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Evidence Based Management of Dental Caries
Understanding the etiology of dental caries
Dr. Wenyuan Shi
Professor and Chair, Oral Biology, UCLA SODProfessor, Microbiology & Immunology, UCLA SOM
Disruptive event/technology
Atomic bomb
Antibiotics
Polio vaccine
Disruptive event in dentistry: sugar
3000 BCE 2000 BCE 1000 BCE 0 1000 CE 2000 CE0
5
10
15
20
25FRENCH
BRITISH
DANISH
(NEOLITHIC TO MODERN ERA)
NEOLITHIC
IRON
ROMAN ERA
COMMON ERA
Disruptive technology in dentistry: fluoride
Cariogenic Tooth Bacteria Decay
Sugars
Fluoride!!!!
What is the next disruptive technology in dentistry?
The first microbes observed
• Anton Van Leeuwenhoek (1632-1723) developed the microscope and was the first to discover oral bacterial flora: “I didn’t clean my teeth for three days and then took the material that had lodged in small amounts on the gums above my front teeth…. I found a few living animalcules..”
Microbial Flora in Oral Cavity
•100,000,000,000,000 bacteria/per mouth
•One of the most complicated microbial flora•>700 species
Microbial Flora in Oral Cavity
Supragingival plaque
Subgingival plaque
G+
G-
Dental Caries
Dental Caries is the localized destruction of the hard tissues of the tooth
What causes dental caries?
• Pre-microbiology (pre-Miller) era
– Dental caries is the death (decay) of a tissue
• Microbiology period (Post-Miller) era
– Dental caries is a microbe related disease
Gryphon Editions: Classics of Dentistry
The microorganisms of the human mouth
W.D. Miller and his "chemico-parasitic" theory
PLAQUEPLAQUE
SUSCEPTIBLE HOST
SUSCEPTIBLE HOST
FERMENTABLE CARBOHYDRATEFERMENTABLE
CARBOHYDRATE
ACID PRODUCTION
ACID PRODUCTION
DEMINERALIZATIONDEMINERALIZATION
Research Commission, American Dental Association, 1939. Consensus
There are “indirect” conditions which predispose to caries, and “direct”, external, or attacking factors which initiate the disease.
Research Commission, American Dental Association, 1939. Consensus
“indirect” conditions: resistance or predisposition to the disease.
a. Heredity – resistant or susceptible
b. Diet – refined food lacks essentials for tooth formation
c. Maintenance of general health – production of “good jaws and teeth”, normal saliva flow
Research Commission, American Dental Association, 1939. Consensus
“Direct” cause:
a. Repeated lodging of fermentable matter between the teeth
b. Recurrent production of acids by undefined oral bacteria which disintegrate the tooth’s enamel
c. Such decalcification enables further action by bacteria on the underlying dentin, which progressively disintegrates.
Are all factors equally important?
Smart experiments by R.J. Fitzgerald
Genetic defect in tooth (new born mice)
Poor immune system (mice with no B/T cells)
Poor saliva flow (mice with salivary grands removed)
Diet (mice eating sugars as major food source)
Poor oral hygiene (mice in dirty, but sterilized cages)
No bacteria inoculation, no cavity
Bacteria inoculation, full of cavity
All other cariogenic factors work through bacteria!Bacteria are the ultimate guilty party!
Host & Teeth
Diet & Time
Cariogenicbacteria
Caries
Isolation of Streptococcus mutans
1924, JK Clark, England
Isolation of an acid-resistant bacterium from human caries lesions, which was
called Streptococcus mutans
Connections between caries & streptococci
- 1943, Belding and Belding published a sketchy description of rats developing caries as a result of inoculation with human streptococci.
- 1954, Orland et al., demonstrated that germ-free rats will not develop caries no matter how much sugar they eat unless they are inoculated with a Streptococcus.
