Effects of Low-dose aspirin and gum diseases

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Effects of long-term use of low-dose aspirin in ex-smokers and periodontitis

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Effects of Low-dose aspirin and gum diseases

  1. 1. Periodontal conditions in relation to low-dose aspirin therapy in ex- and non-smokers by Arthur Drouganis BDS, Grad Cert Dent A thesis submitted for the degree of Master of Dental Surgery (Periodontics) The University of Adelaide Dental School November 1999
  2. 2. Dedication This thesis is dedicated to my loving wife Helen, and my children Vicky, Lambros and Margaret whose support, enthusiasm and tolerance enabled me to complete the work.
  3. 3. TABLE OF CONTENTSAcknowledgments ............................................................................................................xiGlossary of terms...........................................................................................................xiiiSummary.........................................................................................................................xivChapter 1 Introduction....................................................................................................1Chapter 2 Review of the literature..................................................................................5 2.0 SUMMARY OF THE PRESENT UNDERSTANDING OF THE INFLAMMATORY RESPONSE..............................5 2.1 ENDOGENOUS MEDIATORS OF INFLAMMATION...............................................................................8 2.1.1 Histamine: ...................................................................................................................8 2.1.2 Bradykinin: ..................................................................................................................9 2.1.3 Plasmin: .....................................................................................................................9 2.1.4 Complement: ...........................................................................................................10 2.1.5 Platelets:....................................................................................................................11 2.2 EICOSANOIDS ........................................................................................................................12 2.2.1 General properties of eicosanoids.............................................................................13 2.3 ROLE OF EICOSANOIDS IN PERIODONTAL TISSUES.........................................................................15 2.3.1 Biosynthesis of eicosanoids.......................................................................................16 2.3.2 Arachidonic acid pathways: eicosanoid production....................................................17 2.3.3 Catabolism of the eicosanoids...................................................................................24 2.4 THE ROLE OF CYTOKINES IN PERIODONTAL TISSUES.....................................................................25 2.5 CELLULAR EVENTS IN INFLAMMATION........................................................................................31 2.5.1 Macrophage phenotypes...........................................................................................32 2.5.2 Alveolar bone resorption ..........................................................................................33 2.6 NONSTEROIDAL ANTI-INFLAMMATORY DRUGS IN PERIODONTAL DISEASES.......................................34 2.6.1 History of salicylates..................................................................................................34 2.6.2 Physio-chemical properties of aspirin and other salicylates ......................................35 2.6.3 Periodontal studies of the effects of NSAIDs over the last 20 years..........................39 2.7 PERIODONTAL STUDIES WITH ASPIRIN.........................................................................................52 2.7.1 The Waite study: .......................................................................................................52 2.7.2 The Feldman study ...................................................................................................53 2.7.3 The Flemmig study ...................................................................................................54 2.7.4 The Heasman study .................................................................................................55 2.8 SMOKING AND PERIODONTAL DISEASES.......................................................................................56 2.8.1 The periodontal effects of past smoking and smoking dose......................................57 i
  4. 4. 2.9 PERIODONTAL MEASURES..........................................................................................................58 2.9.1 The experimental unit................................................................................................59 2.9.2 Measurement of extent and severity of periodontal attachment loss.........................59 2.10 NULL HYPOTHESES................................................................................................................61Chapter 3 Materials and methods.................................................................................62 3.1. SAMPLE SELECTION.................................................................................................................62 3.2 QUESTIONNAIRE......................................................................................................................64 3.3 ORAL EXAMINATION...............................................................................................................66 3.4 CLINICAL MEASUREMENTS........................................................................................................66 3.4.1 Plaque Index .............................................................................................................66 3.4.2 Calculus ....................................................................................................................67 3.4.3 Bleeding index. ........................................................................................................67 3.4.3 Tooth mobility ...........................................................................................................68 3.4.4 Furcation involvement ...............................................................................................69 3.5 DETAILS OF THE STUDY............................................................................................................69 3.5.1 Periodontal attachment loss (PAL).............................................................................69 3.5.2 Periodontal Pocket Depths (PPD)..............................................................................70 3.5.3 Gingival Recession (GR)...........................................................................................70 3.5.4 Examiner standardisation:.........................................................................................70 3.5.5 Procedure..................................................................................................................70 3.6 STATISTICAL METHODOLOGY....................................................................................................71Chapter 4 Results............................................................................................................72 4.1 INTRA-EXAMINER ERROR..........................................................................................................72 4.2 PROFILE OF STUDY POPULATION:...............................................................................................72 4.3 DEMOGRAPHICS......................................................................................................................73 4.3.1 Age categories of subjects.........................................................................................73 4.3.2 Education status of the subjects................................................................................74 4.3.2 Oral health behaviour................................................................................................75 4.4 TOOTH LOSS...........................................................................................................................76 4.5 THE PERIODONTAL STATUS OF THE STUDY POPULATION................................................................76 4.6 ASSOCIATIONS OF ASPIRIN AND EX-SMOKING WITH VARIOUS MEASURES OF PAL............................78 4.6.1 The associations of aspirin and ex-smoking with mean PAL.....................................78 4.6.2 The associations of aspirin and ex-smoking on the extent and severity of PAL ........79 4.6.3 Associations of aspirin and ex-smoking with the most severe site of PAL (MSS-PAL) .............................................................................................................................................81 4.6.4 Associations of aspirin and ex-smoking with the extreme worst site of PAL (EWS- PAL).....................................................................................................................................82 ii
