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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
Ultrastructure of enamel caries lesion
Objectives:
• Effect of bacterial acids and plaque fluid on the mineral phase of enamel
Erosion
• The concept of critical pH
• Enamel-plaque fluid interaction
Plaque composition: About half of the plaque dry
weight is bacterial and salivary protein. About 25%
of the dry weight is carbohydrates and lipids, and
another 25% is inorganic component.
Plaque Composition
Bacterial and salivary protein – 50%
Carbohydrates and lipids – 20-30%
Extra and intracellular polysaccharides
- Synthesized by bacteria
Carbohydrates in plaque consist of polymers
synthesized by bacteria (glucans, fructans, and
polysaccharides).
- Bacterial attachment and cohesion
- Reservoir of fermentable substrates
Inorganic components – 25%
Ca, P: several times higher than in saliva
Most Ca is non-ionic, becomes ionized as pH drops
Determine rates of enamel dissolution and remineralization
Other ions: K, Na, Mg, and F
Critical point: Dental plaque is responsible for the majority of chemical activities
on the tooth surface.
These extracellular polysaccharides are crucial for
bacterial attachment and cohesion, and serve as a
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 = 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 μg wet weight plaque sample
150 nL plaque fluid
Changes in ionic composition of plaque fluid
cariogenic conditions
Resting plaque fluid: one to several hours after eating
Starved plaque fluid: following overnight fasting
Total organic acids
pH
(mmol/L)
Resting plaque
56.3 - 102.1
5.69 - 6.54
Starved plaque
31.9 - 61.5
6.78 - 7.08
Plaque fluid is the extracellular aqueous phase of
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
centrifugation. Typically a plaque sample of 500
μg wet weight will yield approximately 150 nL of
plaque fluid.
Cariogenic conditions generated by plaque
microorganisms can be seen by changes in the
ionic composition of plaque fluid.
1
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
sugar. Lactic acid is considered to be the main
acid involved in caries formation. 7 min after
sucrose rinse, concentration of lactic acid in
plaque rose to twice the starting point and was
maintained for a period of time, in this case, about
20 min.
Lactic acid: the main acid involved in caries formation
Lactic acid concentrations in plaque fluid following a 2-min 10% sucrose rinse
Acid
(mmol/L)
Lactic
Time (min)
0
7
15
23
17.5
37.5
33.4
18.6
Margolis HC, Moreno EC. Composition and cariogenic potential of dental plaque fluid.
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
Other food ingested
Stephan RM. JADA 1940;27:718-723
Changes in hydrogen-ion concentration on tooth surfaces and in carious lesion.
Stephan RM. JADA 1944; 23:257-266
Intra-oral hydrogen-ion concentrations associated with dental caries activity.
Thickness and age of dental plaque
Stephan curve
The relationship between plaque pH and time after sugar challenge is known as the Stephan curve. In the
40’s, 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.
2
Plaque pH after a glucose challenge
Resting plaque pH:
Constant within each individual, but
differences among groups.
Caries-inactive – resting pH ~ 6.5 - 7
Caries-prone – lower resting pH
What contributes to the differences in resting plaque?
Bacterial composition affects metabolic properties of plaque
Storage form of CHO
energy source when diet is depleted
When the host does not ‘eat’, cariogenic bacteria still produce acids
form storage carbohydrates
In addition to the pH drop, the characteristic of
baseline pH value is also important. Under resting
conditions, plaque pH is quite constant in each
individual. But there are some differences noticeable
from person to person. Caries-inactive individual
usually have a resting pH between 6.5-7, and usually
remain above pH 5 following sugar exposure. In
contrast, the caries-prone group has a lower resting pH
and remains below 5 for a longer period after sugar
exposure.
The difference in pH of resting plaque, however, is not as straight forward. It’s 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’.
What are the differences in plaque fluid between
‘caries-free’ and caries-positive individuals?
‘caries-free’
caries-positive
14.2 + 3.5
2.0 + 0.4
16.5 + 5.4
2.6 + 0.4
59.9 + 4.9
71.4 + 11.3
16.2 + 5.2 *
13.9 + 1.9
6.9 + 0.4
15.6 + 3.6
7.02 + 0.05 *
6.79 + 0.12
1.8 + 0.7
19.9 + 3.5
2.6 + 1.2
20.3 + 4.6
Composition
Na+
Mg2+
K+
(whole plaque) Calcium
P
pH
Acid
Lactic
Acetic
Propionic
5.8 + 1.5
5.8 + 1.5
DS (enamel)
7.11 + 0.66 *
5.42 + 0.68
Margolis HC. Enamel-plaque fluid interaction. Cariology for the Nineties, 1993
The table shows the differences in plaque
fluid between caries-free (CF) and cariespositive (CP) individuals. Starved plaque
fluid (after overnight fasting) from CF and
CP were similar in ionic composition,
except calcium and pH values. Whole
plaque from CF had significantly higher
calcium than plaque from CP. Degree of
saturation of plaque fluid from CF is more
highly supersaturated with respect to
enamel than those from CP. The degree
of saturation will be explained along with
the concept of critical pH.
