© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

J Periodont Res 2014 All rights reserved

JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12245

Review Article

Enamel matrix derivative, inflammation and soft tissue wound healing Miron RJ, Dard M, Weinreb M. Enamel matrix derivative, inflammation and soft tissue wound healing. J Periodontal Res 2014; doi:10.1111/jre.12245. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Over 15 years have now passed since enamel matrix derivative (EMD) emerged as an agent capable of periodontal regeneration. Following thorough investigation, evidenced-based clinical application is now established for a multitude of clinical settings to promote regeneration of periodontal hard tissues. Despite the large number of studies and review articles written on this topic, no single review has compiled the influence of EMD on tissue inflammation, an area of research that merits substantial attention in periodontology. The aim of the present review was to gather all studies that deal with the effects of EMD on tissue inflammation with particular interest in the cellular mechanisms involved in inflammation and soft tissue wound healing/resolution. The effects of EMD on monocytes, macrophages, lymphocytes, neutrophils, fibroblasts and endothelial cells were investigated for changes in cell behavior as well as release of inflammatory markers, including interleukins, prostaglandins, tumor necrosis factor-a, matrix metalloproteinases and members of the OPG-RANKL pathway. In summary, studies listed in this review have reported that EMD is able to significantly decrease interleukin-1b and RANKL expression, increase prostaglandin E2 and OPG expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, as well as increase fibroplasias and angiogenesis by inducing endothelial cell proliferation, migration and capillary-like sprout formation. The outcomes from the present review article indicate that EMD is able to affect substantially the inflammatory and healing responses and lay the groundwork for future investigation in the field.

Soft tissue inflammation

Although the complete report of the inflammatory response that takes place during periodontal disease is out of the scope of this article, a basic understanding of cells and mediators implicated during this process is essential. For a full and complete review on the inflammatory response in periodontal tissues, a number of excellent articles have been

written on this topic over the years (1–5). This review article will present an overview of acute and late phase inflammation and will summarize the mediators and cells involved in both processes. Acute inflammation

The periodontium represents one of the few body sites where bacteria have rapid access to the human body (5).

R. J. Miron1,2, M. Dard3, M. Weinreb4 1

Department of Periodontology, Department of Oral Surgery, University of Bern, Bern, Switzerland, 2Faculty of Dental Medicine, University of Laval, Quebec City, QC, Canada, 3 Department of Periodontology and Implant Dentistry, College of Dentistry, New York University, New York, NY, USA and 4 Department of Oral Biology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv University, Tel-Aviv, Israel

Richard Miron, PhD, Department of Periodontology, University of Bern, Switzerland Tel: +41 31 632-2589 Fax: +41 31 632-4915 e-mail: [email protected] Key words: Emdogain; enamel matrix proteins;

inflammation; periodontal regeneration; periodontal wound healing Accepted for publication October 10, 2014

The periodontal sulcus, an area devoid of a thick epidermal layer, is the host of a number of periodontal pathogens that easily interact with the junctional epithelium by penetrating readily into the underlying connective tissue and disturbing local tissue structures (6). Findings from controlled clinical trials in the United States now estimate that approximately 40% of the adult population is affected by some form of periodontitis (7).

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Typically, in the acute phase of bacterial infection (Fig. 1, Table S1), the intact epithelial barrier of the gingival, sulcular and junctional epithelium prevents bacterial invasion (6). Ideally, the population of B cells and plasma cells that accumulate in the wall of the sulcus produce enough antibodies that target bacteria and amplify phagocytosis by opsonization. At early stages, only few leukocytes are observed near the inflamed sites. When the bacterial infiltration becomes more pronounced, however, proinflammatory mediators synthesized by the junctional epithelium can enter the connective tissue. These include interleukin (IL)-1, IL-8, prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF-a) and matrix metalloproteinases (MMPs) (5). Following these chemotactic signals, gingival vessels are enlarged and facilitate the migration of neutrophils to the site of microbial accumulation. Furthermore, the rate of turnover of the junctional epithelial cells increases, leaving widened spaces for neutrophil diffusion (5). Levels of specific inflammatory markers such as IL-8, the most important chemokine, increase in the gingival tissues and become responsible for recruiting large numbers of neutrophils to the gingival sulcus epithelium (7). Thus, in the initial phase of inflammation, substantial recruitment of neutrophils, proliferation of the epithelial cells and localized secretion of inflammatory enzymes and cytokines occur. These phenomena promote the subsequent recruitment of other inflammatory cells and destruction of the extracellular matrix (Fig. 1, Table S1). Late phase immune response

Although few macrophages are found in healthy gingival (5), late phase inflammation (Fig. 2, Table S2) is marked by an increase in macrophage number and activity during gingivitis and periodontitis (5). Soon after initiation of an inflammatory response, small lymphocytes consisting of B and T cells infiltrate into the tissue in the

presence of antigens and various cytokines (3). B cells soon begin to enlarge and differentiate into plasma cells, producing antibodies against the invading pathogens. Macrophages are then activated through antigen non-specific mechanisms and enhance the overall inflammatory response by producing more inflammatory cytokines, including interferon c, TNF-a, IL-1b, -6, -10, -12 and -15, PGE2 and MMPs. Of these factors, IL-1b, TNF-a and PGE2 are strongly implicated in the pathogenesis of periodontitis (3). Overexpression of these factors thus results in a higher level of collagen breakdown and in particular, an imbalance between levels of MMPs and tissue inhibitors of metalloproteinases (TIMPs), which directly affects the degradation of collagen (3). Furthermore, it is well documented that an increase in inflammatory markers leads to lower expression and synthesis of collagen in fibroblasts, thus further assisting periodontal breakdown (3). The release of IgGs by plasmocytes is then detectable in higher levels in the gingival crevicular fluid and its increase is further associated with development of gingivitis (3). Furthermore, the increase in IL-6 secretion is the driving force behind differentiation of monocytes into multinucleated osteoclasts responsible for bone resorption. An increase in IL-6 levels is also associated with increases in other interleukins leading to further periodontal tissue destruction (3).

