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Historical Background of

Although the concept of the ethical treatment of patients extends back to the time of
Hippocrates (460-377 BC.), the idea that new dental materials must be tested for safety and
efficacy before clinical use is much more recent.

In the Mid 1800s, dentists tried new materials for the first time by putting them into patients'
mouths. 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 amalgams.
 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 are still being challenged and defined today. In most
cases, a committee of clinicians, basic scientists, and laypersons regulate and oversee the testing
of new materials in humans.

Using humans as research subjects today without some previous testing or knowledge of the
biological properties of a material is unethical and illegal. Still, every new material must be
inserted into a human for the first time at some point. Therefore, many alternative tests have
been developed to try to minimize the risks to humans. 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 materials increased.
There was a public outcry against the use of nonconsenting humans as research subjects.
For example :
The use of nonconsenting U.S citizens for radiation experiments by the Department of Energy
The use of institutionalized mentally retarded children for hepatitis research (1963-1366)

Biological testing of materials has evolved significantly over the past 40 years. Initially, most
biological reactions to materials were categorized empirically and relied on animal models.
Many studies between the 1950s and the 1970s involved the use of premolar teeth that were
scheduled for orthodontic extraction.

As cell culture techniques developed, research focused on the mechanisms that affected
biological responses to materials.
In the past decade, new molecular biological and imaging techniques have been applied to assist
our understanding of the biological response to materials. Today, the field of biocompatibility
testing has reached a point where some prediction of biological properties is possible and the
future will likely provide the ability to design materials that elicit customized biological


Histologically, inflammatory response is characterized by edema of the tissue with an infiltration
of inflammatory cells (ie: neutrophils in short-term, monocytes & other lymphocytic cells – in

Current biocompatibility research attempts to determine whether materials may cause or

contribute to inflammation in the host even if no toxicity is evident

Allergic reaction occurs when the body specifically recognizes a material as foreign Reacts
disproportionately to the amount of the material present. Involves all dimensions of the immune
system, including T and B lymphocytes and monocytes or macrophages

Allergic reaction results histologically in an inflammatory response that can be difficult to

differentiate from a nonallergic inflammation or low-grade toxicity. For example, latex may
cause allergy directly by activating antibodies to the material

Type I, II and III reaction tend to occur quickly and are modulated by eosinophils, mast cells or
B lymphocytes that produce antibodies.

Other materials, such as metal ions, MUST interact with a host molecule.


In allergic response, the individual’s immune system recognizes a substance as foreign. Not all
individuals will react to that substance. (Nonallergic) General inflammatory response toward
nickel, will occur for all individuals because it involves NO specific recognition of the substance.

Components of material alter the base-pair sequences of the DNA in cells (MUTATIONS)

It is caused by direct interactions between a substance and DNA or indirectly by alterations in

cellular processes that maintain DNA integrity. Mutation factors include radiation, chemicals and
errors in the DNA replication process.

Mutagens: metal ions (nickel, copper, and beryllium)

Mutagenic, root canal sealers

Resin-based materials have also been identified as mutagenic potential. Note that mutagenicity
does not imply carcinogenicity


Any material used in the body may have local or systemic biological effects

Modulated by substances that are released from material and the biological responses to those

For dental materials, local effects might occur in the pulp of the tooth, in the periodontium, at
the root apex, or in nearby oral tissues (ie: buccal mucosa or tongue). These effects are a
function of the ability of substances to be distributed to these sites, their concentrations and
exposure times that range from second to years. (Ex: nickel)

A function of the distribution of substances released from materials. The substances might gain
access to the body via ingestion and absorption in the gut, inhaled vapor, release at the tooth
apex, or absorption through the oral mucosa. It may occur via simple diffusion/ transport via
lymphatic or blood vessels

Systemic biological response depends on;

1. The duration and concentration of the exposure

2. Excretion rate of the substance

3. The site of the exposure.

For example, mercury (have long life in the body) may accumulate and reach critical levels.
Systemic reactions may also influenced by organs such as the liver


