Diabetes mellitus related bone metabolism and periodontal disease

Abstract

Diabetes mellitus and periodontal disease are chronic diseases affecting a large number of populations worldwide. Changed bone metabolism is one of the important long-term complications associated with diabetes mellitus. Alveolar bone loss is one of the main outcomes of periodontitis, and diabetes is among the primary risk factors for periodontal disease. In this review, we summarise the adverse effects of diabetes on the periodontium in periodontitis subjects, focusing on alveolar bone loss. Bone remodelling begins with osteoclasts resorbing bone, followed by new bone formation by osteoblasts in the resorption lacunae. Therefore, we discuss the potential mechanism of diabetes-enhanced bone loss in relation to osteoblasts and osteoclasts.

OPEN

International Journal of Oral Science (2015) 7, 6372 2015 WCSS. All rights reserved 1674-2818/15 http://www.nature.com/ijos

Web End =www.nature.com/ijos

Ying-Ying Wu1,2, E Xiao2,3 and Dana T Graves2

Diabetes mellitus and periodontal disease are chronic diseases affecting a large number of populations worldwide. Changed bone metabolism is one of the important long-term complications associated with diabetes mellitus. Alveolar bone loss is one of the main outcomes of periodontitis, and diabetes is among the primary risk factors for periodontal disease. In this review, we summarise the adverse effects of diabetes on the periodontium in periodontitis subjects, focusing on alveolar bone loss. Bone remodelling begins with osteoclasts resorbing bone, followed by new bone formation by osteoblasts in the resorption lacunae. Therefore, we discuss the potential mechanism of diabetes-enhanced bone loss in relation to osteoblasts and osteoclasts.

International Journal of Oral Science (2015) 7, 6372; doi:10.1038/ijos.2015.2; published 10 April 2015

Keywords: bone loss; diabetes mellitus; hyperglycemia; inflammation; osseous; osteoblast; osteoclast; periodontitis

DIABETES: INTRODUCTION

Diabetes mellitus is a heterogeneous group of disorders and is characterized by high blood glucose levels.1 Type 1 diabetes mellitus (T1DM) results from an absolute deficiency of insulin, which is most commonly due to auto-immunological destruction of the insulin-producing pancreatic b cells but which can be caused by other etiologies. In type 2 diabetes mellitus (T2DM), muscle, fat and other cells become resistant to the actions of insulin. This results in the activation of a compensatory mechanism that induces b cells to secrete more insulin. T2DM occurs when the compensatory increase in insulin is insufficient to maintain blood glucose levels within a normal physiological range.23 By 2025, 300 million people are projected to be afflicted with diabetes worldwide, with a prevalence of 6.4%.45 The

countries with the most people suffering from diabetes by the year 2025 are predicted to be India, China and the United States. T1DM represents 5%10% of the total number of diabetes cases worldwide6 and is the main type of diabetes in youth, representing 85% or more of all diabetes cases in individuals younger than 20 years of age worldwide.7 On average, males and females are equally affected with T1DM in young populations.8 T2DM accounts for 90% of diabetes cases globally.4 This disorder has traditionally been considered a metabolic disorder of adults; however, it has recently become more common in young adults, adolescents and occasionally, in children.9

PATHOGENIC MECHANISMS OF DIABETES

T1DM is a polygenic autoimmune disease that is characterized by the destruction of insulin-secreting pancreatic beta cells.10 T1DM

typically occurs as a consequence of a breakdown in immune regulation, resulting in an expansion of auto-reactive CD41 and CD81 T cells and auto-antibody-producing B lymphocytes and activation of

the innate immune system, which collaborates to destroy insulin-producing beta cells.11 In an animal model, CD11c1 dendritic cells and ER-MP231 macrophages are the first cells to infiltrate the pancreas of non-obese diabetic mice, at approximately 3 weeks of age. At the same time, potentially pathogenic T cells can be detected surrounding the islet, resulting in peri-insulitis.12 Genetic susceptibility and environmental triggers are thought to contribute to the development of T1DM.13

T2DM is a metabolic disorder that is characterized by hyperglycemia and altered lipid metabolism, which is caused by the inability of islet b cells to secrete adequate insulin in response to varying degrees of insulin resistance caused by over-nutrition, inactivity or obesity.Metabolic defects that contribute to the development of T2DM include an inability of islet b cells to compensate for high glucose levels that are associated with excess food intake, increased glucagon secretion and reduced incretin response, impaired expansion of subcutaneous adipose tissue, hypoadiponectinaemia, inflammation of adipose tissue, increased endogenous glucose production and the development of peripheral insulin resistance.14 Chronic caloric excess is the primary pathogenic event that drives the development of type 2 diabetes in genetically and epigenetically susceptible individuals.1516

