book

1. DENTAL PLAQUE

2. • Most important chapter
• Understanding required
• Health maintenance
• Disease progression

3. OBJECTIVES
• DEFINITION
• Difference between plaque,
calculus , materia alba
• Types of plaque
• Composition of plaque
• Formation of plaque

4. Lecture 2
• Microscopic structure of
plaque
• Concept of biofilm
• Plaque Hypothesis
• Microbial complexes
• Microbial tests for plaque
sample

5. Definition
• Dental Plaque
“is a specific but highly variable structural
entity, resulting from sequential
colonization of microorganisms on tooth
surfaces, restorations & other parts of oral
cavity, composed of salivary components
like mucin, desquamated epithelial cells,
debris & microorganisms, all embedded in
extracellular gelatinous matrix.”
W.H.O ,1961

6. • Dental Plaque can be defined as:
“ soft deposits that form the biofilm
adhering to the tooth surface or
other hard surfaces in the oral
cavity, including removable & fixed
restorations”
Bowen , 1976

7. DIFFERENTIATION:

8. • Materia Alba
“soft accumulations of bacteria and tissue
cells; lack organised structure of dental
plaque; easily displaced by water spray”
• Dental Calculus
‘mineralised dental plaque”

9. DETECTION OF DENTAL PLAQUE
DETECTION
• Visual
• Periodontal Probe or
Explorer
• Disclosing Agents

10. Timeline of plaque
development:
• At birth: sterile
• Hours: facultative aerobic bacteria
• Second day: anaerobic bacteria
• 2 weeks: mature microbiota
• Weaning (> 2 years): 400 different
types of bacteria
• Body contains 10 times more
bacteria than human cells

11. • Open growth system:
communication with the pharynx
• > 500 species in mouth
• In any individual: 150 or more
species at any given time.

12. Adhesion
forces
Removal
forces
1. Mastication
2. Tongue
3. Oral hygiene
4. Washout effect:
saliva
5. Ciliae
1. Soft tissue
2. Hard tissue

13. Niches of plaque
accumulation:
• Supragingival and hard surgfaces:
teeth, implant, restorations.
• Periodontal pocket (hard: root
cementum & soft: pocket epithelium)
• Buccal, palatal and floor of the mouth
epithelium
• Dorsum of the tongue
• Tonsils

14. Natural cleansing
mechanism:
• Gingival crevicular fluid & salivary
flow
• Cleansing effect of mastication and
tongue movement
• Rapid turnover rate of intraoral
epithelial cells
• Host defence mechanisms like
langerhans cells.

15. Hard tissues
• Teeth and implants surface provide
a non shedding surface. It allows
extensive plaque accumulation.
• Provide an ectodermal interruption.
At junctional epithelium the teeth
provide access to bacteria into the
body.
• Port of entry of periopathogens.

16. Macroscopic Structure
• Supragingival Plaque
• Subgingival Plaque
• Marginal Plaque

17. Composition of Plaque
• Microorganisms
• Intercellular Matrix

18. Microorganisms
Bacterial
1gm contains 2X1011 bacteria-500
species
Non-Bacterial
Mycoplasma,
Yeast,
Protozoa,
Viruses

19. Intercellular Matrix
• Accounts for 20% to 30% of the plaque
mass
• Organic and inorganic materials
• Derived from saliva, gingival crevicular fluid,
and bacterial products.

20. Organic Component
• Organic constituents : polysaccharides,
proteins, glycoproteins, and lipid material
• Polysaccharides produced by bacteria-
Dextran: predominant form
• Albumin: originating from crevicular fluid
• Lipid material: debris from the membranes
of disrupted bacterial and host cells and
possibly food debris.