2 key factors that appear to be paramount in determining a materials biocompability

First factor : involves the various types of metal corrosion or other types of material degradation

Second factor : Surface characteristic


First factor : involves the various types of metal corrosion or other types of material
degradation. Corrosion results in the release of substances from a material into the host. The
release can take many forms and may be caused by many factors

Key point : The biocompability of the material DEPENDS to a large degree on the degradation

The biological response to the corrosion products DEPENDS ON the amount, Composition,
Form of these products , as well as their location in tissues.
Corrosion may be VISIBLE or INVISIBLE to the naked eye. Corrosion is determined not only
by a material's composition but also the biological environment in contact with a material .
Biological force that influence corrosion may be specific

An individual ( such as a person in diabetic ketoacidosis)

Common to all individuals (as with occlual forces on materials)

In any cases , it is biological interfaces that creates that for corrosions

This interface is active and dynamic , with the material affecting the body AND the body
affecting the material

Second factor : Surface characteristic

Research has shown that for all materials , the surface is quite different than the interior region of
a material because the surface is the part of a material that the body “ sees” .

The surface composition, roughness , mechanical properties and chemical properties are
critical to the biocompatibility of the material

The surface may also negatively affect the biological response. For most materials, a rough
surface promotes corrosion. If the corrosion products have adverse effects, then roughness is not
desirable. Roughness may also promote the adherence of bacteria AND promote periodontal
inflammation or decay in teeth

The chemical properties of a surface may also hinder the biological response. Studies have
shown that the surfaces of some material chemically attract lipopolysaccharides more than others
, even the surface roughness is the same because , lipopolysaccharide is a key agent in
periodontal inflammation , the presence of this molecule is not desirable.

The boundaries between toxic , inflammatory , allergic and mutagenic reactions are disappearing
as more is known about materials and cells interact, based on the principle that small alterations
in cells of the immune system by materials can have significant biological consequences.

Significant biological consequences OCCUR because of the amplifying nature of immune cells
for example, monocytes control and orchestrate much of the chronic inflammatory and immune
response. To accomplish this role , monocytes secrete many substances that influence and direct
other cells Thus , if a material were to alter a monocyte's ability to secrete these substances , the
biological response would be significantly amplified.
This concept combines the classic areas of toxicology and immunology because the material
causes nonlethal but toxic change in the monocyte so that the immune system cannot function

Immunotoxicity may result from a material causing either an increase or decrease in cellular
function. For example, mercury ions have been shown to increase the glutathione content of
human monocytes in cell – culture , where palladium ions decrease it. Glutathione is important in
maintaning the oxidative stress in cells Mercury causes a decrease in glutathione at higher
concentration as the ions become toxic However , the decrease found for palladium oocur well
belw toxic levels

Biological Response in Dental Environment

(Environmental and Oral Anatomy that
influences the Biological Response)
Several aspects of oral anatomy influence the biocompatibility of dental restorative materials.
The full effects of the oral anatomy on the biocompatibility of materials are not known, hut these
effects will he a major focus of biocompatibility research in coming years.

The anatomy of the tooth, the periodontal attachment and the periapical environment have
profound influences on the biological response to materials, and all are sites of interface between
materials and tissues in dentistry

The enamel-dentin-pulp environment represents a unique symbiosis of mineralized tissues and

cells. The enamel of the tooth is virtually all inorganic material (96 %) arranged in a crystalline
array called enamel rods.

Although the enamel is permeable to some substances, such as the peroxides in bleaching agents,
it is generally not permeable to material components, bacteria, or bacterial products.

The dentin of a tooth, in contrast to enamel, is a mineralized matrix that embeds an organic
network The inorganic content of dentin is lower (70 %),and (organic portion (18 %) is collagen
but also contains other protein components.The nature of the dentin allows bonding to occur
because acids may selectively dissolve the mineralized matrix, but not the collagenous network
embedded in it.

