Rule over the fish in the sea and the birds
in the sky and over every living
creature ... the birds of the heavens and
over every living thing that moves on
the earth. ... God blessed the humans by
saying to them
Defining the use of a material
Methods of measuring biocompatibilty
Biocompatibilty of dental materials
It is the ability of a material to elicit an appropriate
biological response in a given application in the body.
Biocompatibility is not a property of just a material,
but rather a property of how a material reacts with its
The ability of a biomaterial to perform its desired
function with respect to a medical (or dental) therapy,
without eliciting any undesirable local or systemic
effects in the recipient or beneficiary of that therapy,
but generating the most appropriate beneficial
cellular or tissue response in that specific situation,
and optimizing the clinically relevant performance of
DEFINING THE USE OF A MATERIAL
There are several factors that must be considered
when trying to measure the biological response.
The most important factors include
Location of material
The duration of material in the body
Stresses placed on material
Location of material
The location of a material is important to its overall
In general materials that communicate through the
epitheliuim 0r lie completely beneath it will need
closer scrutiny when assessing the biological response
than materials that do not penetrate the epithelium.
Similarly, materials that penetrate tooth enamel will
need more scrutiny than materials than do not.
The duration of material in the
The duration of the material in the body is important
to the biological response.
The duration of the presence of a material is an
important factor, because many interactive effects
between the body and material take some time to
In general, the most stringent tests to measure
biocompatibility are required for materials that are
present for the longest times..
Long durations give sufficient time for the material to
affect the body and for the body to affect the material
in many complex ways
Stresses placed on material
stresses placed on the material are important to the
biological response, These stresses may be physical,
chemical, or thermal in nature.
Short-term, long-term, and fatigue stresses all need
to be considered when assessing the effect of stress on
the biological performance of material.
Although the concept of the ethical treatment of
patients extends back to the time of Hippocrates
(460-377 KC.), the idea that new dental materials
must be tested for safety and efficacy before clinical
use is much more recent.
As late as the mid 1800s,dentists tried new materials
for the first time by putting them into patients'
Many exotic formulations were used. For example,
Fox developed a "fusible metal“ that consisted of
bismuth, lead, and tin, which he melted and poured
into the cavity preparation at a temperature of
approximately 100" C.
Even G.V. Black used patients to test many of his new
ideas for restorative materials, such as early
The current philosophy about testing the biological
properties of dental materials in a systematic way
evolved in the 1960s as the need to protect patients
became politically acute and as the number of new
The concept of protecting the patient as a research
subject is only 30 to 40 years old, and many of the
regulations and ethics in this area still being
challenged and defined today.
Nazi human experimentation
Nazi human experimentations were a
series of medical experiments on large
numbers of prisoners, mainly Jews from
Doctors trial nuremberg code of ethics
In vitro tests
Tests are done in test tube, cell culture dish, or other
wise out side a living organism.
Direct tests: material contacts the cell system
Direct tests can be further subdivided into
Those in which the material is physically present with
Extract from the material contact the cell system
Indirect tests: when there is a barrier of some sort
between the material and the cell system.
Agar overlay method
Millipore filter assay
Dentin barrier tests
1. Quick to perform
2. Least expensive
3. Can be standardized
4. Large scale screening
5. Good experimental control
6. Excellence for mechanism of interaction
1. Relevance to the final in vivo use is questionable
2. Lack if inflammatory and other tissue protection
mechanisms in the in vitro environment
3. Cannot predict the overall biocompatibility of a
Cells used for in vitro tests
1. primary cells: taken directly from animals
and cultured .
A primary cell culture may be composed of mixture
of cell types.
Retains many of the characteristics of cells
2. Continuous cells or cell lines: cell lines have at
least one passage. With each subsequent culture the
cell population becomes more homogenous.
These cells do not retain all in vivo characteristics
Cyto toxicity tests
It can be assessed by the following formula
= Cell death by material
Cell number before the exposure
Cells are plated in a well of a cell culture dish where they
attach. The material is then placed in the best system.
