Biocompatibility of Polymeric Dental

Materials

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Content
• Definition of biocompatibility
• Historical background
• Introduction
• Requirements for dental material biocompatibility
• Methods of measuring biocompatibility
• Biocompatibility of dental polymers
• Different studies on biocompatibility of dental polymers
• References

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 Definition of biocompatibility

• Biocompatibility can be defined as: the properties of materials being

biologically compatible without causing local or systemic responses of
a living system or tissue.

• Another definition refers biocompatibility as: the ability of a material

or a device to remain biologically inert during its functional period.

• Biocompatibility can also be determined as: the ability of a material

to co-exist and perform with a natural substance in a specific biological
application.

• In other point of view, biocompatibility is the relationship between

the material and the organism in which neither of them has unwanted
effects.

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• According to regulatory rules, biocompatibility is a number of tests for
determining the possible toxic effects resulting from contact of the
components of materials and medical devices with the body.

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 Historical background
• 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‘ mouths.
• eg. Fox : fusible metal-bismuth, lead & tin-melted & poured in cavity
preparation at appx.100 º C.

• Even G.V. Black used patients to test many of his new ideas for restorative
materials, such as early amalgams.

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• Concept of protecting patients was developed in early 1960’s where the
current philosophy about testing the biological properties of dental
materials in a systematic way was evolved as the need to protect patients
became politically acute and as the number of new materials increase.

• Regulations & ethics introduced

• Organisations like FDA, ANSI, ADA, and ISO

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 Introduction
• Polymers have aroused a vast and increasing interest in oral medicine due
to their excellent biocompatibility, satisfactory mechanical properties, and
processability.

• Polymers can be divided into two classes, depending on

their source:
Natural polymers: includes chitosan, collagen, fibrin, and agar.
Synthetic polymers: consists mainly of acrylic resin and its derivatives,
polyetheretherketone (PEEK), dendrimer, polylactic acid, etc.

• In addition, polymers can be classified into restoration materials,

accessory materials, tissue regeneration materials, etc., on the basis of
their applications.

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• Although the exact composition of these materials is a trade secret, most
of them are known to contain monomers of acrylate derivatives, initiators
and accelerators of polymerization, and stabilizers.

 Most resin systems used in dentistry are based on methacrylates,

particularly methylmethacrylates (MMA), poly(methyl methacrylate)
(PMMAs), which possesses excellent biomechanical and self-hardening
properties.

 PMMAs are used primarily for dentures and temporary crowns, individual
impression trays and orthodontic devices. PMMAs are classified as heat
curing, chemical (auto) curing, light curing or microwave curing according
to their mode of chemical reaction.

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• Light curing and microwave curing PMMA resins are derived partly from
urethane dimethacrylate (UDMA) and ethylene glycol dimethacrylate
(EGDMA) in addition to PMMA.

 The predominant base monomer used in commercial dental composites is

BIS-GMA, which due to its high viscosity is mixed with other
dimethacrylates, such as TEGDMA (Triethylene glycoldimethacrylate),
UDMA (Urethane dimethacrylate) or other monomers.

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 A common component in most bonding agents in dentistry is 2-
hydroxyethyl methacrylate (2-HEMA) which holds the composite filling to
the residual tooth, creating a chain cross-link during the photo
polymerization process.

 Methacrylate-Based Resin Sealers (MBRS) used as obturation materials for

endodontic treatment of teeth have 4 generations.
- The first generation of MBRS is (poly[HEMA]).
- The second generation is an UDMA resin-based dual-cured sealer.
- The third generation, mostly contain 2-acrylamido-2-methyl-propanesul-
fonic acid (AMPS) as a functional acidic monomer.
- The fourth generation includes an acidic resin monomer,
methacryloyloxyethyl trimellitate anhydride (4-META).

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 Recently, PEEK polymer has been introduced and used as a coating for
dental implant fixture or for manufacturer of whole implant after being
surface modified to enhance the osseointegration with jaw bone.

