Journal of Oral Research & Review. Volume: 4, Issue 2. August 2012.

Biofilm in Endodontics- Redefining the Cause

Umrana Faizuddin *, Vishwanath B. *, Archana Daga **, Chaitanya Biragoni ***,

* Reader, ** P. G. Student, *** Senior Lecturer, Department of Conservative Dentistry & Endodontics, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Hyderabad - 500060, Andhra Pradesh, India. Correspondence to: Dr. Archana Daga , Phone: 9985857248 E-mail: archanadaga4u@gmail.com

ABSTRACT: The main aim of Endodontics is to recognize and removal of etiological factors. Debridement of the root canal by instrumentation, irrigation and removal of biofilm is considered an important factor to prevent and treat endodontic diseases. The root canal morphology provides excellent conditions for a biofilm formation which is one of the main causes for endodontic reinfection. The following review article explores the biofilm formation which commences from adhesion of planktonic microorganisms to a surface followed by colonization, coadhesion, growth and maturation and finally detachment of some microorganisms. Bacterias present in biofilm display a wide range of characteristics that provide a number of advantages over planktonic bacteria.

KEYWORDS: biofilm, quorum sensing, intraspecies communication.

INTRODUCTION: The most common etiology for the pulpal and periradicular pathologies are microorganisms or the microflora. A traditional concept that explains infectious processes occurring in humans suggests that diseases are produced as the result of the aggressive invasion of harmful microorganisms, which battle with the human hosts defenses, triggering mechanisms that release antibodies and immune cells. The impact of such an approach generates a predisposition to search for those most

dangerous microorganisms that can cause/ trigger the most severe damage to the host. In line with this view, infectious processes of the oral cavity were proposed to be caused by a relatively small number of organisms from the diverse collection of species found in the human mouth. (1)

Biofilms are defined as polysaccharide matrix enclosed bacterial populations adherent to each other and /or to surfaces or interfaces. (Costeron & Steewart)(6)Biofilm is defined as a

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Journal of Oral Research & Review. Volume: 4, Issue 2. August 2012.

community

of

micro

colonies

of

basically occur with the same sequence of developmental steps: 1. Deposition of conditioning film 2. Adhesion and colonization of planktonic microorganisms 3. Bacterial growth and biofilm expansion 4. Detachment of biofilm microorganisms into their surroundings. Step 1: Involves adsorption of inorganic and organic molecules to the solid surface which leads to the formation of conditioning film.

microorganisms in an aqueous solution that is surrounded by a matrix made of glycocalyx, which also attaches the bacterial cells to a solid substratum. (Grossman)(7) The complexity and ubiquitous nature of biofilm can be understood by these following examples. The worlds strongest glue (far exceeding any man made type) is found on river rocks (biofilm). British Petroleums latest Alaska oil pipeline shutdown was attributed to corrosion caused by microbacteria that became trapped in a sludge layer of the pipe (biofilm). Chronic Otitis media (prevalent in children) has been recently attributed to the presence of biofilms.

Conditioning film is composed of proteins and glycoproteins which are derived from saliva and gingival crevicular fluid(10) Dental plaque formation on the tooth surface involves

Control of oral biofilms is fundamental to the maintenance of oral health and prevention of dental caries, gingivitis and periodontitis.(2-5) The biofilm mode of growth seems to be advantageous for microorganisms as they form three dimensional structured communities with fluid channels for transport of substrate, waste products and signal molecules.(8)Biofilm formation in root canals, as hypothesized by Svenster and Bergenholtz
(9)

conditioning with salivary pellicle. Step 2: Involves adhesion and colonization of

planktonic microorganisms and its attachment is strengthened by polymer production and unfolding of cell surface structures. Pioneer organism which is involved in the formation of biofilm on the tooth surface is streptococcus followed by subsequent attachment of gram positive and gram negative bacterias. (11) There are numerous factors affecting bacterial attachment which includes pH, temperature variations, flow rate of fluid, nutrients, surface energy of the substrate, bacterial content,

, is probably

initiated at some time after the first invasion of the pulp chamber by planktonic oral organisms after some tissue breakdown.

