| Abstract|| |
The knowledge of biofilm and its eradication from the root canal system are of utmost importance in the clinical practice of an endodontist. Various treatment strategies and protocols have been demonstrated and discussed by numerous clinicians and researchers, on these models, that play an important role in the treatment outcome. Once a biofilm model is developed by considering various factors, several methods can be used to assess the biofilms formed on these models. This review discusses the importance of biofilm models in endodontics, types of biofilm models and factors associated with developing and the methods to evaluate these models.
Keywords: Biofilm models; Enterococcus faecalis; root canal; scanning electron microscopy
|How to cite this article:|
Garg A, Mala K, Kamath PM. Biofilm models in endodontics-A narrative review. J Conserv Dent 2021;24:2-9
| Introduction|| |
Biofilms in endodontics are cultured in order to gain insights about the microbial relations occurring in root canal systems or among microorganisms and host immune cells. At present, these are developed in order to evaluate the efficiency of various irrigating materials, irrigating procedures, and intracanal medicaments used in the field of endodontics. Within a biofilm structure, there are numerous antibacterial resistance mechanisms that act simultaneously, and understanding a few of these resistance mechanisms is vital in the development of biofilm model systems for numerous applications in the field of endodontics.
| Biofilm Models|| |
As clinical trials are considered to be laborious, expensive, and ethically unsafe, it is imperative to initially study existing or new root canal disinfection methods using the laboratory or in vitro models. These models mimic natural biofilms for different experimental purposes.
Factors affecting biofilm models [Flow Chart 1].
Microbial composition of the biofilm
Number of species in the biofilm
Monospecies: Simplicity, standardization, and control are few of the benefits of forming a monospecies biofilm. They are easy to develop, have better reproducibility, and allow high experimental productivity. However, the biological systems are multispecies in nature, and in the infected root canal systems, polymicrobial infection along with complex and widespread microbial communications and metabolic cooperation are typically found.,
Multispecies: These types of polymicrobial species offer more complexity and better likeness to the clinical reality. They show a superior metabolic capability, are stress tolerant, more resilient, and possess a greater challenge toward biofilm eradication. But to simulate models with such species, multiple types of media are required with more handling steps. The procedure becomes more time-consuming and costly and its culture-based evaluation becomes more complex.
Selection of species
Usually, the most commonly found microorganisms in the root canal system are selected. Either Gram-positive or Gram-negative microbial species can be chosen while working on a single species biofilm model. As the root canal space is mostly composed of anaerobic species, a combination of facultative and strict anaerobic bacteria is preferable over the aerobic bacteria. Functioning with strictly anaerobic microbes, however, requires precise experimental model, along with the usage of reducing agent, anaerobic incubation, anaerobic chambers, and maintenance of this anaerobic environment throughout the experiment. Such requirements lead to a complicated procedure when compared to working with aerobic microorganisms.
Enterococcus faecalis is the most commonly used experimental microorganism in the endodontic biofilm model systems.
If a number of recognized organisms are collected together to grow a multispecies biofilm, it is known as defined engineered biofilm, whereas, biofilms consisting of microorganisms which are unknown or have been collected directly from the natural environment, it is known as an undefined natural biofilm.,
The inoculum defines the preliminary inhabitants of the biofilm. The number of cells present in the inoculum must be sufficient enough to allow these cells to attach to the substrate and initiate the growth of biofilms. The most commonly used concentration of microorganisms in biofilms is 108 CFU/mL (ranging from 104 to 1010 CFU/mL). The presence of air bubbles in the canal should be avoided during inoculation. When the tooth sample is completely exposed to the inoculum, the growth of biofilm can also be appreciated on the external surfaces of the tooth.
Five techniques of inoculating the root canal system are:
- Whole tooth or root sample can be positioned in a recipient comprising of the inoculated medium
- Inoculating medium can be introduced in the root canal
- Flow cell can be used, where the medium is continuously driven through the canal
- By the addition of a microbial culture to the medium
- Inoculation occurring naturally with the presence of inhabitant oral flora.
