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2012-02-08 18:53:12

Caries midterm
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  1. White Spot Lesion:
    • 1) plaque begins to release enzymes and acid into underlying enamel  DEMIN
    • 2) The enamel UNDER-NEATH the plaque demins first  bc it reflects light differently, this looks like a chalky white spot (remains UNCAVITATED)
    • 3) Upon application of MI paste or CPP-ACF, Ca2+ and HPO4 are driven back into the enamel and it will begin to heal.
    • 4) Bottom Line: we can remineralize teeth and change the makeup of plaque!!!
    • Quick Note about White Lesions: as long as lesions remain UNCAVITATED, they can be healed with CPP-ACF. That’s partially why a sharp explorer is no longer used to check for caries; you may inadvertently cavitate (make a hole thru) a soft white lesion and prevent any chance of healing it “naturally”.
  2. Caries multi-factorial disease:
    • 1. HOST
    • 2. TIME - driven by the FREQUENCY of the eating (shifts the demineralization <> remin balance)
    • 3. DIET -dependent on DIETARY SUCROSE
    • 4. MICROFLORA - a BACTERIAL DISEASE: S. mutans, lactobacilli, A. ciscosus, S. sobrinus (all acidogenic, acid tolerant)
  3. Caries is modified by?
    • i. Harder tooth structure
    • ii. Inhibits acid production by bacteria
    • SALIVA
    • i. Buffering,
    • ii. Demin  remin balance
    • iii. Low flow of saliva = high risk
    • a. Treat the caries before it even starts (a carious lesion is really the END-STAGE of disease)
    • b. Medical management of caries
  5. CAMBRA:
    • Caries Management by Risk Assessment
    • a. The idea is to TILT the balance toward NO CARIES by decreasing risk and disease.
    • b. Assessment  Determine Risk Status
    • a. Based on how many disease factors you have as well as disease indicators, each person gets labeled at different RISK LEVELS for caries:
    • b. LOW
    • c. MEDIUM
    • d. HIGH
    • e. EXTREME
    • c. Make changes to “Our Present Understanding of Caries”:
    • a. Caries is a BACTERIAL DISEASE Change the microflora with topical chlorhexidine, fluoride, and povidone iodine, Dr. Shi’s cavity-fighting lollipop (kills S. mutans)
    • b. Caries is dependent on DIETARY SUCROSE  reduce dietary sucrose and ADD XYLITOL (a sugar that is metabolized differently by bacteria and doesn’t contribute to biofilm)
    • c. Caries is driven by the FREQUENCY decrease the frequency of eating (CHUG vs SIP), avoid sodas with high acidity (critical pH for acid challenge = 5.5)
    • d. Caries is modified by SALIVA increase salivary flow by mechanical stimulation/vigorous chewing and changing drugs with reduce flow
    • e. Caries is modified by FLUORIDE, CALCIUM, AND PHOSPHATE  add fluoride, Ca2+, & P8
  6. Saliva Deficiency
    • a. Causes: Medication Side effects, stress, dehydration, salivary gland dysfunction, disease, hormonal imbalance, smokine
    • b. Signs: difficulty eating or swallowing, tongue sticking to the roof of your mouth, changes in taste, inadequate denture retention, soft tissue trauma
  7. Saliva Flow
    • i. Stimulated Saliva Flow
    • 1. Normal: >1 ml/min
    • 2. Low: 0.7 -1 ml/min
    • 3. Very Low: <0.7 ml/min
    • ii. Unstimulated Saliva Flow
    • 1. Normal: >0.25 ml/min
    • 2. Low: 0.1 – 0.25 ml/min
    • 3. Very low: <0.25 ml/min
  8. How does a decrease in salivary flow influence the risk for dental caries?
    Saliva acts as a buffer; reducing saliva prolongs the affects of acid
  9. During the formation of hydroxyapatite, what element may occur in the voids created by the disordering of the hydroxyl groups of the hydroxyapatite crystal?
  10. In the delicate balance between remineralization and demineralization, what is the critical pH for most individuals thought to be?
    <5.5 demineralization occurs
  11. In our contemporary concept of dental caries, what set of parameters need to be present in order for dental caries to occur?
    Nutrition, Bacteria, Time, Host
  12. What immunoglobulin is associated with salivary secretions?
  13. What is the general formula for the calcified matrix of enamel?
  14. Who proposed the Chemo-parasitic Theory?
  15. What are the reparative mechanisms for an acellular tissue such as tooth enamel?
    There are no repair mechanisms for enamel.