Enamel as a substrate for dental caries
Enamel substrate
Enamel consists of 96% by weight or 87 % by
volume of mineral. The other 13% by volume is
interprismatic space filled with protein, lipid, and
water that form diffusion channels. Enamel is a
microporous solid that allows a variety of ions to
diffuse in and out.
Enamel: 96% by weight or 87% by volume mineral
13 vol % interprismatic space is diffusion channel
Major mineral component (teeth and bone):
Calcium phosphate crystals ~ Hydroxyapatite Ca10(PO4)6(OH)2
Hydroxyapatite lattice structure
Hydroxyl ions
form
columns of parallelogram
Calcium ions
form
triangle around hydroxyl ion
The major mineral components of teeth (and
bones) are microcrystals of calcium phosphate
with the arrangement of atoms resembling the
mineral hydroxyapatite (HAP).
Phosphates fill space
Nikiforuk G. Understanding Dental Caries. Karger 1985
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.
3
Biological mineral is ‘nonstoichiometric’
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.
≠ Ca10(PO4)6(OH)2
Concentration of the chemical components is different from pure HAP
¾ 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
PostPost-eruptive Maturation
Discussion (group of 5-6)
Newly erupted teeth have relatively greater caries susceptibility
During demineralization, carbonate is lost and excluded after remin
When a tooth is just erupted into the oral cavity, it is
more susceptible to demineralization.
Decrease carbonate & increase fluoride in enamel surface
Less susceptible to demineralization
Why?
= 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
dissolve?
Teeth dissolve when pH is lower than a critical pH
Teeth dissolve when the pH is lower than a
critical pH. A few parameters are important
to better understand the concept of critical
pH: Ksp, IAP, and degree of saturation.
Solubility product (Ksp)
Ksp is the ionic activity products of substance at saturation
Ksp = Concentrations of the component ions
to the power in saturated solution
e.g., HAP Ca5(PO4)3OH ; Ksp(HAP) = [Ca2+]5[PO43-]3[OH-] = 7.36 x 10-60
Higher Ksp =
easier to dissolve
Ksp(enamel) = 5.5 x 10-55
Ksp(carbonated-HAP) = 4.57 x 10-49
easy
Ksp is a constant value
Acidic solution:
When do teeth dissolve in acid?
H+ remove PO43- & OHDecrease [PO4] & [OH] in solution
Apatite mineral dissolves
[PO4] & [OH] rise to maintain the saturation level
4
Solubility product (Ksp) determines the
solubility of substance such as
hydroxyapatite. Ksp is the ionic activity of
the substance at saturation. It is calculated
as product of the concentrations of the
component ions to the power in a saturated
solution.
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 PO43and 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
concentration of available ions in the
solution. For any solution, such as saliva or
plaque fluid, IAP is determined the same way
as Ksp.
Ionic Activity Product (IAP)
Concentration of available ions in the solution, calculated similar to Ksp
Degree of saturation (DS)
Ratio of the ionic product of a substance in the solution (IAP) to its
ionic product at saturation (Ksp ) e.g., for hydroxyapatite (Ca5(PO4)3OH)
1/9
IAP (ionic activity products in solution)
DS =
Ksp (ionic activity products at saturation)
DS = 1 : Saturation condition
DS < 1 : Solution undersaturated WRT mineral
DS > 1 : Solution supersaturated WRT mineral
(WRT = with respect to)
Margolis HC, Moreno EC
Crit Rev Oral Biol Med 1994;5:1-25
Critical pH
The concept of critical pH
= pH at which a solution is just saturated WRT a particular mineral
If the solution pH > critical pH
supersaturated
mineral precipitate
If the solution pH < critical pH
undersaturated
mineral dissolve
Normal condition: Our teeth do not dissolve in saliva or plaque fluid
Saliva and plaque fluid are supersaturated WRT tooth enamel
pH of saliva & plaque fluid > critical pH
Saliva & plaque fluid contain Ca, P, OH
Degree of saturation is the ratio of IAP to its
solubility product at saturation Ksp.
At saturation, DS = 1. Demineralization
occurs when DS < 1, which means that the
solution is undersaturated with respect to the
mineral phase. When DS > 1, the solution is
supersaturated, thus favors remineralization.
IAP > Ksp tooth enamel
The tooth will dissolve when the pH of fluid phase is less than critical pH.
