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Extracellular ATP is a key modulator of alveolar bone loss in periodontitis
Accepted Manuscript Title: Extracellular ATP is a key modulator of alveolar bone loss in periodontitis Authors: Itzhak Binderman, Nasir Gadban, Avinoam Yaffe PII: DOI: Reference:
S0003-9969(17)30148-6 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.05.002 AOB 3881
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
13-9-2016 9-5-2017 10-5-2017
Please cite this article as: Binderman Itzhak, Gadban Nasir, Yaffe Avinoam.Extracellular ATP is a key modulator of alveolar bone loss in periodontitis.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Extracellular ATP is a key modulator of alveolar bone loss in periodontitis. Itzhak Binderman1,2, Nasir Gadban1 and Avinoam Yaffe3 1
Department of Oral Biology, Sackler Faculty of Medicine, Maurice and Gabriela Goldschleger,
School of Dental Medicine, and 2Department of Bio‐Medical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel; 3Department of Prosthodontics, Haddasah Faculty of Dental Medicine, Hebrew University, Jerusalem, Israel. Highlights
• Degradation of marginal gingival fibers by MMPs reduces cellular strains that induces immediate release of ATP from marginal gingival fibroblasts. • eATP interacting with P2X7 purinoreceptors present on gingival fibroblasts, induces the inflammatory cells to secrete cytokines, interleukin IL‐1, TNF and RANKL that trigger alveolar bone loss. • eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3‐ inflammasome activation via P2X7. • Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis.
Abstract Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca+2. Increased extracellular ATP (eATP) by interacting with P2X7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3‐inflammasome activation via P2X7. Then, the inflammatory cells secrete cytokines, interleukin IL‐1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that
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initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis. • Degradation of marginal gingival fibers by MMPs reduces cellular strains that induces immediate release of ATP from marginal gingival fibroblasts. • eATP interacting with P2X7 purinoreceptors present on gingival fibroblasts, induces the inflammatory cells to secrete cytokines, interleukin IL‐1, TNF and RANKL that trigger alveolar bone loss. • eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3‐ inflammasome activation via P2X7. • Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. Keywords: Periodontitis,extracellular ATP,alveolar bone loss; , marginal gingiva detachment Introduction From 5 to 20% of the population is affected by chronic periodontitis, a condition that is the leading cause of teeth loss in adults and increases the risk for several major systemic diseases (Hayashi et al.,2010; Lamster et al.,2008;). A critical event in the initiation of periodontal disease is the colonization of teeth and gums by pathogenic bacterial biofilms that leads to a chronic inflammatory process, due to activation of a host response in the marginal gingival connective tissue (Haffajee and Socransky.,1994; Kornman et al.,1997; Van Dyke and Serhan, 2003). During active stages, there is an initial neutrophilic cellular infiltrate that switches to a predominating monocytic and lymphocytic cellular infiltrate (Hayashi et al.,2010). Both gingivitis and periodontitis start by a bacterial‐induced inflammatory process in the marginal gingiva, however, they differ in their signaling to activate osteoclastic alveolar bone resorption. Although, the etiology of periodontitis is linked to virulent bacteria in the plaque biofilm, it is the immune response of a susceptible host to bacterial challenge that instigates the tissue breakdown (Kirkwood et al.,2007; Teles et al.,2010). Both resident adherent cells of marginal 2
gingiva and the inflammatory cells that invade the gingival tissue, release matrix metalloproteinases (MMPs) which degrade gingival collagen fibrous tissue, leading to its detachment from root cementum, loss of tissue integrity and reduction of cellular strains (Sapna et al.,2014, Binderman et al.,2002). Interestingly, it was reported that the amount of degraded collagen fibers in the marginal gingiva, was highly elevated in periodontitis, significantly less in gingivitis with minor degradation in the healthy marginal gingiva (Seguier et al.,2000). In addition, a distinct correlation between degradation of fibrous tissue by MMPs and increased secretion of inflammatory cytokines in marginal gingiva were revealed (Seguier et al.,2001). The MMPs that are secreted by the inflammatory cells and by local fibroblasts is most important host factor responsible for marginal gingiva collagen and extracellular matrix (ECM) degradation. MMPs are a class of enzymes, responsible for degradation of pericellular substrates, including cell surface receptors, cell–cell adhesion molecules, collagen fibers and almost all structural ECM proteins. The activities of MMPs are generally balanced by endogenous inhibitors such as tissue inhibitors of metalloproteinase (TIMP), and any imbalance between MMP and TIMP levels plays an important role in the disease progression (Kubota, 1996; Kubota et al.,2008; Khokha et al.,2013; Nagarajen et al.,2015). It is therefore, inevitable that degradation of collagenous fibrous tissue of marginal gingiva proceeds the process of alveolar bone resorption in chronic periodontitis. Our approach was to study whether surgical detachment of marginal gingiva from root surfaces, mimics the effect of MMPs on alveolar bone resorption. It was striking to find that surgical detachment of marginal gingiva from root surfaces is signaling alveolar bone resorption in similar fashion and pattern that is described for periodontitis (Yaffe et al.,1995; Binderman et al.,2001). Yet, it is not clear whether collagen degradation or local secretion of inflammatory cytokines in the marginal gingiva, each separately or together are needed to elicit osteoclastic resorption of alveolar bone in periodontitis. Also, little is known by which pathway, the progression of this response in marginal gingiva is signaling alveolar bone resorption underneath gingiva around the extensive periodontal pocket. A.
The molecular pathway from surgical detachment of marginal gingiva toward the alveolar bone resorption and bone loss.
