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Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
Effects of Low-dose aspirin and gum diseases
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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

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

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  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. " 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. 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. 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. 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. 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. 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. 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. 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. • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. (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. 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. 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. 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. 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. • 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. • 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. 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. 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. 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. • 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. • 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
  • 54. tains methylsalicylate) have long been used in the treatment of rheumatism. (Walton et al.1994). Since the early 1800’s, salicin was hydrolysed to glucose and salicylic alcohol, whichwas then converted to salicylic acid. Sodium salicylate was first used in 1875 in the treatmentof rheumatic fever and as an anti-pyretic. The success of this drug prompted Hoffman, achemist employed by Bayer, to prepare acetylsalicylic acid based on the earlier, but forgotten,work of Gerhardt in 1853. The name aspirin is derived from Spiraea, the plant species fromwhich salicylic acid was once prepared (Campbell and Halushka 1996). According to Rains-ford (Rainsford 1984) the Bayer Company enjoyed immense profitability from aspirin by pro-tecting its patents until the beginning of World War I. This was when other companies startedto process aspirin and challenged the Bayer monopoly. In USA the patent office cancelledBayer’s registered rights to the name of aspirin, in 1918 and the US Supreme Court ruled thatthere was no infringement of tradename rights by US companies because Bayer’s aspirin hadbeen over-advertised to such an extent that it had become a common name. At about thesame time in Australia, the Federal government also suspended the Bayer’s patent rights. Apharmacist George Nicholas with a chemist called Smith produced the first Australian aspirinby 1915. Smith later withdrew from the partnership. George Nicholas joined with his brotherand formed the Nicholas Proprietary Ltd. They registered their aspirin under the trademark of“Aspro” (Rainsford 1984).2.6.2 Physio-chemical properties of aspirin and other salicylates .The structure of aspirin is shown in Figure 2.3 (Campbell and Halushka 1996). Aspirin israpidly absorbed from the gastrointestinal tract, partly from the stomach and mainly from theupper small intestine where it is quickly hydrolysed to salicylate (salicylic acid) by esteraseenzymes in the gut wall, blood and liver. 35
  • 55. Figure 2.3 Structure of aspirin (Rang et al. 1996). C O O H O C O C H 3The rate of absorption of aspirin and salicylic acid is governed by: • their physio-chemical properties - aspirin and salicylate are weak acids and have high lipid solubility • the surface area of the gastric and intestinal tract available for absorption • the pH of the gastrointestinal lumen. Absorption of salicylic acid is facilitated at the low pH existing in the stomach. The absorption of aspirin is half to one-third the rate of absorption of salicylic acid because of their different partition coefficients (lipophilicity). Also, the delayed absorption rate of aspirin is governed by the enzymatic hydrolysis by aspirin esterases in the mucosa, ie the spontaneous hydrolysis of aspirin. This is a rate-limiting step. • the rates of gastric emptying and intestinal transit times (which are dependent on the osmolality and acidity of the gastric contents). • the presence of food in the gastrointestinal tract • the physical form of the drug (tablet, capsule or soluble forms).(Rainsford 1994; Campbell and Halushka 1996; Rang et al. 1996).The half-life of aspirin in humans is 20-30 minutes and the half-life of salicylic acid is 2-3hours (Walton et al. 1994). Metabolism of aspirin to salicylic acid occurs by: 36
  • 56. • hydroxylation by liver cytochrome P450 system to gentisic acid or non- enzymatically by Fe(II)-catalysed hydroxyl radical (OH) • conjugation to phenolic or acyl-glucuronides by the enzyme UDP- glucuronosyltransferase. These glucuronides are synthesised in the small intestine, liver, bladder, kidney, lung, gastrointestinal mucosa and spleen. • conjugation of the carboxylate hydroxyl groups or gentisic acids to form salicylurate or gentisturate. These reactions activate acetyl-Co A in mitochondria of the liver and kidney.Various derivatives of salicylic acid have been synthesised for systemic use becausesalicylic acid is irritating to gastric mucosa. These comprise esters of salicylic acid (ofwhich aspirin is one) or salts of salicylic acid. Sodium salicylate is a salt of salicylic acidand has two thirds of the potency of aspirin. Aspirin itself is relatively insoluble, thedrug is nearly always taken orally, and soluble formulations are more efficacious thantablet formations (Seymour et al. 1986; Holland et al. 1988). Aspirin and salicylatecovalently modifies COX-1 and COX-2 by acetylating the active sites of both enzymes,and irreversibly inhibiting COX activity. Aspirin is 10-100 times as potent againstCOX-1 than against COX-2 (Figure 2.4) (Bing et al. 1999).Platelets cannot bio-synthesise cyclo-oxygenase (COX-1) and are therefore susceptible tothe action of aspirin. Aspirin and its derivative acetylsalicylic acid have the sameinhibitory effect on platelet cyclo-oxygenase at low and high doses (Lloyd and Bochner1996; Diener 1998; Müller 1998). A single low-dose of aspirin inhibits platelet cyclo-oxygenase for the life of the platelet (10 days) (Meade et al. 1993). Since aspirin andother NSAIDs are organic acids, they accumulate at sites of infection, an attractivepharmacokinetic property (Campbell and Halushka 1996). 37
  • 57. Figure 2.4 Effects of aspirin on cyclo-oxygenases (Bing et al. 1999).As low-dose aspirin irreversibly inhibits cyclo-oxygenase over the whole lifetime ofplatelets, it is widely used in middle-aged and elderly populations to prevent coronaryartery disease, stroke and peripheral vascular diseases. Additionally the lower doseshave lower gastro-intestinal side effects than high doses (Vane and OGrady 1993;Underwood 1994; Lloyd and Bochner 1996; Diener 1998; Müller 1998). Other NSAIDsare either organic acids or (once absorbed) are converted to organic acids to causereversible COX inhibition. Aspirin and other NSAIDs do not affect the leukotrienepathway. The majority of NSAIDs are non-selective inhibitors of the COX enzymes(Brooks et al. 1991).Most side effects of NSAIDs (gastric/duodenal ulcers, bleeding, cardiovascular and renalfailure) are due to the non-selective inhibition of COX-1 (Sharma and Sharma 1997;Dubois et al. 1998). Longitudinal studies show that 75mg can cause small but significantincrease in gastrointestinal bleeding, the effect doubles with 300mg and there is a 5 foldincrease with 1.5-2.4 g per day (Lloyd and Bochner 1996). New agents have beendeveloped that selectively inhibit COX-2, minimising the systemic side effects. 38
  • 58. However low-dose, slow release or enterically coated aspirins and its low cost ofproduction, make aspirin the highly used non-steroidal anti-inflammatory drug it is today(Vane and Botting 1992; Lloyd and Bochner 1996).2.6.3 Periodontal studies of the effects of NSAIDs over the last 20 yearsThere have been two reviews of NSAIDs and their effects on the gingival and periodon-tal diseases (Howell and Williams 1993; Drisko 1996). Most studies prior to 1990 wereretrospective, making comparisons difficult because of variable drug regimes, poor con-trol of specific medications, dosage or systemic health status of test patients. Overall,subjects taking NSAIDs had lower plaque indices, gingival indices, probing pocketdepths, attachment loss and less bone loss (Howell and Williams 1993). Table 2.6 liststhe current NSAIDs used in periodontal studies.The current review shows that prostanoids and leukotrienes are implicated in a wide range ofevents that are associated with disease, such as platelet aggregation, vasodilation and vasocon-striction, chemotaxis of neutrophils, increased vascular permeability and bone resorption. Inperiodontal diseases, these metabolites seem to be closely associated with connective tissuedestruction and alveolar bone resorption.Table 2.6 The types of NSAIDs (and their classification) used in periodontal studies Acetylated salicylates Aspirin Acetic acid Indomethacin, Sulindac, Ketorlac Propionic acids Ibuprofen, Naproxen, Flurbiprofen, Ketoprofen, Fenamic acid Mefenamic acid, Enolic acids Piroxicam 39
  • 59. The physiochemical properties determine their distribution in the body and differences in these properties may lead to variable therapeutic efficacies (Brooks and Day 1991).The following review discusses the effect of NSAIDs (as inhibitors of cyclooxygenase) on theclinical course of experimental and naturally occurring gingivitis and periodontitis in humans.Table 2.7 details the periodontal effects of NSAIDs in human studies.Table 2.7 Periodontal effects of NSAIDs in human studies Flurbiprofen Study Observations and comments (Williams et al. 1989) A double-blind placebo Individuals taking flurbiprofen had a sig- controlled study using 44 individuals of 24 nificant lower rate of alveolar bone loss months duration (pre-and treatment phase) with compared to baseline and control up to 18 periodontal disease. Gingival inflammation was months but not at 24 months ie returned to assessed every 2 months, standardised pre-treatment baseline values. There was radiographs every 6 months determined the rate no significance in the long-term use of this of alveolar bone loss. The 2-year treatment drug. group was compared to the 6-month baseline. (Heasman et al. 1989) A double-blind split There was a serious flaw in the studys de- mouth experimental gingivitis study with sign. Flurbiprofen is absorbed by the contralateral sides as controls. The study used whole oral mucosa and this could have 24 healthy subjects for 17 days. Each subject systemic effects affecting the control sites cleaned one upper quadrant with 10mM- in split mouth studies. Flurbiprofen solu- flurbiprofen solution every second day. A tion reduced gingival inflammation in ex- follow-up investigation of 6 subjects was perimental gingivitis in both test and con- performed only using placebo irrigation. trols. Due to this flaw, the experiment was modified so that an extra 6 subjects irrigated the whole mouth with a placebo solution and data compared to the flur- biprofen groups; there were significant differences in gingivitis and probing depths. 40
  • 60. Flurbiprofen Study Observations and comments(Heasman and Seymour 1989) A There were no changes in probing pocket depthssingle blind controlled parallel study for all three groups. Systemic flurbiprofenof 27 days duration looking at reduced gingivitis over a 21-day period. Butsystemic effects of flurbiprofen on there are problems justifying long term use ofexperimental gingivitis. There were 100mg/day flurbiprofen due to possible gastric25 healthy volunteers who stopped side effects. Therefore topical application maytooth brushing for 21 days. On day be of more value (as shown by a previous study)22 each subject was randomly (Heasman et al. 1989) bypassing the gastricplaced into one of three groups. side effects.Then assessed for plaque, gingivalindex and pocket depths.(Heasman et al. 1990) A follow-up Flurbiprofen levels in GCF were found to beprospective 28-day study on 5 higher than plasma levels. This led to theindividuals ingesting flurbiprofen assumption that flurbiprofen concentrates in theand then measuring flurbiprofen periodontal tissues.levels in GCF by high performanceliquid chromatography. 41
  • 61. Flurbiprofen Study Observations and comments(Heasman and Seymour 1990) A There were no differences for any clinical pa-controlled cross sectional study of rameters when compared to the control group,92 patients age and sex matched. these results were contrary findings to otherFifty hospital rheumatoid arthritis studies (Waite et al. 1981; Feldman et al. 1983).test patients were on a range of One should question the results of this studyNSAIDs with a 2-30 year duration. since there were a number of serious flaws in theThe controls were 42 non- design and selection of the sample.rheumatoid hospital patients with nohistory of nonsteroidal therapy. The (a) One of the main problems in selectingclinical parameters measured were 6 rheumatoid arthritis patients is that someRamfjord teeth for plaque index, studies have indicated that these individualsgingival index, pocket depth, loss of may have higher severity and extent of peri-attachment, gingival recession, odontal disease (Tolo and Jorkend 1990;gingival crevicular flow and non- Kasser et al. 1997).standardised radiographic analysisof bone loss. (b) With a mean age of 50, females may be en- tering menopause (Payne et al. 1997) these changes may affect the outcome in non- steroidal anti-inflammatory periodontal pro- gression studies. (c) No control of smoking (smoking is a risk factor for periodontitis and osteoporosis) (Jeffcoat 1998) Individuals were taking a number of different NSAIDs and no attempt was made to differenti- ate between them; different NSAIDs have dif- ferent effects on cyclo-oxygenase(Abramson et al. 1992) Double Systemic flurbiprofen decreased GCF-PGE2blind parallel randomised controlled and TxB2 during treatment; these returned tostudy consisting of 21 subjects of normal levels 7 days after cessation of drug.57-day duration. The main aim to These correlated well to beagle dog findingsdetermine the effect of systemic (Williams et al. 1988; Offenbacher et al. 1989)flurbiprofen administration on GCF- and similar findings to topical application ofPGE2 and TxB2 levels flurbiprofen (Heasman et al. 1989). One of the problems acknowledged with this study was that the individuals had a low prevalence of severe gingivitis and periodontitis and it is not known whether the effects of flurbiprofen would be the same in severe cases of disease. 42
  • 62. Flurbiprofen Study Observations and comments(Heasman et al. 1993a) A placebo Radiographically, a significantly number of sitescontrolled clinical trial of 12 months (8%) had more bone gain compared to controlsduration. Assessing the efficacy of (3.3%) but there were no significant differencesflurbiprofen toothpaste as an adjunc- between the test and control groups in plaque,tive measure in non-surgical manage- bleeding scores, probing depths or attachmentment chronic adult periodontitis. loss. The conclusion was that flurbiprofen-con-There were 25 test subjects and 24 taining toothpaste had a small significant effectcontrol subjects and both groups were on bone metabolism. Possible reason discussedage and sex matched. Plaque scores, for these findings was the Hawthorne effect.bleeding scores, crevicular fluid flow,probing pocket depths andattachment levels were measured atbaseline, 3, 6, 9 and 12 months.Radiographs were taken at baselineand 12 months.(Heasman et al. 1994) A double- There were no significant differences betweenblind parallel controlled study of 27 groups for plaque, and gingival crevicular flow.days duration with 47 (male and There was borderline significance in the resolu-female) subjects. Looking at the tion of gingivitis compared to placebo controls.systemic effects of 100mg This reinforced the findings of other studiesflurbiprofen in conjunction with tooth (Waite, et al. 1981; Offenbacher et al. 1987; Of-brushing on experimental gingivitis. fenbacher et al.1989) that not all inflammation is due to cyclo-oxygenase derivatives. 43
  • 63. FlurbiprofenStudy Observations and comments(Brägger et al. 1997). A double blind split After 6 months there were no statisticallymouth placebo controlled clinical trial of 6 significant differences between 4 groupsmonths duration, using flurbiprofen in bone density or bone remodelling. Afollowing flap surgery on 19 subjects with significant difference in probing depthsmoderate to severe adult periodontitis. At and clinical attachment gain was seen inbaseline all patients underwent surgical all groups irrespective if flurbiprofen orperiodontal therapy at 2 sites with >5mm placebo, indicating there was no influencepocket depths and two sites with >5mm of the NSAID on bone metabolism post-pocket depths with no surgical treatment. surgically. The authors stated that a possi-Ten subjects received 150mg/day ble explanation for these results was thatflurbiprofen postoperatively for 30 days 150mg flurbiprofen/day was too low orwith 9 subjects using a placebo during that there may be completely different bi-healing phase. Measurements taken were ological mechanism or pathways involvedof plaque index, gingival index, probing in pathological alveolar bone resorption ofpocket depths, subtraction digital image periodontitis compared to bone healingradiography analysing alveolar bone (repair) following surgery. The non-sig-density, recession, probing attachment nificant results could have been influencedlevels and bleeding on probing. by independent variables such as age and gender (not stated in this study), poor con- trol for smoking and diabetes. These risk factors could have influenced the out- come. 44
  • 64. Ketorolac Study Observations and comments(Jeffcoat et al. 1995) A random, parallel- The flurbiprofen and ketorolac groupsplacebo, double-blind positive control design had significantly less bone loss with sig-of 6-month duration on 55 patients with un- nificant reductions in GCF-PGE2 levelstreated adult periodontitis. The main aim compared to the placebo group. In addi-was to measure the effects of topical ketoro- tion, ketorolac, preserved more bonelac on alveolar bone height, bone mass, peri- than systemic flurbiprofen. Studies (Jef-odontal bone mass, probing attachment lev- fcoat et al. 1988; Heasman et al. 1989;els, plaque, gingival index. The groups were Williams et al. 1989; Jeffcoat et al.balanced for gender. To determine and iden- 1991) on NSAIDs and their effects ontify active alveolar disease sites each patient gingivitis had mixed results . Ibuprofen,was given a radioactive agent orally and a naproxen, ketoprofen, meclofenamatebone scan detector identified these sites. At effects on gingivitis have been minimalbaseline, 3-months and 6-months plaque but appear to affect the progression ofscores, probing depth, attachment levels, bone loss. Topical oxazlopyridine inhib-blood, urine, GCF-PGE2 and IL-1β were col- ited gingivitis and systemic flurbiprofenlected. Digital subtraction radiograph analy- inhibited experimental gingivitis in hu-sis was alsoused. Each patient had 2x/day mans. There was a significant decreaseketorolac mouth rinsing. Three groups were in gingivitis in this study. This study diddeveloped, a placebo rinse group (negative not control for the regular prophylaxis,control), a placebo rinse plus 100mg-flur- oral hygiene and Hawthorne effects onbiprofen tablet group (positive control) and a gingivitis, which could have accounted10% ketorolac rinse and a placebo tablet. for the results. 45