A history of enamel matrix derivative Over 15 years have now passed since EMD was first introduced as a periodontal regeneration agent capable of supporting new periodontal ligament (PDL), cementum and alveolar bone formation. The actions of EMD on periodontal regeneration mimic the normal development of periodontal tissues (8). The histological observation that amelogenin, which until then was considered an enamel-specific protein, is deposited on to the surface of developing tooth roots before

cementum formation, led to the hypothesis that amelogenin might be responsible for the differentiation of periodontal tissues (8). This hypothesis was the basis of a number of preclinical and clinical studies thereafter, which demonstrated a positive effect of EMD on the proliferative and differentiation potential of periodontal tissues (8–14). Based on these observations, the purified amelogenin fractions (including amelogenin fragments and non-amelogenin peptides) was given the working name EMD and this formulation has been the basis for over 600 publications supporting its use in periodontal tissue regeneration. The major components of EMD are amelogenins, a family of hydrophobic proteins derived from different splice variants and controlled postsecretion from the expression of a single gene. They account for more than 95% of the total EMD protein content (15). These proteins self-assemble into supramolecular aggregates, forming an insoluble extracellular matrix that functions to control the ultrastructural organization of the developing enamel crystallites (15). Other proteins found in the enamel matrix include enamelin, ameloblastin (also called amelin or sheathlin), amelotin, apin and various proteinases, which have since also been found in trace amounts in EMD (16,17). The cellular events that take place in various cells in the periodontium following application of EMD are reviewed in two recent articles (18,19). It was demonstrated that EMD possesses a significant influence on behavior of many cell types by mediating attachment, spreading, proliferation and survival as well as expression of transcription factors, growth factors, cytokines, extracellular matrix constituents and other molecules involved in the regulation of bone remodeling (18,19). Despite the wealth of knowledge describing the role of EMD in the regeneration of periodontal tissues, to date no single review has gathered all the studies describing the effects of EMD on inflammation, a critical factor for the etiopathogenesis and

EMD, inflammation and soft tissue healing

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Fig. 1. Acute periodontal inflammation. Accumulation of a periodontal biofilm persists on enamel surfaces and causes inflammation of gingival tissues. Host cells recognize bacterial infiltration and produce large amounts of proinflammatory mediators such as IL-1, IL-8, IL-6, PGE2, MMPs and TNF-a, which cause vasodilation of blood vessels, release of serum proteins into surrounding periodontal tissues and absorption of histamine and neuropeptides into blood vessels. A marked determinant of acute inflammation is characterized by substantial recruitment of neutrophils. IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinases; PGE2, prostaglandin E2; PMN, polymorphonuclear neutrophil; TNF, tumor necrosis factor.

resolution of gingivitis and periodontitis. Therefore, the purpose of this study was to gather systematically all studies that have specifically targeted the actions of EMD on inflammatory cells including monocytes, leukocytes, macrophages, neutrophils and endothelial cells as well as on the release of inflammatory markers such as interleukins, prostaglandins, TNF-a, MMPs and members of the OPG-RANKL pathway.

Search strategy

To gather all available biological data relevant to the topic, a systematic approach was applied by searching databases, including MEDLINE, PubMed, Embase and Cochrane up until December 28, 2013. The initial search criteria for “enamel matrix proteins,” “enamel matrix derivative” or “emdogain” generated 820 initial articles, and thereafter, this search query was searched against each cell

types and each inflammatory marker, including interleukin, prostaglandin, TNF-a, MMP, OPG and RANKL separately. Then manuscripts were selected after careful review and allocated to their appropriate title headings as presented in the remainder of this article. In addition, a secondary search of possible articles relevant to the topic was searched within other databases to identify possible additional studies that may have been missed.

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Fig. 2. Late-phase inflammation, including an immune response. Late phase inflammation involves an increase in macrophage number and activity followed by small lymphocytes (B and T cells) infiltrating into periodontal tissues in the presence of antigens and various cytokines. B cells then begin to enlarge, differentiate into plasma cells and produce high levels of antibodies against infiltrating pathogens. If gingival inflammation persists, monocytes differentiate into osteoclasts and bone resorption takes place causing alveolar bone loss. Together with breakdown of connective tissues, this will lead to eventual periodontal attachment loss and recessions. CAL, clinical attachment loss; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinases; PGE2, prostaglandin E2; PMN, polymorphonuclear neutrophil; TGF, transforming growth factor; TIMP, tissue inhibitors of metalloproteinases; TNF, tumor necrosis factor.