(a) Functional structure of marginal gingiva The normal functional structure of the marginal gingiva consists of bundles of collagen fibers (Sharpey fibers) that extend from the cementum of the cervical part of the root toward the papilla, 3
toward the periosteum that is lining the alveolar bone, and toward the adjacent teeth, underneath layers of gingival epithelial cells (GEC). The fibroblasts and collagen fibers, which extend from the cementum, create a very intimate, physical and strong covalent chemical attachment of fibroblasts to fibronectin‐collagen cementum matrix and PDL fibrous matrix through transmembrane integrin proteins and by intercellular junctional complexes like cadherins, thus, creating a molecular connectivity of strained network of cells (Yamamoto et al.,1998; Binderman et al.,2002). These intercellular and cell‐ ECM attachment apparatus is connected to intracellular cytoskeletal structures, (actin, microtubule and intermediate filaments) through a chain of series of adhesion molecules, creating physiological traction forces that modulate tense connectivity in the tissues. The organized and well regulated cytoskeletal networks serve as structural support (intermediate filaments), as well as enabling the cell to maintain a specified shape and polarity by polymerization and de‐polymerization of actin and microtubule; crucial in transducing and facilitating mechanical interactions with the cell outside environment (Ingber, 2003). Moreover, they exist in a state of isometric tension, and it is because of this internal prestress, the physiological tensile network of adherent cells onto dento‐gingival fibers is a vital control system that maintains normal functions of periodontal tissues (Binderman et al.,2002; Sawhney and Howard, 2004). It was found that the traction forces transmitted through cell‐cell junctions are of greater magnitude than the traction forces at the cell‐ECM adhesions (Maruthamutu et al., 2011), suggesting that within a network of connected cells, the major pathway of force transmission is via cell‐cell rather than via cell‐ ECM adhesions (Figure 1). (b) Activation of extracellular ATP pathway in response to surgical detachment of marginal gingiva. We and others described that an osteoclastic alveolar bone resorption is activated when detachment of marginal gingiva from root cementum in response to fiberotomy or elevation of a full‐thickness flap surgeries were performed (Yaffe et al.,1995; Binderman et al.,2001; Kaynak et al.,2003; Nobuto et al.,2003; Yaffe et al.,2006 ). In several in vivo studies, we observed that in Wistar rats a significant alveolar bone loss happened three weeks after surgical detachment of marginal gingiva from root surfaces (Yaffe et al.,1995; Binderman et al.,2001; Binderman et al.,2002; Binderman et al.,2007). We demonstrated that alveolar bone resorption commences only when the surgery is performed by the coronal approach, in contrast to an apical surgical approach where no significant alveolar bone resorption was seen (Binderman et al., 2001). Immediate excision of the marginal gingiva after elevation of mucoperiosteum flap by coronal approach, prevented alveolar bone resorption. These findings indicate that the signal of alveolar bone resorption comes from the marginal gingiva tissues (Figure 1). 4
We have reported, that in rats, surgical detachment of the marginal gingiva fibrous tissue stimulated an early up‐regulation of the P2X4 receptor for extracellular ATP (eATP) (Binderman et al.,2007). Moreover, the bone loss was significantly reduced (by 50%) by instant and single local application of apyrase, enzyme that degrades extracellular ATP (eATP) or 70% by blocking the P2X purinoreceptors by application of Coomassie Briliant Blue (Binderman et al.,2007), at the surgical site. We conceived the notion that detachment of marginal gingiva from root surfaces is activating the eATP‐P2X pathway that triggers alveolar bone resorption and bone loss in periodontitis. In a recent study, human gingival fibroblasts (HGF) that were grown on films composed of collagen fibers developed a normal strained architecture of HGF (Binderman et al.,2014; Gadban et al.,2015). Once detachment of collagen films was performed an immediate rise of ATP release from human gingival fibroblasts (HGF) occurred, followed by a cellular influx of Ca+2, the upregulation of P2X7 purinoreceptor and an increase in RANKL expression (Gadban et al.,2015). Also, a significant reduction of tensile forces in the local fibroblasts network occurred that was followed by immediate cytoskeleton remodeling and changes in cell shape from strained elongated cells toward more round cells with short processes (Binderman et al.,2014; Gadban et al.,2015; Figure 1). The main factor required for osteoclast maturation is the receptor activator of nuclear factor kappa‐B ligand (RANKL), expressed on surface of osteoblasts. Although, osteoblasts are the major source of RANKL, this molecule may be produced by other cells such as fibroblast and T lymphocytes. It seems therefore that in periodontitis like in surgical detachment of marginal gingiva, an increased expression of RANKL in HGF triggered by eATP‐P2X7 pathway can directly activate osteoclastic alveolar bone resorption and bone loss without being dependent on the inflammatory pathway (Gadban et al., 2015; Binderman et al.,2007; Yu and Ferrier 1994; Binderman et al.,2014) (Figure 2). B. The interaction between eATP–P2X7 pathway and the proinflammatory pathway, in activating alveolar bone loss, in periodontitis. In a recent review, Lim and Mitchell (Lim and Mitchell,2012) described the involvement of purinergic signaling in oral pathophysiology of dental and periodontal tissues. It was recognized that adenosine 5′‐ triphosphate (ATP) and other nucleotides are released from cells following stress or injury (Jiang, 2012). Generally, the elevated eATP appears to be important in triggering cellular responses to trauma (Burnstock and Boeynaems,2014; Franke et al., 2006). Also, it is well established that under pathological conditions that include inflammation and hypoxia an increase of eATP levels occurs, due to active release of ATP, as well as passive leakage from damaged or dying cells, in combination with 5