  • 65. Ibuprofen Study Observations and comments(Taiyeb and Waite 1993) A random, Ibuprofen significantly reduced gingival bleed-split-mouth, controlled prospective ing, colour change and pocketing in the teststudy of 8 weeks duration with 17 indi- group augmenting periodontal treatment. Thisviduals. Investigated the systemic ef- effect diminished rapidly after the drug regimefects of ibuprofen on mild to severe ceased with no significant differences by 8generalised chronic periodontitis. weeks. The authors stated that clinical applica-Plaque, gingival inflammation, BoP tions of ibuprofen were not justifiable and at-and pocket depths were measured. tribute this to the inability of ibuprofen to blockTwo groups were developed both giv- lipoxygenase products which they believed mayen whole mouth oral hygiene instruc- be the more prominent pathway of the two intions. Scaling on one side only ran- early inflammatory reactions, (differentdomly selected, making each patient NSAIDs do have different pharmacokinetic andtheir own control. The test group was pharmocodymamic properties (Brooks and Daygiven 200mg 4x/d of ibuprofen for 14 1991). Further factors which could have had andays. The 2 week regime avoided impact on the outcome was that the sample se-placing excessive demands on patient lected was not controlled for smoking and dis-compliance and minimised GIT side ease severity may have varied between groupseffects. The controls received placebo (at baseline). The authors recognised that thetablets. duration was too short and that the mode of de- livery may have affected the level of the drug in the gingival tissue, therefore affecting its effica- cy. Ibuprofen is of moderate potency on cyclo- oxygenase, explaining the moderate resolution of inflammation. A further hypothesis was that NSAIDs were only effective in active disease sites in the presence of acute inflammation (since increased cyclo-oxygenase and lipoxyge- nase are present in these sites) but not in quies- cent sites. 46
  • 66. Ibuprofen Study Observations and comments(Haffajee et al. 1995) Double blind, longi- Overall, the whole group gained attachmenttudinal comparative study with 98 subjects of 0.34mm with a pocket depth reduction ofaged range 14-71 with evidence of attach- 0.6mm. However a few subjects in eachment loss. All subjects had a minimum of treatment group had a poor response. The20 teeth with at least 4 pockets >4mm and 4 antibiotic groups had significant greatersites with LOA > 3mm. The duration of "gain" in attachment than ibuprofen or place-study was 12 months with 2 month moni- bo groups with no statistically significant dif-toring intervals measuring six sites/tooth of ference between Ibuprofen and placebo.all teeth present. The aim was to assess the Shallow pockets (< 4mm) lost attachment,effects of periodontal surgery and 4 system- intermediate pockets (4-6mm) showed mod-ically administered agents (Augmentin, erate gain, and deep pockets (>6mm) showedtetracycline, ibuprofen and a placebo) on the most gain. Adjunctive use of antibioticsclinical and microbiological parameters of during "active" disease produced more effec-periodontal disease. Two months into study tive outcomes than mechanical instrumenta-individuals with LOA >2.5mm and pockets tion alone. From this study one cannot dis->4mm at one or more sites during longitu- cern if individuals without "active" diseasedinal monitoring (this was taken as sign of would respond in the same way. The resultsdisease progression) were treated by modi- of this study are contrary to past studies onfied Widman flap, scale and cleaning, Ibuprofen (Offenbacher et al. 1992; Taiyeb0.12% chlorhexidine and during the treat- and Waite 1993). The main problems in thisment phase they were randomly placed on study were that there was a small sample sizean agent for 30 days, blind for the whole (6 subjects) in the Ibuprofen group comparedsurgical period. Plaque, gingivitis, BoP, to the antibiotic groups (13 and 10 subjects),suppuration, PPD and PAL were measured. therefore affecting the statistical power and itThe clinician performing the surgery was is difficult doing multiple comparisons (scal-different to clinician measuring all clinical ing, root planing, chlorhexidine rinses, oralparameters in a blind fashion. hygiene and test agents) and drawing conclu- sions on inflammation, bleeding and attach- ment status. No history of smoking was giv- en for subjects. 47
  • 67. Ibuprofen Study Observations and comments(Ng and Bissada 1998) The aim was to There were statistically significant gains ininvestigate the efficacy of a systemic an- CAL for both doxycycline (0.4mm) andtibiotic (doxycycline) and ibuprofen, ad- doxycycline/ibuprofen (0.5mm) groups andministered either separately or as adjunc- significant reduction of pocket depthstive treatment to SRP.A placebo, con- (0.7mm) and gingival index scores. Thetrolled split-mouth study on 32 subjects control group with SRP had CAL gain of(18 males and 14 females) aged 32-72 0.5mm and reduction of 0.4mm pocketyears with generalised moderate adult pe- depths and gingival scores. Although thereriodontitis of 24 weeks duration. At weeks was improvement in the doxycycline and3, 6, 12, 24 plaque, probing depths, (CAL) ibuprofen/doxycycline groups it was onlyclinical attachment levels (with stents) modest in comparison to the improvementwere taken. Intraoral photographs and pe- achieved by SRP. The ibuprofen only groupriapical radiographs were taken 24 weeks had short-lived effects with only a 12-weekfollowing baseline scaling. gain in CAL, and only 6 weeks for reduction of pocket depths. These effects are similar to other findings (Feldman et al. 1983; Williams et al. 1989). Doxycycline achieves high levels in gingival tissues and possesses anti-collagenase activity, while the findings for ibuprofen could have been due to the low dosage. 48
  • 68. Naproxen Study Observations and comments(Johnson et al. 1990) Double blind This was the first human study on the gin-placebo controlled study where 102 gival effects of naproxen. There was no ef-patients were given 500mg naproxen fect on plaque, gingival inflammation orfor 30 days. Investigating the effect of bleeding indices at day 20, but on day 28systemic naproxen on experimental after plaque removal there was statisticallygingivitis. significant improved resolution of gingival inflammation in the naproxen group by day 30. An explanation for these results is that the inflammatory pathway involves many different endogenous mediators that com- pensate for prostaglandin inhibition, name- ly leukotrienes, complement, cytokines and direct stimulation of inflammation by lipopolysaccharides. In addition the au- thors stated that as long as plaque is present NSAIDs are not efficacious in suppressing inflammation due to other products of the arachidonic acid metabolites. Naproxen may have a greater effect in inhibiting the cyclo-oxygenase pathway in bone loss than preventing soft tissue inflammation. 49
  • 69. Naproxen Study Observations and comments(Jeffcoat et al. 1991) A double blind placebo Naproxen decreased the rate ofcontrolled study with 15 patients having refrac- alveolar bone loss in conjunction withtory periodontitis. All patients were treated periodontal therapy. Digitalwith scaling and root planing. Seven patients subtraction radiography showed therewere given 500mg naproxen b.i.d. adjunct ther- was significantly less bone mass andapy for 3 months. Bone loss or gain was mea- height loss in patients taking thesured with digital subtraction radiography, naproxen compared to placebo group.alveolar bone metabolism changes and alveolar This supported the Johnson et al.,bone height was measured radiographically us- (1990) findings that naproxen is moreing intravenous radioactive methylene diphos- efficacious in preventing alveolar bonephate, which is an alveolar bone-seeking radio- loss than gingivitis.pharmaceutical.(Offenbacher et al. 1990) An in vitro study on This study found the following drugshuman pooled gingival tissue samples from 10 to be inhibitors of cyclo-oxygenasepatients with severe periodontitis who had not from the most to the least potent; α-to-received prior treatment with drugs. The aim copherol, ketoprofen, indomethacin,was to see which agents (ketoprofen, in- flurbiprofen, meclofenamate, naprox-domethacin, naproxen, ibuprofen, a-tocopherol, en, docosahexaenoic acid, eicosapen-docosahexaenoic acid eicosapentaenoic acid, taenoic acid, ibuprofen, diflunisal andflurbiprofen, meclofenamate, sulindac and di- sulindac. Naproxen was found to be aflunisal) were efficacious inhibitors of arachi- moderate inhibitor of PGE2 synthesisdonic acid metabolism at different concentra- in vitro. The authors recognised thetions. By measuring the concentration of drug fact that in vitro tests have inherentthat caused 50% inhibition of maximum PGE2 problems and may not predict in vivosynthesis in gingival epithelial and fibroblast effects. Drugs that may be efficaciouscells. The reference agent was indomethacin in preventing inflammatory changes insince it is a moderately potent cyclo-oxygenase arthritis may not be effective inhibitorsinhibitor. of periodontal disease. 50
  • 70. Meclofenamate Study Observations and comments(Reddy et al. 1993) Double blind controlled The meclofenamate groups had significantlyclinical trial of 6-month duration. 22 subjects less bone loss and more gain in attachment(7 male 15 females) with a mean age of 36.5 but the major improvement in the clinical pa-years. The aim was to determine the efficacy rameters was due to mechanical therapy; oralof meclofenamate as an adjunct to scaling and hygiene and or the Hawthorne effect mayroot planing in rapidly progressive periodonti- have contributed as well. The bone gainstis and disease active sites. were similar to those seen in the naproxen study (Jeffcoat et al. 1991) The authors questioned the use of high dose meclofena- mate since there were gastrointestinal side effects. Sulindac Study Observations and comments(Vogel et al. 1983) A randomised As plaque increased, all three groups showed an in-double blind study of 7 weeks duration crease in GCF flow and inflammation. The topicalon 18 male dental students with no pe- steroid significantly inhibited gingival inflammationriodontal disease. Investigating the ef- and systemic sulindac had no effects on GCF flowfect of systemic sulindac on experi- or inflammation clinically and histologically. Sulin-mental gingivitis. All were taken to dac is a cyclo-oxygenase inhibitor, flucinonide in-state of optimal gingival health. At hibits phospholipase limiting the availability ofday 0 of the treatment period the GCF arachidonic acid metabolites from the cyclo-oxyge-flow, gingival inflammation and gingi- nase and lipoxygenase pathway and in addition in-val bleeding were measured on select- hibits immunologic responses. The authors gaveed teeth in the maxillary right quad- three reasons for the findings:rant, then all subjects randomly placedinto 3 groups. One group received a (i) topical steroids may affect the plaque direct-placebo gel and capsule, another group lyreceived placebo gel plus 150mgsulindac capsule 2x/day. The third (ii) the treatment period of 22 days was too shortgroup received a placebo capsule, and for sulindac to have reached a gingival con-0.05% flucinonide gel (a steroid). All centration level to inhibit inflammationoral hygiene ceased for 22 days on themaxillary right quadrant. A biopsy of (iii) topical steroids have a greater concentrationfree gingiva on the mid-buccal surface locally than systemic administration of anof the maxillary right molar was taken agent.for histological assessment Inflammatory mediators peak at 2-3 months from onset and the response of alveolar bone to NSAIDs is delayed, with effects seen between 3-6 months. 51
  • 71. Generally NSAIDs have been shown to reduce alveolar bone loss in humans, but their effecton gingivitis is not clear, perhaps because different host response pathways operate in gingivi-tis and in bone resorption. Since inhibition of COX may cause shunting towards other path-ways eg pathway-2 or 3 (Sharma and Sharma 1997), drugs blocking one biochemical path-way may have a variable effects in gingival tissue (e.g. ATLs), whereas the bone destructionpathway is a more tightly coupled mechanism on which modulation of PGE2 could have moreprofound and significant effects.The ability of NSAIDs to reduce or inhibit bone and periodontal attachment loss are stronglylinked to PGE2 as the major mediator of bone resorption, in association with IL-1β, IL-6 andTNF-α (Offenbacher et al. 1993b). The NSAIDs studies show that the inhibition of COX isadvantageous since it may directly limit the bone resorption activity of PGE2 and modulatethe activity of the other synergistic agonists (Offenbacher et al. 1993b; Page et al. 1997).2.7 Periodontal studies with aspirinFew studies have investigated the effects of aspirin intake on periodontal status (Waite et al.1981; Feldman et al. 1981; Heasman et al. 1990; Flemmig et al. 1996).2.7.1 The Waite study:(Waite et al. 1981)There were 44 subjects participating, with an age range 22–68 years (mean age 45.8 years).The test group consisted of 22 rheumatology patients from a London hospital (13 females and9 males). One test patient was on aspirin, the rest were on indomethacin and/orphenylbutazone. The duration of drug therapy ranged from 1-20 years, no dosage or drugregime data were given. The control group had 22 subjects (14 females and 8 males) whowere office workers. All participants had to have all six Ramfjord teeth present. Subjectswere cross-matched for age and plaque levels. There were no significant differences in 52
  • 72. periodontal attachment loss but gingival indices and pocket depths differed significantly.Subjects taking aspirin, indomethacin and / or phenylbutazone had a lower gingival index(GI), shallower pocket depths, with a pattern for less loss of attachment but not significantlyless. The author proposed that these effects were due to PGE2 inhibition principally based onfindings from past studies. Problems in this study were that the control subjects were from adifferent source, the sample sizes were small, there was no control for age, sex and smokinginfluences and the study relied on the subjects memories of dosage and duration. In addition,only one subject was on aspirin, making it of little value in understanding the effects of aspirinon the periodontium.2.7.2 The Feldman study(Feldman et al. 1983)This was a retrospective study investigating the effects of long term aspirin with and withoutindomethacin therapy on alveolar bone levels in men only. The test group was composed of75 subjects (mean age of 57 years) who had been taking high dose aspirin for more than 5consecutive years for rheumatic problems. The control group consisted of 75 male volunteers(mean age of 58 years) from an ongoing dental longitudinal study. Alveolar bone loss on themesial and distal surface of every tooth was assessed radiographically. Both groups werematched for age and remaining dentition and denture use. The findings from this study werethat: • the test group had significantly fewer interproximal sites with 10% or more alveolar bone loss (33.2) compared to the controls (37.2 sites) • the mean percentage bone loss per individual, although lower in the test group was not statistically significantly different from the control group. 53
  • 73. The authors concluded that the better periodontal conditions were probably due to theinhibition of PGE2. However, it is not possible to differentiate between the effects of aspirinand indomethacin, since some subjects used more than one prescribed medication over the 5year period. Furthermore, the radiographs were not standardised (measurement error mayhave had an effect on the results) and the dosages of NSAIDs were not constant over the fiveyears, (patients took from 1-12 tablets per day). The number of patients taking both aspirinand indomethacin was not reported.2.7.3 The Flemmig study(Flemmig et al. 1996)This was a double blind, placebo controlled, split-mouth study of 30 males and females, aged30-65 with untreated moderate to severe adult periodontitis. Inclusion criteria was a mini-mum of 18 teeth and each quadrant having one 6mm pocket or 4mm PAL. The primary aimwas to investigate the efficacy of 500mg (q.i.d) systemic acetylsalicylic acid as an adjunct toscaling, using GCF-elastase levels as a measure of disease activity. A placebo was given for 6weeks (q.i.d) to all subjects, establishing a baseline. Then a 6-week treatment phase of acetylsalicylic acid was given to the test subjects and the placebo continued for the controls. Mono-therapy of 2g/day of acetyl salicylic acid had no significant effects except in reducing GCF-α1elastase. These findings were contrary to the Waite et al., (1981) findings for gingival in-dex and probing pocket depths. On the other hand, the combination of acetyl salicylic acid(inhibiting inflammatory response) and scaling (reduction in bacterial plaque) resulted in asignificant therapeutic efficacy, approximately equivalent to the sum of each individual thera-py in reducing gingival inflammation, pocket depth and probing attachment loss (p<.001) ahighly significant result. Combined therapy did not significantly increase the efficacy ofacetyl salicylic acid to reduce GCF-α1elastase. These findings indicated a decreased risk of 54
  • 74. periodontal disease progression in subjects taking aspirin since GCF-α1elastase has been posi-tively correlated with connective tissue and alveolar bone destruction (Zafiriopoulos et al.1991).2.7.4 The Heasman study(Heasman and Seymour 1990):This was a retrospective study investigating the effect of long-term use of NSAIDs on theseverity of periodontal disease. The test group comprised 50 rheumatology patients selectedfrom a Newcastle hospital and from general dental practitioners. There were 21 male and 29female patients, with a minimum 2-year duration of NSAID therapy. The control group (16males 26 females) were selected from subjects attending a Dental Hospital for a routine dentalcheck up. Both groups were matched for age, remaining teeth (20- 25) and plaque indices andperiodontal conditions around only the six Ramfjord teeth. No statistically significantdifferences were found between the test and the control groups for plaque index, gingivalindex, probing depth, loss of attachment, recession and alveolar bone loss. There was asignificant difference for gingival fluid flow. Some of the problems with this study were thatthe test group had 72% of its subjects referred from general dental practitioners and thecontrols were recruited from a dental hospital as well as dental practices, creating serioussample selection error or bias, since the majority of subjects in both groups were referred fromdental practitioners. Less than 10% of test and control subjects had greater than 3mm probingdepths i.e. low prevalence of periodontitis. The partial recording of six Ramfjord teeth wouldhave influenced the findings; this study was poorly designed and the results should beinterpreted with caution. 55
  • 75. 2.8 Smoking and periodontal diseasesSmoking is now recognised as the most important cause of preventable death and diseasein the western world (MacGregor 1992). There are over 4000 chemical toxins present incigarette smoke, these include products such as carbon monoxide, oxidating radicals,nitrosamines (carcinogens) and nicotine; over 50% of smokers will die of smoking-related disease. On the other hand, smoking cessation has been associated withsignificant improvement in life expectancy and decreased morbidity (U.S. 1990).Cigarette smoking has been found to be an important risk factor for periodontitis (U.S.Department of Health, 1990). Extensive studies and reviews on the association ofsmoking and dental health demonstrate a clear association between smoking and theprevalence and severity of periodontitis (Haber et al. 1993; Bergström and Preber 1994;Zambon et al. 1996). Smokers have more tooth loss, deeper probing sites, moreattachment loss and more furcation involvement than non-smokers (Ismail et al. 1983;Goultschin et al. 1990; Grossi et al. 1995). The relative risk of developing periodontitisbetween smokers and non-smokers varies from 2.5-14 (Haber et al.1993; Schenkein etal.1995). Generally, 50% of individuals who smoke have an odds ratio of 6-8 in favourdeveloping periodontitis (Salvi et al. 1997).Heavy cigarette smoking (> 30 pack/year history of smoking) is one of the highest risk indica-tors for periodontitis; the greater the exposure in terms of pack years, the greater the amountof alveolar bone loss (Grossi et al. 1996). Smoking is a risk factor for rapidly progressive pe-riodontitis as well as adult periodontitis (Haber and Kent 1992). The exact mechanism of in-creased smoking-related susceptibility to periodontitis is unknown (Johnson 1998). Studiesshow that smoking exerts both local and systemic effects on the oral tissues, vasoconstriction,by nicotine, depression of various arms of the immune system and affects gingival fibroblastfunction and bone metabolism (Preber and Kant 1973; Preber and Bergström 1986; McGuire 56
