4. Saliva, Pellicle, Plaque, Caries4. Saliva, Pellicle, Plaque, Caries

Section 12: Mineralized TissuesSection 12: Mineralized Tissues

3/7/063/7/06

The oral environment:The oral environment:saliva & dental plaquesaliva & dental plaque

General question:General question:How do How do

bacteria, saliva & dietary components bacteria, saliva & dietary components interact with interact with

oral structures (enamel, gingiva, etc.) oral structures (enamel, gingiva, etc.) to produce to produce

the two main dental problems: the two main dental problems:

caries & periodontal diseasecaries & periodontal disease? ?

Saliva functionsSaliva functionsdigestivedigestive

food: dissolved, softened, dispersed, lubricatedfood: dissolved, softened, dispersed, lubricated enzymes: enzymes: -amylase (ptyalin)-amylase (ptyalin)

lipaselipaseprotectiveprotective

washes away microorganisms, toxinswashes away microorganisms, toxins coats epithelial surface (mucus barrier)coats epithelial surface (mucus barrier) minimizes minimizes pH (buffering)pH (buffering) proteins: proteins: antibodiesantibodies

antibacterial enzymes & peptidesantibacterial enzymes & peptidesacquired enamel pellicleacquired enamel pellicle

CaCa 2+2+, P, Pii, F, F –– minimize HA dissolution minimize HA dissolution

aid remineralizationaid remineralization11

Saliva composition Saliva composition (partial list)(partial list) in most cases, in most cases,

concentrations concentrations different different from plasmafrom plasma saliva hypotonicsaliva hypotonic NHNH44

++ probably probably from urea via from urea via hydrolysishydrolysis

most concentrations most concentrations change with change with flow rateflow rate many many with with raterate [P[Pii] ]

at all flow rates, ion product > K'at all flow rates, ion product > K'sp sp for HA if pH > ~for HA if pH > ~ 5.55.5

plasma plasma saliva saliva (GCF)(GCF) low fr*low fr* high fr* high fr* mM mM mM mM mM mM

inorganicinorganicNaNa++ 140140 2020 3030NHNH44

++ 00..0303 00..77 00..22

CaCa2+2+ 11..33 11 22PPii 11 55 22HCOHCO33

–– 2525 22 3030

FF–– 00..001001 00..001001 0 0..001001 pHpH 77..44 6 – 6 6 – 6..55 77..55

*fr = flow rate;*fr = flow rate;low fr ≈ 0.3 mL/min; high fr ≈ 2.5 mL/minlow fr ≈ 0.3 mL/min; high fr ≈ 2.5 mL/min

22

[H[H++] ]

Saliva: organic componentsSaliva: organic components glc & lipidsglc & lipids

low concentration low concentration shows saliva not fuel shows saliva not fuel source for source for microorganismsmicroorganisms

ureaurea diffuses freely across cell membranesdiffuses freely across cell membranes source of nitrogen for microorganismssource of nitrogen for microorganisms some bacteria secrete some bacteria secrete ureaseurease

HH++ + 2 H + 2 H22O + HO + H22NCONHNCONH22 2 NH 2 NH44++ + HCO + HCO33

––

this hydrolysis of urea tends to raise local pHthis hydrolysis of urea tends to raise local pH

plasma plasma saliva saliva low fr*low fr* high fr* high fr*

mM mM mM mM mM mM

glucoseglucose 55 0.04 0.04total lipidtotal lipid 20 20 0.1 0.1

ureaurea 55 5 5 22proteinprotein(mg/100mL)(mg/100mL) 70007000 200200 400400

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Salivary proteins & peptidesSalivary proteins & peptides

protein/peptide ~mg/100mLprotein/peptide ~mg/100mL functionfunction

mucinsmucins 30 30 lubrication, coat surfaceslubrication, coat surfaces (>40% carb.)(>40% carb.)

