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Wound Healing Process of Injured Pulp Tissues with Emdogain Gel
Basic Research—Biology
Wound Healing Process of Injured Pulp Tissues with Emdogain Gel Hikaru Kaida, DDS,* Takafumi Hamachi, DDS, PhD,* Hisashi Anan, DDS, PhD,† and Katsumasa Maeda, DDS, PhD* Abstract This study aimed to investigate the wound healing process of injured pulp tissues with Emdogain gel (EMD). Pulpotomy was performed for the first molars of the mandibles in rats. EMD or Vitapex (VIT)-containing calcium hydroxide was applied to the exposed pulp tissues. The treated teeth were extracted after 7, 14, and 28 days and prepared for histologic examination. In the VIT-treated group, the number of interleukin-1 (IL-1)– expressing macrophages initially increased, followed by that of transforming growth factor–1 (TGF1)– expressing macrophages. The number of cells expressing bone morphogenetic proteins (BMPs) gradually increased with reparative dentin formation. Meanwhile, in the EMD-treated group, cells expressing IL-1 or TGF-1 were few. However, the number of BMP-expressing cells, partly macrophages, increased in the early phase, and large amounts of reparative dentin were observed. This study demonstrated that different healing processes existed for EMD and VIT. BMP-expressing macrophages might play important roles in reparative dentin formation. (J Endod 2008;34:26 –30)
Key Words BMP, dental pulp, Emdogain, macrophage, wound healing
From the *Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan; and †Section of Operative Dentistry and Endodontology, Department of Odontology, Fukuoka Dental College, Fukuoka, Japan. Address requests for reprints to Dr Takafumi Hamachi, Department of Periodontology, Faculty of Dental Science, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 8128582, Japan. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2008 by the American Association of Endodontists. doi:10.1016/j.joen.2007.09.011
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arious materials have previously been used in pulp capping and pulpotomy procedures. Among them, calcium hydroxide has been conventionally used in clinics (1). However, it has been reported that pulp tissues become strongly irritated, and a necrotic layer forms as a result of the alkaline action of calcium hydrate (2). Although reparative dentin is newly formed by calcium hydroxide, its structure is porous (3). The formation of pores increases the risk of bacterial infection (4). Moreover, although the majority of studies with calcium hydroxide– based materials reported hard tissue bridging, Olsson et al (5) claimed that these did not give satisfactory results. For these reasons, the development of biocompatible materials that induce a dentin/pulp complex is preferred. Recently, many studies have reported that new biocompatible materials such as bone morphogenetic proteins (BMPs) (6), osteogenic protein–1 (OP-1) (7), demineralized dentin (8), and mineral trioxide aggregate (MTA) (9) can induce the formation of reparative dentin. Recent research has suggested that Emdogain gel (EMD) (BIORA AB, Malmö, Sweden), which is often used in periodontal regenerative therapy, also has the capacity to induce rapid reparative dentin formation in pulpotomized teeth (10, 11). EMD is made from an enamel matrix derivative secreted from Hertwig’s epithelial sheath during porcine tooth development (12, 13). This enamel matrix derivative contains amelogenin as its major component in addition to other enamel matrix proteins such as enamelins, tuftelin, amelin, and ameloblastin (14 –17). It serves as an important regulator of enamel mineralization (18) and plays an important role during periodontal tissue formation (12, 13). It also stimulates the regeneration of periodontal tissues including the acellular cementum, periodontal ligaments, and alveolar bone by mimicking tooth development. However, the precise mechanisms of periodontal tissue regeneration with EMD remain unknown. Furthermore, the mechanisms of wound healing in the injured pulp tissues with EMD as a pulp-capping material have hardly been demonstrated. Therefore, the purpose of this study was to investigate the wound healing process of injured pulp tissues with EMD. Because it was reported that EMD enhanced alkaline phosphatase (ALP) activity and the expression of bone matrix proteins in osteoblasts (19), ALP staining and immunostaining against dentin matrix protein–1 (DMP-1), which was one of the bone matrix proteins identified from dentin, were initially performed to investigate the status of the injured pulp and reparative dentin. Moreover, we investigated the behavior of inflammatory cells, the expressions of inflammatory cytokine, anti-inflammatory cytokine, and hard tissue formation–activated factor by using immunohistochemical methods. These are considered to be important for bone tissue remodeling (20). Macrophages and T cells, interleukin-1 (IL-1), transforming growth factor–1 (TGF-1), and BMPs were selected as markers. To elucidate the effect of EMD on wound healing of the injured pulp tissues, the results obtained with EMD were compared with those of Vitapex (calcium hydroxide and iodoform paste; Neo-Dental, Tokyo, Japan) (VIT)-containing calcium hydroxide.
Materials and Methods Surgical Procedure A total of 90 mandibular first molars from 45 male Sprague-Dawley rats (age, 5 weeks) were used in this study. All experimental procedures were conducted in accordance with the animal experimental guidelines of Kyushu University. The animals un-
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Basic Research—Biology derwent general anesthesia with pentobarbital sodium. After the first molars were cleaned and disinfected with 3% hydrogen peroxide followed by swabbing of the mouth with 0.2% chlorhexidine gluconate, pulpotomy was performed with a #1/2 round bur rotated by a low-speed electric engine. The size of the cavities was 1 mm in depth and 1 mm in diameter. During cavity preparation, the tooth and cutting instrument were irrigated with sterile saline to prevent impairment from heat. Bleeding was controlled with sterile saline irrigation and the use of cotton pellets. All the procedures resulted in pulp exposure of uniform size. The exposed pulp tissues were covered with Emdogain gel (EMDtreated group, 30 teeth) or Vitapex (VIT-treated group, 30 teeth). The cavities were subsequently sealed with glass ionomer cement (Fuji IX; GC, Tokyo, Japan). Moreover, some teeth were only sealed with glass ionomer cement without the pulp-capping material as control (GICtreated group, 30 teeth). Each group was randomly divided into 3 additional groups, and the animals were killed at 7, 14, and 28 days after treatment. The number of teeth analyzed at each stage in each group was 10.