 For tissue regeneration and regenerative endodontics, both natural and

synthetic polymers are useful for manufacturer of dental scaffolds. Natural
polymers such as: Collagen, Chitosan, Silk protein, Alginate, and
Hyaluronic acid (HA). Synthetic polymers such as: Polylactic acid (PLA),
Polyglycolic acid (PGA), Polyethylene glycol (PEG), Polylactic-co-glycolic
acid (PLGA), and Polycaprolactone (PCL). They are studied for the
biocompatibility.

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• Biocompatibility of dental polymers is an important clinical
issue.

 The dental polymers that is to be used in the oral cavity should be

harmless to all oral tissues—gingiva, mucosa, pulp, and bone.

 It should contain no toxic, leachable, or diffusible substance that can be

absorbed into the circulatory system, causing systemic toxic responses,
including teratogenic or carcinogenic effects.

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 Requirements for Dental Material
Biocompatibility

 Should not be harmful to pulp & soft tissues

 Should not contain toxic diffusible substances

 Should not produce allergic responses

 Should not be carcinogenic

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 Measuring the biocompatibility

In vitro tests Correlation among in vitro,

• Cytotoxicity tests animal, and usage tests.
• Tests for cell metabolism or cell
function
• Tests that use barriers (indirect Using in vitro, animal, and
tests) usage tests together.
• Other assays for cell function
• Mutagenesis assays
Standards that regulate the
Animal tests
measurement of
Usage tests biocompatib- ility.
• Dental pulp irritation tests
• ANSI⁄ ADA specification 41
• Dental implants in bone
• ISO 10993
• Mucosa and gingival usage tests
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 - In vitro tests

• In vitro tests are done in test tube, cell culture dish, or otherwise out side
a living organism.

• These tests require placement of a material or a component of a material

in contact with a cell, enzyme, or some other isolated biological system.
• The contact can be either direct or indirect.

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In vitro tests can be classified into direct or indirect tests:
 Direct tests: The material contacts the cell system without a barrier.
• Direct tests can be further subdivided into:
 Those in which the material is physically present with the cells
 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

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 Advantages:
• 1. Quick to perform
• 2. Least expensive
• 3. Can be standardized
• 4. Large scale screening
• 5. Good experimental control
• 6. Excellence for mechanism of interactions

 Disadvantages:
• 1. Relevance to in vivo use is questionable
• 2. Lack of inflammatory and other tissue protection mechanisms in the in
vitro environment
• 3. Cannot predict the overall biocompatibility of a material
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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 in- vivo.

 2. Continuous cells or cell lines: are primary cells that have been
transformed previously. 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

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1- Cytotoxicity tests
 Idea: Cytotoxicity tests assess cell death caused by a material by
measuring cell number or growth before and after exposure to the
material.

• It can be assessed by the following formula

= Cell death by material

Cell number before the exposure

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 Method: The 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 (no. of cells per unit area)
may be assessed at different distances from the material, and a zone of
inhibited cell growth may be described. Cell density can be assessed
qualitatively, semiquantitatively, or quantitatively.

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 Control materials, should be well defined and commercially available
to facilitate comparisons among other testing laboratories. Control
materials may be:
• Negative control materials (non cytotoxic materials) as: Teflon and cell
culture treated polystyrene
• Positive control materials (cytotoxic materials) as: Plasticized poly vinyl
chloride

 Another group of tests is used to measure cytotoxicity by a change in

membrane permeability. Membrane permeability tests:

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Membrane permeability tests:
 Measure cytotoxicity by:
• The ease with which a dye can pass through a cell membrane, because
membrane permeability is equivalent to or very nearly equivalent to cell
death.
• Change in the membrane permeability to the dye.
 There are two basic types of dyes used:
1. Vital dyes: Neutral red & Na251CrO4
2. Nonvital dyes: Trypan blue & Propodium iodide
 Advantages of membrane permeability tests: is that it could identify cells
that are alive or dead under microscope. This feature is important because
cells may be physically present, but dead (when materials fix the cells).

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2- Tests for cell metabolism or cell function
 Idea: Use the biosynthetic or enzymatic activity of cells to assess
cytotoxicity of the test material.
• Example: Tests that measures DNA synthesis or protein synthesis

 Method: Synthesis of DNA or protein by cells is usually analyzed by

adding radioisotope-labeled precursors to the medium and quantifying
the radioisotope [3H-thymidine or 3H-leucine] incorporated into DNA or
Protein.
 A commonly used enzymatic test for cytotoxicity is the MTT test [3-(4,5-
Dimethylthiazol-2-yl)-2,5 Diphenyltetrazolium Bromide].