PATHOGENIC CHANGES IN BOTH T1DM AND T2DM Hyperglycemia

Hyperglycemia is due to impaired insulin secretion in T1DM and insulin resistance in T2DM. Beta cell destruction in T1DM and inadequate expression of glucose transporter 2 in T2DM are thought to contribute to hyperglycemia.17 Several pathways are thought to exhibit increased activity under hyperglycemic conditions and to contribute to oxidative stress via the polyol pathway,18 the hexosamine

1State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; 2School of Dental Medicine, University of Pennsylvania, Philadelphia, USA and 3Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing, ChinaCorrespondence: Dr DT Graves, School of Dental Medicine, University of Pennsylvania, Philadelphia PA 19104, USAE-mail: [email protected] 24 November 2014

REVIEW

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pathway 1921 and activator of protein kinase C.22 Hyperglycemia also leads to greater activation of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-kB), by protein kinase C in vitro.19

Hyperglycemia also results in the oxidation of sorbitol by NAD1,

thereby increasing the cytosolic NADH:NAD1 ratio and consequently inhibiting glyceraldehyde-3-phosphate dehydrogenase activation.

Advanced glycation end productsAdvanced glycation end products (AGEs) are formed by the nonenzymatic reaction of glucose and other glycating compounds that are derived from glucose and increased fatty acid oxidation. Intracellular hyperglycemia is the primary initiating event in the formation of both intracellular and extracellular AGEs.23 AGEs are derived from the intracellular auto-oxidation of glucose to glyoxal,24

decomposition of the Amadori product (glucose-derived 1-amino-1-deoxyfrutose lysine adducts) and fragmentation of glyceraldehade-3-phosphate and dihydroxyacetone phosphate to methylglyoxal.25

These reactive intracellular dicarbonyls (glyoxal, methylglyoxal and 3-deoxyglucosone) react with the amino groups of intracellular and extracellular proteins to form AGEs. Intracellular proteins that are modified by AGEs exhibit altered function. Extracellular matrix components that have been modified by AGE precursors interact abnormally with other matrix proteins and their receptors on cells. Several AGE receptors are linked to increased inflammation, including receptor for AGE (RAGE). Proteins can be structurally modified by glycosylation, thereby affecting their function. Alternatively, AGE binding to AGE receptors can induce the production of reactive oxygen species, the production of inflammatory cytokines such as tumour necrosis-alpha (TNF-a), and the activation of NF-kB.26

LipotoxicityDue to the presence of long-chain free fatty acids in the plasma, lipotoxicity is often increased in states of insulin resistance, thereby impairing b-cell secretory function 2728 and contributing to b-cell apoptosis 2930 and insulin resistance.31 Muscle cells and hepatocytes are negatively affected by excessive amounts of fatty acids, which cause increased ceramide accumulation, activate inflammatory pathways, and increase the release of reactive oxygen species (ROS) and enhance apoptosis.32

Oxidative stressOxidative stress is central to the development of insulin resistance and diabetic complications.3334 Oxidative stress plays a critical role in diabetic complications. Hyperglycemia leads to the overproduction of superoxides in mitochondria. This increase in superoxide production activates several pathways that contribute to diabetic complications, including polyol pathway flux, increased AGE formation and RAGE expression, and activation of protein kinase C and the hexosa-mine pathway.18 Inflammation induced by increased intracellular ROS also contributes to diabetic complications.35 After ROS, are created, they deplete cellular antioxidant defences, rendering the affected cells and tissues more susceptible to oxidative damage.36 It has been

shown that ROS not only play a role in the destruction of cells and tissues but also function as intracellular second messengers that regulate signal transduction cascades and gene expression. Oxidative stress can also induce the activation of multiple serine kinases, which impair the capacity of insulin to stimulate protein kinase B activation and glucose transport. NF-kB, p38 MAPK and the JNK/SAPK pathway are sensitive to oxidative stress, which is linked to impaired insulin action and the development of the late diabetic complications.33

Immune responseNeutrophils play a crucial role in several autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Some studies have noted the involvement of neutrophils in T1DM; a mild neutro-phil reduction has been associated with T1DM subjects;3738 however,

neutrophil counts in T2DM patients are normal.37 The reduction in circulating neutrophils that is observed in T1DM might be due to impaired neutrophil differentiation and output from bone marrow, increased neutrophil apoptosis or anti-neutrophil-specific antibodies, and increased recruitment into tissues.39 The functional alteration of mononuclear phagocytes has also been reported in diabetic subjects, including altered superoxide (O22) production, defective chemotaxis, and phagocytosis.40 A study has shown that infiltrating monocytes in T1DM subjects spontaneously secrete pro-inflammatory cytokines, which are known to induce and expand Th17 cells.41 Evidence also shows that the classically activated macrophages initiate insulitis and b-cell death in T1DM subjects and play a role in insulin resistance in T2DM by triggering an inflammatory response. In contrast, alternatively activated macrophages exert a protective effect in DM by attenuating tissue inflammation.42

