Methods of detecting microleakage/ orthodontic course by indian dental academy

CONTENTS
 INTRODUCTION
 DEFINITION OF MICROLEAKAGE
 DIFFERENT CAUSES FOR MICROLEAKAGE
 ROUTES OF MICROLEAKAGE
 METHODS OF DETECTING MICROLEAKAGE
 AIR PRESSURE METHOD
 PENETRATION STUDIES
a. Dye penetration
b. Chemical tracers
c. Radio isotopes
d. Neutron activated analysis
e. Bacterial toxins and bacterial products
f. Chemical diffusion technique
 FLUID CONDUCTION STUDIES
a. Fluid transport device
 ELECTRONIC METHOD
a. Electrochemical studies
b. Electronic monitoring of microleakage

 MICROSCOPIC EXAMINATION
a. Scanning electron microscope
b. Replication and SEM
c. Fluorescent microscopy
d. Co focal microscope
 MISCELLANEOUS
a. Artificial caries
b. Marginal percolation
c. Resin infiltration method
 RECENT ADVANCES
a. Nanoleakage
b. Electron micro probe
c. Ion beam milling probe
d. Constant depth form fermentor
 CONCLUSIONS
 REFERENCES

INTRODUCTION
The goal of operative dentistry is to restore the tooth to its form and
function. One of the requisite of ideal restorative material is to adapt itself
to cavity walls. Among the various restorative materials currently used and
inspite of tremendous improvement in means and technology, non of the
material could actually joined chemically with tooth surface. The gap left
between the cavity wall and restorative material place an important role in
prognosis of restorative treatment.
In the past, pulpal reactions to the dental procedure were thought to be
induced by the mechanical irritations like Heat, vibration, galvanization,
etc., or chemical irritation by the restorative materials and its component.
Research have demonstrated that probably bacterial leakage was a greater
threat to the pulp than the toxicity of the restorative material.
A great deal of dental research has lead to development of various
restorative materials and methods that provide us long term clinical success.
All these materials and methods have a single concept in mind that is to
create an interface – a bond which could prevent any type of leakage to
occur between the restoration and the tooth. This clinically undetectable
passage of fluids and microorganisms termed “microleakage” has its effect

not only on the longevity of the restoration but also on the vitally of tooth
itself. Various studies have shown that, it is not the toxicity of the material,
which causes pulpal damage but the colonization of bacteria or their toxins,
which cause the pulpal damage. In addition, it can case discoloration,
hypersensitivity, recurrent caries hastening of marginal break down, all of
which led to pulpal pathology and failure of the restoration. It is interesting
to know that there are different methods of detecting microleakage, but still
conventional methods are only followed. This article presented here is a
summary of various techniques used to study microleakage, which could
give a better insight of the tooth-restoration interface, act as a guide for
future investigation and enhance one with a knowledge of what has been
done and what could be done to detect and prevent microleakage and
provide a better restoration.
Microleakage :
Microleakage can be defined as the clinically undetectable passage of
bacteria and bacterial products, fluids , molecules or ions from the oral
environment along the various gaps present in the cavity restoration
interface. (Kidd 1976)

Microleakage is also depend as the passage of ions, bacteria or
bacterial toxins between the tooth and restorative interface.

The difference causes occurrence of Microleakage are
1) Poor adaptation of restorative materials
2) Its contraction during setting
3) Non adherence to tooth structure
4) Deformation under load
5) Temperature induced volume changes.
For microleakage to occur , the interface is composed of tooth, smear
layer, filling and the cementing media.
3 possible routes of microleakage.
1. Within or via the smear layer
2. Between smear layer and cavity varnish / cement
3. Between cavity varnish / cement and the restoration.