  • 76. et al. 1989; Ah et al. 1994; Preber et al. 1995; Tipton and Dabbous 1995; Salvi et al. 1997;Bando et al. 1998; Johnson 1998; Ryder et al. 1998).2.8.1 The periodontal effects of past smoking and smoking doseThe degree of periodontitis in ex-smokers lies in between current smokers and non-smokers and is dependent on the dosage (pack years) of smoking (Tonetti 1998). Pastsmokers tend to have similar response to treatment as non-smokers (Kaldahl et al. 1996).Current smokers have less probing depth reduction after periodontal treatment than doex- and non-smokers. The response in pocket depth reduction and CAL were similar informer smokers and non-smokers (Grossi et al. 1997). There was no correlation betweenthe number of years of smoking cessation and therapy response, suggesting that there areimmediate periodontal benefits from quitting smoking. A similar finding has beenreported in relation to the success rate of dental implants (Bain and Moy 1993). Smokerswho quit one week prior to implant placement and started smoking again 8 weeks afterimplant placement had failure rates similar to non-smokers. Heavy smokers quittingsmoking in the first 2 weeks following thick free gingival grafting had a comparableresponse in terms of root coverage to non-smokers (Miller 1987); it was also noted thatpatients who smoked < 5 cigarettes/day did not have a negative impact in response to thistype of therapy. Smoking history also affects tooth loss - for a given age, ex-smokers liebetween non-smokers (least tooth loss) and smokers (most tooth loss) (Österberg andMellström 1986). Quitting smoking greatly reduces risk to general health; once a patientquits, one can also expect a normal response to periodontal therapy (although we cannotreverse the past effects of smoking). Unfortunately, smoking behaviour is not easilychanged since nicotine is an addictive drug (Johnson 1998). 57
  • 77. 2.9 Periodontal measuresLindhe et al.(1986) stated that any trial to study periodontal disease to assess the effect ofa certain treatment procedure on human periodontal disease must take into account: • the nature of periodontal disease • the progression of periodontal disease • the measurement of efficacy of therapy • the experimental unit • the goals of periodontal therapy.Plaque and gingival indices are traditional measures used to measure the presence andseverity of gingivitis and plaque (Loe et al.1965; Haffajee et al. 1983; Mombelli etal.1987; Ainamo 1988; Listgarten 1988; Claffey et al. 1990; Haffajee et al. 1991;Armitage 1996; Newbrun 1996). Bleeding on probing, pocket depth and attachment lossare important measures of periodontal diseases (Lindhe et al. 1986; Armitage 1996).They provide useful information regarding the location, presence or absence of diseasedtissues and the absence of these conventional signs is strongly indicative of a healthy,stable periodontium (Lang et al.1986). These commonly used diagnostic procedureshave significant weaknesses. Although inflammation may be indicative of disease, itgives no indication of severity, morbidity, or eventual outcome of periodontal infections.The clinical signs of inflammation are unreliable in differentiating between non-destructive and destructive forms of periodontal disease, current disease activity and thecause of the attachment loss. Loss of periodontal attachment only measures pastepisodes of disease. The parameters measured in this study were gingival bleeding onprobing (indicative of inflammation), gingival recession and periodontal pocketing. 58
  • 78. 2.9.1 The experimental unitIn medical research, the individual or subject forms the unit of examination whereas inperiodontology, recording units may be site specific (Lindhe et al. 1986; Ainamo 1988). Theindications for using periodontal sites as the unit are that different sites: • in the same individual show different patterns of disease severity, progression and lesion morphology • often respond differently to periodontal therapy.2.9.2 Measurement of extent and severity of periodontal attachment lossEpidemiology is the science concerned with the factors that influence the distribution andoccurrence of health, disease and mortality among groups of individuals (ranging in sizefrom small populations to large sub-national aggregates to entire countries). A frequentmeasure of disease used in epidemiology is prevalence - the proportion of individuals ina group who exhibit the condition at a given point in time (Fletcher et al. 1988). Thetotal number of individuals with a given condition is also frequently referred to as theprevalence. Prevalence is estimated from cross-sectional studies.The periodontal status of an individual, let alone a group, is difficult to describeconcisely. To thoroughly characterise periodontal status, epidemiological studies assessnumerous sites in both dental arches for each individual. Epidemiological assessment ofa group of individuals requires a summary of the periodontal status of the individual sitesin the mouth. Development of summaries must deal with the difficult issue of casedefinition as well as the number and types of sites to assess. The effect of falliblemeasurement techniques is also an important issue and will be discussed later. Beforethe prevalence of a "periodontal disease" can be described, the disease must be defined.This requires consideration of severity (such as the amount of inflammation or 59
  • 79. periodontal attachment loss) as well as the extent (the percentage or number of affectedsites). Although all epidemiological studies use some combination of the concepts ofprevalence, extent and severity to describe the periodontal status of a population, variousstudies have assessed these concepts differently. It is difficult to compare the resultsfrom studies that use different measurement methods or case definitions and comparisonscan therefore be misleading.Epidemiological studies have used a variety of indicators and indices as a measure ofperiodontitis. Earlier indices included the Periodontal Index (Russell 1959) and thePeriodontal Disease Index (Ramfjord 1959). These indices were not satisfactory orcompletely accepted; both present a single mean index score for an individual or group,which used an estimate of disease severity, but ignored the extent or distribution of thedisease. The World Health Organisation proposed the use of the Community PeriodontalIndex of Treatment Needs (CPITN) (Ainamo et al. 1982; Cutress et al. 1987; Ainamo1988) as an index of rapid assessment of periodontal treatment needs in a population.However, this index only records the "worst score" for each of the six segments of themouth, and ignores much information obtained from a clinical examination. The CPITNdoes not quantify loss of attachment (Slade and Spencer 1995). Direct measurement ofperiodontal attachment loss has only recently been used in periodontal research, despitehaving been proposed two to three decades ago as an important measure of disease(Ramfjord, 1959; Glavind et al.1967). The most appropriate epidemiological indicator ofperiodontal destruction is based on the measurement of loss of attachment (through itscomponents, gingival recession and pocket depth) (Brown and Garcia 1994; Slade andSpencer 1995). 60
  • 80. 2.10 Null hypotheses1. There is no difference in periodontal attachment loss between ex-smokers and non-smokers irrespective of their aspirin status.2. There is no difference in periodontal attachment loss between low-dose aspirin takers and non-aspirin takers irrespective of their smoking history.Level of significanceRejection of the null hypothesis for all tests of significance was set at a probability valueof alpha = 0.05.Aims of the studySpecific aims:1. The primary aim of this study was to assess the severity and extent of periodontal attachment loss in males over 50 years of age with a history of long-term low- dose aspirin therapy and compare to non-aspirin takers using a cross-sectional design.2. The associated aim of this study was to assess the severity and extent of periodontal attachment loss in males over 50, with a past history of smoking and on long-term low-dose aspirin therapy and compare them to non-smokers using a cross-sectional design.Secondary aims:(i) To assess the case definition of severe attachment loss within the study population.(ii) To extrapolate PAL from the above defined study population to the general male population of the metropolitan Adelaide area(ii) To evaluate the association between education, pension status, and oral hygiene habits with the above primary aims. 61
  • 81. Chapter 3 Materials and methodsThe University of Adelaides Human Research Ethics Committee gave ethical approval for thisstudy. The approval number was: H/35/97.3.1. Sample selectionA self-selected sample of males aged 50 years and over from the general populationliving in Adelaide (state capital) of South Australia made up the study population.Subjects were recruited through local press media advertising (Figure 3.1).Figure 3.1 A copy of an advertisement placed in local press media to recruit subjectsMEN !Are you over 50?Would you like a free Colgate oral care package?Do you have at least 6 teeth of your own?Do you take aspirin regularly?Are you an ex-smoker or a non-smoker?If you answered "YES" to any of these questions, we want you to take part in aresearch project looking into the dental health of MEN ONLY.All you have to do is answer a short questionnaire about your health and have anexamination.HOW? -Ring Thursday, or Friday 8303 3436 for further information and to make anappointment.WHERE? - Colgate Australia Clinical Dental Research Centre, Frome Road,Adelaide.Unfortunately we cant include you if you are a current smoker, or have Rheumaticfever, a heart valve defect, a pace-maker, joint replacement, severe arthritis,cancer, liver or kidney disease. 62
  • 82. Advertisements were placed in the following newspapers: The Advertiser,Messenger.and the Mature Times. Table 3.1 lists the studys inclusion andexclusion criteria.Table 3.1 Inclusion and exclusion criteria INCLUSION CRITERIA: 1.6.3 EXCLUSION CRITERIAMales only Current smokersAll participants must be aged 50 years People requiring antibiotic cover (artificial and over heart valves, congenital heartMinimum of 6 or more natural teeth. defects, rheumatic fever, and jointAll participants had to complete and prosthesis) for dental examinations. sign an ethical consent form. Drug exclusion:Anyone on aspirin therapy for longer People on long term: than 2 years and never smoked - SteroidsAnyone on Aspirin therapy for longer - Non-steroidal anti- than 2 years and an ex-smoker inflammatory drugs (other for longer than 2 years. than aspirin),Anyone not on aspirin therapy and - Dilantin never smoked. ImmunosuppressantsAnyone not on aspirin therapy and ex- - Anti-coagulants smoker for longer than 2 years History of extensive antibiotic therapy in the last three months- Chlorhexidine mouth washes Systemic diseases: People suffering from - Diabetes - Rheumatoid arthritis - Hepatic or renal diseases - Cancer People with pace makers Periodontal surgery within the last five years Less than 6 natural teethSubjects matching the inclusion criteria for both test (aspirin takers ex- and non-smokers) and control groups (non-aspirin takers ex- and non-smokers) were offeredthe opportunity to participate by telephoning the Colgate Australian Clinical DentalResearch Centre (CACDRC) located in the Adelaide Dental Hospital. Receptionstaff conducted a further screening (of inclusion and exclusion criteria) giving a 63
  • 83. brief description of the study over the phone, prior making appointments. The staffunderwent a continuous education program and discussions about the study andgiven strategies to encourage subject participation.When subjects attended their appointment, the reception staff handed them aninformation sheet (Appendix A), an ethics consent form (Appendix B) and aquestionnaire. After the subjects read the information leaflet they signed a consentform which was later signed by the investigator and an independent witness. Oncompletion of the periodontal examination, participants received a "Findings form"(Appendix C), a Colgate Oral Care Kit (containing a toothbrush, toothpaste, dentalfloss and pamphlet on oral care), a copy of the signed consent form and theinformation leaflet.3.2 QuestionnaireThe questionnaire collected demographic information, medical and dental histories. Tosimplify the subjects task and to reduce time, a multiple-choice format was used for allquestions (Appendix D). This format standardised the data and facilitated statisticalanalysis. The questionnaire was self-administered in the CACDRC waiting room toavoid examiners / interviewers bias or influence (Abramson 1974; Bourque and Fielder1995; Fink 1995a). When requested by the subjects to clarify questions, concepts orwhere there was a language/reading difficulty, CACDRC staff gave assistance. Thequestionnaire sought a variety of information (Table 3.2). 64
  • 84. Table 3.2 Aims of questionnaire. • demographics • socioeconomic status • medical history • aspirin history (type, dosage and duration) • smoking history (dosage, duration and term of cessation) denture use • the subjects oral hygiene habits and oral care • past experiences of periodontal treatmentQuestion format and wordings were selected from the W.H.O. Oral Health Surveys BasicMethods (W.H.O. 1997), Australian Bureau of Statistics (Census) (McLennan 1996) and theSouth Australian Dental Longitudinal Study Five year follow-up Questionnaire (Spencer1997).Questions 5 and 6 (see Appendix D) were designed as a final cross-check to exclude subjectswho fell outside the studys selection criteria as well as collecting subjects medication history.History of aspirin use was assessed in questions 7-11. Question 7 elicited if subjects were onaspirin and the type or generic brand of aspirin. Questions 8 & 9 recorded the frequency ofintake and daily intake. Question 10 recorded the dosage in milligrams and question 11focussed on the duration of aspirin therapy, (relying on the subjects recollection). Noinformation was obtained as to whether aspirin intake was by prescription or over thecounter. Smoking history was assessed in questions 12-16 in a similar format, again relyingon the subjects recollection of the time since cessation, age of smoking debut and number ofcigarettes that they used to smoke per day. Dental habits were elicited by question 17-24.Oral hygiene habits were recorded in answers to questions 20-22. Question 23 recorded the 65
  • 85. subjects actual regularity of dental visits. Question 24 measured the subjects regularity ofperiodontal care. Question 25 elicited which subjects had had periodontal surgery.3.3 Oral ExaminationThe objectives of the oral examination were to determine the teeth present and thegingival and periodontal status of the subjects in a descriptive epidemiological form. Theoral examinations were conducted by one dentist, blind to the subjects aspirin andsmoking histories and were based on the methodology used in Slade et al., (1995) andFlemmig et al., (1996) with some modifications to the periodontal assessment as follows: (i) all erupted teeth (including the third molars) were assessed (ii) measurements of gingival recession and periodontal pocket depth were made at six sites around each tooth i.e. mesio-buccal, mid-buccal and disto- buccal, mesio-lingual, mid-lingual and disto-lingual.FDI notation was used during the examination. A custom written computer program wasset up in the CACDRC for nursing staff to directly input all measurements as called outby the examiner during the examination. All survey details were entered at thecompletion of the dental examination by the nursing staff.3.4 Clinical measurementsThe following clinical variables were assessed:3.4.1 Plaque IndexUsing the Silness and Löe plaque index (Silness and Löe 1964), plaque scores of the"Ramfjord teeth" ie 16, 21, 24, 36, 41, and 44 were entered directly into the computerprogram and an overall Plaque Index (PI) score was computed for each subject (Silness 66
  • 86. and Löe 1964). The PI was an ordinal scale from 0-3 and each score quantifiedaccordingly (Newbrun 1996):Table 3.3 Plaque index (Silness and Löe 1964) Gingival area of tooth free of plaque; the surface is tested by running a 0 probe across the tooth surface, if no soft material adheres, then its consid- ered plaque free. No plaque observed in situ by the unaided eye, but plaque is made 1 visible on the point of a probe after the probe has been moved over the tooth surface at the entrance of the gingival crevice. Gingival area is covered by thin to moderate thick layer of plaque 2 visible to the naked eye. Heavy accumulation of soft matter, the thickness of which fills the 3 crevice produced by gingival margin and tooth surface.3.4.2 CalculusA dichotomous index of calculus was recorded per tooth for every tooth present. Nodistinction was made between supra- and sub-gingival calculus. Calculus detectedvisually or by probing was given a value of "1" and if absent, was scored "0", giving areading by tooth, rather than by tooth surface or specific site.3.4.3 Bleeding index.The modified Sulcular Bleeding Index (mSBI) was used as a quantitative measurementof gingivitis see Table 3.4.3 (Mombelli et al.1987)Table3.4 Modified Sulcus Bleeding Index (mSBI). Grade Description ((Mombelli et al.1987; Newbrun 1996) 0 No bleeding when a periodontal probe is passed gently along the gingival margin 1 Isolated bleeding spots visible 2 Blood forms a confluent red line on margin 3 Heavy or profuse bleeding 67
  • 87. This index gives a recording per tooth rather than by a subjects gingival sites. Thesurfaces recorded per tooth were the lingual surfaces of the first and third quadrants andthe buccal surfaces of the second and fourth quadrants. The mSBI entails a gentleprobing action at the gingival crevice/sulcus with a probe angulation of 600 to the longaxis of the tooth (Newbrun 1996). This is a more sensitive indicator of gingivalinflammation and less likely to elicit false-positive bleeding than does probing to thebottom of the pocket (van der Weijden et al. 1994a; van der Weijden et al. 1994b).Gingival bleeding was considered to be a better estimator of the efficacy of oral hygienethan plaque scores, which only indicate the amount of plaque present. Bleeding scoresbetter reflect a subjects day-to-day level of plaque control. The use of the mSBI indexnecessitated the need to standardise the lighting to have a consistent visual assessment ofthe gingiva.3.4.3 Tooth mobilityTooth mobility was recorded for every tooth using the Miller notation (Miller 1950)(Table 3.5). The method used was by placing a mirror handle on the tooth and buccal,lingual and occlusal force applied and recording its movement.Table 3.5 Tooth mobility indexScore Description ( Miller 1950; Grant et al. 1988a)0 No detectable movement when force is applied1 Barely distinguishable movement when a force is applied.2 The crown of tooth moves 1mm in any direction3 Movements of more than 1mm in any direction 68
  • 88. 3.4.4 Furcation involvementFurcation involvement was measured using an ordinal scale (Table 3.6) measuringattachment loss based on horizontal probing characteristics (Hamp et al. 1975; Grant etal. 1988b).Table 3.6 Furcation indexClass 1 The degree of attachment loss involves only the furcation entrance by the periodontal probe or explorer.Class II The degree of attachment loss extends under the roof of the furcation but does not penetrate through and through.Class III The degree of penetration by the probe or explorer is through and through3.5 Details of the study3.5.1 Periodontal attachment loss (PAL)Sterile, non-pressure sensitive NIDR periodontal probes (American Dental, Chicago Il.)were used, to measure gingival recession and periodontal pocket depth at each site. TheNIDR periodontal probe is calibrated into 2mm bands, which can create examiner errorwhen truncating fractional band measurements. Fractional band measurements weretruncated to the lower whole millimetre, including to odd numbers. PAL was calculatedby adding the measurements for pocket depth and recession. Negative measurements ofrecession (like hypertrophy or pseudopockets) were subtracted from probing pocketdepths. Measurements were excluded from the study when the cemento-enamel junction (CEJ)could not be visualised or when pockets could not be probed (for example when largecalculus deposits were present). 69
