Evidence Based Management of Dental...

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!