If the material is not cytotoxic cells will remain attached to
the well and will proliferate over time
If the material is cytotoxic, cells may stop growing, exhibit
cytopathic features or detach from the well
If the material is a solid then the density of cells may
be assessed at different distances from the material,
and a zone of inhibited cell growth may be described
Negative control materials (non cyto toxic materials)-
Teflon and cell culture treated poly styrene
Positive control materials (cytotoxic material)-
Plasticized poly vinyl chloride
Membrane permeability tests
Measure cytotoxicity by the ease with which a dye can
pass through a cell membrane, because membrane
Is equialent to or very nearly equvalent to cell death
Change in the membrane permeability to the dye
1. Vital dyes: Neutral red & Na2CrO4
2.Nonvital dyes: Trypan blue & Propodium iodide
Tests for cell metabolism or cell function
Bio synthetic or enzymatic activity of cells are used to
assess cyto toxicity of the test material
E g. Tests that measures DNA synthesis or protein
Synthesis is analyzed by adding radio isotope labeled
precursors to the medium and quantifying the
radioisotope [H-thymidine or H-leucine]incorporated
into DNA or Protein
Measures the activity of cellular dehydrogenase
MTT is a yellow, soluble molecule
Chemical MTT is used to know the enzymatic
action of cell.
If the cell is able to reduce the MTT the resulting
Formazen formed is proportional to the
Formazen quantified by dissolving it and measuring the
optical density of the resulting solution alternatively the
formazen can be localized around the test sample by light
or electron microscopy
Other formazen generating chemicals NBT, DXTT, WST,
Alaram blue tests quantitatively measure cell proliferation
using a fluorescent indicator that allows the continuous
monitoring of cells over time.
Tests that use barriers
Cyto toxicity tests measure the toxicity when material
is indirect contact with the cell culture
Agar overlay method
Millipore filter assay
Dentin barrier tests
Agar over lay method
Monolayer of cultured cells +neutral red +fresh
Agar forms a barrier between the cells and the material
which is placed in the top of the agar
Nutrients, gas and soluble toxic substances can diffuse
through the agar
Solid test samples or liquid samples absorbed on to filter
paper can be tested with this assay for up to 24hrs
Millipore filter assay
Establish monolayer of cells on the filter
Culture medium placement and this mixture is
allowed to gel over the cells
Filter- monolayer gel is detached and turned over so
that the filter is on top for placement of solid or
soluble test samples for 2 or more hours
After exposure the toxicity in the Millipore filter test
is assessed by the width of the cytotoxic zone around
each test sample
Dentin barrier tests
It shows the improved correlation with the
cytotoxicity of dental materials in usage tests in teeth
Incorporation of dentin disks between the test samples
and the cell assay system
Then the cytotoxicity is measured the testing material
placed on one side of the dentin disk in the devised
used to hold the dentin disk
Cells can also be grown in the collection side of the disks.
Collection fluid is also in the other side.Immortalized
pulpal fibroblats can be used as target cells.
Components of the material may diffuse through the
dentin and effect of medium on cell metabolism can be
To assess the rate of diffusion the collection fluid can e
circulated into and out of the collection
Other assays for cell function
These assays measures cytokine production by
lymphocytes and macrophages, lymphocyte
proliferation, chemotaxis or T-cell rosetting to sheep
red blood cells
Other tests measure the ability of a material to alter
the cell cycle or activate complement
These tests elicit the effect of dental material on a
cells genetic material
Ames test: most widely used short term mutagenesis
Only short term one that is thoroughly validated
Genetically altered bacteria are used as test
organisms. These bacteria cannot grow and form
colonies on a special culture agar, which is histidine-
But as soon as they come into contact with a
mutagenic substance, they begin to grow. The
number of forming colonies is a criterion for the
Styles cell transformation test :
This test on Mammalian cells offers an alternative to
bacterial tests(ames test)
Untransformed fibroblasts normally will not grow
with in a agar gel where as genitically altered cells can
In vitro test systems based on cells include the HPRT
test, in which an alteration of the gene is detected that
encodes for the enzyme HPRT.
in vitro micronucleus test, in which direct
morphological alterations of the chromosomes are
identified (formation of micronuclei)
HPRT- H ypoxanthin- G uanin- P hospho s ribosyl t ransferase
Mice, Rat, Hamsters, Guinea pigs
More relevant than In-vitro tests difficult to interpret
Expensive, Time consuming, Involves significant ethical
1. Mucous membrane irritation test
2. Skin sensitization test
3. Implantation tests
Mucous membrane irritation test
+ve control, test material, -ve controls are all placed
into contact with Hamster cheek pouch tissue or
rabbit oral tissue
Several weeks after test sites are examined and the
gross tissue reactions in the living animals are
recorded and photographed in color
The animals are sacrificed and biopsy specimens are
prepared for histological evaluation
Skin sensitization tests
Materials are injected intradermally to test for
development of skin hypersensitivity reactions,
followed by secondary treatment with adhesive
patches containing the test substance.
