PRESENTED BYASHISH KARODE

MDS STUDENT

Histopathology of Dental CariesHistopathology of Dental Caries

HISTOLOGY : Study of microscopic anatomy of cells and tissues of plants and animals.

HISTOPATHOLOGY: Microscopic examination of tissues in order to study the manifestations of disease.

DENTAL CARIES: Microbial disease of calcified tissues of teeth characterised by demineralisation of inorganic portion and destruction of organic portion.

Dental Caries

It is a microbial disease of the calcified tissues of the teeth,

characterized by demineralization of the inorganic portion

and destruction of the organic substance of the tooth.

Many researches/investigations for more than a hundred years

have been done, still, many aspects of the etiology of this

disease is obscure and efforts at prevention are only partly

successful.

► Dental caries or tooth decay is one of the most common

of all disorders, second only to common cold.

► Dental caries has afflicted more humans longer than any

other disease. It was first appeared about 1400 BC years

ago. From that time to the present, dental caries affected

almost all human populations, at all socioeconomic

levels, and at all ages.

► Some isolated populations like Eskimos, some African

natives, and inhabitants of rural India are “immune” to

dental caries because they are not exposed to western

food habits.

The word ‘caries’ is derived from the Latin word ‘rot’.

It is the most prevalent disease affecting the human race,

Practically spread all over the world,

Affects both sexes and all races, all socio-economic strata and people of all ages,

Starts soon after teeth erupt into the oral cavity

Dental CariesDental Caries

Enamel CariesEnamel Caries Dentin CariesDentin Caries Cementum CariesCementum Caries(Root caries)(Root caries)

Smooth surface cariesSmooth surface caries Pit and fissure cariesPit and fissure caries

SMOOTH SURFACE CARIES

PIT AND FISSURE CARIES

OF ENAMEL: Hard translucent tissue covering the anatomical crowns.

COMPOSITION: Inorganic matter 96% Organic matter 0.2-0.8% Water 1.2-4% STRUCTURE: Hydroxyappatite crystals

laid in form of rods, Striae of Retzius, rod sheath etc.

OF DENTIN: Semi transparent calcified connective tissue penetrated by small canals containing protoplasmic processes belonging to cell which remain outside the tissue in pulp cavity.

COMPOSITION: Inorganic matter 61-73% Organic matter 20.2-22.8% Water 10.8-15.4% STRUCTURE: Hydroxyapatite crystals laid in

form of dentinal tubules, dentin matrix and Tomes fibres etc.

DENTINO ENAMEL JUNCTION:

Has more interprismatic substance, crossing and branching of dentinal tubules, enamel spindles and enamel lamellae.

OF CEMENTUM: Bone like tissue arranged in layers present around the anatomical root

COMPOSITION: Inorganic matter 65-70% Organic matter 35-40% STRUCTURE: Acellular cementum and

cellular cementum.

Causative Factors• Presence of caries pathogens (MS, LB)• Frequent or prolonged exposure to fermentable carbohydrates Potentiating Factors• Low fluoride exposure level• Inadequate salivary flow

Causes of dental caries

• Pre-microbiology era– Dental caries is the death (decay) of a tissue

• Microbiology period era– Dental caries is a microbe related disease

The first microbes observed• Anton Van Leeuwenhoek(1632-1723) developedthe microscope and wasthe first to discover oralbacterial flora: “I didn’tclean my teeth for threedays and then took thematerial that had lodgedin small amounts on thegums above my frontteeth…. I found a fewliving animalcules..”

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

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 acidicconditions while other beneficial bacteria areinhibited. This results in dominance of the plaqueby cariogenic bacteria

2. Acid tolerance (aciduricity)

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

Ecological niche: Human oral cavity “Intentionally designed … to be a

cariogenic organism” (Coykendall 1976) Carciogenic properties

Ability to produce acid (acidogenicity)

Ability to withstand acid conditions (aciduricity), at the expense of benign flora

Ability to adhere to teeth

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 micro flora driven by a change in the local environment (frequentsugar intake etc).