  16. What is the most acidic organic acid produced through glycolysis by plaque bacteria?
    Lactic Acid
  17. What is the approximate percentage of inorganic material in enamel and dentin?
    96% in Enamel, 70% in Dentin
  18. Under normal physiologic conditions what ion is the saliva saturated with?
  19. What enzyme is responsible for the conversion of pyruvate to lactate during bacterial glycolysis?
    Lactate Dehydrogenase
  20. Which dental plaque organism is thought to be a key bacterium in the induction of pit and fissure caries?
    S. mutans
  21. After a short sucrose exposure the plaque pH will fall below the critical pH for approximately how long?
    20 minutes
  22. Which of the glucans are thought to be the most caries promoting?
    Branched (insoluble) Glucans
  23. How are the bacteria that cause dental caries transmitted from one individual to another?
    From care-giver to child
  24. Treatment of Xerostomia
    • i. Increased water intake
    • ii. Change medications
    • iii. Saliva substitutes
    • iv. Lubricating getl intraorally, Vaseline on lips
    • v. Toothpaste without additives
    • vi. DO NOT USE lemon and glycerine swabs/toothettes  turns to alcohol
    • vii. DO NOT UES alcohol containing mouthwashes
  25. Demineralization – Remineralization -> Healing WHITE SPOTS
    • a. Add mineral ions to the oral environment (see pictures of dentinal tubules below)
    • i. Flouride
    • ii. Amorphous Calcium Phosphate (ACP)
    • iii. Recaldent (CPP-ACP)
    • CPP-ACPF truly heals the white spot! Remin thru body of lesion + re-mineralized enamel is more acid resistant (red = fluroroapatite)
  26. Restorations/Replacements
    • a. If all else fails…too late to prevent? To remineralize?
    • b. The best restoration is to never to a restoration at all
    • c. Restorative dentistry is the END-STAGE treatment for dental caries
    • d. Glass Ionomer Restoratives: for transitional restorations
    • i. Can release fluoride and continue to uptake and release fluoride, also great at sealing margins
    • ii. Ex. Fuji IX GP Extra, Fuji II LC
    • e. Glass Ionomer “Sealant”
    • i. Protection for newly and partially erupted molars
    • ii. Remineralization of affected dentin
  27. The Chemo-Parasitic Theory (W.D. Miller – 1890)
    • 1. A unifying theory using an in-vitro caries model
    • 2. Much of his work was based on the work of others:
    • a. Pasteur – discovered microorganisms could convert sugar lactic acid
    • b. Magitot (1867) – showed fermentation of sugars would dissolve tooth enamel
    • c. Underwood and Miles (1881) – observed bacteria in carious lesions; considered caries to be ABSOLUTELY DEPENDANT upon presence of bacteria
    • 3. Miller tied in all these theories together – made association of role of bacteria with the importance of production of organic acid to the decomposition of teeth!
    • 4. Findings:
    • a. Acid present within deeper carious lesions
    • b. Different starchy foods could decalcify teeth when mixed with saliva
    • c. Several types of mouth bacteria could produce enough acid to cause caries
    • d. Lactic acid was identified product (LA is the most destructive and acidic organic acid)
    • e. Different microorganisms invade carious dentin
    • f. Acids produced from sugars found in the mouth could destroy the mineralized tooth.
    • i. Weakness: not able to define the importance of bacterial plaque biofilms and their relationship to the tooth surface.
  28. Plaque-Host-Substrate Theory
    • the modern Concept of Dental Caries
    • 1. Caries is a biofilm-associated disease which is primarily caused by the production of organic acids by the plaque bacteria.
    • 2. The organic acids are result from the fermentation of specific carbohydrates thru glycolysis.
    • 3. This is a MULTI-FACTORIAL DISEASE – only in interaction of biofilm(bacteria) + host(teeth) + presence of carbohydrate(substrate) will caries occur (REVERSIBLE)
    • a. If penetrate dentin, cannot be reversible - @ that point, would have to restore with mechanical means (filling)
    • b. Virulence comes from formation of BIOFILM
    • c. Closely related to the consumption of fermentable carbohydrates
    • d. Infectious and transmissible among animal species (often momchild)
    • e. A particular group of bacteria are most probable agents (Mutans sps)
  29. Infectious Nature of Caries
    • a. Dental Plaque
    • b. Dental Biofilms: form on all exposed dental tissues
    • i. Bacterial colonization is NOT random – occurs thru a highly specific adhesion/receptor interaction
    • ii. Difficult to eliminate and alter
    • iii. Salivary Pellicle: an acellular film containing proteins and carbohydrates that are favorable for bacteria to grow and presents specific binding sites that allow bacteria to attach on (if you don’t have pellicle, there probably won’t be any caries formation
  30. Infectious Nature
    • i. Bacteriological agents
    • ii. Genetic Component?: genetics play little part in caries but there are genetic symptoms that have associated malformaitons of teeth
    • iii. Very transmissible!
