臺灣博碩士論文加值系統

牙周炎是細菌導致的發炎疾病，會造成牙齦腫脹、齒槽骨流失，導致牙齒動搖度增大最終脫落，影響病患生活品質。糖尿病是高盛行率的慢性病，有血糖升高、胰島素抗性增加、傷口不易癒合等症狀，患者組織內會累積糖化終產物。先前的研究指出，糖尿病是牙周病的危險因子，控制糖尿病有助於減緩牙周病的發炎反應。美諾四環素(minocycline)是一種抗生素，其凝膠狀劑型被用於輔助治療牙周病。本研究的目標是評估在糖尿病控制不佳的牙周病患者治療中，在非手術階段在牙齦下使用美諾四環素治療，與不使用美諾四環素而只進行非手術性治療之差異，並分析牙周參數、牙齦溝液中的生物標誌、牙齦組織中糖化終產物及其受器，研究在牙齦下施用美諾四環素對糖化終產物引發的發炎反應之抑制效果。
23位第二型糖尿病控制不佳(糖化血紅素>7.5%)的受試者是由來到台北醫學大學附設醫院牙周病科及雙和醫院牙周病科的患者取樣，隨機分成實驗組 (n=10)與控制組(n=13)。實驗組在牙周統合治療(comprehensive periodontal treatment program, CPTP)流程下，兼施牙齦下使用美諾四環素。控制組不使用美諾四環素，僅進行非手術性牙周統合治療，包含：嚴格之口腔衛生指導、超音波洗牙，及牙齦下根面整平。牙齦溝液與臨床牙周參數在治療前與治療後一、三、六個月取樣，針對牙齦溝液中的可溶性糖化終產物受器(sRAGE)與白細胞介素六(IL-6)以酵素連結免疫分析法(ELISA)定量，在治療後一、三、六個月也同時測量臨床牙周參數包含：牙周囊袋深度、探測出血、牙菌斑指數。同時收集糖尿病控制不佳的牙周病患者(n=13)與非糖尿病且有牙周炎患者病人(n=6)，因為牙周病適應症須以牙周手術方式治療或拔牙，則取手術中切除的牙
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齦組織，以免疫染色測量糖化終產物與糖化終產物受體的強度。
結果顯示，實驗組之平均糖化血紅素在治療六個月後下降2.12%，控制組上升0.12%；控制組之牙周囊袋深度在治療後第三個月及第六個月顯著深於實驗組；兩組之探測出血在治療後皆顯著降低，但組間無顯著差異。免疫染色結果顯示，同為中重度牙周病患者，患有糖尿病的患者表現顯著較強的AGE (p=0.03)和RAGE (p=0.03)。計算相關係數發現，sRAGE和IL-6與各牙周參數及糖化血紅素皆無顯著相關。
結論: 在糖尿病控制不佳的牙周病患者的CPTP療程中合併使用美諾四環素，有助於控制血糖與牙周病。組織切片顯示，糖尿病與非糖尿病患者牙齦組織中有顯著較強的致炎性抗原(AGEs和RAGE)表現，暗示糖尿病患牙齦的代謝產物可能調控牙周發炎反應，是糖尿病患者特有的牙齦組織特徵。

Periodontitis, an inflammatory disease caused by bacteria, leads to gingiva swelling, alveolar bone destruction, thus resulting in increased teeth mobility and shedding of the teeth. Diabetes is a high prevalence of chronic diseases which cause elevated blood sugar, insulin resistance, impaired wound healing and other symptoms, finally leading to advanced glycation end products accumulation. Previous studies have shown that diabetes is a risk factor for periodontal disease, diabetes control helps reduce the inflammation of periodontal disease. Subgingival minocycline administration (SMA) is used as an adjunctive therapy for periodontitis. The objective of this study was to evaluate the difference in the use of SMA in deep pockets during accepting comprehensive periodontal treatment program (CPTP) of patients with poorly controlled type II diabetes (T2DM), and to do CPTP without the use of SMA.