Plaque on the surface of the tooth (enamel)

Lesion Initiation • MS present in low numbers in plaque (carrier state) • Frequent sugar supply gives MS competitive advantage • ECP helps adhere them firmly to tooth surface • Metabolism of sugar to acid by products lowers pH • Few organisms can survive in low pH (aciduric) • MS proportions increase • This lowers the pH further and decreases the number of competing organisms • Once the pH low enough (critical pH, 5.0-5.5) tooth mineral begins dissolving • Mineral loss follows down the grain of the enamel rods

• Saliva dilutes & buffers acid & contains concentrated calcium & phosphate ions • When sugar present, net demineralization rapidly occurs • Between sugar episodes, remineralization slowly occurs • If remin periods exceed demin periods, subsurface lesion will mineralize & arrest • If demin periods exceed remin periods, cavitation will occur • Demin periods exceed remin periods when sugar Intake

is increased in intensity

The surface of a tooth i. e. covered by plaque, which consists mainly of bacteria. Plaque is often found close to the gum, in between teeth, in fissures and at other "hidden" sites.

Demineralization:When sugar and other fermentable carbohydrates reaches the bacteria, they form acids which start to dissolve the enamel - an early caries lesion occurs due to loss of Calcium and Phosphates

Remineralization:When sugar consumption has ceased, saliva can wash away sugars and buffer the acids. Calcium and Phosphates can again enter the tooth. The process is strongly facilitated by fluorides

A CAVITY occurs if the Demineralization "wins" over the Remineralization over time

The first indication of tooth decay are white spots on the enamel caused by the loss of calcium.

If the demineralization process outruns the natural remineralisation process, the lesion grows and a cavity is formed. 

The bacteria may invade the pulp of the tooth, 

causing a consistent tooth pain, especially during the night.

The bacteria may also produce an abscess,

and eventually the tooth may be extracted by the dentist.

SLOWLY PROGRESSING LESIONS GO THROUGH MORE DEMIN/REMIN CYCLES LEADING TO DARK SHALLOW LESIONS

RAPIDLY PROGRESSING LESIONS GO THROUGH FEWER DEMIN/REMIN CYCLES LEADING TO LIGHTER-COLORED, MORE AGGRESSIVE LESIONS

1. A tooth surface without caries.2. The first signs of demineralization.3. The enamel surface has broken down.4. A filling has been made but the demineralization has not been stopped.5. The demineralization proceeds and undermines the tooth.6. The tooth has fractured.

ENAMEL CARIES The early lesion is a white spot which

appears on the surface of the enamel. Caries spreads in zones which are as follows.

Translucent zone Dark zone Body of the lesion Surface zone

The zones seen before complete disintegration of enamel are:Zone 1: Translucent zone,

-lies at the advancing front of the lesion,

-slightly more porous than sound enamel,

-it is not always present

Zone 2: Dark zone,

-this zone is usually present and referred

to as positive zone

-formed due to demineralization.

Zone 3: Body of the lesion,

-found between the surface and the dark zone,

-it is the area of greatest demineralization,

Zone 4: Surface zone,

-relatively unaffected area,-greater resistance probably due to greater degree of mineralization and greater F concentration.

Pit And Fissure Caries:

-lesion begins beneath plaque, with decalcification of enamel

-pit and fissures are often deep, with food stagnation,

-enamel in the bottom of pit or fissure is very thin, so early dentin involvement

frequently occurs.

-here the caries follows the direction of the enamel rods.

-It is triangular in shape with the apex facing the surface of tooth and the

base towards the DEJ.

-when reaches DEJ, greater number of dentinal tubules are involved.

-it produces greater cavitation than the smooth surface caries and there is

more undermining of enamel.

Smooth surface Caries: The earliest manifestation of incipient caries (early caries) of enamel is usually

seen beneath dental plaque as areas of decalcification (white spots).