  • 89. 3.5.2 Periodontal Pocket Depths (PPD)Periodontal pockets depths were measured from the gingival margin crest to the pocketbase. Measurements of gingival recession and periodontal pocket depth were made at sixsites per tooth.3.5.3 Gingival Recession (GR)Recession was measured from cemento-enamel junction to the gingival crest for all 6sites. If the CEJ was not evident these measurements were excluded.3.5.4 Examiner standardisation:Prior to starting the trial, the examiner carried out repeat examinations on six individuals(2-days apart) to establish intra-examiner error, to ensure that the recordings were madewith uniform interpretation and consistency (W.H.O. 1997).3.5.5 ProcedureThe examinations were carried out in the CACDRC in the Adelaide Dental Hospital.After the questionnaire had been completed, the subject was escorted to the dentalsurgery by the nursing staff, introduced to the examiner, and seated on a dental chair.The patient was given a brief explanation of the oral examination procedure with apictorial description of the probing procedure (Berns 1993). The dental chair wasreclined. A mouth mirror and probe were set out for every patient, oral examinationswere performed under overhead illumination. The dental assistant recorded all readingsdirectly onto a computerised dental chart. First, all teeth missing were charted, followedby plaque scores. The next procedure was to record the mSBI on all teeth. Then six sitesper tooth were measured for GR and PPD in the following order: distal-buccal site,direct-buccal site, mesial-buccal site, mesial-lingual site, direct lingual site and the distal- 70
  • 90. lingual site. These recordings were for all maxillary and mandibular teeth present. Thenext recordings were of teeth with supra and subgingival calculus. Recordings offurcation involvement and tooth mobility completed the examination.3.6 Statistical methodologyThe data were analysed using SPSS for Windows® 8.0.0 (SPSS Inc.), using the GeneralLinear Model General Factorial Procedure, incorporating Scheffes test and PearsonsChi-Square tests where appropriate. A statistical model was used to measure the effectsof aspirin and ex-smoking on PAL. The general linear model used was a two-wayANOVA including the interaction term, with age (in years) as a covariate included. If amain effect was found to be not significant, the model was further reduced by removal ofthat term/variable. Tables only present the reduced models. The level of significancewas calculated at alpha= 0.05. This model was applied to all measures of severity andextent of PAL and on two new measures of severity of PAL, the mean of the most severesite (MSS-PAL) per person and the extreme worst site (EWS-PAL) per person. 71
  • 91. Chapter 4 ResultsAll tables appear at the end of this chapter (pages 87 to107).4.1 Intra-examiner error.Repeat examinations were carried out in 6 males ≥50 years of age measuring PAL at 630sites (2 days between examinations) to establish intra-examiner error using the samerecruiting and examination procedures that were used in the subsequent study. Theaccuracy of reproducibility of measurements was analysed using kappa statistics. Table4.1 indicates that there was a very high agreement with a corresponding overall kappacoefficient of 0.94 ± 0.01(se) (values > 0.80 are classified as high (Hunt 1986)).No prior sample size or power calculation planning was done. However, one long-termstudy (Feldman et al.1983) investigated the effects of high dose aspirin on theperiodontium of 75 males. It was decided that an estimated sample size of 100 subjectsper group would probably be sufficient to investigate the independent variables of aspirinand past smoking on PAL. The study commenced in June 1998 and examinations werecompleted by early February 1999.4.2 Profile of study population:A total of 392 subjects participated in the study: they were classified into the followingfour groups according to aspirin and smoking history: Aspirin Never Smoked (ANS) Aspirin eX-Smokers (AXS) No Aspirin Never Smoked (NANS) No Aspirin eX-Smokers (NAXS) 72
  • 92. Table 4.2 shows the number and percentage distribution of subjects within each group.The non-aspirin groups had the highest number of subjects (239). The aspirin groups had153 subjects participating and of these, the ANS group had the lowest number of subjects(51). Demographics and oral health behaviours (dental hygiene and maintenance) wereused to sub-claasify the distribution of subjects within each group.4.3 Demographics4.3.1 Age categories of subjectsThe mean age for all subjects was 63.4 years ages with a range from 50-85 years(Table 4.3). In addition 55.9% (219) were under the age of 65 years and 44.1% (173)were ≥ 65 years. The ANS and AXS groups had the highest mean ages when comparedto NANS and NAXS groups (Table 4.3). The mean age of the ANS group was 68.2years, representing the group with the highest proportion of elderly subjects ≥ 65 years.The non-aspirin groups had more subjects ≤ 64 than ≥ 65 in approximately a 2:1 ratio.One way ANOVA indicated that there were significant differences between the fourgroups for mean age i.e. there was an uneven age distribution between groups. Multiplecomparisons for age differences between groups were analysed using a Scheffés testwhich found significant mean age differences between groups, the NANS being (6.1 ±1.06 years) younger than the AXS group (p=<0.001). Additionally, the NANS groupwas 8.3 ± 1.31 years younger than the ANS group (p <0.001). The mean differencebetween the ANS and the NAXS groups was 5.6 ± 1.32 years, with NAXS being theyounger group (p=0.001) (Table 4.4). A Scheffés analysis on the homogeneity betweentwo groups at a time found that there was no significant difference in mean age betweenthe groups of subjects taking aspirin (ANS ≡ AXS) (p=0.324), no significant difference 73
  • 93. between the non-smoking groups (NANS ≡ NAXS) (p=0.157) and no significantdifference between the ex-smoking groups (NAXS ≡ AXS) (p=0.052) (Table 4.5).As the study targeted males over 50, it was assumed that many participants would be ofor into retirement age. The minimum age qualifying for an aged pension for males inAustralia is 65 years. Pension status was taken as a gross indicator of income, aspensioners are generally of a lower income than non-pensioners. Table 4.6 presents thedistribution of all subjects based on pension status; of all subjects 58.9% werepensioners. The two aspirin groups had the highest number of pensioners (108)compared to non-pensioners (45). Table 4.7 shows denture use in pensioners and non-pensioners: 79.4% of non-pensioners did not wear a denture compared to 58.9% ofpensioners.4.3.2 Education status of the subjects.Of the 392 subjects, 80.9% had at least secondary or higher education. The highesteducated group (tertiary) was the NANS group (41.0%) with the NAXS group having thelowest percentage of tertiary educated subjects (20.5%) (Table 4.8). The ANS grouphad the highest percentage of subjects with the lowest education level (no schooling plusprimary levels) (23.5%).English language skill was a further measure of education. Table 4.9 shows that 94.6%of the study population stated that they spoke English well to very well. Data fromTables 4.8 and 4.9 show that a large proportion of subjects had high education levels andhad good English language skills. These attributes have been shown to help in thesocialisation of subjects and correlate positively with good oral health (Srikandi 1982). 74
  • 94. 4.3.2 Oral health behaviourTable 4.10 shows the distribution of all subjects based on demographics and dentalbehaviours. Pensioners tended to brush more frequently (> 1/day), than non-pensioners.The more highly educated subjects (93.7%) brushed more frequently than once per daycompared to lower educated subjects (6.7%). Subjects who had visited the dentist morefrequently tended to brush more than once per day (92.5%); while 57.9% of subjects whohad not visited the dentist in the last 5 years claimed that they brushed more than onceper day.A measure of the subjects oral health awareness was the time interval since their lastdental visit. Approximately 56.7% of the study population had visited the dentist in thelast 12 months and 33.6% between 1-5 years previously (Table 4.11). Only 9.8% had notseen a dentist for more than 5 years, indicating that this group was reasonably dentallyaware. The group with the lowest rate of dental attendance rate NAXS, with 31% havingnot attended for at least 3 years. The majority of the study population reported that theybrushed their teeth at least once per day, hardly ever used a mouth rinse, flossed less thanonce a week and most had a scale and clean within the last two years.Chi-Square analysis (Pearsons Chi-Square) between pensioners and non-pensionersfound no significant differences between groups in the distribution of tooth brushing,mouth rinses and flossing (p>0.05). There was a significant difference between asubject’s last scale and clean visit with pension status. Overall 60% of subjects had ascale and clean in the last two years, of these, significantly more pensioners (53.7%) hada scale and clean in the last 2 years compared to non-pensioners (45.0%) (p=0.043).There were no significant differences in mouth rinsing habits based on education, butthere were significant differences in tooth brushing (p<0.001), flossing (p=0.04) and last 75
  • 95. scale and clean visit (p<0.001). Subjects who had visited the dentist within the last 12months were significantly more likely to brush once or more per day (p<0.001), flossmore often (p<0.001) and have had a more recent scale and clean than subjects whovisited the dentist less frequently (p<0.001). There were no significant differencesbetween ≤ 64 and ≥ 65 year-olds for all the above parameters (Table 4.10).4.4 Tooth lossAll 392 persons examined were dentate. The association between age and tooth loss isshown in Table 4.12. The mean number of missing teeth for all subjects was 9.5 ± 0.31(se); subjects aged ≥65 had a significantly higher mean number of missing teeth (11.7 ±0.50 se) compared to subjects aged ≤ 64 (7.8 ± 0.37 se) p=0.001. Both ex-smokinggroups had more missing teeth (10.2 ± 0.43 se) than the non-smoking groups (8.6 ± 0.45se) (p=0.010) (Table 4.13). Aspirin had no significant association with the mean numberof missing teeth.4.5 The periodontal status of the study populationTable 4.14 is a descriptive table of the mean plaque scores for each group and by age.The overall mean plaque index as shown in Table 4.14 was 1.4± 0.69 (sd), with the ≤ 64year olds having a mean plaque score of 1.3 ± 0.70 (sd) and 1.6 ± 0.67 (sd) for ≥ 65 yearolds. The younger NANS group had the lowest plaque score (1.2 ± 0.61 sd) compared tothe older NAXS group (1.7 ± 0.69 sd).Table 4.15a shows the range and generic names of aspirin use in the current study. Thetwo primary compositions used were aspirin and acetyl salicylic acid. Thirty foursubjects used acetyl salicylic acid and 119 were on aspirin. Only one subject used thegeneric brand of Ecotrin, which is supplied as a 650mg tablet (this subject took a half a 76
  • 96. tablet per day). The majority of subject (95) were on 300mg of aspirin per day comparedto 57 subjects on 100mg of aspirin per day. The most popular brands were Solprin (63subjects) and Cartia (33 subjects). No information was gathered regarding whetherbrands of aspirin had been switched during the period of intake.The hypothesis that there was no difference in plaque scores between the different age,aspirin and smoking history groups was tested using ANOVA analysis. A two wayANOVA with mean age as the covariate showed there were no significant differences inplaque scores between aspirin takers and non-aspirin takers and so aspirin was removedfrom the model. A further one way ANOVA of smoking history with age as thecovariate showed a significant age association with mean plaque scores, with every one-year increase in age having a significant increase of 0.02 in the mean plaque score(p<0.001) (Table 4.15b). Ex-smoking subjects had significantly higher plaque scores byan average value of 0.14 ± 0.07 (se) than non-smoking subjects (p<0.043) (Table 4.15b).Figure 4.1 shows the distribution of gingival bleeding. Overall, 57.7% of teeth hadassociated gingival bleeding; the ANS subjects aged ≤64 had the lowest percentage ofsites with bleeding 49.7%. However two-way ANOVA analysis found no significantassociations for aspirin (p=0.310) or ex-smoking history (p=0.424) with gingivalbleeding between the four groups.The descriptive Table 4.16 presents the mean percentage of teeth with calculus amonggroups, aspirin takers and the ex-smokers. The inferential analysis (using two-wayANOVA) found no significant associations of aspirin and ex-smoking history with themean percentage of teeth with calculus, other than for age associations. An ordinaryleast square regression was conducted, showing that age was significantly correlated withcalculus, with older subjects having more teeth with calculus across all groups (p=0.042) 77
  • 97. (Table 4.17). Older subjects had significantly more teeth with calculus than youngersubjects but these correlations were minor (0.78% per year increase) (Table 4.17).Two-way ANOVA found no significant associations of aspirin with tooth mobility(p=0.139); no table is shown since there was no statistical association. A further analysis(one-way ANOVA) found that ex-smokers overall had a statistically significantly highermean percentage of teeth with mobility compared to non-smokers (indicating worseperiodontal status compared to non-smokers), independent of age associations (Table4.18).ANOVA analysis showed that there were no significant associations of age (p=0.168),aspirin (p=0.550) and smoking history (p=0.910) on furcation involvement betweengroups.4.6 Associations of aspirin and ex-smoking with various measures of PAL4.6.1 The associations of aspirin and ex-smoking with mean PALThe hypotheses that there were no differences in periodontal attachment loss betweenaspirin takers-non-aspirin takers and ex-smokers-non-smokers was evaluated using meanPAL and ANOVA analysis. The estimated means and test of significance were analysedusing two-way ANOVA evaluated at the mean age covariate (63.4 years). There weresignificant associations of low-dose aspirin and ex-smoking on mean PAL. Ex-smokershad significantly more PAL (2.9 ± 0.07mm) compared to non-smokers (2.6 ± 0.08mm),independent of aspirin history. In addition, low-dose aspirin takers had significantly lessPAL (2.6 ± 0.08mm) compared to non-aspirin takers (2.9 ± 0.06mm) and this associationwas independent of past smoking status (Table 4.19). These results reject the nullhypotheses that there were no statistical differences in PAL between the groups.Controlling for age, this model showed that ex-smoking had an association with mean 78
  • 98. PAL with a magnitude of 0.4mm. At a given point in time, ex-smokers had 0.4mm morePAL than non-smokers while aspirin takers had approximately 0.2mm less PAL thannon-aspirin takers (Table 4.19). The same analysis was performed on mean PAL bydosage and duration of low-dose aspirin; no differences were found (Table 4.20 and4.21).A further analysis of the association of dosage and duration of past smoking on meanPAL was carried out using ANOVA. Tables 4.22a & b show that there were significantassociations between mean PAL and the number of cigarettes smoked and past durationof smoking years. There was significantly more mean PAL in subjects who smokedmore than 10-30 cigarettes per day compared to those smoking <10 cigarettes per day(p=0.013). In subjects who smoked >30 cigarettes per day the association was highlysignificant (p<0.001) compared with subjects who smoked <10 cigarettes per day.Subjects who smoked 10-19/day had more mean PAL by 0.5mm than non-smokers whilethose who had smoked ≥20/day had 0.74mm more mean PAL than non-smokers.Subjects who had smoked ≥5 years had significantly more mean PAL (0.42 mm) thansubjects who had smoked for <5 years (0.35mm) (p=0.036). The majority of ex-smokers(196) had quit smoking for ≥ 5 years; 17 had quit smoking in the last 5 years. There wasno association between mean PAL and the time since quitting smoking (p=0.077) (Table4.22b).4.6.2 The associations of aspirin and ex-smoking on the extent and severity of PALThe general linear model of two-way ANOVA with one covariate was applied tomeasure the associations of low-dose aspirin and ex-smoking at various thresholds ofseverity (≥2 to ≥7mm) of PAL (Table 4.23). Aspirin and ex-smoking had significant 79
  • 99. associations at threshold levels, ≥2 to ≥4 mm mean PAL. Table 4.23 shows that asseverity increased, the number of subjects with severe PAL decreased. At ≥ 5 meanPAL, there was no significant association of aspirin with PAL (p>0.05) while there wasborderline significance (p=0.044) for ex-smoking history.The threshold of ≥5mm and ≥6mm PAL showed no statistically significant correlationbetween aspirin and mean PAL. The values for ≥6mm PAL were not included in thetable since no significant statistical associations were seen. The threshold ≥7mm meanPAL had no significant statistical associations but was included since this measure isindicative of severe attachment loss and is often used in studies of mean PAL. Only 191subjects had mean ≥7mm PAL, probably explaining the lack of significant associationsof aspirin and ex-smoking on severe PAL.The same analysis was performed for the extent of disease. Table 4.24 summarises theassociations of aspirin and ex-smoking using the statistical analysis of the estimatedmean percentages of PAL at ≥2, ≥4 and ≥7mm PAL and the corresponding p-values.Aspirin and ex smoking had significant statistical associations with the extent of PALfrom threshold levels ≥2 to ≥7mm mean percentage PAL. At the low mean percentageof ≥2mm PAL, there was an interaction association of aspirin on ex-smokers and non-smokers. Analysed separately, there was no significant aspirin association with PAL≥2mm in ex-smokers, however there was a significant aspirin association with PAL≥2mm in non-smokers.Table 4.25 summarises the magnitude of the significant statistical associations of aspirinand ex-smoking for severity and extent of PAL. The negative scores for aspirin reflectless PAL at various levels of PAL for severity and extent, independent of smoking 80
  • 100. history. Ex-smokers had a greater magnitude of PAL loss for the same PAL levels thannon-smokers. For example, ex-smokers at ≥ 4mm PAL had more overall mean loss ofPAL (0.2mm) and an extra mean 9.1% of sites with ≥ 4 mm PAL than non-smokers.Aspirin takers overall had less mean loss of PAL (0.1 mm) and less sites (5.1%) at mean≥ 4mm PAL than subjects who had not taken aspirin.Further to this, the mean number of teeth present per subject was 22.5 with a mean totalof 135 sites per person (22.5 x 6 sites). At mean PAL ≥4mm, each ex-smoker had amean of 45 sites (33.3%) with a mean PAL of 4.7mm in severity (Tables 4.23). Aspirintakers had an average of 35 sites (26.2%) of mean PAL ≥ 4mm (Table 4.24), a differenceof 10 sites compared to ex-smokers. Since 6 sites/tooth were measured, this is equivalentto aspirin takers having approximately 2 fewer teeth with ≥4mm PAL than ex-smokers.That is, aspirin takers had significantly less attachment loss compared to non-aspirintakers. Using the same formulation non-aspirin takers had 39 sites involved, orapproximately 1 tooth less with ≥4mm PAL compared to ex-smokers.4.6.3 Associations of aspirin and ex-smoking with the most severe site of PAL (MSS-PAL)In a separate analysis of the data, the most severe site of PAL (MSS-PAL) for each toothwas measured and then averaged per subject. This was used as another observation ofthe severity of PAL (Figure 4.3). These results highlighted a reduced MSS-PALobserved in the AXS subjects compared with NAXS subjects in the range 4.5-6.5mmMSS-PAL. In addition, Figure 4.3 shows that ex-smokers had more MSS-PAL than non-smokers. Figure 4.3 also shows that the ANS and NANS groups had a similar pattern ofMSS-PAL distribution indicating that there were no differences between both groups at 81
