Dental Caries is a Multifactorial Disease

3. Development of Noncavitated Enamel Caries.[Modified from Fejerskov and Clarkson, 1996]

4. Terminology Deposit: something precipitated, delivered, and left, or thrown down, as by a natural process; a natural accumulation Integument: a natural covering (as skin, shell, or rind); any covering coating, enclosure. Pellicle: a thin skin or membrane; film, scum Plaque: a thin flat plate

5. Classification of Surface Coverings: Non-Mineralized Coverings of the Enamel Surface Coverings of embryologic origin Coverings acquired after eruption of teeth

6. Coverings of Embryologic Origin Primary Enamel Cuticle Reduced Enamel Epithelium Primary enamel cuticle- Def: Light micorospic term fore an apaprnet organic layer on the enaeml surface fo underupted teeth and sondered to be the final secretory product of the amelolsets. Electorn microspic observations have failed to subbastanitiate the existance of theprimary eneamel cuticle. organic layers that was the final secretory product of the ameloblasts after the formation of enamel was completed, also called the inner cellular and outer cellular layers of the Nasmyth�s membrane. With the electron microscope no organic structure corresponding to a primary enamel cuticle or the dark layer seen between ameloblasets and enamel by phase-co0ntrast microscopy was ever observed. The absence of a primary enamel cuticle in sections studied with electron microscopy suggests that the appearance of a cuticle with light microscopy is probably an artifact due to an optical phenomenon Reduced Enamel Epithelium and basal lamina. DEF: A few layers of flat cubnnoidfal cells aht are the reamisns of the epitheliala enamel organ afrter the formation of enaeml is complete. The peripheral portion of the reduced enamel epithelium consists of cells derived primarily form the stratum intermedium. Although it is generally agreed that the structures of embryologic origin are lost with or soon after eruption of the teeth, the exact sequence of events accompanying the loss of the embryologic covering of enamel and the acquisition of an acquired coating is not known. Primary enamel cuticle- Def: Light micorospic term fore an apaprnet organic layer on the enaeml surface fo underupted teeth and sondered to be the final secretory product of the amelolsets. Electorn microspic observations have failed to subbastanitiate the existance of theprimary eneamel cuticle. organic layers that was the final secretory product of the ameloblasts after the formation of enamel was completed, also called the inner cellular and outer cellular layers of the Nasmyth�s membrane. With the electron microscope no organic structure corresponding to a primary enamel cuticle or the dark layer seen between ameloblasets and enamel by phase-co0ntrast microscopy was ever observed. The absence of a primary enamel cuticle in sections studied with electron microscopy suggests that the appearance of a cuticle with light microscopy is probably an artifact due to an optical phenomenon Reduced Enamel Epithelium and basal lamina. DEF: A few layers of flat cubnnoidfal cells aht are the reamisns of the epitheliala enamel organ afrter the formation of enaeml is complete. The peripheral portion of the reduced enamel epithelium consists of cells derived primarily form the stratum intermedium. Although it is generally agreed that the structures of embryologic origin are lost with or soon after eruption of the teeth, the exact sequence of events accompanying the loss of the embryologic covering of enamel and the acquisition of an acquired coating is not known.

7. Primary Enamel Cuticle Light microscopic term for an apparent organic layer present on the enamel surface of unerupted teeth and considered to be the final secretory product of the ameloblasts. Electron microscopic observations have failed to substantiate the existence of the primary enamel cuticle.

8. Reduced Enamel epithelium A few layers of flat cuboidal cells that are the remains of the epithelial enamel organ after the formation of enamel is complete.

9. Coverings Acquired after Eruption of Teeth Materia Alba Food Debris Dental Plaque Dental Calculus

10. Materia Alba A soft, white mixture of bacteria, salivary components, desquamated epithelial cells,and disintegrating leukocytes that adheres loosely to the surface of the tooth, dental plaque, or the gingiva.

12. Food Debris Large food particles that can be removed with a stream of water or by rigorous mouth rinsing

13. Dental Plaque A tenacious structure formed on tooth surfaces which contains large numbers of closely packed microorganisms surrounded by salivary components and extracellular material of bacterial origin

15. Dental Calculus

16. Acquired Pellicle Histological Appearance Composition Function

17. Acquired Pellicle An organic film on tooth enamel surfaces formed by selective adsorption to apatitic surfaces of specific glycoproteins of salivary origin.