  5. 5. 4.7 THE ASSOCIATIONS OF VARIOUS CLINICAL PARAMETERS ON MEAN PAL........................................84 4.7.1 Site and tooth variations in recession and pocket depth by mean PAL......................85 4.7.2 Socio-economic factors and periodontal attachment.................................................86Chapter 5 Discussion....................................................................................................115 5.1 PROFILE OF THE STUDY POPULATION.......................................................................................115 5.1.1 Age groupings .........................................................................................................117 5.2 QUESTIONNAIRE....................................................................................................................119 5.2.1 Socio-economic status.............................................................................................120 5.3 PERIODONTAL ATTACHMENT LOSS..........................................................................................121 5.3.1 Age associations with PAL .....................................................................................121 5.4 MEASURING PAL.................................................................................................................122 5.4.1 Case definitions.......................................................................................................122 5.5 OUTCOMES OF ASPIRIN AND PAST SMOKING ON PAL................................................................125 5.5.1 Mean PAL................................................................................................................125 5.5.2 MSS-PAL.................................................................................................................127 5.5.3 EWS-PAL.................................................................................................................127 5.5.4 Plaque .....................................................................................................................128 5.5.5 Gingival bleeding ....................................................................................................129 5.6 COMPARISONS WITH OTHER ASPIRIN STUDIES............................................................................130 5.7 SMOKING AND PAL..............................................................................................................133 5.8 PREVALENCE OF PERIODONTAL ATTACHMENT LOSS...................................................................133 5.9 FUTURE RECOMMENDATIONS...................................................................................................134Conclusions....................................................................................................................135Appendix A....................................................................................................................137Appendix B.....................................................................................................................138Appendix C....................................................................................................................139Appendix D....................................................................................................................140References......................................................................................................................144 iii
  6. 6. TABLESTable 2.1 Interactions of plaque bacteria and their products in inflammation andimmunity............................................................................................................................7Table 2.2 Composition of eicosanoids ...........................................................................12Prostanoids.......................................................................................................................12Table 2.3 Cell sources and actions of prostanoids .......................................................15Table 2.4 Major tissue destructive mediators in periodontitis ...................................26Table 2.5 Neutrophil components and function (Williams et al. 1996)......................31Table 2.6 The types of NSAIDs (and their classification) used in periodontal studies...........................................................................................................................................39Table 2.7 Periodontal effects of NSAIDs in human studies.........................................40Table 3.1 Inclusion and exclusion criteria.....................................................................63Table 3.2 Aims of questionnaire. ...................................................................................65Table 3.3 Plaque index ...................................................................................................67Table3.4 Modified Sulcus Bleeding Index (mSBI).......................................................67Table 3.5 Tooth mobility index......................................................................................68Table 3.6 Furcation index...............................................................................................69Table 4.1 Intra examiner reliability test using kappa statistics..................................87 iv
  7. 7. Table 4.2 The number and percentage distribution of subjects participating in thestudy by group.................................................................................................................87Table 4.3 Distribution of age by group..........................................................................87Table 4.4 A Scheffés analysis of homogeneity between two groups at a time formean age differences.......................................................................................................88Table 4.5 Scheffésa, analysis for homogeneity between subsets..................................88Table 4.6 Demographics on pension status with group specific characteristics........89Table 4.7 Pension status in relation to denture use. ....................................................89Table 4.8 Demographic data on schooling of all subjects with group specificcharacteristics. ................................................................................................................89Table 4.9 A self-evaluation of English language skill...................................................90Table 4.10 Socio-economic factors and dental behaviours..........................................90Table 4.11 Population and percentage distribution of subjects since their last dentalvisit. The time range was from less than one year to never visiting the dentist.......91Table 4.12 Missing teeth by age and group...................................................................92Table 4.13 Missing teeth and smoking history..............................................................92Table 4.14 The mean plaque index per group. ............................................................92Table 4.15a Profile of aspirin use and subject numbers .............................................93Table 4.15b The association of age and past smoking on mean plaque scores withtests of significance..........................................................................................................93 v
  8. 8. Table 4.16 Distribution of mean percentage of teeth with calculus by age, aspirinand ex-smoking. ..............................................................................................................94Table 4.17 The association of age with mean percentage of calculus between groups...........................................................................................................................................95Table 4.18 The association of low-dose aspirin and ex-smoking with the meanpercentages of mobile teeth............................................................................................95Table 4.19 The correlation of low-dose aspirin and past smoking with mean PAL..96Table 4.20 The association of aspirin dosage with mean PAL....................................97Table 4.21 The association of aspirin duration with mean PAL.................................97Table 4.22a The association of past smoking dosage and duration with mean PAL............................................................................................................................................97Table 4.22b The correlation of the number of cigarettes smoked and duration ofsmoking with mean PAL with t-test of significance.....................................................98Table 4.23 Univariate analysis of variance in mean PAL at ≥2, ≥4 ≥ 5 and ≥7mm............................................................................................................................................99Table 4.24 Univariate analysis of variance on mean % PAL at ≥2, ≥4, ≥ 5 & ≥7mm..........................................................................................................................................100Table 4.25 The magnitude of the association of aspirin and smoking history withseverity and extent of PAL at ≥2, ≥4, ≥ 5 & ≥7 mm PAL using the general linearmodel (2-way ANOVA) of analysis..............................................................................101Table 4.26 The correlation of aspirin and past smoking history with MSS-PAL.. .102 vi
  9. 9. Table 4.27 The age class distribution of males 50+ years in metropolitan Adelaide in1996 from census statistics and their appropriate frequency distribution. ............103Table 4.28 The proportional weights given to each group using the percentagefrequency of each class interval from census statistics for metropolitan Adelaide.103Table 4.29 Descriptive statistics of EWS-PAL ...........................................................103Table 4.30 The association of aspirin and past smoking history with EWS-PALusing weighted data.......................................................................................................104Table 4.31 The ratio of aspirin to smoking on various measurements of PAL. ....105Table 4.32 Associations of plaque and age with mean PAL with tests of significance..........................................................................................................................................105Table 4.33 Associations of calculus and age with mean PAL with tests ofsignificance.....................................................................................................................105Table 4.34 Associations of gingival bleeding and age with mean PAL with tests ofsignificance.....................................................................................................................106Table 4.35 Socio-economic factors, oral hygiene patterns and mean PAL (mm)....106Table 4.36 The statistical power values for most ANOVA analyses ........................107Table 4.37 Relative percentage of subjects with medical conditions per group......107Table 4.38 Outcome of age, ex-smoking and aspirin with various indices of PAL. 107 vii
  10. 10. FiguresLeukotrienes.....................................................................................................................12Figure 2.1 Products and pathways of cyclo-oxygenase ...............................................14Figure 2.2 The chemical structures of PGE2 and TxB2 .............................................20Figure 2.3 Structure of aspirin ......................................................................................36Figure 2.4 Effects of aspirin on cyclo-oxygenases .......................................................38Figure 3.1 A copy of an advertisement placed in local press media to recruitsubjects.............................................................................................................................62Figure 4.1 The mean percentage of sites with gingival bleeding (modified bleedingindex)..............................................................................................................................108Figure 4.2 The mean percentage of teeth with calculus ............................................108Figure 4.3 Cumulative distribution of MSS-PAL representing the worst score (site)per tooth per subject, averaged over all subjects. .....................................................109Figure 4.4 Diagrammatic representation of PAL according to smoking and aspirintaking history, showing mean PAL, MSS-PAL and EWS-PAL...............................110Figure 4.5 Cumulative distribution of EWS-PAL. Data were weighted using ageclass statistics for metropolitan Adelaide population................................................111Figure 4.6 Variations of recession and pocket depths by tooth- and jaw type for thewhole study population.................................................................................................112 viii
