Chronic inflammation around painless partially erupted third molars

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Vol. 95 No. 3 March 2003

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY ORAL AND MAXILLOFACIAL SURGERY Interim Editor: Peter E. Larsen

Chronic inflammation around painless partially erupted third molars Mikael Laine, BDS,a,b Irja Venta¨, DDS, PhD,c Tapio Hyrka¨s, DDS, PhD,d Jian Ma, DDS,a,b and Yrjo¨ T. Konttinen, MD, PhD,a.b,e Helsinki, Finland UNIVERSITY OF HELSINKI, CITY OF HELSINKI, FINNISH STUDENT HEALTH SERVICE, AND ORTON RESEARCH INSTITUTE AND THE ORTHOPAEDIC HOSPITAL OF THE INVALID FOUNDATION

Objectives. We sought to assess the histologic host response in chronic, symptomless pericoronitis. Study design. Gingival mucosal (n ⫽ 20) and dental follicle (n ⫽ 20) samples were collected during extraction from patients with pericoronitis and clinically healthy control subjects. Antibodies-recognizing macrophages (CD68), natural killer cells (CD56), T cells (CD2), helper T cells (CD4), suppressor/cytotoxic T cells (CD8), and neutrophils (lactoferrin) were applied in a labelled streptavidin-biotin method by using a DAKO TechMate staining robot. Results. Macrophage was the most numerous kind of cell in pericoronitis, but CD2⫹ T lymphocytes, with a normal CD4/CD8 ratio, were also increased (P ⬍ .01). Neutrophils were not increased and did not show signs of activation. Dental follicles did not contain increased numbers of inflammatory cells. Conclusion. This type of pericoronitis is a chronic/smoldering, rather than an acute/purulent, infection. Because of the chronic and often symptomless nature of pericoronitis, various long-term sequelae may result, which may lead to the need for extraction. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:277-82)

Inflammation in gingival tissues around the third molar and its eventual effect on tooth eruption and local and systemic disease states are poorly understood. A smoldering subclinical inflammation may lead to clinically overt pericoronitis. Such pericoronitis is caused by invading microorganisms,1 but the host response against them has not been studied. This is the first attempt to characterize the inflammatory host cell response, which might be evoked by bacteria and tooth migration in Supported by grants from the Finnish Dental Society and the Academy of Finland, in addition to an EVO Clinical Research Grant (TKIO 601). a Department of Medicine/Inva¨rtes Medicin, University of Helsinki Hospital, Finland. b Institute of Biomedicine/Anatomy, University of Helsinki, Finland. c Department of Special Dental Care, City of Helsinki, Finland. d Finnish Student Health Service, Helsinki, Finland; e ORTON Research Institute and the Orthopaedic Hospital of the Invalid Foundation, Helsinki, Finland. Received for publication Jun 6, 2002; returned for revision Sep 16, 2002; accepted for publication Nov 5, 2002. © 2003, Mosby, Inc. 1079-2104/2003/$30.00 ⫹ 0 doi:10.1067/moe.2003.86

patients without symptoms but with local clinical signs of pericoronitis. Similarly, the eventual host and cellular responses in symptomless third molars without any clinical signs of inflammation have not been studied. Whether such cases are characterized by subclinical but histologically evident inflammation is of great importance. Samples from such individuals were obtained from healthy control subjects undergoing prophylactic third molar extractions. The third molar eruption has been widely studied in a clinical sense,2,3 but the roles of host factors and responses as observed in gingival mucosa and dental follicular tissue have not been elucidated. Inflammation around third molars could be either an acute inflammation dominated by neutrophils or a chronic inflammation characterized by mononuclear inflammatory cells. We sought evidence of inflammation in patients with symptomless (ie, painless) partially erupted third molars and clinical signs of pericoronitis and compared these patients with clinically healthy controls. The methods and markers used have been previously applied in other inflammatory diseases.4,5 The inflammatory cells studied included neutrophils, macrophages, 277

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Table I. Clinical data of the patients with healthy or infected mandibular third molars Healthy/inflamed

Sex

Age (y)

Tooth*

Clinical status

Radiographic status

H H H H H H H H H H

F F F M F F F F F F

48 38 48 38 48 38 48 38 48 38

Partially erupted Retained Retained Retained Partially erupted Retained Retained Retained Retained Retained