Two test methods are recommended using guinea
The maximization test and
The Buehler test
The Maximization test
The investigated substance is at first injected
intradermally into the experimental animal, together
with Freud’s Complete Adjuvans (FCA).
Seven days later, the same substance is applied
topically at the same site for 2 days.
It is intended to amplify the immunological effect by
FCA and, thus, to increase the sensitivity of the test.
Fourteen days after this induction period, the test
substance is applied on a different area of the skin
Subsequently, the skin reaction is assessed after an
appropriate exposure time . It is important that the
substances be applied at a concentration that does
not evoke primarily toxic (irritating) skin reactions.
The Buehler test is similarly executed on guinea pigs
but without the application of FCA. Therefore, the
Buehler test is considered to be more protective for
the animals than the maximization test.
Usage tests may be done in animals or in human
They are distinct from other animal tests because
they require that the material be placed in a situation
identical to its intended clinical use.
The usefulness for predicting biocompatibility is
directly proportional to the fidelity with which the
test mimics the clinical use of the material in every
regard, including time, location, environment, and
For this reason, usage tests in animals usually employ
larger animals that have similar oral environments to
humans, such as dogs, mini-swine or monkeys.
When humans are used, the usage test is termed a
Relevance to use of material is assured
Very time consuming
Major legal/ethical issues
Can be difficult to control
Difficult to interpret and quantify
Dental Pulp Irritation Tests
Pulp compatibility of a material is investigated on
teeth of experimental animals or on human teeth that
have to be extracted for orthodontic reasons.
In both cases, class V cavities are prepared as
atraumatically as possible and are then filled with the
test material. This approach is equivalent to the
future mode of application on patients.
After a period of days to several months, the teeth
removed and histologically prepared, and the pulps
are microscopically evaluated for signs of acute or
chronic inflammation and odontoblast reaction
(including dentin neogenesis)
In addition, the space between test material and the
cavity wall is investigated for bacterial penetration.
These methods can be modified in such a way that
the pulp is exposed or part of the pulp is removed
the material is applied
Assessment: The most important causes of pulp
damage resulting from a restorative procedure (in
addition to cavity preparation) are the following:
• Toxic substances released from the material
• Bacteria and their toxins between the material and
The pulp can react to these irritations in the
Tertiary dentin formation
Dentin sclerosis reduces the dentin’s permeability,
but low-molecular substances may diffuse even
through sclerotic dentin.
However, sound teeth, mostly without obliterated
dentin, are used in pulp/dentin tests.
Thus, there may be a discrepancy between the clinical
situation (below a carious lesion with dentinal
sclerosis) and the usage test with filling materials.
The diffusion of potentially damaging substances
through sclerotic dentin toward the pulp may be
In addition, the target organ of the pulp/dentin test is
the pulp of sound (test) teeth and not the
“predamaged” pulp, as is frequently the case in
A chronic inflammation in the patient’s tooth pulp
may impair the defensive capacity of the pulp,
rendering it more susceptible to toxic material
Periapical Tissue Damage
and Endodontic Usage Test
The literature includes descriptions of animal models
(e.g., primates, dogs) that allow the application of a
given material into the root canal according to
endodontic techniques after a usual root canal
Compatibility is assessed by histologic evaluation of
the periapical tissues.
Assessment: The classic endodontic usage test is
very elaborate and includes the same technical and
ethical problems as the pulp/dentin test using large
Relatively few studies using this test method are
available in the literature.
The presented findings, however, document a good
correlation with clinical observations.
In particular, stimulating effects on special cells can
be determined, such as the influence of calcium
hydroxide compounds on periapical cementoblasts.
Otherwise, implantation tests, in which Teflon tubes
are filled with the experimental material and
subsequently implanted, may be used as alternatives
Such tests are especially useful when assessing the
claimed bioactive effects of test materials.