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MTT test:
 Idea: This test measures the activity of cellular dehydrogenases, which
convert a chemical called MTT, via several cellular reducing agents, to a
blue, insoluble formazen compound.
• MTT is a yellow soluble molecule, 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 enzymatic activity.
• If the dehydrogenases are not active because of cytotoxic effects, the
formazen will not form.
 Production of formazen is 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.

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• Other formazen-generating chemicals; NBT, XTT, WST, Alamar Blue dyes.
• Alaram blue tests, are recently developed indicator dyes that could
quantitatively measure cell proliferation using a fluorescent indicator that
allows the continuous monitoring of cells over time.

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3- Tests that use barriers [Indirect tests]
• Are cytotoxicity tests that measure the toxicity when the material is
indirect contact with the cell culture.
• Idea: Researchers have long recognized that in vivo, direct contact often
does not exist between cells and the materials.
• Separation of cells and materials may occur from keratinized epithelium,
dentin, or extracellular matrix.
• Thus, several in vitro barrier tests have been developed to mimic in vivo
conditions. They include:
 - Agar overlay method
 - Millipore filter assay
 - Dentin barrier tests

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 - Agar overlay method
 Method: A monolayer of cultured cells is established before adding 1% agar
or agarose (low melting temp.) plus a vital stain such as neutral red, --to a
fresh culture media.
(Monolayer of cultured cells + neutral red + fresh culture medium)

• The 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 onto filter paper can be tested
with this assay for up to 24hrs.

 Advantages:
o it correlates positively with the direct-contact assays described previously
and the intramuscular implantation test in rabbits
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o Can provide a best qualitative cytotoxic ranking among materials.
 Disadvantages:
o 1- the agar may not adequately represent the barriers that occur in vivo.
o 2- variability of the agarʼs diffusion properties which make it difficult to
correlate the intensity of color or width of the zone around a material
with the concentration of the leachable toxic products.

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 - Millipore filter assay
 Method: this technique establishes a monolayer of cells on filters made of
cellulose esters.
• The culture medium is then replaced with medium containing about 1%
agar, and this mixture is allowed to gel over the cells.
• Finally, the 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
 Advantages: can provide a best qualitative cytotoxic ranking among
materials.
 Disadvantages: arbitrarily influencing the diffusion of leachable products
from the test material.

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 - Dentin barrier tests
• have shown improved correlation with the cytotoxicity of dental materials
in usage tests in teeth, and are gradually being developed for screening
purposes.
• Studies showed that; Dentin forms a barrier through which toxic materials
must diffuse to reach pulp tissue. And the thickness of dentin correlates
directly with the protection offered to the pulp.
 Method: Incorporation of dentin disks between the test samples and the
cell assay system.
• Subsequent steps are illustrated in figure 6.5
• Then the cytotoxicity is measured.

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 Advantages: directional diffusion between the restorative material and
the culture medium.

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4- Other assays for cell function
• In vitro assays to measure immune function or other tissue reactions have
been used.
• They may show promise for being able to reduce the number of animal
tests required to assess the biocompatibility of dental materials.

• 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.

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• Activation of complement is of particular concern to researchers working
on artificial or “engineeredˮ blood vessels and other tissues in direct
contact with blood.
• Materials that activate complement may generate inflammation or
thrombi, and may propagate a chronic inflammatory response.

• Whereas concerns about these types of response to dental materials are

fewer, it is possible that activation of complement by resins or metals or
their corrosion products may prolong inflammation in the gingiva or pulp.

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5- Mutagenesis assays
• These tests assess the effect of dental material on the cellʼs genetic
material (DNA).
 Ames test: is the most widely used short term mutagenesis test and the
only short term test that is thoroughly validated.
• Genetically altered bacteria (Salmonella) are used as test organisms. These
bacteria cannot grow and form colonies on a special culture agar, which is
histidine deficient.
• 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
mutagenicity.