downregulation of ectonucleotidases (Lazarowski et al.,2003). In addition, eATP is an important mediator of inflammation that is associated with systemic damage and toxicity in vivo (Cauwels et al.,2014). In this capacity, eATP serves as an agonist for P2X and P2Y nucleotide receptors. They can act on all immune cells through a spectrum of P2X ligand‐gated ion channels and G protein‐coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and then the adenosine metabolite exerts additional regulatory effects through its own receptors (Fredholm,2007; Deaglio and Robson,2011). Recently it was shown that eATP is elevated due to its release from inflammatory immune cells, during an inflammatory process (Ruscitti et al.,2015) and vice versa increase of eATP may activate inflammatory process by promoting leukocyte recruitment and NLRP3‐inflammasome activation via P2X7 purinoreceptor, both in vivo and in vitro (Bours et al.,2011 ;Pelegrin et al.,2008). The activation of the purinergic receptor, P2X7, leads to assembly of a complex of proteins, called the inflammasome, which results in activation of caspase‐1 and subsequent processing and secretion of the proinflammatory cytokines, IL‐1β and IL‐18 (Schroder and Tschopp., 2010: Johnsona et al., 2015). Recently, Ramos‐Junior et al., (Ramos‐Junior et al.,2015) found that P2X7 purinoreceptor has a dual role, being critical not only for eATP‐induced IL‐1β secretion but also for intracellular pro‐IL‐1β processing. The processing of the inactive “pro” form into the mature active form (17 kDa) requires the assembly and activation of a caspase‐1‐ activating complex, the inflammasome (Franchi et al.,2009). It should be noted that the inflammasome and its constituents are crucial in the initiation of periodontal disease and several chronic systemic diseases associated with periodontitis (Olsen and Yilmaz, 2016). Also, it is agreed that the proinflammatory and bone‐resorptive characteristics of IL‐1b are associated with the immunopathology of periodontitis, leading to periodontal tissue destruction and bone loss (Hayashi et al.,2010; Haffajee and Socransky.,1994; Kornman et al.,1997; Kirkwood et al.,2007; Hajishengallis and Sahingur, 2014). These findings indicate that eATP interacting with P2X7 is most effective in activating maturation and release of IL‐1β. It was concluded that eATP by interacting with P2X7 receptors triggers the secretion of cytokines like interlukin‐1b (IL‐1b) that contribute to osteoclastic alveolar bone resorption (Sahoo et al.,2011;Hajishengallis and Sahingur,2014). Furthermore, IL‐1 increases prostaglandin synthesis in bone. In addition, after inflammatory stimulus, prostaglandins, such as prostaglandin E2 (PGE2), may also mediate the upregulation of RANKL by activating cell‐surface receptors, thus regulating osteoclast differentiation and activation, leading to bone resorption. In this regard, the production of IL‐1β by Porphyromonas gingivalis infected macrophages, in the presence of eATP has an important role in the pathogenesis of periodontitis. Altogether, eATP by activating the P2X7 receptor has a key role in 6
modulating periodontal immunopathogenesis. It was suggested therefore, that targeting of the P2X7 pathway should be considered in future as therapeutic interventions in periodontitis to reduce and prevent alveolar bone loss (Ramos‐Junior etal.,2015). It seems, that the addition of apyrase that reduces eATP levels is reducing the release of caspase‐1 and mature IL‐ 1β. Also, pretreatment of cells with the P2X7 receptor inhibitor AZ11645373 reduced both caspase‐1 release and the secretion of IL‐1β and IL‐18 (Sahoo et al.,2011). Similar results were also observed with oxidized ATP, another P2X7 inhibitor. Interestingly, eATP could also be secreted directly from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis (Ding et al.,2016). They found that Aggregatibacter actinomycetemcomitans but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum are capable to secrete ATP, in vitro. The eATP induced chemokine expression, suggesting eATP‐ P2X7 receptor interaction, because silencing of P2X7 receptor in periodontal fibroblasts led to significant reduction in bacterial eATP induced chemokine response. These findings provide evidence for bacterial eATP as a novel virulence factor contributing to inflammation during periodontal disease (Jun et al.,2012; Ding et al.,2016). Removing eATP by means of the ATPase apyrase markedly reduced production of both the pro‐inflammatory and cell death inducing cytokines TNF and IL‐1, and the anti‐ inflammatory cytokine IL‐10 (Jun et al.,2012). It is inevitable to point out that gingival epithelial cells (GEC) lining the gingival mucosa are direct targets of invasion by Porphyromonas gingivalis and other bacteria that can multiply and survive within host epithelial cells. The infected GEC can respond to the presence of periodontal bacteria, by producing proinflammatory cytokines, chemokines and upregulate cell adhesion molecules (Hasegawa et al.,2008). They overexpress pro‐IL‐1b, but secretion of the cytokine requires a second stimulus, such as treatment with exogenous ATP, to activate caspase‐1 through the NLRP3 inflammasome (Yilmaz et al.,2010). Also, it is evident that the GEC comprise of P2X7 receptors that are distributed throughout the GEC plasma membrane (Yilmaz et al.,2008). In this regard, recent studies demonstrated that the Porphyromonas gingivalis is producing a nucleoside‐diphosphate kinase (NDK) homolog which is capable to degrade the released ATP, this way inhibiting the eATP‐ P2X7 pathway in the GEC that otherwise would activate the caspase‐1 (Johnsona et al.,2015). It is therefore predicted that ATP that is secreted by GEC or local other bacteria will be degraded by the NDK enzyme secreted by Porphyromonas gingivalis. Thus, such reduction of eATP will support the survival of GEC by suppressing their apoptosis (Yilmaz et al.,2010). Despite of what is described above, the Porphyromonas gingivalis bacteria which can invade also the subgingival cells are involved in the degradation of extracellular matrix proteins such as collagen, through activation of the host MMPs, inactivation of plasma proteinase inhibitors and cleavage of cell surface receptors (How et al., 2016). Furthermore, it 7
