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Biochemistry of saliva and tooth tissues

dorota.luchowska-kocot@umlub.pl

Tooth is a complex system of specialized tissues

Tooth tissues enamel

the hardest part of the tooth acts as a protective tissue that covers the exposed part of a tooth (the crown)

dentin contains collagen is similar in composition to bone is the tissue that forms the mainly mass of a tooth and supports the enamel

dental pulp a soft connective tissue containing nerves and blood vessels nourish the tooth

cementum covers the dentine outside of the root (under the gum line) attached to the bone of the jaw with little elastic fibers

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Enamel the hardest substance in the human body contains the highest percentage of minerals (95% is hydroxyapatite), water and organic material initially composed of high amount of protein; during tooth matures proteins are removed and the voids are backfilled with HAP Remineralisation of teeth - repairing damages of the tooth only to a certain degree The maintenance and repair of human tooth enamel - one of the primary concerns of dentistry.

Dentin is a mineralized connective tissue with an organic matrix of collagenous proteins

contains exclusively genetic type I collagen its poilpeptide chain is rich in glycine (Gly), proline (Pro) and hydroxyproline (Hyp)

composed of mineralized calcium phosphate (calcium phosphate phase called hydroxyapatite (HAP) Ca10(PO4)6(OH)2) 70% - within a matrix of collagen fibrils (20%) and water (10%)

HAP - living mineral that is continually grown, dissolved and remodeled in response to signals of internal (e.g., pregnancy) and external (e.g., gravity, exercise) environment 20-50nm Hydroxyapatite Nanoparticles

Crystalline structure of hydroxyapatite in enamel

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Cementum and dental pulp Cementum

is a specialized bony substance covering the root of a tooth 45% inorganic material (mainly hydroxyapatite), 33% organic material (mainly collagen) 22% water

Cementum is excreted by cementoblasts within the root of the tooth and is thickest at the root apex.

Dental pulp the part in the center of a tooth made up of living soft tissue and cells called odontoblasts commonly called 'the nerve', although it contains many other structures which are not nerves

biofilm consisting of large amounts of various bacteria which forms on teeth if not removed regularly, can lead to dental cavities (caries) or periodontal problems (such as gingivitis) plaque can mineralize along the gingiva, forming tartar microorganisms form the biofilm: almost entirely bacteria (mainly Streptococcus and anaerobes)

Certain bacteria in the mouth in the absence of oxygen produce lactic acid (which dissolves the

calcium and phosphorus in the enamel). This proces - demineralisation, leads to tooth

destruction.

Dental plaque

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Dental caries infectious disease which damages the structures of teeth caused by certain types of acid-producing bacteria which cause the most damage in the presence of fermentable carbohydrates such as sucrose, fructose, and glucose The resulting acidic levels in the mouth affect teeth because a tooth's special mineral content causes it to be sensitive to low pH.

Teeth demineralisation:

10 4)6 2 9 2 4)6 2 2+

optimal pH for remineralization: 6.4-7.8 (pH<6,2 leads to the demineralization ) ph<6,2 leads to the demineralization Mg2+, Mn2+, Zn2+, Cu2+, Si2+ ions increase mineralization

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Saliva

the most important factor maintaining homeostasis in the oral cavity Saliva contains:

fluid produced by the salivary glands gingival crevicular fluid (GCF) transudate - extravascular fluid with low protein content and a low specific gravity exfoliated epithelial cells, white blood cells, buccal cells, serum proteins, cell-free DNA, RNA bacteria and other pathogens

Daily salivary output

During the night: 3 ml/hr. Resting saliva: 30 ml/hr. During the meal: 120 ml/ hr. Under normal physiological conditions: about 1.0 liter per day.

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Salivary secretion: Saliva is secreted by:

salivary glands parotid gland sublingual gland and submandibular gland

small salivary glands scattered in the mucous membrane of the lips, palate, tongue and cheeks.

