the control group (P < .016). After the 3-day period, the MI decreased in the 2 groups. Therefore, the data of the control group were compared with these maximum values (Table 2). Comparison of the control group with the Ca(OH)2 and the MTA groups showed a significantly increased MI (P < .016). However, no such difference could be detected (P > .05) between the Ca(OH)2 and the MTA groups 1, 3, and 7 days after direct pulp capping. A multivariate variant analysis was performed to provide in-depth comparison between the 2 treatment groups (MTA and Ca(OH)2). Dependent variable was rate of tagged cells; independent variables were time interval and treatment group. The analysis demonstrated only a significant dependence of the tagged cells to time interval (P = .02). Distribution of the Tagged Cells The majority of cells in the control group marked with BrdU after 1 day and after 3 days of direct pulp capping in the S phase were fibroblasts and at lower rate endothelial cells. Ho¨hl cells were visible in both treatment groups (MTA and Ca(OH)2) seven days after direct pulp capping (Table 3). The data of the control group were compared with the results for the 2 treatment groups 7 days after direct pulp capping, because the differences between them were then most prominent. No other cells

TABLE 3. Results of the Statistical Evaluation of the Immunohistology: Percent Distribution (mean values and standard deviation) of the Different Cells Marked with BrdU Material

Interval (days)

Fibroblasts, mean ± SD (%)

Endothelial cells, mean ± SD (%)

Without operation Ca(OH)2 MTA Ca(OH)2 MTA Ca(OH)2 MTA

0 1 1 3 3 7 7

90.75  5.52 94.02  7.30 91.46  6.71 68.49  10.41 71.80  8.57 63.33  7.17* 58.35  6.59*

9.24  5.52 5.97  7.30 8.53  6.71 31.50  10.41 28.19  8.57 26.15  5.54* 28.81  4.90*

Ho¨hl cells, mean ± SD (%) 0 0 0 0 0 10.51  4.98* 12.82  3.18*

MTA, mineral trioxide aggregate; SD, standard deviation. *Significant difference compared with the control group without treatment (P < .016).

816

Dammaschke et al.

JOE — Volume 36, Number 5, May 2010

Basic Research—Biology than fibroblasts, endothelial cells, and Ho¨hl cells could be tagged in any section. Fig. 1A shows a stained section of the untreated control group. In comparison, Fig. 1B and C show an exemplary illustration of the immunohistologic tagged cells 3 days after pulp capping; Fig. 1D and E show 7 days after direct pulp capping with Ca(OH)2 and MTA, respectively.

Results One Day and Three Days after Direct Pulp Capping Regarding the distribution between fibroblasts and endothelial cells, statistically no significant differences could be observed between

the 2 treatment groups and the control group (P > .05). Ho¨hl cells could not be marked.

Results Seven Days after Direct Pulp Capping The results for the Ca(OH)2 and MTA groups showed that significantly fewer fibroblasts and significantly more endothelial cells were tagged, when compared with the control group (P < .016). The distribution of fibroblasts and endothelial cells showed no significant difference between the Ca(OH)2 and the MTA groups (P > .016). A significant difference between the 2 treatment groups and the control group was found in the rate of tagged Ho¨hl cells, because no Ho¨hl cells were tagged in untreated teeth (P < .016). Furthermore, the MTA group showed

Figure 1. (A) Light micrograph of an upper rat molar tooth without treatment (control group). Only few cells (red tagged) underwent mitosis. f, fibroblast; c, capillary. Bar = 50 mm; original magnification, 250 (immunohistologic staining). (B) Light micrograph of an upper rat molar tooth after pulp exposure and 3 days of contact with Ca(OH)2. All red tagged cells were in the S phase of the cell cycle. e, endothelial cell. All other red tagged cells were identified as fibroblasts. Bar = 50 mm; original magnification, 250 (immunohistologic staining). (C) Light micrograph of an upper rat molar tooth after pulp exposure and 3 days of contact with MTA. All red tagged cells were in the S phase of the cell cycle. e, endothelial cell. All other red tagged cells were identified as fibroblasts. Bar = 50 mm; original magnification, 250 (immunohistologic staining). (D) Light micrograph of an upper rat molar tooth after pulp exposure and 7 days of contact with Ca(OH)2. All red tagged cells were in the S phase of the cell cycle. h, Ho¨hl cell; f, fibroblast. Bar = 50 mm; original magnification, 250 (immunohistologic staining). (E) Light micrograph of an upper rat molar tooth after pulp exposure and 7 days of contact with MTA. All red tagged cells were in the S phase of the cell cycle. h, Ho¨hl cell; e, endothelial cell. All other red tagged cells were identified as fibroblasts. Bar = 50 mm; original magnification, 250 (immunohistologic staining).