  11. 11. Figure 4.7 Variation of recession and pocket depths by tooth- and jaw type in theAXS group......................................................................................................................112Figure 4.8 Variation of recession and pocket depths by tooth- and jaw type in theNAXS group. .................................................................................................................113Figure 4.9 Variation of recession and pocket depths by tooth- and jaw type in theANS group......................................................................................................................113Figure 4.10 Variation of recession and pocket depths by tooth- and jaw type in theNANS group...................................................................................................................114 ix
  12. 12. Signed StatementThis research report is submitted in partial fulfillment of the requirements of the Degreeof Master of Dental Surgery (Periodontics) in the University of Adelaide.The thesis contains no material which has been accepted for the award of any otherdegree or diploma in any University and that, to the best of my knowledge and belief, thethesis contains no other material previously published or written by another person,except where due reference is made in the text of the thesis.I give consent to this copy of my thesis, when deposited in the University Library, beingmade available for photocopying and loan if accepted for the award of the degree.……………………………………..Arthur Drouganis.November 1999 x
  13. 13. AcknowledgmentsI wish to take this opportunity to thank those people who have assisted me in completingmy candidature. I am particularly grateful to many people but utmost to my wife, andfamily for their patience and understanding throughout this challenging course.I am truly indebted to two individuals. Robert Hirsch my supervisor, a true researcher,for his kindness, knowledge and in particular his insight and wisdom who lent meunconditional support, tempered at times, by considerable forbearance. To BryonKardachi, for his clinical knowledge, expert guidance and for his enthusiasm. Theknowledge I have gained from both of them is, and will be invaluable.My thanks go to the Colgate Australian Clinical Dental Research Centre for the use of itsstate-of-the-art facilities and I am especially grateful to Kerrie Ryan and Jane Burns whogave excellent support and assistance. To Colgate Australia for their generosity insupplying the Oral Care Kits which were given to each participant in the study. Aspecial thank you to Professor Felix Bochner, Department of Clinical and ExperimentalPharmacology, Division of Health Sciences University of Adelaide for his initialguidance.I am deeply grateful to Knute Carter for his meticulous statistical analyses of the data.To Jane Carter for her enthusiasm and ideas on the studyThese people have inspired and encouraged me to ask questions, to learn to reason andthink independently. I truly believe I have been educated.Thank you. xi
  14. 14. " Do not be rash to make friends; but, when once they are made, do not drop them" DIOGENES (412-332 B.C.) A Greek philosopher I can quite honestly say that I have made life time friends. xii
  15. 15. Glossary of termsANS Aspirin Never Smoked groupAXS Aspirin eX-Smoker groupCOX Cyclo-oxygenase, an enzyme that produces the prostanoid and thromboxane mediators of inflammationCytokines Polypeptide mediators released by cells involved in inflammation healing and homeostasisEWS-PAL The extreme worst site of PAL per subject, then averaged across each groupExtent The proportion of tooth sites of an individual with PAL exceeding 1mm and often measured at various threshold valuesGCF Gingival Crevicular FluidIgG Immunoglobulin-GIL-1 Interleukin-1 an inflammatory cytokine involved in inflammation, immunity, tissue breakdown and homeostasisIL-6 Interleukin-6 an inflammatory cytokine involved in inflammation, immunity, tissue breakdown and homeostasisLow-dose aspirin ≤300mg per dayLPS LipopolysaccharideMean PAL The average PAL of all sites per subject, then averaged across each groupMSS-PAL The most severe site of PAL per tooth per person then averaged across each groupNANS No Aspirin Never smoked groupNAXS No Aspirin eX-Smoker groupNSAIDs Non-steroidal anti-inflammatory drugsPAL Periodontal attachment lossPGE2 Prostaglandin-E2. A primary cyclo-oxygenase mediator of inflammationPrevalence The proportion of group who have PAL (ie cases)Severity The degree of PAL averaged per affected tooth sitesTNF-α A proinflammatory cytokine with synergistic effects with other cytokines xiii
  16. 16. SummaryIn the 1970s, Vane proposed that the anti-inflammatory effects of aspirin and aspirin-likedrugs (non-steroidal anti-inflammatory drugs, NSAIDs) were due to inhibition of the enzymecyclo-oxygenase, which stops the production of prostanoids (prostaglandins andthromboxanes). By the early 1980s, high doses of aspirin and other NSAIDs were shown tosignificantly reduce gingivitis, periodontal attachment loss and alveolar bone loss in humans.However, long-term use of these agents in periodontal therapy was not advocated, due to theirside effects and the inconsistent findings between studies. Often test and control groups werenot from the same sample population, results were based on concurrent use of other NSAIDs,dosages and duration varied between groups, and there was no control for smoking effects.Research in the 1990s showed that periodontitis is a multifactorial disease, being dependenton genetic and environmental influences, which modify the host response to the microbialchallenge. One of the primary environmental risk factors for periodontitis is cigarettesmoking. Ex-smokers lie between non-smokers and current smokers with regard to theseverity and extent of periodontal attachment loss and alveolar bone loss; people who quitsmoking respond to periodontal therapy similarly to non-smokers.There is no information in the literature about the periodontal effects of low-dose aspirin onthe periodontium in either non-smokers or ex-smokers. The aim of this study was to assess theperiodontal status of a self-selected sample of men (aged 50 and above), residing inmetropolitan Adelaide, South Australia, with respect to aspirin intake and smoking history.Subjects were targeted by advertisements placed in the local press.Demographic data were collected from information obtained from a self-administeredquestionnaire and periodontal health was assessed by a periodontal examination carried out byone operator, blind to each subject’s aspirin and smoking history. Measurements of pocketdepths and gingival recession were made at six sites of all teeth present and were used to xiv
  17. 17. compute periodontal attachment loss (PAL) for all subjects. Other parameters recorded wereplaque and calculus accumulation, gingival and bleeding indices and tooth mobility.Periodontal assessments were carried out in 392 men, aged 50-85 years. Significant ageeffects were found on PAL but these were of small magnitude in comparison to the significantinfluences that aspirin and ex-smoking had on PAL. The subjects were divided amongst foursub-groups:• aspirin never smoked (ANS),• aspirin ex-smokers (AXS).• no aspirin never smoked (NANS)• no aspirin ex-smokers (NAXS).The extent and severity of PAL was evaluated against a background of age, ethnicity, socio-economic and dentition status. The study population comprised low, middle and highereducational levels and there were no significant distribution differences between the groups.The study population comprised a much higher group of educated subjects when compared tothe general population of Adelaide. Higher educated subjects with good English skillsbrushed more frequently and had a more recent scale and clean than the lower educatedgroups. A measure of subjects’ economic level was their pension status; pensionersrepresenting low income. Approximately 58.9% of subjects were pensioners; there were nosignificant differences in mean PAL between pensioners and non-pensioners.In order to correlate the effects of aspirin and smoking habits on advanced PAL, threemeasures of PAL were used; mean PAL, the most severe site of PAL (MSS-PAL) and theextreme worst site of PAL (EWS-PAL). Mean PAL was the overall mean PAL of all sitesper tooth/per subject/per group. MSS-PAL was the most severe site of PAL of the six sitesper tooth/subject. This method associated the effects of aspirin and ex smoking on advanced xv
  18. 18. PAL by reducing the overwhelming effects of sites with low PAL. EWS-PAL was theextreme worst site of PAL/mouth. The results were as follows: Mean PAL mm ± se MSS-PAL mm ± se EWS-PAL mm ± se ANS 2.5 ± 0.01 3.7 ± 0.13 6.2 ± 0.22 AXS 2.8 ± 0.09 4.1 ± 0.11 7.0 ± 0.18 NANS 2.7 ± 0.08 4.0 ± 0.10 6.8 ± 0.17 NAXS 3.1 ± 0.08 4.4 ± 0.10 7.5 ± 0.17Prevalence was measured using different threshold levels of PAL. Significant positive effectsof aspirin for the extent of PAL were found for all threshold levels. At thresholds of ≥2mmPAL, the prevalence of PAL was approximately 94%. At a moderate threshold of 4mm PAL,28.7% of subjects exhibited PAL ≥4mm with a mean severity score of 4.6 ± 0.03mm (se),indicating that the percentage of subjects with advanced PAL was low particularly at higherthresholds. Controlling for age, ANOVA analysis showed that the prevalence rate of PALwas significantly lower in aspirin takers when compared to non-aspirin takers and theseeffects were independent of smoking history. In addition, ex-smokers had significantly morePAL compared to non-smokers and this effect was independent of aspirin history. Theprevalence of advanced PAL in subjects (using 7mm PAL as a threshold) was found to be2.6% with a mean PAL of 7.7 ± 0.05mm (se).Epidemiological studies (including this one) attribute all PAL to the effects of destructiveperiodontal diseases. No account is given to other causes of PAL such as continuous tootheruption, alveolar dehiscence, cervical enamel projections, cracked or split roots andretrograde periodontitis. Taking these factors into account, the true prevalence of advancedPAL due to periodontitis within the community must be lower than the estimated rate of10-15%.My findings suggest that men aged 50 and above may benefit from taking low-doses ofaspirin daily in order to reduce their risk of PAL. With the reduced severity and extent xvi