Section Preparation Each animal was fixed by intracardiac perfusion of periodatelysine-paraformaldehyde (3% paraformaldehyde, 0.01 mol/L NaIO4, and 0.075 mol/L lysine in 0.025 mol/L phosphate buffer, pH 7.3). The mandibular first molars were dissected with the surrounding jaw bones and post-fixed with the same fixative at 4°C for 12 hours. The samples were then demineralized with 10% ethylenediaminetetraacetic acid in a 7.5% polyvinylpyrrolidone solution (Sigma Chemical Co, St Louis, MO) at 4°C for 14 days. The decalcified specimens were then washed with 0.01 mol/L phosphate-buffered saline (PBS) and freeze-embedded in OCT compound (Miles Scientific, Naperville, IL). Frozen specimens were serially step-sectioned (5 m) parallel to the long axis of the tooth. Subsequently, enzyme activities and immunohistochemical reactions were investigated in the serial sections. Enzyme Histochemistry Nonspecific ALP staining was performed by the method described by Burstone (21), with naphthol AS-BI phosphate (Sigma) as the substrate and Fast Red Violet LB salt (Sigma) as the coupler. The sections were counterstained with methyl green (Sigma). Immunohistochemistry Frozen sections were first incubated with 0.3% H2O2 in methanol for 30 minutes to remove endogenous tissue peroxidase. After rinsing in PBS, the sections were incubated with appropriate primary antibodies such as mouse monoclonal antibody to rat macrophages, ED1 (Serotec Co, Indianapolis, IN); mouse monoclonal antibody to rat T cells, CD5 (Seikagaku Co, Tokyo, Japan); rabbit polyclonal antibodies to rat IL-1 (Endogen, Woburn, MA); rabbit polyclonal antibodies to TGF-1 (Promega Co, Madison, WI); goat polyclonal antibodies to BMP-2 (Santa Cruz Biotechnology Inc, Santa Cruz, CA); goat polyclonal antibodies to BMP-4 (Santa Cruz); and mouse and rabbit polyclonal antibodies to DMP-1 (Takara Bio Inc, Shiga, Japan). Immunoreactivity was developed by using a streptavidin-biotin peroxidase staining system (Nichirei Co, Tokyo, Japan). Throughout the staining, the sections were washed 3 times for 5 minutes each with PBS between each incubation step. Finally, the sections were exposed to DAB (Nichirei) until the color was developed and counterstained with methyl green. In addition, double staining was performed to investigate the relationship between ED1-positive macrophages and the expressions of IL-1, TGF-1, BMP-2, or BMP-4. Frozen sections were first incubated with anti–IL-1, –TGF-1, –BMP-2, or –BMP-4 antibody, and the color was developed by using DAB (Nichirei), as described above. After washJOE — Volume 34, Number 1, January 2008
ing with PBS, the anti-ED1 antibody was placed on the sections and left to react. Immunoreactivity was developed by using Histofine Simple Stain AP (M) (Nichirei). Tetramisole hydrochloride (Sigma) was used to block endogenous ALP activity. After the color was developed, the sections were counterstained with methyl green.
Histometric Analysis Histometric analysis was performed with serial sections taken from near the central part of injured pulp tissues. The width of the necrotic layer and the area of reparative dentin formation were measured with the computer software Scion Image (Scion Co, Frederick, MD). ED1-positive mononuclear cells, CD5-positive cells, IL-1–, TGF1–, BMP-2–, and BMP-4 – expressing cells were counted in a field determined in an ocular grid (7 mm ⫻ 7 mm), which was placed right under the necrotic layer by using a ⫻400 magnification objective lens (Fig. 1). The average values of 10 sections taken from 10 teeth were used as reported results for each stage in each group. Statistical analysis was performed with the Student t test.
Results Histochemical and Immunohistochemical Findings VIT-treated Group Throughout the experimental period, strong ALP activity was observed, and CD5-positive T cells were scattered in the injured pulp tissues. Moreover, the area of the necrotic layer was significant. Seven days after treatment, many neutrophils, ED1-positive macrophages, and IL-1– expressing cells were observed below the necrotic layer. TGF-1– and BMP-expressing cells were few; and reparative dentin was hardly seen (Figs. 2a and 3a). Fourteen days after treatment, many neutrophils, ED1-positive macrophages, and IL-1– expressing cells were still observed below the necrotic layer. However, TGF-1– expressing cells increased below the necrotic layer. DMP-1–positive reparative dentin was added to the surface of the root canal wall, and the number of BMP-expressing cells was increased near the capillaries and the reparative dentin (Figs. 2b, 3b, and 4a–f ).
Figure 1. Method of histometric analysis. Each immunopositive cell was counted in a field determined in an ocular grid, which was placed right under the necrotic layer by using a ⫻400 objective lens.
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Figure 2. ALP staining at 7 (a), 14 (b), and 28 (c) days after VIT treatment and 7 (d ), 14 (e), and 28 (f ) days after EMD treatment. The red sites show ALP activity. ND, new dentin; D, dentin. Bars: 250 m.
Twenty-eight days after treatment, large amounts of DMP-1–positive reparative dentin were observed in the root canal. However, many pores were present in the reparative dentin. The numbers of neutrophils, ED1-positive macrophages, and cells expressing IL-1 or TGF-1 decreased. On the other hand, many BMP-expressing cells were observed near the reparative dentin (Figs. 2c and 3c). In the VIT-treated group, it seemed that macrophages correlated well with cells expressing IL-1 or TGF-1 on the basis of cell shape and location. EMD-treated Group Throughout the experimental period, strong ALP activity was observed, and CD5-positive T cells were scattered in the injured pulp tissues. Moreover, the area of the necrotic layer was narrow, and cells expressing IL-1 or TGF-1 were few. Seven days after treatment, many ED1-positive macrophages were observed below the necrotic layer. DMP-1–positive reparative dentin was observed on the coronal side of the injured pulp tissues, and many
Figure 4. Immunostaining at 14 days in the VIT-treated group (a–f ) and EMDtreated group (g–l). The brown color shows immunopositive cells. IL-1– or TGF-1– expressing cells in the VIT-treated group and BMP-expressing cells in the EMD-treated group correlated with macrophages as assessed by their shape and location. ND, new dentin. Bars: 50 m.
