Advanceshttp://adr.sagepub.com/ in Dental Research
Enamel Matrix Derivative Promotes Reparative Processes in the Dental Pulp Y. Nakamura, L. Hammarström, E. Lundberg, H. Ekdahl, K. Matsumoto, S. Gestrelius and S.P. Lyngstadaas ADR 2001 15: 105 DOI: 10.1177/08959374010150010201 The online version of this article can be found at: http://adr.sagepub.com/content/15/1/105
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Advances in Dental Research can be found at: Email Alerts: http://adr.sagepub.com/cgi/alerts Subscriptions: http://adr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
Downloaded from adr.sagepub.com by guest on July 13, 2011 For personal use only. No other uses without permission.
Enamel Matrix Derivative Promotes Reparative Processes in the Dental Pulp Y. Nakamura 1 , L. Hammarstrom 2 , E. Lundberg3, H. Ekdahl3, K. Matsumoto 1 , S. Gestrelius3, S.P. Lyngstadaas3*'4 'Department of Endodontics, School of Dentistry, Showa University, 2-1I, Kitasenzoku, Ohta-ku, Tokyo 145-8515, Japan; 2 Center for Oral Biology, School of Dentistry, Karolinska Institutet, PO Box 4064, S-14 104 Huddinge, Sweden; 3 Biora AB, Medeon Science Park, Malmo, S-205 12, Malmo, Sweden; 4 Clinical Research Laboratory, Faculty of Dentistry, University of Oslo, PO Box I 109, Blindern, N-03 I 7 Oslo, Norway; •corresponding author,
[email protected] Adv Dent Res 15:105-107, August, 2001
Abstract — During odontogenesis, amelogenins from the preameloblasts are translocated to differentiating odontoblasts in the dental papilla, suggesting that amelogenins may be associated with odontoblast changes during development. In the present study, we have explored the effects of enamel matrix derivative (EMD) on the healing of a pulpal wound. Coronal pulp tissue of permanent maxillary premolars of miniature swine were exposed through buccal class V cavities. The exposed pulp was capped with EMD. The contralateral teeth served as controls and were capped with a calcium hydroxide paste (Dycal®). The cavities were sealed with glassionomer cement. After 2 and 4 weeks, the histology of the teeth was analyzed. In the EMD-treated teeth, large amounts of newly formed dentin-like hard tissue with associated formative cells outlined the pulpal wound separating the cavity area from the remaining pulp tissue. Inflammatory cells were present in the wound area but not subjacent to the newly formed hard tissue. Morphometric analysis showed that the amount of hard tissue formed in EMD-treated teeth was more than twice that of the calcium-hydroxide-treated control teeth (p < 0.001), suggesting that EMD is capable of promoting reparative processes in the wounded pulp more strongly than is calcium hydroxide.
Introduction It is well-documented that an exposed vital pulp can repair 1 by forming a bridge of hard tissue subjacent to the exposed •i sites, and recent advances in the field of dentin formation 1 have opened new vistas for pulp therapy (Kakehashi et al, 1965; for review see Yamamura, 1985; Ranly, 1994; Ranly and Garcia-Godoy, 2000; Tziafas et al, 2000). However, no biological strategy that predictably induces reparative a n d / o r regenerative processes in the dental pulp has so far been introduced for clinical use. One way to approach this problem is to try to mimic the processes that take place during odontogenesis by exploring the effects of factors and substances that have been found to be associated with the initial stages of dentin formation. One of those substances is amelogenin. During crown formation of feline and rodent teeth, amelogenin has been found to be translocated to the differentiating odontoblasts prior to the formation of circumpulpal dentin (Inai et al, 1991; Hammarstrom, 1997). Based on this observation, it has been suggested that amelogenin participates in the final differentiation of odontoblasts and the subsequent dentin formation (Inai et al, 1991). Deposition of amelogenin has also been found to precede cementum formation during normal root development in several mammals, including monkeys. Furthermore, an amelogenin-rich fraction of porcine enamel matrix (EMD) has been successfully used to induce cementum formation and
periodontal ligament regeneration in Macaca monkeys (Hammarstrom, 1997; Hammarstrom et al, 1997). Periodontal ligament cells, cultivated in the presence of EMD, show an increased production of mineralizing extracellular matrix, as well as a marked increase in expression of several growth factors (Gestrelius et al, 1997; Lyngstadaas et al, 2001). A formulation of EMD marketed as Emdogain® has successfully been used to induce regeneration of the periodontal tissues in patients with advanced periodontitis (Heijl et al, 1997; Pontoriero et al, 1999; Sculean et al, 1999). The purpose of the present study was to explore the effects of Emdogain® on experimentally exposed pulp tissue of premolars of adult miniature swine, with special attention to its ability to produce hard-tissue bridging.
