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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. 7