JOE — Volume 36, Number 5, May 2010

MTA for Direct Pulp Capping

817

Basic Research—Biology a similar number of Ho¨hl cells in the S phase when compared with the Ca(OH)2 group (P > .05).

Discussion Rat molar teeth are a valid model to evaluate histologically the outcome of direct pulp capping. Pulp healing in rats is histologically similar to pulp healing in humans after direct pulp capping with Ca(OH)2. Rat molar teeth, including pulp tissue, can be seen anatomically and histologically as well as biologically and physiologically as miniature human molar teeth (30). Furthermore, Kuratate et al (31) already marked proliferating cells with BrdU in rat molar teeth. A proliferation peak was found 3 days after direct pulp capping. Kuratate et al also observed odontoblast-like cells, which appeared after 3 days and which showed an odontoblast-like morphology after 14 days. This is similar to the results found in the present study. The histology of the pulp reaction after pulp capping with MTA when compared with Ca(OH)2 has already been described in detail in previous research (7), and similar results were found by Te´cle`s et al (32) in an in vitro study. Three days after direct pulp capping with MTA or Ca(OH)2, the MI was significantly increased when compared with the control group. This can be explained by the development of granulation tissue after direct pulp capping with Ca(OH)2 and MTA (7). Initially, a distinct superficial necrosis occurred as a result of pulp tissue exposure and direct pulp capping. However, 1 day and 3 days after direct pulp capping the necrosis was less distinct in the MTA group (7). The necrosis induced an inflammation, because necrotic tissue acts like a foreign body in a healthy environment. The necrotic tissue will soon be metabolized and replaced by granulation tissue as a result of a cellular reaction of the surrounding connective tissue. The formation of granulation tissue through the proliferation of capillaries and fibroblasts initiates the reparation of pulp tissue. Thus, the inflammatory reaction is an essential part of the wound healing process (23, 33, 34). The increased MI depends on the state of the inflammatory reaction. Hence, it became apparent that the significant increase of the MI 3 days after pulp capping with Ca(OH)2 or MTA can be explained by the regular healing processes of pulp tissue, ie, formation of granulation tissue. This has been vindicated by the observation that after 3 days only fibroblasts and endothelial cells (ie, typical cells of the granulation tissue) were tagged. Beside fibroblasts and endothelial cells, Ho¨hl cells were found 7 days after direct pulp capping. In case of an injury or a decline of primary odontoblasts, these Ho¨hl cells might have the potency to differentiate to odontoblast-like cells and to form reparative dentin (35–38). Therefore, it can be concluded from the results of the present study that MTA and Ca(OH)2 had a positive effect on the MI of Ho¨hl cells. The MI for tagged Ho¨hl cells was higher after direct pulp capping with MTA when compared with Ca(OH)2, but this difference was not statistically significant. This effect might have an influence on the formation of reparative dentin. Seventy days after direct pulp capping, teeth treated with MTA showed slightly more reparative dentin, when compared with Ca(OH)2. However, this difference was not statistically significant (7). It is well-known that Ca(OH)2 promotes the differentiation of odontoblasts or odontoblast-like cells, which will form a hard tissue bridge in the pulp; Ca(OH)2 contributes actively to the formation of new hard tissue by induction and up-regulation of the differentiation of odontoblast-like cells (39). Furthermore, low concentrated Ca(OH)2 induces the proliferation of pulp fibroblasts (40). It can be assumed that MTA has the same effects because of the release of calcium and hydroxyl ions when in contact with water or tissue fluids. Takita et al (4) compared the effects of MTA and Dycal (hard setting Ca(OH)2 salicylate ester cement) in human pulp cells in an in vitro study. Compared 818