antibacterialantibacteriallysozymelysozyme 11 lysis of gramlysis of gram++ bacteria cell wall bacteria cell walllactoferrinlactoferrin 11 binds iron, thus limiting supply binds iron, thus limiting supply

to bacteria to bacteriaperoxidaseperoxidase 1 1 HH22OO22 + SCN + SCN–– HH22O + OSCNO + OSCN––

thiocyanatethiocyanate hypothiocyanite hypothiocyanite

IgAIgA 2020 major Ab in mucous secretionsmajor Ab in mucous secretionsdefensinsdefensins cationic peptides that bind to & cationic peptides that bind to & disrupt bacterial cell membranes disrupt bacterial cell membranes

44

Salivary proteins (cont'd)Salivary proteins (cont'd)

protein ~mg/100mLprotein ~mg/100mL function function

digestivedigestive-amylase-amylase 5050 hydrolyzes hydrolyzes 1,4 glycosidic bonds1,4 glycosidic bondslipaselipase active at low pHactive at low pH

pellicle formers pellicle formers

proline-richproline-rich 5050 inhibit crystal initiationinhibit crystal initiation proteins (PRP)proteins (PRP) & growth & growth

statherinstatherin 1010 similar to PRPssimilar to PRPs

cystatinscystatins protease inhibitionprotease inhibition

histatinshistatins bacteriostatic; antifungalbacteriostatic; antifungal

55

Saliva in diagnosticsSaliva in diagnosticsnoninvasive, accessiblenoninvasive, accessible currently availablecurrently available

hormones (e.g., cortisol)hormones (e.g., cortisol) antibodies (e.g., HIV, herpes, hepatitis B)antibodies (e.g., HIV, herpes, hepatitis B) DNA, human & microbialDNA, human & microbial drugsdrugs

in developmentin development caries risk assessmentcaries risk assessment

via salivary glycoproteinsvia salivary glycoproteins glyco groups differ in their binding to microbe surfacesglyco groups differ in their binding to microbe surfaces people have different combinations of glycoproteinspeople have different combinations of glycoproteins

that correlate with caries susceptibilitythat correlate with caries susceptibility

66

pH buffering in salivapH buffering in salivabicarbonate system most importantbicarbonate system most important

concentration concentration ↑↑ with with ↑↑ flow rate flow rate under most conditions, its concentration highest among under most conditions, its concentration highest among

saliva bufferssaliva buffers acidic form volatile (disposable)acidic form volatile (disposable)

pKpKaa

HCOHCO33–– + H + H++ H H22COCO33 H H22O + COO + CO22 6.16.1

othersothers P PiiHPOHPO44

2–2– + H + H++ H H22POPO44–– 77

{protein side chains} + H{protein side chains} + H++ H{protein side chains} H{protein side chains}++ 44 -7-7

NHN

NHN

+

H77

Acquired enamel pellicleAcquired enamel pellicle exposure of a cleaned enamel surface to exposure of a cleaned enamel surface to

saliva results in formation of a 1-10 µm filmsaliva results in formation of a 1-10 µm film composition:composition:

mainly Hmainly H22O, protein, CaO, protein, Ca2+2+

salivary proteins adhere to polar HA surface via polar salivary proteins adhere to polar HA surface via polar (especially ionic) interactions(especially ionic) interactions

since proteins anionic, Casince proteins anionic, Ca2+2+ bridging important bridging importantprobable functionsprobable functions

protect against acid attack (local buffering)protect against acid attack (local buffering)facilitate adhesion of gingiva to enamel surfacefacilitate adhesion of gingiva to enamel surfaceassist in remineralizationassist in remineralizationbind microorganismsbind microorganisms

88

Acquired pellicle:Acquired pellicle:scanning electron microscopyscanning electron microscopy

cleaned enamel surfacecleaned enamel surface same surface covered by pelliclesame surface covered by pellicle

from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 1000x)from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 1000x)99

