Furthering Knowledge of the Pathogenesis of Periodontal Disease in the Evaluation of Biomarkers for Monitoring Disease Susceptibility and Activity
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Furthering knowledge of the pathogenesis of Periodontal Disease in the evaluation of biomarkers for monitoring disease susceptibility and activity. A thesis submitted in fulfilment of the requirements of the degree of MPhil Cardiff University 2015 Bindhu Koshy Tissue Engineering and Reparative Dentistry School of Dentistry College of Biomedical and Life Sciences Cardiff University i Summary The development of periodontitis is a multifactorial process initiated by a bacterial-induced inflammation which is further modified by genetic and environmental factors contributing towards an exaggerated host response and associated tissue destruction. Pro-inflammatory mediators such as cytokines play an important role in the resulting inflammation leading to degradation of periodontal connective tissue, several of which may be detected in GCF and may be of diagnostic and prognostic value. However, our limited understanding of the molecular mechanisms involved in the pathogenesis of periodontal disease restrains the effectiveness of current diagnostic and management techniques in assessing true periodontal health, identify susceptible patients, monitor response to therapy or implement the use of biomarkers which may assist in the management of patients with periodontal disease. The refore, this thesis aims to further our understanding of the pathology of periodontal disease through a series of in-vitro and in-vivo studies. In recognising that as a consequence of periodontal disease is the degradation of the extracellular matrix, clinical studies investigated the release of proteoglycan components from the periodontal tissue in patients with chronic periodontitis. Increasing levels of chondroitin sulphate (CS), a proteoglycan metabolite was observed with progressive clinical attachment loss, highlighted periods of activity and inactivity, with only a few sites demonstrating disease activity over a 21 month period. Furthermore, in-vitro studies investigated the cellular synthesis of proteoglycan in a pathological condition by examining the biological effects of P. gingivalis LPS on PDL cells. In the presence of P. gingivalis LPS, an alteration in cell behaviour was observed with an increase in cell proliferation and a decrease in matrix formation, further suggesting that the degradation products detected in GCF such as decorin and biglycan, were as a consequence of tissue destruction and not as a result of repair or remodelling. Collectively, these results highlight the potential of CS present in GCF as a marker of disease activity. Due to the multifactorial nature of the disease, it is highly unlikely that any one marker may provide information that may be of diagnostic as well as prognostic value. Rather, the use of iii a panel of markers may further corroborate the implementation of biomarkers in periodontal disease management. Therefore, in considering the prominent role of cytokines in tissue destruction, this thesis further examined the prognostic value of cytokine profiling in identifying the high risk patient. On cross-sectional evaluation of cytokine profiles in GCF from patients with different types of periodontal disease using bead array technology, a variable response was observed in the severe type, which was suggestive of an imbalance in Th1/Th2/Th17/Treg responses, further affirming the predominant role of an altered host response in disease progression. Further, resident PDL cells were also considered as potential contributors to the variation in response. Consequently, in-vitro studies demonstrated that although TLR receptors were present on PDL cells, no cytokines were released on exposure to P. gingivalis LPS further suggesting that the cytokines detected in GCF were produced as a consequence of an altered host response which brings about progression of disease. In conclusion, the development of rapid, non-invasive, site based risk assessment and comprehensive screening for biomarkers may be possible in the near future as a result of the rapid development of new diagnostic technologies such as microarray and microfluidics along with the use of oral fluids such as GCF. Therefore, in the future enhanced patient assessment may be possible which will enable provision of customized therapies that target treatment at individual level. iv CONTENTS Chapter 1: Review of literature 1.1 Introduction…………………………………………………………………………………..