    • iv. Experiment: gnotobiotic “germ free” animals were given high carbohydrate diets  no caries
    • 1. But when inoculated with bacteria  caries developed
    • 2. Antibiotics  could suppress caries activity
    • 3. Bottom Line: showed you can have high carbohydrate diet but without bacteria, there is no caries!
  31. Experiments of Keyes & Fitzgerald:
    • provided experimental evidence for the infectious nature of caries
    • i. Experiment 1: caged together hamsters with high caries prevalence with hamsters with low caries  both groups got similar caries rates
    • ii. Experiment 2: took fecal pellets and oral plaque swabs and showed that caries-inactive group can become caries-active
    • iii. Experiment 3: caries-resistant offspring delivered by C-section would become caries active when raised by females of caries active group  mom passed onto kids
    • iv. Conclusions:
    • 1. Caries is caused by a microbial infectious agent.
    • 2. This disease is NOT a classical genetic disease that is related to some genetic trait
    • 3. Most likely infection is transmitted from mother to offspring early in life (only when teeth are beginning to develop…S. mutans cannot colonize when there are no teeth present)
    • features of dental biofilms contribute to caries induction and progression
    • a. Non-specific Plaque Hypothesis: Numbers important
    • b. Specific Plaque Hypothesis (what most people believe to be true now): a unique set of organisms need to be present and DO NOT have to be in high numbers
    • i. S. mutans – highly suspect induction of pit and fissure/smooth surface caries
    • ii. A. viscosus/naeslundii – root surface/cemental caries
    • iii. Lactobacillus sps – caries progression and dentinal caries
    • c. You don’t want to completely remove the biofilm because it protects you from more deadly diseases and keeps the other microflora in check.
  33. CHEMISTRY OF TOOTH ENAMEL- General Characteristics
    • i. It’s a TISSUE!!! Hardest tissue in the body (acellular)
    • ii. Has great ability to absorb and reflect light
    • iii. Only formed in one point of your life and must survive over your lifetime in an environment that is not sterile and under great physical stress
    • iv. Unfortunately, it’s the bacterial surface of choice!
  34. Chemical composition of teeth
    • Enamel Dentin/Bone
    • Inorganic Salts 96 wt% 70 wt%
    • Organics 1 20
    • Water 3 10
  35. Inorganic Salts
    • mainly HYDROXYAPATITE, a hydrated calcium phosphate salt
    • 1. Ca10(PO4)6OH2
    • 2. This is NOT stoichiometrically balanced!!!
  36. Organics (1-20 wt%)
    • 1. Remnants of EMP (enamel matrix proteins)
    • 2. Lipids (more so in dentin)
    • 3. Since organic molecules have nitrogen, they can oxidize  yellow color
  37. Water (3-10 wt%)
    • 1. Water content of enamel can vary resulting in changes in appearance of enamel
    • 2. When teeth are dehydrated (like first generation bleaching use to cause), it leaves a white, chalky appearance.
