periodontitis P. MARK BARTOLD, MELI SSA D. CANTLEY & DAVI D R. HAYNES The periodontal diseases range from the relatively benign form of periodontal disease known as gingi- vitis, to chronic and aggressive forms of periodontal disease, all of which not only threaten the dentition but may also be a threat to general health (72). All forms of inammatory periodontal disease are asso- ciated with chronic inammation, resulting in destruction of the periodontal ligament and bone. If left untreated, signicant tissue damage occurs; the affected teeth can become loose and may be lost if the disease continues to be active. The periodontal diseases are very prevalent, with up to 90% of the adult population suffering from gingivitis, 60% having chronic periodontitis and 515% having aggressive periodontitis (72). Histologically and biochemically the periodontal lesions of patients with chronic and aggressive peri- odontitis appear to be similar. While there may be some differences in the cellular inltrate between these two diseases (discussed elsewhere in this edi- tion of Periodontology 2000), the molecular mediators and pathologic processes are generally the same. The only differences between chronic periodontitis and aggressive periodontitis with regard to tissue destruction appear to be perhaps the magnitude, sequelae and control of the response. Otherwise the mechanisms are remarkably similar. While a great deal of focus has been on managing the inammation in the gingival tissues, advances in our understanding of bone metabolism are opening up new avenues of understanding regarding the pathologic bone loss in periodontitis. This knowl- edge, together with the development of novel drugs that can inhibit bone loss destruction, provides us with opportunities to target not only soft tissue inammation but also the destructive bone loss seen in periodontitis. This review aims to demonstrate that, in the future, periodontal treatments will not only target the inammatory response but will also utilize adjunct agents to prevent alveolar bone loss associated with progressing periodontitis. Mechanism of tissue damage in periodontitis Periodontitis (both aggressive and chronic) repre- sents a specic inammatory response to microbial residents of the subgingival biolm. Within the con- ditions known as periodontitis there is considerable variability in terms of clinical manifestation and disease progression rates. This variability may be attributed to differences in the composition of the subgingival microbial ora. However, emerging evi- dence strongly suggests that it is the inammatory response of the host that drives the tissue destruc- tion, and the variability of host responses can ac- count for much of the variability in the clinical manifestation of periodontitis. Hence, although bac- teria are necessary for disease initiation, they are not sufcient to cause disease progression unless there is an associated inammatory response within a sus- ceptible host (73). Effector mechanisms of tissue destruction in periodontitis It is now well accepted that a large consortia of cytokines, cell-signaling molecules and matrix me- talloproteinases are dysregulated and intimately in- volved in the pathogenesis of periodontitis. The task 55 Periodontology 2000, Vol. 53, 2010, 5569 Printed in Singapore. All rights reserved 2010 John Wiley & Sons A/S PERIODONTOLOGY 2000 ahead of us is to identify the various roles of these important biological mediators of inammation and how to control them; for example, their cellular sources, their concentrations, the cells they affect in vivo, the stages in which they are active, the role and concentrations of their inhibitors and, perhaps most importantly, their site of action. Regarding this last point, it is becoming apparent that the mechanisms of soft tissue destruction (gingival and periodontal ligament) and hard tissue destruction (alveolar bone) are quite different. Indeed, the effects of cytokines and cell-signaling molecules on normal and patho- logical cellular process are important, and it is pro- posed that their roles in the pathophysiology of extracellular matrix destruction result from their excessive production, dysregulation or inadequate inhibition (80). Hence, the destruction of soft and hard tissues seen in periodontitis is the result of not only a large number of cytokines but also the sus- tained presence of other effector molecules released by resident and migrating cells. Together these inammatory mediators of inammation are able to induce the cascade of molecular events associated with extracellular matrix degradation and resultant tissue damage. Mechanisms of bone loss in periodontitis A principal feature of inammatory-mediated bone loss in periodontitis is enhanced osteoclast activity without a corresponding increase in bone formation. Osteoclasts are multinucleated cells that are derived from the monocyte macrophage lineage and are considered to be the principal cell responsible for bone resorption (9, 54, 84). Studies in mice lacking osteoclasts have demonstrated the pivotal role of the cells in bone resorption (76, 79). Multinucleated os- teoclasts have been shown to resorb alveolar bone in both animal and human studies of periodontitis. The formation of osteoclasts is driven by cytokines pres- ent in the inamed periodontal tissues, and under- standing this process is central in the development of strategies to control this