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 Stylesʼ cell transformation test:
• This test on Mammalian cells offers an alternative to bacterial tests (Ames
test).
• Idea: Untransformed fibroblasts normally will not grow with in an agar gel,
where as genetically altered cells can grow.
• This characteristic of transformed fibroblasts is the only characteristic that
correlates with ability of cells to produce tumors in vivo.
 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
• 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.
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 - Animal tests

• Animal tests for biocompatibility are usually used in mammals such as

mice, Rats, Hamsters, Guinea pigs, although other animals have been
used.

• Animal tests are distinct from usage tests (which are also often done in
animals) in that the material is not placed in the animal with regard to its
final use.

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 Advantages:
• 1- Allows many complex systemic interactions (ex, an immune
response or a complement activation)
• 2- The biologic responses are more comprehensive than in vitro tests
• 3- More relevant than in vitro tests
 Disadvantages:
• 1- Expensive
• 2- Time consuming
• 3- Involves significant legal ⁄ethical concerns
• 4- Difficult to interpret and control
• 5- The relevance of the test to the in vivo use of material is questionable,
especially in estimating the appropriateness of an animal species to
represent a human.

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1- Mucous membrane irritation test
• Idea: Determines if a material causes inflammation to mucous
membranes or abraded skin.
• Method: test materials, +ve controls, and -ve controls are all placed into
contact with Hamster cheek pouch tissue or rabbit oral tissue.
• After several weeks of contact, the controls and test sites are examined,
and the gross tissue reactions in the living animals are recorded and
photographed in color
• The animals are then sacrificed, and biopsy specimens are prepared for
histological evaluation of inflammatory changes.

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2- Skin sensitization tests
• The materials are injected intradermally to test for development of skin
hypersensitivity reactions.
• This injection is followed by secondary treatment with adhesive patches
containing the test substance.
• If hypersensitivity developed from the initial injection, the patch will elect
an inflammatory response. A spectrum from no reaction to intense
redness and swelling according to the allergenicity of the test material.

 Two test methods are recommended using guinea pigs:

(i) The maximization test
(ii) The Buehler test

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(i) 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.

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(ii) Buehler test
• 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.

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3- Implantation tests
• Are used to evaluate materials that will contact subcutaneous tissue or
bone.
• Amalgams and alloys are usually tested because the margins of the
restorative materials contact gingiva also, dental implants, endodontic and
periodontic treatment materials.
 Short-term implantation tests:
• Is studied by aseptically placing the compounds in small, open-ended,
polyethylene tubes into the tissue.
• The test samples and controls are placed at separate sites and allowed to
remain for 1 to 11 weeks.
• Alternatively, an empty tube is embedded first, and the inflammatory

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• Reaction from surgery is allowed to subside.
• The implant site is then reopened, and the test material is placed into this
healed site or is packed into the tube that was placed previously.
• At the appropriate time, the areas are excised and prepared for
microscopic examination and interpretation.
• The tissue response can be evaluated by normal histological,
histochemical, or immunohistochemical methods.

 Long-term implantation tests:

• For identification of either chronic inflammation or tumor formation.
• Are performed in a manner similar to that of short-term tests except the
materials remain in place for 1 to 2 years before examination.

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 - Usage Tests

• Usage tests may be done in animals or in human study participants.

• Usage tests in animals usually employ larger animals that have similar oral
environments to humans such as dogs, mini-swine, or monkeys.

• They are distinct from other animal tests because they require that the
material be placed in a situation identical to its intended clinical use.

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• 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 placement
technique.
• If humans are used, the usage test is termed a clinical trial.
• In dentistry, dental pulp, periodontium, and gingival or mucosal tissues are
generally the targets of usage tests.

 The most commonly used usage tests are:

- Dental Pulp Irritation Tests
- Periapical Tissue Damage and Endodontic Usage Test
- Dental Implants in Bone
- Mucosa and Gingival Usage Tests

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 Advantages
• 1- Relevance to use of material is assured
• 2- These tests are the gold standard, in that they give the ultimate answer
to whether or not a material will be biocompatible.
 Disadvantages
• 1- Very expensive
• 2- Very time consuming
• 3- Major legal/ethical issues
• 4- Difficult to interpret and control accurately
• 5- The statistical analysis of these tests is often a daunting process

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1- 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.
• Method: In both cases, class V cavities are prepared as a traumatically
and aseptically as possible and are then filled with the test material. This
approach is equivalent to the future mode of application on patients.
• The thickness of remaining dentin should be measured and recorded.
• These methods can be modified in such a way that the pulp is exposed or
part of the pulp is removed before the material is applied.
• Zinic oxide eugenol (ZOE) and silicate cement could be used as –ve and
+ve control materials, respectively.