  • 101. various levels of MSS-PAL, with cross-over occurring at 4mm MSS-PAL and at 5.5mmMSS-PAL.A two-way ANOVA analysis of MSS-PAL using age as a covariate found significantstatistical associations of aspirin and smoking histories (Table 4.26). Subjects on low-dose aspirin had significantly less MSS-PAL (0.33mm) (p=0.013) than non-aspirin takersand ex-smokers had significantly more MSS-PAL (0.44mm) (p<0.001). Figure 4.4represents the magnitude and direction of PAL using mean PAL and MSS-PAL.4.6.4 Associations of aspirin and ex-smoking with the extreme worst site of PAL (EWS-PAL)The distribution of subjects by numbers and age between each group was uneven (Table4.3). The ANS group had 51 subjects; who were older than subjects in the other threegroups. In order to have a better estimate of prevalence of severe periodontal disease andto enable extrapolation to the population at large, the data were weighted. The age classinterval distribution of each group was matched to the age class intervals of Australianmales aged 50 years or more as estimated by the Australian Bureau of Statistics (Census1996) in the 50-59, 60-69, 70-85 age groups. These weighted age class intervals of thestudy population were analysed separately for PAL. The data were analysed on theextreme worst site of PAL (EWS-PAL) per subject.Table 4.27 shows the age class intervals and corresponding percentage distributions ofmales in the general population of Adelaide. These data were used to calculate thecorresponding sample class intervals, weighted for all groups (Table 4.28). Lownumbered age class intervals had heavier weighting; for example in the age class intervalof 50-59 in the ANS group (7 subjects) each had a weight of approximately 2.91compared to the same age group of NANS subjects (63 subjects) who each had a weight 82
  • 102. of 0.77. That is, each subject aged 50-59 years in the ANS group counted as nearlythree. The 70+ age ANS group had a weight factor of 0.70 counting as ¾ of anindividual; the class interval of 60-69 had the least weight fluctuation for all four groups.Table 4.29 presents descriptive data for EWS-PAL. Weighted data was used in a two-way ANOVA with age as the covariate on EWS-PAL. This showed that there was asignificant protective association of low-dose aspirin with EWS-PAL in ex-smokers andnon-smokers (Table 4.30). Figure 4.5 shows the cumulative percentages of EWS-PAL ofall groups. The differences between the ANS and NANS groups were exaggerated whenthe data were weighted (compared with Figure 4.3 MSS-PAL).Figure 4.5 shows that the percentage of subjects with severe attachment loss decreased asPAL increased, and that the association of aspirin with EWS-PAL was positive insubjects who had smoked (AXS) when compared to NAXS subjects, but not for lowMSS-PAL (≤6mm). In addition, both non-smoking groups had less severe EWS-PAL.All subjects had at least one worst site of 4mm EWS-PAL. The NAXS group had 58.2%of subjects with a maximum 7mm EWS-PAL, while AXS had 49.5% of subjects with aworst site of 7mm EWS-PAL. The major differences were seen in the range of 7-11mmPAL. As a whole group, 48.7% subjects had at least one site with 7mm EWS-PALcompared to 6.1% of subjects with a worst score of 11mm EWS-PAL.Ex-smokers had an EWS-PAL averaging 0.75mm more compared to non-smokers whileaspirin takers had 0.54mm lower mean EWS-PAL compared to non-aspirin takers (Table4.31). This could be interpreted as indicating that aspirin takers were approximately 0.21in magnitude better off in EWS-PAL than non-aspirin takers (Table 4.30).The ratio of beneficial associations of aspirin to the detrimental associations of ex-smoking were similar in trend for all measures of severity of PAL, ranging from 59% to 83
  • 103. 75% (Table 4.31). That is, aspirin-takers had a lower PAL compared to ex-smokers by amagnitude of approximately half to three-quarters, depending on the severity index used.4.7 The associations of various clinical parameters on mean PAL.One-way ANOVA with one covariate was used to assess the associations of plaque,calculus, gingival bleeding, mobility and furcation with PAL. Mean PAL (6 sites/tooth/mouth) was used as the dependent variable. The ordinal scales of the plaque score weredichotomised as being present (1, 2 or 3 score) or not present (0 score). There was anassociation between age, plaque and mean PAL for all four groups. The overall meanPAL was 2.8 ± 1.00 (sd) mm. The higher the mean plaque scores the higher the meanPAL (0.48mm), with a 0.01 increase in mean PAL due to age, indicating that oldersubjects and those with plaque present had higher mean PAL (Table 4.32).A one-way ANOVA using the dichotomous scale of calculus showed there was anassociation between calculus with mean PAL for all four groups. The calculuscorrelation with mean PAL was significant but very minor (approximately 0.004),indicating that subjects with calculus had slightly higher mean PAL (Table 4.33).Gingival bleeding (using mBSI) and mobility were assessed as either being present (1, 2& 3) or absent (0). Figure 4.1 shows that the mean percentage of bleeding sites pergroup varied from 56.5% for the ≤65 and 59.3% for >65 year old subjects. The ANS had54.2% of sites with bleeding, the AXS had 57.4%, the NANS had 57.3% and NAXS had60.0% of sites with gingival bleeding. In all four groups, mean PAL significantlyincreased with increasing age and with the increasing percentage of teeth with gingivalbleeding (p < .001). For every one-year increase in age, there was an increase in meanPAL by 0.02 mm. For every percent increase in the percentage of teeth with gingival 84
  • 104. bleeding, there was an extra 0.01mm increase in mean PAL (Table 4.34). The sametrends were seen with furcation involvement and mobility.4.7.1 Site and tooth variations in recession and pocket depth by mean PALThe components of mean PAL (gingival recession and pocket depth) were investigatedfor tooth and jaw variations. Figure 4.6 represents the mean (mm) of gingival recessionand periodontal probing pocket depths for all groups using the mean of 6 sites per tooth.The vertical dimensions of each bar represent the mean PAL per tooth. Maxillary teethare represented with the positive PAL scores; the negative PAL scores represent themandibular teeth. Each tooth type the mean represents the overall mean PAL for thattooth type for both left and right sides of each jaw type. Figures 4.7-4.10 represent theintra-group jaw and tooth variation in mean PAL.Different patterns of PAL were seen within and between groups for tooth and jaw type.In the maxilla, most PAL was seen in 1st, 2nd and 3rd molars with maxillary 1st molarshaving the largest amount of mean PAL (3.2mm) with intra-group variation ranging from3mm for ANS and NANS to 3.4 mm for NAXS and 3.5 mm AXS. The pattern ofattachment loss was different in the lower arch, with the largest mean PAL occurringaround the mandibular incisors. The pattern of mean PAL varied between groups,ranging from a mean PAL of 2.4 mm for AXS to a mean PAL of 3.4 mm for NAXS.The main contributor to mean PAL in maxillary molars was pocket depth (average of2.6mm for pocket depth and 0.5mm recession). Recession contributed to the largercomponent of attachment loss in the mandibular incisors as compared to the maxillarymolars (an average of 2.0mm per pocket depth and 1.1 mm recession).In the AXS group, the principal component of PAL was pocket depth (Figure 4.7).Recession mainly occurred around maxillary and mandibular molars and lower anterior 85
  • 105. teeth. The NAXS group showed a similar pattern as in the AXS group but had a largercomponent of PAL in the lower anteriors (Figure 4.8). The ANS group had a patternsimilar to both smoking groups but the pocket depth component of PAL was less in thisgroup (Figure 4.9). The NANS group had a similar pattern to the ANS group (Figure4.10).4.7.2 Socio-economic factors and periodontal attachmentTable 4.35 summarises the demographic and socio-economic associations with meanPAL. All analyses were performed under the same statistical model (two-way ANOVAwith age as a covariate on the dependant variable of mean PAL). Education hadsignificant statistical associations (p=0.008) on the mean PAL between groups. Thelower educated subjects had more periodontal attachment loss. A further analysis usingthe ANOVA model with education as the covariate found no associations with meanPAL (data not presented).Controlling for age and using the ANOVA model, there were no statistically significantassociations with mean PAL in pensioners and non-pensioners. There was a statisticallysignificant association between mean PAL and the time interval of the last dental visit.There were no statistically significant associations between mean PAL and subjects thatbrushed > 1/day and subjects that brushed < 1/day. There were no statisticallysignificant associations between mean PAL and mouth rinsing. There was a statisticallysignificant association between mean PAL and subjects who practiced interproximalcleaning; subjects who flossed once or more per week had statistically significantly lessmean PAL compared to those who flossed less than once per week or never flossed.Education level and floss use were the two independent factors that were mostsignificantly correlated with PAL. 86
  • 106. Table 4.1 Intra examiner reliability test using kappa statistics Number of sites Kappa value Std errorRecession 630 0.922 0.02Pocket depths 630 0.914 0.02(combined pocket and recession 1260 0.941 0.01measurements)Table 4.2 The number and percentage distribution of subjects participating in the study by group Frequency PercentAspirin never smoked 51 13.0Aspirin ex-smoker 102 26.0Aspirin takers total. 153 39.0No aspirin never smoked 122 31.1No aspirin ex-smoker 117 29.8Non-aspirin takers total 239 60.9Overall Total 392 100Table 4.3 Distribution of age by group Age distribution Numbers Std Age range of males in group Deviation ≤ 64 yrs ≥65 yrs Mean Minimum MaximumANS 19 32 51 68.2* 53 85 7.71NANS 85 37 122 59.9* 50 84 7.67AXS 45 57 102 66.0* 50 85 7.93NAXS 70 47 117 62.6* 50 83 8.12Total 219 173 392 63.4 50 85 8.39One-way ANOVA. SSQ 3427.55, df =3, F =18.40,(*) Significant mean age differences between groups, p < 0.001 87
  • 107. Table 4.4 A Scheffés analysis of homogeneity between two groups at a time for mean age differences(I) (J) Difference (I-J) in Std. Error Sig means (years) (p-value)ANS ANS AXS 2.2 1.35 0.435 NANS 8.3 1.31 < 0.001 NAXS 5.6 1.32 0.001 ANS -2.2 1.35 0.435 AXSAXS NANS 6.1 1.06 < 0.001 NAXS 3.3 1.07 0.021NANS ANS -8.3 1.31 < 0.001 AXS -6.1 1.06 < 0.001 NANS NAXS -2.7 1.02 0.067NAXS ANS -5.6 1.32 0.001 AXS -3.3 1.07 0.021 NANS 2.7 1.02 0.067 NAXSTable 4.5 Scheffésa, analysis for homogeneity between subsets Subsets for alpha = 0.05Group N 1 2 3NANS 122 59.8NAXS 117 62.6 62.6AXS 102 66.0 66.0ANS 51 68.2Significance value 0.157 0.052 0.324Means for all groups in homogeneous subsets are displayed.(a) Uses Harmonic Mean Samples Size = 86.7. 88
  • 108. Table 4.6 Demographics on pension status with group specific characteristics. GROUP Pension or Health Card? ANS AXS NANS NAXS TOTAL Yes N 37 71 61 62 231 % 72.0% 69.6% 50.4% 53.00% 58.9% No N 14 31 61 55 161 % 28.0% 30.40% 49.6% 47.0% 41.1% Total N 51 102 122 117 392 % 100.0% 100.0% 100.0% 100.0% 100.0%N = Number of subjectsTable 4.7 Pension status in relation to denture use. Pension or health card? yes no Total Have a denture yes 41.1% 20.6% 33.2% no 58.9% 79.4% 66.8% Total 100% 100.0% 100.0%Table 4.8 Demographic data on schooling of all subjects with group specific characteristics.Education ANS AXS NANS NAXS TOTAL N % N % N % N % N %No schooling 1 3.9 1 1.0 2 0.8 6 1.7 10 1.5Primary 10 19.6 18 17.6 19 15.6 22 18.8 69 17.6SchoolSecondary 21 41.2 56 54.9 52 42.6 69 59.0 198 50.5SchoolTertiary 18 35.3 27 26.5 50 41.0 24 20.5 119 30.4SchoolTotal 51 100.0 102 100.0 122 100.0 117 100.0 392 100.0N = number of subjects per group% = percentage of subjects per group 89
  • 109. Table 4.9 A self-evaluation of English language skill.How well do you GROUP ANS AXS NANS NAXSspeak English? TOTAL N % N % N % N % N %Very well 37 74.0 83 81.4 84 68.3 76 65.5 281 71.6Well 10 20.0 14 13.7 35 28.5 31 26.7 90 23.0Not well 2 4.0 5 4.9 3 2.5 9 7.8 19 5.10Not at all 1 2.0 1 0.3Total 50 100.0 102 100.0 122 100.0 116 100.0 391 100.0N = number of subjects per group% = percentage of subjects per groupTable 4.10 Socio-economic factors and dental behaviours. Tooth Tooth Use a Hardly Floss Floss Scale Scale brush brush mouth ever more less and and >1/day < 1/day rinse use a than 1/ than 1/ clean ≤ clean ≥ mouth week week 2 years 3 years rinsePension statusPension 201 28 55 229 68 161 123 106non-pension 146 14 31 129 59 102 103 58Education level< primary school 54 21 17 58 14 61 25 50> secondary school 293 21 69 245 113 202 201 114How long since lastdental visit< 1 yr 211 9 56 164 92 129 180 411yr-5yr 114 17 23 108 32 99 45 86> 5 years 22 16 7 38 3 35 1 37Age≤ 64 189 29 52 166 75 144 130 89≥ 65 158 13 34 137 52 119 96 75GroupsANS 44 5 10 39 14 35 23 26AXS 92 10 23 79 37 65 64 38NANS 112 9 25 96 47 75 77 45NAXS 99 18 28 89 29 88 62 55 90
  • 110. Table 4.11 Population and percentage distribution of subjects since their last dental visit. The time range was from less than one year to never visiting the dentist. How long since your last ANS AXS NANS NAXS Total dental visit? Number of 11 42 41 46 140 subjects < 6 months % within 22.4% 41.2% 33.6% 39.3% 35.9% group Number of 14 18 34 15 81 6-11 subjects months % within 28.6% 17.6% 27.9% 12.8% 20.8% group Number of 11 19 23 20 73 subjects 1-2 years % within 22.4% 18.6% 18.9% 17.1% 18.7% group Number of 6 16 13 23 58 subjects 3-5 years % within 12.2% 15.7% 10.7% 19.7% 14.9% group Number of 3 4 6 4 17 subjects 6-10 years % within 6.1% 3.9% 4.9% 3.4% 4.4% group Number of 3 3 5 7 18 subjects > 10 years % within 6.1% 2.9% 4.1% 6.0% 4.6% group Number of 1 2 3 subjects Never % within 2.0% 0.0% 0.0% 1.7% 0.8% group Number of 49 102 122 117 390 subjects Total % within 100.0% 100.0% 100.0% 100.0% 100.0% group 91
  • 111. Table 4.12 Missing teeth by age and group.Age ≤ 64 yrs ≥ 65 yrs Group Total Std Std Std Mean N Mean N Mean N error error errorANS 6.7 0.91 19 11.3 1.10 32 9.6 0.82 51NANS 7.0 0.56 85 11.1 1.11 37 8.2 0.54 122AXS 8.0 0.79 45 12.5 0.87 57 10.5 0.63 102NAXS 9.0 0.71 70 11.5 0.98 47 10.0 0.59 117Group 7.8* 0.37 219 11.7* 0.50 173 9.5 0.31 392Total* significantly different (2-tailed t-test) p < 0.001N = The number of subjectsComputed at alpha = 0.05 levelTable 4.13 Missing teeth and smoking history Mean number Number of Std error of of missing t-test subjects mean teethEx-smokers 219 10.2 0.43(AXS and NAXS) p = 0.01Non-smokers 173 8.6 0.45(ANS and NANSTable 4.14 The mean plaque index per group. ≤ 64 yrs ≥ 65 yrs Group Total mean SD N Mean SD N mean SD NANS 1.3 0.52 19 1.5 0.81 32 1.4 0.72 51NANS 1.2 0.61 85 1.5 0.56 37 1.3 0.61 122AXS 1.4 0.80 45 1.5 0.62 57 1.4 0.70 102NAXS 1.5 0.76 70 1.7 0.69 47 1.6 0.74 117Group Total 1.3 0.70 219 1.6 0.67 173 1.4 0.69 392SD = Standard deviationN = Number of subjects 92
  • 112. Table 4.15a Profile of aspirin use and subject numbers Brand Name Composition Concentration Number of subjects SOLPRIN Aspirin 300 mg 63 CARTIA Acetyl Salicylic acid 100 mg 33 ASTRIX Aspirin 100 mg 18 SPREN Aspirin 300 mg 16 ASPIRIN Aspirin 300 mg 10 CARDIPRIN Aspirin 100 mg 6 DISPRIN Aspirin 300 mg 4 ASPRO Aspirin 300 mg 2 ECOTRIN Acetyl Salicylic acid 650 mg 1 (1/2 tab/day) Total 153 Table 4.15b The association of age and past smoking on mean plaque scores with tests of significance. ANOVA Outcome 95% confidence a (one-way) of on interval Sum of F Std mean plaque df Sig means Squares statistic error Lower Upper scores plaque limit limit score. Intercept 1.28 1 10.09 0.096 0.37 0.26 -0.15 0.88 Age association 6.50 1 2.79 < 0.001 0.02* 0.004 0.007 0.02 Ex-smoking 1.90 1 14.13 0.043 0.14* 0.07 0.005 0.28 association Error 178.90 389 Total 985.15 392a = magnitude of association on mean plaque scores. Estimated mean plaque scores from ANOVA Ex-smoking history Total (score ± se) Yes No Total 1.5 ± 0.05 1.3 ± 0.05 1.4 ± 0.03* Number of subjects is calculated from descriptive Table 4.14. Estimated means from model evaluated at the mean age covariate. Age: p<0.001 Ex-smoking: p =0.043 (*) Age and ex-smoking had significant differences with mean plaque scores 93
  • 113. Table 4.16 Distribution of mean percentage of teeth with calculus by age, aspirin and ex-smoking. Mean % of teeth with calculus ≤ 64 years ≥ 65 years Group total (% ± sd) 26.0 ± 38.01 64.2 ± 64.91 50.0 ± 59.0 ANS (19) (32) (51) 49.3 ± 59.76 55.0 ± 58.5 52.5 ± 58.85 AXS (45) (57) (102) 44.5 ± 54.24 63.7 ± 64.05 50.3 ± 57.80 NANS (85) (37) (122) 54.4 ± 76.90 64.9 ± 74.75 58.6 ± 75.89 NAXS (70) (47) (117) 47.0 ± 62.49 61.2 ± 65.17 53.3 ± 64.00 Group total (219) (173) (392) Ex-smokers yes no Total 51.6 ± 52.5 ± 58.85 50.0 ± 59.00 yes 58.71 (102) (51) (153) 54.4 ± 58.6 ± 75.89 50.3 ± 57.80Aspirin takers no 67.26 (117) (122) (239) 53.3 ± 55.7 ± 68.40 50.2 ± 57.99 Total 64.00 (219) (173) (392) 94
  • 114. Table 4.17 The association of age with mean percentage of calculus between groups Ordinary least 95% squares confidence F Outcome regression of Sum of a Std interval df statisti Sig on mean % of teeth Squares error c estimates Lower Upper with calculus limit limit (% ± se) Intercept 89.27 1 0.022 0.882 3.64 24.56 -44.64 51.92 Age association 16902.92 1 4.16 0.042 0.78 0.38 0.028 1.54 Error 1584392.60 390 Total 2714822.42 392Computed using level of significance at alpha = 0.05.a = magnitude of association on mean plaque scores.Number of subjects as in Table 4.16.Age: p=0.042Age was significantly associated with mean percentage of calculus.Table 4.18 The association of low-dose aspirin and ex-smoking with the mean percentages of mobile teeth. Mean % of Std. mobile N p- value deviation teeth Aspirin takers 3.6 10.52 153 p > 0.05 F = 2.20 Non-aspirin takers 4.2 13.15 239 df = 1 Ex-smokers 5.5 14.89 219 p = 0.004 F= 8.30 Non-smokers 2.0 7.00 173 df = 1 ANOVA (2-way) using mean age covariate. Computed at alpha = 0.05. Number of subjects as in Table 4.16 95
  • 115. Table 4.19 The correlation of low-dose aspirin and past smoking with mean PAL. 95% 2-way Outcomea confidence ANOVA of Sum of F Std df Sig on mean interval mean PAL Squares statistic error PAL (mm) Lower Upper (mm ± se) limit limitIntercept 8.8 1 9.39 0.002 1.10 0.380.39 1.9Age 14.47 1 15.44 < 0.001 0.02* 0.01 0.01 0.04associationsEx-smoking 12.87 1 13.73 < 0.001 0.40* 0.1 0.17 0.57associationsAspirin 3.94 1 4.20 0.041 -0.20* 0.11 -0.43 -0.09associationsError 363.67 388Total 3464.59 392Computed using level of significance at alpha = 0.05.a = magnitude of association on mean PAL from age, ex-smoking & low-doseaspirin.(-) Magnitude of reduced mean PAL Estimated mean PAL Ex-smokers (mm) from ANOVA Yes No Total Yes 2.8 ± 0.09 2.5 ± 0.01 2.6 ± 0.08 (102) (51) (153) Aspirin No 3.1 ± 0.08 2.7 ± 0.08 2.9 ± 0.06 takers (117) (122) (239) Total 2.9 ± 0.07 2.6 ± 0.08 2.8 ± 0.05 (219) (173) (392) Estimated means from model evaluated at the mean age covariate. ( ) number of subjects Mean PAL (mm ± se) The hypotheses 1 and 2 were rejected for all independent variables. There were significant associated differences (*) for age, ex-smoking and aspirin with mean PAL. Age: p<0.001 Ex-smoking p<0.001 Aspirin: p=0.041 96
  • 116. Table 4.20 The association of aspirin dosage with mean PAL. Mean PAL Std error Number of p-value (mm) subjectsDosage≤ 150 mg 2.8 0.08 131* 0.184≥ 300 mg 3.1 0.16 19*No subjects took aspirin between 151-299mg.The maximum dosage was 325mg by one subject.* Three subjects did not enter their dosage on questionnaire.Table 4.21 The association of aspirin duration with mean PAL.Duration Mean PAL Std error Number of p-value (mm) subjects2-5 years 2.7 0.10 816-10 years 2.8 0.11 53 0.817≥ 11 years 2.9 0.31 19Table 4.22a The association of past smoking dosage and duration with mean PAL. Mean PAL Number of Std error p-value (mm) subjects Time sincequit smokingQuit < 5 yrs 3.4 0.24 17 0.077*Quit ≥ 5yrs 2.9 0.08 196Number ofcigarettes / day< 10 / day 3.1 0.16 40 0.164*10 - 19 / day 2.9 0.12 79 0.013*20 - 30 / day 2.7 0.14 58 0.013* > 30 / day 3.4 0.16 42 0.001* Years was smoker < 5 yrs 2.7 0.30 13 0.257* ≥5 yrs 2.7 0.20 36 0.036*(*) Compared to non-smokers 97
  • 117. Table 4.22b The correlation of the number of cigarettes smoked and duration of smoking with mean PAL with t-test of significance. B 95% confidenceNumber of Outcomea Std interval t statistic Sigcigarettes smoked on mean error Lower Upper PAL (mm) limit limitIntercept 3.40 0.16 21.05 < 0.001 3.08 3.72Association of -0.32 0.23 -1.40 0.164 -0.78 0.13smoking<10 cigarettes/dayAssociation of -0.50 0.20 -2.50 0.013 -0.90 -0.11smoking10-19 cigarettes/dayAssociation of -0.74 0.21 -3.50 0.001 -1.16 -0.33smoking > 20 cigarettes/dayDuration ofsmokingIntercept 3.07 0.08 36.58 < 0.001 2.90 3.23Association of -0.35 0.031 -1.14 0.257 -0.96 0.26smoking < 5 yearsAssociation of -0.42 0.20 -2.106 0.036 -0.81 -0.027smoking ≥ 5 yearsa = magnitude of association on mean PAL by years of smoking.(-) Magnitude of lost mean PAL 98