Pro-inflammatory factorsAdipose tissue appears to be a major site for inflammatory mediator production as a result of cross-talk between adipose cells, macrophages, and other immune cells that infiltrate the expanding adipose tissue.43

Inflammatory mediators might play a dual role in T2DM, contributing to hyperglycemia-induced insulin resistance and contributing to diabetic complications.44 Pro-inflammatory factors, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6 and IL-18, are reportedly increased in diabetes mellitus and contribute to insulin resistance by both JNK and the IKKb/NF-kB pathway.4546 Enhanced production of inflammatory cytokines is thought to contribute to insulin resistance and the destruction of beta cells in the pancreas and is thought to be a major factor in the development of diabetic complications.4748

Anti-inflammatory factorsThe role of pro-inflammatory cytokines in the destruction of pancreatic b cells and the development of T2DM has been investigated; however, our knowledge about anti-inflammatory proteins is rather limited, and an imbalance between pro- and anti-inflammatory cytokines might be essential for the development of DM.4950 IL-1 receptor

antagonist is a naturally occurring anti-inflammatory antagonist of the IL-1 family of pro-inflammatory cytokines.51 Evidence shows that blocking IL-1b signals reduces the expression of inflammatory cytokines, and IL-1 receptor antagonist improves glycemic control and counteracts b-cell destruction.52 Acting as a hormone with anti-inflammatory insulin-sensitizing properties, adiponectin reportedly associates with a decrease in the risk of T2DM.53 However, increased circulating levels of adiponectin are found in T1DM patients, this might be explained by the dependence of T2DM on insulin resistance.50

COMPLICATIONS OF DIABETEST1DM and T2DM have many possible long-term complications. Epidemiological studies indicate that the severity of diabetic complications is generally proportional to the degree of hyperglycemia.54

Macrovascular complicationsThe injurious effects of diabetes on the vascular system are traditionally divided into macrovasular and microvascular complications.55

Evidence suggests that diabetic patients are three times more likely

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to suffer cardiovascular events than are non-diabetic subjects.56

Cardiovascular disease is the most common cause of death in diabetic patients.57 The central pathological mechanism in macrovascular disease is atherosclerosis. Atherosclerosis is thought to result from chronic inflammation of the arterial wall in the peripheral or coronary vascular system. One mechanism is the stimulation of lipid oxidation of low-density lipoprotein in diabetes mellitus, which accumulates in the endothelial wall of arteries.

Microvascular complicationsHigh serum glucose levels affect endothelial cells that line blood vessels and cause the basement membrane to become thicker and less effective.58 Microvascular complications are a significant part of diabetic retinopathy, nephropathy and neuropathy.59 Diabetes-enhanced TNF induces the loss of microvascular endothelial cells and pericytes by activating the transcription factor FOXO1.6061

RetinopathyDiabetic retinopathy is clinically classified into non-proliferative and proliferative disease stages. Only intraretinal microvascular changes are present in non-proliferative diabetic retinopathy, whereas new blood vessels are formed on the retina or on the optic disk in proliferative diabetic retinopathy.62 Diabetic retinopathy is characterized by several common and unique features, including thickening of the vascular basement membrane, pericyte and endothelial cell death, microaneurysms, vascular occlusion and pathological neovascularisation, which advance to retinal haemorrhage, retinal detachment, and vision loss.63 TNF has been shown to play an important role in stimulating the early events of diabetic retinopathy.60

NeuropathyDiabetic neuropathy is characterized by progressive neuronal loss, demyelination, and impaired nerve regeneration, with ultimate nerve fibre dysfunction.62 Diabetic neuropathy can affect sensory, motor and autonomic nerve fibres in any part of the body.64 Although diabetic neuropathy has been widely studied for over 20 years, the pathogenesis of this disease remains unclear; diabetic neuropathy is thought to result from the diabetic microvascular injury of small blood vessels that supply nerves, as well as oxidative injury, AGEs and insulin deficiency.65

NephropathyHyperglycemia induces cellular changes in various kidney cell types. Nephropathy is a progressive kidney disease that is caused by the angiopathy of capillaries in the kidney glomeruli and is characterized by glomerular hypertrophy, thickening of basement, tubular and glomerular membranes and the accumulation of extracellular matrix in these membranes; these changes finally cause tubulointerstitial and glomerular fibrosis and sclerosis.66 AGEs, hyperglycemia and vascular inflammation are thought to cause these pathogenic changes.6768

DIABETES-RELATED BONE DISEASESIn addition to the complications mentioned above, diabetes affect bone metabolism. A considerable amount of evidence has accumulated indicating that metabolic and endocrine alterations caused by diabetes affect bone quantity and quality over the last decades of life.69

