DENT 5302 TOPICS IN DENTAL BIOCHEMISTRY

31 March 2008
Outline
Plaque fluid

Stephan curve

Enamel substrate

Enamel plaque fluid interaction

The concept of critical pH

Objectives: Ultrastructure of enamel caries lesion

Effect of bacterial acids and plaque fluid on the mineral phase of enamel
The concept of critical pH Erosion
Enamel-plaque fluid interaction

Plaque Composition
Plaque composition: About half of the plaque dry
weight is bacterial and salivary protein. About 25%
Bacterial and salivary protein 50%

Carbohydrates and lipids 20-30% of the dry weight is carbohydrates and lipids, and
Extra and intracellular polysaccharides
another 25% is inorganic component.
- Synthesized by bacteria
- Bacterial attachment and cohesion
Carbohydrates in plaque consist of polymers
- Reservoir of fermentable substrates

Inorganic components 25% synthesized by bacteria (glucans, fructans, and

Ca, P: several times higher than in saliva
polysaccharides).
Most Ca is non-ionic, becomes ionized as pH drops
Determine rates of enamel dissolution and remineralization These extracellular polysaccharides are crucial for
Other ions: K, Na, Mg, and F
bacterial attachment and cohesion, and serve as a
Critical point: Dental plaque is responsible for the majority of chemical activities
on the tooth surface. reservoir of fermentable substrates when other food
sources become depleted.

Bacteria cells contain carbohydrate in the form of intracellular glycogen-like polymers, which are stored
as granules in the cell. They also function as a reservoir when dietary substrates are depleted.
The inorganic components, especially Ca and P concentrations are several times higher than in saliva.
Most of the Ca found in plaque is non-ionic. As the pH drops, plaque calcium becomes ionized and is
important in determining rates of enamel dissolution and remineralization. Other ions present are, for
example, K, Na, Mg, and F.
Dental plaque is responsible for the majority of chemical activities on the tooth surface.

Plaque Fluid Plaque fluid is the extracellular aqueous phase of

Plaque fluid = extracellular aqueous phase of dental plaque
Provide aqueous medium for diffusion and exchange of substances
between saliva and tooth surface
Separated from plaque by centrifugation
500 g wet weight plaque sample 150 nL plaque fluid
Changes in ionic composition of plaque fluid
Resting plaque fluid: one to several hours after eating
Starved plaque fluid: following overnight fasting
Total organic acids
(mmol/L)
dental plaque. It provides the aqueous medium for
the exchange of diffusing substances between
saliva through plaque and the tooth surface. Plaque
fluid can be separated from plaque by
cariogenic conditions
centrifugation. Typically a plaque sample of 500
g wet weight will yield approximately 150 nL of
pH plaque fluid.

Resting plaque 56.3 - 102.1 5.69 - 6.54 Cariogenic conditions generated by plaque
Starved plaque 31.9 - 61.5 6.78 - 7.08
microorganisms can be seen by changes in the
ionic composition of plaque fluid.

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Two types of plaque fluid reflect the metabolic activity of bacteria: resting plaque and starved plaque.
Resting plaque fluid is obtained one to several hours after eating. Starved plaque fluid is obtained
following overnight fasting.
Resting plaque has a higher organic acid concentration (56.3 to 102.1 mmol/1) than starved sample (31.9
to 61.5 mmol/1). pH in rested samples (pH 5.69 to 6.54) are lower than those found in starved samples
(pH 6.78 to 7.08). The lower pH in the rested samples result from the metabolism of residual energy
sources that are depleted during overnight fasting.

Among the organic acids produced by plaque

bacteria, lactic acid dominates in the presence of
Lactic acid: the main acid involved in caries formation
sugar. Lactic acid is considered to be the main
Lactic acid concentrations in plaque fluid following a 2-min 10% sucrose rinse
acid involved in caries formation. 7 min after
Acid Time (min) sucrose rinse, concentration of lactic acid in
(mmol/L) 0 7 15 23
plaque rose to twice the starting point and was
Lactic 17.5 37.5 33.4 18.6
maintained for a period of time, in this case, about
Margolis HC, Moreno EC. Composition and cariogenic potential of dental plaque fluid. 20 min.
Crit Rev Oral Biol Med 1994;5:1-25

Stephan curve What contributes to the extent of pH drop after glucose challenge?