The periodontal parameters, which included pocket depth (PD), bleeding on probing (BOP), and plaque score (PS); and biomarkers, which included sRAGE and IL-6, in the gingival crevicular fluid were analyzed to investigate the treatment effect of CPTP or CPTP/SMA on periodontal inflammatory caused by advanced glycation end products. 23 subjects with poorly controlled diabetes (A1c>7.5%) were randomly allocated to the experimental and control groups who came to the Department of Periodontology of the Taipei Medical University Shuang Ho Hospital and Department of Periodontology of the Taipei Medical University Hospital. The patients in the experimental group underwent the SMA during accepting CPTP. The patients in the control group were treated with CPTP but without SMA. The nonsurgical CPTP includes: strict oral hygiene instructions, scaling and root planing. Gingival crevicular fluid (GCF) were collected before treatment and first month, third month, and sixth month after CPTP, for detecting the soluble form of receptor for sRAGE in GCF by ELISA. The A1c and periodontal parameters (periodontal pocket depth, bleeding on probing rate, and plaque score) are also measured before treatment and after first month, third month, and sixth month of CPTP. Meanwhile we also collect the systematic healthy subjects with periodontitis (n=6), and T2DM subjects with periodontitis (n=13) who is indicated for periodontal surgery or other dental surgery for gingival tissue biopsy. The gingival tissues were harvested during periodontal surgery and processed for immunohistochemistry staining (IHC) of advanced glycation end products (AGEs) and their soluble receptor (RAGE). According to the result, the mean A1c of experimental group reduced 2.12% after the CPTP treatment for 6 months, while the control group elevate 0.12%. BOP of both groups significantly decreased after treatment (p<0.001), but there were no significant difference in anytime point. PD of CPTP + SMA group after 3 months and 6 months of the treatment were significant lower than the PD of CPTP only group (p<0.05, p<0.001). The results show that the T2DM subjects express stronger AGEs (p=0.03) and weaker RAGE (p=0.03). The correlation coefficient of IL-6 and sRAGE showed no correlation with other biomarker,A1c, and periodontal parameter.
Our results concluded that treating subjects with T2DM and periodontitis with CPTP and SMA is effective for controlling periodontitis and helpful for A1c control. The IHC results proved that there are stronger inflammatory antigens (AGEs and RAGE) expression in gingival tissue of T2DM subjects,
which implied that the gingival tissue of T2DM patients may possess a unique character of positive labelling of AGE and RAGE and may influence a specific immune-response of inflammatory periodontitis.

CONTENTS
中文摘要 .......................................................................................................... 6
ABSTRACT ...................................................................................................... 8
CHAPETER I: INTRODUCTION .................................................................... 11
CHAPETER II: REVIEW OF THE LITERATURE ........................................... 13
2.1 The relationship between periodontitis and T2DM ................................... 13
2.2 Subgingival antibiotic local delivery .......................................................... 14
2.3 Advanced glycation end products (AGEs), receptor of glycation end products (RAGE), and soluble RAGE ............................................................ 15
2.4 Interleukin-6 (IL-6) .................................................................................... 16
CHAPTER III: PURPOSE .............................................................................. 17
CHAPETER IV: HYPOTHESIS ...................................................................... 18
CHAPETER V: MATERIALS AND METHODS .............................................. 19
5.1 Study subjects .......................................................................................... 19
5.1.1 Patient selection .................................................................................... 19
5.1.2 Inclusion Criteria ................................................................................... 19
5.1.3 Exclusion Criteria .................................................................................. 19
5.2 Study design ............................................................................................ 20
5.3 Clinical Examination and Classification .................................................... 21
5.3.1 Probing depth (PD) ............................................................................... 21
5.3.2 Bleeding on probing .............................................................................. 21
5.3.3 Plaque score ......................................................................................... 22
5.3.4 Criteria for Classification of periodontal severity ................................... 22
5.4 Gingival Cervical Fluid (GCF) preparation ............................................... 22
5.4.1 Collection of GCF .................................................................................. 22
5.4.2 Elution and storage of GCF .................................................................. 23
5.5 Assay of sRAGE ...................................................................................... 23
5.6 Immunohistochemistry staining of AGEs and RAGE ............................... 23
5.7 IHC grading system ................................................................................. 24
5.8 Calculations and analysis of data ............................................................. 25
5.9 Experimental Materials ............................................................................ 25
CHAPETER VI: RESULTS ............................................................................ 26
6.1 Descriptive statistical analysis .................................................................. 26
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6.2 A1c ........................................................................................................... 26
6.3 Probing depth ........................................................................................... 27
6.4 Plaque score ............................................................................................ 27
6.5 Bleeding on probing ................................................................................. 28
6.6 sRAGE in GCF ......................................................................................... 28
6.8 IHC of AGE & RAGE ................................................................................ 29
6.9 Correlation coefficient .............................................................................. 30
CHAPTER VII: DISCUSSION ........................................................................ 31
7.1 SMA and periodontal parameter .............................................................. 31
7.2 Non-surgical periodontal treatment and A1c ............................................ 32
7.3 sRAGE quantity in GCF and periodontal parameters .............................. 32
7.4 IL-6 quantity in GCF and periodontal parameter ...................................... 33
7.5 AGE and RAGE expression in T2DM subjects and systematic healthy subjects .......................................................................................................... 34
7.6 Limitation ................................................................................................. 36
CHAPTER VIII: CONCLUSION...................................................................... 37
REFERENCES .............................................................................................. 38
APPENDIX ..................................................................................................... 46

1. Grossi SG, Genco RJ. Periodontal Disease and Diabetes Mellitus: A Two-Way Relationship Annal Periodontol 1998, 3:1:51-61.