18. Histology Acquired Pellicle Acellular, scalloped surface with bacterial ghosts and debris 2 hour, 100-700 nm 24 hours, 50-1,000 nm Can be globular, fibrillar, or granular depending on the nature of the surface it adheres to Sub-surface pellicle

26. Composition Many possible contributors Saliva Bacteria Epithelium Gingival crevicular fluid Hemmorrhage from extraction Exogeneous material such as the diet Oral hygiene agents Dental materials and restorations Also may be affected by the �enamel fluid� that apparently flows from the pulp to the enamel surface

27. Composition of Acquired Pellicle There is a definite contribution of bacterial cell walls to the composition of natural pellicle

28. Function of the Acquired Pellicle Lubricating medium between opposing surfaces of enamel Acquired Pellicle in some instances is necessary for plaque formation. After the formation of pellicle bacteria begin to adhere to the surface of the pellicle Protective function Fluoride Remineralization

29. Composition and Ecology of the Oral Flora Important Concepts about Bacteria Caries is an infectious disease Flora of the mouth Bacterial composition of the mouth

30. Important Concepts About Bacteria Ecology Biofilms Structure, morphology, etc. Bacterial physiology Virulence factors

31. Ecology A study of the interrelationships of organisms with their environment and the factors in the environment that determine the distribution and abundance of organisms Niche: Oral cavity The factors: Water, temperature, food The organisms: Streptococcus, Lactobacillus, Actinomyces, and hundreds of others

32. Ecology Ecosystem: A community and their environment: Tongue, tooth, gingival crevice, saliva Community: Groups of species within each ecosystem: Streptococcus, Lactobacillus Populations: A species present in a community: Streptococcus mutans Organisms: A bacteria present in a popualation: Streptococcus mutans

33. Biofilms A non-random assembly of microorganisms attached to a surface Bacteria in environment exist in two states: Free living Complex unattached communities=Biofilm Characteristics: Slimy(capsule) Exposed to liquid(but undisturbed) Has channels for nutrients Becomes more anaerobic over time

34. Biofilms Advantages Community formed Protected form antibacterial agents Nutrients concentrated Examples?(besides plaque!) Good biofilms vs. bad biofilms How do we control biofilms?

35. Review of Bacterial Structure Morphology(rods=bacillus, spheres=cocci, spirals=spirochetes) Aggregation(filaments, chains/ bunches) Classification(gram+, gram-) Cell wall structure(peptidoglycan, EPS) Lifestyles(aerobic, anaerobic, acidogenic, aciduric)

36. Bacterial Physiology Nutritional requirements: Nitrogen, carbon, sugar, amino acid, lipids, phosphate, sulfate, other ions, iron Energy production: respiration, fermentation CHO lactic, pyruvic, acetic, butyric acid Sucrose glucan(glucosyltransferases)

37. Virulence Factors A bacterial product or strategy that contributes to virulence or pathogenicity of the organism. Types; those that promote colonization, invasion and those that cause damage to the host Examples: adhesins/fimbriae adhere to host cells Bacteriocins kill your neighbors Capsules evade host immune system Exoenzymes proteases, siderophores, toxin

38. Caries Is an Infectious Disease Koch�s postulates Pathogenic vs. nonpathogenic How do we know bacteria cause caries?

39. Koch�s postulates The bacterium should be found in all people with the disease and the bacterium or its products should be found in parts of the body affected by the disease The bacterium's should be isolated from the lesions of an infected person and maintained in pure culture The pure culture, inoculated into a susceptible human volunteer or experimental animal, should produce symptoms of the disease The same bacterium should be re-isolated in pure culture from the intentionally infected human or animal

40. Problems with Koch�s postulates Implies that virulence is independent of the host Some bacteria are nonculturable in pure form Implies that all members of disease-causing species are equally virulent Implies that one bacterium is responsible for the disease Ignores human ethics

41. Modified �Koch�s postulates for virulence factors� The gene(s) for the virulence factor should be found and expressed in all strains of the bacteria that cause the disease Disrupting the gene(s) with a mutation should reduce virulence or introduction of the gene(s) into a bacterial strain that is avirulent now renders it virulent The gene is expressed during infection Antibodies to the gene product are protective

42. Pathogenic vs. Nonpathogenic Bacteria are everywhere Definition of �normal flora� External factors that upset flora leading to disease Diet Host

43. Pathogenic vs. Nonpathogenic Mere presence of S. mutans does not render it �pathogenic� Capability to cause disease ALWAYS present: It�s all about opportunity! Colonization sugar, loss of toothbrush, saliva buffering capacity Caries pH flux, demineralization

