Closing The Doors On Microleakage Biology Essay
Coronal leakage and apical leakage continues to be a topic of great interest, because of the advances in the field of endodontics, clinical failures still exist. Numerous studies have examined this phenomenon, identified many sources of possible contamination and emphasized the role of the clinician in preventing coronal leakage following root canal treatment. Pulpal and periradicular diseases develop when microorganisms and/or their by-products contaminate these tissues. Therefore, a major goal of both preventive and restorative dentistry is to prevent penetration of microorganisms into the coronal pulpal space and root canal system. The root canal system, once invaded, may harbour many species of microorganisms, their antigenic by-products and variable amounts of inflamed or necrotic tissues. This review article discusses the various methods used for assessing root canal sealing capacity.
KEY WORDS- Coronal leakage, Dyes, Dye dilution techniques, Filtration
Innovations in materials, equipments & techniques continue to sophisticate endodontic treatment procedures enhancing the incidence of predictable clinical success. However, inspite of these advances, clinical failures/shortcomings still persist.1 Concept of microleakage having an effect on the outcome of endodontic treatment has been known for more than 100 years2.
Microleakage is defined as the " Diffusion of the bacteria, oral fluids, ions & molecules into the tooth and the filling material interface" or " defined as the clinically undetectable passage of bacteria , fluids, molecules or ions between tooth and the restorative or filling material".3 Many studies emphasize that tooth filling materials are not fixed, inert and impenetrable borders but ‘Dynamic micro crevices which contain busy traffic of bacteria, ions and molecules’.3
This leakage may be clinically undetectable, but is a major factor influencing the long term success of endodontic therapy as it causes many severe biological effects leading to recurrence of the pathology and failure of the root canal treatment. 3
Endodontic therapy endeavours to remove microorganisms, sanitize and seal the root canal space and entombs the bacteria.3Ingress of the bacteria, biofilms and their by-products leaking from the root canal through the apical foramen, accessory canals, furcation canals etc or due to improper coronal seal,are the major pathways of microleakage. It is thus essential to remove bacteria from the root canal system, at the same time avoiding the entry of new bacteria.3
Hence, microleakage is arguably the single most risk factor for failure of endodontic therapy. Thus, closing the doors on microleakage opens the doors to more predictable and successful endodontic outcomes of endodontic therapy.3
LEAKAGE AT MICRON LEVEL (BACTERIAL LEAKAGE)
It can be inferred from the definition of microleakage that, marginal gaps around a restoration permit bacteria to pass into the tooth /restoration interface. This is considered to be bacterial microleakage, which is seen at micron level. Numerous studies have shown that once cariogenic bacteria gain access into the tooth/restorative interface they are able to successfully proliferate along this area with the potential to cause an adverse response from the pulp and recurrent caries.3,4
However, it is still questionable about the marginal gap size around the restorations and occurrence of recurrent caries. It is also reported that recurrent caries rate significantly increases with the extent of wide marginal gap.5 The origin of bacteria which are found at the tooth/material interface is still uncertain and their relation to the development of recurrent infection remains to be established. It is believed that bacteria trapped within the smear layer are able to multiply and cause recontamination of the root canal system through microleakage5.
LEAKAGE AT SUBMICRON LEVEL (NANOLEAKAGE)
It can also be interpreted that endodontic filling materials or restorations with marginal gaps that permit ions and molecules to gain access can have microleakage at nanolevel.14It has been reported that the passage of fluid through dentin is affected by dentin permeability which is markedly influenced by number of factors including volume of dentinal tubules, dentin smear layer, dentin calcification and topical application.
Recently, concept of nanoleakage has focused particularly at the hybrid layer.4,14
CAUSES OF MICROLEAKAGE
Causes can be broadly divided into- Coronal leakage as a cause of failure and Apical leakage as a cause of failure.
Apical leakage as a cause of failure was reported in 1956 by Strindberg.
1) Apical percolation or microleakage due to an inadequate apical seal.
2) Bacteria contaminating the apical portion of infected root canal.
3) Inadequately obturated apical portion of root canal.
4) Breakage of the apical seal during the post space preparation.
5) Leakage of the saliva or fluids between the sealer and the canal walls, particularly if the smear layer is present leading to microleakage.
6) Presence of voids apically between the root canal filling and wall of the canal.