  • 118. Table 4.23 Univariate analysis of variance in mean PAL at ≥2, ≥4 ≥ 5 and ≥7mm. SEVERITY Smoking history Mean PAL Aspirin p- (mm ± se) yes no Total value 3.0 ± 0.71 2.7 ± 0.08 2.9 ± 0.07 yes (102) (51) (153) Aspirin 3.2 ± 0.07 2.9 ± 0.07 3.1 ± 0.05 no 0.019 ≥ 2mm takers (117) (122) (239) 3.1 ± 0.05 2.8 ± 0.06 3.0 ± 0.04 Total (219) (173) (392) p-value of smoking 0.001 4.6 ± 0.05 4.4 ± 0.06 4.5 ± 0.04 yes (102) (51) (153) Aspirin 4.8 ± 0.04 4.5 ± 0.04 4.7 ± 0.34 no 0.027 ≥ 4mm takers (117) (122) (239) 4.7 ± 0.04 4.5 ± 0.04 4.6 ± 0.03 Total (219) (173) (392) p-value of smoking < 0.001 5.7 ± 0.04 5.6 ± 0.05 5.6 ± 0.03 yes (99) (46) (145) Aspirin 5.7 ± 0.04 5.6 ± 0.05 5.6 ± 0.03 no 0.972≥ 5 mm takers (111) (106) (217) 5.7 ± 0.04 5.6 ± 0.05 5.6 ± 0.3 Total (210) (152) (362) p-value of smoking 0.044 7.8 ± 0.08 7.8 ± 0.11 7.8 ± 0.08 yes (53) (24) (77) Aspirin 7.6 ± 0.08 7.6 ± 0.09 7.6 ± 0.07 no 0.053≥ 7mm takers (68) (46) (114) 7.7 ± 0.06 7.7 ± 0.08 7.7 ± 0.05 Total (121) (70) (191) p-value of smoking 0.916ANOVA (2-way).Estimated means from model evaluated at the mean age covariate.( ) number of subjects 99
  • 119. Table 4.24 Univariate analysis of variance on mean % PAL at ≥2, ≥4, ≥ 5 & ≥7mm. Extent Ex-smokers2-way ANOVA of mean % PAL yes no p-value of(% ± se) aspirin 95.9 ± 1.00 yes N/A (102) Aspirin 94.1 ± 0.92≥ 2mma no N/A 0.196 takers (117) 95.0 ± 0.67 Total N/A (219) 89.4 ± 0.42 yes N/A (51) Aspirin 95.3 ± 1.07≥ 2mmb no N/A 0.006 takers (122) 92.4 ± 0.97 Total N/A (173) p-value of Ex-smokers aspirin yes no Total 30.7 ± 1.98 21.7 ± 2.34 26.2 ± 1.85 yes (102) (51) (153) Aspirin 35.8 ± 1.83 26.7 ± 1.82 31.3 ± 1.43 no ≥ 4mm takers (117) (122) (239) 0.035 33.3 ± 1.48 24.2 ± 1.72 28.7 ± 1.14 Total (219) (173) (392) p-value of ex-smoking < 0.001 13.1 ± 1.55 6.4 ± 1.84 9.7 ± 1.45 yes (102) (51) (153) Aspirin 18.7 ± 1.44 12.0 ± 1.43 15.4 ± 1.13 no ≥ 5 mm takers (117) (122) (239) 0.003 15.9 ± 1.16 9.2 ± 1.35 12.6 ± 0.90 Total (219) (173) (392) p-value of ex-smoking < 0.001 2.9 ± 0.55 0.8 ± 0.69 1.87 ± 0.55 yes (102) (51) (153) Aspirin 4.4 ± 0.54 2.2 ± 0.54 3.3 ± 0.43 no ≥ 7mm takers (117) (122) (239) 0.043 3.7± 0.44 1.5 ± 0.51 2.6 ± 0.34 Total (219) (173) (392) p-value of ex-smoking < 0.001( ) = number of subjects.Estimated means from model evaluated at the mean value of the age covariate.(a) Associations of aspirin on subjects who are ex-smokers(b) Associations of aspirin on subjects who have never smokedN/A not applicable due to interaction associations. 100
  • 120. Table 4.25 The magnitude of the association of aspirin and smoking history with severity and extent of PAL at ≥2, ≥4, ≥ 5 & ≥7 mm PAL using the general linear model (2-way ANOVA) of analysis.Severity Overall Mean PAL Aspirin association Ex-smoking (c) (mm) on association on Mean PAL (mm) Mean PAL (mm)Mean PAL at sites 3.0 -0.2 0.3with PAL ≥ 2mmMean PAL at sites 4.6 -0.1 0.2with PAL ≥ 4mmMean PAL at sites 5.6 N/S 0.1with PAL ≥ 5mmMean PAL at sites 7.7 N/S N/Swith PAL ≥ 7mmExtent Overall Mean % Aspirin Ex-smoking PAL Association association (mean %) (mean %)Mean % of all sites 92.4 -5.9 I/Ewith PAL ≥ 2mm (a)Mean % of all sites 95.0 N/S I/Ewith PAL ≥ 2mm (b)Mean % of all sites 28.7 -5.1 9.1with PAL ≥ 4mmMean % of all sites 12.6 -5.6 6.7with PAL ≥ 5mmMean % of all sites 2.6 -1.5 2.2with PAL ≥ 7mm(a) Non-smokers only (b) Ex-smokers only. (c) From Table 4.23.N/S Not significant. N/A not applicable. I/E interaction associationSee Tables 4.19, 4.23 & 4.24 for p-values. 101
  • 121. Table 4.26 The correlation of aspirin and past smoking history with MSS-PAL. B 95%2-way ANOVA F confidence Sum of Std of mean MSS- df statis Sig Mean interval Squares errorPAL (mm ± se) tic outcomea Lower Upper (mm) limit limit 17.2 Intercept 24.61 1 < 0.001 1.98 0.47 1.05 2.91 9 17.1Age association 24.45 1 < 0.001 0.03* 0.01 0.02 0.05 7 Ex-smoking 12.8 18.26 1 < 0.001 0.44* 0.12 0.20 0.69 association 3 Aspirin 8.81 1 6.19 0.013 -0.33* 0.13 -0.59 -0.07 association Error 552.43 388 Total 7171.03 392Computed using level of significance at alpha = 0.05.a = magnitude of association on mean PAL from age, ex-smoking & low-doseaspirin.(-) magnitude of reduced MSS-PAL Estimated mean MSS-PAL (mm) Ex-smokers from ANOVA (mm± se) Yes No Total Yes 4.1 ± 0.11 3.7 ± 0.13 3.9 ± 0.10 (102) (51) (153) Aspirin takers No 4.4 ± 0.10 4.0 ± 0.10 4.2 ± 0.08 (117) (122) (239) Total 4.3 ± 0.08 3.8 ± 0.09 4.0 ± 0.06 (219) (173) (392) Computed using level of significance at alpha = 0.05. Estimated means from model evaluated at the mean age covariate. ( ) number of subjectsThe hypotheses 1 and 2 were rejected for all independent variables. Age, ex-smokingand aspirin were significantly associated (*) with MSS-PAL. 102
  • 122. Table 4.27 The age class distribution of males 50+ years in metropolitan Adelaide in 1996 from census statistics and their appropriate frequency distribution. Australian Bureau Number of males in Percentage Relative of Statistics age metropolitan Adelaide frequency frequency class interval [Census, 1996 #290] 50-59 51,418 39.87% 0.40 60-69 39,741 30.83% 0.31 70-85 37,749 29.84% 0.30 Total 128,908 100% 1.00Table 4.28 The proportional weights given to each group using the percentage frequency of each class interval from census statistics for metropolitan Adelaide Age interval 50-59 years 60-69 years 70+ years Group TotalRelative class interval weight .40 .31 .30 1 51 x 0.40 / 7* 51 x 0.31 / 51 x 0.30 / ANS N=51 = 2.91 22* = 0.72 22* = 0.70 102 x 0.40 / 102 x 0.31 / 102 x 0.30 / AXS N=102 23* = 1.77 44* = 0.72 35* = 0.87 122 x 0.40 / 122 x 0.31 / 122 x 0.30 / NANS N=122 63* = 0.77 48* = 0.79 11* = 3.33 117 x 0.40 / 117 x 0.31 / 117 x 0.30 / NAXS N=117 46* = 1.01 47* = 0.77 24* = 1.46 Group Total 139 161 92 N=392N = The number of subjects per group* = The number of subjects in each class intervalTable 4.29 Descriptive statistics of EWS-PAL Means have weighted age Number of EWS-PAL Standard class intervals values subjects (mm) deviation AXS 102 7.00 1.80 ANS 51 6.30 1.91 Total of aspirin takers 153 6.74 1.86 NAXS 117 7.50 2.22 NANS 122 6.70 2.24 Total of non-aspirin takers 239 7.10 2.26 Total of ex-smokers 219 7.20 2.05 Total of non-smokers 173 6.60 2.16 Group Total 392 7.00 2.12 103
  • 123. Table 4.30 The association of aspirin and past smoking history with EWS-PAL using weighted data. 95% 2-way Mean confidence ANOVA Sum of F Std df Sig outcomea interval EWS-PAL Squares statistic error (mm) Lower Upper (mm ± se) limit limit Intercept 125.21 1 29.75 <0.001 4.02 0.76 2.52 5.51 Age 56.82 1 13.50 <0.001 0.04 0.01 0.02 0.07 associations Ex-smoking 26.31 1 6.25 <0.001 0.75 0.22 -0.96 -0.12 associations Aspirin 52.33 1 12.44 0.013 -0.54 0.21 0.33 1.16 associations Error 1632.73 388 Total 20765.4 392 1Computed using level of significance at alpha = 0.05.a = magnitude of association on mean PAL from age, ex-smoking & low-doseaspirin.(-) magnitude of reduced EWS-PALAll data were weighted using metropolitan Adelaide population age classstatistics (see Table 4.28)Estimated weighted EWS-PAL Ex-smokers from ANOVA (mm± se) Yes No Total 7.0 ± 0.18 6.2 ± 0.22 6.6 ± 0.17 Yes (102) (51) (153) Aspirin takers 7.5 ± 0.17 6.8 ± 0.17 7.1 ± 0.13 No (117) (122) (239) 7.2 ± 0.14 6.4± 0.16 6.9 ± 0.11 Total (219) (173) (392)Computed using level of significance at alpha = 0.05.Estimated means from model evaluated at the mean age covariate.( ) number of subjects 104
  • 124. Table 4.31 The ratio of aspirin to smoking on various measurements of PAL. Severity (mm) ≥ 4 mm mean EWS-PAL Mean PAL MSS-PAL PAL (weighted data) Aspirin -0.13 -0.22 -0.33 -0.54 Smoking 0.22 0.37 0.44 0.75 Ratio 0.59 0.59 0.75 0.72 ≥ 7mm mean PAL not included since there were no significant associations Ratio = aspirin PAL/ex-smoking PAL Table 4.32 Associations of plaque and age with mean PAL with tests of significance. ANOVA 95% confidence Outcomea (one-way) of Sum of F Std interval df Sig on mean PAL Squares statistic error Lower Upper estimates (mm ± se) limit limit Intercept 9.41 1 10.8 0.001 1.18 0.36 0.48 1.89 Age 5.78 1 6.66 0.100 0.01 0.01 0.004 0.026 associations Plaque 40.84 1 47.02 <0.001 0.48 0.07 0.34 0.61 associations Error 337.87 389 Total 3464.59 392Computed using level of significance at alpha = 0.05.a = magnitude of association on scoresN= 392 subjects. Table 4.33 Associations of calculus and age with mean PAL with tests of significance. ANOVA 95% confidence Outcomea (one-way) of Sum of F Std interval df Sig on mean PAL Squares statistic error Lower Upper estimates (mm ± se) limit limit Intercept 12.36 1 13.64 <0.001 1.34 0.37 0.63 2.08 Age 10.24 1 11.29 0.001 0.02 0.01 0.008 0.03 associations Calculus 26.00 1 28.68 <0.001 0.004 .001 0.003 0.01 associations Error 352.71 389 Total 3464.59 392Computed using level of significance at alpha = 0.05.a = magnitude of association on scoresN= 392 subjects. 105
  • 125. Table 4.34 Associations of gingival bleeding and age with mean PAL with tests of significance. ANOVA 95% confidence Outcomea (one-way) of Sum of F Std interval df Sig on mean PAL Squares statistic error Lower Upper estimates (mm ± se) limit limit Intercept 6.43 1 7.03 0.008 1.00 0.38Age associations 12.95 1 14.16 < 0.001 0.02 0.01 0.01 0.03Gingivalbleeding 23.01 1 25.16 < 0.001 0.01 0.001 0.004 0.01associations Error 355.70 389 Total 3464.59 392Computed using level of significance at alpha = 0.05.a = magnitude of association on scoresN= 392 subjects. Table 4.35 Socio-economic factors, oral hygiene patterns and mean PAL (mm). ANOVA (2-WAY) Numbers Means Std error P value No school / Primary 75 3.1 0.11 >0.05 Education Secondary / Tertiary 317 2.7 0.06 0.008 Yes 231 2.9 0.07 Pension status >0.05 No 161 2.7 0.08 Time since last dental < 12 months 221 2.7 0.07 >0.05 visit > 12 months 169 2.9 0.08 1 + / day 347 2.8 0.05 Brushing >0.05 < 1 / day 42 2.9 0.20 < 12 months 148 2.7 0.07 1-2 years 78 2.7 0.11 Time since scale and 3-5 years 87 3.0 0.10 >0.05 clean 6-10 years 30 2.8 0.24 > 11 years 47 2.8 0.19 Never 227 2.8 0.07 Frequency of mouth < 1/ wk 95 2.6 0.09 >0.05 rinse 1 + / week 67 2.9 0.12 Never 144 3.0 0.10 >0.05 Frequency of flossing < 1/ week 119 2.7 0.08 >0.05 1+ /week 127 2.6 0.70 0.002 106
  • 126. Table 4.36 The statistical power values for most ANOVA analyses Levels of power from ANOVA analyses (a) Ex-smoking AspirinSeverity ≥2mm PAL 0.986 0.653 ≥4mm PAL 0.984 0.602 ≥7mm PAL 0.051Œ 0.491ŒExtent ≥2mm PAL 0.794 0.363Œ ≥4mm PAL 0.981 0.550Œ ≥7mm PAL 0.910 0.526ŒMean PAL 0.959 0.533ŒMSS-PAL 0.947 0.699EWS-PAL* 0.940 0.703(*) Weighted values(a) Observed power from ANOVA computed using α = 0.05Œ Low to moderate power of associationTable 4.37 Relative percentage of subjects with medical conditions per group Group Percentage ANS 57.0% AXS 63.7% NANS 18.0% NAXS 30.7%Table 4.38 Outcome of age, ex-smoking and aspirin with various indices of PALPAL unit of Outcome of age per Outcome of ex- Outcome of aspirinmeasure year per group smoking per year per year per group groupMean PAL. 0.02 mm 0.40 mm -0.20 mmMSS-PAL 0.03 mm 0.44 mm -0.33 mmEWS-PAL 0.04 mm 0.75 mm -0.54 mm 107
  • 127. Figure 4.1 The mean percentage of sites with gingival bleeding (modified bleeding index). 100 < 65 65+ Group total 80 Mean % of teeth 60 40 20 0 ANS AXS NANS NAXS Group totalFigure 4.2 The mean percentage of teeth with calculus 100 < 65 yrs > 65 yrs 90 Group total 80 Mean % of teeth 70 60 50 40 30 20 10 0 ANS AXS NANS NAXS Group total 108
  • 128. Figure 4.3 Cumulative distribution of MSS-PAL representing the worst score (site) per tooth per subject, averaged over all subjects. 100 90 ANS AXS 80 NANS NAXS 70 Cumulative % of subjects 60 50 40 30 20 10 0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 10.0 MS S -PAL (mm) 109
  • 129. Figure 4.4 Diagrammatic representation of PAL according to smoking and aspirin taking history, showing mean PAL, MSS-PAL and EWS- PAL. m ean M SS- EW S- PAL PAL PAL (m m ) (m m ) (m m ) n o n s m o k e rs 2 .6 a s p ir in ta k e r s 2 . 6 3 .8 n o n s m o k e rsn o n - a s p ir in ta k e r s 2 . 9 3 . 9 a s p i r in ta k e r s e x -s m o k e rs 2 .9 4 4 .2 n o n - a s p ir in ta k e r s 4 .3 e x - s m o k e rs 6 .6 a s p ir in ta k e r s 6 .8 n o n s m o k e rs 7 .1 n o n -a s p irin ta k e r s 7 .2 e x -s m o k e rs ( n o t to s c a le ) 110
  • 130. Figure 4.5 Cumulative distribution of EWS-PAL. Data were weighted using age class statistics for metropolitan Adelaide population. 100 NAXS 90 AXS 80 NANS 70 ANS 60 Percent 50 40 30 20 10 0 4 5 6 7 8 9 10 11 12 PAL 111
  • 131. Figure 4.6 Variations of recession and pocket depths by tooth- and jaw type for the whole study population. Tooth number and jaw type variation in PAL Mean PAL (mm) per tooth 4.0 2.0 0.0 -2.0 8 7 6 5 4 3 2 1 -4.0 Tooth type: Maxillary teeth (+) Mandibular teeth (-). recession pocketFigure 4.7 Variation of recession and pocket depths by tooth- and jaw type in the AXS group. AXS Group 4.0 Mean PAL (mm) per 2.0 tooth 0.0 8 7 6 5 4 3 2 1 -2.0 -4.0 Tooth type: Maxillary teeth (+) Mandibualr teeth (-) recession pocket 112
  • 132. Figure 4.8 Variation of recession and pocket depths by tooth- and jaw type in the NAXS group. NAXS Group 4.0 Mean PAL (mm) per tooth 2.0 0.0 8 7 6 4 3 2 1 -2.0 -4.0 Tooth type: Maxillary teeth (+) Mandibular teeth (-) recession pocketFigure 4.9 Variation of recession and pocket depths by tooth- and jaw type in the ANS group. ANS Group Mean PAL (mm) per tooth 4.0 2.0 0.0 8 7 6 5 4 3 2 1 -2.0 -4.0 Tooth type: Maxillary teeth (+) Mandibular Teeth (-) Recession Pocket depth 113
  • 133. Figure 4.10 Variation of recession and pocket depths by tooth- and jaw type in the NANS group. NANS Group Mean PAL (mm) 4.0 2.0 per tooth 0.0 8 7 6 5 4 3 2 1 -2.0 -4.0 Tooth type: Maxillary teeth (+) Mandibular teeth (-) recession pocket 114
  • 134. Chapter 5 Discussion5.1 Profile of the study populationThe total number of males residing in metropolitan Adelaide in 1996 (excluding overseasvisitors) was 506,351 of whom 132,382 males were over 50 years of age (Census 1996).As this study targeted older males through media advertising, the subjects who volunteeredwere self-selected (a convenience sample). Therefore the population sample was notrepresentative of Adelaides male population aged 50 and above. It would have been usefulto have had a random sample but resources and time did not allow this to occur. Femaleswere excluded as participants because hormonal/osteoporosis factors could have hadconfounding effects on the periodontium (Genco and Löe 1993; Beck 1996; Bando et al.1998; Jeffcoat 1998).It was considered that there was a high chance of finding the appropriate cohorts, bytargeting older males by advertisement. Sampling frame error was minimised because bothexposed and non-exposed subjects were targeted from the same sampling frame. Thisselection method facilitated the creation of exposed groups and non-exposed groups thatwere similar for age, ethnicity and pension status. Every attempt was made to define thestudy population with regards to variables relevant to PAL. Provided that the "conveniencesample" did not differ too greatly from the general population in terms of those variablesand because PAL is usually prevalent, it can be argued that some degree of extrapolationshould be possible with regard to the periodontal status of the two populations. Based onthis argument, one analysis was performed by applying weight to the data (EWS-PAL)(Tables 4.27, 4.28 & Figure 4.5). This enabled a better separation of groups, especiallysince there was some crossover between the ANS and NANS groups (Figure 4.3), and an 115
  • 135. adjustment for the differing age structures between the groups. Otherwise all otheranalyses were carried out on unweighted data.A large sample frame, confidentiality, gifts and eligibility criteria all help in minimisingresponse bias and promote responses, especially from non-random samples (Fink 1995c). Inthe current study, efforts were made to promote a response from as many males over 50 fromthe general population as possible, by offering a free dental examination and a free ColgateOral Care Kit. Considerable efforts were made to reduce sources of population bias (byadvertising in the two major newspapers with the largest amount of coverage of MetropolitanAdelaide). This selection method only allowed the selection of subjects who could readEnglish (creating a biased sample); this may have accounted for the high percentage ofsubjects with good English skills in the study population compared to the general population.The present study population contained less people with primary or no schooling (19.1%),(Table 4.8) than the figure of 35% from Adelaides general community (Census 1996);30.4% of subjects occupied the upper education level (tertiary education), compared to 13%(Census 1996) for Adelaides general population. The percentage of subjects with asecondary education (50.5%) compared favourably with the 52% for Adelaides generalpopulation.The ANS group had the lowest percentage (13%) of subjects participating in the studycompared to the whole sample (Table 4.8). The subject groupings were dissimilar ineducation and age class distribution. On the other hand, subjects in a higher educated groupcould be expected to have "better" health behaviour, as other studies have shown (Srikandi1982; Mullally and Linden 1992). 116
  • 136. Due to limited time and financial resources, this study relied on press advertisements forrecruitment of subjects; mailed flyers to households in metropolitan Adelaide may havehelped in recruiting a more representative study sample from the general population.5.1.1 Age groupingsIn comparison to Adelaides male population, this study population had lower, and in somecases, higher numbers in each age class interval (50-59, 60-69 and 70+), using census data asa basis for comparison (Tables 4.27 & 4.28). The relative number of subjects in the 50-59year aged class interval for the ANS group was 14% compared to 51% for the NANS groupand 40% from the general population of Adelaide. The distribution of subjects in the classinterval of 60-69 years had the least fluctuation between groups, with a relative frequency ofapproximately 40% compared to 30% for the general population.The mean age of the ANS group was 68.2 years, representing the group with the highestproportion of elderly subjects (Table 4.3). The two groups of aspirin takers had the highestmean ages when compared to non-aspirin takers. Low numbers in a sample biases thedistribution and lowers the power (sensitivity) of the statistical analyses. Low power makesit more difficult to assess the real association of the independent variable(s) (e.g. aspirin andex-smoking), by statistically increasing the probability of Type II errors, making it moredifficult to reject the null hypothesis (Fletcher et al. 1988). The relatively low numbers inthe ANS group could have contributed to the low power in the present study (see Table4.36).This is evident from Table 4.36 that all power values were lower for the aspirin associationscompared to ex-smoking associations, (153 aspirin takers compared to 239 non-aspirintakers) (Table 4.2). The relationship that low numbers could have contributed to the low 117
  • 137. power statistic is also evident for ex-smoking at a severity level of ≥7mmPAL, (121 ex-smokers to 70 non-smokers).At a threshold of ≥5mm mean PAL, there were no statistically significant differencesbetween aspirin takers and non-aspirin takers (Table 4.23). This may have been due to thelow number of aspirin takers (145) i.e. a statistical result rather than a non-physiologicalassociation as there was insufficient statistical power to reject the null hypothesis. However210 ex-smokers did give enough power to give a significant finding at this level of PAL. Athigher thresholds with lower numbers of subjects, both aspirin and ex-smoking, did not havestatistically significant associations with severity of PAL.In assessing the extent of PAL, all thresholds had statistically significant associations withboth aspirin and smoking histories because the ‘N’ values were higher for extent of PALthan for severity values, since it measured the dispersion of PAL (Table 4.25). Howeverthe low to moderate power values highlight the fact that the study population was aconvenience sample and indicates that caution is warranted in extrapolating the findings tothe general population (Table 4.36).There may be more males in the community (with poorer health due to past smoking) whoare taking aspirin giving a relatively larger pool of subjects with these characteristics.Individuals on aspirin with a past history of smoking generally had more systemic illnesses(Table 4.37). This could have accounted for the higher numbers in the AXS group and lowernumbers in the AXS group (less disease experience).There are many reasons for people to volunteer and participate in a study. The AXS subjectsmay have been more informed about the protective associations of aspirin and may havebeen interested to participate. A shortcoming of this study was that no information was 118
