Slide 1: CHAPTER 15 DEFENSE MECHANISM OF GINGIVA The gingival tissue is constantly subjected to mechanical and bacterial aggressions . The saliva, the epithelial surface, and the initial stages of the inflammatory response provide resistance to these actions. The permeability of the junctional and sulcular epithelia and the role of sulcular fluid, leukocytes, and saliva is discribed here. SULCULAR FLUID : In 1950, Waerhaug, Brill and Krausse, elucidated the composition and role of sulcular fluid or gingival crevicular fluid (GCF) in oral defense mechanisms. They introduced filter paper into gingival sulcus of dogs that had previously injected intramuscularly with fluorescien, within 3min the fluorescent material was recovered on the paper strips. This indicated a passage of fluid from the blood stream through the tissues and exiting via the gingival sulcus. Slide 2: METHODS OF COLLECTIONS : Use of absorbing paper strips. Twisted threads placed around and into the sulcus. Micropipettes. Intracrevicular washings. 1. USE OF ABSORBING PAPER STRIPS : a) Placed within the sulcus (intrasulcular method) or at its entrance (extrasulcular method). The placement of the filter paper strip in relation to the sulcus or pocket is important. The Brill technique places it into the pocket until resistnce is encountered. This method introduces a degree of irritation of the sulcular epithelium that can by itself trigger the flow of fluid. To minimize this irritation, Loe and Holm- Pedersen placed the filter strip at the entrance of the pocket or over the pocket entrance. The fluid seeping out is picked up by the strip, but the sulcular epithelium is not in contact with the paper. Slide 3: TWISTED THREADS : Preweighed twisted threads were used by Weinstein et al. The threads were placed in the gingival crevice around the tooth, and the amount of fluid collected was estimated by weighing the sample thread. MICROPIPETTES : The use of micropipettes permits the collection of fluid by capillarity. Capillary tubes of standardized length and diameter are placed in the pocket, and their content is later centrifuged and analyzed. CREVICULAR WASHINGS : Used to study GCF from clinically normal gingiva. One method uses an appliance consisting of a hard acrylic plate covering the maxilla with soft borders and a groove following the gingival margins. It is connected to four collection tubes. Slide 4: The washings are obtained by rinsing the crevicular areas from one side to the other, using a peristaltic pump. A modification of the method uses two injection needles fitted one within the other. During sampling, the inside, or ejection needle is at the bottom of the pocket and the outside, or collecting, one is at the gingival margin. The collection needle is drained into a sample tube by continuous suction. PERMEABILITY OF JUNCTIONAL AND SULCULAR EPITHELIA : Substances that have shown to penetrate the sulcular epithelium include albumin, endotoxin, thymidine, histamine, phenytoin, and horseradish peroxidase. Squier and Johnson reviewed the mechanisms of penetration through an intact epithelium. Intercellular movement of molecules and ions along intercellular spaces appears into be a possible mechanism. Substances taking this route do not traverse the cell membranes. Slide 5: AMOUNT : The amount of GCF collected on a paper strip can be evaluated in a variety of ways. The wetted area can be made more visible by staining with ninhydrin. It is then measured planimetrically on an enlarged photograph or with the help of a magnifying or a microscope. An electronic method has been devised for measuring the fluid collected on a “ blotter “ (Periopaper), employing an electronic transducer. The wetness of the paper strips affects the flow of an electronic current and gives a digital readout. The amount of GCF collected is extremely small. Measurement performed by Cimasoni showed that a strip of paper 1.5mm wide inserted 1mm within the gingival sulcus of a slightly inflammed gingiva absorbs about 0.1mg of GCF in 3minutes. Slide 6: COMPOSITION : Cellular Elements. Electrolytes. Organic compounds. CELLULAR ELEMENTS : Bacteria, desquamated epithelial cells, and leukocytes, which migrate through the