2. Lin SJ, Tu YK, Tsai SC, Lai SM, Lu HK. Non-surgical periodontal therapy with and without subgingival minocycline administration in patients with poorly controlled type II diabetes: a randomized controlled clinical trial. Clin Oral Invest 2012;16:599–609.
3. Gemmell E, Marshall RI, Seymour GJ Cytokines and prostaglandins in immune homeostasis and tissue destruction in periodontal disease. Periodontol 2000. 1997 Jun;14:112-43.
4. Geroldi D, Falcone C, Emanuele E. Soluble receptor for advanced glycation end products: from disease marker to potential therapeutic target. Curr Med Chem. 2006;13(17):1971-8.
5. Singhal S, Pradeep AR, Kanoriya D, Garg V. Human soluble receptor for advanced glycation end products and tumor necrosis factor-α as gingival crevicular fluid and serum markers of inflammation in chronic periodontitis and type 2 diabetes. J Oral Sci. 2016;58(4):547-553.
6. Winer N, Sowers JR (2004) Epidemiology of diabetes. J Clin Pharmacol 44, 397-405.
7. Bennett CM, Guo M, Dharmage SC. HbA(1c) as a screening tool for detection of Type 2 diabetes: a systematic review. Diabet Med. 2007 Apr;24(4):333-43. Epub 2007 Mar 15. Review. Erratum in: Diabet Med. 2007 Sep;24(9):1054.
8. Löe H (1993) Periodontal disease. The sixth complication of diabetes
39
mellitus. Diabetes Care 16, 329-334. 9. Sonnenschein SK, Meyle J. Local inflammatory reactions in patients with diabetes and periodontitis. Periodontol 2000. 2015 Oct;69(1):221-54.
10. Chiu SY, Lai H, Yen AM, Fann JC, Chen LS, Chen HH. Temporal sequence of the bidirectional relationship between hyperglycemia and periodontal disease: a community-based study of 5,885 Taiwanese aged 35-44 years (KCIS No. 32). Acta Diabetol. 2015 Feb;52(1):123-31.
11. Merchant AT, Georgantopoulos P, Howe CJ, Virani SS, Morales DA, Haddock KS. Effect of Long-Term Periodontal Care on Hemoglobin A1c in Type 2 Diabetes. Journal of Dental Research. 2016;95(4):408-415.
12. Costa KL, Taboza ZA, Angelino GB, Silveira VR, Montenegro R Jr, Haas AN, Rego RO. Influence of Periodontal Disease on Changes of Glycated Hemoglobin Levels in Patients With Type 2 Diabetes Mellitus: A Retrospective Cohort Study. J Periodontol. 2017 Jan;88(1):17-25.
13. Wang TF, Jen IA, Chou C, Lei YP. Effects of periodontal therapy on metabolic control in patients with type 2 diabetes mellitus and periodontal disease: a meta-analysis. Medicine (Baltimore).
14. Listgarten MA, Lindhe J, Hellden L. Effect of tetracycline and/or scaling on human periodontal disease. Clinical, microbiological, and histological observations. J Clin Periodontol. 1978 Nov;5(4):246-71.
15. SEGAL, B. M. (1963). Photosensitivity, nail discoloration, and onycholysis: Side effects of tetracycline therapy. Archives of internal medicine, 112(2), 165-167.
