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. ‘mineralised dental plaque”
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
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. Washout effect: saliva Ciliae
Adhesion forces
Removal forces
Soft tissue
Hard tissue
Mastication
Tongue
Oral hygiene
Washout effect: saliva
Ciliae

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. Formation of dental Pellicle
Initial Colonization
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: Brownian motion
Sedimentation of micro- organisms
Liquid flow
Active bacterial movement

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

33. Phase III Firm anchorage Specific interactions: Covalent Ionic
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
RODS
COCCI
FILAMENTS

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 ASSOCIATIONof
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

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,.