Plaque pH after a glucose challenge

Plaque pH after a glucose challenge

Type and amount of CHO available

Bacteria present
Salivary composition and flow
Stephan RM. JADA 1940;27:718-723 Other food ingested
Changes in hydrogen-ion concentration on tooth surfaces and in carious lesion.
Stephan RM. JADA 1944; 23:257-266 Thickness and age of dental plaque
Intra-oral hydrogen-ion concentrations associated with dental caries activity.

Stephan curve
The relationship between plaque pH and time after sugar challenge is known as the Stephan curve. In the
40s, Stephan demonstrated that dental plaque has the ability to produce rapid and substantial decreases
in pH in vivo. The pH rapidly decreases immediately following exposure to a sugar challenge by rinsing
with a glucose or sucrose solution. After reaching a minimum, the pH slowly rises to baseline, usually
in about an hour. A later study by Stephan was even more important. He showed that the period and
extent of pH drop was inversely related to the caries activities of the subjects. The extent of plaque pH
decrease (how low and how long) after glucose challenge is attributed to the type and amount of
carbohydrate available, bacteria present, other food ingested, salivary composition and flow, and
thickness and age of dental plaque.

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 Plaque pH after a glucose challenge

Resting plaque pH:

In addition to the pH drop, the characteristic of
Constant within each individual, but baseline pH value is also important. Under resting
differences among groups.
Caries-inactive resting pH ~ 6.5 - 7 conditions, plaque pH is quite constant in each
Caries-prone lower resting pH
individual. But there are some differences noticeable
from person to person. Caries-inactive individual
What contributes to the differences in resting plaque?
usually have a resting pH between 6.5-7, and usually
Bacterial composition affects metabolic properties of plaque
Storage form of CHO energy source when diet is depleted
remain above pH 5 following sugar exposure. In
When the host does not eat, cariogenic bacteria still produce acids
contrast, the caries-prone group has a lower resting pH
form storage carbohydrates
and remains below 5 for a longer period after sugar
exposure.
The difference in pH of resting plaque, however, is not as straight forward. Its probably due to
bacterial composition that affects the metabolic properties of plaque. For example, the storage form of
carbohydrate that allows them to ferment when external diet is depleted. Therefore cariogenic bacteria
which have storage form of carbohydrate can produce acids when the host does not eat.

The table shows the differences in plaque

What are the differences in plaque fluid between
caries-free and caries-positive individuals? fluid between caries-free (CF) and caries-
caries-free caries-positive positive (CP) individuals. Starved plaque
Composition
Na+ 14.2 + 3.5 16.5 + 5.4
fluid (after overnight fasting) from CF and
Mg2+ 2.0 + 0.4 2.6 + 0.4 CP were similar in ionic composition,
K+ 59.9 + 4.9 71.4 + 11.3
(whole plaque) Calcium 16.2 + 5.2 * 6.9 + 0.4 except calcium and pH values. Whole
P 13.9 + 1.9 15.6 + 3.6
plaque from CF had significantly higher
pH 7.02 + 0.05 * 6.79 + 0.12
Acid
calcium than plaque from CP. Degree of
Lactic 1.8 + 0.7 2.6 + 1.2 saturation of plaque fluid from CF is more
Acetic 19.9 + 3.5 20.3 + 4.6
Propionic 5.8 + 1.5 5.8 + 1.5 highly supersaturated with respect to
DS (enamel) 7.11 + 0.66 * 5.42 + 0.68 enamel than those from CP. The degree
of saturation will be explained along with
Margolis HC. Enamel-plaque fluid interaction. Cariology for the Nineties, 1993
the concept of critical pH.

Enamel substrate
Enamel as a substrate for dental caries
Enamel: 96% by weight or 87% by volume mineral Enamel consists of 96% by weight or 87 % by
13 vol % interprismatic space is diffusion channel
volume of mineral. The other 13% by volume is
Major mineral component (teeth and bone):
Calcium phosphate crystals ~ Hydroxyapatite Ca10(PO4)6(OH)2 interprismatic space filled with protein, lipid, and
Hydroxyapatite lattice structure water that form diffusion channels. Enamel is a
Hydroxyl ions form microporous solid that allows a variety of ions to
columns of parallelogram
diffuse in and out.
Calcium ions form
triangle around hydroxyl ion
The major mineral components of teeth (and
Phosphates fill space
bones) are microcrystals of calcium phosphate
Nikiforuk G. Understanding Dental Caries. Karger 1985
with the arrangement of atoms resembling the
mineral hydroxyapatite (HAP).
This diagram represents the crystal structure of HAP. Hydroxyl ions form columns of parallelogram.
Calcium ions form triangles around hydroxyl ions, and phosphate ions fill the space.