Dental Implants in Bone
Materials used for dental implants are inserted into
the jaw of test animals (intraosseous implants).
For this, penetration of the epithelial barrier,
equivalent to the treatment of patients, is simulated
on experimental animals.
Appropriate animals are, among others, primates,
dogs, miniature pigs, guinea pigs, and rats.
Tissue reaction is assessed histologically, with the
tissue in contact with the implant being particular
A good correlation of these findings with patients’
situations can be expected.
Mucosa and Gingival Usage Tests
Tissue response to materials with direct contact of
gingival and mucosal tissues is assessed by placement
in cavity preparations with subgingival extensions.
The material’s effect on gingival tissues are observed
and responses are categorized as slight, moderate, or
severe, depending on the number of mononuclear
A difficulty with this type of study is the frequent
presence of some degree of pre existing inflammation
in gingival tissue due to the presence of bacterial
plaque, surface roughness of the restorative material,
open or overhanging margins, and over- or under-
contouring of the restoration.
Correlation Among In Vitro,Animal, and
In the field of biocompatibility, some scientists
question the usefulness of in vitro and animal tests in
light of the apparent lack of correlation with usage
tests and the clinical history of materials.
However, lack of correlation is not surprising in light
of differences among these tests.
in vitro and animal tests often measure aspects of
biological response that are more subtle or less
prominent than those observed during a material’s
Furthermore, barriers between the material and
tissues may exist in usage tests or clinical use, but
may not exist in the in vitro or animal tests.
Thus it is important to remember that each type of
test has been designed to measure different aspects of
biological response to a material, and correlation is
not always to be expected.
Using In Vitro, Animal, and Usage Tests
Pyramid testing protocol
Early strategy for the use of biocompatibility tests
The contemporary strategy used in most standards documents.
Two suggested future strategies for biocompatibility testing of materials
Allergic Responses to dental
Allergic contact dermatitis or stomatitis
This is most common adverse reaction to dental
The interval between exposure to the causative agent
and the occurrence of clinical feature varies between
It usually occurs where body surface makes direct
contact with the allergens.
E.g.: - monomers of bonding agent, acrylic
components of dental cements.
Industry workers who handle these materials are also
Allergy to latex Products: -
In 1991, FDA issued a bulletin in respond to the
increasing number of latex-related allergic reactions.
Malten & associates (1976) reported increasing
incidence of hypersensitivity.
March (1998), suggested that the polyether in latex
rubber gloves was causative agent
Dermatitis of the hands (eczema) in the most
common adverse reaction.
Reactions vary from localized rashes and swelling to
wheezing and anaphylaxis.
To avoid these reactions to latex products, vinyl
gloves may be used.
Blink horn and Leggate (1984) and Axelsson et al
(1987) reported adverse reaction to rubber dam
involving respiratory distress, edema, and chest pains.
The definitive diagnostic test for these is patch test.
The suspected allergen is applied to skin with intent
to produce reaction in around 48-96 hrs.
Mercury Controversy: -
Controversy has raged over biocompatibility of
amalgam restorations because of the presence of
Recognized symptoms of chronic mercury poisoning
are weakness, fatigue, anorexia, weight loss, insomnia,
The lowest level of total blood mercury at which non-
specific symptom occur is 35 ng/mL.
Minimizing Dental Iatrogenesis:
Iatrogenesis is defined as the creation of side effect,
problems, or complications resulting from treatment
by a physician or dentist.
Stanley HR (1994) reported that low hand piece speed
(6000-20,000 rpm) with air water spray, a cavity
preparation 2 mm from the pulp, elicits minimal pulp
If preparation is less than 1 mm of the pulp, intensity
of response increases.
Histopathologically, thermal insult results in loss of
cytoplasmic continuity of odontoblasts and
displacement of odontoblast nuclei into the dentinal
tubules due to dehydration.
The generation of heat within the pulp is the most
severe trauma that restorative procedures impart on
If the insult is extensive and cell rich zone of pulp is
damaged, reparative dentin formation may be
The pulp is a tissue of low compliance according to
Goodies et al (1989) because “It is encased in hard
dentinal walls, it consists of a large amount of
connective tissue with a small blood supply and has no
possibility of developing a collateral circulation. For
these reasons, the pulp is vulnerable to thermal damage
during and after extensive restorative procedures.