Increased fracture riskT1DM and T2DM are associated with higher fracture risk. A large case control study in Denmark reported a relative risk for any site fracture of 1.9.70 The relative risk of hip fracture is estimated as 6.9 in T1DM

patients and as 1.38 in T2DM patients.71 Women with T2DM have a threefold higher risk of vertebral fracture compared to women who are non-diabetic.72 In the womens health initiative study, the risk of proximal humerus, foot, and ankle fractures was found to be higher among women with type 2 diabetes than among healthy controls.73

Changed bone mineral densityBone mineral density (BMD) is reduced in T1DM.72,74 Decreased

BMD and diabetic vascular complications are associated with an increased risk of fractures. In T2DM, BMD is equal or increased according a meta-analysis,71 but the fracture risk is increased despite this increase in BMD.75 The paradoxical increase in fracture rate in patients with T2DM with increased BMD might result from an increased rate of falling.76 In addition, decreased bone formation with decreased bone quality is thought to contribute to a higher fracture risk in T2DM. Rodent models also support the hypothesis that diabetic bone has reduced mechanical strength, regardless of bone density.77

Lower bone turnover rateDecreased bone turnover rate might explain the increased bone fragility in patients with diabetes. Older bone is not replaced by new bone with decreased bone turnover rate, thus decreasing bone mechanical strength. This is reflected in studies indicating that diabetic patients exhibit decreased osteoblast activity.78 Low bone turnover accompanied by reduced mineral content was found in T1DM models.79 Leptin receptor-deficient mice, a T2DM model, also exhibit decreased bone turnover.8081 The impact of diabetes on bone resorption has yielded

contradictory findings, with some studies indicating increased osteoclast activity under perturbation.8283

Delayed fracture healingFracture healing is delayed in diabetic patients. A study of 5 966 cases of hip fracture in patients with diabetes reported that these patients required longer in-hospital stays.84 Moreover, diabetic patients undergoing ankle fusing procedures exhibit an increased incidence of non-union outcomes (from 27% to 38%).85 In diabetic mice that were fed a high fat diet, fracture calluses were significantly smaller and had markedly increased adiposity.86 In streptozotocin-induced diabetic mice, fracture calluses exhibited reduced bone formation, which was associated with premature cartilage resorption.87

POTENTIAL MECHANISMS BY WHICH DIABETESAFFECTS BONEBone formation and resorption affect bone, and diabetes mellitus affects both bone formation and resorption.

Effect of diabetes on osteoblastsDiabetes has a significant effect on osteoblasts. It has consistently been noted that diabetes causes a reduction in the number of bone-forming cells.88 One of the mechanisms through which diabetes affects osteo-blasts is increased apoptosis. For example, AGEs induce osteoblast apoptosis through the MAP kinase pathway.89 Diabetes also interferes with bone formation by reducing the expression of transcription factors that regulate osteoblast differentiation.90 In rat models of T1DM and T2DM, osteoblasts exhibit lower alkaline phosphatase activity and mineralized matrix formation.9192 When AGEs are applied to

wounds in normal animals, the rate of healing is reduced by half, suggesting that AGEs, which are present at higher levels in diabetes, contribute to impaired bone healing.93 In addition, the receptor for AGEs, RAGE, is expressed at higher levels in osteoblasts in diabetic

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conditions, thus rendering diabetic animals even more sensitive to the effects of AGEs.93

Inflammation also has a significant effect on bone.94 Elevated levels of inflammatory mediators, particularly TNF, are among the striking features of diabetes.95 Diabetic animals exhibit higher levels of TNF in bone, and enhanced TNF levels are associated with reduced bone healing. Thus, diabetes-enhanced TNF contributes to an increase in bone cell apoptosis, which reduces osseous healing.96 Moreover, diabetes causes a more than two-fold induction of more than 70 genes that directly or indirectly regulate apoptosis during inflammation-induced bone injury and that significantly enhance caspase-8, -9 and -3 activity.97 A significant effect on bone, which was induced by cell death in diabetic animals, was shown by treating mice with a pancaspase inhibitor. Treatment with this inhibitor increased the number of bone-lining cells and enhanced new bone formation.97 Profiling of mRNA expression during diabetic fracture indicates that gene sets that are related to apoptosis are significantly upregulated.98 The increased expression of apoptotic genes is associated with elevated TNF-a levels, increased chondrocyte apoptosis and increased activity of the proapoptotic transcription factor, FOXO1. Inhibition of TNF significantly reduces each of these effects in diabetic fracture healing but has little effect in normoglycemic mice.98

Mesenchymal stem cells represent a precursor pool of osteoblasts, which are bone-forming cells. Inflammation, which is elevated in diabetic bone healing,96 has a significant effect on reducing mesenchymal stem cell (MSC) differentiation.99 Inflammation affects MSC through the induction of NF-kB activation. Increased NF-kB activity interferes with wnt-stimulated MSC differentiation by increasing beta-catenin degradation.100 Moreover, TNF suppresses Osx promoter activation,101 thus interfering with the differentiation of MSCs to osteoblasts because osterix is required during the early steps of differentiation. AGEs also inhibit MSC differentiation. One mechanism by which this occurs involves the upregulation of ROS by AGEs in MSCs, leading to a decrease in MSC differentiation.102103 In human mesenchymal stem

cells and mouse stromal ST2 cells, AGEs suppress the osteogenic differentiation of both cell types by increasing TGF-b expression.104 In a