BMP-expressing cells were observed near the reparative dentin and the necrotic layer (Figs. 2d and 3d). Fourteen days after treatment, many ED1-positive macrophages were still observed below the necrotic layer. However, BMP-expressing cells and the amounts of reparative dentin increased further (Figs. 2e, 3e, and 4g–l). Twenty-eight days after treatment, large amounts of reparative dentin were observed from the coronal to the root canal entrance. Pores, which were seen in the VIT-treated group, were few. The numbers of ED1-positive macrophages and BMP-expressing cells decreased (Figs. 2f and 3f ). In the EMD-treated group, it seemed that macrophages correlated with cells expressing BMP-2 or BMP-4 on the basis of cell shape and location. GIC-treated Group The histochemical and immunohistochemical findings of the GICtreated group were similar to those of the VIT-treated group (data not shown).
Double Staining In the VIT- and GIC-treated groups, ED1-positive macrophages expressing IL-1 or TGF-1 were observed (Fig. 5a and b) (data not shown in the GIC-treated group). On the other hand, ED1-positive macrophages expressing BMP-2 or BMP-4 were partly observed in the EMDtreated group (Fig. 5c and d).
Figure 3. Immunostaining with DMP-1 antibody at 7 (a), 14 (b), and 28 (c) days after VIT treatment and 7 (d ), 14 (e), and 28 (f ) days after EMD treatment. ND, new dentin. Bars: 50 m.
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Histometric Findings The width of the necrotic layer was significantly narrow in the order of EMD-, GIC-, and VIT-treated groups throughout the experimental period (Fig. 6a). In regard to the area of reparative dentin formation, the EMDtreated group showed a significantly larger area than that in the other 2 groups throughout the experimental period. Twenty-eight days after treatment, the VIT-treated group showed a significantly larger area than that in the GIC-treated group (Fig. 6b). In all groups, many ED1-positive cells were observed, and their number decreased in a time-dependent manner (Fig. 6c). JOE — Volume 34, Number 1, January 2008
Basic Research—Biology
Figure 5. Double staining at 14 days in the VIT-treated group: (a) anti-ED1 and anti–IL-1; (b) anti-ED1 and anti–TGF-1. Double staining at 14 days in the EMD-treated group: (c) anti-ED1 and anti–BMP-2; (d ) anti-ED1 and anti– BMP-4. The blue and brown colors show ED1-positive cells and IL-1–, TGF1–, BMP-2–, or BMP-4 – expressing cells, respectively. Bars: 50 m.
TGF-1 were few. However, the number of BMP-expressing cells increased in the early phase, and large amounts of reparative dentin were observed. Mundy (20) has previously described the remodeling of bone tissues. The inflammatory reaction is first expanded by infiltrating inflammatory cells, and inflammatory cytokines such as IL-1 and tumor necrosis factor–␣ are released. Next, cells expressing anti-inflammatory cytokines such as TGF-1 increase, and granulation tissues are formed. Then the number of cells expressing BMPs increases, and the formation of bone tissues is activated. The results in our study suggested that the wound healing process of injured pulp tissues in the VIT- and GIC-treated groups was similar to that of bone tissue remodeling. On the other hand, in the EMD-treated group, the number of BMP-expressing cells increased without an increase in the expressions of IL-1 and TGF-1. We also investigated the expressions of IL-1 and TGF-1 at 1, 3, and 5 days after treatment, and their levels did not change (data not shown). This suggested that the wound healing process of injured pulp tissues with EMD differed from the normal circumstances.
In all groups, the number of CD5-positive cells was few throughout the experimental period (Fig. 6d ). The number of IL-1– expressing cells in the EMD-treated group was significantly lower than that in the other 2 groups throughout the experimental period (Fig. 6e). In the EMD-treated group, cells expressing TGF-1 were very few throughout the experimental period. On the other hand, the number of cells expressing TGF-1 peaked at 14 days in the other 2 groups. The numbers of TGF-1– expressing cells in the VIT- and GIC-treated groups were significantly higher than that in the EMD-treated group at 14 and 28 days (Fig. 6f ). In the EMD-treated group, the number of cells expressing BMP-2 or BMP-4 peaked at 14 days. The number of BMP-2– or BMP-4 – expressing cells in the EMD-treated group was significantly higher than that in the other 2 groups at 7 and 14 days. On the other hand, in the VITand GIC-treated groups, the number of cells expressing BMP-2 or BMP-4 increased in a time-dependent manner (Fig. 6g and h).
