Transcript The oral environment: saliva & dental plaque

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
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