7) Infection and leakage occurring due to improperly cleaned lateral and accessory canals in apical portion.
8) Long term biochemical reaction within the material itself and between the material and surrounding environment.5,6
1) Improper placement of the temporary restoration in between the appointments.
2) Sealer dissolution by saliva.
3) Percolation of saliva into the interface between sealer and root canal walls.
4) Leakage of fluid between the sealer and gutta-percha.
5) Voids or minor flaws in the obturation which are not often detected radiographically, may be responsible for the rapid re-contamination of the root canal system.
6) Delay in the post endodontic restoration following the root canal treatment.
7) Fracture of the coronal restoration or tooth.
8) Recurrent decay or secondary caries present at the margins of the restoration.5,6
METHODS TO DETECT CORONAL AND APICAL LEAKAGE
Methodologies in-vitro are used to estimate sealing quality, generally by measuring microleakage that allows the tracer agent to penetrate the filled canal. An understanding of the leakage patterns of restorative materials can lead to an increased awareness of the mechanism and etiology of microleakage, resulting in the establishment of microleakage pattern. Subsequently, this will have relevance for material selection in dental practice. 7 Various methods used for detection of microleakage are Dye penetration, Fluid filtration, Dye extraction or dissolution method, Bacteria and Toxin infiltration method, Air pressure method, Electrochemical method, Neuron activation method, Radio-isotope method, Metal solution tracers, and Reverse diffusion method. In addition, other three-dimensional method SEM (scanning electron microscopy), TEM (transmission electron microscope) and MCT (micro-computed tomography) are also used.7
The use of organic dyes as tracers is one of the oldest and most common method of detecting leakage in vitro. The methodology uses tooth immersion in various types of dyes, reported for the first time by Grossman in 1939 and is perhaps the most widely used method as it is very easy to perform.18
The phenomena of capillarity is of utmost importance in this passive method used to assess apical and coronal leakage , as the tooth is submerged in the dye that penetrates through any space present between the canal walls and the filling material or tooth restoration interface. Dyes used for this study are Methylene Blue mol wt- 373.9 g mol -1 ,Indin Ink, methyl violet, Hematoxylin, - 303.28 g mol -1 ,Eosin -691.85 g mol -1 ,Basic fuschin, Fluouresence and Rhodanine blue, Procaine brilliant green.8,11,18
In general, this method for detecting microleakage in vitro involves placing a endodontic filling material or coronal restoration in an extracted tooth and immersing it in an dye solution after coating the unfilled parts of the tooth with a waterproof varnish. After an interval of time the specimen is removed, washed and sectioned before visual examination to establish the extent of penetration of dye around the filling material.8,11
There have been wide variations in choice of dye used, either as solutions or particle suspensions of different particle size. The concentration of dye used ranges between 0.5%- 10% while the time of immersion ranges between four hours to 72 hours or more. It was found that different concentrations of dye can vary in penetration time between five minutes to over one hour.8,11
There are various factors that influence dye penetration such as sectioning method, clearing technique, particle molecule size, pH of the dye and the chemical reactivity of the dye (which affects the degree of penetration), presence and absence of smear layer, thermocycling done during experiment, gap between the root filling material and canal wall which may contain air/liquid and entrapment of air in the gap prevent the dye penetration .8, 11
The clearing technique recommended by Okumura in 1927, in which the teeth become transparent after a process of demineralization, dehydration and immersion in methyl saliylilate and provides a three dimensional view of the internal anatomy of the root canals without the loss of dental substance, hence making it easier to view the leakage area or pattern. It is simple, fast and performed with substances low in toxins and does not require complex equipment. This technique makes it easier to observe the lateral and accessory canal and clearly reflects the relation between the sealing material and the apical foramen.20
With regard to dyes, particle molecule size, pH and the chemical reactivity are expected to affect the degree of penetration. Methylene blue-most commonly used dye as it is inexpensive, easy to manipulate, has a high degree of staining and a molecular weight even lower than that of the bacterial toxins. Fluorecent dyes found to be particularly useful as tracers because they are particularly detectable in dilute concentrations, inexpensive and being non-toxic can be used safely for clinical as well as laboratory investigations .Basic fuschin have been shown to bind preferentially with carious dentin. India ink particles with diameter smaller than or equal to 3µm are also widely used as it is unlikely that bacterial invasion would occur in spaces inside the canal where this dye is unable to penetrate.7,8,11
Dentin permeability is an another important factor to be considered as the diameter of the dentinal tubules and the number of tubules per unit surface increase as the tubules converge towards the pulp. It has been suggested that the dentin involvement might be used as the relative indicator of marginal leakage.7,8
The fluid filtration method was developed by Pashely’s group in 1987 in which the sealing capacity is measured by the means of air bubble movement inside a capillary tube. Modified by Wu et al in (1993)10 or use in root canals.It consisted of a filled canal that has its coronal portion connected to the tube filled with water under atmospheric pressure and its apex to 20µl glass capillary tube 170mm long and uniform caliber filled with water. Finally a pressure of 0.1 atmospheric is applied through the coronal part, which forces the water through empty spaces along the root canal (Wu, Wesselink P.F 1994).10, 16 The results are generally expressed in µl/min.