  38. Enamel: mineralized tissue composed of individual hydroxyapatite tightly packed together into prism-like structures
    • i. Enamel Rods: made of tightly packed, highly organized hydroxyapatite crystals
    • ii. Prisms
    • iii. Most important part of the crystal is the center – this is where the two hydroxyl groups reside
    • 1. If the two hydroxyl groups come together unfavorably, then electrons cannot be equally paired  will get void and eventually fill up with water
    • 2. As you get more voids, the structure will become weaker and weaker
    • 3. FLUORIDE ions fill in the voids and can participate in H-bonding  theory on how fluoride treatments help prevent cavities
    • a. Consists of balance of the following eqn: Ca10(PO4)6OH2 [H+] [Ca2+] + [PO4-3] + H2O
    • i. (acid catalyzed equilibrium reaction)
    • ii. If you lower pH, you will drive the equilibrium to ionized version of the salt
    • iii. Dentist job is to make sure there is not enough H+ around to make the acid catalyzed equilibrium ion
    • iv. @ neutral pH, the hydroxyapatite is in the form of the salt, BUT if you increase the acidity of the environment, then you drive the equilibrium towards the individual ions
    • v. Whether or not a solution is saturated with respect to HA can be determined by the solubility produce principle
    • 1. This principle (derived from the Law of Mass Action) states “ that the velocity of a reaction is proportional to the product of the masses of the reacting substances, each raised to the power equal to the number of molecules taking part”
  40. Hydroxyapatite Solubility
    • i. The solubility of hydroxyapatite and other calcium phosphates is greatly affected by pH
    • ii. The concentration of the surrounding solution (i.e. saturation) will also influence the solubility of the salt
    • iii. Artificially Demineralize teeth by:
    • 1. Replacing all saliva with water (i.e. like in a xerostomic patient)
    • 2. Consuming large volumes of carbonated, soft drinks
  41. Enamel Hydroxyapatite
    • is NOT PURE!
    • i. Ion Substitutions
    • 1. Carbonate anion replaces phosphate groups because of size and charge(carbonate makes the hydroxyapatite more acid labile – this is natural)
    • 2. Fluoride anion substituting for hydroxyl (makes crystal more acid resistant)
  42. Demineralization/Remineralization
    • i. Caries occurs when Demineralization dominates over remineralization
    • ii. Demin occurs from the production of organic acids
    • iii. Diffusion of acid into enamel rods
    • iv. Dissociation of hydroxyapatite
    • v. Graph: Stephan Curve – pH shift following acid production (same graph as on pg 2)
    • 1. When you eat and expose the biofilm to carbohydrate, the production of acids begins in 2 mins and you see the pH drop from 6.8-7.2 (neutral)  5.3
    • 2. RED LINE = CRITICAL pH = 5.5  below which the ionized form of the salt (hydroxyapatite) is the major form
    • 3. Shows that you need to eat your sugars quickly so saliva can buffer the acids and you don’t remain below the red line for too long
  43. Bacterial Fermentation
    • i. Many caries-causing bacteria are homofermentative
    • ii. Produce lactic acid  has highest pKa which means its very willing to give up its proton  very very strong acid!
    • iii. The production of organic acids is a function of sugar availability
    • iv. Carbohydrate Transport – regular metabolism
    • 1. Membrane bound complexes
    • a. Phosphoenol pyruvate-phosphate transferase complex (PTS-PEP): main transfer system  can uptake carbs @ a fast rate
    • b. Permease complex
  44. Production of water soluble glucans
    • 1. If CARBS are present in HIGH CONCENTRATION, then bacteria also:
    • a. Makes internal dextrans to STORE the glucose in case of starvation later
    • b. Make EXTRACELLULAR GLUTANS – their own glue, contributes to biofilm
    • 2. Glycosyl transferase (GTF) enzymes: capable of synthesizing long chain polymers when sucrose present  make GLUCANS
    • a. Enzymatic hydrolysis of SUCROSE to FRUCTOSE AND GLUCANS
    • i. If carbohydrates are in excess, use transferase to make glucans
    • ii. BRANCHED (1-4 linkage)  very water INSOLUBLE  bacteria increase their BIOMASS (GLUE)
    • iii. UNBRANCHED (1-6 linkage)  very water soluble
    • b. Mutans group: mainly synthesize branched water-insoluble glucans
    • 3. Glucans: can be branched or unbranched depending upon the bacterial enzyme  it’s formation is IRREVERSIBLE!!!