Effects of enamel matrix derivative on specific cell types Monocytes

Until now, the effects of EMD on monocytes have been investigated in four studies (Table 1). Itoh et al. (20) demonstrated that EMD was able to induce osteoclast formation and function when added simultaneously with RANKL. In a study

by Sato et al. (21), it was found that monocytes exposed to lipopolysaccharide in the presence of EMD exhibited a decrease in TNF-a production (0.10–0.52-fold) and an increase in PGE2 production (1.31–2.71-fold) compared to control cells not treated with EMD. On the other hand, a study by Khedmat et al. (22) found that EMD did not alter the expression of TNF-a or IL-1b as assessed by enzyme-linked immunosorbent assay and did not modify the phagocytic

activity of these cells following treatment with EMD. The effect of EMD on osteoclast differentiation was addressed in a study determining the role of transforming growth factorbeta receptor type 1 (TGF-bRI) kinase activity in osteoclastogenesis in the murine macrophage cell line RAW246.7 (23). The results from this study demonstrated that inhibition of TGF-bRI kinase activity abolished the effect of EMD on osteoclastogenesis induced by RANKL. To date no

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Fig. 2. Late-phase inflammation, including an immune response. Late phase inflammation involves an increase in macrophage number and activity followed by small lymphocytes (B and T cells) infiltrating into periodontal tissues in the presence of antigens and various cytokines. B cells then begin to enlarge, differentiate into plasma cells and produce high levels of antibodies against infiltrating pathogens. If gingival inflammation persists, monocytes differentiate into osteoclasts and bone resorption takes place causing alveolar bone loss. Together with breakdown of connective tissues, this will lead to eventual periodontal attachment loss and recessions. CAL, clinical attachment loss; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinases; PGE2, prostaglandin E2; PMN, polymorphonuclear neutrophil; TGF, transforming growth factor; TIMP, tissue inhibitors of metalloproteinases; TNF, tumor necrosis factor.

Effects of enamel matrix derivative on specific cell types Monocytes

Until now, the effects of EMD on monocytes have been investigated in four studies (Table 1). Itoh et al. (20) demonstrated that EMD was able to induce osteoclast formation and function when added simultaneously with RANKL. In a study

by Sato et al. (21), it was found that monocytes exposed to lipopolysaccharide in the presence of EMD exhibited a decrease in TNF-a production (0.10–0.52-fold) and an increase in PGE2 production (1.31–2.71-fold) compared to control cells not treated with EMD. On the other hand, a study by Khedmat et al. (22) found that EMD did not alter the expression of TNF-a or IL-1b as assessed by enzyme-linked immunosorbent assay and did not modify the phagocytic

activity of these cells following treatment with EMD. The effect of EMD on osteoclast differentiation was addressed in a study determining the role of transforming growth factorbeta receptor type 1 (TGF-bRI) kinase activity in osteoclastogenesis in the murine macrophage cell line RAW246.7 (23). The results from this study demonstrated that inhibition of TGF-bRI kinase activity abolished the effect of EMD on osteoclastogenesis induced by RANKL. To date no

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Eosinophilic round bodies

Gingival fibroblasts

In a study by Kim et al. (27), eosinophilic round bodies (ERBs) were investigated following the injection of various EMD concentrations in the backs of Sprague–Dawley rats (Table 1). The histopathological findings were similar to those developed against a foreign body agent. A high concentration of EMD induces ERBs that consist of a 40 kDa protein, which includes a constituent part of amelogenin. These findings demonstrate that the ERBs (or remaining EMD) may promote mesenchymal cell differentiation into hard tissue forming cells around the EMD injection site via activation of ERBs.

Five studies examining the relationship between EMD and inflammation were derived from experiments using gingival fibroblasts (30–34) (Table 1). In the first study, primary human gingival fibroblasts were treated with TNF-a, EMD or both in a serumfree medium. Extracted RNA was analyzed with an extracellular matrixfocused microarray and quantitative real-time polymerase chain reaction assays. Results showed that TNF-a increased the expression of MMP-1 but EMD decreased it and stimulated the expression of TIMP-3, with little effect on various other forms of MMPs and TIMPs (30). These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, but by curbing MMP expression and stimulating TIMP-3 production. This process may lead to reduced periodontal breakdown caused by an inflammatory reaction induced by bacterial pathogens (30). The second study showed that EMD protects human gingival fibroblasts from TNFinduced apoptosis by inhibiting caspase activation (31). Furthermore, it was also determined by this group that EMD stimulates primary human gingival fibroblast cell proliferation through the ERK cascade by synergistically inducing completion of the cell cycle (33). In a subsequent study by Zeldich et al. (34) it was demonstrated that EMD-induced ERK activation and proliferation were partially due to the Src-dependent, metalloproteinase-mediated transactivation of epidermal growth factor receptor. A study by Weinberg et al. (32), demonstrated that EMD was able to abolish the PGE2-induced inhibition of proliferation in gingival fibroblasts.

Endothelial cells

The use of EMD was tested on isolated human umbilical vein endothelial cells (HUVECs) in two studies (Table 1). Cell proliferation, survival, adhesion and migration activities were assessed by Kasaj et al. (28). Furthermore, the effect of EMD on angiogenesis was assessed in a HUVEC three-dimensional sprouting assay. Results from this study revealed that the use of EMD did not influence cell adhesion or survival but did stimulate proliferation and migration, which was activated via an extracellular regulated kinase (ERK) 1/2 pathway. EMD also induced capillary-like sprout formation from HUVEC spheroids in a dose-dependent manner. These data may suggest the association of EMD with wound healing via increased capillary formation in regenerating tissues (28). In the second study, Bertl et al. (29) tested the in vitro effects of EMD on the proliferation/viability, migration and expression of angiogenic factor and adhesion molecules in HUVECs. The proliferation/viability of HUVECs measured by the MTT assay was stimulated by 0.1 mg/mL EMD and cell migration in the wound healing assay was promoted by EMD at doses of 0.1–50 mg/mL (29). The highest expression level of all three tested genes (ICAM-1, E-selectin and ang-2) was observed at 50 mg/mL EMD (29).

