ENDODONTIC BIOFILM

Biofilm is defined as a sessile multi cellular microbial community characterized by cells that are firmly attached to a surface and embedded in a self produced matrix of extracellular polymeric substances

Biofilms are formed whenever there is free flow of fluid , microorganisms and a solid surface. It is one of the basic survival strategies employed by bacteria

Characteristics of biofilm

Biofilms should possess 1. autopoiesis- ability to self organize 2. homeostasis-resist environmental

pertubations 3.synergy- effective in association than

in isolation 4.community- respond to environmental

changes as a unit rather than single individual

Biofilm protects residing bacteria from environmental threats

Structure of biofilm traps nutrients Displays internal compartmentalization-allows

bacterial species with different growth requirements to survive

Communicate and exchange genetic materials

Ultrastructure of a biofilm

Water channels help in exchange of materials between the cells

Ultrastructure of a biofilm

Basic structure of a biofilm- heterogenous arrangement of microbial cells on a solid surface

Glycocalyx matrix made up of extrapolymeric substance surrounds the microcolonies and anchors the bacterial cell to the substrate

85% of biofilm is made up of matrix and 15% by cells

A fully hydrated biofilm appears like a mushroom shape/ tower shape

Water channels are primitive circulatory system in biofilms

How biofilm forms

First stage of biofilm involves the adsorption of macromolecules in the planktonic phase to surface- a conditioning film forms- (transport of microbe to the substrate surface)

Second stage – adhesion and co-adhesion of microbes and attachment strengthened by polymer production and unfolding of cell surface structures-  (initial non-specific microbial-substrate adherence phase)

Third stage involves the multiplication and metabolism of attached microorganisms -(bacterial growth and biofilm expansion)

Fourth stage involves detachment of biofilm micro organisms

Stages of biofilm formation

Recognition between a suspended cell and a cell already attached to substratum- co-adhesion

Genetically distinct cells recognize and clump together- co-aggregation

Factors influencing biofilm formation

PH, temperature, surface energy of substrate, flow rate of fluid, nutrient availability, bacterial growth stage, surface hydrophobicity

Detachment of biofilm- seeding dispersal

Erosion- continous detachment of single cells and small portions of biofilmSloughing- rapid massive loss of biofilm

Bacterial talk in biofilms

Communications between bacterial cells residing in a biofilm is attained through signaling molecules by a process called as quorum sensing

Quorum sensing is mediated by low molecular weight molecules- autoinducers

Qs leads to

Exchange of genetic materials between species

Antibiotic resistance

Nutrient breakdown

Xenobiotic metabolism

Coordinated behaviour of biofilm

Quorum sensing

Endodontic biofilms

Endodontic biofilms are less diverse than oral microbiota

Root canal environment is more anaerobic

Microbes persist in isthmuses, deltas and apical parts of root canal system- so complete disinfection is not possible

Endodontic films are categorized in to

1.intracanal biofilm

2.extraradicular biofilm

3.periapical biofilm

4.biomaterial centered infection

ENDODONTIC BIOFILM FORMATION

First, there is penetration of the organism in the pulp where it attaches and spreads further along the root canal.

Possibly, it is after biofilm formation that the infectious process gains sufficient power to cause subsequent destruction of the pulpal tissue. At some point in the breakdown process, however, a steady state is reached where the bacterial mass is held up by host defense mechanisms.

The demarcation zone may be inside the root canal near the root canal exit,  at the foramen, or, as demonstrated by scanning electron microscopy (SEM), on the external root surface near the exit of the foramen to the periapical tissue environment.

 

Intracanal Biofilm

Forms on root canal dentin of an infected tooth

Identified by Nair 1987

Cocci, rods , filaments and spirochetes are seen

Morphologically distinct type of bacteria are seen

Eg- E faecalis

Characteristic bacteria-dentine wall relationship

Distinct pattern of organization of microbes

Extraradicular biofilms

Root surface biofilms- formed adjacent to root apex of endodontically treated teeth

Found in teeth with asymptomatic periapical periodontitis and chronic apical abscess with sinus tract

Multispecies in nature- F. nucleatum, Po. gingivalis, and Tannerella forsythensis

Dominated by cocci and short rods with cocci attached to tooth substrate

Calcified extraradicular biofilms are reported

Calcified films lead to delayed periapical healing

Periapical biofilms

Isolated biofilms in the periapical area of endodontically involved teeth

Eg- actiomycosis P. propionicum

The aggregation of Actinomyces cells is influenced by pH, ionic strength, and cell concentration which facilitates biofilm formation

Biomaterial centered biofilm

Bacteria adheres to artificial biomaterial surface and forms biofilms

Usually reveals opportunistic invasion by nosocomial organisms

Eg- coagulase negative staphylococcus, s. aureus, enterococci, streptococci, p.aeruginosa

A-Microbial film on guttapercha

B-E. feacalis on film serum plays a significant

role in biofilm formation

Biomaterial centered infection

Bacterial adherence to a biomaterial surface is also described in three phases: 

Phase 1: Transport of bacteria to biomaterial surface, Phase 2: Initial non-specific adhesion phase, and Phase 3: Specific adhesion phase. 