  45. Bacterial Virulence Factors
    • 1. Bacterial attachment
    • 2. Bacterial acid production
    • 3. Sugar consumption
    • 4. Bacterial production of water insoluble extracellular glucans
  46. vi. Bacterial Virulence Factors
    • 1. Bacterial attachment
    • 2. Bacterial acid production
    • 3. Sugar consumption
    • 4. Bacterial production of water insoluble extracellular glucans
  47. Arch form and Tooth Morphology
    • Human Dentition; these are great bacteria breeding grounds:
    • 1. Deep Grooves or Pits and Fissures
    • 2. Interproximal contacts are great bacteria breeding grounds
    • 3. Heights of contour on GINGIVAL THIRD are NON-Cleansing (a teepee formation, i.e. tip of cusp, is self-cleansing)
    • 4. Crowding and narrow arch forms
  48. Saliva: help prevent caries because of its contents but also helps start caries because of pellicle
    • 1. Produced by major and minor exocrine glands in the mouth
    • a. Major: parotid, sublingual, submandibular  mainly make the protein component of saliva
    • b. Minor: occupy most mucosal surfaces of the mouth  more hydrating and mucin-contating (can trap water and keep tissues hydrated)
  49. Saliva Functions:
    • a. Assisting in food intake and taste  if you are xerostomic, can only taste SALT and nothing else
    • b. Provide natural defense against host microbes (anti-fungal, bacterial, viral)
    • c. Provides protective functions for stability of mineralized tooth structures
  50. Saliva for Food and Nutrition
    • a. PRPs(proline rich proteins) + Mucin  provide lubrication for mucosal tissues
    • b. Bicarbonate anion  provide buffering pH to facilitate taste receptor activity
    • c. Amylase, Lipase, Protease  enzymes which help breakdown food
  51. Secretory IgA
    • i. Dimeric immunoglobulin with a PROTEIN secretory component that helps stabilize the molecule
    • ii. Secreted into oral cavity thru the mucosal epi cells and salivary glands
    • iii. Is produced by small intestinal B cells which migrate to the mouth thru the circulatory system
    • iv. What happens? IgA will attach to bacteria and once swallowed and made its way into the small intestines, Peyer’s Patches will start to make antibody to the bacteria  eventually these antibodies make their way back into the mouth via Bcells/circulatory system and it will attack platonic (free floating bacteria)
    • v. Advantages: modulates biofilm formation by inhibition of bacterial attachment
    • vi. Disadvantage: will NOT KILL, just attaches onto bacteria (signal!), DOES NOT activate complement proteins
  52. Lactoperoxidase: efficiently convert HALIDES and PSUEDO-halides to highly reactive oxidizing molecules (“bleach” – HOCL)
    • i. Found in most exocrine glandular secretions
    • ii. Enzyme that is by-product of milk
    • iii. Lactoperoxidase + hydrogen peroxide + Cl- + OSCN-  OCl+, OSCN+ react with –SH groups denaturing proteins
  53. Lysozyme
    • i. Cleaves 1-4 linkages of NAM-NAG of bacterial peptidoglycan
    • ii. Effective against gram positive bacteria
    • iii. Found in tears, nasal secretions, and exocrine glandular fluid
  54. Lactoferrin
    • i. Binds to Iron, an important metabolic co-factor
    • ii. BACTERIOSTATIC properties
    • iii. Present in most exocrine gland secretions
  55. Salivary Components that provide Stability of Enamel
    • i. Calcium binding proteins: this is primary factor
    • 1. If a tooth is placed in a vial of free-floating calcium phosphate  get PRECIPITATION of amorphous calcium phosphate CRYSTALS
    • 2. Why doesn’t this happen in the mouth? Bc of CALCIUM BINDING PROTEINS
    • a. Because saliva contains high concentrations of Ca-binding protein which will SEQUESTER the free-Ca2+, allows us to maintain HIGH levels of Ca2+ without precipitating into crystals
    • b. Need to have saturation of calcium to maintain HA
    • 3. Mechanism – the key is the trick the system into thinking you have an ORGANIC calcium so that it doesn’t precipitate out into crystal (Inorganic  organic)
    • ii. Proline Rich Proteins
    • iii. Phosphate and Calcium
  56. Caries SUMMARY
    • a. Caries Resistance is related to ALL the salivary factors acting upon the system.
    • b. Caries is a chemically-controlled acid driven dissociation of the inorganic salt known as Hydroxyapatite.
    • c. Caries is caused by the presence of biofilm-associated bacteria which produce organic acids with fermentation of sugars
    • d. Sucrose is required for the production of water soluble bacterial glucans which promote bacterial plaque formation and stability
    • e. Caries is caused by an infectious agent which is TRANSMISSIBLE
    • f. This disease IS NOT the result of a genetically inherited trait!