21. Inorganic Component
• Calcium and phosphorus (Most)
• Trace amounts: sodium, potassium
and fluoride.
• Source -supragingival plaque
(saliva) & subgingival plaque (GCF
& Blood)
• Calculus frequently found in areas
of the dentition adjacent to salivary
ducts

22. PHASES OF
PLAQUE FORMATION

23. 1. Formation of dental Pellicle
2. Initial Colonization
3. Secondary Colonization & Plaque
Maturation

24. Formation of dental Pellicle
• Acquired enamel pellicle forms
rapidly - Early pellicle
• Characterized by an absence of
bacteria and their products.
• Composed of proteins and
glycoproteins.
• Demonstrate a higher content of
threonine, serine, and alanine & less
proline than saliva- selective
adsorption

25. • Electron microscopic -reveals a thin,
amorphous, electron-dense layer
immediately adjacent to the hard
surface- thickness from 1 to 2 nm.
• involves a combination of physical
forces (ionic, hydrophobic, hydrogen
bonding, and van der Waals)

26. • May involve the interaction of
phosphate groups with calcium ions
in saliva to form "bridges"
• Protective functions of early enamel
pellicle: protection , lubrication by
decreasing frictional forces, may
selectively concentrate antimicrobial
substances such as
immunoglobulins, lysozyme, and
cystatins at different oral surfaces.

27. • Formation of later pellicle most likely involves
protein-protein or protein-carbohydrate
interactions,- stereospecific in nature .
• For example, A. viscosus and Streptococcus
mitis produce a neuraminidase that cleaves
terminal sialic acid residues on the
glycoproteins in saliva or early pellicles to
expose galactose residues (Costello et aI.,
1979)
• Collectively, these mechanisms may be
important for the initial colonization

28. Initial adhesion
Phase I : Transport to the surface
Phase II : Initial adhesion
Phase III : Attachment
Phase IV : Colonisation of the surface
and biofilm formation

31. Phase I
• Random contacts:
1.Brownian motion
2.Sedimentation of micro- organisms
3.Liquid flow
4.Active bacterial movement

32. Phase II
• Initial reversible adhesion
• Long range and short range forces:
1.van der Walls attractive forces
2.Electrostatic repulsive forces

33. Phase III
• Firm anchorage
• Specific interactions:
1.Covalent
2.Ionic
3.Hydrogen bonding

35. • Adhesions: specific extracellular
proteinacious components on
microorganisms.
• Complimentary receptors: proteins,
glycoproteins or polysaccharides on
the pellicle surface.

36. • Example:
• S. Sanguis- binds to acidic proline
rich proteins, alpha amylase & sialic
acid
• A. viscosus- fimbriae that contain
adhesins- bind to proline rich
proteins of dental pellicle.

37. Colonisation & Plaque
maturation
• Initial colonisers attach to the tooth
surface- provide substrate for
secondary colonisers to attach.
• They create a favourable
environment for secondary
colonisers to survive.

38. Secondary Colonisers:
• Adhere to bacteria already present in
the plaque mass.
• Prevotella intermedia
• Prevotella loeschii
• Fusobacterium nucleatum
• Porphyromonas gingivalis
• Capnocytophaga

39. Co- aggregation
• Cell to cell recognition of genetically
distinct partner cell types
• Highly specific stereochemical
interaction
• Corn cob formation: long filament
bacteria covered with cocci
• Test tube brush: long filament
bacteria covered with flagellated
small motile rods

40. MICROSCOPIC STRUCTURE

41. MICROSCOPIC STRUCTURE
SUPRAGINGIVAL PLAQUE
• typically demonstrates a stratified organization of
the bacterial morphotypes.
• Gram-positive cocci and short rods predominate at
the tooth surface
• gram-negative rods and filaments ,spirochetes
predominate in the outer surface of the mature
plaque mass.
• Highly specific cell-to-cell interactions are also
evident from the "corncob"

42. CORN-COB STRUCTURE
CORN COB STRUCTURE

43. Thin section of supragingival
plaque
GRAM POSITIVE BACTERIA IN
PALISADING ARRANGEMENT

44. • SUBGINGIVAL PLAQUE
• Gingival crevicular fluid, -contains many
substances that the bacteria may use as
nutrients
• Host inflammatory cells and mediators
have influence on the establishment and
growth of bacteria in this region.
• Distinctions present between tooth-
associated and tissue-associated regions
of subgingival plaque