process. Figure 1 demon- strates the inammatory cytokines involved in periodontal bone resorption. Osteoimmunology It is well established that the immune and inam- matory systems are central to the development of periodontitis. More recently the role of the immune system in bone metabolism and bone resorption has been recognized (104). The relationship between the immune system and bone metabolism has been termed osteoimmunology, and this is a rapidly evolving eld of investigation (3). Osteoimmunology seeks to dene and understand the interactions of immune cells and their cytokines with skeletal cells. Both the immune system and bone share a large number of regulatory cytokines and other molecules in common. It is clear that understanding osteoim- munology will be central for the development of new means to prevent and control pathologic bone loss in diseases such as periodontitis. To date, a number of key regulatory molecules have been identied and these are generally related to the receptor activator of nuclear factor kappaB ligand (RANKL), its receptor, receptor activator of nuclear factor kappaB (RANK), as well as associated signaling molecules and tran- scription factors. The key elements of this system are discussed below. Macrophage colony-stimulating factor Macrophage colony stimulating factor (M-CSF) was one of the earliest signaling molecules identied to play a role in osteoclast development and activation. M-CSF is produced mainly by osteoblasts or bone marrow stromal cells and binds to a receptor on pre-osteoclasts known as cFMS, a member of the tyrosine kinase receptor superfamily. The binding of M-CSF to cFMS results in the activation of several transcription factors, including c-fos, which leads to the initiation of osteoclastogenesis. It appears that the main role of M-CSF is to promote the prolifer- ation and survival of pre-osteoclasts as well as ma- ture osteoclasts (13). M-CSF knockout mice have been found to have an osteopetrotic phenotype as a result of the lack of osteoclasts and thus lack of bone resorption. Administration of M-CSF to these animals reverses the osteopetrotic condition and restores osteoclast development and subsequent bone resorption, conrming the importance of M- CSF in osteoclastogenesis (48). RANK and RANKL RANKL is a key mediator in the process of osteoclast formation. This membrane-bound protein is a member of the tumor necrosis factor superfamily and is expressed by a variety of cells, including osteo- blasts, broblasts and T-cells. During normal bone metabolism, RANKL is expressed by osteoblasts. However, at inammatory sites RANKL is also 56 Bartold et al. expressed by immune cells such as T-lymphocytes (106). The expression of RANKL is also regulated by other modulators of bone metabolism including parathyroid hormone, vitamin D3 and interleukin-11 (51, 61). The binding of RANKL to its receptor RANK on the surface of pre-osteoblasts results in the activation of c-jun terminal kinase and the subsequent activation of nuclear factor-kappaB, leading to osteoclast for- mation. While RANKL is considered to be essential for osteoclast-driven bone resorption, tumor necrosis factor has also been reported to be capable of inducing osteoclast bone resorption in the absence of RANKL (47). However, this nding has been chal- lenged and RANKL is generally accepted as the essential ingredient for osteoclast formation (52). Indeed, RANKL knockout mice demonstrate an osteopetrotic phenotype as a result of the absence of osteoclasts (76). Subsequent administration of RANKL to these animals restores osteoclast for- mation and function, leading to enhanced bone resorption and the development of osteoporosis (51). RANKL also plays an important role in osteoim- munology. The production of RANKL is regulated in response to the presence of inammatory cytokines such as tumor necrosis factor-alpha and interleukin- 1 (12, 39). A number of studies have conrmed a role for RANKL in periodontal bone resorption. Elevated expression of RANKL has been noted in inamed periodontal tissues (16). We have also demonstrated a high expression of RANKL in broblasts and mononuclear cells in inamed periodontal tissues and this appears to be closely associated with sites of bone loss (19). The distribution of RANKL in inamed periodontal tissues associated with bone loss is shown in Fig. 2. Osteoprotegerin Osteoprotegerin is a natural inhibitor of RANKL. It is a soluble tumor necrosis factor receptor-like molecule that acts as a decoy and blocks the binding of RANKL to RANK and thus prevents osteoclastogenesis. Studies on osteoprotegerin knockout mice have shown the animals to have an osteoporotic phenotype (11). However, mice that overexpress osteoprotegerin develop osteopetrosis; this is because of a lack of osteoclast formation and hence a lack of bone resorption (65). Further tumor necrosis factor-mediated bone destruction can be prevented through the administration of osteopro- tegerin, thus reducing the osteoclast numbers (78). Osteoprotegerin is produced by human periodontal ligament cells, gingival broblasts and epithelial cells (44, 86), and its expression is modulated by inammatory cytokines. We have previously dem- onstrated that there is a reduction in osteoproteg- erin levels in the granulomatous tissue adjacent to Fig. 1. Role of inammatory cytokines in periodontal bone resorption. Osteoclast formation occurs locally and on the external surface of the bone via several mecha- nisms. The inammatory cytokines result in the produc- tion of receptor activator of nuclear factor kappaB (RANK) by lymphocytes and broblasts. In addition, the inam- matory cytokines can directly activate monocytes to differentiate into macrophages and pre-osteoclasts. In conjunction with receptor activator of nuclear factor kappaB ligand (RANKL), the inammatory cytokines can act directly on the pre-osteoclasts leading to osteoclast formation. 