44. Virulence Factors of Cariogenic Bacteria Acidogenic (acid production leads to demineralization) Aciduric (must be able to live in acidic environment) Adherent (can adhere tightly to surfaces in mouth) Encapsulated (sticky, prevents neutralization) Superoxide detoxification (stress defense)

45. Dental Caries Is an Infectious Disease Characteristics of infectious disease: Transmissible Treatable with antibiotics(bacterial agent) Are present at disease site Early experiments: Germ-free mice on highly cariogenic diet were caries-free Transfer of Streptococcus from cariogenic mouse to uninfected mouse resulted in caries Gnotobiotic mouse infected with the usual suspects on highly cariogenic diet acquired caries Cohabitation of caries-free mice with cariogenic mice resulted in all cariogenic mice

46. Dental Caries is an Infectious Disease How do we know bacteria cause caries? Germ free mice do not develop caries Antibiotics are effective treatment Oral bacteria can demineralize enamel in vitro Bacteria are isolatable form the carious lesion Caries is transmissible from mother to child Unerupted teeth do not develop caries

47. Etiologic Factors (Summary) Microflora: acidogenic bacteria that colonize the tooth surface Host: quantity and quality of saliva, the quality of the tooth, etc. Diet: intake of fermentable carbohydrates, especially sucrose, but also starch Time: total exposure time to inorganic acids produced by the bacteria of the dental plaque

48. Flora of the mouth Streptococcus mutans Other Streptococcus spp. Lactobacillus Actinobacillus, Actinomyces Others

49. Streptococcus mutans Gram positive cocci Facultative anaerobe Cariogenic Makes extracellular capsule of dextran and / or fructan from sucrose(smooth and rough variants) Little or no fimbriae Able to ferment a variety of sugars including mannitol and sorbitol (homofermentative lactic acid former) Very acid tolerant Adhesins: P1 (anti-P1 antibodies render S. mutans non-cariogenic) Binds to S. sanguis, S. oralis and A viscousus, but not to Veillonella

50. Other Streptococcus spp. Includes S. mitior, S. oralis, S. sanguis. S. Gordonii, S. sobrinus(cariogenic) Most species found in dental plaque Majority of species make IgA1 protease (human IgA1 prevents binding of bacteria to tooth sucrose All make extracellular polysaccharides (glucans) from sucrose via glucoysltransferases S. oralis early colonizer S. sanguis: can metabolize arginine as sole energy source, early colonizer, less aciduric (produce ammonia to neutralize acid) S. salivarius, S. sobrinus: bind to other bacteria

51. Lactobacillus spp. Gram-positive rod Facultative anaerobe Homo- and heterofermenter Grows at low pH Debated role in caries, used in susceptibility tests L. acidophilus, L. casei, L. salivarius

52. Actinobacillus actinomycetemcomitans Gram positive rod Facultative anaerobe Agent of periodontal disease Makes IgA protease Makes a leukotoxin which destroys human and monkey neutrophils(PMN�s) Non-fermenter of carbohydrates

53. Actinomyces viscosous Gram-positive rod Facultative anaerobe Early colonizer Causes root caries in mice Two types of fimbriae: Type 1 binds to saliva, Type 2 binds to Streptococci

54. Fusobacterium nucleatum Gram-negative rod Obligate anaerobe Colony morphology varies with Fusobacterium species Agent of periodontal disease

55. Veillonella atypica Gram-negative cocci Obligate anaerobe Major resident of the human tongue Non-fermenter of carbohydrates

56. Autogenic Succession The adaptation of microbial populations to change in the environment Early dominance of streptococci Shift to anaerobes and filamentous bacteria Streptococci shifts to Actinomyces, followed by and increase in Veillonella and Fusobacterium

61. Animal models Germ free mouse Gnotobiotic mouse Rat, hamster, monkey

62. Bacterial Colonization of the Oral Cavity Flora of tooth Flora of the tongue and epithelial surfaces

63. Flora of the Tooth Smooth surface: S. mutans-very significant S. sanguis Pits and fissures: S. mutans-very significant S. salivarius Lactobacillus spp. very significant Flora of the root surface: Actinomyces spp. Streptococcus spp

64. Flora of the Tongue and Epithelial Surfaces S. salivarius: present in greatest amounts, minor significance in caries S. mitior found on mucous membranes S. Sanquis Veillonella