sulcular epithelium. ELECTROLYTES : Potassium, sodium, and calcium have been studied in GCF. Most studies have shown a positive correlation of calcium and sodium concentrations and the sodium-to-potassium ratio with inflammation. ORGANIC COMPOUNDS : Both carbohydrates and proteins have been investigated. Glucose hexosamine and hexuronic acid are two of the compounds found in GCF. Slide 7: Glucose concerntration in GCF is three to four times greater than that in serum. The total protein content of GCF is much less than that of serum. Metabolic and bacterial products identified in GCF include lactic acid, urea, hydroxyproline, endotoxins, cytotoxic substances, hydrogen sulphide and and antibacterial factors. CELLULAR AND HUMORAL ACTIVITY IN GINGIVAL CREVICULAR FLUID : 1. Analysis of GCF has been identified cell and humoral responses in both healthy individuals and those with periodontal disease. The cellular immune response includes the appearance in GCF of cytokines, but there is no clear evidence of a relationship between them and disease. However, interleukin- 1 alpha (IL- 1) and IL- beta are known to increase the binding of PMNs, and monocytes / macrophages to endothelial cells, stimulate the production of prostaglandin (PGE2) and release of lysosomal enzymes, and stimulate bone resorption. Presence of interferon- alpha in GCF, may have a protective role in periodontal disease because of its ability to inhibit bone resorption activity of IL-1beta. The amount of fluid recoverable from gingival crevices is small, only use of sensitive immunoassays permits the analysis of the specificity of antibodies. Slide 8: The role of antibodies in the gingival defense mechanisms is hard to ascertain, there is a consensus indicating that in a patient with periodontal disease : A reduction in antibody response is detrimental. An antibody response plays a protective role in periodontal disease. CLINICAL SIGNIFICANCE : Gingiva is an inflammatory exudate, its presence in clinically normal sulci, can be explained microscopically due to presence of inflammation. The amount of GCF is greater when inflammation is present and proportional to severity of inflammation. GCF production is not increased due to trauma from occlusion, but is increased due to mastication of coarse foods, toothbrushing and gingival massage, ovulation, hormonal contraceptives, and smoking, circadian periodicity and periodontal therapy. Circadian periodicity : There is gradual increase in GCF amount from 6am to 10pm and a decrease afterwards. Slide 9: Sex hormones : Female sex hormones increase GCF flow, because they enhance vascular permeability Pregnancy, ovulation and hormonal contraceptives increase GCF. Mechanical stimulation : Chewing and vigorous gingival brushing stimulate the flow of GCF. Even minor stimulation represented by intrasulcular placement of paper strips increases the production of fluid. Smoking : Smoking produces an immediate transient but marked increase in GCFflow. Periodontal therapy : There is an increase in GCF production during the healing period after periodontal surgery. Slide 10: DRUGS IN GINGIVAL CREVICAL FLUID : Drugs that are excreted through the GCF may be used advantageously in periodontal therapy. Tetracyclines are excreated through GCF. Metronidazole is another antibiotic that has been detected in human GCF. LEUKOCYTES IN DENTOGINGIVAL AREAS : Leukocytes predominantly found in clinically healthy gingival sulci are PMNs. Appear in small number extravascularly in the connective tissue adjacent to the bottom of the sulcus, move to gingival sulcus, where they are expelled. Leukocytes are present even when histologic sections of adjacent tissue are free of inflammatory infiltrate. Differential count shows, PMNs 91.2% - 91.5%, 24% T- lymphocytes, and 18% mononuclear phogocytes. Slide 11: Mononuclear cells identified are 58% B lymphocytes, 24% T lymphocytes, and 18% mononuclear phagocytes. The ratio of T lymphoctes to B lyphocytes found in GCF is 1: 3. Leukocytes were reported in the gingival sulcus of nonmechanically irritated healthy gingiva. The majority of these cells are variable and have been to have phagocytic and killing capacity. They constitute a major protective