57 Bone loss in periodontitis alveolar bone loss (19), suggesting that the balance between RANKL levels and osteoprotegerin levels regulates the bone destruction observed in perio- dontitis. RANKL osteoprotegerin ratio in inamed periodontal tissues A number of studies to date have analysed the con- centrations and distribution of osteoprotegerin and RANKL in healthy and in inamed periodontal tis- sues. The RANKL osteoprotegerin ratio in inamed periodontal tissues has been found to increase either because of an increase in RANK or a decrease in os- teoprotegerin, or both. Such ndings are consistent with studies investigating the role of RANKL osteo- protegerin in bone resorption in conditions such as rheumatoid arthritis. Not only is the RANKL osteo- protegerin ratio increased at sites of periodontal inammation, but recent reports suggest that this ratio also correlates with disease severity. For exam- ple, the RANKL osteoprotegerin ratio is increased in the gingival crevicular uid obtained from patients with chronic periodontitis or aggressive periodontitis compared to that obtained from patients with gingi- vitis or from healthy patients (8). These ndings identify a promising therapeutic target and have encouraged the development and use of drugs that modulate the RANKL RANK osteoprotegerin axis, leading to an increase in osteoprotegerin and a de- crease in RANKL, consistent with an equilibrium state between bone formation and bone destruction. Intracellular regulators of osteoclasts When RANKL binds to RANK, a number of intracel- lular signaling pathways are activated, including those responsible for producing factors such as tu- mor necrosis factor receptor factor-6 and c-Fos, all of which are involved in osteoclast differentiation and activation (Fig. 1). All of these pathways are also in- volved in the induction and activation of nuclear factor of activated T-cells-1, which is considered to be the master transcription factor for osteoclasto- genesis (105). Tumor necrosis factor receptor factor-6 Tumor necrosis factor receptor factor-6 plays an important role in linking the intracellular events fol- lowing RANK RANKL interactions. An initial step in RANK signaling is the binding of tumor necrosis factor receptor factor adaptor proteins to the cyto- plasmic domain of RANK. This role in osteoclast formation has been demonstrated through tumor necrosis factor receptor factor-6-decient mice, which have been found to have an osteopetrotic phenotype. In these animals, osteoclast formation is impeded as a result of incomplete signaling following RANK RANKL binding (68). Some of the main downstream targets of tumor necrosis factor receptor factor-6 include transcription factors (nuclear factor- kappaB, activator protein-1 and nuclear factor of activated T-cells-1) and various mitogen-activated protein kinases, including p38 stress kinase, c-jun N- terminal kinase, extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinases (Pi3K) protein kinase B (AKT) (108). Nuclear factor kappa-light-chain- enhancer of activated B cells A crucial transcription factor that is needed for suc- cessful osteoclast formation and activation, and which is involved in RANK signaling, is nuclear fac- tor-kappaB. This has been demonstrated by the fact that nuclear factor-kappaB knockout mice develop OPG RANKL Normal gingiva Periodontitis A B C D Fig. 2. Immunolocalization of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in healthy tissue and in inamed (periodontitis) gingival tissues. The amount of osteoprotegerin (A and B) staining in the inamed tissues appears to decrease and the amount of RANKL (C and D) staining appears to in- crease with the presence of inammation in the gingival tissues. 58 Bartold et al. osteopetrosis because of a lack of osteoclasts (42). Following activation by the binding of RANKL to RANK, nuclear factor-kappaB, which is located in the cytoplasm of nonstimulated cells, enters the nucleus and activates the inhibitor of NfkappaB kinase (IkB) kinase complex (97). There are two catalytic sites within the IkappaB kinase (IKK) complex IKK-a (IKK-1) and IKK-b (IKK-2) which are linked to a third regulatory component IKK-c. Of these com- ponents it is the IKK-b that is essential for nuclear factor-kappaB activation through the phosphory- lation of IKK-a, leading to its degradation and subsequent activation of nuclear factor-kappaB (85). Alternatively, IKK-a can activate nuclear factor- kappaB through phosphorylation and proteosome processing of P-100, which generates an active p52 product (4). Both of these pathways are considered to be of central importance for osteoclast formation. Activator protein-1 family The transcription factor complex known as activator protein-1 is activated following RANK RANKL binding. Activator protein-1 is a dimeric complex composed of the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2) and activating transcription fac- tor (ATF) (ATFa, ATF2, ATF3, ATF4, B-ATF) proteins (115). Of these, c-Fos is the major activator protein-1 induced by RANK RANKL binding. Mice decient in c-Fos develop a severe osteopetrotic phenotype as a result of their inability to form osteoclasts (34). Another major component of the activator protein-1 complex is the Jun family of proteins. Unlike c-Fos, mice with conditional knockout of c-Jun and JunB do not show complete inhibition of osteoclast for- mation, and this indicates that these activator pro- tein-1 components may be able to substitute for each other during osteoclastogenesis (115). The crucial role of c-Fos in osteoclastogenesis is further demonstrated by the observation that the induction of nuclear factor of activated T-cells-1 by RANKL does not occur in c-Fos-decient cells (103). The importance of nuclear factor of activated T-cells-1 in osteoclastogenesis is discussed below. Nuclear factor of activated T-cells As detailed above, RANKL RANK binding activates nuclear factor-kappaB, activator protein-1 and mito- gen-activated protein kinses, which are considered to be important in osteoclastogenesis. However, be- cause these pathways can also be activated by inter- leukin-1, which does not induce osteoclastogenesis, other, more specic, pathways of terminal osteoclast differentiation are likely to be specic for osteoclast development. As such, nuclear factor of activated T- cells-1 has been identied as a key intracellular molecule specically involved in the regulation of terminal osteoclast differentiation (4). Nuclear factor of activated T-cells-1 expression relies upon the expression of tumor necrosis factor receptor factor-6, nuclear factor-kappaB and c-Fos, all of which are activated by RANKL. Evidence for a central role of nuclear factor of activated T-cells-1 in osteoclast formation comes from the observations that nuclear factor of activated T-cells-1-decient embryonic stem cells are unable to differentiate into osteoclasts and the expression of ectopic nuclear factor of activated T-cells-1, in the absence of RANKL, can stimulate bone marrow-derived precurser cells to differentiate into osteoclasts (103). Nuclear factor of activated T- cells-1 has been determined to be the end point factor that leads to the expression of osteoclast genes such as the calcitonin receptor (CTR), cathepsin K, tartrate- resistant acid phosphatase (TRAP) and the b3 integrin and osteoclast-associated receptor (OSCAR). Therapeutic approaches to treat pathologic bone loss Conventional Conventional therapies in conditions such as rheu- matoid arthritis which, like periodontitis, involve both soft and hard tissue destruction have included the use of agents such as nonsteroidal anti-inam- matory drugs, glucocorticoids and disease-modifying anti-rheumatic drugs. Unfortunately, while these therapies may reduce the inammation, none of these medications appear to have any direct effect on controlling or reversing bone resorption. This, to- gether with a number of well-documented unwanted (and sometimes life-threatening) side-effects, makes these medications of limited use in the treatment of pathologic bone loss in periodontitis. Tumor necrosis factor-alpha More recently, anti-tumor necrosis factor-alpha therapy has become a well-accepted therapy for the management of rheumatoid arthritis as a result of its rapid onset of action, which reduces joint destruction compared with the medications listed above. Tumor necrosis factor-alpha is produced by activated mac- rophages as well as by many other connective tissue 59 Bone loss in periodontitis cells such as synoviocytes and periodontal bro- blasts. Tumor necrosis factor-alpha has been re- ported to act either directly on osteoclasts (47) or indirectly to induce osteoclast formation through the stimulation of RANKL production by osteoblasts (90). Because tumor necrosis factor-alpha can inuence osteoclast formation in the presence or absence of RANKL it is a potential therapeutic target to control pathologic bone loss. There are currently at least ve tumor necrosis fac- tor-alpha antagonists that are commercially available and approved for use in the treatment of rheumatoid arthritis (Table 1). While effective, these medications are generally used only if more traditional therapies have failed. Hence, by the time that anti-tumor necrosis factor-alpha medications are used, bone resorptionwill already have occurred, thus limiting the effectiveness of anti-tumor necrosis factor-alpha in eliminating bone resorption. Recent evidence indi- cates that even if anti-tumor necrosis factor-alpha is administered at the onset of rheumatoid arthritis, bone damage can still occur (40). Other reports have indicated that treatment with anti-tumor necrosis factor-alpha can result in small increases in bone mineral density, but this may be of limited clinical signicance (87). Nonetheless, bone loss at sites of inammation and in conditions such as osteoporosis are associated with the presence of tumor necrosis factor-alpha, and hence anti-tumor necrosis factor- alpha therapy may have the potential to be benecial for bone loss in inammatory diseases (36, 87). Tumor necrosis factor-alpha has been recognized for some time as an important cytokine in peri- odontal inammation and tissue destruction (74). Overexpression of tumor necrosis factor-alpha has been associated with osteoclastogenesis in patients with periodontitis (10), and tumor necrosis factor antagonists have been