  • 138. obtained as to why the subjects participated, why they were on aspirin, how they obtainedthe aspirin and whether they knew about aspirins effects.Alternatively there may be fewer people in the community on aspirin who have neversmoked. This is probably reflected by the fact the NANS group had the highest number ofsubjects (Table 4.2). Yet another explanation for the lower number of subjects in the ANSgroup is that people who have never smoked but who take aspirin may consider that they arehealthier than others and that they do not need to see a health professional. Better incentivesto participate (other than a free dental check up and a Colgate Oral Care Kit) may have beenneeded to target this group of people.The advertising targeted subjects for all four groups with emphasis on aspirin takers; morespecific advertisements placed for a longer period of time may have resulted in more ANSsubjects volunteering.5.2 QuestionnaireQuestions were worded as in the W.H.O. Oral Health Surveys (W.H.O. 1997) and Australianof Bureau Statistics (McLennan 1996); a multiple-choice format was used (Appendix D).The questionnaire was self administered and unsupervised to avoid interviewer bias andinfluence (Abramson 1974). This format minimised the number of staff required, made itmore likely that all the subjects answered the same questions and sensitive topics likepension status and medical conditions were answered truthfully (Fink 1995b). However, thismethod relied on individuals being able to read and comprehend the questions, eliminatingsubjects who were illiterate, had poor English language skills or who were mentallyhandicapped. Since this study had subjects with high levels of English language skills(Table 4.9) the results obtained from the questionnaire could be assumed to be relativelyvalid. 119
  • 139. Due to financial constraints, questionnaires were not mailed to subjects prior to theirexaminations. This could have avoided errors relating to the subject’s recollection of aspirindosage and duration, and smoking history, which relied on subjects unaided memories; itmay have given the subjects time to seek information from other members of their family ortheir medical practitioner.5.2.1 Socio-economic statusSocioeconomic status has often been associated with the prevalence of periodontal diseases.In general, the higher the socioeconomic status, the greater the awareness of oral healthpractices (less tooth loss and less PAL) (Srikandi 1982; Papapanou 1996). In this study theindicators used for socio-economic status were education and pension status (as the factorindicating income).Education appears to be more closely related to oral health than occupational income level(Richards and Barmes 1971; Srikandi 1982). Since most people feel uneasy or sensitiveabout releasing information about their income, the main indicator used in this study waspension card status. Classification of educational level was based on that used by theAustralian Bureau of Statistics (McLennan 1996) and a similar format for pension card statusas used by the South Australian Dental Longitudinal Study (Spencer 1997). It is assumedthat the higher the education level, the better the socialisation process since socialisation isassociated with "better" health behaviour (Blaikie 1979; Hicks and Newcomb 1981). Thestudy population comprised subjects with middle and higher educational levels and therewere no significant distribution differences among the 4 groups. Higher educated subjectsbrushed more frequently, and had a more recent scale and clean than the lower educatedgroups (Tables 4.10, 4.11). The number of subjects with higher education and good Englishskills was high compared to the general population of Adelaide. These attributes have beenshown to help in the socialisation of subjects and correlate well with good oral health 120
  • 140. (Srikandi 1982). This could mean that this study population might have had less PAL thanthe general population of males aged 50 and over in Adelaide. This would need to beconfirmed in a wider study.The economic variable taken into account was the pension status; pensioners representinglow income. Approximately 58.9% of subjects were pensioners with the two aspirin groupshaving the highest number of pensioners (Table 4.6). In this context, the aspirin groupscould be interpreted as the lower income groups compared to non-aspirin groups. Comparedwith non-pensioners, pensioners were less likely to use a mouth rinse, floss and have a scaleand clean. Surprisingly, more pensioners brushed more than once per day compared to non-pensioners (Table 4.10).5.3 Periodontal attachment loss5.3.1 Age associations with PALPAL increases with increasing age, reflecting cumulative episodes of active PAL overtime (Beck et al. 1990; Brown and Löe 1993; Brown and Garcia 1994; Mullally andLinden 1994; Slade and Spencer 1995; Locker et al. 1998). In the current study,ANOVA analyses were used to assess the statistical associations of age with differentindices of PAL (Table 4.38). The associations of age with PAL were statisticallysignificant, but minor in comparison to the associations with smoking and aspirin history.This finding is in agreement with the epidemiological literature which shows that age isassociated with increasing PAL and alveolar bone loss among older age groups, but thatthis association was clinically insignificant (Burt 1994). Burt concluded that theassociations of age and low level of PAL: "is not sufficient alone to cause tooth loss which is the most clinical important endpoint of periodontal disease". 121
  • 141. PAL measurements are retrospective in cross-sectional studies and therefore cannotdetermine if PAL in the elderly is a function of time or aging (Burt 1994). Many studieshave shown similar results to the current study (Papapanou et al. 1989; (Ismail et al.1990; Haffajee et al. 1991; Mullally and Linden 1992; Locker and Leake 1993b).Other studies have found no association of increasing age and increasing PAL (Albandar1990; Gribic et al. 1991; Brown et al. 1994; Beck et al. 1995; Ship and Beck 1996).ANOVA analyses showed statistically significant associations between age and PAL; themagnitude of age on PAL was small, giving further validity to the independentassociations of aspirin and ex-smoking.In summary, the age relationship with PAL was: (i) an association with PAL rather than a cause of PAL (ii) minor in magnitude, indicating that age was a poor predictor of PAL and not a clinically significant risk factor for PAL.5.4 Measuring PAL5.4.1 Case definitionsOne of the fundamental principles in measuring PAL is to capture the degree ofdestruction of periodontal support. Two methodological characteristics required tomeasure the degree of destruction are concise definitions of "cases" and units ofanalysis. The selection of periodontal sites around the dentition with which tocalculate summary measures is an important methodological issue. Since the numberof sites that could be measured is large, a careful selection of sites that minimises lossof information from partial recording is often necessary. Although a number of studiesinvestigated various combinations of sites to indicate overall periodontal status,additional research is needed to better assess information lost from partial recordings.Prevalence of PAL is potentially underestimated with partial recording; the degree of 122
  • 142. under estimation varies widely, depending on factors such as the severity level atwhich the disease is assessed and its true distribution (extent) within and betweensubjects. Compared with prevalence, the estimates of extent or severity of PAL areless influenced by partial recording (Carlos et al. 1986; Papapanou et al. 1993;Papapanou 1996).Past studies (Alabandar et al. 1999; Anagnou-Vareltzides et al. 1996; Slade andSpencer 1995; Bagramian et al. 1993; Gilbert and Heft 1992; Horning et al. 1990;Brown et al. 1989) have measured the extent and severity of periodontitis usingdifferent baseline measures of PAL. Beck (1990) considered that a severity score of4mm loss of PAL would be serious in young subjects while the same score in oldersubjects would have a different severity value. To identify the subjects with severeattachment loss, the same author used a threshold measure of 4 sites or more with aloss of attachment ≥ 5mm and one of those sites had to have a pocket depth of ≥3mm.These threshold definitions can hardly be described as severe in the everyday clinicalcontext. Partial recordings reduce the number of sites recorded and with few peopleexpressing most of the PAL, these methods underestimate the prevalence and severityof PAL.Other studies (Bagramian et al. 1993; Beck 1990; Gilbert and Heft 1992) have useddifferent definitions for severity of PAL, some looking at the worst site score permouth, others use means set at ≥7 mm PAL as the measure of severe periodontaldisease in older subjects (Gilbert and Heft 1992; Slade and Spencer 1995). A thresholdis set to measure the severity and extent of disease using the mean values of PAL as theunit of measure. Partial and full-mouth recordings are used and it is therefore oftendifficult to compare and analyse the results of different studies. In addition, taking 123
  • 143. mean values of all sites or setting threshold values is not an accurate representation ofthe true attachment loss, since 80-99% of sites have PAL between threshold values of≥1mm-≤4mm PAL (Baelum et al. 1986; Beck et al. 1990; Gilbert and Heft 1992;Mullally and Linden 1992; Slade and Spencer 1995). The very large number ofshallow sites lowers the mean, which lowers the severity and prevalence rates. Datafor deeper sites are swamped by the shallow sites, underestimating the true values ofseverity and extent. In addition there are some major shortcomings when setting highthresholds (≥7mm PAL): • as the number of deeper sites decreases dramatically the power of the statistical analysis is reduced. Therefore, large sample sizes are required. • if one subject had numerous sites of 6mm PAL, they would not be classified as having severe PAL, whereas a subject with one site 7mm PAL would be. Is the presence of one severe site more indicative of severity than or the presence of multiple moderate sites? There is no satisfactory answer to this question.These arguments highlight that epidemiological studies of PAL have no standard casedefinitions for severity of PAL and most are arbitrary and inconsistent (Locker andLeake 1993a; Papapanou 1996).In order to overcome the problems of low subject numbers, the numerous sites withlow mean PAL and to identify subjects with multiple sites with moderate PAL, themost severe site per tooth (MSS-PAL) was developed as a different measure of PAL.This method may be a more relatively valid measure of site specific severe attachmentloss than mean PAL. Other studies have used a subject-specific measure of attachmentloss; the worst site of attachment loss per mouth as a measure of severity, bypassing 124
  • 144. the overwhelming lower value site scores (Beck et al. 1990; Gilbert and Heft 1992;Slade and Spencer 1995). However the limitation of this method is that a smallnumber of people in a given population are affected by the most severe PAL(Papapanou 1996; Locker et al. 1998) while the majority of subjects would have lowvalues of worst scores. Therefore, the number of affected sites are low and thevariation or standard deviation of the values increases making it more difficult to rejectthe null hypothesis. This measure may be more useful in epidemiological studies withvery large samples.5.5 Outcomes of aspirin and past smoking on PALLong term, low-dose aspirin provided a significant protective association by loweringthe severity and extent of PAL in both ex- and non-smokers. Controlling for age,aspirin takers had significantly less PAL than non-aspirin takers in both ex- and non-smokers. Long term, low-dose aspirin use had statistically significant positivecorrelations with different measures of PAL (Tables 4.19, 4.23-25) which were evidentat low and high levels of attachment loss. Figure 4.4 illustrates the study’s findings ina diagrammatic form, highlighting the differences in subjects according to aspirin andsmoking histories using three different measures of PAL. This is the first time theseoutcomes have been reported; the findings could have interesting ramifications interms of the management of periodontal diseases and may give further insights intotheir pathogenesis.5.5.1 Mean PALThe most intriguing finding was that a lower mean PAL was consistently observed inthose subjects who took aspirin, compared with those who did not. The findings weresimilar to those of past NSAID studies (Williams et al. 1989; Jeffcoat et al. 1991; 125
  • 145. Reddy et al. 1993). The data could be interpreted as indicating that aspirin reduced therate of attachment loss. In addition non-periodontal in vitro and in vivo models showthat ATLs reduce pro-inflammatory cytokines (LTB4, IL-8, TNF-α & IL-1β) fromepithelial, endothelial and neutrophil cells and may increase INF-γ, IL-4 and IL-13(Claria 1996; Claria 1995; Serhan 1997; Takano et al. 1997 Chiang et al. 1998;Gronert et al. 1998; Clish et al. 1999;). Therefore ATLs and lipoxins are anti-inflammatory and could have modulating effects on PAL, alveolar bone loss andimmune responses (there are no periodontal studies of the effects of ATLs on PAL todate). These hypotheses needs to be confirmed by prospective human periodontalstudies. Although the significant association between the subject groupings could havebeen related to selection bias, the statistically significant differences and the internalconsistency of the findings indicate that this explanation is unlikely to be valid.Furthermore, the data showed that ex-smokers had higher levels of PAL than non-smokers, in line with the well-documented detrimental association of smoking on PAL(Bergström et al.1983; Bergström et al.1986; Haber et al.1992; Haber et al.1993;Grossi et al.1997) and further supporting the validity of the data.Severity analyses (Table 4.23) showed significantly greater amounts of mean PAL inthe older aged subjects and ex-smokers, with less PAL in subjects who did not smoke.Extent analysis showed significantly greater amounts of mean PAL among ex-smokersthan those who did not smoke. The magnitude of severity and extent on mean PALwas consistently less in aspirin takers than in ex-smokers as shown in Table 4.25 withthe ratio of aspirin to ex-smoking being 3:4 (Table 4.31). 126
  • 146. 5.5.2 MSS-PALOften severity and extent scores are expressed as mean values. The arithmetic mean isthe sum of values divided by the number of events, making the mean a measure ofcentral tendency and not very representative in skewed samples. In the current study,the majority of subjects expressed a large number of sites with low mean PAL values(Table 4.24), skewing the sample towards low mean PAL values. When the data wereanalysed using the most severe site of PAL (MSS-PAL) for each tooth, averaged persubject, the same pattern of PAL was observed in relation to the subjects’ aspirin andsmoking histories. This measure of PAL was chosen in addition to mean PAL toreduce the data from shallow sites, which predominated in this study. The MSS-PALmeasure selected sites with the most attachment loss; the differences between themeasures of PAL are apparent in Fig 4.4. Low-dose aspirin and smoking had highlysignificant associations with the severity of MSS-PAL.5.5.3 EWS-PALWhen analysing the age distribution of subjects within groups the older age distributionof the ANS group had major implications on the extent and severity of disease. Therewas a mean age difference of 8-9 years between ANS and NANS (Tables 4.4 and 4.5)and in addition the ANS group had the lowest number of subjects which may haveinfluenced the statistical analysis. Since the current study population differed from thegeneral population in terms of age of subjects in the different age class intervals, dataweighting was used in another assessment of severe PAL (EWS-PAL) (Tables 4.28and 4.30). This method highlighted the aspirin and smoking differences that were alsoseen in mean and MSS-PAL. The cumulative percentage scores from Figure 4.5showed that all subjects had at least one site of maximum 4 mm EWS-PAL. At 7 mmEWS-PAL, all groups showed a similar trend. As the severity of PAL increased, the 127
  • 147. number of subjects with sites ≥7mm EWS-PAL decreased. These data showed thatsubjects taking low-dose aspirin with or without a history of smoking had significantlyless severe EWS-PAL than non-aspirin takers with or without a history of smoking.Some age class intervals had more subjects assigned to them by the weighting process thanother age class intervals (Table 4.28). For example, the 50-59 age class in the ANS group had7 subjects; when weighted, the final ‘number’ was 21. The same age class interval in theNANS group had 63 subjects and when weighted, the final ‘number’ was 48. Thesehighlights the shortcomings of the weighting process, which on one hand, extrapolates dataobtained from a few subjects and, on the other, disregards data from others. It is the formerthat is most disturbing, because it is assumed that the weighted subjects had identical patternsof PAL as the subjects who had been examined. One must take particular caution inextrapolating current EWS-PAL data to the general population, (because of data weighting)nevertheless the findings followed the pattern of the other analyses (mean and MSS-PAL).5.5.4 PlaqueThere were no significant differences in plaque accumulation between the groups(Tables 4.14 & 4.15). Older subjects had significantly more plaque than youngersubjects in all four groups, as has been reported by others (Holm-Pedersen et al. 1975;Locker and Leake 1993a; Grossi et al. 1995). One study found the reverserelationship, with younger subjects having higher plaque scores and gingival bleeding,although the age differences were not large (van der Velden et al. 1985). A similarstudy using a broader age range and experimental gingivitis found no association ofplaque and gingival bleeding with age (Winkel et al. 1987). Findings from this studyindicate that plaque levels and PAL were not associated, as has been shown in otherstudies (Baelum et al. 1986; Bagramian et al. 1993). 128
  • 148. 5.5.5 Gingival bleedingUsing the mBSI, 57.7% of all the subjects sites had gingival bleeding, with younger subjectshaving less gingival bleeding (Figure 4.1). ANOVA showed that low-dose aspirin and ex-smoking history had no association with gingival bleeding. Smoking in some studies hasbeen shown to inhibit the inflammatory response with less gingival bleeding, less redness andless gingival fluid flow (Preber and Bergström 1986; Danielsen et al. 1990; Grossi et al.1997). Of more significance, there were no differences in gingival bleeding between aspirintakers and non-aspirin takers. This is a surprising finding since the anti-platelet and anti-in-flammatory effects of aspirin could have been expected to reduce gingival inflammation, withincreased gingival bleeding. This result is contrary to some past findings that subjects on sys-temic NSAIDs had reduced gingival indices (Waite et al. 1981; Heasman and Seymour 1989;Johnson et al. 1990). Other studies of NSAIDs (of less than 8 weeks duration) showed nodifferences in gingival inflammation (Vogel et al. 1983; Heasman et al. 1989; Flemmig et al.1996). In longer-term studies of NSAIDs, there was reduced gingival bleeding and colourchange by 2-3 months followed by a gradual return to normal, indicating that the inflammato-ry response has compensating mechanisms (Taiyeb and Waite 1993; Ng and Bissada 1998).It is assumed that by 2-3 months, the leukotriene mediators (LTB4) may compensate for theinhibited COX mediators (PGE2 and TxB2), restoring a normal inflammatory response. Al-ternatively, other factors such cytokines IL-1β, neutrophil/platelet adhesion may affect thecellular and vascular elements of inflammation . Recent studies (Claria 1995; Claria 1996;Clish et al. 1999; Serhan 1997; Takano et al. 1997) investigating the effects of ATLscould account for the non-significant differences in gingival bleeding between aspirin takersand non-aspirin takers. However to date there are no studies investigating the effects of ALTsin the periodontal inflammatory process. 129