mechanism against the extension of plaque into the gingival sulcus. Leukocytes are also found in saliva. The main port of entry of leukocytes into oral cavity is the gingival sulcus. Slide 12: SALIVA : Salivary secretions are protective in nature because they maintain the oral tissues in a physiologic state. Saliva exerts a major influence on plaque by mechanically cleansing the exposed oral surfaces, by buffering acids produced by bacteria, and by controlling bacterial activity. ANTIBACTERIAL FACTOTORS : Saliva contains numerous inorganic and organic factors that influence bacteria and their products in the oral environment. Inorganic factors include ions and gases, bicarbonate, sodium potassium, phosphates, calcium, fluorides, ammonium, and carbon dioxide. Organic factors include lysozyme, lactoferrin, myeloperoxidase, lactoperoxide and agglutinins and antibodies. Lysozyme is a hydrolytic enzyme and works both on gram- negative and gram- positive organisms and targets Veillonella and Actinobacillus actinomycetemcomitans. Slide 13: It repels certain transient bacterial invaders of the mouth. The lactoperoxide – thiocyanate system in saliva are bactericidal to Lactobacillus and Streptococcus. Lactoferrin, is effective against Actinobacillus species. SALIVARY ANTIBODIES : Saliva, like GCF contains antibodies that are reactive with indigenous oral bacterial species. Although immunoglobulins G (IgG) and M (IgM) are present, immunoglobulin A (IgA) is preponderant. Salivary antibodies are synthesized locally because they react with strains of bacteria indigenous to the mouth. The enzymes normally found in the saliva are derived from the salivary glands, bacteria, leukocytes, oral tissues, and ingested substances. Hyaluronidase, lipase, catalase, peroxidase and collagenase are the enzymes present in increased concentrations in the saliva. Slide 14: Proteolytic enzymes in saliva are generated both by host and oral bacteria and contributes to initiation and progression of periodontal disease. High molecular- weight mucinous glycoproteins in saliva bind specificallyto many plaque- forming bacteria. The glycoprotein- bacteria interactions facilitate bacterial accumulation on the exposed tooth surface. Glycoproteins and a glycolipid serve as receptors for the attachment of some viruses and bacteria. Glycoproteins of salivary secretions and components of the epithelial cell surface inhibit antigen sorption and therefore may limit pathologic alterations. SALIVARY BUFFERS AND COAGULATION FACTOR : The maintenance of physiologic hydrogen ion concerntration (pH) at the mucosal epithelial cell surface and the tooth surface is an important function of salivary buffers. In saliva the most important salivary buffer is the bicarbonate- carbonic acid system. Slide 15: Saliva also contains coagulation factors (factors VIII, IX, and X; plasma thromboplastin antecedent [PTA]; and the Hageman factor) that hasten blood coagulation and protect wounds from bacterial invasion. LEUKOCYTES : The saliva contains all forms of leukocytes, of which the principal cells are PMNs. The number of PMNs varies from person to person at different times of the day is increased in gingivitis. PMNs reach the oral cavity by migrating through the lining of the gingival sulcus. Living PMNs in saliva are sometimes reffered to as orogranulocytes , and their rate of migration into the oral cavity is termed the orogranulocytic migratory rate . Slide 16: ROLE IN PERIODONTAL PATHOLOGY : Saliva exerts a major influence on plaque initiation, maturation, and metabolism. Salivary flow and composition also influence calculus formation, periodontal disease, and caries. The removal of the salivary glands in experimental animals increases the incidence of dental caries and periodontal disease and delays wound healing. In humans, an increase in inflammatory gingival diseases, dental caries, and rapid tooth destruction associated with cervical or cemental caries is partially a consequence of decreased salivary gland secretion (xerostomia). Xerostomia may result from a variety of factors, among them sialolitiasis, sarcoidosis, Sjogren’s syndrome, Mikulicz’s disease, irradiation and surgical removal of the salivary glands.