FORMATION OF BIOFILM: The phases for microbial community to develop a biofilm and colonize the

bacterial growth stage, bacterial cell surface charge, and surface hydrophobicity.

environment may sometimes be unusual, but

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Step

3:

Involves

bacterial of

growth

and STRUCTURE BIOFILM The phenotype of biofilm bacteria is distinct from planktonic bacteria due to following reasons: 1. EPS (extrapolymeric saccharide) protects the residing bacteria from environmental threats 2. Structure of biofilm permits trapping of nutrients and metabolites AND CHARACTERISTICS OF

expansion. attracts

Monolayer secondary

microorganisms to form

colonizers

microcolonies. These microcolonies are similar to towers with lateral and vertical growth of microorganisms. Two types of microbial interaction are seen at cellular level during biofilm formation-co-adhesion and

coaggregation.12 Step 4: Involves the deattachment of biofilm

3. Biofilm structures display organized internal compartmentalization 4. Communication between bacterial cells and exchange of genetic materials.

microorganisms into their surroundings which are of two types seeding disposal and clumping dispersal. Seeding dispersal: it is a programmed

Caldwell et al highlighted 4 characteristics of biofilm. 1.Autopoiesis 2.Homeostasis 3. Synergy 4. Communality EPS/ GLYCOCALYX PRODUCTION: The cells within biofilms produce either matrix made up of extracellular polymeric substances EPS(15) or cell surface structures (capsule). EPS protects the biofilm bacteria from

detachment of planktonic bacterial cells caused by local hydrolysis of the extracellular polysaccharide matrix, and conversion of a subpopulation of cells into motile planktonic cells. The de-attached cells are the ones which causes persistent infection.
(13)

Clumping Dispersal: A physical detachment pathway in which a fragment of a microcolony, simply detaches from the biofilm and is carried by the bulk until it lodges in a new location and initiates a new sessile population.

environmental stresses such as UV radiation, pH shifts, osmotic shock and dessication. 16 EPS can also engulf metals, cations and toxins. 17 It also acts as an ion exchange center.
18

Deattachment can be of two types Erosion (continuous deattachment of single cell)and Sloughing (rapid detachment of biofilm) Electron microscopically Biofilms appears as tower or mushroom shaped microcolonies with interspersed channels that are separated from the external environment through which fluids move by convection. 14

The

activity of positively charged antimicrobial agents is retarded by EPS as they are negatively charged. 19 ELECTROSTATIC INTERACTION:

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Electrostatic interaction helps biofilm to sequester trace elements and nutrients.

20

functions of resident bacterial cells within a biofilm. Quorum sensing can also regulate microbial property such as virulence factors and extracellular DNA incorporation.24 Example of interspecies communication includes

Where else water channels facilitates efficient exchange of materials between outer fluid medium and inner fluid medium providing sufficient ecosystem. nutrition
(21)

to

nutrient

deprived

release of peptide molecules by streptococci. AUTO- INDUCER MECHANISM: Auto inducer system 2(AI -2) helps in interspecies communication.
(25)

Bacteria produce tiny filaments

that extend from one cell to another. At a certain stage in biofilm development genes are expressed that allow for the creation of nanowires. Examination of these nanowires has shown that electrical impulses are being transmitted from cell to cell. It is possible that other genes are being turned on and off according to the needs of the colony and the ability of the bacteria to sense its environment. This could be a primitive type of neural network. COMPLEX ARCHITECTURE: Biofilm has overall complex architecture with niches and also it possess different array of lytic enzymes, proteolytic enzymes. Mature biofilm with increased nutrient, pH, metabolic products, signaling molecules and gaseous gradients possesses antimicrobial resistance. Oral biofilm is dominated by anaerobic bacteria because of redox gradient formed within the internal compartmentalization of biofilm.
23

This signal is

released by many gram positive and gram negative microorganisms. Close proximity of microorganisms between them facilitates gene transfer

BIOFILM IN ENDODONTICS: Bacterial biofilm can be seen beyond the apex of the root as bacteria in biofilm survive unfavorable environmental and nutritional conditions.26, canal gains
27

Infectious processes in root power to cause

sufficient

subsequent destruction of the pulpal tissues only after biofilm formation. Biofilm formation in root canals is probably initiated at some time after the first invasion of the pulp chamber by planktonic oral organisms after some tissue breakdown. At this point, the inflammatory lesion frontage that moves successively toward the apex will provide the fluid vehicle for the invading planktonic organisms so these can multiply and continue attaching to the root canal walls. (Hypothesis: Svenster and Bergenholtz) The necrotic pulp tissue becomes a favorable environment for microbial proliferation due to the presence of

QUORUM SENSING: Quorum sensing is intraspecies communication which is mediated by low molecular weight molecules, which can alter the metabolic activity of neighboring cells and coordinate the

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organic residue or nutrients, which act as substrate or culture medium. Gram-negative bacteria are more frequent than Grampositive bacteria. Facultative or strict anaerobic microorganisms are more frequent than aerobic

multispecies bacteria and varying degree of extracellular matrix.