was shown that eATP can cause a release of MMP‐9 and a decrease in tissue inhibitor of metalloproteinase 1 (TIMP‐1) from leukocytes, inducing matrix degradation that vice versa may affect local release of ATP (Gu and Wiley,2006). It seems that in periodontitis, the inflammatory cells, fibroblasts or osteoblasts are dominant in modulating the signaling of alveolar bone resorption and bone loss, either by the MMP pathway or/and through the eATP/P2X7 pathway (Figure 2). Conclusions From animal studies and clinical observations, it is evident that surgical detachment of marginal gingiva is inducing alveolar bone loss in similar pattern as is in periodontitis. We have shown that surgical detachment of strained marginal gingiva fibrous tissue results in immediate drop in cell strain forces and a sharp release of ATP into local extracellular environment activating P2X7 pathway, without intervention of inflammatory cells. In addition, increase of RANKL expression was measured when HGF were detached from collagen fibers, in vitro (Gadban et al.,2015). Chronic periodontitis is initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response. We propose that the critical event in clinical onset of chronic periodontitis is the increased activity of MMPs that outcomes in degradation of fibrous attachment of the marginal gingiva and its detachment from the root surfaces. Then, immediate drop in the cellular strain may induce a sharp release of ATP and change of cell shape due to remodeling of cytoskeleton. The locally released ATP by fibroblasts and by inflammatory cells, interacting with P2X7 receptors on fibroblasts or on osteoblasts is proposed to be the main trigger that activates expression of RANKL, that is inducing differentiation and activity of osteoclasts on the periodontal aspect of alveolar bone crest. In addition, an alternative inflammatory pathway leads to secretion of cytokines like IL‐1 and TNF that are known to activate osteoclastic bone resorption. Furthermore, IL‐1 also stimulates osteoclast activity by increasing production of macrophage colony‐stimulating factor (M‐CSF) and inhibits osteoclast apoptosis ( Ruscitti et al.,2015). Although this review is focusing on importance of eATP in modulating alveolar bone resorption in periodontitis, it should be noted, that cytokines like IL‐1 and PGE2 are capable to activate directly osteoclastic alveolar bone resorption, in response to inflammatory triggers. Yet, eATP is also released from specific bacteria which additively can induce the eATP‐P2X7 pathway, affecting the GEC their main target. However, the Porphyromonas gingivalis which is an important pathogen bacteria
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of periodontitis is capable to release an NDK homolog that is degrading the eATP and therefore preserving the GEC from eATP‐P2X7 signaling. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis (Arulkumaran et al.,2011),(Figure 1 and Figure 2). Legends to Figures: Figure 1: The path toward alveolar bone resorption during periodontitis. Three crucial steps in the development of periodontal disease. (A) Pathogenic bacterial biofilm in the crevice between marginal gingiva and tooth surface, (B) Release and increased activity of MMPs that leads to degradation of connective tissue, (C) Increased levels of extracellular ATP. Figure 2. Extracellular ATP (eATP)‐Key Trigger for Alveolar Bone Resorption. The importance of eATP and its interaction with P2x7 receptors present on GEC, gingival fibroblasts and inflammatory cells. Also the interaction of pathogenic bacteria in regulating the eATP/P2x7 at the GEC and subgingival level in controlling the release and activity of cytokines that activate osteoclastic activity leading to bone loss. References: Arulkumaran, N., Unwin, R.J., & Tam, F.W.K. (2011). A potential therapeutic role for P2X7 receptor (P2X7R) antagonists in the treatment of inflammatory diseases. Expert Opin Investig Drugs. 20(7), 897–915. Binderman, I., Adut, M., Zohar, R., Bahar, H., Faibish, D., & Yaffe, A. (2001).Alveolar bone resorption following coronal versus apical approach in a mucoperiosteal flap surgery procedure in the rat mandible. J Periodontol. 72, 1348–1353. Binderman, I., Bahar, H., & Yaffe, A.(2002). Strain relaxation of fibroblasts in the marginal periodontium is the common trigger for alveolar bone resorption: a novel hypothesis. J Periodontol. 73(10), 1210‐1215. Binderman, I., Bahar, H., Jacob‐Hirsch, J., Zeligson, S., Amariglio, N., Rechavi, G., Shoham, S., & Yaffe, A. (2007). P2X4 is up‐regulated in gingival fibroblasts after periodontal surgery. J Dent Res. 86(2), 181‐185. 9
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Ruscitti, P., Cipriani, P., Carubbi, F., Liakouli, V., Zazzeroni, F., Di Benedetto, P., Berardicurti, O., Alesse, A., & Giacomelli, R.(2015). The Role of IL‐1 in the Bone Loss during Rheumatic Diseases, Review. Mediators of Inflammation, 2015, 1‐10. 782382, Article ID 782382, 10 pages doi.org/10.1155/2015/782382. Sahoo, M., Ceballos‐Olvera, I., del Barrio, L., & Re, F. (2011). Role of the inflammasome, IL‐1β, and IL‐18 in bacterial infections. Sci World J., 11, 2037–2050. Sapna, G., Gokul, S., & Bagri‐Manjrekar, K. (2014). Matrix metalloproteinases and periodontal diseases. Oral Dis. 20(6), 538‐550. doi: 10.1111/odi.12159. Epub 2013 Jul 15. Sawhney, R.K., & Howard, J. (2004). Molecular dissection of the fibroblast‐traction machinery. Cell Motil Cytoskeleton 58,175–185. Seguier, S., Godeau, G., & Brousse, N. (2000). Collagen fibers cells in healthy and diseased human gingival tissues. A comparative and quantitative study by immunohistochemistry and automated image analysis. J Periodontol, 71, 1079–1085. Seguier, S., Gogly, B., Bodineau, A., Godeau, G., & Brousse, N.2001. Is collagen breakdown during periodontitis linked to inflammatory cells and expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival tissue? J Periodontol., 72, 1398–1406. Schroder, K., & Tschopp, J. (2010). The inflammasomes. Cell, 140, 821–832. Teles, R., Sakellari, D., Teles, F., Konstantinidis, A., Kent, R., Socransky, S., & Haffajee, A. (2010). Relationships among gingival crevicular fluid biomarkers, clinical parameters of periodontal disease, and the subgingival microbiota. J. Periodontol. 81, 89–98. Van Dyke, T.E., & Serhan, C.N.(2003). Resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases. J Dent Res. 82, 82–90. Yaffe, A., Fine, N., Alt, I., & Binderman, I. (1995). The effect of bisphosphonate on alveolar bone resorption following mucoperiosteal flap surgery in the mandible of rats. J Periodontol. 66, 999‐ 1003. Yaffe, A., Bahar, H., & Binderman, I.(2006). Topical application of drugs influencing cytoskeleton and cell contractility affects alveolar bone loss in rats. J Periodontol. 77(5), 826‐831. Yamamoto, T., Domon, T., Takahashi, S., Islam, N., Suzuki, R., & Wakita, M.(1998). The structure and function of periodontal ligament cells in acellular cementum in rat molars. Ann Anat. 180, 519–522. 13
Yilmaz, O., Yao, L., Maeda, K., Rose, T.M., Lewis, E.L., Duman, M., Lamont, R.J., & Ojcius, D.M. (2008).Cell Microbiol. 10(4), 863–875 doi: 10.1111/j.1462‐5822.2007.01089. Yilmaz, O., Sater, A.A., Yao, L., Koutouzis, T., Pettengill, M., & Ojcius, D.M. (2010) ATP dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis. Cellular microbiology 12, 188–198. Yu, H., & Ferrier, J.(1994). Mechanisms of ATP‐induced Ca2+ signaling in osteoclasts. Cell Signal. 6, 905–914.