  19. 19. of PAL in ex-smokers taking aspirin, it is tempting to speculate that subjects withperiodontitis may benefit significantly by taking low-dose aspirin to reduce theirperiodontal and cardiovascular risks, irrespective of their smoking history. Furtherresearch should aim to establish whether patients with periodontitis would benefit fromtaking low-dose aspirin as an adjunct to periodontal therapy and whether low-doseaspirin modulates the effects of periodontitis in females and current smokers. xvii
  20. 20. Chapter 1 IntroductionDestructive periodontal diseases are multifactorial in origin; the interplay between lifestylefactors, the social environment and the dental biofilm determine an individual’s susceptibility(Clarke and Hirsch 1995). Inflammatory as well as immunological responses are activated bythe many components of dental biofilm which constitutes the microbial challenge to the host(Miyasaki 1996; Wilson and Kornman 1996; Darveau et al. 1997). The vascular and cellularresponses occurring in inflammation are controlled by the release of endogenousinflammatory mediators (Page and Schroeder 1976; Page 1991; Genco et al. 1994;Offenbacher 1996). There is an extensive list of endogenous inflammatory mediators knownto be involved in the regulation of the inflammatory response. In periodontal tissues, thesemediators are the link between health, tissue damage, inflammation and immunity (Page andSchroeder 1976; Offenbacher et al. 1990; Page 1991; Offenbacher et al. 1993a; Offenbacheret al. 1993b; Genco et al. 1994; Wilson and Kornman 1996).One of the first and major pathways of tissue destruction in inflammatory periodontaldiseases is the synthesis and release of eicosanoids. Eicosanoids are formed frommembrane polyunsaturated fatty acids (mainly arachidonic acid), which include theprostaglandins, prostacyclins, thromboxane A2 and the leukotrienes (Rang et al. 1996).Eicosanoids are not found preformed in cells like histamine, but are generated de novofrom cell plasma membrane phospholipids when tissues are damaged (Salmon and Higgs1987; Davies and MacIntyre 1992). They control many physiological and pathologicalprocesses and are the most important mediators and modulators of the immuno-inflammatory pathways (Rang et al. 1996). In response to microbial virulence factors,damaged gingival tissues produce phospholipids, which become the substrate forphospholipase. This enzyme synthesizes and releases free arachidonic acid (Howell and 1
  21. 21. Williams 1993) which may be synthesized into either prostanoids or leukotrieneproducts. These are associated with platelet aggregation, vasodilatation, chemotaxis ofneutrophils, increased vascular permeability and alveolar bone resorption. Prostanoidsare produced from arachidonic acid by cyclo-oxygenase (COX) which occurs inneutrophils, macrophages, mast cells, fibroblast, lymphocytes keratinocytes, osteoblastsand platelets (Howell and Williams 1993; Offenbacher 1996; Wiebe et al. 1996).Leukotrienes are products produced by lipoxygenase and are restricted to neutrophils,eosinophils, monocytes/macrophages and mast cells (Salmon and Higgs 1987).The predominant prostanoid product in immuno-inflammatory responses in periodontaldiseases is prostaglandin E2 (PGE2) (Howell and Williams 1993; Offenbacher et al.1993b). PGE2 is considered to be one of the key components in the pathogenesis ofperiodontitis (Page 1991). A large portion of periodontal pathology is attributed to PGE 2,especially in association with other proinflammatory cytokines (IL-1, IL-6, IL-8 andTNF-α) (Alexander and Damoulis 1994; Mathur and Michalowicz 1997; Soskolne 1997;Ellis 1998; Okada 1998). The principal sources of PGE2 in periodontal tissues aremacrophages, monocytes and fibroblasts (Fu et al. 1990).In the 1970s, Vane (1971) advanced the hypothesis that the anti-inflammatory effects ofaspirin-like drugs lay in their ability to inhibit prostanoid synthesis (prostaglandins andthromboxanes). Among its actions, aspirin irreversibly inhibits COX which exists in twoforms (Smith 1992; Meade et al. 1993; Vane 1994; Sharma and Sharma 1997; Dubois etal. 1998):• COX-1 is found in all cells as a constitutive enzyme, which produces the prostanoids that regulate normal homeostasis (e.g. regulating vascular responses and coordinating the actions of circulating hormones). 2
  22. 22. • COX-2 is the inflammatory cyclo-oxygenase that is induced only by inflammatory stimuli, releasing prostaglandin E2 (PGE2). Platelets do not contain COX-2.In the early 1980s, the effects of aspirin and other nonsteroidal anti-inflammatory drugs(NSAIDs) on periodontal attachment loss started to be investigated in humans. Peopletaking high doses of aspirin or other NSAIDs were found to have significantly lowerplaque scores, less gingival inflammation, less attachment and bone loss than the controls(Waite et al. 1981; Feldman et al. 1983; Williams et al. 1989; Jeffcoat et al. 1991;Heasman et al. 1993b; Howell 1993; Offenbacher et al. 1993b; Flemmig et al. 1996;Offenbacher 1996). NSAIDs were considered to have modified the host responses byinhibiting PGE2 production and therefore reducing bone and periodontal attachment loss.Unfortunately many factors in these studies were not controlled, such as age, sex, poorcomparison or control groups (sampling frame error), smoking and systemic disease.Furthermore, most human studies were retrospective and often relied on the subjectsrecollection of dosage and duration, and more than one NSAID was often usedconcurrently. The majority of aspirin studies used patients suffering from rheumatoidarthritis who were taking high daily doses (650mg->3gm/day). These confoundingfactors made comparisons between studies difficult and resulted in conflicting outcomeswith respect to plaque indices, gingival indices, periodontal attachment loss and alveolarbone loss.Low-dose aspirins ability to irreversibly inhibit cyclo-oxygenase over the whole lifetimeof platelets has made it a widely used anti-thrombogenic agent in middle-aged andelderly populations to prevent coronary artery disease, stroke and peripheral vasculardiseases, with low gastro-intestinal side effects (Vane and OGrady 1993; Underwood1994; Lloyd and Bochner 1996; Diener 1998; Müller 1998). Low-dose aspirin has 3
  23. 23. decreased the incidence of heart attacks and stroke by up to 50% (Vane 1994). Low-dose aspirin can inhibit thromboxane A2 production by platelets equipotently as candoses > 300mg. In Australia, the maximum benefit/risk ratio dose used is 100-150mg ofaspirin per day (Lloyd and Bochner 1996).Smoking is recognised as the most important cause of preventable death and disease in thewestern world (MacGregor 1992) and there is a clear association between smoking and theprevalence and severity of PAL (Bergström and Floderus-Myrhed 1983; Haber et al. 1993;Bergström and Preber 1994; Zambon et al. 1996). The greater the exposure in terms of packyears, the greater the amount of PAL and alveolar bone loss (Grossi et al. 1996; Grossi et al.1997).To-date, no studies have investigated the effects of long-term low-dose aspirin on PAL.Since there is a large pool of people in the community taking low-dose aspirin daily formany years, this study was undertaken to correlate PAL with aspirin and smokinghistories. In particular, the aim of this study was to gather descriptive epidemiologicaldata relating to the extent and severity of periodontal attachment loss in an adult malepopulation within metropolitan Adelaide specifically targeting men with and without ahistory of long-term low-dose aspirin therapy, with or without a history of smoking.Data from this study could also provide information relating to oral hygiene habits,dental attendance, socio-economic factors, tooth loss and attachment loss patterns in anelderly population. 4
  24. 24. Chapter 2 Review of the literature2.0 Summary of the present understanding of the inflammatory response.Periodontal diseases are mostly chronic infections characterised by a destructiveinflammatory process affecting the supporting tissues of the tooth, with subsequentpocket formation and resorption of the alveolar bone (Offenbacher 1996). The intent ofthis review is to place the current understanding of the regulatory mechanisms thatinfluence the inflammatory response in perspective, focussing on prostaglandins asimportant elements of the inflammatory process and as major mediators of periodontalattachment loss (PAL) and alveolar bone loss (Offenbacher 1996; Gemmell et al. 1997;Page et al. 1997).Inflammation is the normal response of the body to infection, tissue injury or insult; it israpid and provides a first line of defence. It is initially a nonspecific host response,eliciting the same reaction irrespective of the nature of the insult. The insult may bemicrobial, physical or chemical in nature, and all initiate a series of local processes toneutralise, limit the spread and eradicate the insulting agent(s) (Lakhani et al. 1993;Offenbacher 1996; Gemmell et al. 1997; Page et al. 1997). Inflammation is divided intoacute and chronic forms based on the duration of the response and the predominantinflammatory cell type. Whether acute or chronic, the process may be modified by manyenvironmental and host factors; such as the pathogenicity and virulence of the microbialchallenge, nutritional status, host immune status, use of antibiotics, anti-inflammatorydrugs and / or surgical/non-surgical therapy (Lakhani et al. 1993; Miyasaki 1996;Wilson and Kornman 1996; Page et al. 1997). These responses are characterised bydilatation of the local blood vessels, increased permeability of capillaries, plasmaexudate, with the chemotactic accumulation of neutrophils, monocytes/macrophages, 5