E. faecalis, Str. sanguinis, Streptococcusintermedius, Streptococcus pyogenes, Staphylococcus aureus form biofilm on GP points.

F. nucleatum, Propionibacterium acnes, Po. gingivalis, and Pr. intermedia do not form biofilm on Gutta-Percha(GP) points.

Eradication Of Biofilms

SODIUM HYPOCHLORITE Effective against biofilms containing p.intermedia,

peptostreptococcus micros, s.intermedius and fusobacterium

Disrupts oxidative phosphorylation and inhibits DNA synthesis of bacteria

The antibacterial effectiveness and tissue dissolution capacity of aqueous hypochlorite is a function of its concentration, and so is its toxicity

Fresh hypochlorite consistently reaches the canal system, and concentration of the solution may thus not play a decisive role

 One of the methods to improve the efficacy of sodium hypochlorite was to use heated solution. This improves their immediate tissue-dissolution capacity. 

Ultrasonic activation of sodium hypochlorite has also been advocated, as this would “accelerate chemical reactions, create cavitational effects, and achieve a superior cleansing action”

CHLORHEXIDINE DIGLUCONATE Effective against gram positive and gram negative

bacteria Denatures bacterial cell wall causes leakage of

intracellular organisms Substantivity effect Eg- e. fecalis

IODINE Bactericidal, fungicidal, virucidal and sporicidal Attacks proteins , nucleotides and fatty acids resulting

in cell death

Despite its usefulness as a final irrigant, chlorhexidine cannot be advocated as the main irrigant in standard endodontic cases, because (a) chlorhexidine is unable to dissolve necrotic tissue remnants, and (b) chlorhexidine is less effective on Gram-negative than on Gram-positive bacteria

Eradication Of Biofilms

EDTA Extracts bacterial proteins by combining with cell

envelope proteins and results in bacterial cell death Inhibits growth of bacteria and ultimately destroys them

by starvation EDTA chelates with metallic ions. Chelators may detach

biofilms adhering to root canal walls. An alternating irrigating regimen of NaOCl and EDTA may be more efficient in reducing bacterial loads in root canal systems than NaOCl alone 

TETRACLEAN More effective than MTAD against E. faecalis Contains cetrimide for antimicrobial properties

MTAD Tetracycline- Bacteriostatic broad spectrum

antibiotic Low ph, calcium chelator Substantivity Promotes healing Citric acid- removes smear layer Detergent- decreases surface tension Kills most E. faecalis strains High binding of doxycycline- prolongs antibacterial

effect

CALCIUM HYDROXIDE Ineffective in killing E. feacalis on its own Effective when combined with 2% chx Combination completely eliminates E. faecalis

ULTRASONIC ACTIVATED IRRIGATION Improves root canal cleaning and shaping- isthmus and

deltas cleaning

OZONE Ozone in 0.1-0.3 ppm is able to kill bacteria after 15- 30

mins of contact time

OZONEULTRASONIC IRRIGATION

LASERS Induce thermal effect producing an alteration in the bacterial

cell wall- change in the osmotic gradients and cell death ER- YAG irradiation reduces the number of viable cells Eg- A. naeslundi, E feacalis, P. acnes, F. nucleatum

PHOTOACTIVATED DISINFECTION Combination of photosensitizer solution and low power laser

light Photosensitizer selectively accumulated in the target cell is

activated by a visible light of appropriate wave length

ENDOACTIVATOR SYSTEM- debrides deep lateral anatomy , removes smear layer and dislodges simulated biofilm

Biofilm Detection

The forces of interaction among bacterial cells and between bacterial cells and substrates has been studied by atomic force microscopy –AFM

Micromanipulators have been used to sample individual cells or biofilm compartments.

Laser-based optical tweezers are noninvasive and non-contact tools that can probe the interaction between microscopic objects such as bacteria and collagen.

Fourier transform infrared (FTIR) spectroscopy is used to characterize the chemical composition of mature biofilm structures qualitatively and quantitatively.

Solid-state nuclear magnetic resonance (NMR) is a powerful analytical tool to study the constituents of bacterial biofilm, as well as to obtain metabolic information in planktonic cells, adherent bacterial cells, and in situ biofilm bacteria

Recent advances in micromanipulator-assisted analysis, green fluorescent protein (GFP) tagging, confocal laser scanning microscopy (CLSM), flow cytometry, and fluorescence in situ hybridization (FISH) have made biofilm characterization very comprehensive.