shown to inhibit the inam- matory response and bone loss in experimental periodontitis (5). However, more recently, results from studies investigating the effects of tumor necrosis factor-alpha inhibitors on periodontal parameters have produced conicting results (23, 32, 62, 71, 75). While the currently available anti-tumor necrosis factor-alpha agents have shown some promise, further studies are needed to determine their true efcacy relative to their expense and side effects as an adjunct to periodontal treatment. Interleukins Apart from tumor necrosis factor-alpha a large number of other inammatory cytokines are involved in inammatory diseases associated with bone loss and have therefore become logical targets for the development of therapeutic agents (Table 1). One of the rst cytokines to be targeted was interleukin-1 as a result of its key regulatory role in bone resorption in diseases such as rheumatoid arthritis and periodontitis. In addition to interleukin- 1, agents targeting interleukin-3, interleukin-6, interleukin-15 interleukin-12 and interleukin-23 have been studied (6, 26, 70, 92, 119). These anti-cytokine agents provide new opportu- nities to modulate host responses in inammatory diseases. In particular, most seem to inuence the secondary effects of cytokines on RANKL expression and therefore may not inuence osteoclast-mediated bone resorption directly. Future efforts in this area, to ensure more effective control of pathologic bone resorption, should try to target those cytokines that directly inuence osteoclast formation and function. Surprisingly few studies have investigated the ef- fect of interleukin antagonists, such as those listed in Table 1, on periodontitis. One study investigating inammation and tissue loss in a nonhuman primate model of periodontitis using human soluble inter- leukin-1 receptor type 1 as an inhibitor of interleu- kin-1 reported that the inhibition of interleukin-1 has a signicant effect on the reduction of inammation, connective tissue attachment loss and bone resorp- tion (22). Given the emerging use of these agents as anti-inammatory and anti-resorptive agents, further investigations in experimental models of periodonti- tis are warranted. Antiresorptive therapies It is apparent that despite the use of the above medications, which generally target inammation and its suppression, bone loss can still progress. In light of this, agents that specically target bone turnover have been investigated. Bisphosphonates The bisphosphonates act through their ability to in- hibit osteoclast activity and have been used in a variety of bone disorders including osteoporosis, tu- mor-associated osteolysis, arthritis and periodontitis (28, 37). The actions of bisphosphonates can differ, with some being more suited to the management of systemic bone loss and others being better for con- trolling focal or local areas of bone loss. As most bisphosphonates act directly on osteoclasts and have 60 Bartold et al. Table 1. Medications currently available for controlling inammation bone resorption Tumor necrosis factor-alpha antagonists Adalimumab Product name: Humira Manufacturer: Abbott Laboratories Description: human monoclonal antibody Cetrolizumab pegol Product name: Cimzea Manufacturer: UCB (Union chimique belge) Description: humanized tumor necrosis factor-alpha antibody Entanercept Product name: Enbrel Manufacturer: Amgen and Wyeth Description: Tumor necrosis factor receptor (p75): Fc1IgG construct Golimumab Product Name: Simponi Manufacturer: Centocor Fully human monoclonal tumor necrosis factor-alpha antibody Iniximab Product name: Remicade Manufacturer: Centocor Schering-Plough elsewhere Description: chimeric monoclonal antibody Inhibitor of cytokines from the tumor necrosis factor superfamily Atacicept Product name: BLys Manufacturer: Human Genome Sciences Description: a recombinant fusion protein that binds and neutralizes B-lymphocyte stimulator and a proliferation-inducing ligand Anti-cytokine agents Anakinra Product name: Kineret Manufacturer: Amgen Description: a recombinant, nonglycosylated form of the human interleu- kin-1 receptor antagonist (IL-1Ra) Canakinumab Product name: Ilaris Manufacturer: Novartis Description: recombinant, human anti-hu man-interleukin-1 monoclonal antibody that belongs to the IgG1 j isotype subclass. Tocilizumab Product name: RoActemra Manufacturer: Roche Description: interleukin 6 (IL-6) receptor-inhibiting monoclonal antibody AMG714 Product name: AMG714 Manufacturer: Novartis Description: human monoclonal antibody against interleukin-15 Ustekinumab Product name: Stelara Manufacturer: Centacor Description: human monoclonal antibody. It is directed against interleukin-12 and interleukin-23 61 Bone loss in periodontitis little effect on inammation (95), they are often used in combination with anti-inammatory agents for the management of inammation-associated bone loss. A number of studies have been carried out to assess the potential for bisphosphonates to be used in the management of periodontal bone loss (33, 53, 82, 94, 101, 102, 114). Although some of these studies have demonstratedanimprovement inalveolar bone height following bisphosphonate treatment and conven- tional periodontal treatment, the improvements were generally quite modest and of questionable clinical signicance. Overall, the data published to date do not support the use of bisphosphonates for the manage- ment of