Materials & Methods Surgical procedure
Twenty-two maxillary premolar teeth from 4 adult miniature swine were used. Animals were divided into two groups with 2 (n = 5 teeth/EMD-treated, n = 5 teeth/Ca(OH)2 -treated) or 4 wks (n = 6 teeth/EMD-treated, n = 6 teeth/Ca(OH)2-treated) of observation time, respectively. The animals were anaesthetized with an intramuscular injection of 10 mL Ketalar® and 8 mL pentobarbital i.v. The surgical sites were infiltrated with Xylocain® (20 mg/mL) and adrenalin (12.5 |jLg/mL). Each tooth pulp was exposed by trepanation through a 2-mm-diameter buccal class V cavity by means of sterile burs and sterile saline spray. Bleeding was controlled with sterile cotton pellets. The exposed pulp tissue was then covered with EMD (EMDOGAIN® Gel; 30 mg/mL in propylene-glycol-alginate [PGA]; BIORA AB, Malmo, Sweden) or calcium hydroxide (Dycal®, DENTSPLY, Konstanz, Germany), and subsequently each cavity was filled with glass-ionomer cement (GC Fujii II , GC Corporation, Tokyo, Japan). Histological examination
At 2 and 4 wks after surgery, the animals were killed by an intracardial bolus injection of 50 mL sodium pentobarbital in ethanol. Following death, all experimental teeth and the adjacent alveolar bone were removed en bloc and fixed in cold 4% neutral buffered formaldehyde for 24 hrs. The specimens were then demineralized in 12.5% EDTA and subsequently embedded in paraffin. After serial sectioning (6 |xm), every second section was stained with hematoxylin and eosin. Series of sections containing coronal pulp tissue were then analyzed in a light microscope equipped for histometry. Quantitative analyses of new hard tissues
The amount of new hard tissue formed adjacent to the cavity in each experimental tooth was assessed in every fourth section (n = 5), covering the central part of the experimental wound. The areas covered by newly formed hard tissue in these sections were measured by means of digital histometry equipment (Olympus Microimage®, Media Cybernetics, L.P., Silver Spring, MD, USA). The apical cut-off distance was set at 3 mm from the bottom of the experimental cavity. Keywords Dental pulp, dentinogenesis, enamel matrix derivative, pulpal wound healing. Presented at the International Meeting on Signaling Mechanisms in Dentin Development, Regeneration, and Repair: from Bench to Clinic, held at Thessaloniki, Greece, November 10-11, 2000
Downloaded from adr.sagepub.com by guest on July 13, 2011 For personal use only. No other uses without permission.
105
Figs. 1 and 2 — Micrographs showing premoiar teeth 2 wks after treatment with EMD or Ca(OH)2. Fig. 1 shows tooth treated with EMD. The pulp wound has features of a classic active wound, i.e., a necrotic superficial layer overlying a narrow zone of chronic inflammatory cell infiltrate. The new dentin-like tissue (ND) is formed at the border between the wound tissue and the healthy subjacent pulp tissue. Fig. 2 shows tooth treated with Ca(OH)2. Normal-appearing pulp tissue w i t h o u t inflammatory cells can be observed adjacent to the pulpal wound, resembling an inactive wound typical of a chemical burn. No new dentin or odontoblasts were observed in or close to the wound in these teeth.
off the traumatized part of the pulp, including the inflammatory cells (Figs. 3, 4). The pulp tissue subjacent to the bridge appeared normal and free of inflammation. A distinct layer of cells resembling odontoblasts with inserting tubules could be observed abutting the newly formed hard tissue. In calcium hydroxide controls, only a small amount of new dentin was observed in association with the exposed site at 4 wks post-surgery, secreted onto the primary dentin. There were no signs of inflammation in the Ca(OH)2-treated teeth (Fig. 5). Quantitative analyses of newly formed hard tissue, calculated as the sum of areas covered by new dentinlike tissue in the 5 most central sections from each experimental cavity, revealed a significant (p < 0.001) increase in hard-tissue formation in EMD-treated teeth when compared with Ca(OH) 2 treatment (Fig. 6). After both 2 and 4 wks, the amount of newly formed hard tissue in the EMD-treated teeth was more than twice that of the controls.
