1. Section 12: Mineralized Tissues
4. Saliva, Pellicle, Plaque, Caries
Dental fluorosis is only a problem when exposure is during enamel formation.
3/7/06

2. 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?
Looking at molecular aspect of how enamel, gingival tissue, period tissues are affected by microorganisms, by the solvent like saliva, gingival clavicular fluid, dietary factors like sucrose that many others contribute, how do they get together and cause the problems. 1

3. 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
Ca 2+, Pi, F – minimize HA dissolution aid remineralization
Touched on saliva and review some of the things mentioned, divide the role of saliva into two main categories, digestive one where saliva is solvent or dispersant, also supplies enzymes for digestion although both are considered minor factors in the digestive process. More important is the group of effects, protective where dissolution and washing away of materials so that they don’t accumulate is important as a protector of the soft tissue of oral cavity. Mucous material also important effect. At the molecular biochemical level, fact that components cannot prevent or diminish or minimize changes in pH can also occur, also a valuable contribution. There are several proteins that have protective effects, 1

4. Saliva composition (partial list)
in most cases, concentrations different from plasma
saliva hypotonic
NH4+ probably from urea via hydrolysis
most concentrations change with flow rate
many ­ with ­rate
[Pi] ¯
at all flow rates, ion product > K'sp for HA if pH > ~ 5.5
plasma saliva
(GCF) low fr* high fr* mM mM mM
inorganic Na NH Ca
Pi 1 5 2
HCO3– F–
pH –
*fr = flow rate; low fr ≈ 0.3 mL/min; high fr ≈ 2.5 mL/min
Representative list of components. Like most biological fluids, saliva is derived from blood plasma, but very different in composition. The process by which saliva is made is something that will be covered in more detail in physiology. Plasma is the source, also added this here, the plasma has a composition of the gingival clavicular fluid, another fluid that plays are role in oral problems or prevention. Here the composition of this fluid, is pretty much the same as plasma, a filtrate of plasma, doesn't have same cell composition as blood but other components are pretty much the same. Nutrient source. You can see the concentration of things like sodium is much lower in saliva, in general hypotonic solution compared to plasma, fluoride is exception but concentration is low. In ammonia, normally very low in plasma, not something found in freshly secreted saliva. These numbers apply to whole saliva, its been subject to the action of bacteria causing the ammonia concentration not go up. Another important factor with regard to components of saliva is concentrations change as flow rate changes. Resting conditions, low flow rate, these two columns represent low flow rate conditions and high flow rate conditions. Going from low to high flow rate, calcium concentration doubles, phosphate goes in the opposite direction, going down by more than half, very important change in flow rate, consequence is bicarbonate going from low concentration to a number that is higher than that of plasma. Going along with that, see that pH resting condition is acidic but going to alkaline side as flow rate increases. Several substances, sodium and others not shown, calcium and bicarbonate go up in concentration as flow rate goes up, phosphate goes down, and given that pH goes up, hydrogen ion concentration goes down with increasing flow rate. as mentioned before, look at these numbers, calcium and phosphate numbers, ion product of those exceeds the solubility product for hydroxyapatite under the usual conditions, if the pH is slightly acidic for alkaline side. Under conditions described here, have supersaturating situation at all times.
[H+] ¯ 2

5. Saliva: organic components
glc & lipids
low concentration shows saliva not fuel source for microorganisms
urea
diffuses freely across cell membranes
source of nitrogen for microorganisms
some bacteria secrete urease
H H2O + H2NCONH2 ® 2 NH HCO3–
this hydrolysis of urea tends to raise local pH
plasma saliva
low fr* high fr* mM mM mM
glucose
total lipid
urea
protein (mg/100mL)
Organic components of saliva. Notice that the concentration of such tings as glucose, lipid concentration are low compared to plasma, these are here to make the point that saliva as such without dietary components, saliva is not a good source of energy. Urea is an organic component hat is of some significance under resting conditions. Goes down as flow rate increases. Urea is one of the small molecules, polar that goes everywhere, hard to contain it, that’s why it ends up in saliva. One effect that is inevitable when bacteria are around is urea are hydrolyzed by urease, enzyme that we don’t have in our cells, we have no reason to hydrolyze it so we don’t have it. Microorganisms to grow need nitrogen, and when scarce they can use this ammonia produced this way and has adverse effect in any place where bacterial growth is not good. The beneficial effect is that you can see it consumes a hydrogen ion. This reaction to the extent that it occurs has a pH increasing effect, consumes hydrogen ions that might be produced elsewhere. This presence of urea consumes hydrogen ions but supplies nitrogen in the form of ammonia. Protein concentration is much lower than that of plasma, also rises with flow rate. Proteins are of some value with buffering and protection 3