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Biological mineral is nonstoichiometric Ca10(PO4)6(OH)2
Concentration of the chemical components is different from pure HAP
Substitution of three primary constituents with
- carbonate
- other trace elements (impurities): F, Na, Cl, Mg, K, Zn, Si, Sr
Dental mineral is carbonated HAP
Carbonate (CO3)2- substitute (PO4)3- or 2 (OH)-
Carbonate ions disturb the regular array of ions in the crystal lattice
More soluble in acid than pure HAP
Biological minerals like tooth enamel are
'nonstiochiometric', the concentration of the
chemical components is different from pure HAP.
This is because the substitution of three primary
constituents with carbonate and other trace
elements, or by surface absorption and the
presence of mineral deficient apatites. Current
concept looks at enamel as a carbonated HAP.
Carbonate ions substitute either 1 phosphate or 2
hydroxyl ions. Carbonate disturbs the regular
array of ions in the crystal lattice, so the
carbonated HAP is much more soluble in acid.

Post-
Post-eruptive Maturation
Discussion (group of 5-6)
Newly erupted teeth have relatively greater caries susceptibility

When a tooth is just erupted into the oral cavity, it is During demineralization, carbonate is lost and excluded after remin

more susceptible to demineralization. Decrease carbonate & increase fluoride in enamel surface

Why? Less susceptible to demineralization

= post-eruptive maturation

Formula of tooth mineral

(Ca)10-x(Na)x(PO4)6-y(CO3)z(OH)2-u(F)u

Carbonate and fluoride play an important role in enamel maturation. During demineralization,
carbonates dissolve easily and are excluded from the newly formed remineralized mineral. As enamel
matures the level of carbonate on the surface decreases and fluoride increase. This may explain the
relative caries susceptible of newly erupted teeth and less susceptible to the caries process of mature
teeth. The formula of our tooth mineral is: (Ca)10-x(Na)x(PO4)6-y(CO3)z(OH)2-u(F)u
When do teeth
Teeth dissolve when pH is lower than a critical pH When do teeth dissolve in acid?
dissolve?
Teeth dissolve when the pH is lower than a
Solubility product (Ksp)
critical pH. A few parameters are important
Ksp is the ionic activity products of substance at saturation
Ksp = Concentrations of the component ions to better understand the concept of critical
to the power in saturated solution pH: Ksp, IAP, and degree of saturation.
e.g., HAP Ca5(PO4)3OH ; Ksp(HAP) = [Ca2+]5[PO43-]3[OH-] = 7.36 x 10-60
Solubility product (Ksp) determines the
Higher Ksp =
Ksp(enamel) = 5.5 x 10-55
easier to dissolve Ksp(carbonated-HAP) = 4.57 x 10-49 solubility of substance such as
easy

Ksp is a constant value

hydroxyapatite. Ksp is the ionic activity of
Acidic solution: H+ remove PO43- & OH-
the substance at saturation. It is calculated
Decrease [PO4] & [OH] in solution as product of the concentrations of the
Apatite mineral dissolves
[PO4] & [OH] rise to maintain the saturation level
component ions to the power in a saturated
solution.

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For example: Ksp of hydroxyapatite [Ca]5[PO4]3[OH] is 7.36 x 10-60; Kenamel is 5.5 x 10-55, Kcarbonated-HAP
is 4.57 x 10-49. The higher (less negative power) Ksp, the easier for the mineral to dissolve. Therefore,
carbonated apatite dissolve easiest, follow by enamel and hydroxyapatite, respectively. This makes
sense, because enamel is in between carbonated apatite and hydroxyapatite.
Ksp is a constant value, which means that in an acidic solution where protons remove some of the PO43-
and OH-, [PO4] and [OH] concentrations are reduced. Therefore apatite mineral dissolves to increase the
concentration of PO43- and OH- ions to maintain the saturation level.