Parotid glands secret clear, watery, serous fluid which is rich in amylase. Sublingual glands secret primarily mucus and most viscous saliva. Submandibular glands produce primarily serous fluid with some mucus.

Salivary secretion: The first stage primary saliva: saliva produced in the acinar cells isotonic to plasma rich in NaCl

The second stage final saliva: active absorption of Na +, Ca2 +, Cl- and HPO4

2- ions passive secretion of HCO3

- and K+ ions osmotic gradient causes the passive transport of water. Ions resorption faster than secretion, small water penetration ultimately saliva is hypotonic

At very high flow: final saliva composition approaches to the composition of the priamary saliva. Secretion of HCO3

- anions determines an increased pH of the saliva flowing through the ducts.

AQP5 - Aquaporin 5 is a water channel prote, plays a role in the generation of saliva, tears and pulmonary secretions.

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Components of saliva:

Water: 99%

Organic components: enzymatic and non-enzymatic proteins

low molecular nitrogen compounds lipids

Inorganic components: cations and anions

Organic compounds proteins: Mucins:

one of the major proteins of the oral cavity belong to the class of glycoproteins increased quantity of mucin in the saliva increases its density and viscosity Mucins function:

Tissues coating protecvive coating about soft and hard tissues, primary role in formation of acquired pellicle concentrates anti-microbial molecules at mucosal interference

Lubrification increases lubricating qualities (film strength) Anti-bacterial:

aggregarion of bacterial cells bacterial adhesion

A sugar component of mucins is sialic acid (N-acetylneuraminic acid).

polipeptide chain

sugar residue

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Organic compounds proteins: Proline reach proteins, PRPs

inhibitor of calcium phosphate crystal growth p pellicle (0.1- surface)

Statherins: prevent precipitation or crystallization of supersaturated calcium phosphate in ductal saliva and oral fliud cause demineralization of early carious lesion lubricant

Organic compounds proteins:

Histatins small histidine-rich proteins potent inhibitor of bacteria and Candida albicans growth - histatins molecule has a positively charged side chain that binds to the negative charges of the cell membrane phospholipids of microorganism (bacterium or fungus)

it leads to loss of microogranism membrane integrity various ions and organic compounds (eg. ATP) "run away" from the microbial cell this results in damage and consequent microorganism cell death

natural metalloproteinase inhibitors (histidins have the ability to bind Zn2+ and Cu2+ ions - activators of metalloproteinases - enzymes that degrade the extracellular matrix) remineralization function

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Organic compounds proteins: Cystatins

inhibitor of harmful cysteine-proteases protective against unwanted proteolysis (bacterial proteases lysed leukocytes), inhibit proteases in periodontal tissues inhibit the precipitation of calcium phosphates in the saliva play an important role in the prevention of periodontal diseases alleviate inflammation in the oral cavity occur mainly in submandibular salivary secretions

Lactoferrins glycoprotein that has the ability to bind Fe3+ ions (some of the oral bacteria require these ions to growth)

Organic compounds proteins: Immunoglobins: IgA

represents 90% of the antibodies in the saliva of produced by plasma cells in the salivary gland epithelium and the epithelial cells of oral mucosa affects phagocytosis of streptococci by leukocytes

IgM is secreted in part by the salivary gland, and a part comes from the tissue fluid

IgG represent 70% of the antibodies in the gingival fluid comes to saliva from gingival pockets and intercellular space through inflamed mucosa

IgA and IgG have the ability to agglutinate Streptococcus mutans facilitating their removal from the mouth

with swallowing saliva. Therefore they influence reducing the adhesion of bacteria to the epithelium of the cheeks and tooth

enamel.

