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Section 12: Mineralized Tissues 4. Saliva, Pellicle, Plaque, Caries 3/7/06 The oral environment: saliva & dental plaque General question: How do bacteria, saliva & dietary components interact with oral structures (enamel, gingiva, etc.) to produce the two main dental problems: caries & periodontal disease? Saliva functions digestive food: dissolved, softened, dispersed, lubricated enzymes: a-amylase (ptyalin) lipase protective washes away microorganisms, toxins coats epithelial surface (mucus barrier) minimizes DpH (buffering) proteins: antibodies antibacterial enzymes & peptides acquired enamel pellicle 2+, Pi, F – Ca minimize HA dissolution aid remineralization 1 Saliva composition (partial list) in most cases, plasma saliva concentrations (GCF) low fr* high fr* different mM mM mM from plasma inorganic + Na 140 20 30 saliva hypotonic + NH 0.03 0.7 0.2 4 + NH4 probably Ca2+ 1.3 1 2 from urea via Pi 1 5 2 hydrolysis HCO3– 25 2 30 F– 0.001 0.001 0.001 most concentrations pH 7.4 6 – 6.5 7.5 change with flow rate *fr = flow rate; low fr ≈ 0.3 mL/min; high fr ≈ 2.5 mL/min many with rate + [Pi] [H ] at all flow rates, ion product > K'sp for HA if pH > ~ 5.5 2 Saliva: organic components plasma glc & lipids low concentration shows saliva not fuel source for microorganisms urea glucose total lipid urea protein (mg/100mL) mM 5 20 5 7000 saliva low fr* high fr* mM mM 0.04 0.1 5 2 200 400 diffuses freely across cell membranes source of nitrogen for microorganisms some bacteria secrete urease H+ + 2 H2O + H2NCONH2 2 NH4+ + HCO3– this hydrolysis of urea tends to raise local pH 3 Salivary proteins & peptides protein/peptide ~mg/100mL function mucins 30 lubrication, coat surfaces (>40% carb.) antibacterial lysozyme 1 lysis of gram+ bacteria cell wall lactoferrin 1 binds iron, thus limiting supply to bacteria peroxidase 1 H2O2 + SCN– H2O + OSCN– thiocyanate IgA defensins 4 20 hypothiocyanite major Ab in mucous secretions cationic peptides that bind to & disrupt bacterial cell membranes Salivary proteins (cont'd) protein ~mg/100mL digestive a-amylase lipase pellicle formers proline-rich proteins (PRP) statherin cystatins histatins 5 function 50 hydrolyzes a1,4 glycosidic bonds active at low pH 50 inhibit crystal initiation & growth similar to PRPs 10 protease inhibition bacteriostatic; antifungal Saliva in diagnostics noninvasive, accessible currently available hormones (e.g., cortisol) antibodies (e.g., HIV, herpes, hepatitis B) DNA, human & microbial drugs in development 6 caries risk assessment via salivary glycoproteins glyco groups differ in their binding to microbe surfaces people have different combinations of glycoproteins that correlate with caries susceptibility pH buffering in saliva bicarbonate system most important concentration ↑ with ↑ flow rate under most conditions, its concentration highest among saliva buffers acidic form volatile (disposable) pKa – HCO3 others Pi + H+ H2CO3 H2O + CO2 6.1 HPO42– + H+ H2PO4– {protein side chains} + H+ H{protein side chains}+ HN 7 N HN N + H 7 4 -7 Acquired enamel pellicle exposure of a cleaned enamel surface to saliva results in formation of a 1-10 µm film composition: 2+ mainly H2O, protein, Ca salivary proteins adhere to polar HA surface via polar (especially ionic) interactions since proteins anionic, Ca2+ bridging important probable functions protect against acid attack (local buffering) facilitate adhesion of gingiva to enamel surface assist in remineralization bind microorganisms 8 Acquired pellicle: scanning electron microscopy cleaned enamel surface 9 same surface covered by pellicle from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 1000x) Dental plaque, a bacterial biofilm pellicle becomes plaque upon bacterial colonization adhesion of bacteria initially, adhesion is superficial like most proteins, bacteria surface has net negative charge, so Ca2+ is important as bridging agent some have specific attachment sites on surface (adhesins) later, bacteria proliferate & modify plaque sl 11 salivary proteins (mucins, etc.) bind & are modified: e.g., anionic sugars (sialate) removed plaque polysaccharide formation: with sucrose present, bacteria direct synthesis of sl 12 mutans, dextrans (glucans, i.e., polyglucoses) levans (polyfructose) 10 Mucins: bacteria-induced modification mol wt ~ 106 ~800 short (disaccharide) side chains ~ very hydrophilic, extended structure (anionic sialates) Modification ~ x H 2O sialidase x sialidase, secreted by oral bacteria, transforms mucins ~ ~ protein products are: less hydrophilic ~ less soluble ~ folded, aggregated part of the enamel pellicle & plaque matrix, where 11 they can be nutrients for bacteria galNAc sialate (neg. charged) Plaque polysaccharides functions