Ionic activity product (IAP) is the

Ionic Activity Product (IAP)
concentration of available ions in the
Concentration of available ions in the solution, calculated similar to Ksp
solution. For any solution, such as saliva or
Degree of saturation (DS) plaque fluid, IAP is determined the same way
Ratio of the ionic product of a substance in the solution (IAP) to its as Ksp.
ionic product at saturation (Ksp ) e.g., for hydroxyapatite (Ca5(PO4)3OH)
1/9
Degree of saturation is the ratio of IAP to its
IAP (ionic activity products in solution)
DS = solubility product at saturation Ksp.
Ksp (ionic activity products at saturation)
At saturation, DS = 1. Demineralization
DS = 1 : Saturation condition
occurs when DS < 1, which means that the
DS < 1 : Solution undersaturated WRT mineral

DS > 1 : Solution supersaturated WRT mineral

solution is undersaturated with respect to the
(WRT = with respect to)
Margolis HC, Moreno EC
mineral phase. When DS > 1, the solution is
Crit Rev Oral Biol Med 1994;5:1-25
supersaturated, thus favors remineralization.

Critical pH
The concept of critical pH
= pH at which a solution is just saturated WRT a particular mineral
Critical pH is the pH at which a solution is
If the solution pH > critical pH supersaturated mineral precipitate just saturated with respect to a particular
If the solution pH < critical pH undersaturated mineral dissolve mineral. If the pH of the solution is above
Normal condition: Our teeth do not dissolve in saliva or plaque fluid the critical pH, the solution is supersaturated
Saliva and plaque fluid are supersaturated WRT tooth enamel
pH of saliva & plaque fluid > critical pH
and mineral will precipitate. If the pH of the
Saliva & plaque fluid contain Ca, P, OH IAP > Ksp tooth enamel solution is less than the critical pH, the
The tooth will dissolve when the pH of fluid phase is less than critical pH. solution is undersaturated and mineral will
dissolve until the solution becomes saturated.
Critical pH of carious formation in enamel ~ 4.5-
4.5-5.5
The effect of pH can be counteracted by an
Coincide with pH when plaque bacteria ferment carbohydrates
HAP is undersaturated & FAP is supersaturated increase in concentration of ionic species
(e.g., Ca2+) in the solution to restore the
equilibrium.
For example, the pH of saliva and plaque fluid are normally higher than the critical pH of tooth enamel.
The level of Ca, P and OH ions in saliva and plaque fluid is supersaturated with respect to tooth enamel
at that pH. In other words, IAP of saliva and plaque fluid is higher than Ksp for hydroxyapatite. Our
teeth do not dissolve in saliva or plaque fluid unless the pH is reduced to less than the critical pH.
Critical pH of caries formation in enamel is often referred to as pH between 4.5-5.5. This range
coincides with pH of acids formed when plaque bacteria ferment carbohydrates. At this pH range, HAP
is undersaturated while fluroapatite (FAP) is supersaturated.

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 Fluorapatite (FAP) is less soluble than
demineralization hydroxyapatite (HAP). FAP dissolves at pH
pH 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 4.5 and HAP at pH 5.5 Therefore, between pH
FAP 4.5-5.5, HAP is undersaturated and FAP is
Critical pH
HAP
supersaturated, i.e, HAP dissolves and FAP
deposit caries erosion precipitates to form subsurface lesion (initial
pH 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 caries lesion). If the pH was so low that FAP
remineralization was undersaturated, an erosive defect will be
formed.
Carious lesion forms at pH 4.5 - 5.5
Erosion lesion forms when pH < 4.5

Critical pH is not a fixed value, it depends on

the levels of calcium and phosphate in plaque
fluid. This diagram is solubility isotherms of
Critical pH is not a fixed value
HAP and FAP. The solubility isotherm depicts
Solubility isotherm
pH 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 points when a compound precipitates from a
100
solution at a given pH and ion concentration, in
10
this case, calcium ions. Solubility isotherms
calcium (mol/l)

1
HAP represent the just saturated condition. Above
0.1

FAP the curve is supersaturated, below the curve is

0.01

oral fluid undersaturated with respect to each mineral.