periodontitis. Moreover, with the emergence of osteonecrosis of the jaws being reported in people undergoing bisphosphonate treatment (25, 50), the wisdom of using these medications for the mana- gement of periodontitis has been questioned (2, 28). Hormone replacement therapy Hormonal control of bone metabolism is well rec- ognized, and the use of hormone replacement therapy for the management of postmenopausal osteoporosis has been shown to be benecial (20). However, whether hormone replacement therapy is a benecial treatment for other bone-resorptive con- ditions is debatable. Indeed, the use of hormone replacement therapy for the management of rheu- matoid arthritis has produced conicting results (98, 116). Hormone replacement therapy in postmeno- pausal women may produce some slight improve- ment in their periodontal condition but generally such improvements appear to be small and of debatable value (35, 58). Therefore, given the poten- tially large number of signicant side-effects associ- ated with hormone replacement therapy, it is difcult to see this being a recommended management strategy for periodontitis. RANKL RANK interactions As detailed above, the RANKL RANK osteoproteg- erin axis is central to the regulation of bone metab- olism. Under normal homeostasis there is a balance between bone resorption and bone formation. This Table 1. Continued Rilonacept Product name: Araclyst Manufacturer: Cigna Description: a dimeric fusion protein blocks interleukin-1 beta and, to a lesser extent, interleukin-1 alpha from interacting with cell-surface receptors. AIN457 Product name: AIN457 Manufacturer: Novartis Description: fully human IgG1j monoclonal anti-interleu- kin17 antibody that selectively neutralizes interleukin-17A B-cell targets Rituximab Product name: Rituxin Manufacturer: Genetech Description: monoclonal anti-CD20 Inhibitor of costimulation Abatacept Product name: Orencia Manufacturer: Bristol-Meyers Squibb Description: fusion protein CTLA-4 with immunoglobulin Inhibitor of RANK RANKL Denosumab Product name: Denosumab Manufacturer: Amgen Description: a fully human monoclonal antibody that specically targets RANKL CTLA4, cytotoxic T-lymphocyte antigen 4; RANK, receptor activator of nuclear factor kappaB; RANKL, receptor activator of nuclear factor kappaB ligand. 62 Bartold et al. appears to be mediated via the RANK RANKL os- teoprotegerin axis, whereby excessive bone formation may be related to an excess of osteoprotegerin or to a reduction in RANKL levels (i.e. an increase in the osteoprotegerin RANKL ratio). By contrast, a de- crease in the osteoprotegerin RANKL ratio will be associated with pathologic bone loss. A number of studies have addressed these ratios in periodontitis (16) and, in general, the ratio of RANKL osteopro- tegerin is increased owing to an increase in the level of RANKL and to a decrease in the level of osteo- protegerin (19). Thus, the RANKL RANK pathway is an attractive target for the treatment of pathological bone loss. In an early study, osteoprotegerin fused to the human IgG constant region was injected into postmeno- pausal osteoporotic women and it was found that a single injection of this osteoprotegerin led to a rapid, signicant and sustained reduction in bone turnover (7). This demonstrated that the osteoprotegerin- mediated inhibition of RANKL has the potential to reduce bone loss in osteoporosis. Since this study was carried out, a number of studies have investigated the inhibition of RANKL by other mechanisms in a number of conditions involving pathologic bone loss. Denosumab is a monoclonal antibody to RANKL that has been investigated in women with osteoporosis and rheumatoid arthritis (17, 55). Studies to date indicate that RANKL inhibitors, such as denosumab, can lead to increased bone mineral density and de- creased bone resorption. Currently, the use of deno- sumab and similar agents in periodontitis has been restricted to animal intervention studies (43, 45, 107, 111). In these studies, inhibitors of RANK-mediated osteoclastogenesis led to a protective effect on pathologic bone loss in experimental periodontitis lesions. Notwithstanding these encouraging results, there are concerns that the use of RANK RANKL inhibitors can inhibit both physiological and inammatory bone resorption and may have an un- wanted systemic effect on bone. Cathepsin K inhibitors The enzyme cathepsin K is a cysteine proteinase of the papain superfamily, which is selectively ex- pressed in osteoclasts and plays a pivotal role in the degradation of bone matrix. It is the only known mammalian proteinase that can solubilize both type I and II collagens by cleavage of the telopeptide region. Cathepsin K knockout mice develop an osteopetrotic phenotype as a result of the lack of bone resorption. Cathepsin K has been identied in periodontal tis- sues and in gingival crevicular uid. Increased con- centrations of cathepsin K have been detected in the gingival crevicular uid from patients with perio- dontitis, which correlated with an increased con- centration of RANKL, suggesting that both contribute to osteoclastic bone destruction in periodontal dis- ease (27, 66). Accordingly, cathepsin K has been viewed as an attractive target for modulating bone resorption. Indeed, animal (nonhuman primate) studies have demonstrated that the inhibition of cathepsin K induces a reduction in bone resorption (99). A number of prototype cathepsin K inhibitors have now