Vertical Distoangular Mesioangular Mesioangular Vertical Mesioangular Vertical Mesioangular Mesioangular Mesioangular

I I I I I I I I I I

M F M F F F F M F F

26 27 19 22 23 20 26 23 22 23 ៮ ⫽ 23 X 23 22 22 30 26 22 26 26 27 20 ៮ ⫽ 24 X

48 38 48 48 48 38 48 48 38 38

Partially Partially Partially Partially Partially Partially Partially Partially Partially Partially

Distoangular Vertical Vertical Vertical Vertical Mesioangular Vertical Vertical Vertical Mesioangular

erupted erupted erupted erupted erupted erupted erupted erupted erupted erupted

*38, left mandibular third molar; 48, right mandibular third molar.

natural killer cells, T cells, helper T cells, and suppressor/ cytotoxic T cells. These cells represent the main cells involved in acute and chronic inflammation and the most important cellular sources of tissue-destroying proteolytic enzymes and osteoclast-activating cytokines.

MATERIAL AND METHODS Patients and samples The protocol for collection of human samples was approved by the local ethical committee of the Finnish Student Health Service, Helsinki, Finland. All patients were students at the University of Helsinki, Finland. Informed consent was obtained from all patients. While the patients were under local anesthesia with lidocaine and adrenaline (Table I), 2 experienced oral surgeons (I.V., T.H.) extracted the third molars and took samples. Patients with pericoronitis (n ⫽ 10) who were symptom-free but had clinical signs of inflammation (eg, swelling and redness) underwent third molar extraction. Healthy control subjects with retained or partially erupted symptomless third molars and without clinically evident inflammation (n ⫽ 10) underwent a prophylactic extraction (Table I). Tissue specimens were snap-frozen, stored at ⫺70°C, and embedded in OCT compound (Sakura, Zoeterwoude, The Netherlands) before cutting.

Immunohistochemistry Six-micrometer cryostat sections were mounted on Dako Capillary Microscope slides (TechMate; Dako, Glostrup, Denmark), fixed in cold acetone at 4°C for 5 minutes, and washed in Tris-buffered saline (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.5) and distilled water followed by staining at 22°C in a Dako TechMate Horizon Immunostainer robot. The primary antibody was diluted with Dako ChemMate antibody diluent for 25 minutes. The concentrations of the antibodies used were as follows: rabbit antihuman lactoferrin immunoglobulin (Ig) 1.85 ␮g/mL,6,7 monoclonal mouse antihuman IgG1 ␬ against CD68 0.16 ␮g/mL,8 CD56 0.67 ␮g/mL,9 CD2 0.3 ␮g/mL,10 CD4 0.08 ␮g/mL,11 and CD8 0.33 ␮g/mL.11 Biotinylated secondary antibodies (biotinylated goat antimouse or goat antirabbit Ig) were placed in buffered solution containing carrier protein and sodium azide for 25 minutes. Peroxidase blocking solution was used 3 times for 3 minutes each time. Peroxidase-conjugated streptavidin was used for 25 minutes, and substrate working solution containing 3,3diaminobenzidine tetrahydrochloride was used for 5 minutes. Between steps the sections were washed with Tris-buffered saline (20 mM Tris, 150 mmol/L NaCl, pH 7.5). After staining, the sections were counterstained with hematoxylin and mounted in Diatex solution (Becker Industrifa¨ rg AB, Ma¨ rsta, Sweden). Nor-

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Fig 1. A, CD68 (macrophages); B, lactoferrin (neutrophils); C, CD2 (T cells); D, CD4 (helper T cells); E, CD8 (suppressor/ cytotoxic T cells) in the gingival mucosa of patients with symptomless but clinically and histopathologically evident pericoronitis. F, Control stains with mouse immunoglobulin (Ig) G1 ␬ or rabbit Ig (not shown) did not have any immunoreactivity (hematoxylin counterstaining; original magnification ⫻400).