0.001

0.0001
Current concepts on the theories of the mechanism of action of fluoride.
ten Cate JM. Acta Odontol Scand 1999;57:325-9.
Persons with low concentration of calcium and
phosphate in saliva and plaque fluid, the upper
limit of critical pH of enamel may be as high
as 6.5. Plaque fluid with high calcium and
phosphate content may have the critical pH
close to 5.

Ultrastructure of enamel caries

Ultrastructure of enamel caries lesion When enamel crystallites are subjected to acid,
Crystal damage from acid:
- Surface etching
two main types of crystal damage are
- Central defect or hairpin observed. One is surface etching, the other is
central or core defect forming a hollow center.
The crystal core is more susceptible to acid
Crystal core has more dislocations or lattice defects
Higher carbonate content
because there are more dislocations or lattice
defects, and some suggest that the carbonate
Dissolving crystals are smaller
Increased intercrystalline space
concentration is higher. This crystal damage
has a hairpin appearance when viewed under
Larger crystal at prism periphery electron microscope. Dissolving crystals are
from remineralization
smaller and the intercrystalline space is
increased. But crystals at the prism periphery
are larger, which is a result of remineralization.

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 1.
Surface
zone

2.
The larger crystals are also found in the
2 3 1 2 3 4
Body of
lesion 1 4 surface zone and the dark zone, one of the
evidence that remineralization take place in
3.
Dark zone these 2 zones.
Larger crystals in
surface zone and dark zone
4.
Translucent
zone Indication of remineralization

Sound
enamel

Range of crystal size in each zone of early enamel lesion

acid corrosion'
Erosion, or sometimes called (chemical)
Loss of dental hard tissue through chemical etching and dissolution 'corrosion', is the loss of dental hard tissue through
by acids of non-bacterial origin

Endogenous acid: gastric acid, gingival crevicular fluid chemical etching and dissolution by acids of non-
Gastroesophageal reflux disease, vomiting
Exogenous acid: diet, medicine, industry bacterial origin. Source of acid and be
Frequent and prolonged ingestion endogenous, from gastroesophageal reflux disease
of acidic fruits, fruit juices and
acidic beverages (GERD), or exogenous from medication or food.
3/4 of a bottle of white wine
Every evening for 34 years
Frequent and prolonged ingestion of acidic fruits,
Sipping over a 3 hours after dinner
Wine pH ranges about 3-4.
fruit juices and acidic beverages has been reported
Dental consumption due to wine consumption. Mandel L. JADA 2005;136:71-75 as causing dental erosion. In this case, a woman
drinks 3/4 of a bottle of white wine every evening
Can acidic food and drinks soften enamel surface?
Enamel samples alternately immersed, 5 sec each, in food or drink
for 34 years, sipping over a 3 hours period after
and in artificial saliva for 10 cycles.
dinner. pH of wine ranges about 3-4.
300

250 In this study, we want to know if acidic food and

* *
Enamel Hardness

200
drinks soften tooth surface. Enamel samples were
150 * Before

100
After
dipped in food or drink alternating with artificial
50

0
saliva for 5 sec each, 10 cycles. That's not much
Cola Sports drink Orange Drinking Lemon-
juice yogurt grass soup time at all, but there was dramatic decrease in
pH 2.74 3.78 3.75 3.83 4.20

S. Wongkhantee et al., J Dent 2006;34:214-220.

enamel hardness in certain drinks.
Effect of acidic food and drinks on surface hardness of enamel, dentine, and tooth-coloured filling materials.

Recommended references Diagram showing effect of increase Ca on degree of saturation of plaque

fluid with respect to enamel
1. Zero DT. Dental Caries Process. Dent Clin North Am 1999;43(4):635-664.
2. Featherstone JD. The science and practice of caries prevention. J Am Dent
Assoc 2000;131:887-899.
3. Gordon Nikiforuk. Understanding Dental Caries 1. Etiology and
Question:
Mechanisms, Basic and Clinical Aspects. Basel; New York: Karger 1985.
Chapters 4 &10. Which line represent
4. Margolis HC, Moreno EC. Composition and cariogenic potential of dental individuals with higher
plaque fluid. Crit Rev Oral Biol Med 1994;5:1-25. tendency for caries
5. Margolis HC. Enamel plaque fluid interactions. In WH Bowen and LA formation?
Tabak (Eds) Cariology for the nineties. University of Rochester Press
1993:173-186.