T2DM mouse model, the diabetic mouse has fewer MSCs, and these MSCs exhibit poor ability to target the injury site.105 In a T1DM rat,

more numerous apoptotic cells were present in hyperglycemic bone marrow, and the size of the osteoprogenitor pool was significantly reduced.92 Thus, elevated levels of TNF and the effects of AGEs might interfere with the production of osteoblasts that are formed by inhibiting the differentiation of MSC to osteoblasts.

Effect of diabetes on osteoclastsHuman studies of diabetes mellitus generally indicate that osteoclastogenesis is enhanced. Humans with T2DM exhibit increased circulating levels of tartrate-resistant acid phosphatase, which is indicative of increased osteoclast activity.82 In patients with T1DM or T2DM, poor glycemic control leads to increased bone resorption and bone loss.78 In

patients with T2DM, levels of the bone resorption marker tartrate-resistant acid phosphatase are increased in serum, indicating increased osteoclastic function, which might be due to the diabetic polyol pathway.106 However, some studies indicate that bone resorption markers are decreased in individuals with T2DM.107

Animal studies generally indicate that osteoclast activity is increased in diabetic patients.108109 In streptozotocin-induced T1DM rats, cathepsin K expression is elevated compared to a control group, indicating increased osteoclast activity.110 In T2DM rats, osteoclastic bone resorption is enhanced compared to normoglycemic controls.111 In

diabetic mice, TNF-a, macrophage-colony stimulating factor, receptor activator of nuclear factor kappa-B ligand (RANKL) and vascular endothelial growth factor-A are upregulated; this upregulation can directly promote osteoclast differentiation and activation.87,112

Patients with T2DM exhibit elevated mitochondrial ROS levels, which promote RANKL-mediated osteoclast differentiation and function.113

Increased fatty acid levels in patients with diabetes mellitus can induce osteoclastogenesis by TNF-a.114 T2DM mice exhibited enhanced osteoclast formation in response to M-CSF and RANKL.82 In vitro

studies suggest that hyperglycemia predisposes patients to increased osteoclast formation.115 AGEs might also increase osteoclast activity.116 Mice that lack the receptor for AGE, RAGE, exhibit increased bone mass and decreased osteoclast numbers compared to wild-type mice,117 supporting the concept that AGEs contribute to osteoclast formation in patients with diabetes.

PERIODONTAL DISEASE: INTRODUCTIONPeriodontitis is one of the most widespread oral diseases and is characterized by the loss of connective tissues within the periodontium and the destruction of alveolar bone support.118119 Severe periodontitis,

which can result in tooth loss, is found in 5%20% of most adult populations worldwide. The latest data from the 2009 and 2010 National Health and Nutrition Examination Survey cycle estimates that over 47% American adults have had periodontitis.120 An epidemi

ology study shows that almost 25% of adults in Australia aged 3554 years have moderate or severe periodontitis, and 34% of 30- to 39-year-old adults living in Pomerania have periodontitis.121 Children and adolescents can have any of the several forms of periodontitis, such as aggressive periodontitis, chronic periodontitis and periodontitis as a manifestation of systemic diseases.122 However, estimates of the global prevalence of periodontitis differ based on the distribution of the disease and the methodologies used to assess it.123

PATHOGENIC MECHANISMS OF PERIODONTAL DISEASE Microorganisms

The chronic inflammatory condition of periodontitis is induced by pathogenic biofilms or dental plaque, which accumulates on the tooth surface. Over 500 bacterial species have been detected in periodontal plaque; however, the composition of the causative bacterial species is still under debate.123126 Red complex Gram-negative bacteria, com

prising Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, have been proposed as the primary aetiological agents of periodontitis.127 Recent studies have identified Filifactoralocis and many species of Spirochetes as being closely linked to periodontitis.128

As the best characterized periopathogen, P. gingivalis is a minor constituent of the total microbiota but can trigger changes in the amount and composition of the oral commensal microbiota that can disrupt homeostasis and cause inflammatory periodontal bone loss.129 Gram-

positive bacterial and oral commensal bacteria might also play a significant role in the development of periodontitis 128,130131.