16. Greenstein G, Polson A. The role of local drug delivery in the management of periodontal diseases: a comprehensive review. J
40
Periodontol. 1998 May;69(5):507-20. 17. Lu HK, Chei CJ. Efficacy of subgingivally applied minocycline in the treatment of chronic periodontitis. J Periodontal Res. 2005 Feb;40(1):20-7.
18. March CJ, Mosley B, Larsen A, Cerretti DP, Braedt G, Price V, Gillis S, Henney CS, Kronheim SR, Grabstein K (1985). "Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs". Nature. 315 (6021): 641–7.
19. Yoshimura, Teizo, et al. "Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)." The Journal of immunology 139.3 (1987): 788-793
20. Lindemann RA, Economou JS, Rothermel H. Production of interleukin-1 and tumor necrosis factor by human peripheral monocytes activated by periodontal bacteria and extracted lipopolysaccharides. J Dent Res. 1988 Aug;67(8):1131-5.
21. Graves DT, Cochran D. The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. J Periodontol. 2003 Mar;74(3):391-401.
22. Takahashi K, Takashiba S, Nagai A, Takigawa M, Myoukai F, Kurihara H, Murayama Y. Assessment of interleukin-6 in the pathogenesis of periodontal disease. J Periodontol. 1994 Feb;65(2):147-53.
23. Reinhardt RA, Masada MP, Kaldahl WB, DuBois LM, Kornman KS, Choi JI, Kalkwarf KL, Allison AC. Gingival fluid IL-1 and IL-6 levels in refractory periodontitis. J Clin Periodontol. 1993 Mar;20(3):225-31.
41
24. Reinhardt RA, Masada MP, Kaldahl WB, DuBois LM, Kornman KS, Choi JI, Kalkwarf KL, Allison AC. Gingival fluid IL-1 and IL-6 levels in refractory periodontitis. J Clin Periodontol. 1993 Mar;20(3):225-31.
25. Graves DT, Li J, Cochran DL. Inflammation and Uncoupling as Mechanisms of Periodontal Bone Loss. Journal of Dental Research. 2011;90(2):143-153.
26. Yang S, Madyastha P, Ries W, Key LL. Characterization of interferon gamma receptors on osteoclasts: effect of interferon gamma on osteoclastic superoxide generation. J Cell Biochem. 2002;84(3):645-54.
27. Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation. 2006 Aug 8;114(6):597-605.
28. Singh VP, Bali A, Singh N, Jaggi AS. Advanced Glycation End Products and Diabetic Complications. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology. 2014;18(1):1-14.
29. Chilelli NC, Burlina S, Lapolla A. AGEs, rather than hyperglycemia, are responsible for microvascular complications in diabetes: a "glycoxidation-centric" point of view. Nutr Metab Cardiovasc Dis. 2013 Oct;23(10):913-9.
30. Ramasamy R, Vannucci SJ, Yan SS, Herold K, Yan SF, Schmidt AM. Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiology. 2005 Jul;15(7):16R-28R.
31. Raucci, Angela, et al. "A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the
42
membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10)." The FASEB Journal 22.10 (2008): 3716-3727. 32. Koyama H, Yamamoto H, Nishizawa Y. RAGE and Soluble RAGE: Potential Therapeutic Targets for Cardiovascular Diseases. Molecular Medicine. 2007;13(11-12):625-635. doi:10.2119/2007-00087.Koyama.
33. Nawroth PP, Stern DM. Soluble forms of RAGE: an index of vascular stress? A commentary on "Soluble RAGE in type 2 diabetes: association with oxidative stress". Free Radic Biol Med. 2007 Aug 15;43(4):506-10.
34. Wu TL, Tsai CC, Wang YY, Ho KY, Wu YM, Hung HC, Lin YC. The association between the RAGE G82S polymorphism, sRAGE and chronic periodontitis in Taiwanese individuals with and without diabetes. J Periodontal Res. 2015 Dec;50(6):881-9.
35. van Steenberghe D(1), Rosling B, Söder PO, Landry RG, van der Velden U, Timmerman MF, McCarthy EF, Vandenhoven G, Wouters C, Wilson M, Matthews J, Newman HN. A 15-month evaluation of the effects of repeated subgingival minocycline in chronic adult periodontitis. J Periodontol. 1999 Jun;70(6):657-67.
36. Newton JT, Asimakopoulou K. Managing oral hygiene as a risk factor for periodontal disease: a systematic review of psychological approaches to behaviour change for improved plaque control in periodontal management. J Clin Periodontol. 2015 Apr;42 Suppl 16:S36-46.