The first change seen histologically is the loss of inter-rod substance of

enamel with increased prominence of the rods.

-this is followed by the loss of mucopolysaccharides in the organic substance.

-presence of transverse striations of the enamel rods,

- accentuated incremental lines of Retzius

as it goes deeper, the caries forms a triangular pattern or cone shaped lesion

with the apex towards DEJ and base towards the tooth surface.

Finally, there is loss of enamel structure, which gets roughened due to

demineralization, and disintegration of enamel prisms.

intact surface layer

incremental growth linesstriae of Retzius

Early lesion with intact enamel

5-10% mineral loss, zone

of intact enamelLesion body 60%

or more mineral loss

translucent zone – 5-10%

mineral loss

normal enamel

The events of the dentinal caries are as follows:

Defense reaction of the pulpodentinal complex

1. Seclerosis2. Reactionary dentine formation3. Sealing of the dead tracts

Carious destruction1. Demineralization2. proteolysis

CARIES OF DENTIN

Begins with the natural spread of the caries process along the DEJ and rapid

involvement of the dentinal tubules. The dentinal tubules act as tracts leading to

the pulp (path for micro-organisms).

Early Dentinal Changes:

-initial penetration of the dentin by caries dentinal sclerosis,

-calcification of dentinal tubules and sealing off from further penetration by

micro-organisms,

-more prominent in slow chronic caries.

Dentinal sclerosis

Cariouslesion

Dentin reaction to caries

Behind the transparent sclerotic zone, decalcification of dentin appears.

In the earliest stages, when only few tubules are involved,

microorganisms may

be found penetrating the tubules Pioneer Bacteria.

This initial decalcification

involves the walls allowing

them to distend as the

tubules are packed with

microorganisms. Each

tubule is seen to be packed

with pure forms of bacteria,

eg., one tubule packed with

coccal forms the other

tubule with bacilli.

As the microorganisms proceed further they are distanced from the

carbohydrates substrate that was needed for the initiation of the

caries.

Thus the high protein content of dentin must favour the growth of the

microorganisms.

Therefore proteolytic organisms might appear to predominate in the

deeper caries of dentin while acidophilic forms are more prominent in

early caries.

Advanced Dentinal Changes ;-decalcification of walls, confluence of the dentinal tubules,

-tiny “liquefaction foci”, described by Miller are formed by the focal

coalescing and breakdown of dentinal tubules. These are ovoid areas of

destruction parallel to the course of the tubules which filled with necrotic

debris and increase in size by expanding.

The adjacent tubules are distorted and their course is bent due to this

expansion.

The destruction of dentin by decalcification and then proteolysis occurs

in numerous focal areas- leading to a necrotic mass of dentin of a

leathery consistency.

-clefts present in the carious dentin that extends at right angles to the

dentinal tubules, accounts for the peeling off of dentin in layers while

excavating.

Shape of the lesion is triangular with the apex

towards the pulp and the base towards the

enamel.

Zone 1; Zone of Fatty Degeneration of Tome’s

Fibers,(next to pulp)

-due to degeneration of the odontoblastic

process. This occurs before sclerotic

dentin is formed and makes the tubules

impermeable.

Zone 2; Zone of dentinal sclerosis,

-deposition of Ca salts in the tubules.

Zone 3; Zone of decalcification of dentin

Zone 4; Zone of bacterial invasion

Zone 5; Zone of decomposed dentin due to

acids and enzymes.

Root Caries

Root caries as defined by HAZEN, is a soft, progressive lesion

that is found anywhere on the root surface that has lost its

connective tissue attachment and is exposed to the environment.

-the root surface must be exposed to the oral environment before

caries can develop here.

-Plaque and micro-organisms are essential for the cause and

progression of the lesion, mostly Actinomyces,

-micro-organisms invade the cementum either along the Sharpey’s

fibers or between the bundles of fibers.