Modern high-throughput techniques indicate that the subgingival microbial profiles from periodontitis patients differ according to age, pocket depth, gender and race.128,132133 Interestingly, bacteria com

munity diversity has been shown to increase with a history of period-ontitis, which runs counter to most bacterial infectious processes, which are generally associated with decreased diversity.128

Host responseAlthough bacteria are necessary for periodontal disease to occur, a susceptible host is also needed.123 The inflammatory process occurring

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in periodontitis is characterized by the infiltration of leukocytes, which limit the level of bacterial invasion and can be harmful to the periodontal tissue.134 Periodontal ligament and bone destruction is thought to be caused by a disruption of the homeostatic balance between the host response and bacteria, which causes inflammation in close proximity to bone.129,134135 The process is thought to involve

the host immune response to bacterial infection through the stimulation of osteoclastogenic factor production by immune cells, which then help to cause the periodontitis-related bone loss. Our laboratory has recently shown that the production of factors by osteoblasts and osteocytes also contributes to osteoclast formation and activity in periodontal disease (unpublished data). Evidence that the host response plays a critical role has also shown by studies in which treatment with a prostaglandin inhibitor reduced periodontitis-related bone loss136 and studies involving the inhibition of inflammatory cytokines, such as IL-1 and TNF.137138 Thus, periodontitis is a complex disease in which multiple causal risk factors play simultaneous and interactive roles; these risk factors include the immune-inflammatory status and genetic background of individuals, and the presence of environmental stressors and/or systemic diseases, such as diabetes.139141

Effects of diabetes on periodontitisDiabetes and chronic periodontitis are chronic diseases that have long been considered to be biologically linked.142143 In fact, diabetes is one

of the primary risk factors for periodontitis.144145 Cross-sectional and longitudinal studies identified that the risk of periodontitis is approximately 34 times higher in people with diabetes than in non-diabetic subjects.144 Periodontitis was found in 57.9% of T1DM patients and in15.0% in controls without diabetes.146 In another study on the periodontal status in children and adolescents with T1DM, a prevalence of20.8% of gingivitis and 5.9% of periodontitis was observed.147 Patients with T2DM were also at higher risk of having severe forms of period-ontitis compared with non-diabetic subjects.148 A study in African

Americans found that 70.6% of patients with T2DM had moderate periodontitis, and 28.5% had a severe form of the disease; this value is significantly higher than the prevalence of 10.6% among control subjects without diabetes.149 A direct relationship exists between the level of glucose control and the severity of periodontitis.146,150 The odds

ratios of T2DM with periodontal destruction in comparison to nondiabetics are 1.97, 2.10 and 2.42 in well, moderately and poorly controlled diabetics, respectively.151

EFFECTS OF DIABETES ON PERIODONTIUM TISSUES Gingiva/gingivitis

Periodontitis is preceded by various stages of gingival inflammation, which are referred to as gingivitis. The prevalence of gingivitis in children and adolescents with T1DM is nearly twice that observed in children and adolescents without diabetes.152 Evidence shows that the gingival index was 1.54 in a 5- to 9-year-old diabetic group and 1.14 in the control group; however, in the corresponding 10- to 14-year-old group, the gingival index was 1.98 in diabetic subjects and 1.17 in control subjects.153 Furthermore, gingival bleeding index is significantly correlated with age and the level of glycosylated haemoglobin.154 Similarly, the rates of gingival inflammation in adults with T2DM are higher than those in adults without diabetes. Nearly 64% of patients with T2DM have gingivitis; however, only 50% of subjects without diabetes have the disease.152 The degree of metabolic control of diabetes is an important factor in the development and progression of gingivitis; good control significantly reduces the prevalence of gingivitis.155156 In animal models, diabetes leads to the

increased production of TNF in the epithelium and connective tissue.157 Periodontal infection causes an increase in epithelial cell and connective tissue fibroblast apoptosis, which is significantly enhanced by diabetes through a caspase-3-dependent mechanism.157 This is

significant because increased diabetes-enhanced inflammation and apoptosis are thought to negatively impact the gingiva by causing a loss of epithelial barrier function and inhibiting repair processes.158159

Animal studies have demonstrated that high levels of TNF-a can stimulate the expression of pro-apoptotic genes, which induce apoptosis.96,160

In vivo studies have demonstrated that the inhibition of TNF-a reduces connective tissue cell apoptosis.161

Periodontal ligament/loss of attachmentPeriodontitis involves the loss of tooth-supporting structures comprising connective tissue attachment and bone. A statistically significant association is observed between diabetes and loss of attachment, and the level of glycemic control is an important determinant of this relationship.146,162 The odds ratio of T1DM patients with attachment

loss is 3.84 compared with non-diabetic subjects.163 In addition, more than 25% of T1DM patients with poor metabolic control exhibit sites with clinical attachment loss of o5 mm, compared with 10% of subjects with good metabolic control.152 Subjects with T2DM also exhibit a significantly higher number of sites with clinical attachment loss of o3 mm as well as loss of o5 mm. Furthermore, the prevalence of sites with moderate to severe attachment loss is proportional to the duration of diabetes.150,164