  25. 25. eosinophils, basophils and mast cells to the site of injury or infection (Kay 1970;Bienenstock et al. 1986; Faccioli et al. 1991; Page et al. 1997). The chemotactic factorsare both chemotactic and cell activating, leading to increased cell numbers and / oraffinity of adherence receptors on the surfaces of both endothelial and inflammatory cells(Page 1991; Page et al. 1997). The expression of adhesion receptors enables themigration of inflammatory cells from the circulation into the sites of injury (Page 1991;Page et al. 1997), where they actively eliminate the noxious agent and participate withresident tissue cells in wound healing and tissue remodelling (Miyasaki 1996; Wilsonand Kornman 1996; Page et al. 1997). In addition to the cellular response, plasmaconstituents including complement and immunoglobulins are poured into the sites ofinflammation (medications are also transported to these sites by the plasma orinflammatory exudate).The host through the neutrophils and macrophages has the capacity to destroy allbiological structures (Williams et al. 1996). In the process of containing the microbialchallenge, host defences can cause bystander tissue destruction which can be moreoffensive than the original insult (Page et al. 1997; Okada 1998). The damage is eitheressential, such as the removal of collagen allowing room in the tissue for aninflammatory cell infiltrate, or the damage may be bystander damage (accidental) in theprocess on containing the microbial challenge. "Bystander damage" is a common featureof chronic inflammatory diseases such as rheumatoid arthritis, tuberculosis, andemphysema. Loss of periodontal attachment in periodontitis is caused by bystanderdamage from the host response to the microbial plaque (Williams et al. 1996; Page et al.1997). 6
  26. 26. Inflammatory reactions consist of two components, the inflammatory exudate (theplasma component) and the cellular response. Both responses are activated by the manyconstituents of dental plaque biofilm which constitutes the microbial challenge to thehost in periodontal diseases (Miyasaki 1996; Wilson and Kornman 1996; Darveau et al.1997). Aerobic and anaerobic bacteria found in the gingival crevice or periodontalpockets release a variety of products that can cause the onset of vascular changes, leadingto acute inflammation. These products include metabolic acids, extracellular enzymes,volatile sulphur compounds, lipoteichoic acid and lipopolysaccharides.Table 2.1 Interactions of plaque bacteria and their products in inflammation and immunityAny stimulus that damages host cells or other components will trigger inflammation, and theresulting inflammation helps activate an immuno-inflammatory response against foreign orantigenic material present. Conversely, humoral immune reactions will activate aninflammatory reaction at the site where the antibody binds to the antigen (Williams et al.1996).Bacterial products Effects • activate complementWhole bacteria • activate neutrophils and macrophages • are antigenicMost peptides and proteins secreted by • chemotactic for neutrophils andbacteria macrophages • damage host cells • degrade connective tissue matrixEnzymes • activate and degrade complement • degrade antibodies • are antigenic • activates complement • damages some host cellsLipopolysaccharide (LPS) • activates neutrophils and macrophages • are antigenicPolysaccharide plaque matrix and • polyclonal B-cell activatorBacterial capsule • are antigenicOther toxins, acids, reducing agents and • damage host cellsmetabolites • are antigenic 7
  27. 27. Table 2.1 summarises the interactions of plaque products and their effects on inflammationand immunity. These factors can directly or indirectly damage sulcular epithelium andunderlying connective tissue, disrupt microvasculature and initiate an inflammatory response(Darveau et al. 1997). Some aspects of the inflammatory response are clearly distinct but theprecise role played by many of the mediators has not been completely clarified (Page andSchroeder 1976; Page 1991; Genco et al. 1994; Offenbacher 1996).2.1 Endogenous mediators of inflammationThe inflammatory exudate flowing from the gingival tissues into the gingival crevice orperiodontal pocket consists of blood components and host defence mediators which cancontain the microbial challenge, or they themselves act as a source of nutrients for themicrobes. The rate of gingival crevicular fluid flow generally reflects the severity of theinflammation, the increased volume of inflamed tissue and the greater surface area ofpockets (Williams et al. 1996). The initial host response to the bacterial challenge ischaracterised by the release of a number of vasoactive and antimicrobial factors:2.1.1 Histamine:This mediator of acute inflammation is present in mast cells. Histamine may be releaseddirectly either by: (a) bacterial mediators such as lipopolysaccharide and enzymes (trypsin like or proteases) which activate the complement pathway (alternate pathway) eventually releasing C3a and C5a or (b) direct complement activation (C3a and C5a), or interleukin-1 and other factors from endothelial cells, neutrophils and lymphocytes. In addition, 8
  28. 28. antibody-antigen complexes can activate complement (through the classic pathway) releasing C3a and C5a.These mediators activate the release of mast cell granules, which increase vascularpermeability (in capillaries and venules), and characteristically are the major mediator ofacute short-lived inflammatory responses.2.1.2 Bradykinin:With tissue and vascular injury, serum Hageman factor (Factor XII of the coagulationcascade) activates the release of bradykinin, a nonapeptide (a long-lived vasodilator)(Rang et al. 1996; Wilson and Kornman 1996). Bradykinin often follows the release ofhistamine and is capable of increasing vascular permeability (Nisengard and Newman1996). Bradykinin induces:• continued exudation and crevicular fluid flow• bone resorption in organ cultures via the prostaglandin cyclo-oxygenase pathway (Newman et al. 1976; Nisengard and Newman 1996).2.1.3 Plasmin:Plasminogen enzyme is a normal constituent of plasma proteins. It is converted toplasmin by the action of plasminogen activator (also called kallikrein). When theintrinsic coagulation system is activated, the fibrinolytic system is activated through theaction of plasminogen activators. Activation of Hageman factor (XII) begins a cascadeof reactions in which it catalyses the reaction of circulating plasminogen to plasmin.Plasmin is a multi-functional protease enzyme that digests fibrin and fibrinogen(fibrinolysis) and other plasma proteins namely clotting factors II, V, VII and many othertissue proteins. Plasmin is also an activator of several matrix metalloproteinases (Okada 9
  29. 29. 1998). Plasmin derived lysis of the fibrin clot generates fibrin degradation products thatinduce vascular permeability and trigger the complement system with the formation ofC3a and C5a components causing the release of histamine from mast cells. These fibrinproducts are chemotactic to other inflammatory cells (Walter and Grudy 1993).2.1.4 Complement:Specific antibody and complement are two very important antimicrobial factors in GCF.Activation of complement is one of the first host defences after injury, with these effects:• vasodilation and increased blood flow (by C2, C3a and C5a)• activate mast cells to release histamine (by C3a, C5a)• augment opsonisation (C3b) of bacteria by antibodies and allow some antibodies to kill bacteria or by phagocytosis• chemoattractant to neutrophils and macrophages (C3a, C5a) and trigger the release of prostaglandins, leukotrienes and enzymes into the tissue• cause pores to open in the membranes of pathogens causing cell lysis (C5-9) (Dennison and Van Dyke 1997).Bacteria in the gingival sulcus can activate the complement system via two majorpathways (Page 1991; Offenbacher et al. 1993a):The classical pathway: Activation of this system occurs rapidly. This pathway is activated by antigen- antibody complexes (Dennison and Van Dyke 1997). Complement C1qrs binds to the Fc component of IgG or IgM antibodies. This is fixed to the bacterial 10
  30. 30. receptor via the Fab region of immunoglobulin activating a cascade of enzymic reactions to release C3 the precursor of C3A, C3b, C5a, C5b-9 which cause lysis of cell membranes or functional alterations to promote phagocytosis (Lakhani et al. 1993; Offenbacher 1996).The alternative pathway:Activation of this cascade does not involve immunoglobulin. Activation occurs directlyby bacterial surface lipopolysaccharide (LPS) and endotoxin (from gram-negativeanaerobes). This pathway also involves a series of reactions to release the precursorcomplement protein C3 and produce the cleavage products C3a and C5a to C9 (Lakhaniet al. 1993; Offenbacher 1996).The central event in both pathways is activation or splitting of C3 to C3b which becomesattached to the activating stimulus (usually bacterial surfaces or antigen-antibodycomplexes). Whichever pathway is activated, large amounts of C3a and C3b componentare released, fixing to the inflammatory stimulus and resulting in increased histaminerelease, vascular permeability, chemotaxis to phagocytes (promoting phagocytosis), andpromotion of blood clotting. Complement can cause bystander damage since a smallamount may bind to host cells causing lysis or triggering neutrophils to attack.2.1.5 Platelets:Platelet adhesion and granule release plays an important role in the early development of thevascular and cellular aspects of the inflammatory process (Walter and Grudy 1993). Releaseof granules from platelets can also help initiate vascular permeability. Mediators releasedfrom platelets include serotonin, a number of coagulation factors and thromboxane A2(TxA2), all of which are pro-inflammatory. Platelet-derived growth factor (PDGF) is derived 11
  31. 31. from the platelet α−granules that contribute to the repair process (anabolic) following inflam-matory responses or damaged blood vessels (Walter and Grudy 1993). Other anabolic effectsof PDGF are down regulation of alkaline phosphatase and promotion of proliferation of fi-broblasts and periodontal regeneration (Okada 1998).2.2 EicosanoidsCellular disturbances (e.g. from cell damage, LPS, complement, thrombin, bradykinin andantigen-antibody complexes) cause enzymes known as phospholipases to generatearachidonic acid from the cell membrane phospholipids. Arachidonic acid metabolites are asmall group of lipids known collectively as eicosanoids. Eicosanoids are not found pre-formed in cells like histamine, they are generated de novo from cell membrane phospholipids.They control many physiological processes and are the most important mediators andmodulators of the inflammatory reaction (Campbell and Halushka 1996). The prostanoids,and in particular prostaglandins, are produced from arachidonic acid by cyclo-oxygenase thatoccurs in neutrophils, macrophages, mast cells, fibroblasts, lymphocytes, keratinocytes,osteoblasts and platelets (Offenbacher 1996). Prostanoids encompass all cyclo-oxygenaseproducts (Table 2.2). The predominant prostanoid product of the inflammatory response indestructive periodontal diseases is thought to be PGE2 (Howell and Williams 1993).Table 2.2 Composition of eicosanoids Eicosanoids Prostanoids Leukotrienes All cyclo-oxygenase products All lipoxygenase products • prostaglandins • thromboxane • prostacyclins 12