Substrate of the biofilm
Dentine is the most obvious choice to be used as a substrate material for the growth of biofilm, as it signifies the biofilm's natural habitat. Dentine acts as a very particular substrate material which is extremely difficult to mimic or copy due to its very precise composition. The bacteria adhere to the proteinaceous part of the dentine and very rarely to the mineral component of the dentine matrix.
Nonbiological materials can also be used as a substrate for the biofilm. The development of biofilms on these materials varies completely from that of dentine as the initial bacterial interaction with these substrate materials is different and complex.
- Resin (polymethyl methacrylate) root canal model
- Methacrylate-based root canal model
- Polyethylene glycol-modified polydimethylsiloxane.
The use of bovine teeth to perform laboratory studies in endodontics has also gained immense popularity over the past few years. This is attributed to the fact that human extracted teeth are increasingly difficult to find. Bovine teeth are freely available and are similar to human teeth in terms of the morphology, chemical composition, physical properties, and the tubule diameter.
Root canal geometry
Single-rooted teeth are preferred over multirooted teeth. The development of biofilm species in unprepared canal space best denotes the clinical condition. However, the unprepared root canal systems can be a little tedious to inoculate, especially the narrower canals, as the inoculating media has to be introduced completely within the root canal space. As a result, shaping and widening of root canals is required before biofilm formation. It is also preferable to eliminate the smear layer so as to expose the dentinal surface before inoculating the medium into the canal space.
While working on simulated root canals comprising of two halves, an adequate seal should be formed between the two halves. It is also advisable to have a tooth model with a closed apex since the eradication of the intracanal biofilm requires thorough irrigation.
The chemical composition of the substrate material plays an essential role in the initial adherence of microorganisms and the biofilm structure. The pretreatment of the substrate should be carried out before inoculating the canal space so as to promote the initial adhesion and biofilm growth.
- Collagen type I – Identified by Streptococcus species and functions as an adhesion substrate
- Bovine serum albumin – Improves the adherence of microorganisms to the canal walls of both human and artificial teeth
- Mucin – provides additional protein to promote biofilm formation, e.g., Streptococcus mutans.
Before growing a biofilm, the model, regardless of the substrate material, should be devoid of any microorganisms, therefore, necessitating the need for sterilization. Autoclave/high-pressure steam, ethylene oxide, gamma irradiation, and gaseous hydrogen peroxide are some of the methods used for sterilization.
According to the guidelines given by the CDC, the extracted teeth being used for research purposes should be autoclaved at 121°C for 40 min. Even though substrate sterilization is essential for the growth of biofilms, some sterilization methods adversely affect the structure and integrity of the substrate material. Various studies have concluded that sterilization by autoclaving following the CDC protocols has resulted in the reduction of dentine microhardness. Thus, gamma irradiation is preferred.
Biofilms can be cultured for ≤1 week or for several weeks. Contrasting differences can be seen in the biofilms in terms of its thickness/biomass, its cell number, and its antibacterial resistance when immature biofilm growth is compared to the mature or “old” biofilms. The maturation phase of the biofilms is an essential factor in biofilm-based research, however it is challenging to describe generalizable guidelines concerning the optimal/relevant incubation time of biofilms. A period of 2–3 weeks is considered as an optimal time for biofilm maturation.
A medium that correctly enhances and supports the biofilm growth of the inoculated species should be selected. The media chosen should be very nutritive in composition, i.e., it should promote the growth of all inoculated bacterial species.,
- Brain–heart infusion (BHI) is the most commonly used medium for biofilm development
- Tryptic soy broth (TSB), A. C. broth, Todd Hewitt broth, and artificial saliva are also used as growth media
- Saline solution, devoid of any nutrients, has also been reported but it does not resemble the clinical conditions.