46. Thin section of plaque in a deep
pocket
FILAMENTS
RODS
COCCI

47. DENTAL PLAQUE AS BIOFILM

48. Plaque-A Biofilm
1.Structure-microcolonies of bacteria in
matrix
2.Exopolysaccharides
produced by bacteria
50-90% of dry wt
integrity of biofilm
buffer

49. 3.Physiological Heterogenicity
Same sp.-different physiologic states in biofilms
4.Quorum Sensing
“regulation of expression of specific genes”-
intercellular communication
distinct properties
Prosser,1999
5.Attachment of Bacteria- fimbrae & fibrils

50. 6.Increased antibiotic Resistance
Bacteria in biofilms more resistant
Slow growth
Decreased diffusion
Accumulation of enzymes- lactamases,dehydrogenases
Alteration of genes.

51. METABOLIC INTERACTIONS

52. Microbial Complexes
microbiota into groups or complexes, -
appear to occur together
transition from a healthy oral environment
to gingivitis and to periodontal disease is
triggered by a specific 'set' or 'complex' of
bacterial species
Socransky and Haffajee and
colleagues,1998

53. THE VARIOUS COMPLEXES

54. Porphyromonas gingivalis, Treponema
denticola, and Tannerella forsythia: the 'red
complex‘
Several characteristics make them prime candidates
as pathogens in the clinical destruction of periodontal
tissues:
1. occur concomitantly with the clinical signs of
periodontal destruction;
2.appear closely 'linked' topologically in the
developing biofilm;
3. in vitro studies demonstrate their ability to produce
a number of outer membrane-associated proteinases

55. Association of Plaque
microorganisms with Periodontal
diseases
• Early 1900-papers on specific organisms
• Mid 1930s-”bacteria-dental orphans”
Belding & Belding 1936

56. “Non Specific Plaque Hypothesis”
Walter Loeche, 1976
“Specific Plaque Hypothesis”
Walter Loeche , 1976
A.actinomycetemcomitans-LAgP
Newman,Socransky,1977
“Specific Plaque Hypothesis revisited”
Specific strains

57. MICROORGANISMS IN SPECIFIC
PERIODONTAL DISEASES

58. • Total no. of bacteria per gram of plaque-
twice in periodontally diseased sites
• Healthy sites-gram +ve facultative rods &
cocci
J Slots ,1977
• Protective species-S.Sanguis,C.Ochracea
Dzink JL ,1985

59. • Plaque induced Gingivitis- Harold Loe, 1965
Gram positive -
S.Sanguis,S. intermedius,S.Oralis,A. viscosus etc
Gram negative-
F.nucleatum,P.intermedia,Capnocytophaga
• Pregnancy induced gingivitis
P. intermedia

60. • Chronic periodontitis
Spirochetes,
gingivalis,
B.forsythus,
A.actinomycetemcomitans
P. micros,
Treponema

61. • Localized Aggressive Periodontitis –
A.actinomycetemcomitans Socransky, 1977
P. gingivalis,
F. nucleatum

62. • Necrotizing Periodontal
diseases
P. intermedia
• Abcesses of Periodontium
F.nucleatum
P. intermedia,
P. gingivalis,
P. micros

63. CRITERIA for ASSOCIATION
of
PERIODONTAL PATHOGENS

64. KOCH’S POSTULATES
In I870s, Robert Koch’s criteria for causative agent in
human infections.
1. Be routinely isolated from diseased individuals
2. Be grown in pure culture in the laboratory
3. Produce a similar disease when inoculated into
susceptible laboratory animals
4. Be recovered from lesions in a diseased laboratory
animal

65. SIGMUND SOCRANSKY’S CRITERIA FOR
PERIODONTAL PATHOGENS
1. Be associated with disease, as evident by increases in
the number of organisms at diseased sites
2. Be eliminated or decreased in sites that demonstrate
clinical resolution of disease with treatment
3. Demonstrate a host response, in the form of an
alteration in the host cellular or humoral immune
response
4. Be capable of causing disease in experimental animal
models
5. Demonstrate virulence factors responsible for enabling
the microorganism to cause destruction of the
periodontal.tissues