…1 1.2 Global periodontal tissue epidemiology……………………………………………………..5 1.3 The periodontal tissues in health……………………………………………………………..6 1.3.1 Extracellular matrix of the periodontium…………………………………………….11 1.3.1.1 Proteoglycans……………………………………………………………12 1.3.1.2 Glycoproteins…………………………………………………………...24 1.3.1.3 Proteins associated with bone and cementum……………………...25 1.4 Turnover in the periodontium………………………………………………………………...26 1.5 Aetiology of periodontal disease…………………………………………………………….29 1.6 Pathogenesis of periodontal disease………………………………………,……………….31 1.6.1 Role of P. gingivalis in the pathogenesis of periodontal disease…………… 32 1.6.1. 1 Receptors for LPS…………………………………………………………………35 1.6.1.1.1 CD14 receptors……………………………………………………… 36 1.6.1.1.2 TLR receptors………………………………………………………….40 1.7 Histopathological and immunopathological changes leading to periodontal tissue destruction……………………… ..…………… 1.7.1 Cytokines of the advanced lesion…………………………………………..…... .46 1.7.2. Role of T- helper subsets……………………………………………………… ....49 1.7.2.1 Pro-inflammatory cytokines of T- cell lineage in host response…...…51 1.7.2.1.1 Th1 subset……..…………………………………………..51 1.7.2.1.2 Th2 subset……...………………………………………….5 2 1.7.2.1.3 Th17 subset……..…………………………………………52 1.7.2.1.4 Treg…………………………………………………………53 1.8 Degradation of periodontal tissue 1.8.1 Connective tissue destruc tion………………………….………………………55 1.8.2 Bone resorption………………………………………….………………………56 1.9 Resolution of inflammation………………………………………….……………………...58 1.9.1 Anti- inflammatory cytokines…………………………………………………….59 1.10 Gingival crevicular fl uid……………………………………………… ……………………64 1.11 Biomarkers for elucidation of pathology …………………………………………………..66 1.11.1 Bacterial products in GCF……………..………………………………………66 1.11.2 Inflammatory and im mune markers ……………….…………………………67 1.11.3 Hydrolytic and cytos olic enzymes……….……………………………………67 1.11.4 Markers of connective tissue degradation…...………………………………68 1.12 Aims…………………………………………………………………………………… …….69 v Chapter 2: An increase in proteoglycan metabolites in GCF represents periods of disease activity: a longitudinal study in patients with chronic periodontitis. 2.1 Introduction……………………………………………………………………………… 70 2.2 Materials and Methods…………………………………………………………………. 75 2.2.1 Longitudinal monitoring of proteoglycan metabolites in GCF…..76 2.2.1.1 Validation of methodology 2.2.1.1.1 Inclusion/Exclusion criteria………………….77 2.2.1.1.2 Sample collection…………………………….77 2.2.1.1.3 Measurements of clinical parameters………78 2.2.1.1.3.1 Florida Probe…………………………78 2.2.1.1.3.2 Investigator calibration………………79 2.2.1.1.3.3 Longitudinal assessment of periodontal health……………………79 2.2.1.1.4 Evaluation of release of sGAG in GCF during collection period…………………………………………………….. ……… 79 2.2.1.1.5 Cellulose Acetate Electrophoresis……………………………...80 2.2.1.1.6 Assessment of methods for GAG quantification……………....81 2.2.1.1.7 Statistical An alysis…………………………………………….....82 2.3 Results…………………………………………………………………………………....84 2.3.1 Investigator calibration………………………………………………..84 2.3.2 Assessment of methods for quantification of G AGs……………....84 2.3.3 Longitudinal analysis of proteoglycan metabolites in GCF with disease severity……………………………………………………….85 2.3.4 Cross-sectional analysis of proteoglycan metabolites in GCF with disease severity…………………………………………...99 2.4 Discussion……………………………………………………………………………….101 Chapter 3: P. gingivalis LPS inhibits the reparative and regenerative processes during periodontal disease by influencing PDL cell behaviour and altering the biological events associated with matrix synthesis. 3.1 Introduction……………………………………………………………………………….....108 3.2 Materials and Method s……………………………………………………………………..113 3.2.1 Extraction of LPS from P. gingivalis ………………………………………….113 3.2.2 Characterisation of LPS……………………………………………………….114 3.2.2.1 SDS- Polyacrylamide Gel Electr ophoresis…………………..115 3.2.2.2 Staining with Coomassie Brilliant Blue for detection of protein c ontamination………………………………………….116 3.2.2.3 S taining with Silver for characterisation of LPS……………..116 3.2.2.4 Staining with Ethidium Bromide for assessing contamination w ith nucleic acids………………………………117 3.2.2.5 LAL assay……………………………………………………..….117 vi 3.2.3 Isolation of PDL cells……………………………………………………………118 3.2.4. Characterization of PDL cells………………………………………………….119 3.2.5 Determination of seeding densit y using MTS assay………………………...120 3.2.6 Determination of sub-toxic levels of LP S on cell viability…………………...121 3.2.7 Effect of sub-toxic levels of LPS on cell proliferation………………………..122 3.2.8 Effect of sub-toxic levels of LPS on alkali ne phosphatase activity………...123 3.2.9 Effect of subtoxic levels of LPS on matrix formation………………………...124 3.2.9.1 RT- PCR……………………………………………………........124 3.2.9.1.1 RNA extraction……………………………………....124 3.2.9.1.2 Quantification of RNA………………………. ……..126 3.2.9.1.3 Reverse transcription of total RNA………………..126 3.2.9.1.4 Polymerase chain reaction………………………...127 3.2.9.1.5 Visualisation of PCR products on Agarose gels…129 3.2.9.2 Western bl ot analysis………………………………………… 129 3.2.9.2.1 Protein extraction…………………………… 129 3.2.9.2.2 Protein quantification us ing BCA assay…..130 3.2.9.2.3 Protein separation using SDS- PAGE……..130 3.2.9.2.4 Western