Nitrogen cylinder Pressurized buffer reservoir
Advantages in comparison with dye penetration method- Samples are not destroyed, results are recorded automatically, thus providing quantative measurements, avoids operators errors, results are precise, as small volumes can be recorded, more sensitive than dye penetration in detecting empty spaces along the canal, system sensitivity can be adjusted by altering the pressure used or the diameter of micropipette. 8,19
Orucoglu et al (2005)7 developed a new computerized fluid filtration meter based on light refraction at the starting and ending positions of air bubbles movements inside micropipette. It has some advantages over the conventional ones with the computer control and digital air pressure arrangement. Addtionally, the bubbles can be observed by laser diodes which are computer controlled rather than visual findings.7
DYE EXTRACTION METHOD
In this method, the teeth are dissolved in acids that release all the dye from the interface and optical density of the solution is measured by spectrophotometer.
It is fast and carried out with an equipment. This method takes into consideration the porosity of the interface between the filling material and the root. Technique is based on quantative measurements of liquid passing through these interface. The dye extraction method presents an advantage over the fluid filtration method because the values tend to diminish over a time, as the water penetrates all the irregularities until a plateau is reached.17
BACTERIAL & TOXIN INFILTRATION METHOD
Earliest first such study was done by Fraser in 19298 when he tested cements and restorative materials to determine whether they would allow bacteria to pass through or around them. Later they investigated the marginal seal of acrylic restorations placed in broth cultures. The filling materials were subsequently removed and the dentinal shavings from the base of the cavity were cultured. These provided purely qualitative results depending mainly upon the presence and absence of bacteria in part of the dentin examined 8.
Mortensen, Boucher and Ryge in 1965 developed a method for isolating the filled crown of a tooth from its root using a plastic tube sealed with epoxy resin. Broth inoculated with bacterial culture was placed over the crown , while the sterile broath was placed in contact with the root of the tooth. Microleakage was diagnosed if the sterile broth turned cloudy 8. It has been claimed that the bacterial penetration studies are more clinically related to leakage, as they are associated with caries process and recurrent decay.
AIR PRESSURE METHOD
Introduced by Harper in 1912.7 This technique is based on introduced of compressed air into the pulp space of restored tooth while investigating the delivery of air bubbles at the restoration margin which are placed in fluid. Harper constructed class II amalgam restoration in a steel dye, delivered air under pressure to the floor of the cavity and examined restoration under water. Microscopic examination of the release of air bubbles from the margins of the submerged restoration provided a subjective view of the margin seal. The advantages are that it is a non destructive test and quantative analysis is possible by measuring the loss of pressurized air. This method proved to be a valuable technique for comparing the sealing properties of different amalgams as well as cements. Main limiting factors of this method of the study is that it could only detect leakage pathways that were complete from the floor to the margin of the cavity. It is also difficult to detect leakage results as air flow may pass through restoration tooth cracks or because of marginal gap. Though the samples are not destroyed , but still can’t be used in future to determine the leakage as the interface is exposed to great pressure at a given point in time that some material lost 7,8, 10.