-spread laterally, since cementum is formed in concentric layers.

-after decalcification of cementum, destruction of matrix occurs

similar to dentin with ultimate softening and destruction of this

tissue.

-invasion of micro-organisms into the dentinal tubules, finally

leading to pulp involvement.

-the rate is slower due to fewer dentinal tubules than crown area

Dark brown pigmented carious lesion , insensitive to painful stimuli in which caries progression is halted.

ZONE 1: Surface layer- Brown in colour and of

leathery consistency. ZONE 2: Pigmented zone- Hard, dark

brown in colour, forming main bulk with presence of coalesced bacteria bodies

ZONE 3: Sclerotic layer-Hard, white zone having highly calcified tubules with absence of bacteria.

Caries occuring beneath or around the existing restoration.

Histologically can occur as:1. Outer lesion 20%2. Wall lesion 11.9%3. Both leisions 60% cases

IN ENAMEL: A triangular outer lesion is seen which continues with the dentinal lesion along the dentinal wall. Both lesions are walled by dark zone.

IN DENTIN: Appearance can be-1. Superficial demineralization of cavity walls 2. Subsurface demineralization without visible

changes of wall surface3. Subsurface demineralization with increased

mineralization of surface layer4. Alternating zones of demineralization and

remineralization.

Dental caries is the result of the metabolic activities of bacteria in microbial communities on teeth termed dental biofilms (often referred to as dental plaque)

Hence, the presence of microbial communities on the tooth surface is a prerequisite for caries lesions to develop

Although there are different opinions as to how and which microorganisms produce carious lesions, it is agreed that caries cannot occur without microorganisms

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The prevalence of dental caries (i.e., the percentage of persons with >1 decayed, missing, or filled teeth) in permanent teeth increases with age, from 26% among persons aged 5--11 years to 67% among persons aged 12--17 years and 94% for dentate adults (with >1 natural teeth) aged >18 years.

Complex ecology of the oral cavity. 300 – 400 species are indigenous oral flora. History:

Miller (1880): Little knowledge about bacteria. Clarke (1924): First who associate bacteria with

dental caries

oFirst to isolate MS from human dental caries

oFirst to produce caries in extracted teeth.

Orland (1955): Used animals to induce dental caries using MS.

1924: Clark recovered Gram-positive cells

1960: Fitzgerald & Keyes found caries-conducive streptococci to be Streptococcus mutans

What is virulence?The ability of a bacterium to cause

infection.

Virulence factors: Two types: Those that promote bacterial colonization and

invasion of the host tissue Those that cause damage of the host tissue.

The bacterium should be found in people with the disease

The bacterium should be isolated from the lesions of infected person

Pure culture, inoculated into a susceptible individuals or animals should produce the disease

Same bacterium should be re-isolated from intentionally infected animals or humans.

Virulence is within the bacterium and is independent of the host

Isolation and growth of bacterium is necessary: Yet, some pathogens not yet cultured

Nos. 2 & 4: assume that all members of the same species are virulent

No. 3: Ethics with human subjects, Yet some pathogens from humans can not cause the same effect in animals.

BACTERIAVIRUSFUNGIPROTOZOAMYCOPLASMA

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Natural microflora exists in harmonious

relationship with the host

Bacteria

Gram positive cocci

Gram positive Rods and

filaments

Gram negative cocci

Gram negative rods

1. Streptococcus

2. Enterococcus

1. Actinomyces

2. Eubacterium

3. Lactobacillus

4. Propionibacterium

1. Neisseria2. Veillonella

1. Haemophilus2. Eikenella3. Capnocytopha

ga4. Actinobacillus5. Porphyromona

s6. fusobacterium 82

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Fungi

Candida

Viruses

CMV

Coxsackie A2,4,5,6,8,9,10 and 16

Hepatitis

HIV

ProtozoaTrichomonas tenaxEntamoeba gingivalis

Mycoplasma M. salivariusM. pneumoniaeM. hominis

An organism must be acidogenic An organism must be aciduric An organism must exhibit tropism for

teeth An organism must utilize refined sugar

(sucrose)

