2010 Dental Lecture 48-Biochemistry of Dental Caries

Biochemistry of Tooth development and Formation of Dental Carious Lesions

• Dr. Kumar• Lecture 48

Section of a human molar

Macromolecules associated with calcification in mammals

Component Cartilage Bone Dentine Enamel

Collagen I + + + -

GLA Proteins (Gamma carboxy glutamate)

(osteocalcin, osteopontin, osteonectin, osteogenin

+ + + -

Glycoproteins + + + +

Phosphoproteins + + + +

Proteoglycans + + + ND

Components of Calcified Oral Tissues

Tissue Inorganic components

Protein Components Other components

Cementum 45% 33 % protein

Mainly Collagen type I and III

Water 22%

Dentin 70% 20% (collagen I, b-FGF, osteocalcin, osteonectin, osteogenin, osteopontin

Phosphoproteins, Dentin specific proteins (DSPs)

10% (Glycoproteins, proteoglycans) phospholipids, water

Enamel ~95% (5 %) Ameloblastin, amelogenin,enamelins, Tuftelins

<1 %

Origin of Oral Cavity Cell Types

Pulp cavity: Filled with soft connective tissue and contains blood vessels which enter the tooth from a hole in the root. It contains pre-odontoblasts, fibroblasts, macrophages, T-lymphocytes. The pulp is called “the nerve” of the tooth

Cementoblasts: Derived from periodontal ligament cells (PDL cells) and fibroblastic progenitor cells

Odontoblasts: Most likely derived from mesoderm as embryonic mesenchymal cells reside in pulp cavity from which these cells are derived. Active all during life of the organism

Ameloblasts: Ectodermal origin. Differentiate to mature ameloblasts which die off after laying down the enamel

Cells producing dentin and enamel

Higher magnification view of ameloblasts

Cementoblasts

Transmission electron micrographs illustrating (a) an osteoblast, (b) an osteocyte, (c) a bone-lining cell, (d) AEFC-forming cementoblasts, (e) a CIFC-forming cementoblast, and (f) a cementocyte. AEFC, acellular extrinsic fiber cementum; CIFC, cellular intrinsic fiber cementum; and EF, extrinsic fibers.

CementumCementum is a hard connective tissue that is derived from ectomesenchyme

Embryologically, there are two types of cementum:Primary cementum: It is acellular and develops slowly as the tooth erupts. It covers the coronal 2/3 of the root and consists of intrinsic and extrinsic fibers (PDL).Secondary cementum: It is formed after the tooth is in occlusion and consists of extrinsic and intrinsic (they derive from cementoblasts) fibers. It covers mainly the root surface.Functions of Cementum: It protects the dentin (occludes the dentinal tubules)It provides attachment of the periodontal fibersIt reverses tooth resorption Cementum is composed of 90% collagen I and III and ground substance.50% of cementum is mineralized with hydroxyapatite. Thin at the CE junction, thicker apically.

Dentin Proteins1. Collagen type I2. FGF beta: Occurs in dentine in inactive form and is activated during

active dentin formation3. Osteopontin: Highly acidic glycoprotein of MW 44,000-probably binds

Ca++ during calcification.4. Osteonectin: Osteonectin is a glycoprotein in the bone that binds

calcium. It is secreted by osteoblasts and odontoblasts initiating mineralization and promoting mineral crystal formation. Osteonectin also shows affinity for collagen in addition to bone mineral calcium

5. Osteogenin: Also known as bone morphogenic protein6. Osteocalcin: Osteocalcin is a noncollagenous protein found in bone and

dentin. It is secreted by osteoblasts and thought to play a role in mineralization and calcium ion homeostasis. It has been stipulated that osteocalcin may also function as a negative regulator of bone formation, although its exact role is unknown.

7. Dentin specific proteins

Dentin Specific proteins• Several extracellular matrix (ECM) proteins have been isolated

from dentin and shown to be synthesized by odontoblasts.

• Highly phosphorylated proteins (HP), the phosphophoryns, are specifically found in dentin and are synthesized only by odontoblasts. Phosphophoryns are probably involved in controlling the site and/or the rate of circumpulpal (calcified tissue surrounding the pulp cavity) mineralization.

• Other moderately phosphorylated (MP) and low phosphorylated (LP) proteins have also been detected in dentin, but have not been studied extensively.

• Dentin specific sialoprotein-a marker of dentin mineralization

Western immunoblots with anti-DSP antibody. DSP in both dentin and bone were detected as a 95-kDa band on 5-15% SDS-PAGE. Lane 1,

molecular marker; lane 2, 0.3 µL of dentin D3; and lane 3, 60 µL of bone D3. Note that the density of the 95-kDa band in lane 3 is much lower than

that in lane 2.J DENT RES, Vol. 81, No. 6, 392-394 (2002)

RT-PCR products generated from tooth germ and calvaria RNA. Lanes 1 and 11 are DNA markers (bp). Lanes 2 (DSP), 4 (DPP), and 6 (DSPP) are samples from the RT-PCR products generated from tooth germ with the 35-cycle protocol. Lanes 3 (DSP), 5 (DPP), and 7 (DSPP) are samples generated from calvaria RNA with the 35-cycle procedure. Lanes 8 (DSP), 9 (DPP), and 10 (DSPP) are from calvaria RNA with the 40-cycle procedure. Please note that the band densities of lanes 8, 9, and 10 (calvaria, 40-cycle) are similar to those of lanes 2, 4, and 6 (tooth germ, 35-cycle), respectively.