Zach and Cohen (1965) reported 15% of irreversible
pulpal damage in monkeys for a temperature elevated
C, 60% for a temperature elevated to 110
100% for a temperature elevated to 16.60
In a cavity preparation with a diamond bur, the entire
surface of the bur is in contact with the tooth surface
thus generating frictional heat but in the case of TC bur
the flutes themselves may allow a slight cooling action
with a greater cutting efficiency.
According to Cohen, ‘Blushing’ of teeth during or
after cavity or crown preparation is attributed to
frictional heat. Coronal dentin develops a pinkish hue
very soon after dentin is cut. This represents vascular
stasis and is reversible
Pulp responses to specific agents:
These are used in non-vital and vital teeth.
These agents contain peroxides
These agents may be in contact with teeth for several
minutes to severe hours.
Peroxides can penetrate the intact enamel and reach
Occurrence of tooth sensitivity is very common with
the use of these agents.
Bleaching agents will also damage the gingiva, if not
Swerdlow and Stanley (1962) reported that the pulp
response to amalgam placement is due to
Little pulpal response is elicited when cavity is
prepared with high-speed air-water spray technique
However, when cavity is restored with amalgam the
pressures of condensation will intensify the response
Boremark and associates (1968) showed that
radioactive mercury reached the pulp in humans after
6 days if no cavity liner was used.
Implantations tests show that low copper amalgams
are well tolerated, but high copper amalgam cause
Liners are suggested to avoid pulpal reaction.
Amalgam based on gallium rather than mercury have
been developed that are free of mercury.
The following pulp reactions may occur immediately
after application/condensation of amalgam in deep
cavities with a remaining dentin thickness (RDT) of
less than 0.5 mm
• Reduced number of odontoblasts
• Odontoblast nuclei in dentin tubules
• Dilated capillaries
• Slight to severe inflammatory cell infiltration in the
Pulp reaction 1 month after application of an amalgam
filling. Dilated blood vessels close to the predentin; otherwise,
no noteworthy alterations. Distance between pulp and
cavity is 0.52 mm
Visible light-cure Resin composites:
The level of the pulp response to resin composite
restorations is especially intensified in deep cavity
preparations when an incomplete curing of resin
permits a higher concentration of residual
unpolymerized monomer to reach the pulp.
Visible light-cured systems were developed to provide
greater depth of cure, shorter curing time, less
porosity and more wear resistant composite
A more conservative cavity preparation with
incremental placement of the resin composite is
highly recommended to minimize the pulp response.
No pulp damage is to be expected if resin-based
composites or adhesives are applied in shallow or
medium cavities, even after prior acid-etching of the
dentin (total etch/total bonding technique).
In these situations, adhesives may serve as sealants
and thus as protection against potentially penetrating
In deep cavities, however, especially if microexposure
of the pulp cannot be excluded, the use of a calcium
hydroxide preparation applied on the deepest part of
the cavity is still recommended.
If a calcium hydroxide suspension is used for this
purpose, then it should be covered by suitable glass
Zinc Phosphate Cement
If zinc phosphate is used instead of ZOE to cement a
crown or inlay, the phosphate cement is forced into
the dentinal tubules
After 3-4 days, it creates a wide spread three-
dimensional inflammatory lesion involving all the
coronal pulp tissue.
A young tooth with wide – open dentinal tubules is
more susceptible to intense response than an older
tooth, which has produced sclerotic and reparative
dentin that block’s the tubules.
Zinc phosphate cements elicits strong to moderate
cytotoxic reactions that decrease with time after
setting Leaching of zinc ions and a low pH is cause of
Initial pH on setting is 4.2 at 3 minutes
The best protection against phosphoric acid
penetration is provided by coating the dentin with
two coats of an appropriate varnish, a dentin-bonding
agent, or a thin wash of calcium hydroxide.
Calcium hydroxide plugs the dentin tubules and
neutralizes acids; hydrophilic resin primers infiltrate
the collagen mesh produced by acid-etching of the
dentin and seal the patent dentin tubules.