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Figure 1: The path toward alveolar bone resorption during periodontitis. 15
Figure 2. Extracellular ATP (eATP)‐Key Trigger for Alveolar Bone Resorption.
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S0003-9969(17)30148-6 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.05.002 AOB 3881
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
13-9-2016 9-5-2017 10-5-2017
Please cite this article as: Binderman Itzhak, Gadban Nasir, Yaffe Avinoam.Extracellular ATP is a key modulator of alveolar bone loss in periodontitis.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Extracellular ATP is a key modulator of alveolar bone loss in periodontitis. Itzhak Binderman1,2, Nasir Gadban1 and Avinoam Yaffe3 1
Department of Oral Biology, Sackler Faculty of Medicine, Maurice and Gabriela Goldschleger,
School of Dental Medicine, and 2Department of Bio‐Medical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel; 3Department of Prosthodontics, Haddasah Faculty of Dental Medicine, Hebrew University, Jerusalem, Israel. Highlights
• Degradation of marginal gingival fibers by MMPs reduces cellular strains that induces immediate release of ATP from marginal gingival fibroblasts. • eATP interacting with P2X7 purinoreceptors present on gingival fibroblasts, induces the inflammatory cells to secrete cytokines, interleukin IL‐1, TNF and RANKL that trigger alveolar bone loss. • eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3‐ inflammasome activation via P2X7. • Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis.
Abstract Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca+2. Increased extracellular ATP (eATP) by interacting with P2X7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3‐inflammasome activation via P2X7. Then, the inflammatory cells secrete cytokines, interleukin IL‐1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that
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initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis. • Degradation of marginal gingival fibers by MMPs reduces cellular strains that induces immediate release of ATP from marginal gingival fibroblasts. • eATP interacting with P2X7 purinoreceptors present on gingival fibroblasts, induces the inflammatory cells to secrete cytokines, interleukin IL‐1, TNF and RANKL that trigger alveolar bone loss. • eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3‐ inflammasome activation via P2X7. • Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. Keywords: Periodontitis,extracellular ATP,alveolar bone loss; , marginal gingiva detachment Introduction From 5 to 20% of the population is affected by chronic periodontitis, a condition that is the leading cause of teeth loss in adults and increases the risk for several major systemic diseases (Hayashi et al.,2010; Lamster et al.,2008;). A critical event in the initiation of periodontal disease is the colonization of teeth and gums by pathogenic bacterial biofilms that leads to a chronic inflammatory process, due to activation of a host response in the marginal gingival connective tissue (Haffajee and Socransky.,1994; Kornman et al.,1997; Van Dyke and Serhan, 2003). During active stages, there is an initial neutrophilic cellular infiltrate that switches to a predominating monocytic and lymphocytic cellular infiltrate (Hayashi et al.,2010). Both gingivitis and periodontitis start by a bacterial‐induced inflammatory process in the marginal gingiva, however, they differ in their signaling to activate osteoclastic alveolar bone resorption. Although, the etiology of periodontitis is linked to virulent bacteria in the plaque biofilm, it is the immune response of a susceptible host to bacterial challenge that instigates the tissue breakdown (Kirkwood et al.,2007; Teles et al.,2010). Both resident adherent cells of marginal 2
gingiva and the inflammatory cells that invade the gingival tissue, release matrix metalloproteinases (MMPs) which degrade gingival collagen fibrous tissue, leading to its detachment from root cementum, loss of tissue integrity and reduction of cellular strains (Sapna et al.,2014, Binderman et al.,2002). Interestingly, it was reported that the amount of degraded collagen fibers in the marginal gingiva, was highly elevated in periodontitis, significantly less in gingivitis with minor degradation in the healthy marginal gingiva (Seguier et al.,2000). In addition, a distinct correlation between degradation of fibrous tissue by MMPs and increased secretion of inflammatory cytokines in marginal gingiva were revealed (Seguier et al.,2001). The MMPs that are secreted by the inflammatory cells and by local fibroblasts is most important host factor responsible for marginal gingiva collagen and extracellular matrix (ECM) degradation. MMPs are a class of enzymes, responsible for degradation of pericellular substrates, including cell surface receptors, cell–cell adhesion molecules, collagen fibers and almost all structural ECM proteins. The activities of MMPs are generally balanced by endogenous inhibitors such as tissue inhibitors of metalloproteinase (TIMP), and any imbalance between MMP and TIMP levels plays an important role in the disease progression (Kubota, 1996; Kubota et al.,2008; Khokha et al.,2013; Nagarajen et al.,2015). It is therefore, inevitable that degradation of collagenous fibrous tissue of marginal gingiva proceeds the process of alveolar bone resorption in chronic periodontitis. Our approach was to study whether surgical detachment of marginal gingiva from root surfaces, mimics the effect of MMPs on alveolar bone resorption. It was striking to find that surgical detachment of marginal gingiva from root surfaces is signaling alveolar bone resorption in similar fashion and pattern that is described for periodontitis (Yaffe et al.,1995; Binderman et al.,2001). Yet, it is not clear whether collagen degradation or local secretion of inflammatory cytokines in the marginal gingiva, each separately or together are needed to elicit osteoclastic resorption of alveolar bone in periodontitis. Also, little is known by which pathway, the progression of this response in marginal gingiva is signaling alveolar bone resorption underneath gingiva around the extensive periodontal pocket. A.