Discussion In all experimental groups, a strong ALP activity and DMP-1– positive reparative dentin were observed in the injured pulp tissues. ALP is a marker of hard tissue–forming cells, and DMP-1 plays an important role in odontoblast differentiation and matrix mineralization (22). We assumed that mineralized reparative dentin formation was induced in all experimental groups. However, the amounts of reparative dentin in the EMD-treated group were significantly larger than those in the other 2 groups, and there were many differences between the EMD-treated group and the other 2 groups for the structure of reparative dentin and the process of reparative dentin formation. In the VIT-treated group, the irritation of pulp tissues was significant, and the newly formed reparative dentin was porous, as shown by previous reports (2, 3). Many neutrophils and macrophages were observed below the necrotic layer. The number of IL-1– expressing macrophages initially increased, followed by that of TGF-1– expressing macrophages. Cells expressing BMPs gradually increased in number with reparative dentin formation. The findings of the GIC-treated group were similar to those of the VIT-treated group. Although the width of the necrotic layer was narrow compared with that of the VIT-treated group, there were small amounts of reparative dentin formation 28 days after treatment. It seemed that these differences were based on calcium hydroxide contained in VIT. On the other hand, in the EMD-treated group, the width of the necrotic layer was narrow. Although a large number of macrophages were observed below the necrotic layer, cells expressing IL-1 or JOE — Volume 34, Number 1, January 2008
Figure 6. Histometric analysis: (a) width of the necrotic layer; (b) area of reparative dentin formation; (c) number of ED1-positive cells; (d ) number of CD5-positive cells; (e) number of IL-1– expressing cells; (f ) number of TGF1– expressing cells; (g) number of BMP-2– expressing cells; (h) number of BMP-4 – expressing cells. Yellow bars ⫽ VIT-treated group; Blue bars ⫽ EMDtreated group; White bars ⫽ GIC-treated group; 夝 ⫽ P ⬍ .05; 夝夝 ⫽ P ⬍ .01.
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Basic Research—Biology IL-1, an inflammatory cytokine, has several functions such as the activation of bone resorption, the activation of immunocytes, and the production of prostaglandin or collagenase. These effects are greatly concerned with the extension of inflammatory reactions and tissue breakdown (23). TGF-1 inhibits the production of inflammatory cytokines (24) and promotes the production of extracellular matrix and the proliferation capacity of osteoblasts and odontoblasts (25, 26). These effects result in the suppression of inflammatory reactions and promote wound healing. Recently it has been reported that EMD suppresses the inflammatory cytokine production by immunocytes (27) and contains TGF-–like molecules (28). EMD might create a favorable environment for promoting wound healing in the injured pulp tissues. BMPs, which belong to the TGF- superfamily, can independently induce ectopic hard tissue formation (29, 30). The use of BMPs has been attempted in various regeneration fields. In the EMD-treated group, the number of BMP-expressing cells increased in the early phase, and large amounts of reparative dentin were observed. It has been reported that BMPs promote pulp cells to differentiate into odontoblasts (31), and EMD contains BMP-like molecules (32). BMP-expressing cells and BMP-like molecules in EMD might cooperate to promote odontoblast differentiation and reparative dentin formation. Interestingly, BMP-expressing cells were partly macrophages because of their shape and location in the EMD-treated group. Furthermore, double staining demonstrated the existence of BMP-expressing macrophages. Although there is little information about whether macrophages express BMPs, Champagne et al (33) suggested that macrophages expressing BMPs play an important role in healing of hard tissues. EMD might directly or indirectly induce BMP-expressing macrophages. It is necessary to examine in vitro and in vivo which substances contained in EMD induce BMP-expressing macrophages. Recently a study that applied EMD to human pulp tissues was reported (34). Although the postoperative symptoms were less and the amounts of reparative dentin formation were markedly larger than in teeth treated with calcium hydroxide, there are still unresolved problems. In conclusion, this study demonstrated that there were differences in the wound healing process between EMD and VIT-containing calcium hydroxide when used as a pulp-capping material. The role of macrophages in the wound healing process was greatly associated with these differences. BMP-expressing macrophages induced by EMD might play important roles in reparative dentin formation.
Acknowledgments This study was partly supported by a Grant-in-Aid for Scientific Research, 16209056 and 18209057, from the Ministry of Education, Science, Sports and Culture of Japan. We are also grateful to the members of the Section of Periodontology, Faculty of Dental Science, Kyushu University.
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6. Nakashima M, Akamine A. The application of tissue engineering to regeneration of pulp and dentin in endodontics. J Endod 2005;31:711– 8. 7. Jepsen S, Albers HK, Fleiner B, Tucker M, Rueger D. Recombinant human osteogenic protein-1 induces dentin formation: an experimental study in miniature swine. J Endod 1997;23:378 – 82. 8. Nakashima M. Dentin induction by implants of autolyzed antigen-extracted allogeneic dentin on amputated pulps of dogs. Endod Dent Traumatol 1989;5:279 – 86. 9. Barrieshi-Nusair KM, Qudeimat MA. A prospective clinical study of mineral trioxide aggregate for partial pulpotomy in cariously exposed permanent teeth. J Endod 2006;32:731–5. 10. Ishizaki NT, Matsumoto K, Kimura Y, Wang X, Yamashita A. Histopathological study of dental pulp tissue capped with enamel matrix derivative. J Endod 2003;29:176 –9. 11. Nakamura Y, Slaby I, Matsumoto K, Ritchie HH, Lyngstadaas SP. Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative. Calcif Tissue Int 2004;75:243–52. 12. Hammarström L. Enamel matrix, cementum development and regeneration. J Clin Periodontol 1997;24:658 – 68. 13. Bosshardt DD, Nanci A. Hertwig’s epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in porcine teeth. J Clin Periodontol 2004;31:184 –92. 14. Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU. Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 1980;255:9760 – 8. 15. Ogata Y, Shimokawa H, Sasaki S. Purification, characterization, and biosynthesis of bovine enamelins. Calcif Tissue Int 1988;43:389 –99. 16. Cerný R, Slaby I, Hammarström L, Wurtz T. A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains. J Bone Miner Res 1996;11:883–91. 17. Krebsbach PH, Lee SK, Matsuki Y, Kozak CA, Yamada KM, Yamada Y. Full-length sequence, localization, and chromosomal mapping of ameloblastin: a novel toothspecific gene. J Biol Chem 1996;271:4431–5. 18. Robinson C, Brookes SJ, Shore RC, Kirkham J. The developing enamel matrix: nature and function. Eur J Oral Sci 1998;106:282–91. 19. He J, Jiang J, Safavi KE, Spångberg LS, Zhu Q. Emdogain promotes osteoblast proliferation and differentiation and stimulates osteoprotegerin expression. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:239 – 45. 