These procedures eliminate 90% of the severity of
the adverse pulp responses, making them similar to
those of polycarboxylate cement
They cause slight to moderate response after 3 days.
They are recommended only in cavities with intact
Zinc Oxide Eugenol cements
ZOE is recommended as a nontoxic reference
substance in respective
Cox CF et al 1987 stated that eugenol from ZOE fixes
cells, depresses cell respiration and reduces nerve
transmission with direct contact
ZOE may form a temporary seal against bacterial
It inhibits the synthesis of prostaglandin and
Interaction of eugenol with vallinoid receptors on
nerve cells playing an important role in nociception
Glass Ionomer Cement
When GIC first introduced, the pulpal response were
classified as bland, moderate, less irritating than
silicate cement, zinc phosphate cement.
The blandness of GIC is attributed to absence of
strong acids of toxic monomers.
Polyacrylic acid and polyacids are much weaker than
phosphoric acid and possess higher molecular weight
that limit their diffusion through dentinal tubules to
Tobias and other (1978), found that glass ionomer
cements were less irritating than zinc phosphate
cement, equivalent in irritancy to polycarboxylate
cement and more irritating than zinc oxide cement.
Smith and Rusa (1986) compared the initial activity of
GIC with zinc polycarboxylate and zinc phosphate
cements and found a general rise in pH for all
cements during first 15 minutes. However, the initial
reactions of GIC’s were slower.
It is recommended that if there is less than 0.5-mm
residual dentin or a pulp exposure, an appropriate
lining of calcium hydroxide should be placed prior to
the placement of a glass ionomer
Resin – based composite cements (Dual-Cure)
These are low viscosity resin cements, proper
restoration seating with less pressure reduces the
possibility for luting voids beneath stress bearing where
fracture are most likely to occur.
Pameijir and Stanley (1992) compared the pulp
responses to dual-cured (light cured and chemically,
self-cured) agents. Only when the dual-cure resin
cement received no visible light energy did the average
pulp response levels exceed the accepted level of
Conditioning (etching) agents: -
Before a resin composite or a GIC restorative material
is placed, surface contaminants must be removed to
permit the micro mechanical attachment or the ionic
exchange of the dental material with the tooth
Brannstrom and Nordenvall (1977) noted no
significant difference between dentinal surface
conditioned for 15 seconds and those conditioned for
2 seconds and thus recommended shorter
Brannstrom (1981), showed that conditioning of
dentin and removal of smear layer allows the ingress
of bacteria and the outward flow of dentinal fluid
within the tooth – material inter facial region
resulting in biofilm formation that interfaces with
Some scientists recommend that smear layer showed
remain but in modified form, where as some other
propose that the smear layer be completely removed.
Bowen and colleagues (1982) introduced mordanting
solution (acidified ferric oxalate), that appeared to
dissolve the original smear layer and replace it with a
more uniform ‘artificial’ (altered) smear layer.
With the use of less concentrated acids with higher
molecular weights and shorter time intervals for
conditioning, pulp response is minimized.
Bonding agents do not appear to be toxic
To enhance bonding to composite, a fast setting
visible light cured, low viscosity (unfilled) resin
primer is applied that infiltrates the demineralized
dentin surface and the exposed collagen mesh to form
The plugging of the dentinal tubules prevents the
penetration of toxic components to the pulp from
Influence of Patient Age on Pulp response
As permanent teeth endure the effects of abrasion,
caries and restorative procedure, the pulp becomes
reduced in size because of deposition of secondary
dentin, pulp stones and clarifications.
At age 55 years, the volume of pulp tissue is one fifth
that at age 25 years and contains only one fifth of its
former blood supply (Stanley, 1990)
If an inflammation develops in pulp of an aging
patient, that pulp has less defense in resolving a lesion
and resisting infection.
Pulps Responses to Clinical Procedures:
Some clinicians observed that postoperative pulpits
could be reduced if restorations are luted with
temporary cement before final cementation thus
waiting for formation of reparative dentin.
More practical approaches are (1) the application of
cavity liner, coating or base. (2) Sealing the dentin by
infiltrating the conditional dentin with a primer.
Incomplete bonding or resin penetration into the
collagen mesh of acid-etched dentin can lead to fluid
ingress along gaps wider than 1 μm, which is referred
to as microleakage.