(30)

Fusobacterium

nucleatum, Porphyromonas gingivalis and Tannellera forsythensis were detected

associated with extraradicular biofilm by using PCR based 16s rRNA gene assay. 31 PERIAPICAL BIOFILM: Periapical microbial biofilms are isolated biofilms in the periapical region of

microorganisms, and the presence of bacilli and filaments is equivalent to that of cocci. Endodontic categories 1. Intracanal biofilm 2. Extra radicular biofilm 3. Periapical biofilm 4. Foreign body centered biofilm INTRACANAL BIOFILM: Intracanal biofilm are microbial biofilm formed on the root canal dentine of infected tooth. First identification of biofilm was earlier reported by Nair 1987 under transmission electron microscopy. Major bulk of the biofilm can be of various

endodontically infected teeth. They can be seen even in the absence of root canal infections. Bacterias present in such biofilm should have the capacity to overcome host defense mechanisms and result in periapical lesions.(32)Actinomyces species and P.

propionicum are associated with periapical lesions. presence Actinomyces of yellow species granules show the

commonly

referred as sulphur granules. Microscopically these granules appear as ray fungus. The aggregation of Actinomyces cells are

organisms existed as loose collections of cocci, rods, filaments and spirochetes apart from this bacterial condensations were seen as palisade structure similar to dental plaque seen on tooth surface.
(28)

influenced by pH, ionic strength and cell concentration.

(33)

Aggregation

facilitates

biofilm formation. FOREIGN BODY CENTERED BIOFILM: Foreign body centered biofilm is seen when bacteria adheres to an artificial biomaterial surface and forms biofilm structures.
34

The extracellular matrix

material of bacterial origin was also found. EXTRARADICULAR BIOFILM: Extra radicular biofilm are root surface biofilms formed on root surface adjacent to the root apex of endodontically infected teeth.(29) In a study of cases resisting treatment (refractory endodontic cases) Tronstad et al examined root tips of surgical extracted teeth under SEM and found structureless smooth biofilm with

it is

also known as biomaterial centered infection. It is a major complication associated with prosthesis and also in an implant supported prosthesis. Biomaterial centered infection reveals opportunistic invasion by nosocomial organisms. Takemura N et al suggested Gram

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positive facultative anaerobes have the ability to colonize and form extracellular polymeric matrix surrounding gutta-percha, serum plays a crucial role in biofilm formation.
(35)

electron

microscopy

shows

complete

destruction of endodontic biofilms for a depth of 1 mm inside a root canal after plasma treatment for 5 min. Plasma emission

Studies

have suggested that extraradicular microbial biofilm and biomaterial centered biofilm are related to refractory periapical disease.
(36)

spectroscopy identifies atomic oxygen as one of the likely active agents for the bactericidal effect.

Biofilm can be identified by various methods like Environmental SEM, Confocal Microscopy, Special fluorescent stains (FISH techniquefluorescence in situ hybridization) LASERS Er:YAG laser have produced excellent results due to its capacity for ablating hard tissue with very less thermal effects. They are considered ERADICATION OF BIOFILM: IRRIGATING SYSTEMS: The structural dense organization of the biofilm within the polymeric matrix restricts the penetration of any agent into them. Biofilm are found to be resistant to amoxicillin, doxycycline and metronidazole. (37) PHOTODYNAMIC THERAPY (PAD) PAD is a unique combination of a to be effective tool for the removal of apical biofilm

photosensitizer solution and low-power laser light. The photosensitizer, which is mostly colored, adheres to or gets absorbed by

Sodium hypochlorite is an effective irrigant to destroy all forms of Enterococcus faecalis including its biofilm form. (38) Chlorhexidine 2% gel or liquid form is effective to eliminate Enterococcus faecalis from the superficial layers of dentinal tubules up to 100

microbial cells. The low-power laser will destruct the target area and inactivate the microbial invaders. The photosensitizer then binds to microbial cell walls or even enters the cells. Further, the laser light activates the photosensitizer and creates a cascade of energy transfer and variable chemical

micrometer. (40)

reactions in which singlet oxygen and free 1-minute use of ultrasonically activated radicals play an important role. PAD needs a maximum of 150 seconds. PAD is effective against Enterococcus faecalis, Streptococcus intermedius, Fusobacterium micros, nucleotum, Prevotella

irrigation, followed by root canal cleaning and shaping has been shown to improve canal and isthmus cleanliness in terms of necrotic debris/biofilm removal. Plasma dental probe is effective for tooth disinfection. Scanning

Peptostreptococcus intermedia)

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CONCLUSION: The application of the biofilm concept to endodontic microbiology will play a crucial role in helping us to understand, not only the pathogenic potential of the root canal microbiota, but also the basis for new approaches for disinfection. Microorganisms adaptation under different disease conditions as well as how biofilms are organized in root canals are important issues to be addressed on the road to obtain a clear understanding of how the root canal bacteria resist endodontic treatment measures .
16. 15. 14. 13. 12. 11.

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