Therefore, most dentin bonding agents attempt to penetrate the undissolved collagen matrix. The
dentin also has about 12 % water, which is important because many resin restorative materials
are hydrophobic and must be designed to wet the dentin.
When the enamel is removed during restoration of the tooth, fluid flows toward the enamel.
However, evidence indicates that this outward flow is not sufficient to eliminate the inward
diffusion of bacteria, bacterial products or material components into the pulp.

The odontoblasts are sealed togethere with tight cellular junctions that limit the diffusion of
substances past the odontoblastic layer.

Caries may lead to infection and destruction of odontoblasts or they may stimulate deposition of
additional dentin, depending on the relative rate of interaction with these cells.

The gingiva normally extends above the level of the cementum and forms a potential space
against the enamel called- periodontal pocket.

The periodontal pocket is the site of development of periodontal disease, which can destroy the
junctional epithelium, periodontal ligament and supporting alveolar bone.

Many dental restorative are near in the periodontal attachment area, the biocompatibility of these
materials may influence the normal periodontal architecture, the potential disease process.

Periodontal pocket is a unique microenvironment that allow concentration of components from

materials to reach higher levels than are seen in the rest of the oral cavity.

Special Biological Interfaces with Dental Materials

The use of materials to restore damaged or lost tooth structure creates specialized environments
in which the biocompatibility of the material is of central importance to the long-term survival of
the restoration.

In the previous section, several areas of specialized oral anatomy were presented within
formation about their relevance to biocompatibility In this section two specialized interfaces, the
dentin-resin interface and the implant-bone interface , are explored in mole detail as examples of
how the biological interaction between materials and the body can influence both.

The composite nature of dentin allows the mineralized matrix to be dissolved away by acids
while preserving the collagen network.

If the network does not collapse, which may happen if the dentin is desiccated, it may be
embedded by a resin containing material, thereby mechanically bonding the resin material to the

The dentin bonding process is complex. However, the interface of the resin material with the
collagenous network has a profound influence on the biocompatibility of the material.

If the resin material does not penetrate the collagenous network or debonds from it as the resin
shrinks during polymerization, a gap will form between the resin and the dentin.
The shrinkage may also occur with enamel. Although this gap is only a few microns wide, it is
wide enough to permit bacteria and oral fluids to percolate from the pulp outward or from the
oral cavity inward. This leakage has traditionally been termed microleakage.

The biocompatibility of a restoration is altered by the leakage process which may cause a
number of undesirable events:

It may allow bacteria or bacterial products to reach the pulp and cause infection.

b) It may encourage the breakdown of the material. This breakdown then exposes the body to
products of the material and increases the gap, thereby promoting more leakage.

c) The leakage may discolor the margins of the restoration, making the tooth restoration
complex aesthetically unacceptable and possibly resulting in a decision to repair or
replace the restorative material.

Although nanoleakage age probably does not allow bacteria to bacterial products to penetrate the
marginal gaps of the restoration and the pulp, fluid exchange most likely occurs that can degrade
the resin or the collagen network that has been incompletely embedded with resin, thereby
reducing the longevity of the dentin-resin bond.

It is unclear whether leakage is major factor in the biological response to dental materials, the
clinician must also be concerned about immune response in the pulp and periapical tissues that
may occur independently of leakage phenomenon.

The success of endosseous dental implants relies on the ability of the materials to promote
osseointegration and allow a close approximation of bone with the material. This interface must
sustain the forces placed on it durin the normal use of the implant ( from biting, clenching,
chewing.etc. ) The ability of a material to allow osseointegration is closely related to its
biocompatibility. In dentistry, relatively few materials allow osseointegration. These include
commercially pure titanium, titanium-aluminium-vanadium alloy, tantalum, several types of

Materials that allow osseointegration have very low degradation rates, and they tend to form
surface oxides that promote bony approximation. The mechanisms by which these oxides
encourage or allow bone formation without intervening fibrous tissue are not known.