6. 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 hypothiocyanite
IgA 20 major Ab in mucous secretions
defensins cationic peptides that bind to & disrupt bacterial cell membranes
Several proteins in saliva target various bacteria. Lysozyme, secretions in general, mucous secretions in tears, all of them that are exposed to outside world, target gram positive bacteria, stunt growth and proliferation. More recent protein is the lactoferrin by binding up iron, limits supply of bacteria when iron is in short supply. Another enzyme that has a antibacterial effect is perioxidase converting hydrogen peroxide to hypothiocyanite, toxic to some bacteria, and hydrogen peroxide is also toxic. Thiocyanate as component of saliva is relatively recent discovery. Mention again antibody IgA secreted by that process, then another set of recently characterized materials, not proteins but peptides that have an antibacterial effect, disrupting the membranes. Digestive proteins put on for completeness. 4

7. Salivary proteins (cont'd)
protein ~mg/100mL function
digestive
a-amylase 50 hydrolyzes a1,4 glycosidic bonds
lipase active at low pH
pellicle formers
proline-rich 50 inhibit crystal initiation
proteins (PRP) & growth
statherin 10 similar to PRPs
cystatins protease inhibition
histatins bacteriostatic; antifungal
mucin concn based on "Basic & Applied Dental Bchm" p. 370
Classified as components of pellicle and their roles, some of the effects are shown. PRP limit the formation of crystals which would make them anti-calculus components. Inhibition of protease or some bacteriostatic effect. 5

8. Saliva in diagnostics noninvasive, accessible currently available
hormones (e.g., cortisol)
antibodies (e.g., HIV, herpes, hepatitis B)
DNA, human & microbial
drugs
in development
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
Tabak, JDE 12/01 AAAS innov- ations report, 3/05
The use of saliva as a source or easily accessible fluid through which diagnostic procedures can be done. Some of the new things relevant to dental practice is use of proteins in saliva to access risk of carries for individual people. These proteins seem to be different and correlated with the caries risk of individuals. The idea is by characterizing the mix of these proteins in saliva, would be possible to classify individuals in terms of susceptibility to caries, dictates several therapeutic characteristics. 6

9. 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– + H+ « H2CO3 « H2O + CO2­ 6.1
others Pi HPO42– + H+ « H2PO4– 7
{protein side chains} + H+ « H{protein side chains}
Reminder of how buffering works, of the components in saliva, bicarbonate is the most important, get more of bicarbonate as flow rate increases, so the reaction, equilibria for bicarbonate CO2 system, in context of product of metabolism, important that carbon dioxide as acidic component is something that is volatile, can be gotten rid of , can get rid of more the faster it is produced. In most cases buffers only limit pH because they bind up the hydrogen ions, in this system its disposing of the acidic form and is much more valuable, despite the fact that pKa is not in the optimal range as in the case of saliva, it’s a better buffer. Here’s the phosphate system, protein side chains such as the histidine side chain, basic form, acidic form on the left, also bind with and buffer protons. Carboxyl groups on proteins, proteins have lots of carboxyl group side chains that have pKas that are more acidic, these two come into play as it gets more acidic. Carboxyl groups are generally to good as buffers in the blood. In the oral cavity, low pH will be offset by protons. 7

10. Acquired enamel pellicle
exposure of a cleaned enamel surface to saliva results in formation of a 1-10 µm film
composition:
mainly H2O, protein, Ca2+
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
Clin Cariol ~1um p.89 10um p.41
Exposure of enamel to saliva, get this material forming on the surface as soon as its cleaned, the pellicle, containing an extract of saliva, different concentrations of water protein and ions compared to saliva, those are the sources of the materials. We have these interactions that are a consequence of fact that hydroxyapatite surface is polar, and so you have ionic interactions. Calcium, not on in the mineral but form solution is considered important component of this in so far as it being positively charged, bridging two negative components. Pellicle, role is considered established in so far as buffering ,based on proteins and other components in pellicle, offset pH changes. Adhesion of gingival to the surface of enamel is considered valuable thing, then as a source of substances which can help remineralization, calcium and the like, that would be beneficial effect, mixed bag would be ability to bind microorganisms. 8