Discussion
In the experiments reported here, Dycal®-treated teeth were free from inflammation, but the treatment seemed to produce an inactive type of wound typical of chemical burns. Such wounds are known to heal slowly without going through the classic stages of ordinary wound healing. As previously reported, the absence of inflammation could also result from Figs. 3, 4, 5 — Micrographs showing premoiar teeth 4 wks after treatment with EMD or Ca(OH)2. Fig. 3 shows the antibacterial environment caused tooth treated with EMD. The pulp wound now shows features of classic wound healing, i.e., a superficial layer by the highly alkaline Ca(OH) 2 or scab, consisting of extracellular matrix proteins and cell remnants, overlying a zone of chronic inflammatory cell infiltrate. Subjacent to the healing wound, a bridge of new dentin-like tissue (ND) is forming, sealing off the (Stanley and Lundy, 1972; Tronstad, wound from the healthy pulp. Fig. 4 shows a higher magnification of Fig. 3. The new hard tissue is bordered 1974). At 4 wks after surgery, a small with odontoblast-like cells, and tissue subjacent to the forming bridge is healthy and free of inflammatory cells. amount of new, reactive dentin was Fig. 5 shows tooth treated with Ca(OH)2. Normal-appearing pulp tissue without inflammatory cells can be formed in the otherwise healthy pulp observed adjacent to the inactive pulpal wound. A small amount of new dentin is formed along the pre-existing tissue, but no proper regeneration of dentin walls adjacent to the wound. C = experimental cavity. D = dentin. ND = newly formed dentin-like tissue. P = pulp tissue. Sections stained with H&E. Scale bar = 1 mm (Figs. 1, 2, 4) or 0.5 mm (Figs. 3, 5). the odontoblast layer was observed. These findings demonstrate that even though preparations of Ca(OH)2, such Results as Dycal®, are regarded as tissue-friendly and protect against infections and inflammation, they are not capable of specifically Two weeks after surgery, a necrotic layer overlying a zone inducing dentin regeneration or repair. moderately infiltrated with chronic inflammatory cells was In EMD-treated teeth, the pulp wound showed features of observed adjacent to the experimental cavities in EMD-treated classic wound healing, i.e., a superficial layer or scab, consisting teeth. Newly formed hard-tissue nodules resembling dentin of extracellular matrix proteins and necrotic cell remnants, outlined by a distinct layer of odontoblast-like cells bordered overlying a zone of chronic inflammatory cell infiltrate. Subjacent the wound (Fig. 1). to the healing wound, a bridge of new hard tissue was forming, In teeth treated with Ca(OH) 2 , there were no signs of sealing off the wound from the healthy pulp tissue. The pulp inflammation at this stage, and normal-appearing connective tissue subjacent to this new hard tissue was invariably free of all tissue was demonstrated in close contact with the capping signs of inflammation. Moreover, a layer of odontoblast-like cells material. However, neither odontoblast nor new dentin was had formed, abutting the newly formed mineralized tissue. present directly adjacent to the pulpal wound in these teeth These findings suggest that EMD has the potential to promote (Fig. 2). However, in some controls, small amounts of new formation of a dentin-like hard tissue when applied onto a dentin were observed produced by existing odontoblasts onto coronal pulpal wound. The effect of EMD resembles the effects of the primary dentin close to the experimental cavity. a scab during dermal wound healing which promotes the classic Four weeks after surgery, a large amount of new dentinwound-healing cascade and the subsequent tissue regeneration like tissue bridged the full width of the cavity. The bridge or repair. The newly formed hard tissue resembled tubular formation was observed in the vital pulp at some distance dentin that was delineated by odontoblast-like cells. from the most apical part of the experimental cavity, sealing Downloaded from adr.sagepub.com by guest on July 13, 2011 For personal use only. No other uses without permission.
106
Nakamura et al.
Adv Dent Res 15:105-107, August, 2001
References New dentin formation
Fig. 6 — Chart expressing the total amount of new hard tissue formed after 2 and 4 wks in experimental teeth following EMD or Ca(OH)2 application and cavity sealing. The amount of new hard tissue is presented in mm 2 , measured by histometry in serial sections. At both stages, the amount of new dentin-like tissue formed in EMD-treated teeth was significantly higher than in the Ca(OH)2-treated teeth, n = 11 at all time points. Error bars give ± SD. p < 0.001 for both series.