Some materials, such as the so-called bioglass ceramics, promote integration between the bone
and the material with no intervening space at all. When this integration occurs, the material is
said to biointegrate with the bone.
Biointegration appears to require a degradation of the ceramic to promote bone formation,
although the specific reactions are not well understood.

No known desirability of osseointegration over biointegration has been established. Like all
biocompatibility phenomena, osseointegration and biointegration are dynamic processes that
may be altered by changes in the host, fatigue of the materials, or function of the implant. It is
also important to understand that neither osseointegration nor biointegration mimic the normal
ligamentous connection between a tooth root and the alveolar bone

The Oral Immune System

As has been noted previously, the immune system plays an important role in the biological
response to any material.

The immune system in the oral environment appears to behave somewhat differently in oral
epithelium and connective tissue than in the rest of the body, and the biological responses to
materials in the mouth may not always parallel those seen in other locations.

Studies performed on guinea pig models have shown that oral exposure to certain allergenic
metals such as chromium or nickel may actually incduce immunological tolerance to these

However, if the initial exposure occurs in other locations (including the gastrointestinal tract),
allergy often develops. Themechanisms that control tolerance versus allergy to dental materials
are not known, nor is the potential of these types of reactions to occur in humans.

However ; it is important to remember that the oral environment is not always equivalent in
structure or function to other areas of the body and that these differences may alter the biological
response to materials.

Current Biocompatibility Issue in Dentistry

The use of latex rubber dam exposes the patient and the dental personnel to latex.

In 1980’s, because of AIDS, dental personnel started to wear gloves to avoid the risk of its
transmission. Since then, latex hypersensitivity increased dramatically.

Surveys have shown that :

 42 % have adverse reactions to occupational materials, most of which were related to

dermatomes of the hands and fingers.
 Adverse reactions in 3.7 % of adult patients and 5.7 % od pediatric patients were
associated with latex gloves.

 8.8 % of the adult patients experienced hypersensitivity reaction used latex gloves to

 The hypersensitivity to product with latex represents latex allergy to accelerators and
antioxidants used in latex processing.

 Dermatitis is the most common allergy.

 It occurs when gloves and rubber dams come in contact with mucous membrane.

 This exposure could result in angioneurotic edema,chest pain and rashes on neck and
chest of a severely allergic person.

 Ashmathic reactions and other respiratory reaction have also been reported because of
components of latex released into the air.

Processing latex :

Latex are made of a white, milky sap harvested from a rubber tree. Ammonia is added to
preserve it. Ammonia hydrolyzes and degrades the allergens in sap. Vulcanization occur. (liquid
latex hardened into rubber through the use of sulfur compounds and heat. Final process, the latex
is soaked inhot water to release allergen from rubber products.

A common component of dental alloys, used for crowns, partial dentures, removable partial
dentures and some orthodontic appearance. It is the most allergenic metal known.

Hypersensitivity among women is more common because of chronic exposure through jewelry
although the incident among men is increasing. Its possible that people become sensitive to
nickle after having a nickle containing alloy in their mouth, and nickel are known to have high
allergy frequency. As to why it is high, it is unknown. (maybe genetic component)

Nickle has other adverse biological effect :

 Nickle subsulfide is a documented respiratory carcinogen in humans, but this compound

is unknown in dentistry.

 Nickle ion documented mutagen in humans, but there is no evidence that nickle ions can
cause carcinogenesis intraorally.
 Nickle ions also shown to be non-specific inducers of inflammatory reaction along with
cobalt and mercury.

Used in Ni-Cr alloys with conc of 1 wt% to 2 wt% to increase the cast ability of these alloys and
lower their melting range.

The use of beryllium in dental alloys is controversial because of its biological effects.

Beryllium is a documented carcinogen in either metallic or ionic state, although there are no
studies showing that dental alloys with beryllium can cause cancer in humans.The acidic
environment enhance beryllium released from Ni-Cr alloys.