Dammaschke et al.

with the control group, MTA stimulated significantly the proliferation of cells within 12 days, whereas Dycal showed no such effect. The amount of calcium ions released by MTA was significantly higher when compared with Dycal. When the cell cultures got in contact with calcium ions at different concentration levels, the cell proliferation counts mirrored these different levels. Takita et al suggested that the release of calcium ions from MTA might induce the proliferation of human pulp cells as well (4). Other in vitro studies showed that MTA is able to stimulate several types of cells such as fibroblasts (41) and osteoblast-like cells (42, 43). Furthermore, MTA was able to induce the differentiation of pulp cells into odontoblast-like cells and the formation of reparative tertiary dentin. MTA probably stimulated mineralization by up-regulation of bone morphogenetic protein (44). In vitro MTA promotes the production of mineralization matrix genes, mRNA, and a protein expression of cellular markers, which play a role in the mineralization process (45). In comparison with an untreated control group, MTA might induce a significant increase of MDPC-23 cells in the S phase and the G2 phase and of OD-21 cells in the S phase of the cell cycle in vitro. However, MTA did not influence the apoptosis of these cells. Therefore, it can be concluded that MTA induces the proliferation, but not apoptosis, of pulp cells. This might explain the regenerative processes observed after direct pulp capping with MTA in vivo (46).

Conclusions The present immunohistologic study in rodents undertaken 1–7 days after direct pulp capping showed that MTA produced similar good results when compared with Ca(OH)2. MTA promotes the formation of granulation tissue within 3 days, which is a precondition for pulp tissue healing. Furthermore, 7 days after direct pulp capping with MTA, Ho¨hl cells were observed, which is identical to Ca(OH)2 and might play a role in hard tissue formation. We are grateful to Dr. Alison Dougall, Consultant for Medically Compromised Patients, Division of Special Care Dentistry, Dublin Dental School and Hospital, Trinity College, Dublin for kindly reviewing the manuscript.

Acknowledgments The authors thank Priv-Doz. Dr. rer. medic. Dr. phil. RudolfJosef Fischer, Institute for Medical Informatics and Biomathematics (Westphalian Wilhelms-University, Mu¨nster, Germany) for his advice on the statistical analysis of this study. We are grateful to Dr. Alison Dougall, Consultant for Medically Compromised Patients, Division of Special Care Dentistry, Dublin Dental School and Hospital, Trinity College, Dublin for kindly reviewing the manuscript.

References 1. Dammaschke T, Gerth HUV, Zu¨chner H, Scha¨fer E. Chemical and physical surface and bulk material characterization of white ProRoot MTA and two Portland cements. Dent Mater 2005;21:731–8. 2. Holland R, de Souza V, Nery MJ, Otoboni Filho JA, Bernabe´ PFE, Dezan Junior E. Reaction of dogs’ teeth to root canal filling with mineral trioxide aggregate or a glass ionomer sealer. J Endod 1999;25:728–30. 3. Faraco Ju´nior IM, Holland R. Response of the pulp of dogs to capping with mineral trioxide aggregate or a calcium hydroxide cement. Dent Traumatol 2001;17:163–6. 4. Takita T, Hayashi M, Takeichi O, et al. Effect of mineral trioxide aggregate on proliferation of cultured human dental pulp cells. Int Endod J 2006;39:415–22. 5. Al-Hezaimi K, Al-Hamdan K, Naghshbandi J, Oglesby S, Simon JHS, Rotstein I. Effect of white-colored mineral trioxide aggregate in different concentrations on Candida albicans in vitro. J Endod 2005;31:684–6.