Throughout the incubation period, the growth media should be continuously refreshed so as to provide the biofilm with additional nutrients and to eliminate waste products and dead cells. This constant replenishment allows a continuous growth of biofilms under set experimental conditions during the incubation period. However, if the medium is not refreshed periodically, the concentration of metabolic waste and planktonic cells increases due to the scarcity of fresh nutrients. This causes stress on the growing species leading to a decline in the metabolic activity of the biofilm cells.,
| Confirmation of a Biofilm|| |
After taking into consideration all the above discussed factors affecting the biofilm growth, it is essential that after the incubation period, the resultant biofilm is confirmed and characterized. Various properties of biofilm such as thickness, its canal coverage, and composition should be evaluated while confirming the presence of biofilms. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and microscopes are the visual techniques used to confirm the presence of biofilms.
| Types of Models|| |
Biofilm models can be classified into four types (in vitro, ex vivo, nonmammalian in vivo models, and mammalian in vivo biofilm models) which are used to cultivate various microorganisms; however, in endodontics, mostly in vitro biofilm models are developed and used.
In vitro models
Basic in vitro models have been influential in providing solutions to the questions regarding the formation, structure, and physiology of biofilms. As stated earlier, both anaerobic and aerobic situations can be created in such models to promote in vitro biofilm formation.
The in vitro experimental biofilm model systems are commonly used to:
- Evaluate the attachment/adherence of definite microbial organisms to a biomaterial surface (gutta percha or resin)
- Examine the nature and pattern of initial biofilm development on a specific substrate (Dentine)
- Evaluate the interaction among numerous biofilm species and host immune cells
- Investigate the efficiency of antibacterial agents and various antibacterial treatment strategies.
- Static biofilm models: It involves the rapid screening of biofilm species, biomass formation, and biofilm-forming ability, as well as the composition of extracellular matrix (ECM). Few of the most common and popular models are used, e.g., microtiter plates and colony-forming model
- Dynamic biofilm models: In these models, used culture comprising of various wastes such as metabolic by-products, dead or planktonic cells are continuously substituted by the fresh media. e.g., flow cell system and chemostat
- Microcosms: Microcosms are more refined models that closely resemble clinical conditions. These models include numerous microbial species and utilize substrates from the clinical environment, such as inclusion of saliva and hydroxyapatite to model biofilms or layering abiotic surfaces using human cells so as to resemble clinical situations. Ideally, both static and dynamic systems can be converted into microcosms.
Model 1: Flow cell system
It is considered as one of the most commonly used dynamic models. The flow cell system is made up of a flow cell, a reservoir for nutrients, a peristaltic valve, and a waste container. The flow cell has a rectangular glass coverslip enclosed with a rubber seal and a Delrin polyacetal resin extension. The flow channel consists of 8–10 spherical recesses. Every individual cell contains an inoculated coupon. Each flow cell is packed with fresh THB medium from the reservoir by decreasing the atmospheric pressure near the waste area of the flow cell by starting the peristaltic pump. As soon as the cell is filled with the medium, the peristaltic pump is switched off and the overnight inoculum is introduced into the bioreactor cell. The laminar flow is maintained at 20 ml/h to flush the inoculum from the flow cell, thus leading to biofilm growth.
Model 2: Cellulose nitrate membrane
An alternative technique of biofilm formation involves the growth of species on cellulose nitrate membranes with dimensions of 0.2 μm pore size and 13 mm in diameter. In this method, the nitrate membrane is positioned in 5% sheep blood (defibrinated) with BHI broth agar plates to culture both aerobic and facultatively anaerobic microorganisms. In order to cultivate strict anaerobes, the membrane can also be placed in 5% defibrinated sheep blood with fastidious anaerobe agar plates. These membranes are then inoculated with the test microorganisms (20 μl of each microbial suspension). Following inoculation, the agar plates are incubated at 37°C under suitable gaseous conditions. The aerobic and the facultative anaerobic microorganisms are incubated in a chamber comprising of CO2, whereas the strict anaerobes are placed in a chamber enclosing an anaerobic atmosphere of 10% hydrogen, 10% carbon monoxide, and 80%nitrogen gases.