mal mouse IgG1 ␬ and rabbit serum Ig fraction diluted with Dako ChemMate antibody diluents were used as negative staining controls at the same concentrations as the primary antibodies. Assessment of the number of immunoreactive cells Cell counts were performed on serial sections examined at ⫻320 magnification with a 10 ⫻ 10 eyepiece graticule (Graticules Ltd, Tornbridge, Kent, England)

calibrated with a stage micrometer as described earlier.4 Two hundred to 400 cells from 2 to 5 randomly selected fields of each of the 320 stained sections were counted. Those who counted the cells were blinded to the coded specimens. These codes were first opened after the counting results had been recorded and the data had been frozen, before the samples were grouped for subsequent statistical analysis. Counting was independently performed by 2 experienced technicians (M.L., J.M.), with similar results. The results for each individ-

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Fig 2. Cell counts for different inflammatory cells in the inflamed gingival mucosa of patients with pericoronitis (white bars), compared with those of healthy control subjects (striped bars), disclosed significant differences for CD68, CD2, CD4, and CD8 staining (P ⬍ .02). All data are expressed as means ⫾ SEM. LF, Lactoferrin.

ual case indicated the mean count. The number of positive cells in the sample was calculated according to the following equation: N ⫽ P ⫻ C ⫻ A where N equaled the number of immunolabelled cells in the sample, P equaled the percentage of immunolabelled cells of all cells, C equaled the density of cells per millimeter squared in inflammatory cell infiltrates, and A equaled the area of the sample invaded by inflammatory cell infiltrates. Statistics The normal distribution of the variables was confirmed by using Wilks’ W test. Statistical significance was evaluated by using the 2-group unpaired Student t test. All data are expressed as means ⫾ SEM. RESULTS Gingival tissue around the third molar was obtained from symptom-free patients with pericoronitis who had signs of inflammation, such as swelling and redness. Control samples were usually obtained from retained third molars, but in 2 cases, partially erupted third molars were used. These molars, however, were symptomless and without any clinical signs of inflammation. CD68⫹ macrophages were the most numerous cells found in patients with pericoronitis in the lamina propria—particularly in the vicinity of the basal lamina (Fig 1, A). There was a difference between patients with pericoronitis and control subjects in this respect (P ⬍

.01; Fig 2; Table II). CD68 staining did not reveal epithelial cells or inflammatory cells in the epithelium. In contrast to the frequency of CD68⫹ macrophages, neutrophils were found to be present only in low numbers (Fig 1, B). CD2⫹ T cells were more frequent in the lamina propria of infected gingival mucosa than in clinically healthy gingival mucosa (Fig 1, C). The CD4/CD8 ratio was approximately 2:1. Epithelium contained occasional CD8⫹ T lymphocytes. CD4⫹ cells were mainly confined to the infiltrates below the basal lamina, whereas CD8⫹ cells were scattered throughout the lamina propria (Fig 1, D and E). CD2⫹, CD4⫹, and CD8⫹ cells were more frequent in patients with pericoronitis than in controls (P ⬍ .01, Fig 2; Table II). Individual CD56⫹ natural killer cells (data not shown) and lactoferrin-positive neutrophils were scattered throughout the papillary and reticular lamina propria, both in patients with pericoronitis and controls. The epithelium did not contain CD56⫹ natural killer cells or lactoferrin-positive neutrophils. Dental follicles did not differ between the patients with pericoronitis and the control subjects. Staining controls confirmed the specificity of the staining. DISCUSSION These findings suggest that macrophages in particular and, to a lesser extent, lymphocytes play an important role in the cellular host response against the invading microorganisms in pericoronitis.1 Interestingly, in so-called autoimmune diseases such as Sjo¨ gren’s syndrome, T lymphocytes clearly dominate macrophages in the inflammatory cell infiltrates in the target tissue.12,13 This finding is compatible with the macrophage-mediated, slowly moldering infectious character of pericoronitis, as opposed to the lymphocyte-mediated autoimmune character.1 In addition, lactoferrin-positive neutrophils were relatively few. Macrophages, as the name implies, are usually recognized for their phagocytic potential. However, it has already long been recognized that they are very potent secretory cells that can produce numerous proinflammatory factors and proteolytic enzymes, which are likely to play a role in local tissue pathoses and, eventually, the migration of the third molars as well. Lactoferrin-positive neutrophils were also present, but not in increased numbers. Interestingly, lactoferrin was strictly confined to the neutrophil cytoplasm. Lactoferrin is contained in the secondary or specific neutrophil granules, which are believed to be particularly labile. They release their contents, including lactoferrin and neutrophil collagenase-2 (MMP-8), early on degranulation/neutrophil activation. This finding further confirms the chronic nature of pericoronitis. Because