MICROLEAKAGE AND METHODS OF DETECTING IT.
Various methods to detect microleakage :
I. Air pressure method
II. Penetration studies
a. Dye penetration
b. Chemical tracers
c. Radio isotopes
d. Neutron activated analysis
e. Bacterial toxins and bacterial products
f. Chemical diffusion technique
III. Fluid conduction studies
a. Fluid transport device
IV. Electronic method
a. Electrochemical studies
b. Electronic monitoring of microleakage
V. Microscopic examination
a. Scanning electron microscope
b. Replication and SEM
c. Fluorescent microscopy
d. Confocal microscope

VI. Miscellaneous
a. Artificial caries
b. Marginal percolation
c. Resin infiltration method
VII. Recent advances
a. Nanoleakage
b. Electron micro probe
c. Ion beam milling probe
d. Constant depth form fermentor
1. AIR PRESSURE METHOD
This method was introduced by Harper 19121. A class II amalgam
restoration in a steel dye was constructed and air under pressure was
delivered to the floor of cavity. The restoration was then examined under
water. Microscopic examination of the release of air bubbles from the
margin of the restoration revealed microleakage.
2. PENETRATION STUDIES :
A] DYES :
They are organic tracers used in dentistry to detect microleakage.
They may be solutions or particles of suspension.

Dye leakage can be evaluated in any of the following ways.
a. Passive
The restored teeth are exposed to die under constant atmospheric
pressure, for required period of time.
b. Vaccum or negative
The restored teeth are exposed to the die under negative pressure.
c. Positive pressure
The teeth are placed in a container with a mix of freshly prepared
eposy resin and the dye. The container is then put in an autoclave and the
pressure is increased to required level.
d. Centrifuge.
In this method the restored teeth are placed in glass vias immersed in
dye and placed in the centrifuge for required amount of time.
B] RADIOACTIVE ISOTOPES :
The widespread use of isotopes was baaed partly on the inherent
ability of isotopes to penetrate more deeply than the dye of tracers that had
been used previously and partly on the fact that autoradiography technique
permitted the detection of minute amounts of tracers that otherwise could
not be commonly visualized2. The isotopes used are : Ca45 , c14, I31, S35,
Na22 , P32, Rb86 and C14 (radio active sucrose TC99. The roots and

crowns of the extracted teeth are painted with varnish except for the surface
immediately adjacent to experimental restoration. Then the sealed teeth are
immersed in the isotope solution for several hours. After removal from the
isotope solution the teeth are subjected to prolonged rinsing. The teeth are
then cut into longitudinal sections through the restoration. The cut surfaces
are applied to a photographic film. The resulting auto radiographic indicates
the presence and location of any radioactive isotopes that has penetrated
between the restoration and the cavity wall.
C] BACTERIAL STUDIES
Bacteria have also been used in the study of microleakage. This is more
realistic than dye and isotope diffusion method because the size of most dye
molecules and isotopes is infinitesimal in comparison with bacteria. The
filled teeth are placed in broth culture, the filling materials are removed and
dentinal shavings from the base of the cavity of cultured (Developed by
poss, William and Falcetti 1985). Another method is isolating the filled
crown of a tooth from its root using plastic tube sealed with epoxy resin. A
broth inoculated with a bacterial culture was placed in contact with the root
of the tooth. Microleakage is diagnosed if the sterile broth turned cloudy.
We can evaluated both apical and coronal leakage by this method3 .
Examples of bacteria used are enterococcus fecalis and streptococcus.

D] BACTERIAL TOXINS AND BACTERIAL PRODUCTS:
Mostly the soluble bacterial factors, which are released by organisms
penetrated more efficiently and rapidly than the bacterial cells.Various
materials like Lipopolysacchrides and cell wall materials such as dextran
have shown to provoke inflammatory relations to dental pulp.
E] NEUTRON ACTIVATION ANALYSIS:
This method was introduced by Going et al (1968). This method can
be used both invivo and invitro4,5.In this method the teeth are isolated with
a latex isolator i.e. the clinical crown was isolated and 2ml of manganese
(non – radioactive ) was injected into the flexible latex bag for a soaking
period of one hour. The test teeth were exposed to a pulsed neutron flux.
Irradiation at 1 megawatt for two minutes provided good counting of gamma
ray emission were measured with the scintillation detector and a germanium
crystal lined to a gamma ray spectrometer. A graph of counts versus channel
numbers is plotted so that data in form of numerical printout could be
converted into total counts. The calculated uptake expressed as micrograms
of manganese per tooth.
F] CHEMICAL TRACERS