Model 3: Clegg et al.
Dentin sections are prepared after which the smear layer is removed. Dentin sections are sterilized and then soaked for 24 h in a dish of the patient's filter-sterilized saliva which leads to the formation of a pellicle layer. Specimens are then positioned in wells containing tissue culture plates in which TSB is added. Microbial species from the patient samples are suspended in Amies transport solution using ultrasonics for 15 s after which the specimens are incubated in anaerobic conditions for a period of 7 days to allow biofilm development.
Model 4: Turbidity of the culture medium
In this model, the bacterial species strain is placed in BHI broth (7 ml– 8 ml) and incubated at a temperature of 37°C for 24 h. The biological markers are cultured on the surface of BHI agar after which the bacterial cell samples are resuspended in a saline medium to achieve a final concentration of about 3 × 108 cells/ml. The experimental model (substrate) is sterilized in 5% sodium hypochlorite (NaOCl) for 20–30 min. About 5 ml of sterile BHI broth and 5 ml of inoculum consisting of E. faecalis are inoculated. The root canals are dried using paper point strips and refilled with distilled water. Each sample is collected by using three paper points individually transported and immersed in 7 mL of Letheen Broth (LB; Difco Laboratories, Detroit, MI, USA), followed by incubation at 37°C for 48h. The microbial formation is evaluated by determining the turbidity of the culture medium.
Model 5: Microtiter plate-based systems
These static systems are constantly used as biofilm model systems. It performs different tests simultaneously which is ideal in screening methods of disinfection and removal of biofilms. Various stains such as crystal violet, tetrazolium salts like resazurin, XTT, or dimethyl blue, and nucleic acid stain like SYTO9 can be used in these systems. This system allows the assessment of more than one antibacterial organism within the same test. Furthermore, this system can be upgraded and used along with flow devices.
Model 6: Modified Robbins device
In the modified Robbins device, there is constant development of biofilm exposed to fluid flow. Hydroxyapatite or silicone discs are used as substrates for cultivating biofilms with or without the addition of agents supporting or preventing the bacterial growth. This system allows for the assessment of more than one antibiofilm species within the same experimental method.
Model 7: Microfluidic device
Microfluidic device has gradually gained popularity for growing biofilms, as this model closely simulates the clinical scenarios. Analysis of single-cell resolution of the biofilm species under firmly controlled and strict conditions can be performed. This model also allows the evaluation of chemical assays using very less amount of liquid medium on a small chip.
Model 8: Chemostat
Chemostats are dynamic model systems that are used to grow biofilms on the substrate material which is completely immersed within the chemostat cell. The most important characteristic of this model is that the bacterial biofilms can be formed under constant environmental conditions (temperature, pH) at a steady rate.
Biofilm assays are used to characterize factors such as:
- Number and type of microorganism
- Vitality (dead/living cells)
- Thickness (monolayered or multilayered)
- Structure (homogeneous, irregular, dense, and porous)
- Surface topography (peaks and valleys).
Different techniques of evaluating biofilms [Figure 1]
|Figure 1: Various techniques for biofilm evaluation: (a) Culture technique (b) Calorimetric technique (c) Scanning electron Microscopy, (d) CLSM E. Microscopic technique F. Molecular technique(PCR)|
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- Quantification techniques:
- Culture techniques
- Colorimetric techniques
- Molecular methods.
- Microscopic techniques
- Physical methods
- Biochemical methods.