The molecular pathway from surgical detachment of marginal gingiva toward the alveolar bone resorption and bone loss.
(a) Functional structure of marginal gingiva The normal functional structure of the marginal gingiva consists of bundles of collagen fibers (Sharpey fibers) that extend from the cementum of the cervical part of the root toward the papilla, 3
toward the periosteum that is lining the alveolar bone, and toward the adjacent teeth, underneath layers of gingival epithelial cells (GEC). The fibroblasts and collagen fibers, which extend from the cementum, create a very intimate, physical and strong covalent chemical attachment of fibroblasts to fibronectin‐collagen cementum matrix and PDL fibrous matrix through transmembrane integrin proteins and by intercellular junctional complexes like cadherins, thus, creating a molecular connectivity of strained network of cells (Yamamoto et al.,1998; Binderman et al.,2002). These intercellular and cell‐ ECM attachment apparatus is connected to intracellular cytoskeletal structures, (actin, microtubule and intermediate filaments) through a chain of series of adhesion molecules, creating physiological traction forces that modulate tense connectivity in the tissues. The organized and well regulated cytoskeletal networks serve as structural support (intermediate filaments), as well as enabling the cell to maintain a specified shape and polarity by polymerization and de‐polymerization of actin and microtubule; crucial in transducing and facilitating mechanical interactions with the cell outside environment (Ingber, 2003). Moreover, they exist in a state of isometric tension, and it is because of this internal prestress, the physiological tensile network of adherent cells onto dento‐gingival fibers is a vital control system that maintains normal functions of periodontal tissues (Binderman et al.,2002; Sawhney and Howard, 2004). It was found that the traction forces transmitted through cell‐cell junctions are of greater magnitude than the traction forces at the cell‐ECM adhesions (Maruthamutu et al., 2011), suggesting that within a network of connected cells, the major pathway of force transmission is via cell‐cell rather than via cell‐ ECM adhesions (Figure 1). (b) Activation of extracellular ATP pathway in response to surgical detachment of marginal gingiva. We and others described that an osteoclastic alveolar bone resorption is activated when detachment of marginal gingiva from root cementum in response to fiberotomy or elevation of a full‐thickness flap surgeries were performed (Yaffe et al.,1995; Binderman et al.,2001; Kaynak et al.,2003; Nobuto et al.,2003; Yaffe et al.,2006 ). In several in vivo studies, we observed that in Wistar rats a significant alveolar bone loss happened three weeks after surgical detachment of marginal gingiva from root surfaces (Yaffe et al.,1995; Binderman et al.,2001; Binderman et al.,2002; Binderman et al.,2007). We demonstrated that alveolar bone resorption commences only when the surgery is performed by the coronal approach, in contrast to an apical surgical approach where no significant alveolar bone resorption was seen (Binderman et al., 2001). Immediate excision of the marginal gingiva after elevation of mucoperiosteum flap by coronal approach, prevented alveolar bone resorption. These findings indicate that the signal of alveolar bone resorption comes from the marginal gingiva tissues (Figure 1). 4
We have reported, that in rats, surgical detachment of the marginal gingiva fibrous tissue stimulated an early up‐regulation of the P2X4 receptor for extracellular ATP (eATP) (Binderman et al.,2007). Moreover, the bone loss was significantly reduced (by 50%) by instant and single local application of apyrase, enzyme that degrades extracellular ATP (eATP) or 70% by blocking the P2X purinoreceptors by application of Coomassie Briliant Blue (Binderman et al.,2007), at the surgical site. We conceived the notion that detachment of marginal gingiva from root surfaces is activating the eATP‐P2X pathway that triggers alveolar bone resorption and bone loss in periodontitis. In a recent study, human gingival fibroblasts (HGF) that were grown on films composed of collagen fibers developed a normal strained architecture of HGF (Binderman et al.,2014; Gadban et al.,2015). Once detachment of collagen films was performed an immediate rise of ATP release from human gingival fibroblasts (HGF) occurred, followed by a cellular influx of Ca+2, the upregulation of P2X7 purinoreceptor and an increase in RANKL expression (Gadban et al.,2015). Also, a significant reduction of tensile forces in the local fibroblasts network occurred that was followed by immediate cytoskeleton remodeling and changes in cell shape from strained elongated cells toward more round cells with short processes (Binderman et al.,2014; Gadban et al.,2015; Figure 1). The main factor required for osteoclast maturation is the receptor activator of nuclear factor kappa‐B ligand (RANKL), expressed on surface of osteoblasts. Although, osteoblasts are the major source of RANKL, this molecule may be produced by other cells such as fibroblast and T lymphocytes. It seems therefore that in periodontitis like in surgical detachment of marginal gingiva, an increased expression of RANKL in HGF triggered by eATP‐P2X7 pathway can directly activate osteoclastic alveolar bone resorption and bone loss without being dependent on the inflammatory pathway (Gadban et al., 2015; Binderman et al.,2007; Yu and Ferrier 1994; Binderman et al.,2014) (Figure 2). B. The interaction between eATP–P2X7 pathway and the proinflammatory pathway, in activating alveolar bone loss, in periodontitis. In a recent review, Lim and Mitchell (Lim and Mitchell,2012) described the involvement of purinergic signaling in oral pathophysiology of dental and periodontal tissues. It was recognized that adenosine 5′‐ triphosphate (ATP) and other nucleotides are released from cells following stress or injury (Jiang, 2012). Generally, the elevated eATP appears to be important in triggering cellular responses to trauma (Burnstock and Boeynaems,2014; Franke et al., 2006). Also, it is well established that under pathological conditions that include inflammation and hypoxia an increase of eATP levels occurs, due to active release of ATP, as well as passive leakage from damaged or dying cells, in combination with 5