Osteoclasts

The effects of EMD on osteoclast formation were reported by one in vitro study (35) (Table 1). Otsuka et al. investigated the effect of bioactive fractions from EMD in mouse bone marrow cells for TRAP-positive

multinucleated cell formation and expression of RANKL in osteoblastic cells. The study concluded that EMD induces the formation of osteoclasts and that this effect was driven through the production of RANKL (35). Periodontal ligament cells

The effect of EMD on interleukin expression has been investigated in two studies (Table 1). Nokhbehsaim et al. (36) found that EMD downregulates significantly the expression of IL-1b and cyclooxygenase (COX)-2 after 1 d of treatment and that of IL-6, IL-8 and COX-2 after 6 d in normal cell culture conditions. In addition, it was shown that, in an inflammatory environment, the antiinflammatory actions of EMD were significantly enhanced at 6 d. In the presence of low biomechanical loading, EMD caused a downregulation of IL-1b and IL-8, whereas high biomechanical loading significantly abrogated the anti-inflammatory effects of EMD at both time points tested in this study (36). Similarly, it was also shown that the in vitro combination of a bovine derived bone graft (BioOss) with EMD significantly decreased the expression of IL-1b in both PDL cells and osteoblasts by twofold (37).

Influence of enamel matrix derivative on inflammatory mediators Interleukins

The expression/release of interleukins following exposure to EMD has been monitored in a number of in vitro studies (Table 2). Petinaki et al. (24) were the first to test EMD with peripheral blood lymphocytes and found that no changes in IL-2 or IL-6 levels were noticed after application of EMD for 3 d. In primary human osteoblasts, Jiang et al. (38) observed up to a twofold increase in IL-6 mRNA levels after 24, 48 and 72 h exposure to EMD. Similarly, in another study, PDL cells exposed to EMD also demonstrated a twofold

EMD, inflammation and soft tissue healing increase in IL-6 production after 72 h (39). Lee et al. (40) also found in two osteoblastic cell lines (MG63 human, MC3T3 mouse) that IL-6 production increased 4–10-fold following application of EMD. Myhre et al. (41) tested the effects of EMD in a whole blood model and found a twofold decrease in IL-8 production. In a study by Gundersen et al. (42) to assess the effects of EMD on systemic inflammation, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg per min). EMD did not modify the systemic IL-1b or IL-6 concentrations. Furthermore, experimental periodontitis in rats was created by elevating a full-thickness gingival flap and ligating silk threads around the first molars of the mandible. After 14 d the expression of IL-1b was significantly decreased at 14 d in animals treated with EMD when compared to control animals (43). Kaida et al. (44) tested the effects of EMD on the inflammation of injured pulp tissues. In this study, EMD significantly reduced the expression of IL1b over sixfold (44). In a monocyte culture system, EMD did not change the expression of IL-1b concentrations as assessed by Khedmat et al. (22). Nokhbehsaim et al. (36) found that EMD downregulated significantly the expression of IL-1b and COX-2 in PDL cells at 1 d and of IL-6 and IL-8 at 6 d in normal cell culture conditions. Similarly in another study of EMD combined with a bone graft, EMD significantly decreased IL-1b expression (37).

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systemic TNF-a levels in seven pigs receiving a prophylactic EMD bolus injection followed by continuous infusion. Khedmat et al. (22) found that EMD did not change the expression of TNF-a or IL-1b in monocytes as assessed by enzyme-linked immunosorbent assay and did not modify the phagocytic activity of these cells following treatment with EMD.

Prostaglandins

The effect of EMD on the release of prostaglandins has been investigated in a number of studies (Table 3). Jiang et al. (38) demonstrated that prostaglandin G/H synthase 2 was significantly increased 1.5 times compared to the control following treatment with EMD in osteoblasts. Schwartz et al. (45) demonstrated in an osteoblast cell model that EMD did not change the production of PGE2. In a subsequent study, chondrocytes were isolated from the resting zone and growth zone of the costochondral growth plate cartilage of adolescent rats and stimulated with EMD. The obtained results demonstrated a fivefold increase in PGE2 production, specifically in immature cells (46). Mizutani et al. (47) also demonstrated that EMD only slightly increased PGE2 production by 1.3fold in human osteoblasts. Sato et al. (21) demonstrated in monocytes exposed to EMD an increase in PGE2 production (1.31–2.71-fold) compared to controls not treated with EMD.

OPG and RANKL

The effect of EMD on OPG and RANKL (affecting osteoclast differentiation) was investigated in nine studies (Table 3). He et al. (48) were the first to show that EMD slightly increased OPG expression (1.5-fold) in mouse MC3T3 pre-osteoblasts. Lee et al. (40) also demonstrated a slight but significant increase in OPG in mouse osteoblasts following exposure to EMD. A study of Takayanagi et al. (49) demonstrated that EMD decreased RANKL expression in human PDL cells by 50% while OPG levels remained unchanged. Galli et al. (50) demonstrated an increase of 50% in OPG levels and a decrease of 50% in RANKL levels when human alveolar osteoblasts were exposed to EMD. EMD was able to stimulate up to a twofold increase in OPG production in PDL cells for short periods (51). EMD enhanced OPG gene expression and protein synthesis, and inhibited RANKL gene expression and soluble RANKL synthesis in MC3T3 mouse osteoblasts (52). MG-63 human osteoblasts were cultured on titanium surfaces and demonstrated a sevenfold increase in