Introduction by Korn field – 1953 . This method involves the use of
two colourless cdompounds to produce an opaque precipitate. The silver
nitrate method of measuring microleakage is an acceptable technique. A
50% silver nitrate solution is used to immerse the invitro specimen, it is then
reacted with a photographic developed such as benzene , diol
(Hydroquinone). Processing or Developing techniques vary widely with
developing times, which varied between 8 and 16 hours. Douglas (1988)
measured leakage along the tooth interface using single transverse section
cut at the center of each restoration and observed under a stereomicroscope
incorporating a calibrated scale.
G] CHEMICAL DIFFUSION TECHNIQUE.
Described by Crisp , this method requires preparation of a polyester
disc (Fig. 1a, 1b, 1c, ). The specimen is placed in the center of the circular
polythyne tube and set in a polyster mounting resin. The junction of the
enamel / resin interfaces is sealed by applying coats of varnish or nail polish.
The apparatus has a U shaped tube with two arms. The mounted specimen is
clamped between the 0 – rings in the right hand arm with the uncut tooth
surface lower most. 10 ml of deionized water is added to the right hand arm
and the other arm is filled with CaCl2

Solution until the levels of liquid are equal in both the arms. To
minimize evaporation, a cork lined with thin polyethylene sheeting is used to
seal each arm. At various intervals, 2 ml of liquid is withdrawned from the
right hand arm with a pipette. The level in the right hand arm is maintained
by addition of 2 ml of deionized water counting calcium ions in the
withdrawn liquid is liquid is carried out in an inductively coupled plasma
(ICP) instrument. Any calcium present is detected and this is indicative of a
leakage that has occurred.
3. FLUID CONDUCTION STUDIES
This method was developed by Pashley. The teeth are sectioned after
the placement of a restoration of restoration. The section after the placement
of a restoration. The section teeth are connected to a plastic tube. This
connection is closed tightly by twisting a piece of stainless steel wire. The
plastic tube is filled with deionized water. A standard glass capillary tube is
connected to the plastic tube at the outlet of the specimen. Using a syringe
the water is sucked back approximately 3 mm into the open end of the glass
capillary, reacting an air bubble in the capillary. The whole set up is then
placed in water bath at a contrast temperature. Using syringe, the air bubble
is adjusted to a suitable position within the capillary, a required head speed
pressure is applied from the inlet side o force the water through the void

along the filling, thereby displacing the air bubble in the capillary tube. The
volume of fluid of transport is measured by observing the movement of the
air bubble. The displacement of air bubble is recorded as the fluid transport
result (F) which is expressed in ml / day

4. ELECTRONIC METHOD :
a) Electrochemical Studies
It is conductimetric technique developed by Jacobson and Von
Fraunhoter in 1975. This study was to evaluate changes in the dimension of
the cavity wall or restoration interphase using an electrochemical cell wall.
Because glass has a similar coefficient of thermal expansion to the tooth
substance glass tube of 4 mm internal diameter and 15 mm of length was
chosen for the study. The two or cavity is formed by a nickel – plated mass
electrode. After inserting the material, 4 mm of the glass tube is immersed in
a 1% motion of lactic acid. A lead from the brass electrode is connected to
one terminal of cut and this is further connected through a series of
resistance to a reference secrete that is formed by a nickel – plated mass rod.
The circuit is completed when the misplace between the test material and
pass is occupied by electrodes when there is a leakage of the solution. Thus
the massage is elevated.

B) ELECTRONIC MONITORING OF MICROLEAKAGE.
This technique allows a real – time electronic numbering of micro
flow. This permits several reading to be obtained from specimens over a
period of time and electronic monitoring enables information to be generated
continuously over long period in rest time.
The microleakage measuring system consists of two main parts.
a) Constant pressure reservoirs
b) Micro-pressure sensor
The constant pressure head is provided by a stainless steel pressure
vessel. This is connected through valves to an electronic pressure sensor to
monitor the pressure and a manual air pump. This allows precise control of
pressure in the vessel and the valve of the pulp allows a constant pressure to
be supplied to the specimen, while under test. The micro pressure sensor
consists of piezo – sensitive power supply. This provides an output of 0.175
V / mm of Hg pressure change at sensor. Voltage changes are constantly
monitored on a chart recorder. Pressure changes as small as 0.05 mm of Hg
can be detected routinely. Microleakage measurements are made inside a
length of clear polyvinyl chloride tubing with known inside and outside
diameter, connected between a pressure reservoir and the pressure sensor. A