Although this gap is only a few microns wide, it is
enough to permit bacteria to penetrate this interfacial
space, since the average size of a Streptococcus
bacterium is only about 1 μm in diameter.
The bacteria that migrate to the pulp may initiate an
infection of pulp tissue. The gap also promotes
material breakdown along the unsupported margin.
If the resin penetrates the collagen network of dentin
but does not penetrate it completely, then a much
smaller gap (less than 0.1 μm in most cases) will exist
between the mineralized matrix of dentin and the
collagen–resin hybrid layer.
This much smaller gap has been claimed to allow
nanoleakage, which probably does not allow bacteria
or bacterial products to penetrate the marginal gaps
of the restoration and the pulp.
However, fluid exchange most likely occurs, and this
may degrade the resin or the collagen network that is
incompletely embedded with the resin, thereby
reducing the longevity of the dentin–resin bond.
When the nerve endings in the odontoblastic layer
and predentin, are injured by a restorative procedure,
the healing process induces an enormous out growth
of dendrites contributing to hypersensitivity.
Approximately, 21 days are required for complete
regeneration of the nerve ending.
If symptoms disappear over time (7-12 weeks), this
may mean sufficient reparative dentin has formed to
block the tubules and eliminate postoperative
If symptoms persists, it may be due to
Degradation of micro mechanical bond
Shrinkage of resin during polymerization
Exposure of patent dentinal tubules etc.
If patient experiences prolonged sensitivity and the
composite is replaced with other material, the
symptoms may soon disappear. This may result due to
sealing of involved tubules.
Powell and colleagues (1990, 91), using teeth from
adolescents and a few adults, found that the most
severe postoperative pain occurred in females.
Calcium hydroxide has been mainly used in pulp
capping, pulpotomy, root amputation, apexificaiton
The cement is alkaline in nature. The high pH is due
to presence of free hydroxyl ions in the set cement.
The pH ranges from 11-13.
The following zones of tissue reaction can be observed
histologically after application of calcium hydroxide for
direct pulp capping:
Zone of obliteration (early changes, caustic effect,
area of superficial debris)
Zone of coagulation necrosis
The line of demarcation
Early stages of dentin bridge formation
Calcification of the bridge
The calcium-hydroxide-triggered coagulation
necrosis seems to be a stimulus that is sufficient to
initiate healing in the subjacent vital pulp tissue.
This process will then initiate the differentiation of
cells to odontoblast-like pulp cells (secondary
odontoblasts), which will finally result in a bridging.
Tunnel defects may cause a problem. These
defects, which are located in newly formed dentin,
create tunnels and thereby open communications
between the calcium hydroxide and the pulp and may
act as access for bacteria.
This problem underscores that a tight restoration and
sealing of the cavity is decisive for the success of a
direct pulp capping. Bacterial infection is the most
important reason for failure of a direct pulp capping
Only highly purified gutta-percha should be used in
patients with a latex allergy. If necessary, synthetic
gutta-percha points can be applied (e.g.,
It may be concluded from these data that
thermomechanical compaction (condensation),
specifically at a higher rotational speed
(>10,000/min), may damage the periodontal tissues.
Patients with an allergy to eugenol (or to fragrances)
should not be treated with materials containing
eugenol, isoeugenol, or Peru balm.
Data show that ZOE sealers are characterized by a
moderate local toxicity, which is significantly increased
if paraformaldehyde is added.
A number of case reports document that
paraformaldehyde-containing ZOE sealers may cause
an aspergillosis of the maxillary sinus when the root
canals of upper posterior teeth are overfilled and the
sealers are pressed into the maxillary sinus
Epoxy-based sealers are initially toxic, but toxicity
considerably declines when the materials are set,and
then no tissue reactions, or only slight ones, are
Epoxy-based sealers are initially toxic, but toxicity
considerably declines when the materials are set, and
then no tissue reactions, or only slight ones, are
CH sealers are characterized by a low toxicity, which
occurs only in the initial period after application.
There is clear indication that these materials may
stimulate the formation of hard tissue.
However, an inferior marginal adaptation together
with microleakage due to increased solubility is a
potential risk to be considered for this group of
As a consequence of pathologic changes in the dental
pulp, the root canal system can harbor numerous
As irritants are released from the root canal system
into the periradicular tissue, granulation tissue
proliferates and replaces normal periradicular tissues.