entered clinical trials for the management of osteoporosis (21). To date there are no reports on the effect of cathepsin K inhibitors on periodontal bone loss. IKK-b inhibition As detailed earlier, IKK-b is activated following the binding of RANK to RANKL. As such, this process is a target for the inactivation of nuclear factor-kappaB activation because IKK-b is essential for nuclear fac- tor-kappaB activation by proinammatory cytokines (85). Oral administration of a selective potent inhib- itor of IKK-b has demonstrated both anti-inamma- tory and anti-bone-resorbing effects in an animal model (91). Hence, the dual effect of inhibiting inammation and inhibiting bone resorption indi- cates some potential for these drugs. To date there are no reports on the effect of inhibition of IKK-b on periodontal bone loss. Alternative approaches to modulating bone resorption Vitamin D Vitamin D, which is derived from dietary sources and the action of sunlight, is essential for bone formation. A chronically low intake of vitamin D can lead to a negative calcium balance, leading to an increased loss of calcium from bone. There are numerous studies conrming that vitamin D deciency is associated with bone loss (49). In addition to its role in bone homeostasis, vitamin D has anti-inamma- tory and immunomodulatory properties (117). On the basis of these functions, modifying vitamin D levels is an attractive way of regulating bone loss in periodontitis (38). However, to date the use of vita- min D in periodontics has received little attention. In a recent study, it was noted that patients who took 63 Bone loss in periodontitis vitamin D and calcium supplements had slightly better clinical periodontal parameters than those who did not (63). It was concluded that larger lon- gitudinal studies were required to fully understand the signicance of this relationship. Statins Statins 3-hydroxy-3-methylglutaryl-CopA (HMG- CoA) reductase inhibitors have been widely used to prevent cardiovascular disease through controlling lipid metabolism. In addition to their capacity to re- duce serum cholesterol levels, statins also possess signicant anti-inammatory properties (64). One of the most common statins is simvastatin, which has shown interesting results regarding the control of alveolar bone loss by simvastatin. Although two studies have questioned any benecial effect of simvastatin on periodontal bone loss (67, 88), several studies have demonstrated protective features with concerning periodontitis and alveolar bone loss (57, 69, 93, 112). Most recently the dual effect of statin medications on the periodontium was demonstrated to be dependent on the inammatory status of the periodontal tissues (89). Overall, these studies indi- cate that statins may have some benecial thera- peutic benets for the periodontium through their immunomodulatory, anti-inammatory and reduced bone-resorptive actions but conrmation awaits more denitive studies. Novel approaches to the management of bone resorption With the expanding knowledge in bone biology and the mechanisms involved in the regulation of bone metabolism and periodontal pathology, new ap- proaches for the management of periodontal bone loss may be developed. Kinase inhibitors Kinase inhibitors are potential new targets for peri- odontal therapies. They are low-molecular-weight peptides that are relatively inexpensive and have efcacy similar to that of other, more expensive, biological agents, but with reduced risks (92). Kinases are intracellular molecules involved in signal trans- duction during inammation. Following binding of a ligand to a cell-surface receptor, numerous cyto- plasmic kinases are activated that then modulate the activity of transcription factors and thus regulate gene expression. Two types of kinases can be tar- geted: mitogen-activated protein kinases (p38, ERK and c-jun N-terminal kinase) and tyrosine kinases (Janus Kinase 3 [JAK-3] and Syk). A number of oral drugs that target these complexes have been tested in animal models of periodontitis and found to reduce inammatory cytokine production and osteoclast formation and thus protect against inammatory- mediated alveolar bone loss (46, 83). Wnt pathway A number of reports have indicated that the Wnt canonical pathway and the transcription factor acti- vator protein-1 are important for the regulation of osteoprotegerin production in osteoblasts (29). It has been proposed that the net production of osteopro- tegerin in these cells depends on the interplay be- tween the activation by the Wnt canonical pathway and the suppression by the transcription factor acti- vator protein-1 (100). Within this process, beta-cate- nin plays an important role in the Wnt signaling pathway and in bone remodeling (30). The relation- ship between Wnt signalling and activator protein-1 in osteoprotegerin production has been demon- strated in the periodontal ligament and in gingival broblasts (100). It was shown that beta-catenin could enhance interleukin-1alpha-induced osteopro- tegerin production, and activator protein-1 sup- pressed interleukin-1alpha-induced osteoprotegerin production in periodontal ligament cells. Further- more, a high expression of c-Fos (a component of the activator protein-1 transcription dimeric complex) has been reported in periodontal ligament cells compared with gingival broblasts, and this suggests a role for periodontal ligament broblasts in alveolar bone resorption