20. Mundy GR. Bone remodeling and its disorders. London: Martin Dunitz Ltd, 1999:1–11. 21. Burstone MS. Enzyme histochemistry. New York: Academic Press, Inc, 1962: 190 –220. 22. Massa LF, Ramachandran A, George A, Arana-Chavez VE. Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry. Histochem Cell Biol 2005;124:197–205. 23. Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunol 1989;44:153–205. 24. Musso T, Espinoza-Delgado I, Pulkki K, Gusella GL, Longo DL, Varesio L. Transforming growth factor beta downregulates interleukin-1 (IL-1)-induced IL-6 production by human monocytes. Blood 1990;76:2466 –9. 25. Hock JM, Canalis E, Centrella M. Transforming growth factor-beta stimulates bone matrix apposition and bone cell replication in cultured fetal rat calvariae. Endocrinology 1990;126:421– 6. 26. Melin M, Joffre-Romeas A, Farges JC, Couble ML, Magloire H, Bleicher F. Effects of TGFbeta1 on dental pulp cells in cultured human tooth slices. J Dent Res 2000;79:1689 –96. 27. Myhre AE, Lyngstadaas SP, Dahle MK, et al. Anti-inflammatory properties of enamel matrix derivative in human blood. J Periodontal Res 2006;41:208 –13. 28. Kawase T, Okuda K, Yoshie H, Burns DM. Anti-TGF-beta antibody blocks enamel matrix derivative-induced upregulation of p21WAF1/cip1 and prevents its inhibition of human oral epithelial cell proliferation. J Periodontal Res 2002;37:255– 62. 29. Urist MR. Bone: formation by autoinduction. Science 1965;150:893–9. 30. Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res 1971;50:1392– 406. 31. Saito T, Ogawa M, Hata Y, Bessho K. Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts. J Endod 2004;30:205– 8. 32. Takayama T, Suzuki N, Narukawa M, Tokunaga T, Otsuka K, Ito K. Enamel matrix derivative stimulates core binding factor alpha1/Runt-related transcription factor-2 expression via activation of Smad1 in C2C12 cells. J Periodontol 2005;76:244 –9. 33. Champagne CM, Takebe J, Offenbacher S, Cooper LF. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2. Bone 2002; 30:26 –31. 34. Olsson H, Davies JR, Holst KE, Schröder U, Petersson K. Dental pulp capping: effect of Emdogain Gel on experimentally exposed human pulps. Int Endod J 2005; 38:186 –94.
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Wound Healing Process of Injured Pulp Tissues with Emdogain Gel Hikaru Kaida, DDS,* Takafumi Hamachi, DDS, PhD,* Hisashi Anan, DDS, PhD,† and Katsumasa Maeda, DDS, PhD* Abstract This study aimed to investigate the wound healing process of injured pulp tissues with Emdogain gel (EMD). Pulpotomy was performed for the first molars of the mandibles in rats. EMD or Vitapex (VIT)-containing calcium hydroxide was applied to the exposed pulp tissues. The treated teeth were extracted after 7, 14, and 28 days and prepared for histologic examination. In the VIT-treated group, the number of interleukin-1 (IL-1)– expressing macrophages initially increased, followed by that of transforming growth factor–1 (TGF1)– expressing macrophages. The number of cells expressing bone morphogenetic proteins (BMPs) gradually increased with reparative dentin formation. Meanwhile, in the EMD-treated group, cells expressing IL-1 or TGF-1 were few. However, the number of BMP-expressing cells, partly macrophages, increased in the early phase, and large amounts of reparative dentin were observed. This study demonstrated that different healing processes existed for EMD and VIT. BMP-expressing macrophages might play important roles in reparative dentin formation. (J Endod 2008;34:26 –30)
Key Words BMP, dental pulp, Emdogain, macrophage, wound healing
From the *Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan; and †Section of Operative Dentistry and Endodontology, Department of Odontology, Fukuoka Dental College, Fukuoka, Japan. Address requests for reprints to Dr Takafumi Hamachi, Department of Periodontology, Faculty of Dental Science, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 8128582, Japan. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2008 by the American Association of Endodontists. doi:10.1016/j.joen.2007.09.011
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arious materials have previously been used in pulp capping and pulpotomy procedures. Among them, calcium hydroxide has been conventionally used in clinics (1). However, it has been reported that pulp tissues become strongly irritated, and a necrotic layer forms as a result of the alkaline action of calcium hydrate (2). Although reparative dentin is newly formed by calcium hydroxide, its structure is porous (3). The formation of pores increases the risk of bacterial infection (4). Moreover, although the majority of studies with calcium hydroxide– based materials reported hard tissue bridging, Olsson et al (5) claimed that these did not give satisfactory results. For these reasons, the development of biocompatible materials that induce a dentin/pulp complex is preferred. Recently, many studies have reported that new biocompatible materials such as bone morphogenetic proteins (BMPs) (6), osteogenic protein–1 (OP-1) (7), demineralized dentin (8), and mineral trioxide aggregate (MTA) (9) can induce the formation of reparative dentin. Recent research has suggested that Emdogain gel (EMD) (BIORA AB, Malmö, Sweden), which is often used in periodontal regenerative therapy, also has the capacity to induce rapid reparative dentin formation in pulpotomized teeth (10, 11). EMD is made from an enamel matrix derivative secreted from Hertwig’s epithelial sheath during porcine tooth development (12, 13). This enamel matrix derivative contains amelogenin as its major component in addition to other enamel matrix proteins such as enamelins, tuftelin, amelin, and ameloblastin (14 –17). It serves as an important regulator of enamel mineralization (18) and plays an important role during periodontal tissue formation (12, 13). It also stimulates the regeneration of periodontal tissues including the acellular cementum, periodontal ligaments, and alveolar bone by mimicking tooth development. However, the precise mechanisms of periodontal tissue regeneration with EMD remain unknown. Furthermore, the mechanisms of wound healing in the injured pulp tissues with EMD as a pulp-capping material have hardly been demonstrated. Therefore, the purpose of this study was to investigate the wound healing process of injured pulp tissues with EMD. Because it was reported that EMD enhanced alkaline phosphatase (ALP) activity and the expression of bone matrix proteins in osteoblasts (19), ALP staining and immunostaining against dentin matrix protein–1 (DMP-1), which was one of the bone matrix proteins identified from dentin, were initially performed to investigate the status of the injured pulp and reparative dentin. Moreover, we investigated the behavior of inflammatory cells, the expressions of inflammatory cytokine, anti-inflammatory cytokine, and hard tissue formation–activated factor by using immunohistochemical methods. These are considered to be important for bone tissue remodeling (20). Macrophages and T cells, interleukin-1 (IL-1), transforming growth factor–1 (TGF-1), and BMPs were selected as markers. To elucidate the effect of EMD on wound healing of the injured pulp tissues, the results obtained with EMD were compared with those of Vitapex (calcium hydroxide and iodoform paste; Neo-Dental, Tokyo, Japan) (VIT)-containing calcium hydroxide.