  57. Why is microbiology relevant for Dentistry?
    • a. Acids cause enamel/dentin demineralization (caries)
    • b. Bacteria are the main acid producer in the mouth
  58. Plaque-Host Substrate Theory
    • a. 3 Factors (can’t have one without the other) to make caries:
    • i. Host (Teeth and saliva)
    • ii. Substrate (Diet and Time)
    • iii. Plaque Bacteria
    • b. If you didn’t have plaque, you could eat all the sugar you wanted and never develop caries! Therefore, bacteria is required for caries development
  59. Willoughby Dayton Miller + Experiments
    • a. Scientific pioneer in dentistry, worked with Koch
    • b. Proposed combination of specific microorganisms and carbohydrate consumption as cause for tooth decay
    • c. Tried to apply Koch’s postulates to dental caries
    • d. Published a famous book: Microorganisms of the Human Mouth
    • e. Miller tried to isolate the causative bacteria (according to Koch’s Postulates) but was not successful…instead he make recommendations for keeping the teeth clean which we still use today
    • i. “cleanse teeth often to remove plaque”
    • ii. “delimit intake of sugar”
    • iii. “use antimicrobials to treat”
  60. Cariogenic Species
    • a. Most popular Suspects: Lactic Acid Producing Bacteria
    • i. Mutans streptococci (caries initiation)
    • 1. S. mutans
    • 2. S. sobrinus
    • ii. Lactobacilli (caries progression)
    • 1. L. acidophilus
    • 2. L. rhamnosus
    • 3. L. fermentum
    • iii. Actinomyces (early caries lesions and root caries)
    • 1. A. neaslundii
    • 2. A. odontolyticus
  61. First Isolation of Cariogenic Bacterium
    • 1. J.K. Clarke discovered S. mutans in 1924
    • 2. Isolated form human cariogenic lesion  able to produce caries
    • 3. …and this discovery was forgotton until 1960’s  Keyes & Fitzgerald (discovered that dental caries is TRANSMISSIBLE and an INFECTIOUS DISEASE)
  62. Isolation of Lactobacilli
    • 1. First implicated by Bunting
    • 2. Rediscovered by Jay in 1947
    • 3. Lactobacilli were found to colonize existing lesions but were absent during early lesion formation – opportunistic but NOT causative
  63. Discovery of Actinomyces
    • 1. Originally implicated in perio disease, associated with caries in 70s/80s
    • 2. Can ferment carbohydrates and produce acids
    • 3. Produce NH3+ to control environmental pH (you may think that producing base is caries-preventative but it’s not enough base to bring the pH above the critical level; it’s mainly for the bacteria to be able to survive in the mouth)
    • 4. Linked to early stages of demineralization and root caries
  64. Virulence Factors of Cariogenic Bacteria
    • 1. Acidogenicity
    • 2. Aciduricity
    • 3. Glucan Formation
    • 4. Adhesins
    • acid production
    • i. Ability to lower pH below the critical pH for enamel/dentin demineralization
    • ii. Inhibits growth of many “commensal” non-acid producing bacteria
    • acid tolerance
    • i. Allows cariogenic bacteria to thrive under acidic conditions
    • ii. Can result in dominance of cariogenic species in plaque
    • i. Long-chained, branched extracellular polysaccharide that is WATER INSOLUBLE
    • ii. Allows tight adherence of cariogenic bacteria to the tooth surface
    • iii. Enhances resistance to mechanical removal
    • iv. Enhances resistance to antimicrobial treatments
    • i. “Unspecific” surface adhesion: fimbraie/pilli
    • ii. Adhesion to specific surface molecules
    • 1. Attachment of surfaces
    • 2. Attachment to other bacterial species
  69. The Evolution of Oral Microbiology Research 1950 – 1960’s
    • i. Culture based bacterial isolation
    • 1. Procedures
    • a. Plating of salivary and plaque samples
    • b. Development of selective plating methods
    • c. Biochemical characterization
    • d. Microscopic examination
    • 2. Limitations: more than 700 oral bacterial species identified BUT only 100 can be cultivated  why? Very complex organisms that often live off other species so can’t be grown in isolation
    • ii. Germ-free animal models for disease (“gnotobiotic “ – to show that the microorganisms could develop disease)
    • iii. Advanced microscopy techniques
    • 1. Electron Microscopy Transmission: important invention so that we can see “inside” the cell by slicing slide by slide
    • 2. Electron Microscopy Scanning : allows you to look at the surface structure
  70. The Evolution of Oral Microbiology Research Molecular Approaches to Oral Biology
    • i. Checkerboard DNA-DNA hybridization
    • 1. i.e. to test bacterial species against dental plaque samples
    • 2. Run probed in one direction and then back and forth:
    • a. Whenever there is HYBRIDIZATION  will light up
    • b. A DARKER spot indicates heavier load or better match
    • ii. Culture in-dependant approaches  16S rRNA gene sequencing
    • 1. 16S is highly conserved and so you can ID bacteria based on this
    • 2. When matching species, the more DIFFERENCES and FARTHER APART the genes are, the more unrelated they are
    • iii. Whole genome information:
    • 1. scan for genes that encode for metabolic capacities, virulence features (in this way, scientists can study virulence without even growing the organism itself!)