Microbial culture techniques
The biofilms grown on the experimental substrate are enumerated by counting the CFU of the bacterial species attached on the surface directly. The CFU analysis provides essential information on the number of live bacteria adhered on the experimental material or multiplying within the biofilm ultrastructure. However, this technique can only detect bacteria capable of undergoing cell division to form biofilm colonies at a constant rate and whose development necessities are maintained by the various culture media used. In some cases, CFU is also measured from the supernatant collected after removing the biofilms from the experimental substrate by performing the centrifugation/sonication procedures.
Colorimetric assay is classified as a semi-quantitative method which rapidly quantifies the biofilm growth. This method is based on the dye (crystal violet) application of the bacterial cells in a biofilm. In this technique, alcohol or sodium dodecyl sulfate (surfactant) is used to disrupt the biofilms following the dye application, and this expelled dye is measured in terms of intensity with a spectrophotometer. This method is popular among those bacterial strains which produce thick biofilms; however, they are unable to differentiate between strains of weak biofilm producers and nonbiofilm producers. The drawback of this technique is that it does not reflect the true extracellular matrix present in the biofilm structure but only quantifies the number of bacteria in the biofilm.
The most essential method to assess biofilm formation on histological or in vitro samples is light microscopy. To evaluate the structure of biofilms, its type, distribution, viability of bacterial species, and their adherence to substrates, various microscopic techniques have been described. In this method, a fluorescent (e.g., propidium iodide) or nonfluorescent dye (e.g., safranin) is used to stain the bacterial biofilm and the stained areas can be seen as a bluish-green to blue color. The quantification of bacteria on the substrate is carried out using high-resolution light microscopes. Fluorescent microscopes without the use of fluorescent probes can also be used to visualize bacterial cells. This uses plasmid-encoded green fluorescent protein and is a rapid, simple to use, and inexpensive method.,
Various microscopic methods to assess biofilms are as follows:
Scanning electron microscopy
For long, SEM has proven to an efficient mainstay for the analysis of biofilm formation. This technique gives a more detailed information on the structure and the environment of the biofilms. This is because, SEM offers a higher magnification and resolution in comparison to any other technique. In this method, a beam of electrons is focused on the sample surface leading to image production. These electrons react with the atoms present in the sample, producing images containing information regarding the composition and topography of the biofilm. The major drawback of this method is the need for extensive preparation of the microbial sample which includes steps such as fixation, dehydration, drying, and sputtering. Hence, the SEM technique provides a detailed three-dimensional examination of the biofilm, but the original biofilm structure comprising of a large volume of EPS is not appreciated.
Environmental scanning electron microscopy
A potent alternative to the conventional SEM method is the environmental SEM (ESEM) which is a relatively recent technique producing images of the sample species in their natural hydrated form at a higher resolution. This original form of the sample is achieved by preserving the EPS matrix of the biofilms. However, the presence of a low signal-to-noise ratio and the translucent appearance of the EPS leads to an image of lesser resolution. Thus, a combination of ESEM and SEM can, therefore, provide essential information on numerous biofilm species, cellular content, and the EPS matrix.
Epifluorescent microscopy is utilized to analyze the viability of cells, biofilm structural organization, microcolony formation, pH of biofilm species, and the chemical composition of the biofilm structure. Biofilms formed on substrate materials are generally applied with fluorescent dye staining and then observed under an epifluorescent microscope. Dual species biofilms are applied with two separate fluorescent probes for both organisms and then focused and viewed with the help of an epifluorescent microscope under two extreme wavelengths. Two separate images and the background biofilms are produced which are then combined to form a fresh image that shows both the microorganisms together.
Confocal laser scanning microscopy
To explore biofilm structures of 50–200 mm thickness, CLSM is an essential method of analysis as it overcomes certain drawbacks of the conventional microscopic methods. The analysis of composition, structure, pH gradients, and microhabitats of a biofilm can be done with the help of specific color probes., For analysis of biofilms, in vitro, a fluorescent dye (LIVE/DEAD BAC light) is routinely used. The LIVE/DEAD microbial viability kit (Molecular Probes, Eugene) consists of two different dyes (propidium iodide and SYTO 9) in two different vials used in a ratio of 1:1 to stain the biofilm. On viewing it under CLSM, the dormant cells appear red in color, whereas the live/active cells appear green.