Materials and Methods Surgical Procedure A total of 90 mandibular first molars from 45 male Sprague-Dawley rats (age, 5 weeks) were used in this study. All experimental procedures were conducted in accordance with the animal experimental guidelines of Kyushu University. The animals un-
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Basic Research—Biology derwent general anesthesia with pentobarbital sodium. After the first molars were cleaned and disinfected with 3% hydrogen peroxide followed by swabbing of the mouth with 0.2% chlorhexidine gluconate, pulpotomy was performed with a #1/2 round bur rotated by a low-speed electric engine. The size of the cavities was 1 mm in depth and 1 mm in diameter. During cavity preparation, the tooth and cutting instrument were irrigated with sterile saline to prevent impairment from heat. Bleeding was controlled with sterile saline irrigation and the use of cotton pellets. All the procedures resulted in pulp exposure of uniform size. The exposed pulp tissues were covered with Emdogain gel (EMDtreated group, 30 teeth) or Vitapex (VIT-treated group, 30 teeth). The cavities were subsequently sealed with glass ionomer cement (Fuji IX; GC, Tokyo, Japan). Moreover, some teeth were only sealed with glass ionomer cement without the pulp-capping material as control (GICtreated group, 30 teeth). Each group was randomly divided into 3 additional groups, and the animals were killed at 7, 14, and 28 days after treatment. The number of teeth analyzed at each stage in each group was 10.
Section Preparation Each animal was fixed by intracardiac perfusion of periodatelysine-paraformaldehyde (3% paraformaldehyde, 0.01 mol/L NaIO4, and 0.075 mol/L lysine in 0.025 mol/L phosphate buffer, pH 7.3). The mandibular first molars were dissected with the surrounding jaw bones and post-fixed with the same fixative at 4°C for 12 hours. The samples were then demineralized with 10% ethylenediaminetetraacetic acid in a 7.5% polyvinylpyrrolidone solution (Sigma Chemical Co, St Louis, MO) at 4°C for 14 days. The decalcified specimens were then washed with 0.01 mol/L phosphate-buffered saline (PBS) and freeze-embedded in OCT compound (Miles Scientific, Naperville, IL). Frozen specimens were serially step-sectioned (5 m) parallel to the long axis of the tooth. Subsequently, enzyme activities and immunohistochemical reactions were investigated in the serial sections. Enzyme Histochemistry Nonspecific ALP staining was performed by the method described by Burstone (21), with naphthol AS-BI phosphate (Sigma) as the substrate and Fast Red Violet LB salt (Sigma) as the coupler. The sections were counterstained with methyl green (Sigma). Immunohistochemistry Frozen sections were first incubated with 0.3% H2O2 in methanol for 30 minutes to remove endogenous tissue peroxidase. After rinsing in PBS, the sections were incubated with appropriate primary antibodies such as mouse monoclonal antibody to rat macrophages, ED1 (Serotec Co, Indianapolis, IN); mouse monoclonal antibody to rat T cells, CD5 (Seikagaku Co, Tokyo, Japan); rabbit polyclonal antibodies to rat IL-1 (Endogen, Woburn, MA); rabbit polyclonal antibodies to TGF-1 (Promega Co, Madison, WI); goat polyclonal antibodies to BMP-2 (Santa Cruz Biotechnology Inc, Santa Cruz, CA); goat polyclonal antibodies to BMP-4 (Santa Cruz); and mouse and rabbit polyclonal antibodies to DMP-1 (Takara Bio Inc, Shiga, Japan). Immunoreactivity was developed by using a streptavidin-biotin peroxidase staining system (Nichirei Co, Tokyo, Japan). Throughout the staining, the sections were washed 3 times for 5 minutes each with PBS between each incubation step. Finally, the sections were exposed to DAB (Nichirei) until the color was developed and counterstained with methyl green. In addition, double staining was performed to investigate the relationship between ED1-positive macrophages and the expressions of IL-1, TGF-1, BMP-2, or BMP-4. Frozen sections were first incubated with anti–IL-1, –TGF-1, –BMP-2, or –BMP-4 antibody, and the color was developed by using DAB (Nichirei), as described above. After washJOE — Volume 34, Number 1, January 2008
ing with PBS, the anti-ED1 antibody was placed on the sections and left to react. Immunoreactivity was developed by using Histofine Simple Stain AP (M) (Nichirei). Tetramisole hydrochloride (Sigma) was used to block endogenous ALP activity. After the color was developed, the sections were counterstained with methyl green.