Dental plaque, a bacterial biofilmDental plaque, a bacterial biofilmpellicle becomes plaque upon bacterial colonizationpellicle becomes plaque upon bacterial colonizationadhesion of bacteriaadhesion of bacteria

initially, adhesion is superficialinitially, adhesion is superficial like most proteins, bacteria surface has net negative charge, like most proteins, bacteria surface has net negative charge,

so Caso Ca2+2+ is important as bridging agent is important as bridging agent some have specific attachment sites on surface (some have specific attachment sites on surface (adhesinsadhesins))

later, bacteria proliferate & modify plaquelater, bacteria proliferate & modify plaque salivary proteins (mucins, etc.) bindsalivary proteins (mucins, etc.) bind

& are modified: & are modified: e.g.e.g., anionic sugars (sialate) removed, anionic sugars (sialate) removed plaque polysaccharide formation:plaque polysaccharide formation:

with sucrose present, bacteria direct synthesis ofwith sucrose present, bacteria direct synthesis of mutans, dextrans (glucans, i.e., polyglucoses)mutans, dextrans (glucans, i.e., polyglucoses) levans (polyfructose)levans (polyfructose)

1010

sl 11sl 11

sl 12sl 12

Mucins: bacteria-induced modificationMucins: bacteria-induced modification mol wt ~ 10mol wt ~ 1066

~800 short (disaccharide) ~800 short (disaccharide) side chainsside chains

very hydrophilic, extended very hydrophilic, extended structure (anionic sialates)structure (anionic sialates)

ModificationModification sialidase,sialidase, secreted by oral secreted by oral

bacteria, transforms mucinsbacteria, transforms mucins protein products are:protein products are:

less hydrophilicless hydrophilic less solubleless soluble folded, aggregatedfolded, aggregated part of the enamel pellicle & plaque matrix, wherepart of the enamel pellicle & plaque matrix, where

they can be nutrients for bacteriathey can be nutrients for bacteria

galNAcgalNAcsialatesialate(neg.(neg.charged)charged)

sialidasex H2O

~ ~

~~

x

~~

1111

Plaque polysaccharidesPlaque polysaccharides functions for oral microorganismsfunctions for oral microorganisms

fuel sourcefuel source adhesive surfaceadhesive surface anaerobic environment (cariogenic)anaerobic environment (cariogenic)

synthesissynthesis catalyzed by bacteria-secreted enzymes (catalyzed by bacteria-secreted enzymes (sucrases*sucrases*)) extracellular (amount not limited by bacteria's cell volume)extracellular (amount not limited by bacteria's cell volume) sucrose main sourcesucrose main source

(activated precursor)(activated precursor) breakdownbreakdown

monosaccharidesmonosaccharidesremoved by removed by hydrolases:hydrolases:dextranase, mutanase, dextranase, mutanase, levanaselevanase

GG-F + G-G-G~ F + -F + G-G-G~ F + GG-G-G-G~-G-G-G~

dextran or mutandextran or mutansucrasesucrase

sucrosesucrose dextran dextran

(G)(G)nn

fructosefructose dextrandextran

(G)(G)nn +1+1

G-G-FF + F-F-F~ G + + F-F-F~ G + FF-F-F-F~-F-F-F~

levanlevansucrasesucrase

sucrosesucrose levan levan

(F)(F)nn

glucoseglucose levan levan

(F)(F)nn +1+1

1212 * * akaaka glycosyl transferases, e.g., glucosyl [fructosyl] transferases glycosyl transferases, e.g., glucosyl [fructosyl] transferases