  32. 32. 2.2.1 General properties of eicosanoidsEicosanoids are found almost in every tissue and body fluid and have the followingproperties: • they are mediators derived from membrane phospholipids • they are effector molecules which are formed from polyunsaturated fatty acids (lipids), mainly arachidonic acid. These include the prostaglandins, prostacyclins, thromboxane A2 and the leukotrienes. • their production increases in response to diverse stimuli and they produce a broad spectrum of biological effects. • these lipids contribute to a number of physiological and pathological processes including inflammation, smooth muscle tone, haemostasis, thrombosis, parturition, and gastrointestinal secretion. • several classes of drugs, most notably the nonsteroidal anti-inflammatory drugs (and in particular aspirin), are therapeutically active because they block the formation of eicosanoids. (Salmon and Higgs 1987; Davies and MacIntyre 1992; Campbell and Halushka 1996; Rang et al. 1996).General effects of prostanoids vary and the type of response elicited is related to specifictarget cell receptors (Table 2.2). Their effects are: i. production of fever, pain and inflammation (Campbell and Halushka 1996). ii. bone resorption by PGEs (Davies and MacIntyre 1992) 13
  33. 33. iii. PGE2 stimulates cAMP formation in cells, phospholipase C, and calcium influx in osteoblastsiv. PGEs also have insulin-like effects on carbohydrate metabolism and exert parathyroid hormone-like effects that result in mobilisation of calcium ions from bone (Campbell and Halushka 1996). v. stimulation of the release of adrenal steroids (ACTH & growth hormone), and of erythropoietin from the kidney (Davies and MacIntyre 1992; Campbell and Halushka 1996).vi. prostaglandins (PGE2, PGD2, PGA2) and prostacyclins (PGI2) are potent vasodilators, while PGG2, PGH2 and TXA2 are powerful vasoconstrictors (Campbell and Halushka 1996).Figure 2.1 shows the products and pathways of cyclo-oxygenase.Figure 2.1 Products and pathways of cyclo-oxygenase (Salmon and Higgs 1987). 14
  34. 34. Table 2.3 Cell sources and actions of prostanoids (Davies and MacIntyre 1992; Campbell and Halushka 1996). Receptor Prostanoid Effect Derived from type vasodilatation inhibition of platelet aggregationPGD2 DP mast cells relaxation of gastrointestinal muscle uterine contraction myometrial contraction increase in cytoplasmic calcium ionsPGF2a FP Corpus luteum vasoconstrictor of pulmonary arteries and veins vasodilatation inhibition of platelet aggregation renin release tubular reabsorption of sodium ions vascularProstacyclin IP increase cAMP epithelium vasoconstriction platelet aggregation bronchial-constriction increase of cytoplasmic calcium ions bone resorption increase in cAMP increases vasodilation increases vascular permeability contraction of bronchial and smooth muscle bronchial-dilation most nucleated EP1, stimulation of intestinal fluid secretions cellsPGE2 EP2 relaxation of gastrointestinal smooth especially EP3 muscle monocytes contraction of intestinal muscle and macrophages inhibition of gastric acid secretion inhibition of lipolysis inhibition of autonomic neurotransmitter release contraction of uterus decrease of cAMP in adipose cells2.3 Role of eicosanoids in periodontal tissuesProstanoid products in the periodontal tissue are primarily mediators of inflammationand tissue destruction (Offenbacher et al. 1993b; Offenbacher 1996). In view of thelarge number of compounds that belong to the eicosanoid family, this section focuses on 15
  35. 35. the main mediator of periodontal inflammation and tissue destruction, i.e. theprostaglandins and in particular PGE2. In periodontal tissues the actions of PGE2 induce(Birkedal-Hansen 1993; Offenbacher et al. 1993b):• vasodilation and increased vascular permeability in the gingival plexus.• matrix-metalloproteinases (MMP) secretion from macrophages, monocytes and fibroblasts stimulating connective tissue breakdown.• increases cAMP in macrophages• interacts with IL-1 and TNF-α to enhance their effects.• modulation of platelet and leucocyte reactivity• inhibition of T cell proliferation• lysosomal enzymes release from neutrophils• generation of toxic oxygen radicals from neutrophils• histamine release from mast cells.• inhibition of macrophage/monocyte and lymphocyte activation• generation and secretion of other cytokines.• osteoclastic bone resorption i.e. increased severity of periodontal diseases (PGE2 has a major role in periodontitis as a long-lived potent mediator of bone resorption interfering with the bone remodelling coupling mechanism between osteoblasts and osteoclasts) (Of- fenbacher 1996; Wiebe et al. 1996; Gemmell et al. 1997; Page et al. 1997; Schwartz et al. 1997; Ueda et al. 1998).2.3.1 Biosynthesis of eicosanoids.The main source of the eicosanoids is arachidonic acid, a 20-carbon polyunsaturated fattyacid found in the phospholipids of cell membranes and to a lesser extent, in theglycerides of cell membranes (Davies and MacIntyre 1992). The initial and rate-limiting 16
  36. 36. step to eicosanoid production is the liberation of arachidonate from the membranes(Rang et al. 1996), either by a one-step process involving phospholipase A2 (PLA2)directly or the indirect two step process involving either phospholipase C anddiacylglycerol lipase or phospholipase D.Phospholipase D is an important signal transducer that induces phagocytosis byphagocytic cells. There are intracellular and extracellular forms of phospholipase A2. Itis mainly the intracellular form that is implicated in the generation of inflammatorymediators; it generates arachidonic acid and platelet activating factor (PAF), anotherpowerful mediator of inflammation (Campbell and Halushka 1996; Rang et al. 1996).The anti-inflammatory action of the glucocorticoids (adrenal hormones e.g. steroids) ismainly due to the fact that they inhibit the formation of PLA2, inhibiting the induction ofcyclo-oxygenase within the cell and thus reducing free arachidonic acid (Campbell andHalushka 1996; Rang et al. 1996).2.3.2 Arachidonic acid pathways: eicosanoid productionThere are three pathways for synthesis of eicosanoids from arachidonic acid (Campbelland Halushka 1996; Sharma and Sharma 1997).Pathway 1This involves COX (also known as prostaglandin synthetase). Many stimuli acting ondifferent cell types can liberate arachidonic acid, for example: •thrombin on platelets •C5a on neutrophils •bradykinin on fibroblasts •antigen-antibody reactions on mast cells F 17
  37. 37. Free arachidonic acid is metabolised by COX to generate the endoperoxide products(PGG2/PGH2) which are unstable at normal physiological pH and temperature and arepivotal in the formation of other products (Salmon and Higgs 1987; Davies andMacIntyre 1992). These products are either: •enzymatically converted into either prostaglandins, prostacyclins or thromboxanes (collectively called prostanoids). •converted to hydroxy fatty acid (HHT) and malondialdehyde (MDA) by enzymatic or non-enzymatic pathways (Salmon and Higgs 1987; Davies and MacIntyre 1992).COX is bound to the endoplasmic reticulum and primarily has two functions: •to produce cyclic endoperoxide PGG2 •to convert PGG2 to another cyclic endoperoxide PGH2.The next steps in arachidonate metabolism vary according to the cell-type secretingvarious mediators, each eliciting different physiological functions (Fu et al. 1990; Ranget al. 1996): •platelets only produce thromboxane A2 mediator •vascular endothelium produces prostacyclin mediator •macrophages/monocytes, fibroblasts produce PGE2 •mast cells produce PGD2The principal source of PGE2 in periodontal tissues is from macrophages/monocytes andfibroblasts (although most nucleated cells can produce PGE2) (Fu et al. 1990). There aretwo mechanisms whereby PGE2 is produced by macrophages/monocytes.(a) Bacterial LPS induced PGE2 release LPS will bind to LBP (a LPS binding protein found in serum) forming a complex which binds to the high affinity CD14 receptor of macrophages/monocytes, triggering high intracellular cAMP levels (with very low levels of LPS). This 18
  38. 38. stimulates the release of PGE2, TNF-α and IL-1β. Bacterial antigen-antibody (IgG) or C3b can elicit the same reaction (Offenbacher et al. 1993b; Offenbacher 1996).(b) Host induced PGE2 TNF-α and IL-1β have an autocrine effect on the secretory macrophage/monocyte and a paracrine effect on the residential fibroblast cells which elicit PGE2, perpetuating the inflammatory response and activating an immune response.COX exists in two forms:•COX-1, a constitutive enzyme found in all cells i.e. it is always present at a constantconcentration in cells but may increase by 2-4 fold upon physiological stimulation,producing low levels of mediators that are necessary for the maintenance of normaltissue integrity and function. COX-1 produces the prostanoids (PGI2/6-keto-PGF1α &TXB2) that regulate normal homeostasis (Sharma and Sharma 1997; Dubois et al. 1998).•COX-2 is the pro-inflammatory enzyme that is induced by inflammatory stimuli only;its activity increases 10-80 times following injury or insult. Inflammatory stimuli (egLPS) or ligands (eg cytokines) bind to inflammatory cells and eventually induce theprostanoid mediators of inflammation (Seibert and Masferrer 1994; Seibert et al. 1994;Gierse et al. 1995; Sharma and Sharma 1997; Dubois et al. 1998).There are approximately 10 prostaglandins; all have a cyclopentane ring (five carbonring) between carbon 8-12 (Figure 2.2). Prostaglandins are named alphabetically from A-J, with three members in each group (except PGI). These are numbered 1, 2 or 3(representing the double bonds on the prostaglandin molecule). For example PGE2 has 19
  39. 39. two double bonds between carbon 5-6, and 13-14. The prostacyclins have only twomembers, (PGI2 and PGI3).The thromboxanes (Tx) are closely related to the prostaglandins and are synthesised fromPGH2 (Figure 2.2). These molecules contain an oxane ring (a six carbon ring with anoxygen atom) instead of a cyclopentane ring. Thromboxane A2 (TxB2) is a potentvasoconstrictor and triggers platelet aggregation, causing thrombus formation (Sharmaand Sharma 1997; Dubois et al. 1998).Figure 2.2 The chemical structures of PGE2 and TxB2 (Davies and MacIntyre 1992). 20