pressure reservoir and the pressure sensor. A bolus of water is injected into
the sensor end of the tube before the specimen is inserted fro this end.
Tofflemire matrices are used to seal the tubing around the specimen
and also the pressure sensor. A constant air pressure is then applied to the
water bolus to force through any microspaces around the root filling and
changes in air pressure on the sensor side of the specimen are recorded.
When the readings complete a microsyringe is used to inject 1 ml of water
into the tube between the sensor and specimen to calibrate the system. The
calibration measures the pressure changes associated with the introduction of
water. Using this proportionality factor, the changes in pressure measures as
the result of microleakage can be converted to volume of leakage per unit
time. The apparatus sensitivity is typically 0.005ml / min.

5. MICROSCOPIC EXAMINATION
a) Scanning Electron Microscopy (SEM)
The object to be viewed is prepared so that it permits a thin layer of
heavy metal, such as gold or palladium, to be deposited on the specimen’s
surface. A common method for metal layering requiring the specimen to be
coated with carbon provide an electrically conducting base. The heavy
metal such as gold or gold palladium alloy, which serves as source of
secondary electrons is attached to the carbon. As a beam of electrons scans
the surface of the object. Some are reflected (Back scatter electrons) and
others are ejected (Secondary electrons) from the heavy metal cost. These
electrons are captured by electron detectors that are interpreted and
displayed as a three dimensional image. SEM is usually used to measure gap
formation that occurs between the restorations and the walls and the floor of
the reparations. The defects at the submicron level can be observed at
required magnification such as x 200 or x 1000. Final evaluation is made in
the microphotographs.
b) Replication Scanning Electron Microscopy :
This method of SEM is improved by use of replicas. This allows
change in the size of marginal defects to be followed on a longitudinal basis

and can be applied clinically in vitro. In vivo technique replicas are made of
the experimental restoration and directly after the baseline finishing and at
required intervals of time. These surfaces are cleaned with a surface active
cavity cleanser, followed by cleaning with a 5% of NaOCl solution. The
liquids are continuously renewed during the last 60 seconds period of
scrubbing with cotton pellets. Each stage cleaning, which are repeated twice
is followed by washing with water. Replica impressions, are then made of
buccal and lingual sections with a vinyl silicone impression material.
Negative impressions are replicated with epoxy resins to obtain positive
cast. The casts are prepared for SEM by mounting on metal stubs and
coating with gold by standard evaporation teeth or spluttering.
Replicas are examined with SEM.
Confocal Microscopy
This is a Laser Scanning microscope, which enables in achieving
multiple scanning. The advantage here is that it permits optical sectioning
of the specimens thereby artifacts due to manual sectioning
Fluorescent Microscopy

They use UV radiation on selected dye, which are capable of
absorbing these radiations at one wavelength and emitting at a different
wavelength.

HOW DOES A CONFOCAL MICROSCOPE WORK?
A laser is used to provide the excitation light (in order to get very high
intensities). The laser light (blue) reflects off a dischroic mirror. From there,
the laser hits two mirrors which are mounted on motors; these mirrors scan
the laser across the sample. Dye in the sample fluoresces, and the emitted
light (green) gets descanned by the same mirrors that are used to scan the
excitation light (blue) from the laser. The emitted light passes through the
dichroic and is focused onto the pinhole. The light that passes through the
pinhole is measured by a detector, i.e., a photomultiplier tube.
So there never is a complete image of the sample – at any given
instant, only one point of the sample is observed,. The detector is attached to
a computer which builds up the image, one pixel at a time. In practice, this
can be done perhaps 3 times a second, for a 512 x 512 pixel image. The
limitation is in the scanning mirrors. Our confocal microscope (from Noran)
uses a special Acoustic Optical Deflector in place of one of the mirrors, in
order to speed up the scanning. This uses a high – frequency sound wave in
a special crystal to crate a diffraction grating, which deflects the laser light
(actually, the first diffraction peak is used, with the zeroth – order peak
being thrown away). By varying the frequency of the sound wave, the AOD