    • 2. lead to genetic manipulation
    • iv. Human Oral Microbiome  currently underway, to sequence all plaque bacteria!
  71. Caries in PRIMARY TEETH
    • i. S. mutans only predominates in subjects with caries in DENTIN
    • ii. Actinomyocese and Veilonella sps seemed to be the main culprit
  72. Caries in PERMANENT TEETH
    • i. Streptococcus is the main player for Caries
    • ii. Veillionella sps is the most ABUNDANT species  but its also abundant in healthy subjects
    • 1. So is it Cariogenic?
    • 2. NO, it’s ANTI-CARIOGENIC because it can metabolize lactic acid to CO2 and water!
    • e. Graph: Prevalence of Cariogenic Lesions in Different Lesion Types
  73. Specific Plaque Theory (Loesche 1976)
    • i. Within a diverse plaque community, only a few species actively contribute to disease
    • ii. Focused on controlling disease by targeting a limited number of organisms for txn
  74. Non-specific Plaque Theory (Theilade 1986)
    • i. Considered that disease as the outcome of the overall activity of the total plaque microflora
    • ii. A heterogeneous mixture of “any” microorganisms could play a role in disease rather than specific species
  75. Ecological Plaque Hypothesis (Marsh 2003)
    • i. Even in healthy plaque, you have a few cariogenic species that are kept in check by other bacteria
    • ii. It’s not until a major ecological pressure (i.e. no saliva flow) hits the community that you shift the balance towards disease (is REVERSIBLE!)
    • iii. Picture: the Ecological Development of Dental Caries
    • iv.
    • -Demineralization is reversible
    • - BUT if you keep on eating sugar, you never go back above the critical pH and you stay in the ionized calcium form
  76. How does Dental Plaque Develop?
    • i. First Layer: Salivary pellicle (forms immediately after you’ve had cleaning)
    • 1. Pellicle is a double-edged sword: need it to help the tissue stay moist and protect against acid but also provides area for bacteria to attach
    • 2. Thin film formed by salivary components  lubricates enamel/dentin tissue, protective against acids
    • 3. Certain proteins in the pellicle can serve as receptors for bacterial attachment
    • a. Parotid Saliva Agglutinin (S. mutans)
    • b. Mucin MG2 (S. oralis and S. sanguinis)
    • ii. Primary Colonizer can directly attaché to surfaces
    • iii. Secondary Colonizer can attach to primary colonizers
  77. Biofilm Development
  78. Dental Plaque (Oral Biofilm) Formation Other Imaging Techniques
    • a. Confocal Laser Scanning Microscopy: allows to look INSIDE the biofilm
    • b. Species-Specific Labeling with Fluorescent In-Situ Hydridization (FISH): can use to compare Colocalization
  79. Biofilm Development – Interspecies Interactions
    • a. Nutrition – Metabolic Interactions
    • i. Host Factors:
    • 1. Sucrose (diet)
    • 2. Proteins (Saliva, crevicular fluid)
    • ii. Cross-feeding between bacterial species  one species will eat the metabolic by-product of another!
    • 1. Lactic acid (streptococci and Veillonella)
    • 2. Peptides/AA
    • 3. Fatty Acids
  80. Interspecies Competition
    • i. Metabolic
    • 1. Acid production by one organism inhibits growth of acid-sensitive species
    • 2. Acid consumption of amnomia production as a counter measure to neutralize pH
    • ii. Chemical/Biological warfare
    • 1. Hydrogen peroxide production
    • 2. Bacteriocin production
    • a. Narrow spectrum (means only kills either gram + or – but not both) antimicrobial small proteins typically active against related organisms
    • b. Well-regulated production
    • c. Streptococci have greatest bacteriocin
  81. Complexity of Interspecies Interactions between Streptococci
    • i. Competition between S.mutans (Sm) and S. sanguinis (Ss)
    • ii. S. mutans produces MUTACIN to kill Ss but Ss retaliates by producing HYDROGEN PEROXIDE
    • Sm -> Ss: Sm colonized first so it kills Ss
    • Ss -> Sm: Ss colonized first so it kills Sm
    • Sm + Ss: if colonize @ the same time, they decide to work together – how?