Developed by Jacobson and Von Fraunhofer in .18 In an attempt to develop a technique that can access restorative microleakage longitudinally, the electrochemical methodology was introduced using a "CONDUCTIMETRIC TECHNIQUE " in which cavity wall/ restoration interface was incorporated into a electrochemical unit.18, 21
Glass tube is 4mm of internal diameter set into lengths of 15mm, because the glass tube has a similar coefficient of thermal expansion to that of tooth substance. The floor of the "cavity" is formed by a nickel-plated brass electrode. After inserting the material, 4 mm of the glass tube is immersed in 1% solution of lactic acid. A lead from a brass electrode is connected to one terminal of a 5 V power source while the other terminal of the circuit is connected through a series of resistances to a reference electrode that is formed by the nickel-plated brass rod. The circuit is completed when the interspace between the test material and the glass is occupied by electrodes.11,18,21
Apical leakage test system for the electrochemical method.
Any change in the current passing through this electrochemical cell reflects changes in the dimensions of the interspace. Since the dimensions of the cavities are constant, the dimensional changes of the materials themselves are observed through measurements of the pontential change in the experimental circuit. This technique has been used in endodontic research and it has proved sensitive and the results could be quantified. It is destructive to the tooth structure and cannot be used in vivo conditions. Also it does not take into consideration the dielectric properties of the restorative material or that these may change with time as the setting reaction continues. Therefore, these methods lack clinical realism11.
NEURON ACTIVATION ANALYSIS
Neuron activation analysis has been used to study microleakage both in vitro and in vivo (Going, Meyers & Prussin 1968) Tooth/root restored are soaked in radioactive solution of Mn55, Vanadium, Indium104 Teeth are placed in nuclear reactor and exposed to pulsed neutron flux.7,8 Nonradioactive material within becomes radioactive emitting gamma rays & number of radioactive counts is proportional to the uptake of Mn/specimen. Emission is measured by Scintillation method.
Disadvantages are high cost, serial sections that were made to define the path and depth of tracer penetration, may create a radiation hazard. It was also noted that presence of manganese, either in the restorative material or in the tooth , caused variably of the results .Origin of leakage was not well defined. It failed to indentify whether leakage is at tooth/ restoration interface or due to the uptake of restoration 7,8.
Another common method for detecting leakage patterns has involved the use of radioactive isotopes. First used by Armstrong & Simon in 1951.9,10 The tracers that have been used are Ca45 , C14 ,I131, S35, Na22. 9,10
In general, Ca45 in the form of calcium chloride at a concentration of 0.1mCi/ml, has been the most popular isotope to be used , because it is a low-energy beta emitter & does not readily penetrate enamel found that Ca45 showed deep penetration in defects. Involves the use of extracted restored teeth. Roots and crowns of the teeth painted with varnish except for the surface immediately near the experimental restoration. This is to prevent leakage through the root canal, cracks in the enamel or exposed dentin, which can obliterate the true picture of the marginal adaption. Sealed teeth immersed in isotope solution for several hours. After removal, teeth are subjected to prolong rinsing. Longitudinal sections of teeth are made through the restoration. Cut surfaces applied to photographic film . Resulting autoradiographs indicate the presence and location of any radioactive isotope that has penetrated between restoration and cavity wall. Results are evaluated by subjective scoring . Disadvantages are that it is destructive to tooth, 2-D image, Ca45 has the affinity with tooth surface and restoration so there are more increased measurement errors & isotopes are able to pass through tooth structure resulting in misinterpretation of leakage 7,8
METAL SOLUTION TRACERS
Metal solutions have been commonly used as tracers to express the tooth/restoration gaps This technique involves the use of atleast two colourless chemicals to produce a coloured precipitate may not occur if only one chemical or the smaller of the two chemicals exist . Early chemical tracing of leakage was introduced by KORNFIELD (1953)8 where he described a technique for the assessment of leakage around acrylic restorations by incorporating lead glass in the acrylic ; so that when immersed in a solution of barium sulphide area on the marginal discolouration indicated leakage.8
Silver nitrate used for measuring leakage is an acceptable technique. It is however a severe test because the silver ions extremely small when compared to the size of typical bacterium and thus is more penetrative. Therefore, it may be assumed that any system that prevents the leakage of silver ions will also prevent the leakage of bacteria .Recently, a solution of 50% silver nitrate has been most frequently used in conjunction with photo-developing solution (hydroquinone) to produce a precipitate at the restoration/ tooth interface. This combination has been widely used as a dental leakage drying technique. The silver stain technique gave a discreate, high contrast marking of the restoration /dentin interface. 16
Advantages are as follows
1) More objective measurements .