Alveolar bone/loss of boneDiabetes potentiates the severity of periodontitis and accelerates bone resorption. The percentage of sites with bone loss in poorly controlled T1DM individuals is 44% compared to 28% and 24% in well-controlled and non-diabetic subjects, respectively.165 Animal studies also demonstrate that alveolar bone loss in rats with STZ-induced T1DM with periodontitis was threefold higher than in normal rats.166167 The

risk and degree of alveolar bone loss is positively correlated with lack of metabolic control.168169

MECHANISMS OF DIABETES ENHANCED PERIODONTAL BONE LOSSThe process of bone remodelling starts with the resorption of bone by osteoclasts, followed by new bone formation by osteoblasts in the resorption lacunae. Under physiological conditions, these two activities are coupled; however, the two processes are uncoupled in pathological processes.118,134 Diabetes affects osteoclast and osteoblasts in

the periodontium in different ways, such as by increasing the expression of inflammatory mediators and RANKL/osteoprotegerin (OPG) ratios and by enhancing the levels of AGEs and ROS (Figure 1).

Effect of diabetes on osteoclasts in periodontitisDiabetes has been shown to enhance osteoclast formation in inflamed areas. Rats with T2DM exhibit a two- to four-fold increase in osteoclast number after bacterial infection by oral inoculation of a periodontal pathogen ligature that induces periodontitis compared with control rats.111,157,170 Rats with T1DM and periodontitis also exhibit a

two- to four-fold increase in the number of osteoclasts compared to non-diabetic rats with periodontitis.171

A higher degree of inflammation and a more persistent inflammatory response following periodontitis are reported in rats with T1DM and T2DM in response to the same inoculation of periodontal pathogen.172173 Diabetes impairs the resolution of periodontal

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Diabetes

Periodontal disease

Increased RANKL/OPG

Increased AGEs

Increased ROS

Increased inflammation( TNF,IL-1b,IL-6)

Enhanced PDL and osteoblast apoptosis

Reduced bone formed

Enhanced osteoclastogenesis

Aveolar bone loss

Bacterial products Host response

Figure 1 A potential mechanism of diabetes-related alveolar bone loss in periodontal disease. Diabetes increases the RANKL/OPG ratios and enhances the expression level of AGEs, ROS and inflammatory mediators, which induce osteoblast apoptosis and osteoclastogenesis. This cascade of events contributes to both increased bone resorption and reduced reparative bone formation leading to greater alveolar bone loss in periodontal disease that is caused by bacterial pathogens. AGE, advanced glycation end product; IL, interleukin; OPG, osteoprotegerin; PDL, periodontal ligament; RANKL, receptor activator of nuclear factor kappa-B ligand; ROS, reactive oxygen species; TNF, tumor necrosis factor.

inflammation. The importance of resolving inflammation has been demonstrated by treating animals with periodontitis with resolvins174

or by treatment of diabetic animals with TNF inhibitors.160,170

Diabetes-enhanced TNF has been shown to prevent the downregulation of genes that are associated with host defence, apoptosis, cell signalling and activity, and coagulation/homeostasis/complement.175

Similarly, patients with periodontitis and diabetes were found to have significantly higher levels of local inflammatory mediators such as IL-1b, TNF-a and prostaglandin E2, which result in more prolonged osteoclast formation and activity.176 Enhancement of IL-17 and IL-23 in periodontitis in subjects with T1DM and overexpression of IL-1b and IL-6 in patients with T2DM have been reported; and these conditions result in osteoclastogenesis and a prolonged duration of inflammatory responses.177178 Patients with T2DM and periodontal

disease exhibit increased levels of TNF-a and IL-6, which are also associated with increased dyslipidemia and lipid peroxidation.179

These results suggest that there is an important association between T2DM, dyslipidemia and the severity of the local inflammatory response to bacteria.179

The interaction of RANKL with receptors (RANK) on the surface of osteoclasts is one of the most potent inducers of osteoclast formation and activity, and OPG inhibits osteoclast formation binding to RANK and then blocks the activity of RANKL.169170,180 A number of studies

focusing on osteoclastogenesis-related factors have reported an elevated expression of RANKL and TNF in diabetes-associated periodontal tissues.180 Studies with animals suggest that RANK-RANKL/OPG ratios and the level of other inflammatory cytokines, such as TNF, are critical mediators of the enhanced osteoclastogenesis in diabetes with periodontal disease.170171,181 TNF levels and the RANKL/OPG ratio in

periodontitis sites in humans are negatively influenced by poor glycemic control in subjects with diabetes.179,182

Diabetes enhances the formation of AGEs in the periodontium and increases the expression of RAGE.183 Gingiva AGEs are increased in both T1DM- and T2DM-associated periodontitis; however, there is evidence that subjects affected with T1DM presented a significantly higher percentage of AGE-positive cells in the epithelium and fibro-blasts than did subjects affected with T2DM.184 Osteoclast-like cells express RAGE, which serves as a positive factor that regulates osteoclast formation.117 AGE accumulation and the interaction of AGEs with RAGE may contribute to osteoclastogenesis via an increased expression of the receptor activator of RANKL and OPG downregulation.185 It has been shown that the AGE-RAGE interaction on monocytes activates transcription factor NF-kB, which alters the phenotype of monocytes/macrophages and results in the increased production of pro-inflammatory cytokines.186