Tumor necrosis factor-a

In a study by Sato et al. (21), it was found that human monocytes exposed to lipopolysaccharide in the presence of EMD exhibited a decrease in TNFa production (0.10–0.52-fold) compared to controls not treated with EMD (Table 3). Myhre et al. (41) found in whole blood following exposure to EMD a decrease of twofold in TNF-a release. In a study by Gundersen et al. (42), EMD did not modify

Table 2. Effects of EMD on the expression of various interleukins Author (reference)

Cell source

Petinaki et al. (24) Jiang et al. (38) Lyngstadaas et al. (39) Lee et al. (40) Myhre et al. (41) Gundersen et al. (42) Fujishiro et al. (43) Kaida et al. (44) Khedmat et al. (22) Nokhbehsaim et al. (70,77) Miron et al. (37)

Peripheral blood lymphocytes Osteoblasts – human PDL cells – human MG63 – human, MC3T3 – mouse Whole blood Prophylactic EMD bolus injection – pig Rat periodontal therapy Pulpal tissues Monocytes – humans PDL cells – human PDL cells, osteoblasts – humans

IL-1b

nd 29 69 nd 59 29

EMD, enamel matrix derivative; IL, interleukin; PDL, periodontal ligament.

IL-2

IL-6

nd

nd 29 29 4–109

IL-8

IL-10

2 9 decrease

nd

nd decrease decrease decrease decrease

2 9 decrease

5 9 decrease

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Eosinophilic round bodies

Gingival fibroblasts

In a study by Kim et al. (27), eosinophilic round bodies (ERBs) were investigated following the injection of various EMD concentrations in the backs of Sprague–Dawley rats (Table 1). The histopathological findings were similar to those developed against a foreign body agent. A high concentration of EMD induces ERBs that consist of a 40 kDa protein, which includes a constituent part of amelogenin. These findings demonstrate that the ERBs (or remaining EMD) may promote mesenchymal cell differentiation into hard tissue forming cells around the EMD injection site via activation of ERBs.

Five studies examining the relationship between EMD and inflammation were derived from experiments using gingival fibroblasts (30–34) (Table 1). In the first study, primary human gingival fibroblasts were treated with TNF-a, EMD or both in a serumfree medium. Extracted RNA was analyzed with an extracellular matrixfocused microarray and quantitative real-time polymerase chain reaction assays. Results showed that TNF-a increased the expression of MMP-1 but EMD decreased it and stimulated the expression of TIMP-3, with little effect on various other forms of MMPs and TIMPs (30). These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, but by curbing MMP expression and stimulating TIMP-3 production. This process may lead to reduced periodontal breakdown caused by an inflammatory reaction induced by bacterial pathogens (30). The second study showed that EMD protects human gingival fibroblasts from TNFinduced apoptosis by inhibiting caspase activation (31). Furthermore, it was also determined by this group that EMD stimulates primary human gingival fibroblast cell proliferation through the ERK cascade by synergistically inducing completion of the cell cycle (33). In a subsequent study by Zeldich et al. (34) it was demonstrated that EMD-induced ERK activation and proliferation were partially due to the Src-dependent, metalloproteinase-mediated transactivation of epidermal growth factor receptor. A study by Weinberg et al. (32), demonstrated that EMD was able to abolish the PGE2-induced inhibition of proliferation in gingival fibroblasts.

Endothelial cells

The use of EMD was tested on isolated human umbilical vein endothelial cells (HUVECs) in two studies (Table 1). Cell proliferation, survival, adhesion and migration activities were assessed by Kasaj et al. (28). Furthermore, the effect of EMD on angiogenesis was assessed in a HUVEC three-dimensional sprouting assay. Results from this study revealed that the use of EMD did not influence cell adhesion or survival but did stimulate proliferation and migration, which was activated via an extracellular regulated kinase (ERK) 1/2 pathway. EMD also induced capillary-like sprout formation from HUVEC spheroids in a dose-dependent manner. These data may suggest the association of EMD with wound healing via increased capillary formation in regenerating tissues (28). In the second study, Bertl et al. (29) tested the in vitro effects of EMD on the proliferation/viability, migration and expression of angiogenic factor and adhesion molecules in HUVECs. The proliferation/viability of HUVECs measured by the MTT assay was stimulated by 0.1 mg/mL EMD and cell migration in the wound healing assay was promoted by EMD at doses of 0.1–50 mg/mL (29). The highest expression level of all three tested genes (ICAM-1, E-selectin and ang-2) was observed at 50 mg/mL EMD (29).

Osteoclasts

The effects of EMD on osteoclast formation were reported by one in vitro study (35) (Table 1). Otsuka et al. investigated the effect of bioactive fractions from EMD in mouse bone marrow cells for TRAP-positive

multinucleated cell formation and expression of RANKL in osteoblastic cells. The study concluded that EMD induces the formation of osteoclasts and that this effect was driven through the production of RANKL (35). Periodontal ligament cells

The effect of EMD on interleukin expression has been investigated in two studies (Table 1). Nokhbehsaim et al. (36) found that EMD downregulates significantly the expression of IL-1b and cyclooxygenase (COX)-2 after 1 d of treatment and that of IL-6, IL-8 and COX-2 after 6 d in normal cell culture conditions. In addition, it was shown that, in an inflammatory environment, the antiinflammatory actions of EMD were significantly enhanced at 6 d. In the presence of low biomechanical loading, EMD caused a downregulation of IL-1b and IL-8, whereas high biomechanical loading significantly abrogated the anti-inflammatory effects of EMD at both time points tested in this study (36). Similarly, it was also shown that the in vitro combination of a bovine derived bone graft (BioOss) with EMD significantly decreased the expression of IL-1b in both PDL cells and osteoblasts by twofold (37).