2) Quantative measurements could be collected for which parametric statistical analysis is appropriate.
3) Special tests on silver indicated that finely divided silver may be coloured silver, gray, blue, yellow or brown and that the suspension of silver particles depend upon the size of deposition of particles.
Disadvantages are the problem of chemical tracer technique is that it involves the use of many chemicals and the microleakage result is dependent on the chemical reactions. Use of silver nitrate was criticized in its clinical relevance, because of its molecular size. On the other hand it was found that silver nitrate deposition was essentially affiliated with collagen fibrils. It seems that silver ions are highly active and therefore they are easily converted into silver metal, which can act as a stable drying agent. 7,8
REVERSE DIFFUSION METHOD
Developed in 1979 in the university of Florida.7,8 A known number of counts of radioactive material is deposited on prepared tooth surface Radioactive material is dried. The tooth is restored and immersed in an appropriate liquid. Microleakage (diffusion) is reversed i.e, radioisotope leaks from the tooth to medium which is termed as reverse diffusion
ADVANTAGES- 1)Non destructive test.
2)Amount of leakage at any point in time can be completed as percentage from original quantity deposited.
3)Samples from medium are taken at various intervals.
DISADVANTAGES- If the radioactive material becomes bound to cavity then it will not be able to leak. Such a lack of leakage rather will be due to inability of the radioactive material to leak and is not due to the sealing ability of restoration.7,8
ARTIFICIAL DENTAL CARIES
Artificial caries like lesions have been produced in vitro using either bacterial cultures or chemical systems – The Acidified Gel Technique.
In 1967 Ellis & Brow, using a bacterial technique to produce artificial secondary caries at the interface of amalgam restoration and the tooth, linked the development of carious lesion to microleakage.8 Microleakage has also been associated with spread of secondary caries .The first investigators to describe the production of caries-like lesions by the acid-gel technique were Muhlemann (1960), Von Bartheld (1961), Silverstone (1968).8 The acid-gel technique developed for the production of secondary carious lesion around the amalgam fillings has also been applied to the study of composites.
The lesions produced by this technique are studied in polarized light & two parts are described as Outer lesion –results from the primary attack of the enamel surface from the adjacent to the restoration. Cavity wall lesion-formed by microleakage of ions from the acidified gelatin around the restorations.8
Advantages of this technique-
1)Microleakage can be linked directly with one of its consequences, namely the development and spread of secondary caries.
2)Quantification of the results are possible where depth of the lesion penetration is chosen as measurable parameter.
3)Degree of mineralization may also be assessed quantitatively or semiquantatively. 4)Eliminates the external variables associated with the formation of natural caries. 5)Efficient in creating a carious lesion within a short period of time and the viscosity of gel stimulates a layer of plaque. 8
SEM (SCANNING ELECTRON MICROSCOPY)
Use of SEM provides a means of direct visual observation of the adaption of materials to the tooth structure because of its high magnification & depth of focus. Many workers have used SEM to measure gap formation that has occurred between restorative material & tooth structure Method of SEM can be improved by the use of replicas. This allows change in the size of marginal defects to be followed on a longitudinal basis & can be applied clinically. Replicas may be repeated many times at different intervals & this does not change the structure being evaluated. Also avoids specimen shrinkage & some of the other artifacts usually associated with preparation of biological tissue for SEM examination.8
1) It has been pointed out that the SEM technique can be criticized for its potential for introducing errors and artifacts related to drying, cracking, distortion and sectioning (Kidd 1976).8
2)Technique is limited to the evaluation of teeth outside the oral environment.
3)It is also difficult to quantify the SEM results.
4)Extremely expensive. 8
There are no biological absolutes; there are however varying degrees and definitions of success. Microleakage is an important cause of failure of treatment. It is essential that due regard be paid to the prevention of such leakage, both during and after root-canal therapy, by paying careful attention to the sealing to the tooth. Prevention of microleakage in endodontically treated teeth is most important for patients who rely on the combined expertise and quality care of dentist /endodontist colleagues.
Thus, Closing the door on microleakage opens the door to more predictable and successful endodontic outcomes
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