JOE — Volume 36, Number 5, May 2010

Basic Research—Biology 6. Holland R, Otoboni-Filho JA, de Souza V, Nery MJ, Bernabe´ PFE, Dezan Junior E. Mineral trioxide aggregate repair of lateral root perforations. J Endod 2001;27: 281–4. 7. Dammaschke T, Wolff P, Sagheri D, Stratmann U, Scha¨fer E. Mineral trioxide aggregate for direct pulp capping: a histological comparison with calcium hydroxide in rat molar teeth. Quintessence Int 2010;41:e20–e30. 8. Pitt Ford TR, Torabinejad M, Abedi HR, Bakland LK, Kariyawasam SP. Using mineral trioxide aggregate as a pulp-capping material. J Am Dent Assoc 1996; 127:1491–4. 9. Aeinehchi M, Eslami B, Ghanbariha M, Saffer AS. Mineral trioxide aggregate (MTA) and calcium hydroxide as pulp-capping agents in human teeth: a preliminary report. Int Endod J 2003;36:225–31. 10. Dominguez MS, Witherspoon DE, Gutmann JL, Opperman LA. Histological and scanning electron microscopy assessment of various vital pulp-therapy materials. J Endod 2003;29:324–33. 11. Queiroz AM, Assed S, Leonardo MR, Nelson-Filho P, Silva LAB. MTA and calcium hydroxide for pulp capping. J Appl Oral Sci 2005;13:126–30. 12. Iwamoto CE, Adachi E, Pameijer CH, Barnes D, Romberg EE, Jefferies S. Clinical and histological evaluation of white ProRoot MTA in direct pulp capping. Am J Dent 2006;19:85–90. 13. Accorinte MLR, Holland R, Reis A, et al. Evaluation of mineral trioxide aggregate and calcium hydroxide cement as pulp-capping agents in human teeth. J Endod 2008; 34:1–6. 14. Asgary S, Eghbal MJ, Parirokh M, Ghanavati F, Rahimi H. A comperative study of histologic response to different pulp capping materials and a novel endodontic cement. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:609–14. 15. Min K-S, Park H-J, Lee S-K, et al. Effect of mineral trioxide aggregate on dentin bridge formation and expression of dentin sialoprotein and heme oxygenase-1 in human dental pulp. J Endod 2008;34:666–70. 16. Nair PNR, Duncan HF, Pitt Ford TR, Luder HU. Histological, ultrastructural and quantitative investigations on the response of healthy human pulps to experimental pulp capping with mineral trioxide aggregate: a randomized controlled trial. Int Endod J 2008;41:128–50. 17. Costa CAS, Duarte PT, de Souza PP, Giro EM, Hebling J. Cytotoxic effects and pulpal response caused by a mineral trioxide aggregate formulation and calcium hydroxide. Am J Dent 2008;21:255–61. 18. Sawicki L, Pameijer CH, Emerich K, Adamowicz-Klepalska B. Histological evaluation of mineral trioxide aggregate and calcium hydroxide in direct pulp capping of human immature permanent teeth. Am J Dent 2008;21:262–6. 19. Shayegan A, Petein M, Vandeen Abbeele A. The use of beta-tricalcium phosphate, white MTA, white Portland cement and calcium hydroxide for direct pulp capping of primary pig teeth. Dent Traumatol 2009;25:413–9. 20. Cotton WR. Pulp response to cavity preparation as studied by the method of 3Hthymidine audioradiographic. In: Finn FB, ed. Biology of the dental pulp organ: a symposium. Birmingham, AL: University of Alabama Press; 1968:69–101. 21. Casasco A, Casasco M, Calligaro A, et al. Cell proliferation in developing human dental pulp. A combined flow cytometric and immunohistochemical study. Eur J Oral Sci 1997;105:609–13. 22. Stanley HR. Cells of the dental pulp. Oral Surg Oral Med Oral Pathol 1962;15: 849–58. 23. Gottrup F, Andreasen JO. Wound healing subsequent to injury. In: Andreasen JO, Andreasen FM, eds. Textbook and color atlas of traumatic injuries to the teeth. 3rd ed. Copenhagen: Blackwell Munksgaard; 1994:13–76. 24. Sveen OB, Hawes RR. Differentiation of new odontoblasts and dentin bridge formation in rat molar teeth after tooth grinding. Arch Oral Biol 1968;13:1399–412.