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• At the end of the study (after several days to months), the teeth are
removed and sectioned for microscopic examination.
• And the pulps are microscopically evaluated for signs of acute or chronic
inflammation and odontoblast reaction (including dentin neogenesis).
• The inflammatory reactions are classified according to the intensity of the
response into mild, moderate, and severe.
• Limitations: The target organ of the pulp/dentin test is the pulp of
sound (test) teeth and not the “preinflamed” pulp, as is frequently the
case in patients.
• Inflamed pulps may respond differently than normal pulps to liners,
cements, and restorative materials.
• Recently, usage tests on teeth with induced pulpitis, allow evaluation of types and
amount of reparative dentin formed and will probably continue to develop.

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2- Periapical tissue damage and endodontic usage test
• Method: This test is done by application of a given material into the root
canal according to endodontic techniques after a usual root canal
preparation.
• The most common animal models are primates and dogs.
• The compatibility is assessed by histologic evaluation of the periapical
tissues.
• Significance: the stimulating effects on special cells can be determined,
such as the influence of calcium hydroxide compounds on periapical
cementoblasts.
• These tests are especially useful in assessing the claimed bioactive effects
of test materials.

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3- Dental implants in bone
• Method: Materials used for dental implants are inserted into the jaw of
test animals (intraosseous implants).
• Appropriate animals are: primates, dogs, miniature pigs, guinea pigs, and
rats.
• Tissue reaction is assessed histologically, with the tissue in contact with
the implant being of particular interest.
• A good correlation of these findings with patients’ situations can be
expected.

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4- Mucosa and gingival usage tests
• Idea: Because various dental materials contact gingival and mucosal
tissues, the tissue response to these materials must be measured.
• Method: These materials are placed in cavity preparations with
subgingival extensions. The material’s effects on gingival tissues are
observed at 7 days and again after 30 days.
• Tissue responses are categorized as: slight, moderate, or severe,
depending on the number of mononuclear inflammatory cells.
• A difficulty with this type of test 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.

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 Correlation Among In Vitro, Animal, and
Usage Tests
• 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
clinical use.
 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.
 Another example is the inflammatory response of the gingiva at the
gingival and interproximal margins of restorations that also could
accumulate plaque and calculus.

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 However, animal tests are of great value in demonstrating the cytotoxic
effects of materials and evaluating materials that will be used with
alveolar bone and apical periodontal connective tissue.
• 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.
 To compare the results of the three tests (in vitro, animal, and usage tests)
we can place the test material in one of the three different tests and
compare the results.

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 Using In Vitro, Animal, and Usage Tests
Together
• Recently, scientists, industry, and the government have recognized that
the most accurate and cost-effective means to assess biocompatibility of a
new material is a combination of in vitro, animal, and usage tests.
• Implicit in this philosophy is the concept that no single test will be
adequate to completely characterize biocompatibility of a material.
• The ways in which these tests are used together, however, are
controversial and have evolved over many years as knowledge has
increased and new technologies were developed.
 Early and contemporary combination schemes (Fig.1&2), proposed a
pyramid testing protocol in which all materials were tested at the bottom
of the pyramid and materials were weeded out as the testing continue
toward the top of the pyramid. Only materials that pass the first tier of
tests were graduated to the next tier and so on.

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• However, The major problem in both of these testing strategies, is the
inability of the early tests to accurately predict problems with the
materials. Thus, good materials might be screened out and poor materials
might be advanced.
 Two newer testing schemes (Fig. 3; A &B) have evolved in the past 5 years,
suggest a different pyramid testing protocol. With several new and
important ideas:
• 1- All tests (in vitro, animal, and usage) continue to be of value in assessing
biocompatibility of a material during its development and even in its
clinical service.
• 2- They incorporate the philosophy that assessing the biocompatibility of a
material is an ongoing process as the roles of materials change and the
technologies for testing improve.