37. Syrjälä AM(1), Kneckt MC, Knuuttila ML. Dental self-efficacy as a determinant to oral health behaviour, oral hygiene and HbA1c level among diabetic patients. J Clin Periodontol. 1999 Sep;26(9):616-21.
43
38. Rudick, Courtney P.; Miyaomoto, Takanari; Lang, Melissa; Agrawal, Devendra K. Assessing Periodontal Disease by Measuring Molecular Biomarkers of Inflammation in Gingival Crevicular Fluid. The Free Library. 2018 American Dental Hygienists'' Association 26 Dec. 2018
39. Zizzi A, Tirabassi G, Aspriello SD, Piemontese M, Rubini C, Lucarini G. Gingival advanced glycation end-products in diabetes mellitus-associated chronic periodontitis: an immunohistochemical study. J Periodont Res 2013; 48: 293–301.
40. Basta G, Schmidt AM, De Caterina R. Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes. Cardiovasc Res. 2004 Sep 1;63(4):582-92.
41. Byun K, Yoo Y, Son M, Lee J, Jeong GB, Park YM, Salekdeh GH, Lee B. Advanced glycation end-products produced systemically and by macrophages: A common contributor to inflammation and degenerative diseases. Pharmacol Ther. 2017 Sep;177:44-55.
42. Katz J, Bhattacharyya I, Farkhondeh-Kish F, Perez FM, Caudle RM, Heft MW: Expression of the receptor of advanced glycation end products in gingival tissues of type 2 diabetes patients with chronic periodontal disease: a study utilizing immunohistochemistry and RT-PCR. J Clin Periodontol 2005; 32: 40–44.
43. Rajeev K, Karthika R, Mythili R, Krishnan V, Nirmal M. Role of receptors of advanced glycation end-products (RAGE) in type 2 diabetic and non-diabetic individuals with chronic periodontal disease: an immunohistochemical study. J Investig Clin Dent. 2011 Nov;2(4):287-92
44. Rajeev K, Karthika R, Mythili R, Krishnan V, Nirmal M. Role of receptors
44
of advanced glycation end-products (RAGE) in type 2 diabetic and non-diabetic individuals with chronic periodontal disease: an immunohistochemical study. J Investig Clin Dent. 2011 Nov;2(4):287-92. 45. Kurtiş B, Develioğlu H, Taner IL, Baloş K, Tekin IO. IL-6 levels in gingival crevicular fluid (GCF) from patients with non-insulin dependent diabetes mellitus (NIDDM), adult periodontitis and healthy subjects. J Oral Sci. 1999 Dec;41(4):163-7.
46. Zhou M, Rong R, Munro D, et al. Investigation of the effect of type 2 diabetes mellitus on subgingival plaque microbiota by high-throughput 16S rDNA pyrosequencing. PLoS One. 2013;8(4):e61516.
47. Goodson JM, Gunsolley JC, Grossi SG, Bland PS, Otomo-Corgel J, Doherty F, Comiskey J. Minocycline HCl microspheres reduce red-complex bacteria in periodontal disease therapy. J Periodontol. 2007 Aug;78(8):1568-79.
48. Jabaudon M, Blondonnet R, Pereira B, Cartin-Ceba R, Lichtenstern C, Mauri T, Determann RM, Drabek T, Hubmayr RD, Gajic O, Uhle F, Coppadoro A, Pesenti A, Schultz MJ, Ranieri MV, Brodska H, Mrozek S, Sapin V, Matthay MA, Constantin JM, Calfee CS. Plasma sRAGE is independently associated with increased mortality in ARDS: a meta-analysis of individual patient data. Intensive Care Med. 2018 Sep;44(9):1388-1399.
49. Nakashima A, Carrero JJ, Qureshi AR, Miyamoto T, Anderstam B, Bárány P, Heimbürger O, Stenvinkel P, Lindholm B. Effect of circulating soluble receptor for advanced glycation end products (sRAGE) and the proinflammatory RAGE ligand (EN-RAGE, S100A12) on mortality in
45
hemodialysis patients. Clin J Am Soc Nephrol. 2010 Dec;5(12):2213-9. 50. Lang NP, Adler R, Joss A, Nyman S. Absence of bleeding on probing. An indicator of periodontal stability. J Clin Periodontol. 1990 Nov;17(10):714-21.