11. Acquired pellicle: scanning electron microscopy
cleaned enamel surface same surface covered by pellicle
EM of cleaned enamel surface, within seconds, material that forms on the surface, this group corresponds. See how rapidly its covered over with material that is known as the pellicle.
from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 1000x) 9

12. 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
salivary proteins (mucins, etc.) bind & are modified: e.g., anionic sugars (sialate) removed
plaque polysaccharide formation: with sucrose present, bacteria direct synthesis of
mutans, dextrans (glucans, i.e., polyglucoses)
levans (polyfructose)
sl 11
Pellicle does not have microorganisms as part of its structure but as soon as bacteria colonize this material, it becomes known as dental plaque, get these bacteria that have to bind, otherwise tend to be washed away, there are proteins known as adhesins that some bacteria have on their surface that are complementary to components of the pellicle proteins and in so doing, they adhere more successfully.
Another factor that bacteria in plaque are going to do, esp if there is sucrose is to make plaque polysaccharides, mutans, dextrans, or fructose is the monomer then levans, by way of review. This is something covered in carb section
sl 12 10

13. Mucins: bacteria-induced modification
mol wt ~ 106
~800 short (disaccharide) side chains
very hydrophilic, extended structure (anionic sialates)
Modification
sialidase, secreted by oral bacteria, transforms mucins
protein products are:
less hydrophilic
less soluble
folded, aggregated
part of the enamel pellicle & plaque matrix, where they can be nutrients for bacteria ~ ~
x H2O
galNAc
sialate
(neg.
charged)
sialidase x ~ ~
Old slide as reminder that mucins have this structure, very polar structure as a consequence of these side chains. Short chains of saccharides, terminal sialate is most significant because its negatively charged, that gives the mucins a hydrophilic property, and extended structure, spread out as consequence of charge repulsion, later if the proteins become modified, bring in a protein that humans don’t contribute, but bacteria do, sialidases removes negative charges and this results in a protein that is modified so as to be less hydrophilic, less extended, and also less soluble, more likely to aggregate with one another and on surfaces like the enamel. Another factor is these proteins, as they contribute to formation of plaque, they are polypeptides of amino acids, as bacteria hydrolyze these, can serve as source of nutrients for bacteria. 11

14. 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 (activated precursor)
breakdown
monosaccharides removed by hydrolases: dextranase, mutanase, levanase
dextran or mutan sucrase
G-F + G-G-G~ F + G-G-G-G~
sucrose dextran (G)n
fructose dextran (G)n +1
Preexisting polymer, polyglucoses, you can add another glucose from sucrose, in this reaction catalyzed by these enzymes known as sucrases, glycosyl transferases also, either glycosyl transferase is glucose is transferred or fructosyl transferase. Either case, these plaque polysaccharides can serve as places to which the bacteria can adhere and also as source of monosaccharides, a stored fuel form. These polysaccharides can be converted to monosaccharide b set of enzymes that hydrolyze monomers of dextranase etc.
levan sucrase
G-F + F-F-F~ G F-F-F-F~
sucrose levan (F)n
glucose levan (F)n +1 12
* aka glycosyl transferases, e.g., glucosyl [fructosyl] transferases

15. Dental plaque: scanning EM
after accumulation of polysaccharides
surface colonized by bacteria
EM of a surface that shows bacteria have been added to the mix and in this pic see combinations of bacteria like this one, others are embedded in this matrix, less likely to be removable by solvent, saliva action. The material after you add polysaccharides, can barely see the bacteria, when you compare the two, have a material on the right that makes a better home for bacteria
from Jenkins, "Physiology & Biochemistry of the Mouth" (magnification ~ 5000x) 13