Critical pH of carious formation in enamel ~ 4.54.5-5.5
Coincide with pH when plaque bacteria ferment carbohydrates
HAP is undersaturated & FAP is supersaturated
Critical pH is the pH at which a solution is
just saturated with respect to a particular
mineral. If the pH of the solution is above
the critical pH, the solution is supersaturated
and mineral will precipitate. If the pH of the
solution is less than the critical pH, the
solution is undersaturated and mineral will
dissolve until the solution becomes saturated.
The effect of pH can be counteracted by an
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.
5
Fluorapatite (FAP) is less soluble than
hydroxyapatite (HAP). FAP dissolves at pH
4.5 and HAP at pH 5.5 Therefore, between pH
4.5-5.5, HAP is undersaturated and FAP is
supersaturated, i.e, HAP dissolves and FAP
precipitates to form subsurface lesion (initial
caries lesion). If the pH was so low that FAP
was undersaturated, an erosive defect will be
formed.
demineralization
pH
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
FAP
Critical pH
deposit
pH
6.5
6.0
HAP
caries
5.5
5.0
erosion
4.5
4.0
3.5
3.0
remineralization
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
HAP and FAP. The solubility isotherm depicts
points when a compound precipitates from a
solution at a given pH and ion concentration, in
this case, calcium ions. Solubility isotherms
represent the ‘just saturated’ condition. Above
the curve is supersaturated, below the curve is
undersaturated with respect to each mineral.
Critical pH is not a fixed value
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
100
HAP
1
0.1
FAP
0.01
calcium (mol/l)
10
oral fluid
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,
two main types of crystal damage are
observed. One is surface etching, the other is
central or core defect forming a hollow center.
The crystal core is more susceptible to acid
because there are more dislocations or lattice
defects, and some suggest that the carbonate
concentration is higher. This crystal damage
has a hairpin appearance when viewed under
electron microscope. Dissolving crystals are
smaller and the intercrystalline space is
increased. But crystals at the prism periphery
are larger, which is a result of remineralization.
Crystal damage from acid:
- Surface etching
- Central defect or hairpin
• Crystal core has more dislocations or lattice defects
• Higher carbonate content
• Dissolving crystals are smaller
• Increased intercrystalline space
Larger crystal at prism periphery
from remineralization
6
1.
Surface
zone
2.
Body of
lesion
2
1
3
1
4
2
The larger crystals are also found in the
surface zone and the dark zone, one of the
evidence that remineralization take place in
these 2 zones.
3 4
3.
Dark zone
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
Erosion, or sometimes called (chemical)
'corrosion', is the loss of dental hard tissue through
chemical etching and dissolution by acids of nonbacterial origin. Source of acid and be
endogenous, from gastroesophageal reflux disease
(GERD), or exogenous from medication or food.
Frequent and prolonged ingestion of acidic fruits,
fruit juices and acidic beverages has been reported
as causing dental erosion. In this case, a woman
drinks 3/4 of a bottle of white wine every evening
for 34 years, sipping over a 3 hours period after
dinner. pH of wine ranges about 3-4.
‘acid corrosion'
Loss of dental hard tissue through chemical etching and dissolution
by acids of non-bacterial origin
Endogenous acid: gastric acid, gingival crevicular fluid
Gastroesophageal reflux disease, vomiting
Exogenous acid: diet, medicine, industry
Frequent and prolonged ingestion
of acidic fruits, fruit juices and
acidic beverages
3/4 of a bottle of white wine
Every evening for 34 years
Sipping over a 3 hours after dinner
Wine pH ranges about 3-4.
Dental consumption due to wine consumption. Mandel L. JADA 2005;136:71-75
Can acidic food and drinks soften enamel surface?
Enamel samples alternately immersed, 5 sec each, in food or drink
and in artificial saliva for 10 cycles.
300
Enamel Hardness
250
*
200
150
In this study, we want to know if acidic food and
drinks soften tooth surface. Enamel samples were
dipped in food or drink alternating with artificial
saliva for 5 sec each, 10 cycles. That's not much
time at all, but there was dramatic decrease in
enamel hardness in certain drinks.
*
*
Before
After
100
50
0
Cola
pH
2.74
Sports drink
3.78
Orange
juice
Drinking
yogurt
Lemongrass soup
3.75
3.83
4.20
S. Wongkhantee et al., J Dent 2006;34:214-220.
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
Mechanisms, Basic and Clinical Aspects. Basel; New York: Karger 1985.
Chapters 4 &10.
4. Margolis HC, Moreno EC. Composition and cariogenic potential of dental
Question:
Which line represent
individuals with higher
plaque fluid. Crit Rev Oral Biol Med 1994;5:1-25.
tendency for caries
formation?
5. Margolis HC. Enamel – plaque fluid interactions. In WH Bowen and LA
Tabak (Eds) Cariology for the nineties. University of Rochester Press
1993:173-186.
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