(Newburn, 1983)

Enamel

Pit & fissure

Dentin, Root

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Mutans Streptococci S. mutans

S. sobrinus

Viridans

Streptococci S. mitis

S. salivarius

S. sanguinis

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Pit and fissure caries- most common carious lesions found in

humans Fissures provide mechanical retention for

the bacteria. S mutans, S salivarius, S sanguis, L acidophilus, L casei, A viscous, A nalsundii, Actinomyces israelii develop fissure lesions

Smooth surface caries-

A limited number of organisms have proved able to colonize smooth surfaces in large enough numbers to cause decay in test animals.

Streptococcus mutans is very significant in this respect.

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Root caries-

In rodents, gram – positive filamentous rods, including actinomyces species have been associated with this type of lesion

Strains of Nocardia and S. sanguis may also cause root caries

In cross-sectional studies of plaque overlying carious root surfaces, mutans streptococci, alone or in combination with lactobacilli, have been isolated more frequently or in higher proportions than on sound root surfaces

(Billings et al., 1985; Brown et al., 1986; Fure et al., 1987; Keltjens et al., 1987; Bowden et al., 1990; van Houte et al., 1990)

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Deep dentinal caries-

Because the environment in deep dentinal lesions is different from that at other locations the flora here is also different

The predominant microbe- lactobacillus

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Secondary caries resembles Pit & Fissure caries

3 associated microbes- S. mutans Lactobacillus Actinomyces viscous

The presence in significant nos. of these bacterias in dentin of Secondary caries indicates their role in formation & development of Secondary carious lesions(Gonzalez- Cabezas; 1999).

Definite relationship between S. mutans & Secondary caries

(Fontana et al;1996) 94

Irrespective of the age of plaque and the diet, the predominant organisms are gram-(+)ve cocci of the genus streptococcus which form about 50% of the total CFU recovered from young plaque

These streptococci have been divided into various groups based on their colonial morphology and physiological characteristics

Oral streptococci are isolated on Mitis-Salivaris Agar, a selective medium that permits isolation from mixed flora 95

In 1924 Clarke isolated a streptococcus that predominated in many human carious lesions

He named them streptococcus mutans because of its varying morphology

Characteristics of S. mutans include Nonmotile Catalase negative Gram positive Cocci in short or medium chains. Opaque, cushion shaped colony on Mitis–

Salivarius bacitracin Agar Colony surface resembles frosted glass 96

Adherence to teeth Extracellular polysaccharide synthesis

( glucan ) Intracellular polysaccharide synthesis Acidogenicity Aciduricity

TYPES: (Coykendall, 1989) S. anginosus : important in purulent infections S. bovis : found in patients with colon cancer S. mitis : similar to sanguis but doesn’t

ferment any sugar S. mutans : seven species (9 species – recent

data) S. salivarius : in saliva, rare in infections S. sanguis : causes endocarditis S. vestbularis : new species from oral cavity.

Species Serotype Arg Raf Mel H2O2 Aero Baci Source

S mutans c, e, f - + + - + - Human

S rattus b + + + - + - Rats

S cricetus a - + + - - + Rats

S sobrinus d, g - - - + + - Human

S ferus c - - - - - + Rats

S macacae c - + - - - + Monkey

S downei h - - - - - + Monkey

S orisus d pigs S orisus dentisuis p k

Three factors: Ability to adhere to other bacteria and tooth

surface Ability to rapidly metabolize nutrients (CHO) Ability to tolerate acidic environment.