16. Anaerobic acid production
as polysaccharide accumulates, relatively porous meshwork fills up, becoming less porous
[O2] becomes limiting
bacterial metabolism becomes more anaerobic
nonvolatile acids produced
pH drops
HA K'sp becomes > ion product
HA dissolution occurs
bacterium
glucose aerobic pyruvate ® CO 
lactate + H+
formate + H+
acetate + H+ propionate+ H+ butyrate + H+
lactic acid 
formic acid 
acetic acid 
propionic acid 
butyric acid 
The effect of having polysaccharides in foods making the metabolism a much less oxygen supported. Glucose to pyruvate to carbon dioxide is aerobic, get lots of energy this way, the end product is carbon dioxide that doesn’t acidify environment but if there isn't enough oxygen then pyruvate will be anaerobically converted to lactate, and some of the other acids will also be produced. All of the acids share the common property of if they build up, combine with hydrogen ion, diffuse from bacterium, and when they get out, acidify the environment. Acid forms shown, but these dissociate and make the environment very acidic. Supersaturation condition translates into undersaturated condition, and you have the hydroxyapatite solubility dissolution takes place and demin solution takes place outdoes the remin, and eventually carious lesion formation occurs.
anaerobic
(demin > remin)
greater than 14

17. 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
glc sucrose frc
glycosyl transferases
PEP
pyr
PEP
pyr
PEP
pyr
phosphoryl group donor: PEP
In bacteria, when mono or disaccharides get into the microorganism, phosphorylation is by PEP instead of ATP. We use ATP, als ados disaccharide gets across, we don’t take in disaccharides in any form, we need monosaccharide. Once inside it produces pyruvate, lactate. Outside production of saccharides by transferase producing polymers, sources of adhesion and also are ways that fuel can be stored up and later broken down.
glc 6-P sucrose 6-P frc 1-P
adapted fr S6L2
glycolysis, etc.
pyruvate lactate etc. 15

18. pH dependence of HA solubility
stoichiometry of the dissolution reaction:
Ca10(PO4)6(OH) H+ ® 10 Ca H2PO4– + 2 H2O
K'sp steeply dependent on [H+]: 4
[Ca][Pi] > K'sp
[Ca][Pi] < K'sp
Property of mineral in enamel. The plotting of solubility, as function of pH illustrates how strongly dependent solubility is on pH. Rationalize that with balanced equation, showing solid state, each one of those, 14 hydrogen ions in order to be transferred into liquid state, solution dissolved state. That’s what goes on when dissolution of biomineral takes place, in formation of carious lesion, remodeling of bone. Net change at chemical level. Reminder of how pH dictates the solubility. Liquid state is calcium and phosphate, if its greater than sign, pointing in direction of tendency of what will happen. This is the supersaturating condition. When we get to pH known as critical pH, between 5 and 6, then the situation where two factors are equal is attained. Equilib at a single pH, lower than that, the ion product is less then Ksp, undersaturating condition, solid state is going to ward dissolved state.
K'sp of HA 2
[Ca][Pi] = K'sp 8 7 6 5 pH 16
critical pH

19. pH change & plaque carbohydrates
catabolism of carbohydrate causes acidification (acid challenge)
acidification reversed by saliva components
exposure time & pH change depend on plaque type 7
thick
polysaccharide-
containing plaque
thin plaque
As a function of time, connection with metabolism of sugar. What is plotted is pH at the surface of enamel as a function of time after carbohydrate sugar is added. This enables the point that you will get some pH drop as consequence of increased metabolism of carbs. Glycolysis goes faster when there are more carbs around. If the plaque is thin, then the pH will drop but reach some minimal level and because of saliva, the pH reverses and retains original pH level. Minimal in terms of hydroxyapatite. If plaque is thicker with polysaccharide, pH drop is greater, goes below critical pH, and here dissolution tendency present. Sugars run out eventually and buffers over come it. 6
pH at enamel surface
critical pH 5
add 30 60
sugar
time, min 17