Enamel ProteinsAmeloblastin: the function of ameloblastins is believed to be in controlling the

elongation of enamel crystals and generally directing enamel mineralization during tooth development.

Amelogenin: The latest research indicates that this protein regulates the initiation and growth of hydroxyapatite crystals during the mineralization of enamel. In addition, amelogenins appear to aid in the development of cementum by directing cells that form cementum to the root surface of teeth. It is hydrophobic

Tuftelin: is an acidic phosphorylated glycoprotein found in tooth enamel. This protein is formed for a short time during amelogenesis. The function of tuftelins is under contention, but it is proposed that it acts to start the mineralization process of enamel during tooth development. Highly hydrophilic

Enamelin: is a protein found in developing tooth enamel. About 30 % of developing enamel consists of protein of which enamelins comprise <1%. Enamelin is not a proteolytic enzyme and does not degrade other enamel proteins. Two enamelins of MW 63 and 67 Kda have been purified. These strongly bind to hydroxyapatite. Mechanism of their action is not clear.

Solubility of recombinant and native amelogenins. (a) Solubility of rM179 and rM166 in 0.05 M potassium phosphate buffer, 0.15 M ionic strength, 25°C, as a function of pH. (b)

Solubility of the 25K, 23K, and 20K porcine amelogenins under the same conditions. (Reprinted from Tan et al., 1998, with permission.) The 25K, 23K, and 20K proteins are

analogous to P173, P161, and P148, respectively.

Proposed model for the onset of nanosphere aggregation at pH 8. At low temperatures (room temperature and below), amelogenin assembles into nanospheres with a dense (hydrophobic) core surrounded by a shell of (hydrophilic and negatively charged) chain segments.

Parallel arrays of enamel crystals (monkey) and in cross-section within orthogonal enamel rods (courtesy of Dr. Ziedonis Skobe).

Amelogenesis imperfectaAn autosomal dominant or x-linked disorder in which there is faulty development of the dental enamel owing to agenesis (incomplete development), hypoplasia (failure of the organ to grow), or hypocalcification of the enamel. It is marked by enamel that is very thin and friable and frequently stained in various shades of brown.

Ameloblastic Carcinoma Ameloblastic Fibroma

Enamel Proteins

• The genes for amelogenin can be used in sex determination of samples from unknown human origin. Using primers specific for intron I of this gene, PCR amplification of the X chromosome gives a 106 bp fragment and Y chromosome gives a 112 bp

fragment.

Male Female

Saliva and Dental CariesM. Lenander-Lumikari*. V. Loimaranta

Department of Cariology and Turku Immunology Centre, Institute ofDentistry, University of Turku, Lemminkaisenkatu 2, FIN - 20520 Turku,

Finland, Corresponding author, [email protected] Dent Res 14:40-47, December, 2000

Caries is a unique multifactorial infectious disease. Our understanding of etiological factors, the progress of the disease, and the effectiveness of prophylactic procedures have led us to believe that we understand the disease. However, we still have too few answers to many questions: "Why can we not predict who will get the disease?" "Why do we not become immunized?" "How much saliva is enough?" or "Which salivary components are protective?" and "Which salivary components predispose for caries?" It is generally accepted, however, that saliva secretion and salivary components secreted in saliva are important for dental health. The final result, "caries to be or not to be", is a complex phenomenon involving internal defense factors, such as saliva, tooth surface morphology, general health, and nutritional and hormonal status, and a number of external factors-for example, diet, the microbial flora colonizing the teeth, oral hygiene, and fluoride availability. In this article, our aim is to focus on the effects of saliva and salivary constituents on cariogenic bacteria and the subsequent development of dental caries.

Salivary Enzymes in Normal Function and Abnormal Pathology

• α-Amylase—normally present in saliva—important in digestion

• Lysozyme • Hyaluronidase• Chondrosulfatase• Aryl Sulfatase• Neutral Protease• Collagenases

Dental CariesParotid Gland

Lysozyme

Oral Microbe Cell Wall Polysaccharides(polymers of NAG and NA-muramic acid)

HyaluronidaseMucinase

ChondrosulfataseArylsulfatase

Salivary Enzymes-Role in Dental Caries

• Components of Dentine are:– Organic matrix consisting of collagen, glycoproteins,

proteoglycans, dentin specific proteins

– Inorganic matrix consisting of hydroxyapatite

• In Oral Cavity– Starch Dextrins Monosaccharides

α-amylase β-amylase

Monosaccharides Lactic acid under anaerobic glycolysis

Ca3(PO4)2 + H+ + 4 H2O 3Ca(OH)2 + 2H3PO4

Functions of Released Enzymes• Components of dentin—Collagen, Hyaluronic acid,

Sulfated glycoproteins, Hydroxyapatite

• Hyaluronic Acid NAG + Glucuronic Acid

• HU

• Sulfated Glycoproteins NAG sulfate + Glucuronic

Acid

• AS, CS

Acids produced breakdown the hydroxyapatite crystal lattice

Resorption of Organic matrix

Abnormal Dentin Structure

Exposed Collagen type I in a

Abnormal Hydroxyapatite Crystal lattice

Human Collagenase type I Bacterial collagenase

¾ and ¼ Chains of collagen

Type IV Collagenase Neutral Proteinase

Amino acids and peptides

Hormonal factors in Dental Caries

• Salivary Clearance capacity-higher the flow-better flushing and lower caries levels

• Antibiotics and other drugs decrease salivary flow rate-higher rate of caries

• Salivary buffer effect-greater in men than in women –women have higher incidence of caries

• Post menopausal women have higher incidence of caries-hormone replacement therapy improves the condition