Beryllium containing particles that are inhaled may cause a chronic inflammatory condition
called berylliosis. (alveoli inflammed with lymphocytes and macrophages)

Mercury and Amalgam

Mercury has three forms :

 Metal

 Inorganic ion

 Organic ion such as methyl or ethyl mercury

Metallic mercury gains access to body through skin and or a vapour through lungs. Studies have
been done to determine whether mercury exposure to a person comes from or other sources, and
whether or not they contribute to our health problems.

A study states that amalgam surface (depending on the amount) could expose a person to
mercury conc with minimum observable effect.

The amount needed = 450 – 530

The amount of amalgam surface used to cover, say, the entire 32 teeth would be 190.

Despite markedly elevated blood, plasma, and urine level of mercury, no renal impairment has
been noted. In summary, there are no data to show that mercury from dental amalgam is harmful.

The ability of a chemical to act as hormone estrogen does in the body.
The concern about estrogen in dentistry center around around a chemical called bisphenol A

The release of this subs might alter the normal cellular development or maintenance if the BPA
has estrogenic effect.

Other biological effect of resin

The primary risk appear to be allergy . It is highest among dental personnel because of frequent
exposure to unpolymerized. The allergenicity of methylmetacrylate is well documented. The use
of gloves is not effective in preventing contact because most monomers pass easily through
gloves. The allergic reaction is primarily contact dermatitis, with the resins acting as haptens via
delayed hypersensitivity.

Defining use of the material

-The function or use of a material has important influence on the induced biological response

Factors to consider:
Duration material being in the body
Stresses on the material

Materials that communicate through epithelium or lie completely beneath will need closer
scrutiny than those that do not penetrate the epithelium

Materials that penetrate tooth enamel will need more scrutiny than those that do not
Advantages of IN VITRO : 
-generally cost less than animal or usage test,
- can be standardized ,
-are well suited to large scale screening ,
- can be tightly controlled to address specific scientific questions.

Disadvantages of IN VITRO : 
- questionable relevance to the final in vivo use of the material and lack of inflammatory and
tissue protective mechanism in the vitro environment .

Usage test : 
-Are tests performed on humans or animals

-The usefulness of usage test for preceding 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 placement technique. For this reason usage tests in animals
usually employ larger animals that have similar oral environments to humans , such as dogs or
monkeys .
-If humans are used the usage test is a clinical trail.
Advantages : 
- relevance. These test are gold standard in that they give the ultimate answer to whether or not
material will be biocompatible

Disadvantages :
Extremely expensive , last for long periods , involve many ethical and legal concerns and are
exceptionally difficult to control and interept accurately
-limited animals can be used

In dentistry dental pulp , periodontium and gingival or mucosal tissues are generally the targets
of usage test.

Animal test
-material is placed in intact organism (rats, mice, hamsters, ferrets, guniea pigs, sheeps,
monkeys, dogs, pigs, baboons,cats)

- difference from in vivo test: animal test use an intact animal rather than cells or tissues

-difference from usage test: animal test expose the animal to the material without regard to the
material's final use (grinding amalgam to feed animal to test for toxicity)

- further divided into short term or long term systemic toxicity; exposure to intact or abraded
membranes; immune sensitization or bone response; mutangenicity; carcinogenicity; other
special conditions
The biological responses in animal tests are more comprehensive and maybe more relevant than
in vitro test and these features are major advantages of these test
they can be difficult to interpret and control, are expensive, may be time consuming and often
involve significant ethical concerns and paperwork.

-Furthermore, the relevance of the test to the in vivo use of a material can be quite unclear,
especially in estimating the appropriateness of an animal species to represent a human.


In vitro, animal,and usage test have to be used together to measure biocompatibility.
There are three phases in testing: primary, secondary and usage
Primary test: performed initially on testing of new material, in vitro to measure system, but may
sometimes include animals to measure system toxicity
Secondary test: are almost always conducted in animals to observe immune response (dermal
irritation, chronic toxicity, response upon implantation)

Usage phase:similar to secondary test, but conducted in clinically relevant situations

Materials must pass first and second phase to proceed to usage test phase

However,mjor et al challenged the tests because:

in vitro and animal tests did not necessarily predict the results of usage test or successful clinical
experience. ZOE ( zinc oxide eugenol cement kills direct cell culture, but had been used for years
clinically without resulting in pulpal damage)

And that if the primary tests were too strict, potentially good materials may be screened out; too
insensitive, will waste time and money and endager living when put to usage tests.