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 Early combination schemes
• Tests at the bottom of the
pyramid were “unspecific
toxicityˮ tests of any type (in vitro
or animal) with conditions that
did not necessarily reflect those
of the materialʼs use.
• The next tier shows specific
toxicity tests that presumably
dealt with conditions more
relevant to the use of material.
Fig. 1, Early strategy for the use of
• The final tier was a clinical trial of biocompatibility tests
the material.

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Later combination schemes
• Tests at the bottom of the
pyramid were primary tests (in
vivo and in vitro tests) that not
necessarily related to the use of
the material.
• Secondary tests are more
advanced biological tests that
may be partially related to the
use of the material.
• Usage tests are either a clinical
trials in humans or done in Fig. 2, The contemporary strategy
animals used in most standards documents

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Two suggested future strategies for
biocompatibility testing of materials
- A) the pyramid scheme is retained.
The primary and secondary tests will
play a continuing (but decreased)
role as the progress of the testing
continue.
- B) the usage test has the most
stature and the most common
progression of the tests is from
primary to secondary to usage, but
the need to go through several
iterations between testing types is
acknowledged. (Fig. 3; A &B) future strategies

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 Standards that regulate the measurement of
biocompatibility
• Standardization is a difficult and lengthy process, made more difficult by
disagreement on the appropriateness and significance of particular tests.
• One of the early attempts to develop a uniform test for all materials was
developed by Dixon and Rickert in 1933, in which the toxicity of most
dental materials in use at that time was investigated by implanting the
materials into pockets in subdermal tissue, than biopsy specimens was
examined microscopically after 6 months.
• Followed other attempts to standardize the techniques in 1959 and 1976.
 In 1972 the ADA Council on Dental Materials, Instruments, and Equipment
approved specification No. 41 for recommended standard practices for
biological evaluation of dental materials.
• The committee that developed this document recognized the need for
standardized methods of testing and for sequential testing of materials to
reduce the number of compounds that would need to be tested clinically.

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 ANSI/ADA Specification 41
• The ANSI/ADA (American National Standards Institute/American Dental
Association) specification 41 used: initial, secondary, and usage tests. As
shown in (Figure 3; B).
• The initial tests, include in vitro assays for cytotoxicity, hemolysis,
mutagenesis and carcinogenesis at the cellular level, and in vivo acute
physiological distress and death at the level of the organism.
• Based on the results of these initial tests, promising materials are tested
by one or more secondary tests in small animals (in vivo) for inflammatory
or immunogenic potential (e.g., dermal irritation, subcutaneous and bony
implantation, and hypersensitivity tests).
• Finally, materials that pass secondary tests and still hold potential are
subjected to one or more in vivo usage tests, (first in larger animals, often

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• primates, and finally, with Food and Drug Administration approval, in
humans).
• The ANSI/ADA specification No. 41, 1982 addendum, added two assays for
mutagenesis—the Ames test and the Styles’ cell transformation test.

Fig. 3; B

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 ISO 10993
• The international standards organization (ISO 10993) was published in
1992.
• The original ISO 10933 contained 12 parts, each dealing with a different
aspect of biocompatibility testing for all types of medical and dental
devices and materials.
• The standard divided tests into initial and supplementary tests to assess
the biological reaction to materials.
• Initial tests are tests for cytotoxicity, sensitization, and systemic toxicity.
Some of these tests are done in vitro, others in animals in non usage
situations.
• Supplementary tests assessed; chronic toxicity, carcinogenicity, and
biodegradation, are done in animal systems, many in usage situations.

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• Although, guidelines for the selection of tests are given in part 1 of the
standard and are based on how long the material will be present; whether
it will contact body surface only, blood, or bone; and whether the device
communicates externally from the body, The ultimate selection of tests for
a specific material is left up to the manufacturer, who must present and
defend the testing results.

• A recent version of ISO standard is now available from the international

organization of standardization.
• ANSI/ADA Specification No. 41 was also recently revised to confirm the
ISO standard, and was reaffirmed in 2001as ANSI/ADA Specification 41-
1979 (reaffirmed 2001).