ROS are known to be one of causes of diabetes-related periodontitis. The invading bacteria trigger the release of inflammatory cytokines, leading to increases in the number and activity of neutrophils, which release ROS in periodontitis.187 During bone resorbtion, osteoclasts that contain NADPH-oxidase actively produce superoxide.188

Simultaneously, neutrophils in diabetic patients produce more super-oxide than neutrophils from normal subjects.26 The imbalance between ROS production and antioxidant defences result in increased oxidative stress.189 In addition, the formation of AGEs also increases oxidative stress in periodontium tissue. It has been shown that certain ROS (such as superoxide and hydrogen peroxide) activate osteoclasts and promote osteoclast formation.190 A related process, lipid peroxidation, is also associated with increased periodontal disease-T2DM

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and a greater inflammatory response in periodontal tissues in humans.179,191

Effect of diabetes on osteoblasts in periodontitisEvidence exists that both diabetes and bacterial infection in period-ontitis enhance the apoptosis of osteoblastic cells, thereby reducing osseous coupling.161,192 It is likely that both the innate and adaptive

immune responses that are induced by bacterial infection cause osteoblastic cell loss.96,193 Diabetes also increases the loss of periodontal ligament (PDL) cells that is induced by periodontal infection by increasing the apoptosis of these cells.111,194 This loss is significant

because the PDL is a rich source of cells that are capable of differentiating into osteoblasts. Studies in diabetic animals indicate that diabetes causes a more than 2-fold induction of genes that regulate the apoptosis of osteoblasts and fibroblasts following bacterial infection and a fivefold increase in osteoblast apoptosis.97,195 The apoptosis of

osteoblasts is a significant component of the failure of diabetic animals to form new bone after the induction of periodontal disease, as shown by a significant increase in bone formation when diabetic animals are treated with a specific blocker of apoptosis following periodontal infection.161

Diabetes leads to the upregulation of pro-apoptotic factors for osteoblasts, including advanced TNF-a, AGEs and the formation of ROS, each of which can contribute to apoptosis.161 Animals with T1DM and T2DM exhibit higher levels of TNF-a in response to a bacterial stimulus than normoglycemic controls.170,184 Enhanced

TNF-a levels have been directly linked to cellular changes in diabetes-enhanced periodontitis.196 TNF-a impairs the function of osteoblasts by blocking the differentiation of osteoblasts when inflammation is thought to be present. Furthermore, TNF-a can induce apoptosis by binding to TNF receptor-1, which triggers the initial events in apoptosis.158

It has been shown that increased bacterial infection-induced alveolar bone loss in diabetic subjects is accompanied by the enhanced expression of RAGE and inflammatory AGEs in gingival tissue.197

AGEs have been shown to interfere with osteoblast differentiation and to induce the apoptosis of osteoblasts in diabetes via the mitogen-activated protein kinase and cytosolic apoptotic pathway.89

Elevated levels of AGEs are found in the periodontium of diabetics, and the AGERAGE interaction results in an increase in pro-inflammatory cytokine expression and induces osteoblast apoptosis.186,198199

CML-collagen, an AGE that is found in bone and serum, stimulates the apoptosis of bone-lining cells in vivo and in various osteoblastic cell cultures, which is mediated by RAGE.200

ROS production is another mechanism by which diabetes increases apoptosis. Persistent inflammation and hyperglycemia leads to cellular ROS accumulation, which is linked to diabetic complications.199,201 Moreover, increased oxidative stress in periodontal tissue has been shown to induce osteoblast apoptosis.202 It has been

demonstrated that ROS causes the activation of caspase-3,203 which

mediates osteoblast apoptosis.

CONCLUSIONIn summary, diabetes mellitus and periodontal diseases are closely associated and exhibit similarities to other chronic diseases. Persistent hyperglycemia leading to exaggerated immune-inflammatory responses that are induced by periodontal pathogens is likely to be responsible for the greater risk and severity of periodontal disease in diabetics.134,199 Moreover, severe periodontitis that results in alveolar bone loss is likely to involve the effect of inflammation on both

osteoclasts and osteoblasts. Diabetes has an important effect on enhancing osteoclastogenesis and on increasing osteoblast apoptosis. Interestingly, the effect of diabetes on bone loss and coupled bone formation is likely to involve the impact of diabetes on both the innate and adaptive immune responses.47,134

ACKNOWLEDGEMENTSThe authors would like to thank Sunitha Batchu for help in preparing this manuscript and Megha Dalalfor for technical assistance. This work was funded by the National Institute of Dental and Craniofacial Research (grant no. DE021921).

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