  40. 40. Pathway 2The leukotrienes (LOX)The second pathway for arachidonic acid metabolism is via the lipoxygenase (LOX)pathway (Figure 2.1) to provide the parent molecule hydoperoxyeicosatetraenoic acid(HPETE). The HPETEs are then further metabolised to leukotrienes, hepoxilins,trioxilins and lipoxins (Sharma et al. 1997). To date there are six HPETEs (5,8,9,11,12and 15-HPETEs) and each is formed by its corresponding enzyme (Sharma 1997).Lipoxygenases are soluble enzymes found in the cytosol of cells of lung, platelets, mastcells and leucocytes. The main enzyme in this group is 5-lipoxygenase; it converts 5-HPETE to leukotrienes, 12-LOX converts 12-HPETE to hepoxilins and trioxilins, 15-LOX converts 15-HPETE to lipoxins. Lipoxygenases differ in their specificity accordingto the hydroperoxy group (-OOH) on arachidonic acid, and tissues differ in thelipoxygenase(s) that they contain. For example, platelets have only 12-lipoxygenase andsynthesise 12-(HPETE) whereas leucocytes contain both 5-LOX and 12-LOX andproduce both 5-HPETE and 12-HPETE (Rang et al. 1996) (see Figure 2.1). Arachidonicacid is enzymatically reduced to hydroxy acids (HPETEs).The HPETEs are unstable intermediate metabolites (like PGG2 or PGH2) and are furthermetabolised by a variety of enzymes. In the leukotriene pathway 5-HPETE isenzymatically to converted leukotriene-A4 (LTA4) which is unstable, but pivotal in theformation of other leukotrienes. LTA4 is enzymatically hydrolysed to LTB4 or non-enzymatically to di-hydroxy acids (di-HETEs). Additionally, LTA4 can be converteddirectly to the precursor of cysteinyl-leukotriene LTC4, which is further metabolised toLTD4, LTE4, and LTF4. LTB4, LTC4, and LTD4 are also known as the "slow reacting 21
  41. 41. substance of anaphylaxis" (SRS-A) (Lewis et al. 1990; McMillan et al. 1992; Salmon etal. 1987; Snyder et al. 1989).LTB4, LTC4, and LTD4 are the most potent leukotrienes. LTB4 is a powerful chemotacticagent for neutrophils and macrophages (acting in picogram amounts) and are importantin the early stages of inflammation. It causes up-regulation of membrane adhesionmolecules of neutrophils, increasing the production of toxic oxygen products and therelease of granule enzymes. It can stimulate proliferation and/or cytokine release frommacrophages (Abramson et al. 1989; Lewis 1990; Rang 1996; Samuelsson 1983).Arachidonic acid or other polyunsaturated fatty acids may be further metabolised bylipoxygenases to other oxygenated derivatives of polyunsaturated fatty acids (Rang1996). A recent addition to these compounds is the lipoxins which were first isolatedin 1984 (Serhan et al. 1984) and generated from within various cells or during cell-cellinteractions. Lipoxins are generated from one of three pathways which can operateindependently or simultaneously (Serhan 1997).(a) A 15-LOX initiated pathway: This enzyme is found in eosinophils, macrophages, monocytes and epithelial cells, under cytokine (IL-1β, TNF-α) and LPS control and regulated by IL-4 and IL-13 (two anti-inflammatory cytokines) (Levy et al. 1993; Nassar et al. 1994; Serhan 1997). Once these cells are stimulated, arachidonic acid is converted to 15-HPETE or 15-HETE in the donor cell which serve as a substrate for 5-LOX in the recipient cell (generally neutrophils) which is converted to lipoxins (by transcellular metabolism) causing vasodilation, leucocyte regulation and blocking leukotriene metabolism (Serhan 1997). 22
  42. 42. (b) 5-LOX initiated pathway: This is generally a platelet-neutrophil interaction. This pathway involves 5-LOX within neutrophils which converts arachidonic acid to 5-HPETE to LTA4 and platelet 12-LOX induces lipoxin biosynthesis (Romano et al. 1993; Romano et al. 1992; Serhan 1997).(c) Aspirin-triggered lipoxins (ATLs). Aspirin has the ability to irreversibly inhibit COX-1 and COX-2 by acetylating an essential serine residue site in both enzymes. The acetylated COX enzymes cannot produce prostaglandins, however more recent medical research shows that acetylated COX-2 in endothelial or epithelial cells converts arachidonic acid to 15-HETE (Claria et al. 1995; Claria et al. 1996). The 15-HETEs are released from these cells by cell to cell adherence i.e. to leucocytes (especially neutrophils) and further metabolised via transcellular pathways by 5-LOX of leucocytes to form 15-epimeric-lipoxin (15-epi-LX) metabolites (Claria 1995). The 15-epi-LX metabolites are also termed aspirin triggered lipoxins (ATLs). Endothelial and epithelial cell COX-2 when induced by pro-inflammatory cytokines (IL-1β, TNF-α) and LPS in the presence of aspirin can shunt arachidonic metabolism to synthesise 15-epi-LX molecules. The 15-epi-LX molecules serve as “stop signals” (i.e. to evoke anti-inflammatory effects) causing vasodilation, inhibiting neutrophil adhesion to endothelial cells (diapedesis), chemotaxis and cell proliferation (Claria 1996; Claria 1995; Clish et al. 1999; Serhan 1997; Takano et al. 1997). 23
  43. 43. From these recent findings it may be that lipoxin production may be an important anti-inflammatory event, especially by ATLs. However most studies on lipoxins use in vitro or in vivo models. However further research is needed to understand the biofeedback regulatory mechanisms involved in converting from a pro- inflammatory eicosanoid phenotype to an anti-inflammatory phenotype. Currently there is no research on lipoxin (especially ATL) involvement in the gingival or periodontal inflammatory process. Nevertheless ATLs may be a further anti-inflammatory (beneficial) pathway provided by aspirin.Pathway 3This pathway involves the cytochrome P450 group of enzymes (present in endoplasmicreticulum) breaking down arachidonic acid to HETEs and DiHETEs.The predominant pathways in eicosanoid production are pathways I and 2 (Sharma andSharma 1997).2.3.3 Catabolism of the eicosanoidsA number of intra-cellular enzymes are involved in the catabolism and inactivation ofmost eicosanoids. There are prostaglandin-specific enzymes that rapidly inactivate theprostaglandins and their metabolites are excreted in the urine. About 95% of PGE2,PGE1 and PGF2a are inactivated during their first passage through the pulmonarycirculation. The half-life of most prostaglandins is less than a minute in the circulation(Campbell and Halushka 1996; Rang et al. 1996). Leukotriene products are inactivatedby oxidative pathways or degraded in the kidneys, lungs and liver (Campbell andHalushka 1996). 24
  44. 44. 2.4 The role of cytokines in periodontal tissuesTable 2.4 shows the major mediators thought to be involved in the pathogenesis ofperiodontitis (Schenkein 1999). Arachidonic acid metabolites have been discussed insection 2.3, the next section only discusses the interleukins and Tumour Necrosis Factoralpha (TNF-α). All these mediators primarily interact with each other and have impacton the pathogenesis of periodontal diseases, but prostaglandins are the major mediatorsinvolved in tissue destruction in conjunction with the following cytokines.The term cytokine means “cell protein”. Cytokines direct and regulate inflammation andwound healing (Page et al. 1997; Okada 1998). Subgroups of cytokines are: • the interleukins which carry complex and detailed messages between leucocytes, • the growth factors which trigger myelopoesis, leucocyte mitosis and cell differentiation • chemokines which trigger cell recruitment • interferons, lymphocyte activating molecules (Birkedal-Hansen 1993; Offenbacher 1996).Cytokines, lymphokines and monokines have autocrine (self-regulate the cells producingthe cytokine), paracrine (modulate distant cells not producing the cytokine) andintracrine (actions within a cell) effects on target cells (Okada 1998). All cytokinesactivate target cells by binding to specific receptors on their cell membranes. Thisreceptor-ligand coupling triggers cellular activation of the target cell, modifying the cellsactivity (Birkedal-Hansen 1993; Offenbacher 1996), e.g. IL-1 binds to fibroblasts totrigger the release of collagenase to degrade collagen in the immediate environs. Ofteninflammatory cytokines trigger the secretion of specific enzymes, lipids, bioactive 25
  45. 45. amines and reactive oxygen metabolites that serve as effector molecules (Alexander andDamoulis 1994). Periodontal tissue destruction is via mobilisation and activation ofmacrophage/monocytes, lymphocytes and fibroblasts. The modulation of these events isvia catabolic cytokines and inflammatory mediators.Table 2.4 Major tissue destructive mediators in periodontitis Interleukins (1, 6, 8) Tumour Necrosis Factor alpha (TNF-α) Arachidonic Acid Metabolites (PGE2)Interleukin-1 (IL-1)Interleukin-1 is a polypeptide, which has diverse roles in immunity, inflammation, tissuebreakdown and homeostasis. It is synthesised by macrophages, monocytes,lymphocytes, endothelial cells, fibroblasts, keratinocytes and brain cells. In theperiodontal tissues, macrophages predominantly secrete IL-1, Il-1α and Il-1β. Bothforms bind to the same cell receptors of many cell types and in various densities(Alexander and Damoulis 1994; Offenbacher 1996; Mathur and Michalowicz 1997;Soskolne 1997; Ellis 1998; Okada 1998; Schenkein 1999)Properties of IL-1: • increases adhesion molecules on fibroblasts, immunocytes (stimulates proliferation of keratinocytes and endothelial cells) • enhances fibroblast synthesis of collagenase, fibronectin and PGE2 • induces the production of matrix metalloproteinases (MMPs) in periodontitis. • elevates the levels of pro-collagenase in gingival and periodontal ligament fibroblasts. • stimulates plasminogen activator in gingival fibroblasts, resulting in the 26
  46. 46. generation of plasmin which is a naturally occurring activator of several matrix metalloproteinases. • activates both T- and B-cells. It also promotes B-cell activation, proliferation, clonal expansion and antibody secretion. It “primes” macrophages and neutrophils by up-regulating receptors for complement and immunoglobulins. • T-cells regulate the immune response by increasing or decreasing IL-1 secretion. T-cells release gamma interferon (IFN-γ) which enhances secretion of IL-1 and PGE2 from LPS-stimulated macrophages. Therefore IFN-γ serves to up-regulate the inflammatory response. (Page 1991; Birkedal-Hansen 1993; Alexander and Damoulis 1994; Mathur and Michalowicz 1997; Soskolne 1997; Ellis 1998; Okada 1998; Page 1998) • IL-1β and IL-1α are potent connective tissue catabolic stimulators. They directly stimulate bone resorption, and trigger the release of PGE2 from fibroblasts and macrophage/monocytes. PGE2 (Seymour et al. 1993; Tatakis 1993).Interleukin-6 (IL-6)IL-6 influences immune and inflammatory responses and the main sources are fromstimulated fibroblasts, endothelial cells, macrophages, T and B-cells and keratinocytes.IL-6 shares many biological properties with IL-1 and has been found to: • be in higher concentrations levels in inflamed sites than healthy sites • stimulate eicosanoid production • stimulate MMP production 27