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Table II. Histomorphometric results (cell numbers per mm2) of cell subtype and a comparison between controls and patients with pericoronitis Results

CD68

LF

CD2

CD4

CD8

Control SEM Patients with pericoronitis SEM P value

77.0 17.7 230.4 36.7 .0014

28.5 9.8 65.4 20.1 NS

49.0 14.3 110.7 18.8 .0176

20.4 5.9 52.8 9.0 .0074

21.1 7.2 71.2 18.2 .0195

LF, Lactoferrin-positive cells; NS, not significant.

this type of pericoronitis is symptomless, it may go undetected and contribute to various long-term sequelae. It can form a focus of bacteremia and a source of odontogenic infections, particularly in immunosuppressed patients. The eventual role of such infectious and inflammatory foci in cardiovascular diseases would be interesting to evaluate.14,15 Pericoronitis most commonly manifests in the partially erupted vertical lower third molars.2,16 The presence of partially erupted third molars, especially those in the vertical position, might be a good basis for extraction. The present results suggest that the dental follicle does not play a role in the host defense/response against infection; rather it is a structure that is primarily responsible for tooth development.17 Macrophages/ mononuclear phagocytes are recognized because of their phagocytic abilities, but they are also important secretory cells. Macrophages secrete substances such as neutral endoproteinases collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9), and collagenase-3 (MMP-13).18-20 Macrophage-derived cytokines (eg, interleukins [IL-1, IL-6] and tumor necrosis factor–␣) regulate osteoclasts.21 Macrophages themselves are effectively activated by bacterial endotoxins/lipopolysaccharides.22-24 The eventual role of inflammation in the migration and eruption of third molars has not been assessed in detail. Further research will have to address the eventual in situ production of proteinases and osteoclast-stimulating cytokines of potential relevance to third molar migration and eruption. The present data reveal an increase in macrophages in the gingival tissues of patients with pericoronitis in comparison with the tissues of clinically healthy controls. Macrophages are characteristic of a chronic inflammation. It can be concluded that pericoronitis in such patients is chronic in nature. Because this type of infection is often relatively symptomless, it can remain undetected during a normal dental check-up if control pantomographic radiographs are not taken. If it persists, this type of pericoronitis can become complicated so that antibiotics and extraction become necessary. A

smoldering chronic infection anywhere in the human body is potentially harmful. If a prophylactic extraction is performed, it could be wise to do it before the patient turns 25 years old (ie, before root development becomes complete). Surgical extraction of a partially erupted third molar from a young patient is a relatively safe procedure. However, the potential complications and the probability of the spread of infection over the patient’s lifetime are unclear. Therefore, further studies are necessary to demarcate the situations in which extraction is necessary. We would like to thank Eija Kaila for superb technical assistance. REFERENCES 1. Peltroche-Llacsahuanga H, Reichhart E, Schmitt W, Lutticken R, Haase G. Investigation of infectious organisms causing pericoronitis of the mandibular third molar. J Oral Maxillofac Surg 2000;58:611-6. 2. Venta¨ I, Turtola L, Murtomaa H, Ylipaavalniemi P. Third molars as an acute problem in Finnish university students. Oral Surg Oral Med Oral Pathol 1993;76:135-40. 3. Venta¨ I, Ylipaavalniemi P, Turtola L. Long-term evaluation of estimates of need for third molar removal. J Oral Maxillofac Surg 2000;58:288-91. 4. Konttinen YT, Reitamo S, Ranki A, Segerberg-Konttinen M. T lymphocytes and mononuclear phagocytes in the skin infiltrate of systemic and discoid lupus erythematosus and Jessner’s lymphocytic infiltrate. Br J Dermatol 1981;104:141-5. 5. Konttinen YT, Tolvanen E, Visa-Tolvanen K, Reitamo S, Forstrom L. Inflammatory cells in sarcoid granulomas detected by monoclonal antibodies and an esterase technique. Clin Immunol 1983;26:380-9. 6. Konttinen YT, Reitamo S. Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears. Histochemistry 1979;62:55-64. 7. Afeltra A, Caccavo D, Ferri GM, Addessi MA, De Rosa FG, Amoroso A, et al. Expression of lactoferrin on human granulocytes: analysis with polyclonal and monoclonal antibodies. Clin Exp Immunol 1997;109:279-85. 8. Pulford KA, Rigney EM, Micklem KJ, Jones M, Stross WP, Gatter KC, et al. KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections. J Clin Pathol 1989;42:414-21. 9. Nitta T, Yagita H, Sato K, Okumura K. Involvement of CD56 (NKH-1/Leu-19 antigen) as an adhesion molecule in natural killer-target cell interaction. J Exp Med 1989;170:1757-61. 10. Plunkett ML, Sanders ME, Selvaraj P, Dustin ML, Springer TA. Rosetting of activated human T lymphocytes with autologous