Influence of enamel matrix derivative on inflammatory mediators Interleukins

The expression/release of interleukins following exposure to EMD has been monitored in a number of in vitro studies (Table 2). Petinaki et al. (24) were the first to test EMD with peripheral blood lymphocytes and found that no changes in IL-2 or IL-6 levels were noticed after application of EMD for 3 d. In primary human osteoblasts, Jiang et al. (38) observed up to a twofold increase in IL-6 mRNA levels after 24, 48 and 72 h exposure to EMD. Similarly, in another study, PDL cells exposed to EMD also demonstrated a twofold

EMD, inflammation and soft tissue healing increase in IL-6 production after 72 h (39). Lee et al. (40) also found in two osteoblastic cell lines (MG63 human, MC3T3 mouse) that IL-6 production increased 4–10-fold following application of EMD. Myhre et al. (41) tested the effects of EMD in a whole blood model and found a twofold decrease in IL-8 production. In a study by Gundersen et al. (42) to assess the effects of EMD on systemic inflammation, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg per min). EMD did not modify the systemic IL-1b or IL-6 concentrations. Furthermore, experimental periodontitis in rats was created by elevating a full-thickness gingival flap and ligating silk threads around the first molars of the mandible. After 14 d the expression of IL-1b was significantly decreased at 14 d in animals treated with EMD when compared to control animals (43). Kaida et al. (44) tested the effects of EMD on the inflammation of injured pulp tissues. In this study, EMD significantly reduced the expression of IL1b over sixfold (44). In a monocyte culture system, EMD did not change the expression of IL-1b concentrations as assessed by Khedmat et al. (22). Nokhbehsaim et al. (36) found that EMD downregulated significantly the expression of IL-1b and COX-2 in PDL cells at 1 d and of IL-6 and IL-8 at 6 d in normal cell culture conditions. Similarly in another study of EMD combined with a bone graft, EMD significantly decreased IL-1b expression (37).

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systemic TNF-a levels in seven pigs receiving a prophylactic EMD bolus injection followed by continuous infusion. Khedmat et al. (22) found that EMD did not change the expression of TNF-a or IL-1b in monocytes as assessed by enzyme-linked immunosorbent assay and did not modify the phagocytic activity of these cells following treatment with EMD.

Prostaglandins

The effect of EMD on the release of prostaglandins has been investigated in a number of studies (Table 3). Jiang et al. (38) demonstrated that prostaglandin G/H synthase 2 was significantly increased 1.5 times compared to the control following treatment with EMD in osteoblasts. Schwartz et al. (45) demonstrated in an osteoblast cell model that EMD did not change the production of PGE2. In a subsequent study, chondrocytes were isolated from the resting zone and growth zone of the costochondral growth plate cartilage of adolescent rats and stimulated with EMD. The obtained results demonstrated a fivefold increase in PGE2 production, specifically in immature cells (46). Mizutani et al. (47) also demonstrated that EMD only slightly increased PGE2 production by 1.3fold in human osteoblasts. Sato et al. (21) demonstrated in monocytes exposed to EMD an increase in PGE2 production (1.31–2.71-fold) compared to controls not treated with EMD.

OPG and RANKL

The effect of EMD on OPG and RANKL (affecting osteoclast differentiation) was investigated in nine studies (Table 3). He et al. (48) were the first to show that EMD slightly increased OPG expression (1.5-fold) in mouse MC3T3 pre-osteoblasts. Lee et al. (40) also demonstrated a slight but significant increase in OPG in mouse osteoblasts following exposure to EMD. A study of Takayanagi et al. (49) demonstrated that EMD decreased RANKL expression in human PDL cells by 50% while OPG levels remained unchanged. Galli et al. (50) demonstrated an increase of 50% in OPG levels and a decrease of 50% in RANKL levels when human alveolar osteoblasts were exposed to EMD. EMD was able to stimulate up to a twofold increase in OPG production in PDL cells for short periods (51). EMD enhanced OPG gene expression and protein synthesis, and inhibited RANKL gene expression and soluble RANKL synthesis in MC3T3 mouse osteoblasts (52). MG-63 human osteoblasts were cultured on titanium surfaces and demonstrated a sevenfold increase in

Tumor necrosis factor-a

In a study by Sato et al. (21), it was found that human monocytes exposed to lipopolysaccharide in the presence of EMD exhibited a decrease in TNFa production (0.10–0.52-fold) compared to controls not treated with EMD (Table 3). Myhre et al. (41) found in whole blood following exposure to EMD a decrease of twofold in TNF-a release. In a study by Gundersen et al. (42), EMD did not modify

Table 2. Effects of EMD on the expression of various interleukins Author (reference)

Cell source

Petinaki et al. (24) Jiang et al. (38) Lyngstadaas et al. (39) Lee et al. (40) Myhre et al. (41) Gundersen et al. (42) Fujishiro et al. (43) Kaida et al. (44) Khedmat et al. (22) Nokhbehsaim et al. (70,77) Miron et al. (37)

Peripheral blood lymphocytes Osteoblasts – human PDL cells – human MG63 – human, MC3T3 – mouse Whole blood Prophylactic EMD bolus injection – pig Rat periodontal therapy Pulpal tissues Monocytes – humans PDL cells – human PDL cells, osteoblasts – humans

IL-1b

nd 29 69 nd 59 29

EMD, enamel matrix derivative; IL, interleukin; PDL, periodontal ligament.