Keyes, 1960Searching for the infectious elements
that cause dental caries
- The experiment:
- Caries-inactive animals caged with one another;
- Caries-inactive animals caged with caries-active animals;
- Caries-inactive animals inoculated with plaque from caries-active animals;
Results
Caries-inactive
Caries-active
Caries-active
Conclusion: Caries is transmitted through plaque
Keyes, 1960Searching for the infectious elements
that cause dental caries
- Caries-inactive animals caged with one another;
- Caries-inactive animals caged with caries-active animals;
- Caries-inactive animals inoculated with plaque from caries-active animals;
Results
Caries-inactive
Caries-active
Caries-active
Conclusion: Bacteria are the cause of caries
Addition of antibiotics
Caries-inactive
Caries-inactive
Caries-inactive
Keyes and Fitzgerald, 1962’s
Re-isolation of “Mutans streptococci”:
• Streptococcus mutans (human) (same species Clark isolated in England in 1924)
• Streptococcus sobrinus (human)
• Streptococcus rattus (rats)
• Streptococcus cricetus
• Streptococcus ferus
• Streptococcus macacae
• Streptococcus downeii
The “cariogenic bacteria” – bacteria associated with dental caries
Actinomyces – early colonizers• A. odontolyticus
• A. naeslundii genospecies 2
• A. isrealii
• A. gerensceriae
Lactobacilli (L. casei) – caries progression
Mutans streptococci (S. mutans) – caries initiation
The virulence factors of cariogenic bacteria
1. Acid production (acidogenicity)
• Lower the pH to below 5.5, the critical pH. Drives the dissolution of calcium phosphate (hydroxyapatite) of the tooth enamel
• Inhibit the growth of beneficial bacteria, promote the growth of aciduric bacteria.
• Further lower the pH, promote progression of the carious lesion
• Allows the cariogenic bacteria to thrive under acidic conditions while other beneficial bacteria are inhibited. This results in dominance of the plaque by cariogenic bacteria
2. Acid tolerance (aciduricity)
The virulence factors of cariogenic bacteria
• Allows the cariogenic bacteria to stick onto the teeth and form a biofilm
3. Glucan formation
• Glucan mediated biofilms are more resistant to mechanical removal
• Bacteria in these biofilms are more resistant to antimicrobial treatments
The virulence factors of cariogenic bacteria
Carbohydrates (Sucrose)
Cariogenic bacteria such as S. mutans
Glucans/Levans Acids
Dental Caries
Plaque formation Demineralization
New problem: everybody has S. mutans!
Fra
cti
on
Number of S. mutans in Saliva(104)
1 10 20 50
0
0.27
Why not every body who has S. mutans develop dental caries?
• S. mutans is not present in high portions
• Acid produced is neutralized urea or ammonia produced by other bacteria in the plaque
• S. mutans is away from the tooth surface so acid produced is diffused
The ecologic plaque hypothesis
Both pathogenic and commensal (non-harmful) bacteria exist in a natural plaque. At sound site, the pathogenic bacteria may exist in low numbers to cause any clinical effect, or they may exist in higher numbers, but the acid produced is neutralized by the action of other bacteria. Disease is a result of a shift in the balance of the residence microflora driven by a change in the local environment (frequent sugar intake etc).
The ecological development of dental caries
Acid producing bacteria
Sugar
Acids
Base producingBacteria: S. sanguis, S. oralis
Neutral pH
Remineralization
Health
More sugar
Lower pH
Inhibition of beneficial bacteriaOvergrowth of cariogenic bacteria
Demineralization
Caries
Many questions remain…
• Is S. mutans the major cariogenic bacterium?
• How many cariogenic bacteria we need to test in order to have accurate risk assessment?
• How to integrate microbiological index into existing caries diagnosis procedures?
• What will happen if we get rid of S. mutans in plaque?