Recently, several studies have reported that various odontogenic proteins can induce reparative dentin formation. Biologically active molecules—such as the bone morphogenic proteins (BMPs) (Nakashima, 1990, 1994; Rutherford and Gu, 2000) and the osteogenic protein (OP-1) (Rutherford et al., 1993)—or biomatrices, such as the demineralized dentin (Nakashima, 1989), have all been proposed to be specific inducers of dentin formation. When these substances were brought into close contact with vital pulp tissue, enhancement of the normal sequence of morphogenic events in the repairing dental pulp was invariably observed, i.e., rapid fibrodentin matrix formation and subsequent reparative dentinogenesis. These reports suggest that several bioactive proteins, like growth factors and matrix molecules, have potential as pulpcapping materials. The mechanism(s) underlying the induction of new hardtissue formation by EMD is still not known in detail. However, recent laboratory findings suggest that EMD induces an intracellular cyclic-AMP signal in exposed cells. In mesenchymal cells, this intracellular signal is followed by autocrine growth factor secretion in a well-orchestrated cascade (Lyngstadaas et al., 2001). Following the release of growth factors, e.g., PDGF and TGF-pi, the EMD-exposed cells proliferate and mature into extracellular-matrix-secreting cells. Similar mechanisms may also be at play when the pulpal wound is healing in the presence of EMD.
Gestrelius S, Andersson C, Johansson AC, Persson E, Brodin A, Rydhag L, et al. (1997). Formulation of enamel matrix derivative for surface coating. Kinetics and cell colonization. / Clin Periodontol 24:678-684. H a m m a r s t r o m L (1997). Enamel matrix, cementum d e v e l o p m e n t and regeneration. / Clin Periodontol 24:658-668. Hammarstrom L, Heijl L, Gestrelius S (1997). Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. / Clin Periodontol 24:669-677. Heijl L, Heden G, Svardstrom G, Ostgren A (1997). Enamel matrix derivative (EMDOGAIN) in the treatment of intrabony periodontal defects. / Clin Periodontol 24:705-714. Inai T, Kukita T, Ohsaki Y, Nagata K, Kukita A, Kurisu K (1991). Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs. Anat Rec 229:259-270. Kakehashi S, Stanley HR, Fitzgerald RJ (1965). The effects of surgical exposures of dental pulps in germfree and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 20:340-349. Lyngstadaas SP, Lundberg E, Ekdahl H, Andersson C, Gestrelius S (2001). Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative. / Clin Periodontol 28:181-188. Nakashima M (1989). Dentin induction by implants of autolyzed antigen-extracted allogeneic dentin on amputated pulps of dogs. Endod Dent Traumatol 5:279-286. Nakashima M (1990). The induction of reparative dentine in the amputated dental pulp of the dog by bone morphogenetic protein. Arch Oral Biol 35:493-497. Nakashima M (1994). Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic proteins (BMP)-2 and -4. / Dent Res 73:1515-1522. Pontoriero R, Wennstrom J, Lindhe J (1999). The use of barrier membranes and enamel matrix proteins in the treatment of angular bone defects. A prospective controlled clinical study. / Clin Periodontol 26:833-840. Ranly DM (1994). Pulpotomy therapy in primary teeth: new modalities for old rationales. Pediatr Dent 16:403-409. Ranly DM, Garcia-Godoy F (2000). Current and potential pulp therapies for primary and young permanent teeth. / Dent 28:153-161. Rutherford RB, Gu K (2000). Treatment of inflamed ferret dental pulps with recombinant bone morphogenetic protein-7. Eur ] Oral Sci 108:202-206. Rutherford RB, Wahle J, Tucker M, Rueger D, Charette M (1993). Induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 38:571-576.
Conclusion The characteristic formation of new hard tissue, observed in this study, suggest that EMD can induce or promote intrinsic reparative processes in the dental pulp. However, more investigations are needed to address the specific mode of action and characteristics of EMD-promoted reparative hardtissue formation in wounded dental pulps.
Acknowledgments The authors thank Ms. Beata Fabi for her kind help with the Figures, and Ms. Ulla-Britt Carlsson for excellent assistance during the animal procedures.
Sculean A, Reich E, Chiantella GC, Brecx M (1999). Treatment of intrabony periodontal defects with an enamel matrix protein derivative (Emdogain): a report of 32 cases. Int J Periodont Rest Dent 19:157-163.
Stanley HR, Lundy T (1972). Dycal therapy for pulp exposures. Oral Surg Oral Med Oral Pathol 34:818-827.
Tronstad L (1974). Reaction of the exposed pulp to Dycal treatment. Oral Surg Oral Med Oral Pathol 38:945-953.
Tziafas D, Smith AJ, Lesot H (2000). Designing new treatment strategies in vital pulp therapy. / Dent 28:77-92. Yamamura T (1985). Differentiation of pulpal cells and inductive influences of various matrices with reference to pulpal wound healing. / Dent Res 64:530-540.
Downloaded from adr.sagepub.com by guest on July 13, 2011 For personal use only. No other uses without permission.
Adv Dent Res 15:105-107, August, 2001
EMD Promotes Pulpai Healing
107