JOE — Volume 36, Number 5, May 2010

25. Torabinejad M, Hong CU, Pitt Ford TR, Kettering JD. Cytotoxicity of four root end filling materials. J Endod 1995;21:489–92. 26. Camilleri J, Montesin FE, Papioannou S, McDonald F, Pitt Ford TR. Biocompatibility of two commercial forms of mineral trioxide aggregate. Int Endod J 2004;37:699–704. 27. Hørsted-Bindslev P, Vilkinis V, Sidlauskas A. Direct pulp capping of human pulps with a dentin bonding system or with calcium hydroxide cement. Oral Surg Oral Med Oral Pathol 2003;96:591–600. 28. Bosq J, Bourhis J. La bromode´xyuridine (BrdU): analyse des prolife´rations cellulaires. Ann Pathol 1997;17:171–8. 29. Nowakowski RS, Lewin SB, Miller MW. Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population. J Neurocytol 1989;18:311–8. 30. Dammaschke T. Rat molar teeth as a study model for direct pulp capping research in dentistry. Lab Anim 2010;44:1–6. 31. Kuratate M, Yoshiba K, Shigetani Y, Yoshiba N, Ohshima H, Okiji T. Immunohistochemical analysis of nestin, osteopontin, and proliferating cells in the reparative process of exposed dental pulp capped with mineral trioxide aggregate. J Endod 2008;34:970–4. 32. Te´cle`s O, Laurent P, Aubut V, About I. Human tooth culture: a study model for reparative dentinogenesis and direct pulp capping materials biocompatibility. J Biomed Mater Res B Appl Biomater 2008;85:180–7. 33. Greenhalgh DG. The role of apoptosis in wound healing. Int J Biochem Cell Biol 1998;30:1019–30. 34. Hart J. Inflammation 1: its role in the healing of acute wounds. J Wound Care 2002; 11:205–9. 35. Baume LJ. The biology of pulp and dentine: a historic, terminologic-taxonomic, histologic-biochemical, embryonic and clinical survey. Basel: Karger; 1980:119–23. 36. Goldberg M, Six N, Decup F, et al. Application of bioactive molecules in pulpcapping situations. Adv Dent Res 2001;15:91–5. 37. Goldberg M, Six N, Decup F, et al. Bioactive molecules and the future of pulp therapy. Am J Dent 2003;16:66–76. 38. Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med 2004;15:13–27. 39. Schro¨der U. Evaluation of healing following experimental pulpotomy of intact human teeth and capping with calcium hydroxide. Odontol Revy 1972;23:329–40. 40. Torneck CD, Moe H, Howley TP. The effect of calcium hydroxide on porcine pulp fibroblasts in vitro. J Endod 1983;9:131–6. 41. Bonson S, Jeansonne BG, Laillier TE. Root-end filling materials alter fibroblast differentiation. J Dent Res 2004;83:408–13. 42. Nakayama A, Ogiso B, Tanabe N, Takeichi O, Matsuzaka K, Inoue T. Behaivior of bone marrow osteoblast-like cells on mineral trioxide aggregate: morphology and expression of type I collagen and bone-related protein mRNAs. Int Endod J 2005;38:203–10. 43. Tani-Ishii N, Hamada N, Watanabe K, Tsujimoto Y, Teranaka T, Umemoto T. Expression of bone extracellular matrix proteins on osteoblast cells in presence of mineral trioxide. J Endod 2007;33:836–9. 44. Yasuda Y, Ogawa M, Arakawa T, Kodowaki T, Saito T. The effect of mineral trioxide aggreagte on the mineralization ability of rat dental pulp cells: an in vitro study. J Endod 2008;34:1057–60. 45. Thomson TS, Berry JE, Somerman MJ, Kirkwood KL. Cementoblasts maintain expression of osteocalcin in the presence of mineral trioxide aggregate. J Endod 2003;29:407–12. 46. Moghaddame-Jafari S, Mantellini MG, Botero TM, McDonald NJ, No¨r JE. Effect of ProRoot MTA on pulp cell apoptosis and proliferation in vitro. J Endod 2005; 31:387–91.

MTA for Direct Pulp Capping

819