Fluorescence in situ hybridization
The fluorescence in situ hybridization (FISH) technique targets 16S rRNA sequences in the bacteria by utilizing probes. It analyses the three-dimensional arrangement of Gram-negative and Gram-positive bacteria. In endodontics, FISH finds its importance in the visualization and identification of bacterial species from the periradicular lesions of asymptomatic root-filled teeth. To study the FISH stained cells in biofilms, CLSM is the preferred method because of its noninvasive visualization of cells.
Conventional physical features of biofilm such as thickness, weight (dry or wet), area, and density measurement are used to quantify biofilm formation.
- Thickness measurement under light microscopy: Experimental biofilm is positioned and focused adequately on the microscope. The microscopic lens is then focused on the surface of substrate material, commonly in areas with not any biofilm. The difference in the fine adjustment settings is used to determine the thickness and the density
- Manual-gauge needle technique with an electronic probe to calculate biofilm thickness
- Cryosection: Determines the biofilm width or thickness and also reveals the layering of embedded microbial cells.
Biochemical methods: Thickness and extracellular matrix
This method is commonly used to determine the extracellular matrix and thickness of the biofilm species. Biofilm thickness indicates the quantity of microbial species present in a particular area. This measurement of microbial thickness is considered to be a quick method and involves measuring of both the dry and wet weights of the whole biofilm sample, determining the cellular composition, observation of the cellular activities or viable/live cells, etc. A technique that is used for the early recognition of active bacterial cells is based on metabolic activity. Adenosine triphosphate bioluminescence is a commonly used technique to determine the metabolic activity of the bacterial cells.
Molecular biological methods
Molecular biological assays provide an enormous amount of genetic information on biofilm species. The main aim of majority of these methods is to develop standard assays in order to evaluate factors affecting biofilm growth and its adherence.
- ELISA: It is a very popular and sensitive method used in the detection of antigens and antibodies in a biofilm sample. It is accomplished using either the direct or indirect assay. ELISA is also utilized as an alternative technique to measure biofilm biomass and also the production of proteins in biofilms,
- PCR: The PCR technique allows the exponential amplification of short DNA sequences. This method is based on thermal cycling and enzymatic replication of the DNA. PCR is mostly used as a qualitative tool so as to detect the presence or absence of a specific bacterial DNA.
| Other Advanced Methods|| |
- Atomic force microscopy
- Laser-based optical tweezers
- FTIR spectroscopy
- Nuclear magnetic resonance.
Summary of culturing and identifying E. faecalis in the root canal [Table 1].
|Table 1: Summary of culturing and identifying the most common microorganism associated with the root canal system, i.e., Enterococcus faecalis|
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[Table 2]: A basic outline to develop an in vitro biofilm model for research purposes in the field of endodontics.
|Table 2: A basic outline to develop an in vitro biofilm model for research purposes in the field of endodontics|
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| Conclusion|| |
Endodontic microorganisms reside inside the infected canal space as substrate-adherent biofilm species. The endodontic biofilm is a highly complex, organized unit and it is a big challenge to duplicate its features and physical characteristics in laboratory models, however, not impossible. In order to resemble the in vivo situation or clinical conditions, various in vitro biofilm model systems are being utilized in the field of endodontics for numerous bacterial studies. However, a convincing choice of the best study model which can be utilized for specific research purpose is yet to be achieved.
The authors suggest that future studies and experiments should focus on the complexity of the root canal biofilm and analyze models to re-evaluate eradication of the biofilm species from the root canal space in addition to investigating the effect of new antibacterial agents on such multifaceted biofilms.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Kundabala Mala
Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Mangalore, Affiliated to Manipal Academy of Higher Education, Manipal, Mangalore - 575 001, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]