  • 149. The lack of significant differences in gingival bleeding between groups may support these as-sumptions. This current study may have benefited in measuring GCF-mediators as more ac-curate measurements of gingival inflammation.5.6 Comparisons with other aspirin studiesIt is not possible to compare the present study with past studies on the association ofaspirin on the periodontium because no other studies have investigated low-doseaspirin. In addition, previous studies have a wide disparity in drug combinations, drugdoses, difficulty in controlling for specific medications or the physical health status ofthe subject, subject selection and, in some studies, the very low numbers of subjects.Subjects in previous studies that have investigated the associations of aspirin onperiodontal conditions using doses that ranged from 650mg to >3 gm per day (Waite etal. 1981; Feldman et al. 1983; Flemmig et al. 1996).Waite et al., (1981) found a significant decrease for gingival index and pocket depth insubjects (suffering from rheumatoid arthritis) receiving NSAID therapy. Although therewas a trend towards less PAL in aspirin takers, it was not significant. Possible reasons forthe non-statistical difference on loss of attachment in the Waite et al. study were that thenumber of test subjects was low (22), and that the study was biased as the controls werenot from the same population. The uneven distribution of the duration of drug therapy(this was not controlled for) was another factor to account for. Furthermore, only onesubject took aspirin while the rest were on different NSAIDs. The dosage regime varied,some subjects took the drugs as required for pain (or continuously) others took differentNSAIDs simultaneously. In addition, no account of smoking history was provided;smoking effects could have overwhelmed the aspirin associations. 130
  • 150. The present study did find that low-dose aspirin had significant associations with PAL.The differences in attachment loss findings between studies was possibly due to thestringent inclusion criterion of long-term duration of aspirin therapy (minimum of 2years) in the present study, while the subjects in the Waite study had been on NSAIDtherapy for a minimum of one year, and this studys exclusion of subjects suffering fromrheumatoid arthritis. Some authors believe that there is an increased prevalence andseverity of periodontal disease in rheumatoid patients which may have influenced theWaite (and other) results (Tolo and Jorkend 1990; Kasser et al. 1997). Furthermore,females were included by Waite et al.(1981) which may have added the confoundingeffect of gender. The large age range (22 – 68) of the test group in the Waite study mayhave affected the prevalence of PAL (less PAL).Feldman et al. (1983) carried out a cross-sectional periodontal radiographic study of 75males (from a rheumatology clinic) taking aspirin (650mg-3.9g per day) for at least 5years. Controls were 75 males from an ongoing longitudinal study. The aspirin groupwas matched to the control group for age, dentition similarity and similarity of remainingteeth (10 teeth minimum). Aspirin takers had significantly fewer sites, which exhibited10% or more interproximal bone loss than the controls. The average percentage ofbone loss per dentition was lower in the group taking aspirin, although the differencewas not statistically significant. It is difficult to compare these findings with thosefrom the present study because of the different methods used to measure PAL, and thehigh doses of aspirin that subjects had been taking in the Feldman study (650mg-3.9gper day). The current study did not investigate radiographic changes in alveolar bone. Alongitudinal study using radiographs on subjects taking low-dose aspirin with or withoutperiodontal treatment would be an important extension of the present study. 131
  • 151. Flemmig et al.(1996) found that acetylsalicylic acid (2g per day) in conjunction withscaling in 30 patients with moderate-severe periodontitis resulted in a synergistictherapeutic efficacy approximately equivalent to the sum of each therapy, significantlyreducing gingival inflammation, pocket depth and probing attachment loss. However,this study did not control for age and sex, it used high doses of aspirin over only 6weeks and there was no control for smoking.According to Flemmig et al.(1996): “The clinical effects of systemically administered NSAID (aspirin) do not appear to extend beyond the termination of the medication, either when administered alone, or in combination with scaling. Thus, significant benefits of systemically administered NSAID (aspirin) on periodontal health can only be expected from a long term regimen”.Although these comments apply particularly to the high doses of aspirin taken over ashort time, they are pertinent in relation to the present findings.The findings from this current study contradict the findings of Heasman et al.(1990) whoevaluated the collective associations of various NSAIDs in a group of hospitalrheumatology patients with non-rheumatology patients used as controls. Males andfemales were studied and no statistically significant differences were found between theexposed and non-exposed groups for plaque index, gingival index, probing depth, loss ofattachment, recession and alveolar bone loss. The non-statistical differences may havebeen due to the fact that the exposed and non-exposed subjects came from differentsample frames and less than 10% of test and control subjects had probing depths greaterthan 3mm. 132
  • 152. 5.7 Smoking and PALSmoking is strongly associated with PAL (Bergström and Floderus-Myrhed 1983; Haberand Kent 1992; Haber et al. 1993). The amount of periodontal damage is associated withthe number of pack years and duration of smoking (Ismail et al. 1983; Goultschin et al.1990; Grossi et al. 1995; Grossi et al. 1997; Salvi et al. 1997). People who quit smokinglie between non-smokers and current smokers in terms of periodontal conditions (Grossiet al. 1997).Current smokers were excluded from the present study because of the knowndifferences in the dynamics of attachment loss between ex- and current smokers(Grossi et al. 1997). In addition, the inclusion of current smokers in the study wouldhave necessitated the recruitment of an extra 2 groups of subjects (current smokerstaking and not taking aspirin) making the study too large to undertake. In the presentstudy, ex-smokers had more PAL than non-smokers, confirming previous studies.Even though 89% of the ex-smokers had quit for at least 5 years, the adverse effects ofsmoking on PAL were apparent, reflecting the increased susceptibility of smokers toPAL and the cumulative nature of attachment loss. Ex-smokers taking low-doseaspirin had significantly less PAL than ex-smokers who did not take aspirin. Thepowerful association of low-dose aspirin, with reduced PAL was emphasised by thisfinding since it occurred in both non- and ex-smokers. The findings give credence tothe hypothesis that aspirin lowered PAL.5.8 Prevalence of periodontal attachment loss.In the NAXS group, 38% of subjects had mean MSS-PAL of ≥5mm, while AXS had26% of subjects with a MSS-PAL of ≥5mm. The percentage of subjects with severe PALwas low. These findings correspond with other reports (Committee of Research Science 133
  • 153. and Therapy 1996; Locker et al. 1998). For example in a study by Ismail et al.(1987),34% of subjects had PAL ≥ 7 mm but only 7% of sites had ≥ 7mm PAL.However, it is erroneous to assume that the attachment loss observed in this and otherepidemiological studies was solely due to the effects of destructive periodontaldiseases (periodontitis). Epidemiological studies do not discriminate between thevaried causes of PAL, which may masquerade as periodontitis, or be present inaddition to periodontitis. These causes of PAL include continuous tooth eruption(Clarke et al. 1986; Clarke and Hirsch 1992; Newman 1999), dehiscence, cervicalenamel projections, cracked or split teeth and retrograde periodontitis (Bergenholtz andHasselgren 1998) and result in over-estimation of the prevalence of periodontitis in allepidemiological studies (Clarke and Hirsch 1992). The prevalence of severeperiodontal diseases may be less than the general accepted prevalence rate of 10-15%within populations.5.9 Future recommendationsWith the reduced severity and extent of PAL in ex-smokers taking aspirin, it istempting to speculate that current smokers who are unable to quit may also benefitfrom taking low-dose aspirin to reduce their periodontal and cardiovascular risks.Further work needs to be done to establish whether patients with periodontitis wouldbenefit from taking low-dose aspirin as an adjunct to periodontal therapy and whetherlow-dose aspirin modulates the effects of periodontitis in susceptible, untreatedpopulations. It would be of interest to investigate the associations of aspirin with PAL ina prospective study. Studies of similarly aged females and younger subjects would alsobe beneficial. 134
  • 154. It would be of considerable benefit to follow up the same subjects (by converting thisstudy into a longitudinal study), which would yield further epidemiological data inrelation to aspirin and ex-smoking associations with PAL. Prospective studies on low-dose aspirin would be useful to investigate: • its use as an adjunct to conventional periodontal treatment • the associations of low-dose aspirin with the periodontal status of current smokers • the associations in women of similar age groups • the associations on young subjects • the its associations in implant casesConclusions • Long term (≥2 years), low-dose (≤350mg) aspirin may infer a significant protective benefit on the periodontium, as both ex- and non-smokers had reduced severity and extent of PAL. • Controlling for age, both ex- and non-smoking aspirin takers had significantly less PAL than non-aspirin takers. • Subjects on long term, low-dose aspirin had significant correlations with different measures of PAL, which were evident at both low and high levels of attachment loss. • The present study population was highly dentally aware and this may have been due to the higher than normal education levels.This is the first time these associations have been reported; the findings could haveinteresting ramifications in terms of the management of periodontal diseases and maygive further insights into their pathogenesis. 135
  • 155. The most intriguing finding was that a significantly lower mean PAL was consistentlyobserved in those subjects who took aspirin, compared with those who did not. Oneinterpretation of the data is that aspirin reduced the rate of attachment loss. Thishypothesis needs to be confirmed by a prospective study. Although the significantdifferences between the subject groupings could have been related to selection bias, thesignificant statistical outcomes and the consistency of the findings indicate that thisexplanation is unlikely. Furthermore, the data showed that ex-smokers had higherlevels of PAL than non-smokers, in line with the well-documented detrimentalassociation of smoking on PAL and further supporting the validity of the data.With the lower levels of PAL in ex-smokers taking aspirin, it is tempting to speculatethat current smokers who are unable to quit may benefit from taking low-dose aspirinto reduce both their periodontal and cardiovascular risks. Enterically coated aspirinhas few side effects (Axon and Huskisson 1992), has proven benefits in protectingagainst vascular diseases and is inexpensive, giving extra weight to the advice thatpatients take aspirin to reduce their risk of PAL. Further work needs to be done toestablish whether patients with periodontitis can benefit from taking low-dose aspirinas an adjunct to periodontal therapy and whether low-dose aspirin modulates theeffects of periodontitis in susceptible, untreated populations. 136
  • 156. Appendix A The University of Adelaide Department of Dentistry Information sheet for participants in the research project "Periodontal conditions in smokers and non-smokers on long term, low-dose aspirin therapy.”Purpose of the studyHealthy gums mean that your teeth are well supported in your jaw. Although we knowthat gum diseases can start when people do not clean their teeth properly and when theyhave risk factors (for example, smoking), there is still much to learn about why somepeople are more likely to develop gum diseases than others.This research project aims to find out whether people who are taking small amounts ofaspirin every day have less gum disease than people who are not taking aspirin regularly.In order to find this out, we need to look at the health of the gums in people who are bothsmokers and non-smokers and aspirin takers or not.What is involved?• Details about your past and present medical and dental history will be recorded. Of importance it will be necessary for you to tell us whether you are on aspirin medication, what type and how much you are taking.• Your smoking history - whether you are a smoker and if so how many cigarettes are smoked per day and for how long you have been smoking.• A single-visit check up of the health of your gums. This will include a measure of the amount of inflammation (redness, bleeding) of the gums and measuring any areas of more severe gum disease using an instrument (periodontal probe) which slides down into the space between the tooth and gum.• This examination will take about 10 minutes.What are the benefits to me?• You will be informed if there are any conditions which need attention.• The information obtained in this study of several hundred people may be useful in helping us work out what makes some people more likely to develop gum diseases than others and whether aspirin treatment can help prevent gum disease.Are there any risks?The risks of being part of this study are very low. The clinical examination involves thesame steps used in a thorough dental check-up; there is minimal discomfort duringperiodontal probing.You may withdraw from the study at any time. All information you give us will betreated with the utmost confidentiality.Please contact the following person if you have any questions:Dr Arthur Drouganis 137
  • 157. Appendix B THE UNIVERSITY OF ADELAIDE CONSENT FORMSee also Information Sheet attached.1. I (please print) hereby consent to take part in the research project entitled: Periodontal conditions in ex-smokers and non-smokers on long term, low- dose aspirin therapy.2. I acknowledge that I have read the Information Sheet entitled: “Periodontal conditions in ex-smokers and non-smokers on long term, low-dose aspirin therapy.”3. I have had the project, so far as it affects me, fully explained to my satisfaction by the research worker. My consent is given freely.4. Although I understand that the purpose of this research project is to improve the quality of medical care, it has also been explained that my involvement may not be of any benefit to me.5. I have been given the opportunity to have a member of my family or a friend present while the project was explained to me.6. I have been informed that, while information gained during the study may be published, I will not be identified and my personal results will not be divulged.7. I understand that I am free to withdraw from the project at any time and that this will not affect medical advice in the management of my health, now or in the future.8. I am aware that I should retain a copy of this Consent Form, when completed, and the relevant Information Sheet.SIGNED DATE _________NAME OF WITNESS (Please print)SIGNED DATE _________I, have described to (Please print)the nature of the procedures to be carried out. In my opinion she/he understood theexplanation.SIGNED DATE _________STATUS IN PROJECT Examiner 138
  • 158. Appendix COral Health Report FormUniversity of AdelaideDepartment of Dentistry.NAME……………………………….. DATE…………….I Dr A Drouganis, conducting a periodontal study from the Department of Dentistryat the University of Adelaide, have examined the above named participant.The following conditions were noted: Dental Caries …………………………………………………………….. Periodontal Pocketing (4mm or more) …………………………….. Mobility (greater than class III) …………………………………….. Calculus / Gingivitis …………………………………………………….. Inflamed / abnormal oral mucosa …………………………………….. Other…………………………………………… None of the above conditionsFrom the findings of this examination I have advised the patient that in my opinion,he/she should seek dental advice/care: Immediately In the near future During a regular recall visit in the future.Examiner: Dr. A Drouganis. B.D.S., MDS Postgraduate student.Signature: ________________________________________ Date: _________ 139
  • 159. Appendix DQuestionnaire ID. NumberSurname: Dr/Mr/Mrs/Miss/Ms__________________________________________________ Preferred Name: __________________________________________________ Address: __________________________________________________ Suburb: _____________________ Post code _____________________ Age: _____________________Is there a phone number we could contact you on for further information in relation tothis project, if needed? Ph:_____________________________________Please tick your answer to each question. It should take 10 - 15 minutes to complete.Q1. In which country were you born? 1- Australia 2- England 3- Scotland 4- Italy 5- Greece 6- New Zealand 7- Viet Nam 8- Other – please specify? ______________________Q2. Education: What level of education did you achieve? No schooling 1- Primary 2- Secondary 3- Tertiary 4-Q3. How well do you speak English? 1- Very well 2- Well 3- Not well 4- Not at allQ4. Are you on a Pension or on Health a Concession Card? 1 - Yes Go to Q 5 2 - No Go to Q 4 MEDICAL HISTORY 140
  • 160. Medical History:Q5. Do you have or have suffered from any of the following conditions: Please tick: Yes NoHeart murmurHeart / vascular disordersRheumatic FeverRheumatoid ArthritisDiabetesLiver or Kidney diseaseCancer therapyHepatitis / HIV / AIDSExcessive bleedingAntibiotic therapyQ6. Are you taking any medications or drugs other than aspirin: 1 – Yes 2 - No If yes, please specify name of condition(s). __________________________ ______________________________________________________________ Name of Drug(s). _________________________________________ Dosage: _________________________________________ ASPIRIN HISTORYQ7. Do you take prescribed aspirin? 1 - Yes 2 - No Go to Q 12 If yes, what type or brand do you use?_________________________________Q8. How often do you take aspirin? Please tick appropriate box. 1 - 1/day 2 - 2/day 3 - 3/dayQ9. How many tablets of aspirin do you take each time? 1 - ½ /tab 2 - 1 tab 3 - 2 tabs 4 - Greater than 2 tabsQ10. How much aspirin is in each tablet ____________ mg/tab.Q11. How long have you been taking aspirin? Please tick. 1 - 2-5 yrs 2 - 6-10 yrs 3 - 11-20 yrs 4 - greater 20 yrs SMOKING HISTORYQ12. Have you ever smoked cigarettes regularly? 1 - Yes 2 - No Go to Q 17Q13. Do you currently smoke cigarettes? 1 - Yes Go to Q15 2 - NoQ14. If you have quit smoking when did you quit? 1 - Quit 2-5 yrs ago 141
  • 161. 2 - Quit 5 or more yrs agoQ15. How many cigarettes did you or are you currently smoking in a day? Please tick. 1 - Less than 10 / day 2 - 10-19 / day 3 - 20 –30 / day 4 - More than 30 / dayQ16. How many years had you been a smoker? 1 - Less than 5 years 2 - 5-10 years 3 - Greater than 10 years DENTAL HISTORYQ17. Do you have more than 10 natural teeth? 1 Yes 2 - NoQ18. Do you have a denture or false teeth for your upper jaw? 1 - Yes 2 - NoQ19. Do you have a denture or false teeth for your lower jaw? 1 - Yes 2 - NoQ20. How often do you brush your natural teeth? 1 - Never 2 - Twice a day or more 3 - Once a day 4 - 4-6 times/ week 5 - 1-3 times / week 6 - less once / week 7 - Intermittently or hardly everQ21. How often do you use a commercial mouth rinse? 1 - Never 2 - Twice a day or more 3 - Once a day 4 - 4-6 times a week 5 - 1-3 times / week 6 - less than once / week 7 - Intermittently or hardly everQ22. How often do you use dental floss or a special brush to clean the spaces between yourteeth? 1 - Twice a day or more 2 - Once a day 3 - 4-6 times a week 4 - 1-3 times / week 5 - less than once a week 6 - Intermittently or hardly ever 7 - NeverQ23. How long has it been since you have seen your dentist, hygienist, about your teeth andgums? 1 - Less than 6 month 2 - Last 6-12 months 3 - 1-2 years 142
  • 162. 4 - 3-5 years 5 - greater than 5 years 6 - greater than 10 years 7 - NeverQ24. Have you had your teeth scaled or cleaned? 1 - Never 2 - In the last 6mths 3 - In last 6-12 months 4 - In last 1-2 years 5 - In last 3-5 years 6 - Greater than 6-10 years 7 - Greater than 11 years.Q25. Have you ever had gum treatment or surgery? 1 - Yes 2 - NoQ26, How long ago?Write the number of years ago. ………………….. 143
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