IL-2

IL-6

nd

nd 29 29 4–109

IL-8

IL-10

2 9 decrease

nd

nd decrease decrease decrease decrease

2 9 decrease

5 9 decrease

10

Miron et al.

EMD is also packaged and sold under the trademark name Xelmaâ for the treatment of hard-to-heal ulcers such as venous leg ulcers, diabetic foot ulcers and pressure ulcers, by functioning as a temporary matrix that improves cell adhesion and subsequent tissue/wound healing. Preclinical studies demonstrated that this by-product of EMD is able to increase, in dermal fibroblasts, both VEGF production over threefold

(78) and proliferation (32,79) and augment fibroblast-driven collagen matrix remodeling (80). Although the scope of this article is not to discuss the clinical effectiveness of Xelmaâ for the treatment of hardto-heal ulcers, a list of published case studies and conference proceedings are presented in Table 5 demonstrating other potent therapeutic uses of EMD for soft tissue regeneration (81–89).

Table 5. Use of an enamel matrix derivative sold under the trademark name Xelmaâ for the treatment of various hard-to-heal ulcers

Lesion Venous leg ulcers

Author (reference) Chadwick et al. (81) Huldt-Nystrom (82) Huldt-Nystrom et al. (82) Romanelli (83)

Romanelli et al. (84) Hampton et al. (85) Vowden et al. (86)

Diabetic foot ulcers

Vowden et al. (87) Vowden et al. (88) Romanelli et al. (84)

Pyoderma gangrenosum

Chadwick et al. (81) Romanelli et al. (84)

Mixed etiology ulcers

Chadwick et al. (81) Vowden et al. (86)

Rheumatoid ulcers

Chadwick et al. (81) Vowden et al. (86)

Pressure ulcers

Vowden et al. (86)

Title The use of amelogenin protein in the treatment of hard-to-heal wounds. Treatment of infected hard-to-heal venous leg ulcers with amelogenin in conjunction with topical silver dressings. Xelmaâ, an advanced wound treatment for venous ulcers: a European perspective. Quality of life assessments with the EuroQol instrument (EQ-5D) in a clinical trial of an advanced therapy using amelogenin in patients with chronic venous leg ulcers. Amelogenin, an extracellular matrix protein, in the treatment of venous leg ulcers and other hard-to-heal wounds: experimental and clinical evidence. An evaluation of a matrix replacement treatment in intractable wound. Experience with the use of an amelogenin-based extracellular matrix substitute in the management of a variety of complex hard-to-heal chronic wounds. Effect of amelogenin extracellular matrix protein and compression on hard-to-heal venous leg ulcers. The effect of amelogenins (Xelma) on hard-to-heal venous leg ulcers. Amelogenin, an extracellular matrix protein, in the treatment of venous leg ulcers and other hard-to-heal wounds: experimental and clinical evidence. The use of amelogenin protein in the treatment of hard-to-heal wounds. Amelogenin, an extracellular matrix protein, in the treatment of venous leg ulcers and other hard-to-heal wounds: experimental and clinical evidence. The use of amelogenin protein in the treatment of hard-to-heal wounds. Experience with the use of an amelogenin-based extracellular matrix substitute in the management of a variety of complex hard-to-heal chronic wounds. The use of amelogenin protein in the treatment of hard-to-heal wounds. Experience with the use of an amelogenin-based extracellular matrix substitute in the management of a variety of complex hard-to-heal chronic wounds. Experience with the use of an amelogenin-based extracellular matrix substitute in the management of a variety of complex hard-to-heal chronic wounds.

Conclusions and potential future research The purpose of the present review was to analyze the currently available literature reporting data on the effects of EMD on the inflammatory process with particular interest in the different cell populations and mediators involved in inflammation and soft tissue wound healing/resolution. The role of EMD was investigated in numerous inflammatory cell types with specific accent on cell markers and cytokines (Fig. 3, Table 6). It was found that EMD was able to significantly decrease the expression of IL-1b in four of six studies and decrease the production of IL-8 in two studies dealing with whole blood isolates and human PDL cells. No conclusive changes in the expression of IL-2, -6 and -10 were observed following application of EMD. The influence of EMD on the expression of TNF-a demonstrated mixed results. Two studies demonstrated a significant decrease in the expression of TNF-a and two others showed no difference (Table 3). In contrast, the production of PGE2 was significantly upregulated in three of four studies (Table 3). The results regarding the effects of EMD on bone resorption suggest that EMD inhibits bone resorption by increasing the expression of OPG in six of seven studies and decreasing the expression of RANKL by approximately 50% in three of four studies (Table 3). Aside from playing critical roles in altering the expression of proinflammatory markers, EMD also exhibited potent effects on various cell types (Table 1). EMD increased the proliferation and migration of T lymphocytes, increased tissue and bacterial debridement, induced endothelial proliferation and formation, as well as promoted angiogenesis and vessel-like formation (Table 6). Currently, there remain many areas of research that lack full understanding in the resolution of inflammation. Key markers such as resolvins and bradykinins remain at the forefront of research; however, the influence of