Dental plaque: scanning EMDental plaque: scanning EMsurface colonized by bacteriasurface colonized by bacteria

from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 5000x)

after accumulation of polysaccharides

13

Anaerobic acid productionAnaerobic acid production as polysaccharide accumulates, relatively porous meshwork as polysaccharide accumulates, relatively porous meshwork

fills up, becoming less porousfills up, becoming less porous [O[O22] becomes limiting] becomes limiting bacterial metabolismbacterial metabolism

becomes more becomes more anaerobicanaerobic

nonvolatile acids nonvolatile acids producedproduced

pH dropspH drops HA K'HA K'spsp becomes > becomes >

ion product ion product HA dissolution HA dissolution

occursoccurs

glucose aerobic pyruvate CO2 lactate + H+

formate + H+

acetate + H+

propionate+ H+ butyrate + H+

lactic acid lactic acid formic acid formic acid acetic acid acetic acid

propionic acid propionic acid butyric acid butyric acid

anaerobic

bacteriumbacterium

(demin > remin)(demin > remin)14

Summary of bacterial carbohydrate metabolismSummary of bacterial carbohydrate metabolismoral bacteria use carbs fororal bacteria use carbs for

fuel, including fuel storage (plaque polysaccharides)fuel, including fuel storage (plaque polysaccharides) adhesive scaffolding (plaque polysaccharides)adhesive scaffolding (plaque polysaccharides) carbon source for biosynthesiscarbon source for biosynthesis

1515

fructans glucans

glc sucrose frc

glc 6-P sucrose 6-P frc 1-P

pyruvatelactate

etc.

phosphorylgroup donor:PEP

glycosyl transferasesglycosyl transferases

PEPPEP

pyrpyrPEPPEP

pyrpyr

PEPPEP

pyrpyr

glycolysis, etc.

pH dependence of HA solubilitypH dependence of HA solubility

stoichiometry of the dissolution reaction:stoichiometry of the dissolution reaction:

CaCa1010(PO(PO44))66(OH)(OH)22 + 14 H + 14 H++ 10 Ca 10 Ca2+2+ + 6 H + 6 H22POPO44–– + 2 H + 2 H22OO

K'K'spsp steeply dependent on [H steeply dependent on [H++]:]:

8 7 6 5

2

4

K's

p o

f H

A

pH critical pH

[Ca][P[Ca][Pii] ] << K' K'spsp[Ca][P[Ca][Pii] ] >> K' K'spsp

[Ca][P[Ca][Pii] ] == K' K'spsp

16

pH change & plaque carbohydratespH change & plaque carbohydrates

catabolism of carbohydrate causes acidificationcatabolism of carbohydrate causes acidification(acid challenge)(acid challenge)

acidification reversed by saliva componentsacidification reversed by saliva components exposure time & pH change depend on plaque typeexposure time & pH change depend on plaque type

3030 6060time, mintime, min

77

66

55

pH a

t en

amel

sur

face

pH a

t en

amel

sur

face

thinthinplaqueplaque

addaddsugar

critical pHcritical pH

thickthickpolysaccharide-polysaccharide-containing plaquecontaining plaque

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carious lesion usually has more demineralization carious lesion usually has more demineralization below surfacebelow surface

surface HA less soluble due to surface HA less soluble due to higher Fhigher F -- content near surface content near surface

limiting factors: supplying Hlimiting factors: supplying H++,,removing Caremoving Ca2+2+, Pi, Pi

HH++/Ca/P/Ca/Pii flow limited by spaces flow limited by spaces ((porespores) between HA crystals) between HA crystals

porepore s:s: 1-2% of enamel surface1-2% of enamel surface 10-20 Å10-20 Å

open systemopen system needed to supply needed to supplyacid, remove products:acid, remove products:

carbohydrate carbohydrate H H++ HA HA Ca Ca2+2+ + P + Pii

aerobicaerobic CO CO22 (volatile acid) (volatile acid)

Acid supply, mineral ion removalAcid supply, mineral ion removal

HA(F)

HA HA

HA(F)

H+Ca

2+ + Pi

anaerobic

in out

18

Biochemistry of caries: summaryBiochemistry of caries: summary

factors favoring formation of carious lesion:factors favoring formation of carious lesion:

1.1. source of fermentable carbohydratesource of fermentable carbohydrate

2.2. polysaccharide-rich plaquepolysaccharide-rich plaque this limits diffusion of Othis limits diffusion of O 22 in, acid out in, acid out metabolism becomes more anaerobicmetabolism becomes more anaerobic

glycolysisglycolysis: produces lactic acid: produces lactic acidother pathwaysother pathways: produce acetic acid, etc.: produce acetic acid, etc.