Histometric Analysis Histometric analysis was performed with serial sections taken from near the central part of injured pulp tissues. The width of the necrotic layer and the area of reparative dentin formation were measured with the computer software Scion Image (Scion Co, Frederick, MD). ED1-positive mononuclear cells, CD5-positive cells, IL-1–, TGF1–, BMP-2–, and BMP-4 – expressing cells were counted in a field determined in an ocular grid (7 mm ⫻ 7 mm), which was placed right under the necrotic layer by using a ⫻400 magnification objective lens (Fig. 1). The average values of 10 sections taken from 10 teeth were used as reported results for each stage in each group. Statistical analysis was performed with the Student t test.
Results Histochemical and Immunohistochemical Findings VIT-treated Group Throughout the experimental period, strong ALP activity was observed, and CD5-positive T cells were scattered in the injured pulp tissues. Moreover, the area of the necrotic layer was significant. Seven days after treatment, many neutrophils, ED1-positive macrophages, and IL-1– expressing cells were observed below the necrotic layer. TGF-1– and BMP-expressing cells were few; and reparative dentin was hardly seen (Figs. 2a and 3a). Fourteen days after treatment, many neutrophils, ED1-positive macrophages, and IL-1– expressing cells were still observed below the necrotic layer. However, TGF-1– expressing cells increased below the necrotic layer. DMP-1–positive reparative dentin was added to the surface of the root canal wall, and the number of BMP-expressing cells was increased near the capillaries and the reparative dentin (Figs. 2b, 3b, and 4a–f ).
Figure 1. Method of histometric analysis. Each immunopositive cell was counted in a field determined in an ocular grid, which was placed right under the necrotic layer by using a ⫻400 objective lens.
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Figure 2. ALP staining at 7 (a), 14 (b), and 28 (c) days after VIT treatment and 7 (d ), 14 (e), and 28 (f ) days after EMD treatment. The red sites show ALP activity. ND, new dentin; D, dentin. Bars: 250 m.
Twenty-eight days after treatment, large amounts of DMP-1–positive reparative dentin were observed in the root canal. However, many pores were present in the reparative dentin. The numbers of neutrophils, ED1-positive macrophages, and cells expressing IL-1 or TGF-1 decreased. On the other hand, many BMP-expressing cells were observed near the reparative dentin (Figs. 2c and 3c). In the VIT-treated group, it seemed that macrophages correlated well with cells expressing IL-1 or TGF-1 on the basis of cell shape and location. EMD-treated Group Throughout the experimental period, strong ALP activity was observed, and CD5-positive T cells were scattered in the injured pulp tissues. Moreover, the area of the necrotic layer was narrow, and cells expressing IL-1 or TGF-1 were few. Seven days after treatment, many ED1-positive macrophages were observed below the necrotic layer. DMP-1–positive reparative dentin was observed on the coronal side of the injured pulp tissues, and many
Figure 4. Immunostaining at 14 days in the VIT-treated group (a–f ) and EMDtreated group (g–l). The brown color shows immunopositive cells. IL-1– or TGF-1– expressing cells in the VIT-treated group and BMP-expressing cells in the EMD-treated group correlated with macrophages as assessed by their shape and location. ND, new dentin. Bars: 50 m.
BMP-expressing cells were observed near the reparative dentin and the necrotic layer (Figs. 2d and 3d). Fourteen days after treatment, many ED1-positive macrophages were still observed below the necrotic layer. However, BMP-expressing cells and the amounts of reparative dentin increased further (Figs. 2e, 3e, and 4g–l). Twenty-eight days after treatment, large amounts of reparative dentin were observed from the coronal to the root canal entrance. Pores, which were seen in the VIT-treated group, were few. The numbers of ED1-positive macrophages and BMP-expressing cells decreased (Figs. 2f and 3f ). In the EMD-treated group, it seemed that macrophages correlated with cells expressing BMP-2 or BMP-4 on the basis of cell shape and location. GIC-treated Group The histochemical and immunohistochemical findings of the GICtreated group were similar to those of the VIT-treated group (data not shown).
Double Staining In the VIT- and GIC-treated groups, ED1-positive macrophages expressing IL-1 or TGF-1 were observed (Fig. 5a and b) (data not shown in the GIC-treated group). On the other hand, ED1-positive macrophages expressing BMP-2 or BMP-4 were partly observed in the EMDtreated group (Fig. 5c and d).
Figure 3. Immunostaining with DMP-1 antibody at 7 (a), 14 (b), and 28 (c) days after VIT treatment and 7 (d ), 14 (e), and 28 (f ) days after EMD treatment. ND, new dentin. Bars: 50 m.
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Histometric Findings The width of the necrotic layer was significantly narrow in the order of EMD-, GIC-, and VIT-treated groups throughout the experimental period (Fig. 6a). In regard to the area of reparative dentin formation, the EMDtreated group showed a significantly larger area than that in the other 2 groups throughout the experimental period. Twenty-eight days after treatment, the VIT-treated group showed a significantly larger area than that in the GIC-treated group (Fig. 6b). In all groups, many ED1-positive cells were observed, and their number decreased in a time-dependent manner (Fig. 6c). JOE — Volume 34, Number 1, January 2008
Basic Research—Biology
Figure 5. Double staining at 14 days in the VIT-treated group: (a) anti-ED1 and anti–IL-1; (b) anti-ED1 and anti–TGF-1. Double staining at 14 days in the EMD-treated group: (c) anti-ED1 and anti–BMP-2; (d ) anti-ED1 and anti– BMP-4. The blue and brown colors show ED1-positive cells and IL-1–, TGF1–, BMP-2–, or BMP-4 – expressing cells, respectively. Bars: 50 m.