changes the angle of the diffracted light, helping scan the sample quickly,
allowing us to take 512 x 480 pixel images 30 times per second. If you want
to look at a smaller field of view, our confocal microscope can go even
faster (up to 480 frames per second, although I personally find that 240
frames per second is a good practical limit).
6. MISCELLANEOUS
a) Artificial Caries
This can be produced in vitro using either bacterial cultures or a
chemical system i.e, the acidified get techniques as first described by
Muheleman in 1960 . The surface of enamel is subjected to a constant attach
of hydrogen ions while the get acts as a diffusion barrier for dissolved
material. The lesions produced by this technique are studied in polarized
light and two parts are seen .
a) An outer lesion
b) A cavity wall lesion
Polarized light microscopy is used to determine the extent of
demineralization of cavity walls adjacent to the restorations. Quantification
of results is possible where depth of lesion is chosen a measurable parameter
and the degree of demineralization may also be assessed quantitatively.

B) Marginal percolation:
This includes study of opening and closing of the margins of
restorations when subjected to temperature changes both in the mouth and in
extracted teeth. The restorations are cooled to 90
c in ice water and are
observed under a microscope as the teeth are warmed by fingers. When
drops of water are seen to extrude from the margins of the restorations as
temperature increases, indicates, microleakage of the restorations. This
occurs because of a difference in coefficient of thermal expansion between
the teeth and the restorations and by thermal expansion of fluid that occupies
the cervices between teeth and restorations.
C) Resin infiltration Method:
Developed mainly to assess the marginal sealing ability to the root
canal wall and the root canal filing material at different levels. The
specimens are coated with nail polish to within 2 mm of the end of the apex.
The resin is prepared dissolving 1.4 gms of resorcinol in 2 ml of 40%
formaldehyde solution and the pH adjusted to 8.2 with aqueous potassium
hydroxide immediately before use. Ice is placed to prevent overheating and
premature polymerization . The teeth are immersed I resorcinol
formaldehyde resin of 5 days of 40
c and resin is allowed to polymerize
completely for 4 days at room temperature. Samples are removed; the nail

polish is completely removed. They are then embedded in epoxy resin and
sectioned horizontally at 1.5 mm (level 1 ) 2.5mm (level II), 3.5 mm (level
III) . From the anatomical apex, sections are immersed in HCL for few
minutes in order to color the resin . All cross sections are transilluminated
and viewed at a magnification of x 25 with stereomicroscope and
photographs are taken for image analysis . Dark brown coloured resin areas
at the gap at each level are measured using a personal image analyzing
system.
CONSTANT DEPTH FILM FERMENTOR
(CDFF):
CEFF is one of the recent method of detecting microleakage. The
leakage of bacteria in to the tooth restoration interfere is studied here. CDFF
is basically a system to create as invivo situation. This generates a large
number of biofilms. Prepared samples are placed into the CDFF over which
the biofilms are generated. At periodic intervals the samples are taken up for
analysis. The penetration of bacteria from the biofilms can be analyzed by
SEM and vital staining to estimate the microleakage.
Thermal cycling and load cycling

All the specimens before they are tested for microleakage undergo
thermal cycling or load cycling or both in order to stimulate the oral
conditions.
Thermal cycling:
It is defined as the invitro process of subjecting a restoration and tooth
to temperature extremes that are similar to those found in the oral cavity.
Load Cycling :
Jorgenson introduced the term mechanical percolation to demonstrate
mechanical factors in the oral environment that might produce asymmetric
pressure on fillings and on the micro spaces between the filling and the tooth
structure.

CONCLUSION
Though many methods are available for study of microleakage, the
general tendency is seen among researchers around the world to stick to the
conventional method i.e., dye penetration. The time has come where in
newer methods, which are more accurate should be sued so as to attain a
more precise value.

REFERENCE
1. Art and science of operative Dentistry – Sturdevant , 4th
edn.
2. Phillip’s science of Dental Materials – Kenneth J. Anusavice, 10th
Edn.
3. Notes on Dental Materials – E.C. Combe
4. Detection of Microleakage around Dental restoration – J of Operative
Dentistry , 1997.
5. In vitro dentinal penetration by traces used in microleakage studies I E
.J – 1998 (31)
6. A Fluorescent dye method for demonstrating leakage around
restoration J. of Dent Res(1966)

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