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 Biocompatibility of specific dental polymers

• In clinical use of dental polymers, allergic reactions and chemical

irritations have frequently been reported. The incompatibility of dental
materials must be keep in mind especially in patients with oral lesions.

 Denture base polymers induced undesirable reactions have been

attributed to substances leaching from these materials, particularly
unreacted residual monomers.

• When the water permeate into matrix, the leachable unreacted

monomers diffuse out, increased in saliva and may cause adverse
reactions on oral mucosa.

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 Resin-based dental materials are tended to release so many compounds
into aqueous or organic solvents. The contents of composite or adhesive
resin are capable of being leached from the set material. Extraction is
more extensive in organic solvents or alcohol than water.

• Substantial amounts of TEGDMA and HEMA may be released by

polymerized composite resins into water.

• Bis-GMA, UDMA, TEGDMA, EGDMA, DEGDMA, 1,6-hexanediol di-

methacrylate, MMA camphoroquinone, 4-N,N-dimethylaminobenzoic
acid, ethyl ester, and varied other substances have been also defined in
minor concentrations in aqueous extracts.

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• These monomers can modify cellular metabolism at subtoxic
concentrations. The modifications may be responsible for clinical and
subclinical effects.

 Hydrophilic monomers, such as TEGDMA were identified in higher

proportions in aqueous extraction media than BisGMA.

• Furthermore the hydrophilic monomers HEMA and TEGDMA were the

only ones to be able to diffuse through the dentin into the pulp space at
high concentrations that may cause harmful effects to the pulpal
homeostasis and repair.

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 Bis-GMA is the most toxic dental monomer. The cytotoxic mechanism is
modification of the lipid layer of the cell membrane which changes
membrane permeability.

• Water-soluble methacrylic acid produced by the hydrolysis of bis-GMA,

can lead to cytotoxicity by increasing the release of tumor necrosis factor
alpha.

 An amphoteric monomer HEMA can displace water in dentin and easily

diffuse through the dentin. Adhesive systems containing HEMA could have
an increased cytotoxicity. HEMA is an effective mediator of apoptotic cell
death. It could also decrease cellular proliferation and lead to apoptosis,
likely via DNA damage.

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 Dental adhesives and composite components may modify the succinate
dehydrogenase activity of primary cells or immortal cell lines.

 When evaluating biocompatibility of PMMA resins and their leached out

components. PMMAs are prevalently used as denture base materials and
liners. Respecting leaching of elutes from PMMA resins, they may cause
local or systemic toxicities, microbial side effects, oral mucosa and gingival
irritations, allergic reactions, mutagenicity and carcinogenicity.

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 Recently, it is shown that 4-Acryloyloxyethyl trimellitate
anhydride/methyl methacrylate-tri-nbutylborane (4-META/MMA-TBB) is
more biocompatible than other luting materials, because of its nontoxic
polymerization properties.

• (i) The 4-META/MMA-TBB resin underwent fast and highly rated

polymerization with lower free radical production than PMMA or
MDPDMA resins.
• (ii) Production of free radicals from 4-META/MMA-TBB was as low as from
glass ionomer cement.
• (iii) The percentage of viable dental pulp cells was quite higher on
MDPDMA and 4-META/MMA-TBB resin than on glass ionomer cement.
• (iv) META/MMA-TBB resin added to its uniquely convenient biochemical
property during polymerization.

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 Vascular actions of dental polymers

• HEMA and TEGDMA monomers incorporated in most dentin bonding

agents may induce vasodilation that might impair pulpal healing by
promoting hemorrhage in iatrogenic pulp microexposures.

• Moreover, there is a possibility that these compounds also can be released

from dental appliances and reach the systemic circulation to produce
effects on other blood vessels. the key mechanism behind the vasodilatory
action of dental polymers is through the calcium antagonistic action.

• further studies, are essential to understand the exact mechanism of the

vasodilatory effect of dental polymers and to fully realize their implications
in clinical dental practice.