282 Laine et al

11.

12. 13.

14. 15. 16. 17. 18.

erythrocytes. Definition of the receptor and ligand molecules as CD2 and lymphocyte function-associated 3 (LFA-3). J Exp Med 1987;165:664-76. Ledbetter JA, Evans RL, Lipinski M, Cunningham-Rundles C, Good RA, Herzenberg LA. Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man. J Exp Med 1981;153:310-23. Konttinen YT. In situ characterization of the cellular infiltrate in labial and palatine salivary glands in Sjo¨ gren’s syndrome. Scand J Rheumatol 1981;10:321-30. 5Fox RI, Carstens SA, Fong S, Robinson CA, Howell F, Vaughan JH. Use of monoclonal antibodies to analyze peripheral blood and salivary gland lymphocyte subsets in Sjo¨ gren’s syndrome. Arthritis Rheum 1982;25:419-26. Kinane DF, Lowe GD. How periodontal disease may contribute to cardiovascular disease. Periodontol 2000 2000;23:121-6. Valtonen VV. Role of infections in atherosclerosis. Am Heart J 1999;138:S431-3. Halverson BA, Anderson WH. The mandibular third molar position as a predictive criteria for risk for pericoronitis: a retrospective study. Mil Med 1992;157:142-5. Ten Cate AR, editor. Development of the tooth and its supporting tissues. In: Oral histology. Development, structure, and function. St Louis: Mosby; 1994. p. 58-80. Campbell EJ, Cury JD, Shapiro SD, Goldberg GI, Welgus HG. Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases. J Immunol 1991;146:1286-93.

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY March 2003 19. Welgus HG, Senior RM, Parks WC, Kahn AJ, Ley TJ, Shapiro SD, et al. Neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation. Matrix Suppl 1992;1:363-7. 20. Lindy O, Konttinen YT, Sorsa T, Ding Y, Santavirta S, Ceponis A, et al. Matrix metalloproteinase 13 (collagenase 3) in human rheumatoid synovium. Arthritis Rheum 1997;40:1391-9. 21. Konttinen YT, Xu J-W, Pa¨ tia¨ la¨ H, Imai S, Waris V, Li T-F, et al. Cytokines in aseptic loosening of total hip replacement. Curr Orthop 1997;11:40-7. 22. Erwing L-P, Kluth DC, Walsh GM, Rees AJ. Initial cytokine exposure determines function of macrophages and renders them unresponsive to other cytokines. J Immunol 1998;161:1983-8. 23. Rawadi G, Zugaza JL, Lemercier B, Marvaud JC, Popoff M, Bertoglio J, et al. Involvement of small GTPases in Mycoplasma fermentans membrane lipoprotein-mediated activation of macrophages. J Biol Chem 1999;274:30794-8. 24. Valledor AF, Comalada M, Xaus J, Celada A. The differential time-course of extracellular-regulated kinase activity correlates with the macrophage response toward proliferation or activation. J Biol Chem 2000;275:7403-9. Reprint requests: Yrjo¨ Konttinen, MD, PhD Biomedicum PO Box 700 00029 HUS, Finland [email protected]