65. Microbial Aspects of Dental Caries Plaque biology Diagnosis Prevention and treatment Current research

66. Plaque Biology How plaque forms Contributions of diet Contributions of host

67. ��..yet notwithstanding, my teeth are not so cleaned theory (rubbing with rag and salt) but what here sticketh or groweth between my front ones and my grinders a little white matter which is as thick as if twere batter���I then most always saw, with great wonder, that in the said matter there were many very little living animalcules, very prettily a-moving��-Anthony Von Leeuwenhoek(1632-1723)

68. How plaque forms Acquired pellicle formation Primary colonizers: S. sanguis, S. oralis, S. salivarius, S. mitis, Actinomyces Secondary colonizers: S. mutans Plaque composition (diet, hygiene, saliva, host immunological experience) Bacterial multiplication plaque build-up

69. Adhesion A mechanism through which cells become attached tenaciously to other cells or to surfaces. Adhesion is considered important in the process of bacterial colonization.

70. Agglutination Bacterial Cells under the influence of specific molecules or cell mechanisms adhere together to form clumps and therefore exhibit agglutination behavior

71. Bacteriocins Antibacterial compounds produced by some streptococci against specific bacteria that may inhibit attachment or growth of the organism in the immediacy of the bacteriocinogenic streptococci.

72. Glucosyltransferase

75. Extracellular Polysaccharide Polymers produced extracellularly by plaque bacteria using sucrose or some other sugar as a substrate. Examples of extracellular polysaccharides are glucans and fructans. Surface Receptors

76. Formation and Development of Dental Plaque Initial Colonization first eight hours rapid, selective Rapid Bacterial growth 8 hours - 2 days after prophylaxis Remodeling 2 days and continues indefinitely, organisms remain constant, with increasing organization

77. Contributions of Diet Sugar Simple sugars diffuse acid into plaque Bacteria produce acid from sugar Brief sugar exposure leads to rapid decrease in plaque pH Repeated sugar consumption leads to demineralization of tooth

78. Roles of Saliva(Host) Attachment (PRPs) Nutrition (alpha amylase) Aggregation and clearance Antibacterial (sIgA, lysozyme, lactoferrin) Buffering agent (demineralization)

79. Major antimicrobial proteins of human whole saliva Immunoglobulins Secretory immunoglobulin A Immunoglobulin G Immunoglobulin M Nonimmunoglobulin proteins Lysozyme Lactoferrin Salivary peroxidase system (enzyme: SCN�-H2O2) Myeloperoxidase system (enzyme: SCN�/halide-H2O2) Agglutinins Parotid saliva glycoproteins Mucins Secretory immunoglobulin A Beta-microglobulin Fibronectic Histidine-rich proteins (histatins) Proline-rich proteins

81. Microbial Composition of Plaque Supragingival plaque/tooth interface condensed microbial layer body of the plaque plaque surface Fissure gram+ cocci and short rods microorganisms and food particles fewer morphological types Subgingival

84. Chemical Composition of Plaque 80% water, 20% solids Protein 40-50% by weight of plaque, carbohydrates 13-18%, lipids 10-14% Similar to washed streptococcal cells, but higher in protein and lipid components, due to intracellular matrix, higher lipid content is from accumulation of gram- organisms

85. Carbohydrates Glucose predominates Arabinose, ribose, galactose, fucose Extracellular polymers(glucans, fructans) or heteropolysacharrides......all produced by microorganisms reservoir of fermentable carbohydrate for plaque metabolism, function in adherence and coherence glucans(dextrans, mutans, amylose type) mutans skeletal link fructans(levans, inulin type)

86. Proteins From saliva, bacteria or gingival fluid amylase lysozyme IgA(from gingival crevicular fluid) IgG(from gingival crevicular fluid) albumin Glucosyltransferase, glucan hydrolase, hyaluronidase, phosphatases, proteases

87. Inorganic Components Calcium, phosphate, fluoride in higher concentration than in saliva fluoride 14-20ppm(in saliva .01ppm)

88. Metabolism of Dental Plaques Glycolysis Concept of Critical pH Clinical Data on Plaque pH in Relation to Caries Properties of Cariogenic Plaque

89. Stephan�s Curve Response

90. Diagnosis Bacterial identification methods Caries activity tests

91. Procedures Obtain plaque sample (high variability) Preservation and dispersion Transport Culture

92. Bacterial identification methods DNA probes RFLP (restriction fragment length polymorphism) PCR (polymerase chain reaction) Ribotyping Bacteriocin typing Fluorescent antibodies

93. Requirements for Caries Activity Tests Accurate Simple Inexpensive Quick Experimentally valid

94. Caries Activity tests Lactobacillus count Streptococcus count Buffer capacity test (pH of saliva) Do they predict future caries activity?