in periodontitis. Because the Wnt pathway seems to form an important link between inammation and bone metabolism, it provides a novel target for treating bone-erosive conditions by changing the balance between bone formation and bone resorption. A glycoprotein that can inhibit the Wnt pathway is Dickkopf-1 (DKK-1). Osteopenia and osteoporosis have been reported when DKK-1 is over- expressed in osteoblasts (31, 77). In addition, an in- crease in the number of erosive lesions in arthritis has been reported to be related to DKK-1. Recent pre- clinical studies have shown efcacy in treating bone erosion when DKK-1 has been neutralized or the Wnt beta-catenin signaling has been enhanced. Use of a DKK1-neutralizing antibody led to an increase in the amount of trabecular bone, an increased number 64 Bartold et al. of osteoblasts and increased osteocalcin levels. It was proposed that DKK1 may regulate osteolytic bone disease and disease progression by impeding the Wnt-regulated differentiation of osteoblasts. The resultant increased production of interleukin-6, in turn, leads to increased osteoclastogenesis through increased RANKL:osteoprotegerin ratios (77). Protease-activated receptor 2 agonists antagonists Protease-activated receptors are a group of related G protein-coupled receptors which are activated following proteolytic cleavage of their extracellular domain and play important roles in chronic inam- mation (18, 60, 113). To date, four protease-activated receptors have been identied, of which protease- activated receptor 2 has received particular attention regarding bone resorption. Activation of this protein results in the release of a variety of prostanoids and cytokines, including interleukin-6 and interleukin-8 (59). A potential role for protease-activated receptor 2, which can be activated by Porphyromonas gingi- valis gingipains, in periodontitis has been suggested on the basis of its expression by alveolar bone os- teoblasts, gingival broblasts and gingival epithelial cells (1, 59, 110). However, there is conicting evi- dence regarding the role of protease-activated receptor 2 in osteoclastogenesis and bone resorption. For example, one study has shown that protease- activated receptor 2 activation can inhibit bone resorption through inhibiting osteoclast differentia- tion (96). A recent study found that protease-acti- vated receptor 2 null mice infected with P. gingivalis did not demonstrate any signs of periodontal bone resorption, in contrast to wild-type mice (118). Sev- eral other studies have demonstrated that protease- activated receptor 2 activation is associated with in- creased production of interleukin-6 and associated periodontal destruction and that a synthetic prote- ase-activated receptor 2 agonist could lead to in- creased interleukin-8 production, which also led to periodontal destruction (59, 110). Additional studies have supported a role for protease-activated receptor 2 antagonists in inhibiting inammation and sub- sequent pathological bone resorption (24, 41). While further studies are needed to determine whether agents such as protease-activated receptor 2 antag- onists are effective in reducing alveolar bone loss, protease-activated receptor 2 remains a potential novel target to control unwanted bone resorption in a number of diseases. Histone deacetylase inhibitors Histone deacetylases are a group of enzymes emerging as potential targets for a number of dis- eases, including those involving pathological bone resorption (14). The properties of histone deacety- lases include inhibition of angiogenesis, inhibition of the production of proinammatory cytokines and also protective effects on bone. Histone deacetylases act by removing the acetyl groups from histone proteins that condense chromatin and regulate protein expression (81). Histone deacetylase inhibi- tors lead to the hyperacetylation of histones and thus modify the expression of genes, leading to stimulation of the cell cycle inhibitors, down-regu- lation of immune stimulators, a reduction in the levels of inammatory cytokines and the suppres- sion of osteoclast-mediated bone resorption (15, 81). Histone deacetylase inhibition may also result in the acetylation of other nonhistone proteins and this may have a variety of effects, including regulating the transcription of genes. The regulation of nuclear factor-kappaB by histone deacetylases seems to be a critical feature of their role in osteoclast develop- ment and inammation (109). Histone deacetylase inhibitors have been studied in animal models of rheumatoid arthritis where they were shown to reduce bone destruction (56). How- ever, to date there are no reports on the use of his- tone deacetylases for periodontitis. Nonetheless, histone deacetylase inhibitors appear to have the potential to provide another therapeutic module for the management of pathological bone loss and therefore further studies are warranted on these agents. Conclusion While this volume of Periodontology 2000 is generally directed at the comparative biology of chronic peri- odontitis and aggressive periodontitis, there is little evidence to suggest that the mechanisms of bone loss are any different between these two diseases. Therefore, this review has covered the principal mechanisms involved in osteoclast-mediated bone resorption and provided an overview of current and future treatment prospects. 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