Evidence Based Management of Dental Caries

Transcription

1 Evidence Based Management of Dental Caries Understanding the etiology of dental caries Dr. Wenyuan Shi Professor and Chair, Oral Biology, UCLA SOD Professor, Microbiology & Immunology, UCLA SOM

2 Disruptive event/technology Atomic bomb Antibiotics Polio vaccine

3 Disruptive event in dentistry: sugar (NEOLITHIC TO MODERN ERA) COMMON ERA FRENCH BRITISH DANISH NEOLITHIC IRON ROMAN ERA BCE 2000 BCE 1000 BCE CE 2000 CE

4 Disruptive technology in dentistry: fluoride

5 What is the next disruptive technology in dentistry? Sugars Cariogenic Bacteria!!! Tooth Decay Fluoride!

6 The first microbes observed Anton Van Leeuwenhoek ( ) 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..

7 Microbial Flora in Oral Cavity 100,000,000,000,000 bacteria/per mouth One of the most complicated microbial flora >700 species

8 Microbial Flora in Oral Cavity G+ Supragingival plaque G- Subgingival plaque

9 Dental Caries Dental Caries is the localized destruction of the hard tissues of the tooth

10 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

11 The microorganisms of the human mouth Gryphon Editions: Classics of Dentistry

12 W.D. Miller and his "chemico-parasitic" theory SUSCEPTIBLE HOST PLAQUE FERMENTABLE CARBOHYDRATE DEMINERALIZATION ACID PRODUCTION

13 Research Commission, American Dental Association, Consensus There are indirect conditions which predispose to caries, and direct, external, or attacking factors which initiate the disease.

14 Research Commission, American Dental Association, 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

15 Research Commission, American Dental Association, 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.

16 Are all factors equally important?

17 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!

18 Host & Teeth Diet & Time Cariogenic bacteria Caries

19 Isolation of Streptococcus mutans 1924, JK Clark, England Isolation of an acid-resistant bacterium from human caries lesions, which was called Streptococcus mutans

20 Connections between caries & streptococci , Belding and Belding published a sketchy description of rats developing caries as a result of inoculation with human streptococci , 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.

21 Keyes, 1960 Searching 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 Cariesinactive Cariesactive Cariesactive Conclusion: Caries is transmitted through plaque

22 Keyes, 1960 Searching 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 Addition of antibiotics Cariesinactive Cariesactive Cariesactive Cariesinactive Cariesinactive Cariesinactive Conclusion: Bacteria are the cause of caries

23 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

24 The cariogenic bacteria bacteria associated with dental caries Mutans streptococci (S. mutans) caries initiation Actinomyces early colonizers A. odontolyticus A. naeslundii genospecies 2 A. isrealii A. gerensceriae Lactobacilli (L. casei) caries progression

25 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

26 The virulence factors of cariogenic bacteria 2. Acid tolerance (aciduricity) 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

27 The virulence factors of cariogenic bacteria 3. Glucan formation Allows the cariogenic bacteria to stick onto the teeth and form a biofilm Glucan mediated biofilms are more resistant to mechanical removal Bacteria in these biofilms are more resistant to antimicrobial treatments

28 Dental Caries Carbohydrates (Sucrose) Cariogenic bacteria such as S. mutans Glucans/Levans Acids Plaque formation Demineralization

29 New problem: everybody has S. mutans! 0.27 Fraction Number of S. mutans in Saliva(10 4 )

30 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

31 The ecologic plaque hypothesis Both pathogenic and commensal (nonharmful) 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).

32 The ecological development of dental caries Caries Acid producing bacteria More sugar Acids Lower ph Demineralization Inhibition of beneficial bacteria Overgrowth of cariogenic bacteria Sugar Neutral ph Base producing Bacteria: S. sanguis, S. oralis Remineralization Health

33 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?

34 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

35 Better detection of demineralization Laser inference microscopy Laser fluorescence detection Laser confocal microscopy Atomic force microscopy

36 In-situ/real-time Imaging Of Demineralization With Laser Interference Microscopy Enamel chip with a grown plaque Quantitative imaging analysis

37 In-situ/real-time Imaging Of Demineralization With Laser Interference Microscopy

38 Better detection of acid productions ph sensitivity dyes Two photon microscopy Micro-electrode Planar ph sensor Combined NMR/confocal microscopy

39 Combined NMR/confocal microscopy

40 Using NMR-Microscope to study carbohydrates within plaque

41 Detailed molecular understanding of dental plaque

42 Scanning EM image of oral microflora

43 Atomic force microscopy Imaging alive bacteria with EM quality S. mutans Fuso 1.4µm A. a S. sanguis

44 Confocal microscopy for 3-D imaging

45 3D imaging of S. mutans in dental plaque Green: S. mutans cells

46 Imaging S. mutans in vivo Roper Bioscience Lumazone

47 Microbial flora in human

48 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).

49 Bacterial genera found in the oral cavity (with culture methods) Gram-positive Cocci (more dominant in numbers) Abiotrophia Enterococcus Peptostreptococcus Streptococcus Staphylococcus Stomatococcus Rods Actinomyces Bifidobacterium Corynebacterium Eubacterium Lactobacillus Propionibacterium Pseudobamibacter Rothia Gram-negative Cocci Moraxella Neisseria Veillonella Rods Actinobacillus (Bacteriodes) Haemophilus Campylobacter Johnsonii Cantonella Leptotrichia Capnocytophaga Porphyromonas Centipeda Prevotela

50 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

51 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

52 DNA hybridization and microarrays mrna cdna Bacteria

53 Checker board hybridization

54 DNA microarray

55 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

56 Human genome project Oral bacteria genome projects Metagenomics of oral cavity

57 Interesting finding No. 1 Human genome only has 200,000 genes. Each oral bacterium has genes, with over 1 million bacterial genes in oral cavity Plaque is a well organized microbial community

58 S. sanguinis S. gordonii S. mitis S. oralis S. Pioneer colonizers salivarius

59 Interesting finding No. 2 Oral bacteria have the ability to count the population size

60 Interesting finding No. 3 Extensive chemical warfare between bacterial species Competition/coexistence between S. mutans & S. sanguinis Sm Ss Sm Ss Sm Ss

61 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!