95. Caries Activity Tests

96. Current Research Methods Physical models Animal models

97. Fields of Study Bacterial composition of plaque, biofilm characteristics Sugar, fluoride, pH effects on plaque formation and composition Bacterial adherence

98. Physical models Mice culture chemostat Artificial mouth In vitro model Intra-oral

99. Animal Models Dental characteristics similar to humans(rat, monkey) Success depends on animal�s susceptibility, applicable results to humans Diet, animal care important Procedures: illicit immune response with vaccine, infect with specific bacteria, alter diet etc., standard scoring methods

100. Control of Dental Plaque Agents that act against the microflora Agents interfering with bacterial attachment attacking plaque matrix components altering the tooth surface Mechanical removal of plaque

101. Prevention and Treatment Vaccines Antibiotics/ Antimicrobials Other

102. Immunology Humoral: IgG Blood (serum) Mucosal Immunity: IgA Location: salivary glands, gut(Peyer�s patches), breast milk, tears, secretions Types IgA1(CH0), IgA2(lipids) Mode of action: neutralization of enzymes or toxins by agglutination, inhibition of attachment or colonization The immune system remembers

103. Use of Antibiotics/ antimicrobials in Dental Caries Safe for intraoral use (safe if swallowed) Active over range of pH, buffered saliva Acceptable taste Able to penetrate and be retained in plaque Mouthrinse, gel, dentifrice, topical varnish

104. Antibiotics/ Antimicrobials Bacteriostatic vs. Bactericidal MIC=minimum inhibitory concentration Testing a possible agent Absorption characteristics? Mechanism Goal: control rather than eliminate plaque

105. Concerns with Antibiotics Abuse (doctors, patient) Antibiotic resistant bacteria Disrupt flora- opportunistic infections Decrease efficiency of other producers Schedule of delivery Substantivity

106. Other Antibiotics/ Antimicrobials Chlorhexidine (bis biguanides) Triclosan (phenolics) Others (phosphates, iodine) Fluoride Combination therapy Oxygenating agents peroxide perborate Quaternary ammonium compounds

107. Prevention of Caries by Plaque Control: The Use of Chlorhexidine in the Control of Caries Indications Mechanism of action, dosage and delivery Side effects staining parotid gland swelling desquamation of gingiva long-term effects Methods of application

108. Other preventatives/ treatments Lower plaque pH Interfere with attachment Attack plaque matrix Sugar substitutes (Xylitol)

109. Oral Hygiene and Dental Caries Effect of Toothbrushing on Dental Caries Effect of Flossing on Dental Caries Professional Prophylaxis

110. Control of Dental Plaque Mechanical removal toothpick toothbrush (1498) commercial development of toothbrushes 18th century little change in design

111. Plaque Detection FD & C no. 3 (erythrosine) tablet, gel or solution Two tone dye (erythrosine and malachite green) Fluorescein sodium

112. Toothbrush Design Lateral profile Cross-sectional profile Bristle shape Bristle firmness Nylon versus natural Bristle actions Handle design Powered toothbrushes

113. Toothbrushing and Toothbrushing Techniques Is there an ideal toothbrush Natural vs. Nylon Handle, head profile and shape Manual vs. Powered in effectiveness and motions used during brushing Methods for use Why are different amounts of time needed by different individuals for toothbrushing? Modification of toothbrushing techniques applicable to special patient care, patients using prostheses and those under orthodontic care

114. Toothbrushing Methods Horizontal Fones Leonard Stillman Charters Bass The Rolling Stroke

115. Toothbrushing Frequency Time Frequency

116. Supplemental Brushing Tongue brushing Special needs Abutment teeth Orthodontic appliances

117. Prevention of Caries by Plaque Control: Does a Clean Tooth Decay Advice to Patients

118. Auxiliary Measures Dental floss Dental floss holder Dental floss threader Knitting yarn Pipe cleaner Gauze strip Interdental tip stimulator

119. Dental Floss Remove plaque and debris that adhere to teeth, restorations, orthodontic appliances, fixed prostheses and pontics, gingiva and the interproximal embrasures and around implants Polishes the surfaces as it removes the debris Massages the interdental papillae

120. Dental Floss Aides in identifying the presence of subgingival calculus deposits, over hanging restorations, or interproximal carious lesions May be used as a vehicle for the application of polishing or chemotherapeutic agents to interproximal and subgingival areas Reduces gingival bleeding

121. Interproximal Plaque Removal Interproximal brushes Dental floss Water jets Toothpicks