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 Different studies on biocompatibility of dental
polymers
1-

Introduction
• Flowable resin composites have been indicated as bioprotective materials
on orthodontic mini-implant heads to prevent the implants from
traumatizing soft tissues.
• However, the leachable components from the resins, such as bisphenol A-
diglycidyl dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate
(TEGMA), and urethane dimethacrylate (UDMA) found in most resins,
have been shown to have a definite cytotoxic effect.
• Because of the proximity of miniimplants to the gingiva and other oral
tissues, a similar effect could occur in these tissues.
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Materials and Methods:
• Forty-eight male Wistar rats were divided into four groups (n = 12).
• Group Control (polyethylene), Group Wave, Group Top Comfort, and
Group Filtek.
• The animals were sacrificed after time intervals of 7, 15, and 30 days and
tissues were analyzed under optical microscopy for inflammatory infiltrate,
edema, necrosis, granulation tissue, multinucleated giant cells, and
collagen formation.
• The biocompatibility and degree of conversion were evaluated by the
Kruskal-Wallis and Dunn tests.

73
Results:

74
2-

Introduction:
• This master thesis seeks to explore the possible hazards dental patients
are exposed to when receiving treatment with resin-based materials.
• Dentists work with polymer resin-based materials every day. Composite
filling materials, bonding, resin based glassionomer cements and polymers
for prosthodontic and orthodontic applications come into this category.
• All of these materials contain highly reactive substances in advance of
polymerization.
• The most common dental monomers incorporated are; MMA, HEMA,
EGDMA, UDMA, TEGDMA, Bis-GMA, and Bisphenol A.

75
Aims:
• The aims of the present study were to investigate the probability of
occurrence of adverse reactions to resin based materials in dental
practice, and to seek for actions that can be done to diminish this
problem.
Materials and methods:
• Composite and acrylic resin discs were prepared and tested using in vitro,
animal, and usage tests.
• The results were summarized in the following table.

76
Results:

77
78
3-

Introduction:
• Acrylic resin (PMMA – Polymethylmethacrylate) is a widely used
material in dentistry; in temporary crowns, denture fabrication, reline
and repair, orthodontic appliances, splints for orthognathic surgery and
others, this material should have the appropriate physical, chemical and
biological properties, and a satisfactory biocompatibility is essential.
The aim of this study:
• Was to evaluate the cytotoxicity caused by the denture base acrylic
resin used, and its components.

79
Materials and methods:
• Different methods are used for cytotoxicity analysis. Among them, the
most common is the MTT test, XTT assay, Enzyme-linked immunosorbent
assay (ELISA)
Results:
• According to the results obtained, there is not a non-cytotoxic acrylic resin
available on the dental market.
• Regarding the polymerization method, the auto-polymerized resin is more
cytotoxic than heat-polymerized resin.
• The cytotoxic effect is dose dependent and depends on the amount of
residual monomers. Residual monomers are responsible for inflammatory
reactions of tissues in contact with the acrylic resin.

80
 References

1- Craig΄s Restorative Dental Materials

2- Kanerva, L., Cross-reactions of multifunctional methacrylates and acrylates. Acta Odontol
Scand, 2001. 59(5): p. 320-9.
3- Schmalz, G. and D. Arenholt Bindslev, Biocompatibility of dental materials. 2009, Berlin:
Springer. XVI, 379 s.
4- Fleisch, A.F., et al., Bisphenol A and related compounds in dental materials. Pediatrics,
2010. 126(4): p. 760-8.
5- Tillberg, A., B. Stenberg, and A. Berglund, Reactions to resin-based dental materials in
patients--type, time to onset, duration, and consequence of the reaction. Contact
Dermatitis, 2009. 61(6): p. 313-9.
6- Marino, R., et al., Burning mouth syndrome: the role of contact hypersensitivity. Oral Dis,
2009. 15(4): p. 255-8.
7- Vamnes, J.S., et al., Four years of clinical experience with an adverse reaction unit for
dental biomaterials. Community Dent Oral Epidemiol, 2004. 32(2): p. 150-7.
8- Lygre, G.B., et al., Reporting on adverse reactions to dental materials--intraoral
observations at a clinical follow-up. Community Dent Oral Epidemiol, 2003. 31(3): p. 200-6.

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Thank you

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