20. Acid supply, mineral ion removal
carious lesion usually has more demineralization below surface
surface HA less soluble due to higher F - content near surface
limiting factors: supplying H+, removing Ca2+, Pi
H+/Ca/Pi flow limited by spaces (pores) between HA crystals
pore s: 1-2% of enamel surface Å
open system needed to supply acid, remove products:
carbohydrate H+ ® HA ® Ca2+ + Pi
aerobic CO2 (volatile acid)
Ca 2+ + Pi H+
HA(F)
HA(F)
Hydrogen ions must get to place were dissolution happens, hydrogen ions must get to mineral in order to participate in the reaction that will cause dissolution. In order for reaction to proceed, products of the reaction need to be removed as well. The spaces are the regions where hydrogen ions must diffuse into and also recall that surface hydroxyapatite that has more fluoride is less soluble, less subject to dissolution. Arrows show diffusion of hydrogen ions that get to subsurface and produce dissolution. In addition, calcium and phosphate produced in the liquid state needs to diffuse out of this solid and be washed away. Need supply of hydrogen ions and removal of calcium and phosphate. Carbohydrate and aerobic metabolism producing carbon dioxide that has to be gotten rid of. As metabolism is anaerobic, hydrogen ions are not as removable, stick around, and get into hydroxyapatite and calcium and phosphate diffuse out and result is loss of mineral. HA HA
anaerobic in
out 18

21. Biochemistry of caries: summary
factors favoring formation of carious lesion:
source of fermentable carbohydrate
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.
pathway to remove dissolved Ca 2+ & Pi
if ions not removed, solution soon becomes saturated & net dissolution stops
Need fermentable carbs. Fermentation implies anaerobic metabolism. Carbohydrate produces products that are not volatile or incompletely. If polysaccharides are plentiful, more anaerobic, carbon dioxide does not get out as well. Need a way to get rid of calcium and phosphate, or situation would become saturated, you don’t get dissolution. 19

22. Caries summary (cont'd)
factors favoring caries (cont’d)
total time of exposure to low pH
HA structure, composition
high substitution with Mg 2+, CO32–, citrate; these substitute ions increase solubility
low F – content
limited flow of saliva
limited availability of F – to
inhibit demineralization
facilitate remineralization
The more time of exposure, and how low pH gets is contributory. Structure of hydroxyapatite, ion exchange for calcium and phosphate make the mineral more soluble whereas fluoride is the opposite effect. Saliva as contributor as source of remineralization and also as pH buffer is important factor as well. Fluoride if its not around, wont facilitate remineralization process. 20

23. Calculus: composition
composition (dry weight)
80% mineral: Ca2+, Pi, Mg2+, 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
Calculus contains many of the things that other minerals do. Its much less organized than hydroxyapatite, get calcium and phosphate and other mineral components, macromolecules would be from matrix. Whatever bacteria might have been around when remin takes place would also be there as well. Depending on whether this mineral is supragingival or subgingival, main source of components is saliva in the first case and with subgingival, fluid is plasma, GCF, contains either no cells or if mild inflammation, neutrophils. A plasma which is a rich source of nutrients for bacteria, good buffering capacity. As potential source of nutrients, source of minerals because plasma is also super saturated. 21

24. Calculus formation
mineral formation reactions Ca2+ + H2PO4– + H2O + OH – ® CaHPO4.2H2O
3 Ca 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)
Reactions that produce mineral. Like formation, biomineralization in forming hydroxyapatite have the pH dependence. Hydroxide is on the left side, in general, high pH is favorable towards formation of these minerals. Conversely, low pH would tend to shift them to the left or slow down the formation. There are several substances that have been investigated as sources. In addition to formation of calculus, couple proteins in saliva, deposit on enamel surface, have calculus forming inhibitory properties. Added to toothpaste is PPi, binding of calcium, lowers concentration of calcium. 22

25. 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
Some products in metabolism of plaque have adverse effects on tissue proteins. In other cases, effect is indirect, and products of plaque metabolism set in motion host response. Inflammation is there to minimize effect of infections and other traumatic events. It does have some problems, several substances that bacteria make and classify them 23

26. 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
As small molecules. Acid have a role, because anaerobic conditions foster accumulation of acid, not necessarily lactic acid. Bacteria that are implicated in periodontal diseases are more anaerobic, and so acids are considered important. Bases, amines, if you produce acid and base, they would neutralize one another, but typically excess of one or the other is produced in any given local area. Location and local effects are important. What they do, high concentrations would be what you would expect for proteins that are subject to unfolding. Any pH deviating from normal causes proteins to unfold. Sometimes its reversible, get pH to normal, proteins reassume function. When proteins are chemically modified as a result of these small molecules get effects that are persistent and not reversible, and sometimes the proteins are recognized as antigenic, foreign proteins, sets in motion the immune system that starts targeting one’s own proteins. 24

27. Next time: 5.
Periodontal disease
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