EMD, inflammation and soft tissue healing

11

Table 6. Summary of the inflammation-modifying changes induced by EMD 1. Decrease in IL-1b, IL-8 production, no difference in IL-2, IL-6 and IL-10 2. Increase in PGE2, no difference in TNF-alpha 3. Increase in OPG, decrease in RANKL 4. Increased proliferation and migration of T lymphocytes 5. Enhances wound healing by bacterial and tissue debris clearance 6. Promotes mesenchymal cell differentiation into hard tissue forming cells 7. Induces endothelial cell proliferation (angiogenesis) and microvascular cell differentiation 8. Decreases bacterial pathogen counts 9. Promotes wound healing in skin lesions 10. Little collected evidence on changes in MMP or TIMP production EMD, enamel matrix derivative; IL, interleukin; MMP, matrix metalloproteinases; PGE2, prostaglandin E2; TIMP, tissue inhibitors of metalloproteinases; TNF, tumor necrosis factor.

Fig. 3. Inflammation-modifying changes induced by EMD. Following application of EMD, decreased production of IL-1b and IL-8 (1) and increased levels of PGE2 (2) are observed with little differences in TNF-a expression. EMD also substantially changes the OPG/RANKL balance by increasing OPG and decreasing RANKL levels, resulting in diminished osteoclast formation/activity (3). EMD also increases the proliferation and migration of T lymphocytes (4), which enable tissue debridement by macrophages (5). Furthermore, EMD promotes mesenchymal cell differentiation into hard tissue forming cells and improves periodontal ligament cell regeneration (6). Microvascular cell differentiation and angiogenesis are improved following EMD application (7) and studies demonstrate that EMD also lowers bacterial numbers (8), resulting in a reduced inflammatory state. EMD, enamel matrix derivative; IL, interleukin; PGE2, prostaglandin E2; PDGF, platelet-derived growth factor; PMN, polymorphonuclear neutrophil; TGF, transforming growth factor; TNF, tumor necrosis factor.

12

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EMD on the expression of these molecules in any cell type has never been investigated. Furthermore, the influence of EMD on vasoactive molecules such as histamine and leukotrienes, which induce augmentation of the permeability of vessels, remains unstudied. There are no studies on the influence of EMD on IgG production and on activation/expression of other key mediators of inflammation such as C5a, C3b components of the complement cascade. There are numerous secondary mediators of inflammation such as vasoactive amines, the kinin system, fibrinolysis system, coagulation system, arachidonic acid cascade, platelet activation and nitric oxide production that remain unstudied following application of EMD. The effect of EMD on the regeneration of the junctional epithelial layer is limited with no in vitro or in vivo studies having investigated this relationship. However, EMD was found to inhibit the in vitro proliferation of gingival keratinocytes (32). This effect can serve hard tissue regeneration by favoring the growth, and later differentiation, of mesenchymal cells within the defect area. Indirect in vivo evidence that EMD inhibits the proliferation of junctional epithelial cells are several animal studies suggesting that clinical application of Emdogain results in a shorter postoperative junctional epithelium (90,91). Furthermore, it is known that leukocyte migration involves a process by which endothelial cells are typically activated by inducing receptor activation of ICAM-1 and LFA-1 to facilitate chemotaxis to sites of lesions by diapedesis into extravascular spaces. The effect of EMD on this process also remains unstudied. Interestingly, the resolution of inflammation in periodontal tissues necessitates a decrease in bacterial colonization. Apart from increasing the speed and quality of soft tissue healing, EMD has also been demonstrated to decrease bacterial numbers from both in vivo and in vitro studies (92–96). Thus, the ability of EMD to limit aggressors of the body such as bacteria of the subgingival biofilm and limit their potential to

induce tissue destruction and necrosis is a clear advantage in the resolution of inflammation. Thus, it may also be interesting to monitor the effects of EMD on wound healing of regular oral mucosal tissues not implicated with the periodontium. As EMD is also sold under the trademark name Xelmaâ for hard-to-heal leg ulcers, it also gives potential uses for healing of the oral mucosa following various or potentially hard-to-heal wounds or ulcers in the oral cavity. The present review article also found that the majority of studies investigating the expression of proinflammatory markers are derived from PDL cells and osteoblasts (Tables 1–3). Unfortunately, this is not the major cell source of these markers in gingival tissues (97). It has also recently been demonstrated that RANKL, an osteoclast differentiation marker principally expressed by osteocytes and osteoblasts, is also expressed by B and T lymphocytes in the bone resorptive lesion of periodontal disease (97). It was found in the present review article that all studies investigating RANKL expression following exposure to EMD are derived from cells other than bone cells. This substantial limitation requires a number of future studies investigating how EMD may affect inflammation in entire periodontal tissues as opposed to specific cell types. Future research aimed at determining the effects of EMD on the expression of RANKL and OPG in B and T lymphocytes is necessary, with a prominent need to study this relationship with highly specific in vivo systems. In conclusion, although it is evident that EMD affects many pathways and expression of various inflammatory mediators, much research is still required in this field. As the understanding of periodontal healing and disease resolution develops, new promising targets within the inflammatory process can be identified.

Conflict of Interest The authors received no form of external funding for the present review article. Michel Dard is

employed by Institut Straumann, Basel, Switzerland. Richard Miron and Miron Weinreb report no conflict of interest.

Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Summary of early reactions that take place during acute inflammation Table S2. Summary of late reactions that take place during an immune inflammatory response

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