TGF-1 were few. However, the number of BMP-expressing cells increased in the early phase, and large amounts of reparative dentin were observed. Mundy (20) has previously described the remodeling of bone tissues. The inflammatory reaction is first expanded by infiltrating inflammatory cells, and inflammatory cytokines such as IL-1 and tumor necrosis factor–␣ are released. Next, cells expressing anti-inflammatory cytokines such as TGF-1 increase, and granulation tissues are formed. Then the number of cells expressing BMPs increases, and the formation of bone tissues is activated. The results in our study suggested that the wound healing process of injured pulp tissues in the VIT- and GIC-treated groups was similar to that of bone tissue remodeling. On the other hand, in the EMD-treated group, the number of BMP-expressing cells increased without an increase in the expressions of IL-1 and TGF-1. We also investigated the expressions of IL-1 and TGF-1 at 1, 3, and 5 days after treatment, and their levels did not change (data not shown). This suggested that the wound healing process of injured pulp tissues with EMD differed from the normal circumstances.
In all groups, the number of CD5-positive cells was few throughout the experimental period (Fig. 6d ). The number of IL-1– expressing cells in the EMD-treated group was significantly lower than that in the other 2 groups throughout the experimental period (Fig. 6e). In the EMD-treated group, cells expressing TGF-1 were very few throughout the experimental period. On the other hand, the number of cells expressing TGF-1 peaked at 14 days in the other 2 groups. The numbers of TGF-1– expressing cells in the VIT- and GIC-treated groups were significantly higher than that in the EMD-treated group at 14 and 28 days (Fig. 6f ). In the EMD-treated group, the number of cells expressing BMP-2 or BMP-4 peaked at 14 days. The number of BMP-2– or BMP-4 – expressing cells in the EMD-treated group was significantly higher than that in the other 2 groups at 7 and 14 days. On the other hand, in the VITand GIC-treated groups, the number of cells expressing BMP-2 or BMP-4 increased in a time-dependent manner (Fig. 6g and h).
Discussion In all experimental groups, a strong ALP activity and DMP-1– positive reparative dentin were observed in the injured pulp tissues. ALP is a marker of hard tissue–forming cells, and DMP-1 plays an important role in odontoblast differentiation and matrix mineralization (22). We assumed that mineralized reparative dentin formation was induced in all experimental groups. However, the amounts of reparative dentin in the EMD-treated group were significantly larger than those in the other 2 groups, and there were many differences between the EMD-treated group and the other 2 groups for the structure of reparative dentin and the process of reparative dentin formation. In the VIT-treated group, the irritation of pulp tissues was significant, and the newly formed reparative dentin was porous, as shown by previous reports (2, 3). Many neutrophils and macrophages were observed below the necrotic layer. The number of IL-1– expressing macrophages initially increased, followed by that of TGF-1– expressing macrophages. Cells expressing BMPs gradually increased in number with reparative dentin formation. The findings of the GIC-treated group were similar to those of the VIT-treated group. Although the width of the necrotic layer was narrow compared with that of the VIT-treated group, there were small amounts of reparative dentin formation 28 days after treatment. It seemed that these differences were based on calcium hydroxide contained in VIT. On the other hand, in the EMD-treated group, the width of the necrotic layer was narrow. Although a large number of macrophages were observed below the necrotic layer, cells expressing IL-1 or JOE — Volume 34, Number 1, January 2008
Figure 6. Histometric analysis: (a) width of the necrotic layer; (b) area of reparative dentin formation; (c) number of ED1-positive cells; (d ) number of CD5-positive cells; (e) number of IL-1– expressing cells; (f ) number of TGF1– expressing cells; (g) number of BMP-2– expressing cells; (h) number of BMP-4 – expressing cells. Yellow bars ⫽ VIT-treated group; Blue bars ⫽ EMDtreated group; White bars ⫽ GIC-treated group; 夝 ⫽ P ⬍ .05; 夝夝 ⫽ P ⬍ .01.
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Basic Research—Biology IL-1, an inflammatory cytokine, has several functions such as the activation of bone resorption, the activation of immunocytes, and the production of prostaglandin or collagenase. These effects are greatly concerned with the extension of inflammatory reactions and tissue breakdown (23). TGF-1 inhibits the production of inflammatory cytokines (24) and promotes the production of extracellular matrix and the proliferation capacity of osteoblasts and odontoblasts (25, 26). These effects result in the suppression of inflammatory reactions and promote wound healing. Recently it has been reported that EMD suppresses the inflammatory cytokine production by immunocytes (27) and contains TGF-–like molecules (28). EMD might create a favorable environment for promoting wound healing in the injured pulp tissues. BMPs, which belong to the TGF- superfamily, can independently induce ectopic hard tissue formation (29, 30). The use of BMPs has been attempted in various regeneration fields. In the EMD-treated group, the number of BMP-expressing cells increased in the early phase, and large amounts of reparative dentin were observed. It has been reported that BMPs promote pulp cells to differentiate into odontoblasts (31), and EMD contains BMP-like molecules (32). BMP-expressing cells and BMP-like molecules in EMD might cooperate to promote odontoblast differentiation and reparative dentin formation. Interestingly, BMP-expressing cells were partly macrophages because of their shape and location in the EMD-treated group. Furthermore, double staining demonstrated the existence of BMP-expressing macrophages. Although there is little information about whether macrophages express BMPs, Champagne et al (33) suggested that macrophages expressing BMPs play an important role in healing of hard tissues. EMD might directly or indirectly induce BMP-expressing macrophages. It is necessary to examine in vitro and in vivo which substances contained in EMD induce BMP-expressing macrophages. Recently a study that applied EMD to human pulp tissues was reported (34). Although the postoperative symptoms were less and the amounts of reparative dentin formation were markedly larger than in teeth treated with calcium hydroxide, there are still unresolved problems. In conclusion, this study demonstrated that there were differences in the wound healing process between EMD and VIT-containing calcium hydroxide when used as a pulp-capping material. The role of macrophages in the wound healing process was greatly associated with these differences. BMP-expressing macrophages induced by EMD might play important roles in reparative dentin formation.
Acknowledgments This study was partly supported by a Grant-in-Aid for Scientific Research, 16209056 and 18209057, from the Ministry of Education, Science, Sports and Culture of Japan. We are also grateful to the members of the Section of Periodontology, Faculty of Dental Science, Kyushu University.
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