66. EVIDENCE SUPPORTING ROLE OF
MICRORGANISMS AS PATHOGENS IN
PERIODONTAL DISEASE; SOCRANSKY’S
CRITERIA
CRITERION A.actinomycetemcomitans P. gingivalis
ASSOCIATION Increased in LAgP
Increased in chronic
periodontitis lesions
Detected in tissues of
LAgP
Increased in
periodontitis lesions
ELIMINATION Supressed or eliminated in
successful therapy in
recurrent lesions
Supressed or
eliminated in
successful therapy
in recurrent lesions

67. HOST RESPONSE Increased serum &
local antibody levels in
LAP
Increased systemic &
local antibody levels in
local periodontitis
ANIMAL STUDIES Capable of inducing
disease in gnotobiotic
rats
Found to be important
in experimental mixed
infections & in
periodontitis in the
cynomolgus monkey
VIRULENCE
FACTORS
Host tissue cell
invasion , leukotoxin,
collagenase,
endotoxin, fibroblast
inhibiting factor etc.
Host tissue cell
adherence &
invasions,
collagenase,
fibrinolysin, H2S etc.

68. MICROBIOLOGICAL TESTS FOR PLAQUE SAMPLES
• Microscopic identification
• Microbiological culturing
• Enzymatic assays
• Immunoassays
• Nucleic acid probes
• Polymerase chain reaction assays

69. MICROSCOPIC IDENTIFICATION
• Light Microscope
• Scanning Electron
Microscope
• Transmission Electron
Microscope

70. MICROBIOLOGICAL CULTURING
• Culture methods -the gold standard
• Available for the positive identification of many
putative periodontopathogenic microorganisms
• Use of selective and non-selective media
• One unique advantage - permits the assessment of
antibiotic sensitivity.
• Disadvantages- inability to detect low levels of
microorganisms, high cost, labor intensiveness,
prolonged time ,difficulty in growing several
bacterial species

71. IMMUNOASSAYS
• Immunofluorescence microscopy,
enzyme-linked immunosorbent assay
(ELISA), membrane assays and latex
agglutination assays.
• Advantages- include fairly low detection
thresholds, relatively low cost, rapid, are
somewhat quantitative
• Disadvantages- do not permit evaluation
of antibiotic sensitivity of the flora.

72. ELISA
• Partially purified, inactivated antigens
pre-coated onto an ELISA plate
• Patient serum which may contain
antibodies.
• Anti-human immunoglobulin coupled to
an enzyme. -second antibody, -binds to
human antibodies
• Chromogen or substrate which changes
color when cleaved by the enzyme
attached to the second antibody.

73. ELISA
Negative ELISA Test Positive ELISA Test

74. NUCLEIC ACID PROBES
• Consist of nucleic acid sequences labeled with a
radioactive or enzymatic-colorimetric marker
• Bind to complementary nucleic acid sequences on
corresponding microorganisms.
• Newer-synthetic oligonucleotides (also known as
16s rRNA probes)
• Oligonucleotide probes have greatest specificity
and lowest cross-reactivity as they target genes
specific to a bacterial species.
• Advantages-greater sensitivity than culture
methods,viability of microorganisms is not a
requirement

75. POLYMERASE CHAIN REACTION
• A molecular biological technique for high-yield
replication of DNA.
• Allows to synthesize vast numbers of copies of
minute samples of DNA
• Modification of the original PCR technology, "real-
time" PCR, permits detection of specific
microorganisms & also its quantification
• PCR assays, used in combination with synthesized
16S rRNA probes -enable the detection of virtually
any microorganism in a plaque sample

76. ENZYMATIC ASSAYS
• Enzymatic assay - detects bacteria that possess
trypsin-like enzymes such as T. Forsythensis,
treponema denticola and P. Gingivalis.
• Plaque sample containing any combination of these
three bacteria- placed on a paper strip impregnated
with a colorless substrate n-benzoyl-dl-arginine-2-
naphthylamide (BANA)
• BANA substrate breakdown produces a blue-black
color
• Disavantages-test unable to distinguish between
relative proportions of the three bacteria,cannot
identify the presence of other oral microorganisms,.