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Histopathology & microbiology of dental caries
 

Histopathology & microbiology of dental caries

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    Histopathology & microbiology of dental caries Histopathology & microbiology of dental caries Presentation Transcript

    • PRESENTED BY ASHISH 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) 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..”
    • 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 acidic conditions while other beneficial bacteria are inhibited. This results in dominance of the plaque by 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 (frequent sugar 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 lines striae of Retzius
    • Early lesion with intact enamel 5-10% mineral loss, zone of intact enamel Lesion 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. Seclerosis 2. Reactionary dentine formation 3. Sealing of the dead tracts  Carious destruction 1. Demineralization 2. proteolysis
    • ARIES OF DENTIN gins with the natural spread of the caries process along the DEJ and rapid olvement of the dentinal tubules. The dentinal tubules act as tracts leading to e pulp (path for micro-organisms). rly Dentinal Changes: itial penetration of the dentin by caries dentinal sclerosis, alcification of dentinal tubules and sealing off from further penetration by icro-organisms, ore prominent in slow chronic caries. Dentinal sclerosis
    • Carious lesion 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 surface 3. Subsurface demineralization with increased mineralization of surface layer 4. 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 73
    • 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.
    • BACTERIA VIRUS FUNGI PROTOZOA MYCOPLASMA 81 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. Streptococc us 2. Enterococc us 1. Actinomyces 2. Eubacterium 3. Lactobacillu s 4. Propionibact erium 1. Neisseria 2. Veillonella 1. Haemophilus 2. Eikenella 3. Capnocytopha ga 4. Actinobacillus 5. Porphyromona s 6. fusobacterium82
    • 83 Fungi Candida Viruses CMV Coxsackie A2,4,5,6,8,9,10 and 16 Hepatitis HIV Protozoa Trichomonas tenax Entamoeba gingivalis Mycoplasma M. salivarius M. pneumoniae M. 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
    • 87
    • 88
    • • Mutans Streptococci • S. mutans • S. sobrinus • Viridans Streptococci • S. mitis • S. salivarius • S. sanguinis
    • 90 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. 91
    • 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) 92
    • 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 93
    •  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 (α-1,3-linked) “Mutan” – polymerized by GtfB **Soluble form (α-1,6-linked) “Dextran” – polymerized by GtfD GtfC responsible for a mixture of α-1,3 & α-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 Year First Tooth 19 33 6.8 +/- 1.4 mo. 26 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. 113
    •  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 114
    • 115  It is a gram positive, non motile, non spore forming organism occurring as rods and filaments. It is a good plaque former  All speciesof actinomycesferment glucose, producing mostly lactic acid, lesser amountsof acetic and succinic acid, and tracesof formic acid.  Most interest hascentered on A viscosusand A naeslundii becauseof their ability to induceroot
    • 116 All speciesof actinomycesferment glucose, producing mostly lactic acid, lesser amountsof acetic and succinic acid, and tracesof 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