Saliva: Lysozyme IgA: (IgA protease), (IgA deficiency)

Bacterial proteins: Ag I/II family: Adhere to saliva proteins Adhesin Fimbrial adhesion: Adhere to saliva pellicle glucan binding (GBP)

Pellicle glycoprotein

PBP*

(Adapted from Slots & Taubman, 1992)

*PBP: Pellicle Binding Protein

Initial Attachment

S. mutans

+Sucrose Glucose Fructose

GTF-S** GTF-I*

(Adapted from Slots & Taubman, 1992)

*Insoluble form (-1,3-linked) “Mutan” – polymerized by GtfB**Soluble form (-1,6-linked) “Dextran” – polymerized by GtfDGtfC responsible for a mixture of -1,3 & -1,6

GTF Binding Glucans

Glucans

(Adapted from Slots & Taubman, 1992)

Pellicle Glycoprotein

Glucan Binding Protein

Glucans

Aggregation

S. mutansS. mutans

S. mutans

Through cell membrane, extrusion of protons: Membrane ATPase hydrolyze ATP molecules Hydrolysis of one ATP, results in extrusion of

three protons This results in elevation of cytoplasmic pH. When pH decreases, ATPase activity increases

4-folds.

Based on ability of S. mutans to synthesize insoluble glucan.

S. mutans have 3 genes: gtfB encodes GTF-I enzyme: insoluble glucan gtfC encodes GTF-SI enzyme: insoluble glucan gtfD encodes GTF-S enzyme: soluble glucan

Strain Gtase Adherence%

MT8148 I,SI/S 72.8 2.6

B29 /SI/S 16.3 1.0

B29 I/SI/S 46.9 5.9

B58 I/ /S 9.6 1.0

B58 I/SI/S 69.9 1.8

B32 / /S 1.4 0.4

(Fujiwara et al., 1996)

Sterile mouth at birth

S. sanguis and S. mutans colonize teeth

Number of bacteria increases in the presence of:

Sucrose Caries Teeth

ACQUISITION OF S. mutans

Birth 5 YearFirst Tooth 19 336.8 +/- 1.4 mo.

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MS

N=38

Caufield et al., J Dent Res. 72:37-45, 1993.

Important facts: Difficult to change S. mutans strain(s) High number of S.mutans strains and isolates. One (or more) strain (isolates) is/are present

in the mouth.

GENETIC VARIATIONS OF S.mutans

Lactobacilli are - Gram (+)ve Non-spore forming Rods Grow best under microaerophilic

conditions. Represents about 1% of the oral flora. Favorite habitat of lactobacilli is in the

dentin of deep carious lesions. Relatively low affinity for tooth surface.

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Preferentially colonize the dorsum of the tongue

Does NOT play a major part in initiation, but important in progression

With established low pH the number of lactobacilli increases and the number of S. mutans decreases

Contribute to the demineralization of the teeth once lesions are established

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It is a gram positive, non motile, non spore forming organism occurring as rods and filaments. It is a good plaque former

All species of actinomyces ferment glucose, producing mostly lactic acid, lesser amounts of acetic and succinic acid, and traces of formic acid.

Most interest has centered on A viscosus and A naeslundii because of their ability to induce root caries.

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All species of actinomyces ferment glucose, producing mostly lactic acid, lesser amounts of acetic and succinic acid, and traces of formic acid

Anaerobic bacteria

Taxonomically distant from streptococci but have very similar sugar metabolism and can produce lactic acid.

Although well-known as gut inhabitants, it is only quite recently that their occurrence in the mouth and possible association with caries has been recognized

This change in viewpoint is because introduction of molecular detection methods the development of a selective medium using

mupiromycin to suppress growth of other bacteria 117

S.Mutans has the central role in etiology of dental caries

If We understand the dental caries microbiology well, we will be able to treat patients differently

It is of paramount value that the term “dental caries” not be equated with “cavities” by dentists. The lesion is not the disease, but the effect of the disease. The disease does not occur without infection by cariogenic bacteria

To prevent, detect, and manage caries throughout life one must not be restrictively focused on the end result of the disease, cavities 118

Thank you