3. pathway to remove dissolved Ca 2+ & Pi

if ions not removed, solution soon becomes saturatedif ions not removed, solution soon becomes saturated & net dissolution stops & net dissolution stops

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Caries summary (cont'd)Caries summary (cont'd)

factors favoring caries (cont’d)factors favoring caries (cont’d)

4.4. total time of exposure to low pHtotal time of exposure to low pH

5.5. HA structure, compositionHA structure, composition high substitution with Mghigh substitution with Mg

2+2+, CO, CO332–2–, citrate;, citrate;

these substitute ions increase solubility these substitute ions increase solubility low Flow F

–– content content

6.6. limited flow of salivalimited flow of saliva

7.7. limited availability of limited availability of FF –– to to

inhibit demineralizationinhibit demineralization facilitate remineralizationfacilitate remineralization

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Calculus: compositionCalculus: composition

composition (dry weight)composition (dry weight)80% mineral: Ca80% mineral: Ca2+2+, P, Pii, Mg, Mg2+2+, carbonate, carbonate

amorphous Ca phosphate, HA amorphous Ca phosphate, HArest: plaque matrix, bacteria (fossilized)rest: plaque matrix, bacteria (fossilized)

main sources of componentsmain sources of componentssupragingivalsupragingival: saliva: salivasubgingivalsubgingival: gingival crevicular fluid (GCF),: gingival crevicular fluid (GCF),

essentially plasma containing neutrophils essentially plasma containing neutrophils

21

Calculus formationCalculus formationmineral formation reactionsmineral formation reactions

CaCa2+2+ + H + H22POPO44–– + H + H22O + OHO + OH –– CaHPO CaHPO44

..2H2H22OO

3 Ca3 Ca2+2+ + 2 H + 2 H22POPO44–– + 4 OH + 4 OH –– CaCa33(PO(PO44))22 + 4 H + 4 H22OO

formation favored by formation favored by high pHhigh pH essentially mineralized plaqueessentially mineralized plaque formation inhibited by formation inhibited by

pellicle proteins pellicle proteins ((see slide 5see slide 5)) statherinstatherin proline-rich proteinsproline-rich proteins

pyrophosphate (PPi)pyrophosphate (PPi) (a component of toothpastes) (a component of toothpastes)

22

Gingivitis & periodontal diseaseGingivitis & periodontal diseaseunlike caries, where acids are prime culprit, unlike caries, where acids are prime culprit,

damaging substances more varied chemicallydamaging substances more varied chemically result of complex interaction of plaque components result of complex interaction of plaque components

& host tissues& host tissuesproducts of plaque metabolismproducts of plaque metabolism

cause tissue damage directlycause tissue damage directlystimulate host defensive response: stimulate host defensive response: inflammationinflammationinflammationinflammation componentscomponents

counteract bacterial actions & productscounteract bacterial actions & products also cause damage to host molecules, cellsalso cause damage to host molecules, cells

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Bacterial products & their effectsBacterial products & their effects small moleculessmall molecules

local production oflocal production of acids, especially under anaerobic conditionsacids, especially under anaerobic conditions amines (bases),amines (bases),

especially NH especially NH33 (e.g., from urea hydrolysis) (e.g., from urea hydrolysis)effects mainly oneffects mainly on host proteinshost proteins

unfolded (denatured)unfolded (denatured) chemically modifiedchemically modified become antigenic (recognized as foreign)become antigenic (recognized as foreign) become nonfunctionalbecome nonfunctional

24

Next time:Next time:

5.

Periodontal diseasePeriodontal disease

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