for oral microorganisms fuel source adhesive surface anaerobic environment (cariogenic) synthesis catalyzed by bacteria-secreted enzymes (sucrases*) extracellular (amount not limited by bacteria's cell volume) sucrose main source dextran or mutan (activated precursor) sucrase G-F + G-G-G~ F + G-G-G-G~ breakdown 12 monosaccharides removed by hydrolases: dextranase, mutanase, levanase sucrose dextran fructose (G)n +1 (G)n G-F + F-F-F~ sucrose levan dextran levan sucrase G + F-F-F-F~ glucose levan (F)n (F)n +1 * aka glycosyl transferases, e.g., glucosyl [fructosyl] transferases Dental plaque: scanning EM surface colonized by bacteria after accumulation of polysaccharides from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 5000x) 13 Anaerobic acid production anaerobic as polysaccharide accumulates, relatively porous meshwork fills up, becoming less porous [O2] becomes limiting bacterial metabolism becomes more bacterium anaerobic glucose aerobic nonvolatile acids pyruvate CO2 produced pH drops lactic acid lactate + H+ HA K'sp becomes > formic acid formate + H+ acetic acid acetate + H+ ion product propionic acid propionate+ H+ HA dissolution butyric acid butyrate + H+ occurs (demin > remin) 14 Summary of bacterial carbohydrate metabolism oral bacteria use carbs for fuel, including fuel storage (plaque polysaccharides) adhesive scaffolding (plaque polysaccharides) carbon source for biosynthesis fructans glucans glycosyl transferases glc PEP pyr glc 6-P 15 sucrose PEP pyr sucrose 6-P glycolysis, etc. pyruvate lactate etc. frc PEP pyr frc 1-P phosphoryl group donor: PEP pH dependence of HA solubility stoichiometry of the dissolution reaction: Ca10(PO4)6(OH)2 + 14 H+ 10 Ca2+ + 6 H2PO4– + 2 H2O K'sp steeply dependent on [H+]: K'sp of HA 4 [Ca][Pi] > K'sp [Ca][Pi] < K'sp 2 [Ca][Pi] = K'sp 8 16 7 pH 6 5 critical pH pH change & plaque carbohydrates pH at enamel surface catabolism of carbohydrate causes acidification (acid challenge) acidification reversed by saliva components exposure time & pH change depend on plaque type 17 7 thin plaque thick polysaccharidecontaining plaque 6 critical pH 5 add sugar 30 time, min 60 Acid supply, mineral ion removal carious lesion usually has more demineralization below surface 2+ + Pi Ca surface HA less soluble due to + H higher F - content near surface limiting factors: supplying H+, removing Ca2+, Pi HA(F) HA(F) + H /Ca/Pi flow limited by spaces (pores) between HA crystals pores: 1-2% of enamel surface 10-20 Å HA open system needed to supply acid, remove products: carbohydrate 18 aerobic anaerobic H+ HA Ca2+ + Pi in CO2 (volatile acid) out HA Biochemistry of caries: summary factors favoring formation of carious lesion: 1. source of fermentable carbohydrate 2. polysaccharide-rich plaque this limits diffusion of O 2 in, acid out metabolism becomes more anaerobic glycolysis: produces lactic acid other pathways: produce acetic acid, etc. 3. pathway to remove dissolved Ca 2+ & Pi 19 if ions not removed, solution soon becomes saturated & net dissolution stops Caries summary (cont'd) factors favoring caries (cont’d) 4. total time of exposure to low pH 5. HA structure, composition high substitution with Mg 2+, CO32–, citrate; these substitute ions increase solubility low F – content limited flow of saliva – 7. limited availability of F to 6. inhibit demineralization facilitate remineralization 20 Calculus: composition composition (dry weight) 2+ 2+ 80% mineral: Ca , Pi, Mg , carbonate amorphous Ca phosphate, HA rest: plaque matrix, bacteria (fossilized) main sources of components supragingival: saliva subgingival: gingival crevicular fluid (GCF), essentially plasma containing neutrophils 21 Calculus formation mineral formation reactions Ca2+ + H2PO4– + H2O + OH – CaHPO4.2H2O 3 Ca2+ + 2 H2PO4– + 4 OH – Ca3(PO4)2 + 4 H2O formation favored by high pH essentially mineralized plaque formation inhibited by pellicle proteins (see slide 5) statherin proline-rich proteins pyrophosphate (PPi) (a component of toothpastes) 22 Gingivitis & periodontal disease unlike caries, where acids are prime culprit, damaging substances more varied chemically result of complex interaction of plaque components & host tissues products of plaque metabolism cause tissue damage directly stimulate host defensive response: inflammation inflammation components counteract bacterial actions & products also cause damage to host molecules, cells 23 Bacterial products & their effects small molecules local production of acids, especially under anaerobic conditions amines (bases), especially NH3 (e.g., from urea hydrolysis) effects mainly on host proteins unfolded (denatured) chemically modified become antigenic (recognized as foreign) become nonfunctional 24 Next time: 5. Periodontal disease Open next slide-set