Removal of irritants from the root canal system and
its total obturation results in repair of the
periradicular tissue to its normal architecture.
Grossman indicated that ideal root canal filling
material should meet the following requirements-
It should seal the canal laterally as well as apically
It should not shrink after being inserted
It should be impervious to moisture
It should be bacteriostatic or at least not encourage
It should not irritate periradicular tissue.
It should be neither mutagenic or carcinogenic
The sealers form a fluid – tight seal at the apex by
The minor interstices between solid material and the
The patent accessory canals.
A minimal reaction was found when the canal was
not overfilled with sealant.
When the teeth were overfilled with sealant, there
was persistent chronic inflammatory response.
Gulati et al 1990 stated that cytotoxicity of zinc oxide
eugenol might be attributed to the fact that
formed hydroxide in contact with tissue fluids and
released free eugenol which was responsible for
Oswald and Cohn reported systemic distribution of
lead contained in N2 – filled root canals in liver,
kidney, adrenal gland and spleen.
Mittal et al 1999 attributed the toxicity of Sealapex to
polymeric resin and for Endoflas FS to presence of
eugenol and para monochlorophenol
AH26 resin sealer cause moderate to severe toxicity.
This toxicity is due to release of formaldehyde during
the initial setting reaction between bisphenol A resin
and hexamethylene tetramine. (Span Burg et al 1993).
Although all root canal sealers leak to some extent,
there is a critical level of leakage that is unacceptable
for healing and may result in endodontic failure.
Leakage may occur at the interface of dentin and
sealer, at interface of solid core and sealer, through
the sealer itself etc.
If the apical surface can be blocked principally by a
solid core material success is improved over time.
In most studies when obturation was done without
sealers the leakage results were enormously greater.
Without question, all the materials used to seal root
canal irritate periradicular tissue if allowed to escape
from the canal.
Apical filling with dentin chips
In advertently, dentin chips may produce an apical
plug when the materials are compacted
Dentin chips produced by endodontic procedure may
occlude apical foramen.
To condense dentin chips deliberately constitutes a
biologic seal rather than a mechanical chemical seal.
Such plug can prevent overfilling and can restrict the
irrigating solution and obturating material to the
M. Mittal, Satish Chandra and Shallen Chandra
Comparative tissue toxicity evaluation of four
Journal of Endodontics.Vol.21, No.12, 1995, 622-62.
They evaluated the response of the tissue
histologically to four root canal sealers Zinc Eugenol,
Tubliseal, Sealapex and Endoflas. By injecting them
into the subcutaneous connective tissue of dorsal
surface of rats
This study concluded all the sealers caused some
inflammation that decreased with time. Overall,
Sealapex showed least inflammatory reaction
compared with other sealers.
Kuo-wei Tai, and Yu-Chao Chung.
Cytotoxicity evaluation of perforation repair materials
on human periodontal ligament cells in vitro.
Journal of Endodontics: Vol. 26, No.7, 2000, 395-97
They evaluated the cytotoxicity of amalgam, light-
cured composite resin and light cured glass ionomers
using cultured human periodontal ligament cells.
This study concluded that both the type of the
material and time of contact affected the cell viability
and proliferation of the cultured cells.
Composite resin exhibited most cytotoxic effects
followed by glass ionomer and amalgam during 14-day
“Tissue reactions to two root canal sealers containing
Oral Surg Oral Med Oral Pathol Oral Radiol Endod :
60 : 1985 : 661-665.
He investigated tissue response to tooth roots filled
with N2 or Endomethasone root canal sealents in the
premolar teeth of dogs. Periapical inflammation was a
common finding with all the materials. It was most
severe with Tubliseal (control) and least severe with
R.S. Tobias et al
“Pulpal response to two semihydrous glass ionomer
International Endodontic Journal, 1991., 24 : 95-107
This study compared the pulpal responses to two
semihydrous glass ionomer luting cement (STA and
ZIN) with those previously obtained for a anhydrous
glass ionomer luting cement Aquacem.
ZIN contained zinc oxide in the formulation, study
was conducted in vivo in ferrets. ZIN was associated
with minimal pulpal response whereas STA was
similar to Aquacem.