Alternative paradigms have been proposed

Standards that regulate measurement of

ANSI/ADA Document 41(Document no 41 for Reccomended
Standard Practices for Biological Evaluation of Dental Materials)

- requires biological testing of all medical devices

-Dental materials are considered devices as opposed to drugs

-Devices are required to show safety only

Uses linear prardigm

Divides testing into initial, secondary, and usage tests
* initial( in- vitro assays for cytotoxicity, red blood cell membrane
lysis,mutangenesis,carcinogenesis, animal tests for system toxicity by oral ingestion)
*secondary(animal tests for inflammatory or immune responses)
*usage tests (pulpal and bone response)

-Does not give suggestion of tests

ISO 10993
-divides tests into initial and supplementary to acess biological reactions to materials

-initial tests are for cytotoxicity, sensitization and systemic toxicity; supplementary tests are for
chronic toxicity, carcinogenicity and biodegradation

-initial test may be in vitro or animal test; supplementary test are performed on animals or
humans ( in this standard, usage test are part of supplementary test)

-gives suggestions on what tests be used, based on how long the material will be present, if it will
contact the body surface, blood or bone, or whether it communicates externally when placed

-not restricted to dental materials

Standards: advantages and disadvantages
has advanced the understanding of biocompatibility and protected the public
-most standards cannot keep up with new scientific information development as a compromise
between manufacturers, academicians and public must be worked out
-Arbitrary in nature as cut off point assigned often rely on acceptability and do not have
scientific basis
-Unavailability of certain techniques on a global scale must be considered
-The manufacturer selects the tests and defend the selection and submits to the ANSI/ADA then
FDA for approval of the material

-Restricted to dental materials

Clinical Guidelines For Selecting

Biocompatible Materials
Clinicians will likely be continually overwhelmed by marketing information or many new
materials, claiming of their mechanical performance

Unconditional biocompability will be often claimed by manufacturers

Knowledge of biocompability issues and some common sense allows clinicians to make
reasonable judgments about biological safety

Define the use of the Material

An important consideration biological safety is how the material will be used. The use of a
material plays a crucial role in its biocompability. Clinicians should consider whether materials
proposed use is new, whether it has been tested in its proposed use. If the material is to be used in
a new way or in a new environment, more caution advised.

The composition of the material is to be considered secondly. Studies shown repeatedly that very
small changes in composition or processing of a material can alter its biocompability. Clinicians
should ask whether the materials composition is different from previous products. Also, whether
the processing of the material has changed If so, caution is advised in applying previous
biological data to the new situation

Clinicians should not be satisfied with nondescript statements Eg. Material has been tested for
biocompability with no problems. If clinical trials are available, make sure that the conditions
and duration of the test are relevant. The quality of any usage test depends on the fidelity of
reproducing clinical use

Clinicians should makes sure that testing conditions were as relevant as possible, and look for
multiple types of tests under different clinically relevant conditions. A well-controlled
comparison with existing materials is always preferable to an isolated test on one material
Think in terms of Risk and Benefit
 In the end, no material be 100% safe or risk-free

 Rarely all data be available to adequately define the risks of using a material

 Clinician must rely on clinical judgment, common sense, and the data available to make a

 Clinician must always recognize that the use of materials in the body requires a risk-
banefit analysis

 The degree of risk assumed must be carefully weighed against possible benefits

 Each clinician will have to adapt a philosophy about the degree of risk he/she is willing to
assume on patients behalf

 The risk must be communicated throughly and clearly to patient so they can decide
whether the benefits outweigh the risks

 This communication is the essence of informed consent

 In dentistry, the process is more important than in evaluating the biological effects of