downregulation of ectonucleotidases (Lazarowski et al.,2003). In addition, eATP is an important mediator of inflammation that is associated with systemic damage and toxicity in vivo (Cauwels et al.,2014). In this capacity, eATP serves as an agonist for P2X and P2Y nucleotide receptors. They can act on all immune cells through a spectrum of P2X ligand‐gated ion channels and G protein‐coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and then the adenosine metabolite exerts additional regulatory effects through its own receptors (Fredholm,2007; Deaglio and Robson,2011). Recently it was shown that eATP is elevated due to its release from inflammatory immune cells, during an inflammatory process (Ruscitti et al.,2015) and vice versa increase of eATP may activate inflammatory process by promoting leukocyte recruitment and NLRP3‐inflammasome activation via P2X7 purinoreceptor, both in vivo and in vitro (Bours et al.,2011 ;Pelegrin et al.,2008). The activation of the purinergic receptor, P2X7, leads to assembly of a complex of proteins, called the inflammasome, which results in activation of caspase‐1 and subsequent processing and secretion of the proinflammatory cytokines, IL‐1β and IL‐18 (Schroder and Tschopp., 2010: Johnsona et al., 2015). Recently, Ramos‐Junior et al., (Ramos‐Junior et al.,2015) found that P2X7 purinoreceptor has a dual role, being critical not only for eATP‐induced IL‐1β secretion but also for intracellular pro‐IL‐1β processing. The processing of the inactive “pro” form into the mature active form (17 kDa) requires the assembly and activation of a caspase‐1‐ activating complex, the inflammasome (Franchi et al.,2009). It should be noted that the inflammasome and its constituents are crucial in the initiation of periodontal disease and several chronic systemic diseases associated with periodontitis (Olsen and Yilmaz, 2016). Also, it is agreed that the proinflammatory and bone‐resorptive characteristics of IL‐1b are associated with the immunopathology of periodontitis, leading to periodontal tissue destruction and bone loss (Hayashi et al.,2010; Haffajee and Socransky.,1994; Kornman et al.,1997; Kirkwood et al.,2007; Hajishengallis and Sahingur, 2014). These findings indicate that eATP interacting with P2X7 is most effective in activating maturation and release of IL‐1β. It was concluded that eATP by interacting with P2X7 receptors triggers the secretion of cytokines like interlukin‐1b (IL‐1b) that contribute to osteoclastic alveolar bone resorption (Sahoo et al.,2011;Hajishengallis and Sahingur,2014). Furthermore, IL‐1 increases prostaglandin synthesis in bone. In addition, after inflammatory stimulus, prostaglandins, such as prostaglandin E2 (PGE2), may also mediate the upregulation of RANKL by activating cell‐surface receptors, thus regulating osteoclast differentiation and activation, leading to bone resorption. In this regard, the production of IL‐1β by Porphyromonas gingivalis infected macrophages, in the presence of eATP has an important role in the pathogenesis of periodontitis. Altogether, eATP by activating the P2X7 receptor has a key role in 6
modulating periodontal immunopathogenesis. It was suggested therefore, that targeting of the P2X7 pathway should be considered in future as therapeutic interventions in periodontitis to reduce and prevent alveolar bone loss (Ramos‐Junior etal.,2015). It seems, that the addition of apyrase that reduces eATP levels is reducing the release of caspase‐1 and mature IL‐ 1β. Also, pretreatment of cells with the P2X7 receptor inhibitor AZ11645373 reduced both caspase‐1 release and the secretion of IL‐1β and IL‐18 (Sahoo et al.,2011). Similar results were also observed with oxidized ATP, another P2X7 inhibitor. Interestingly, eATP could also be secreted directly from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis (Ding et al.,2016). They found that Aggregatibacter actinomycetemcomitans but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum are capable to secrete ATP, in vitro. The eATP induced chemokine expression, suggesting eATP‐ P2X7 receptor interaction, because silencing of P2X7 receptor in periodontal fibroblasts led to significant reduction in bacterial eATP induced chemokine response. These findings provide evidence for bacterial eATP as a novel virulence factor contributing to inflammation during periodontal disease (Jun et al.,2012; Ding et al.,2016). Removing eATP by means of the ATPase apyrase markedly reduced production of both the pro‐inflammatory and cell death inducing cytokines TNF and IL‐1, and the anti‐ inflammatory cytokine IL‐10 (Jun et al.,2012). It is inevitable to point out that gingival epithelial cells (GEC) lining the gingival mucosa are direct targets of invasion by Porphyromonas gingivalis and other bacteria that can multiply and survive within host epithelial cells. The infected GEC can respond to the presence of periodontal bacteria, by producing proinflammatory cytokines, chemokines and upregulate cell adhesion molecules (Hasegawa et al.,2008). They overexpress pro‐IL‐1b, but secretion of the cytokine requires a second stimulus, such as treatment with exogenous ATP, to activate caspase‐1 through the NLRP3 inflammasome (Yilmaz et al.,2010). Also, it is evident that the GEC comprise of P2X7 receptors that are distributed throughout the GEC plasma membrane (Yilmaz et al.,2008). In this regard, recent studies demonstrated that the Porphyromonas gingivalis is producing a nucleoside‐diphosphate kinase (NDK) homolog which is capable to degrade the released ATP, this way inhibiting the eATP‐ P2X7 pathway in the GEC that otherwise would activate the caspase‐1 (Johnsona et al.,2015). It is therefore predicted that ATP that is secreted by GEC or local other bacteria will be degraded by the NDK enzyme secreted by Porphyromonas gingivalis. Thus, such reduction of eATP will support the survival of GEC by suppressing their apoptosis (Yilmaz et al.,2010). Despite of what is described above, the Porphyromonas gingivalis bacteria which can invade also the subgingival cells are involved in the degradation of extracellular matrix proteins such as collagen, through activation of the host MMPs, inactivation of plasma proteinase inhibitors and cleavage of cell surface receptors (How et al., 2016). Furthermore, it 7
was shown that eATP can cause a release of MMP‐9 and a decrease in tissue inhibitor of metalloproteinase 1 (TIMP‐1) from leukocytes, inducing matrix degradation that vice versa may affect local release of ATP (Gu and Wiley,2006). It seems that in periodontitis, the inflammatory cells, fibroblasts or osteoblasts are dominant in modulating the signaling of alveolar bone resorption and bone loss, either by the MMP pathway or/and through the eATP/P2X7 pathway (Figure 2). Conclusions From animal studies and clinical observations, it is evident that surgical detachment of marginal gingiva is inducing alveolar bone loss in similar pattern as is in periodontitis. We have shown that surgical detachment of strained marginal gingiva fibrous tissue results in immediate drop in cell strain forces and a sharp release of ATP into local extracellular environment activating P2X7 pathway, without intervention of inflammatory cells. In addition, increase of RANKL expression was measured when HGF were detached from collagen fibers, in vitro (Gadban et al.,2015). Chronic periodontitis is initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response. We propose that the critical event in clinical onset of chronic periodontitis is the increased activity of MMPs that outcomes in degradation of fibrous attachment of the marginal gingiva and its detachment from the root surfaces. Then, immediate drop in the cellular strain may induce a sharp release of ATP and change of cell shape due to remodeling of cytoskeleton. The locally released ATP by fibroblasts and by inflammatory cells, interacting with P2X7 receptors on fibroblasts or on osteoblasts is proposed to be the main trigger that activates expression of RANKL, that is inducing differentiation and activity of osteoclasts on the periodontal aspect of alveolar bone crest. In addition, an alternative inflammatory pathway leads to secretion of cytokines like IL‐1 and TNF that are known to activate osteoclastic bone resorption. Furthermore, IL‐1 also stimulates osteoclast activity by increasing production of macrophage colony‐stimulating factor (M‐CSF) and inhibits osteoclast apoptosis ( Ruscitti et al.,2015). Although this review is focusing on importance of eATP in modulating alveolar bone resorption in periodontitis, it should be noted, that cytokines like IL‐1 and PGE2 are capable to activate directly osteoclastic alveolar bone resorption, in response to inflammatory triggers. Yet, eATP is also released from specific bacteria which additively can induce the eATP‐P2X7 pathway, affecting the GEC their main target. However, the Porphyromonas gingivalis which is an important pathogen bacteria
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of periodontitis is capable to release an NDK homolog that is degrading the eATP and therefore preserving the GEC from eATP‐P2X7 signaling. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis (Arulkumaran et al.,2011),(Figure 1 and Figure 2). Legends to Figures: Figure 1: The path toward alveolar bone resorption during periodontitis. Three crucial steps in the development of periodontal disease. (A) Pathogenic bacterial biofilm in the crevice between marginal gingiva and tooth surface, (B) Release and increased activity of MMPs that leads to degradation of connective tissue, (C) Increased levels of extracellular ATP. Figure 2. Extracellular ATP (eATP)‐Key Trigger for Alveolar Bone Resorption. The importance of eATP and its interaction with P2x7 receptors present on GEC, gingival fibroblasts and inflammatory cells. Also the interaction of pathogenic bacteria in regulating the eATP/P2x7 at the GEC and subgingival level in controlling the release and activity of cytokines that activate osteoclastic activity leading to bone loss. References: Arulkumaran, N., Unwin, R.J., & Tam, F.W.K. (2011). A potential therapeutic role for P2X7 receptor (P2X7R) antagonists in the treatment of inflammatory diseases. Expert Opin Investig Drugs. 20(7), 897–915. Binderman, I., Adut, M., Zohar, R., Bahar, H., Faibish, D., & Yaffe, A. (2001).Alveolar bone resorption following coronal versus apical approach in a mucoperiosteal flap surgery procedure in the rat mandible. J Periodontol. 72, 1348–1353. Binderman, I., Bahar, H., & Yaffe, A.(2002). Strain relaxation of fibroblasts in the marginal periodontium is the common trigger for alveolar bone resorption: a novel hypothesis. J Periodontol. 73(10), 1210‐1215. Binderman, I., Bahar, H., Jacob‐Hirsch, J., Zeligson, S., Amariglio, N., Rechavi, G., Shoham, S., & Yaffe, A. (2007). P2X4 is up‐regulated in gingival fibroblasts after periodontal surgery. J Dent Res. 86(2), 181‐185. 9
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Figure 1: The path toward alveolar bone resorption during periodontitis. 15
Figure 2. Extracellular ATP (eATP)‐Key Trigger for Alveolar Bone Resorption.
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