New Scientific Advancement That Allow Us To Address Above Questions
• Better detection of demineralization
– high sensitivity, noninvasive, real time
• Better detection of acids within plaque
– real time, in situ, high sensitivity
• Detailed molecular understanding of plaque
– In situ, real time, molecular level, genome level
Better detection of demineralization
• Laser inference microscopy
• Laser fluorescence detection
• Laser confocal microscopy
• Atomic force microscopy
In-situ/real-time Imaging Of Demineralization With Laser Interference Microscopy
Enamel chip with a grown plaque Quantitative imaging analysis
In-situ/real-time Imaging Of Demineralization With Laser Interference Microscopy
Better detection of acid productions
• pH sensitivity dyes
• Two photon microscopy
• Micro-electrode
• Planar pH sensor
• Combined NMR/confocal microscopy
Combined NMR/confocal microscopy
Using NMR-Microscope to study carbohydrates within plaque
Detailed molecular understanding of dental plaque
Scanning EM image of oral microflora
Atomic force microscopyImaging alive bacteria with EM quality
1.4µm
S. mutans
A. a
Fuso
S. sanguis
Confocal microscopy for 3-D imaging
3D imaging of S. mutans in dental plaque
Green: S. mutans cells
Imaging S. mutans in vivo
Roper Bioscience™ Lumazone™
Microbial flora in human
How to isolate and identify oral microorganisms
• Culture based
– take saliva or plaque
– dilute and plate on appropriate plates
– grow to single colonies
– identify by microscopic and biochemical methods (Bergy’s manual).
Gram-positive
Cocci (more dominant
in numbers)
Abiotrophia
Enterococcus
Peptostreptococcus
Streptococcus
Staphylococcus
Stomatococcus
Gram-negative
CocciMoraxella
Neisseria
Veillonella
RodsActinomyces
Bifidobacterium
Corynebacterium
Eubacterium
Lactobacillus
Propionibacterium
Pseudobamibacter
Rothia
RodsActinobacillus (Bacteriodes) Haemophilus
Campylobacter
Johnsonii
Cantonella Leptotrichia
Capnocytophaga Porphyromonas
Centipeda Prevotela
Desulfovibrio
Bacterial genera found in the oral cavity (with culture methods)
How to isolate and identify oral microorganisms
DNA/RNA based:
16S DNA cloning and sequencing 1. use 2 oligonucleotide primers universal to ALL bacteria
2. PCR amplification of the total saliva or plaque DNA pool 3. clone the PCR product and sequence 4. Phylogenetic analysis using computer algorism.
Checker-board or microarray hybridization 1. use oligonucleotide probes of known DNA sequence to make a
DNA chip 2. isolate total DNA from the saliva or plaque 3. label the DNA with radioisotop or fluorescent dyes 4. Hybridize to the chip
5. image analysis
Phylogenetic tree of bacterial species or phylotypes identified by 16s DNA sequencing from the samples from tongue dorsa of healthy and halitosis subjects.
Red clones: halitosis
Bacteria
mRNA cDNA
DNA hybridization and microarrays
Checker board hybridization
DNA microarray
Summary of 16S RNA/DNA study
• Both culture and DNA/RNA-based techniques are used for identification and quantification of oral microorganisms
• Overall, there are ~700 species exist in the oral cavity
• ~20% of these 700 species have been cultivated
• Both Gram-positive and Gram-negative exist
• Most anaerobic or facultative anaerobic
Oral bacteria genome projects
Metagenomics of oral cavity
Human genome project
Interesting finding No. 1
Human genome only has 200,000 genes.
Each oral bacterium has 2000-6000 genes, with over 1 million bacterial genes in oral cavity
Plaque is a well organized microbial community
S. sanguinis S. gordoniiS. mitis
S. oralis
S. salivarius
Pioneer colonizers
Interesting finding No. 2Oral bacteria have the ability to count the population size
Sm Ss Sm Ss Sm Ss
Competition/coexistence between S. mutans & S. sanguinis
Interesting finding No. 3Extensive chemical warfare between bacterial species
Some lights• Is S. mutans the major cariogenic bacterium?
– Responsible for ~70% acids generated in plaque
• How many cariogenic bacteria we need to test in order to have accurate risk assessment?
– S. mutans/Lactobacilli combo provides good prediction
• How to integrate microbiological index into existing caries diagnosis procedures?
– This afternoon’s lecture!
• What will happen if we get rid of S. mutans in plaque?
– This afternoon’s lecture!