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Organic compounds enzymes:

Lysozyme present in numerous organ and most body fluids oral lysozyme is derived from at least 4 sources:

minor and major salivary glands phagocytic cells gingival crevicular fluid

has anti-microbial activity by inhibition of bacterial adhesion to tooth surface and inhibition of glucose uptake and acid production. has muramidase activity (lysis of peptidoglycan layer). damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins

Organic compounds enzymes: lpha-amylase

-1,4-glycosidic bonds in the starch activated by chloride ionsthe optimum pH for its action: 6-7 is helpful in removing the carbohydrate fibers located between teeth It is responsible for the formation of a glycoprotein complex of pellicle. This complex is formed directly on the washed teeth. On the pellicle the plaque (responsible for most of oral diseases dental caries, periodontitis) is formed. Alfa-amylase has high affinity for bacteria and it binds to them. isoenzyme of alpha-amylase present in saliva

Increased activity of the enzyme

in the blood can indicate diseases of the salivary glands or pancreas!

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Organic compounds enzymes:

Lingual lipase secreted by the Von Ebner's glands at the root of the tongue in the first days after birth takes part in the digestion of emulsified lipids contained in milk little importance in the digestion of lipids in other periods of life

Organic compounds antioxidant enzymes: 1. System of salivary peroxidases:

Lactoperoxidase

catalyzes the hydrogen peroxide (H2O2) oxidation of several acceptor molecules:

reduced acceptor (SCN-) + H2O2 oxidized acceptor (OSCN-) + H2O

OSCN ions inhibit the activity of bacterial hexokinase

Source of the hydrogen peroxide usually is the reaction of glucose with oxygen in the presence of the enzyme glucose oxidase that takes place in saliva.

Lactoperoxidase system is effective in killing of aerobic and certain anaerobic microorganisms.

Myeloperoxidase

released in inflammatory processes

oxidizes (with simultaneous reduction of H2O2) chlorine to hypochlorous which has antiviral and antibacterial properties

2. Catalase:

capable of defending against reactive oxygen species

decomposes the hydrogen peroxide into water and oxygen: 2 H2O2 2O + O2

Saliva is the first line of defence against reactive oxygen species (free radicals).

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Non-protein nitrogenous substances

Uric acid concentration in saliva is 40-240 mmol/L small molecule antioxidant (approx. 70-80% of the antioxidant activity of saliva)

Urea metabolic product of salivary glands important in the action of the buffer system in saliva - ammonia, which is derived from urea decomposition, involves (reemoves) an excess of H+ ions

Creatinine, amino acids may be derived from blood to saliva are transferred by the salivary glands

Non-protein organic substances Carbohydrates

most salivary carbohydrates bind to proteins forming glycoproteins higher concentrations are found in diabetics

Lipids simple and phospholipids, cholesterol occur in small amounts in the secretions of the salivary glands may be obtained from the cell membranes of the salivary glands

Steroid hormones their presence and concentration in saliva depends on the concentration in the blood

Opiorphin a peptide, compound with analgesic properties

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Inorganic components of saliva: their content in saliva is not constant come mainly from the blood (with the exception of HCO3

-) occur in ionic form

CATIONS: Na+, K+

are involved in the transport of active compounds through cell membranes. the presence of Na+ in hydoxyapatites increases the solubility of enamel in acids Na+ regulates salivary osmotic tensiom

Ca2 + builds hard tissue involved in the maturation of enamel and remineralization of initial damage activator of certain enzymes in saliva

Mg 2+ involved in creating the structure of the teeth an activator of certain enzymes in saliva content of Mg2+ in the hydroxyapatite increases the solubility of enamel in acid

Inorganic components of saliva: ANIONS: Cl-

regulates osmotic tension of saliva -amylase

F- affects the structure and processes of remineralization of enamel. antibacterial properties

I- plays a role in the defense mechanisms mainly by the presence in the peroxidase system

HCO3-

creates the strongest buffer in the saliva buffer system: bicarbonate/carbonic acid PO4

3-, HPO42-, H2PO4

- involved in the maturation of enamel remineralization of initial damage form a buffer system phosphate/phosphoric acid play an important role in the formation of tartar (calculus)

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The role of saliva: lingual lipase, salivary amylase

water

cystatin, histatins, PRPs, statherin, PO4

3-, HPO42-, H2PO4

-, F-, Ca2+

mucin, histatin, peroxidases, immunoglobulins, cystatins, lactoferrins, lysozyme mucins, statherins

PRPs

antioxidant function

peroxidases, catalase, uric acid

buffering function

PO43-, HPO4

2-, H2PO4, HCO3

-, urea

Moisturizing function of saliva: - facilitates the formation and swallowing bites, chew food - reduces the deleterious effects of mechanical, chemical, thermal

and biological injuries of mucous membranes.