    • Maybe they talk together!
  82. Synergistic Interactions
    • a. Cooperative metabolic interactions
    • b. Cooperative enzymatic actions
    • c. Cooperative integration into the biofilm community
  83. Bacterial Interactions & Biofilm Architecture
    • a. Species distribution is NOT RANDOM
    • i. Metabolic interactions
    • ii. Specific interspecies coaggregation
    • 1. Specific cell to cell recognition between species
    • 2. Cells attach to each other via specific structures
    • iii. Intra/Interspecies signaling
    • 1. Small proteins/peptides  CSP (competence)
    • 2. Quorum sensing via Al-2 (luxS)  for biofilm formation, bacterioicin production
    • a. i.e. when you make mutants without this gene, biofilms have a hard time forming
    • b. Bacteria have the ability to “count” their population size!
    • iv. Interspecies Competition
  84. Exchange of Genetic Material
    • a. The Evidence: discovered that many bacteria that were not related to each other had small stretches of DNA that were similar  HORIZONTAL GENE TRANSFER
    • b. The Mechanisms
    • i. DNA availability
    • 1. Cell lysis
    • 2. Regulated DNA release?
    • 3. Donor cell (conjugation)
    • ii. DNA Uptake
    • 1. Transformation
    • 2. Conjugation
    • 3. Transduction (phages)
    • c. Host Factors
    • i. Provision of nutrition via oral fluids: glycol and heme-containing proteins & minerals
    • ii. Salivary proteins as enabler of initial surface adhesion
    • iii. Defense responses: antibacterial peptides and immune responses (which are limited in saliva)
  85. Modern Approaches in Dentistry
    • A. Disease PREVENTION!
    • a. Move from surgical to medical approach
    • b. From treatment to prevention
    • c. From professional care to self-care
    • B. Is there cure for Caries? Here are some theories, but they don’t all work………
    • a. Inhibition of Adherence  this is difficult because by just having the pellicle on your tooth, you’re inviting the bacteria in
    • b. (Passive) Immunization  saliva has a very poor antibody concentration; only has IgA and IgA can only attach/cover bacteria but doesn’t actually kill it
    • c. Replacement therapy  patients are generally very resistant to having genetically altered bacteria placed into their mouth
    • d. Disruption of signaling
    • e. Complete Plaque Removal? This is not a perfect solution, only solves the problem short term. Even if you remove all the plaque, because the saliva provides a pellicle the plaque will eventually quickly come back!
    • f. Selective Pathogen Elimination
    • i. “Restoring Homeostasis” – first you remove the cariogenic pathogens AND the plaque  the plaque that naturally regrows will NOT have the pathogen in it  therefore, a healthy plaque that prevents other cariogenic species from growing is a good barrier (not all plaque is bad!)
    • ii. Targeted removal of S.mutans from biofilm using short antimicrobial peptides ( Dr. Shi’s Lab!)
    • i. CFU = colony forming units
    • ii. 2_1G2 and C16G2 = short antimicrobial peptides that target S. mutans
    • iii. Conclusion: S.mutans does grow back but it NEVER grows to the same levels as they were before  keep the concentration down!
    • iii. Protective Biofilm Growth: the experiments below show that a BIOFILM IS ENOUGH to block S. mutans growth can be a mechanism for anti-caries campaign
    • iv. Interference with Quorum Sensing: by removing the ability of bacteria to sense each other, the colonizes first rapidly overgrow and in the quick depletion of nutrients, many die off
  86. A Medical Approach to Dentistry:
    • based on assessment of what of bacteria are in your mouth, you can create tailored treatments for people who are at high, medium, and low risk for caries
    • a. Current Advances in Diagnostics
    • i. Species – Specific Detection of cariogenic species in saliva and plaque
    • 1. Mono-clonal antibody (MAbs) bacterial detection techniques: for all of them, you can scan your sample with the antibody for a specific bacteria (in plaque and saliva!)
    • a. Linkage to fluorescent dyes  FLUORSCENCE
    • b. Linkage to color particles or enzymes  COLOR
    • c. Linkage to latex beads  AGGLUTINATION
    • d. Limitations: unfortunately these procedures are not very efficient for chairside!
    • 2. Specific DNA Probes
    • 3. Instant Membrane Strip Test for S. mutans detection: indicates the levels of S. mutans in the mouth
    • b. The FUTURE: Dentist-Scientists who capitalize on research advances to revolutionize current dental practice!