  47. 47. • stimulate B-cells into Ig-secreting plasma cells. • be a potent stimulator of IgG1. • plays a major role in regulating bone turnover and is essential for bone loss caused by oestrogen deficiency (menopause) • act as a paracrine and/or autocrine factor in bone resorption in pathologic states, by stimulating osteoclasts and activating bone resorption (Page 1991; Alexander and Damoulis 1994; Offenbacher et al. 1996; Mathur and Michalowicz 1997; Soskolne 1997; Ellis 1998; Okada 1998; Schenkein 1999).Interleukin-8 (IL-8)Produced by leucocytes and keratinocytes in response to LPS, IL-1 or TNF-α, with thefollowing properties: • proinflammatory • strong chemoattractant to neutrophils. • selectively stimulate MMP in macrophages keratinocytesLocal tissue destruction in periodontitis or inflamed gingiva may be due to thecontinuous and excessive IL-8 levels that in turn mediate chemotactic and activationeffects on neutrophils and production of MMPs. IL-8 may also attract and induce T-cellproliferation (Alexander and Damoulis 1994; Mathur and Michalowicz 1997; Soskolne1997; Ellis 1998; Okada 1998).Tumour necrosis factor α (TNF-α)This proinflammatory cytokine is mainly secreted by monocytes and macrophages; it hasthe following properties: 28
  48. 48. • induces secretion of collagenase by fibroblasts • induces resorption of cartilage and bone • induces periodontal tissue breakdown in periodontitis • in resting macrophages it induces synthesis of IL-1 and PGE2 • activates osteoclasts and thus induces bone resorption although both forms of IL-1 are at least 10 times more potent on a molar level than TNF-α in the induction of bone demineralisation • has synergistic effects with IL-1 in bone resorption actions • lipopolysaccharide (LPS) from gram negative bacteria can initiate the production of TNF-α from macrophages/monocytes (Alexander and Damoulis 1994).Periodontal homeostasis represents a delicate balance between anabolic and catabolicactivities (Offenbacher 1996). Myriads of cytokines are involved in tissue turnover andthe maintenance of the integrity of the periodontium; of interest are the interleukins,prostaglandins, interferons and colony stimulating factors which mediate inflammatoryand immune responses (Williams et al. 1996). Cytokines in association with PGE2 arethought to lead to alveolar bone resorption, inhibition of bone formation and synthesis ofcollagenase by gingival fibroblasts which degrades matrix collagen (Page 1991;Alexander and Damoulis 1994; Offenbacher et al. 1996; Mathur and Michalowicz 1997;Ellis 1998; Okada 1998; Ueda et al. 1998).One of the significant advances in periodontal research in the last 20 years has been thefinding that normal residential cells of the periodontium can be induced to a catabolicstate by exposure to LPS, IL-1, TNF-α and PGE2 and participate in tissue destruction(Reynolds and Meikle 1997; Schwartz et al. 1997). In periodontal health, fibroblastgenes for collagen synthesis and TIMPs are turned on while the genes for MMPs areturned off. During periodontitis, the reverse applies, with fibroblasts producing also 29
  49. 49. IL-1β. This cytokine may cause autocrine stimulation with more IL-1β being secreted,or affect other target cells such as monocytes/macrophages, epithelial and endothelialcells (paracrine stimulation) to further enhance the production of the MMPs and PGE2(Reynolds and Meikle 1997; Schwartz et al. 1997; Schenkein 1999).Clinically healthy gingival and periodontal tissues express a number of anabolic growthfactors: • epidermal growth factor (EGF) • platelet-derived growth factor (PDGF) • transforming growth factor (TGF-β) - this is a superfamily of proteins and contains a number of bone morphogenic proteins (BMPs) • insulin-like growth factor (IGF) • cementum-derived growth factor (CGF) • inflammatory cytokines such as (IL-1, IL-6, and TNF-α) are in low concentrations compared to inflamed sitesThese anabolic molecules are involved in the rebuilding of the extracellular matrix by: • chemoattracting fibroblasts, periodontal ligament cells and bone generating cells • stimulating cells from a stable (nondividing) cycle to undergo mitosis and thus increasing the number of stromal cells • inducing cell differentiation of connective tissue mesenchymal cells to matrix secreting cells (Bartold et al.1998).Many of these molecules are incorporated into newly formed extracellular matrices,which they induce. During wound healing or repair, macrophages are drawn to thesesites by clotting factors. These macrophages respond differently than the pro-inflammatory macrophages which are chemo-attracted to sites by bacterial products orcomplement products (Bartold et al.1998). 30
  50. 50. 2.5 Cellular events in inflammationThe acute inflammatory response is characterised by the presence of neutrophils(Miyasaki 1991; Miyasaki et al. 1994; Miyasaki 1996) which constitute approximately90% of total circulating leucocytes and are the bodys first line of defence againstmicrobes (Van Dyke and Hoop 1990). Neutrophils have at least three types ofcytoplasmic membrane-enclosed granules: primary granules or azurophil granules,secondary granules or specific granules and tertiary or secretory granules (Table 2.5).These granules can release a large variety of enzymes that can degrade host tissue(collagenase, elastase, β-glucuronidase); this is a part of normal tissue homeostasisresulting in remodelling or healing. In addition these granules have a large number ofantimicrobial substances which can kill ingested microorganisms once phagocytosed.Table 2.5 Neutrophil components and function (Williams et al. 1996).Granule Function (all function under anaerobic and aerobic conditions) Granule component EffectPrimary granules Cellular myeloperoxidase Microbial killing(azurophil) Lysosome Cationic proteins Histamine release + enhances phagocytosis Acid hydrolases Antibacterial β−Glucoronidase α−Μannoxidase Neutral protease Elastase Exacerbates and mediates in inflammation CathepsinSecondary (specific) Lysosome Hydrolysis of cell wallgranules Alkaline phosphatase proteoglycans Collagenase Collagen degradation Vitamin B12 binding proteins Lactoferrin BactericidalTertiary (secretory) Gelatinase Replenish cell surfacegranules Alkaline phosphatase receptor expression and adhesion 31
  51. 51. 2.5.1 Macrophage phenotypesSince the host defences cannot inactivate biofilm completely, the inflammatory responsein periodontitis is longstanding and chronic. The presence of the macrophage signalschronicity; the neutrophil/macrophage characterises chronic inflammation while thepresence of macrophage/lymphocytes characterises the immune response (Williams et al.1996). The distinction between chronic inflammation and immunity is not that clear-cut.Macrophages have an important role in antigen processing as parts of the development ofan immune response and a subset of these cells have phagocytic capacity (Page et al.1997). Macrophages arise from bone marrow in a functional, immature condition, buteventually differentiate in the tissues. Macrophage phenotypes may phagocytosebacteria, modulate the clearance of damaged tissue debris during inflammation, modulatetissue remodelling, and trap and present antigens to lymphocytes (helper T-cells) toinduce the immune response. (Miyasaki 1996; Page et al. 1997).Macrophages are capable of synthesizing cytokines that contribute to healing and repair(anabolic) but can also have pro-inflammatory effects (catabolic) in the presence of achronic microbial challenge. (Page 1991; Seymour 1991; Birkedal-Hansen 1993;Offenbacher et al. 1993b; Genco et al. 1994). Macrophages represent 5-39% ofinfiltrating cells in inflamed periodontal tissues (Toppal et al. 1989; Zappa et al. 1991;Okada 1998).The macrophage is the key cell in directing whether anabolic or catabolic changes occurwithin the periodontal tissues. The catabolic changes occurring in the gingival andperiodontal tissues are due to the presence of the proinflammatory macrophages whichhave distinctive properties: 32
  52. 52. • reactive oxygen metabolites, including the superoxide anion(O2-), hydrogen peroxide (H2O2), the hydroxyl ion (OH-), and hypochlorous acid (HOCl-). All are bactericidal but can also be toxic to host cells (Klebanoff 1992; Alexander and Damoulis 1994). • arachidonic acid metabolites such as PGE2 and LTB4 and these can be produced in large amounts to create an inflammatory reaction (Samuelsson 1983; Salmon and Higgs 1987; Davidson 1992; Offenbacher et al. 1993b). • secreting the proinflammatory cytokines IL-1, IL-6, and TNF-α (Offenbacher 1996)In the periodontium, macrophage PGE2 has many regulatory effects: • decrease adherence and migration of macrophages, • under the influence of IL-1β and TNF-α , inhibits the genes controlling the synthesis of collagen and non-collagenous matrix proteins and tissue inhibitors of metalloproteinases (TIMPs) in fibroblasts • stimulates the synthesis and release of matrix metalloproteinases (e.g. collagenase) • it is the major mediator of pathological bone resorption • suppresses leucocyte function • along with cytokines IL-1, TNF-α and interferon-γ (INF-γ) it can regulate IgG production (where high concentrations of PGE2 inhibit antibody production and low concentrations act synergistically with IL-4 and enhance IgG production (Miyasaki 1996; Page et al. 1997; Reynolds and Meikle 1997).2.5.2 Alveolar bone resorptionKnowledge about alveolar bone resorption has lagged behind the understanding andresearch on connective tissue breakdown. There are, however, some well establishedfacts about bone remodelling and resorption (Schwartz et al. 1997): 33
  53. 53. • bone resorption-formation is a tightly coupled process and PGE2, IL-1, TNF-α are known mediators of bone loss. • IL-6 mediates the formation osteoclasts to resorb bone • activities between osteoblasts, osteoclasts and osteocytes are highly integrated and coordinated with one another. • there are biofeedback loops between these cells, where osteoblasts produce local factors that induce osteoclastic activity and vice versa. • cell control is regulated by circulating factors such as steroid hormones, parathyroid hormone, calcitonin and vitamin D.More information is needed to fully understand this area of periodontal pathogenesis.This overview of the inflammatory response is brief; the reaction of the inflammatoryresponse is very complex, and is integrated with the immune system. The aim of thisreview was to show the major inflammatory pathways and their roles in the overallpathologic process.2.6 Nonsteroidal anti-inflammatory drugs in periodontal diseases.The main anti-inflammatory agents are the glucocorticoids and the non-steroidal anti-inflammatory drugs (NSAIDs). Most of these drugs have anti-inflammatory, analgesiceffects and antipyretic effects which are related to their inhibition of the actions of COX(Vane 1971) and thus the inhibition of prostaglandins and thromboxanes (Heasman andSeymour 1989; Heasman et al. 1989; Williams et al. 1989; Heasman et al. 1990;Czuszak et al. 1996).2.6.1 History of salicylatesAspirin (acetylsalicylic acid) is the most widely used medicinal agent in the Western world(Vane et al 1992; Rainsford 1994). Natural products that contain precursors of salicylic acid,such as willow bark (which contains the glycoside salicin) and oil of wintergreen (which con- 34

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