4. Saliva, Pellicle, Plaque, Caries Section 12: Mineralized Tissues 3/7/06.
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Transcript of 4. Saliva, Pellicle, Plaque, Caries Section 12: Mineralized Tissues 3/7/06.
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
33
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
17
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
19
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
20
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
23
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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