Protective function of saliva Salivary proteins which covers the thin shell (called pellicle) surfaces of the teeth and mucous membranes. The pellicle includes:

amino acids and proteins selectively adsorbed on the tooth surface by the interaction between the saliva proteins and hydroxyapatite of tooth enamel. lactoferrin, lysozyme, amylase.

Pellicle role: prevents continuous deposition of salivary calcium phosphate protects the tooth from the acids produced by oral microorganisms after consuming carbohydrates forms in seconds after a tooth is cleaned or after chewing.

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Buffering function of salivaAt pH 6.4 - 7.8 saliva is supersaturated solution of calcium phosphates After a small demineralisation lost minerals may return from the saliva of the hard tissues of the tooth

10 4)6 2 10 2+ + 6 43- + 2 -

(The balance between saliva and enamel.)

Acidification of the environment (eg. due to fermentation of sugars by enzymes) increases the solubility of enamel (saliva reduces the degree of saturation of calcium phosphate, saliva becomes unsaturated solution). in the acidic medium: phosphate groups of hydroxyapatite may be associated with a proton:

10 4)6 2 9 2 4)6 2 2+ (Connection of two H+ corresponds to the release of one Ca2+ ion grom crystal apatite - demineralization of enamel.)

Binding of Ca2+, PO43-, F- (toothpaste) induces an acid-resistant fluoroapatite in

place of the primary hydroxyapatite (remineralization of enamel).

Salivary buffers:

Buffer systems maintain the acid-base balance by neutralizing organic acids (from food and produced by cariogenic bacteria). The most important salivary buffers are:

bicarbonate buffer (higher concentration in stimulated saliva): HCO3

- + H3O+ H2CO3 + H2O phosphate buffer (higher concentration of unstimulated saliva):

HPO42- + H3O+ 2PO4 2- + H2O

saliva buffer capacity - mainly responsible is bicarbonate buffer insufficient buffering capacity of saliva towards acids development of caries, dissolving of the hydroxyapatite

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Saliva as a diagnostic material:

advantages saliva as a diagnostic material: availability and non-invasive way of sampling indicators of the degree of risk of dental caries: amount of secreted saliva, buffer capacity, content of microorganisms Saliva is used for determining the concentration of:

steroid hormones, drugs (benzodiazepines, theophylline, cyclosporine, paracetamol, gentamicin, cisplatin) in toxicology to determine the concentration of the metals (lithium, cadmium, gallium) addictive substances (drugs and their metabolites, such as amphetamine, cocaine , methadone, phencyclidine, marihuana, opiates) - in the presence of saliva a few minutes after use and is keptfor 24 hours alcohol - is detected in saliva after 20 minutes of ingestion and maintained for 12 hours

diagnosis of certain diseases, including cancer, cardio-vascular, autoimmune, infectious (HIV, measles, rubella, viral infection of the liver) and gastrointestinal diseases forensic medicine

Gingival crevicular fluid: Physiological transudate formed in the gingival sulcus Percentage composition plasma-alike The main source of immune cells in the oral cavity The biochemical analysis of the fluid - non invasive means of assessing the response in oral disease. Mesered parameters:

bacterial enzymes, bacterial degradation products, connective tissue degradation products, host mediated enzymes, inflammatory mediators, extracellular matrix proteins higher levels of these parameters can occur in gingival crevicular fluid in oral diseases

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Thank you for your attention.