INTRODUCTION: Although a wide spectrum of materials, devices exists in diverse surgical disciplines as ophthalmology, cardiology, neurology, orthopedics, and dentistry they all have one thing in common. They must have intimate contact with human tissues, providing real, classical physical interface. The term biocompatibility has come into existence with reference to the study of interaction of various materials with human tissues. DEFINITION: “It is the ability of a material to elicit an appropriate biological response in a specific application”. William D,F 1987 KEY CONCEPTS OF BIOCOMPATIBILITY Biomaterials are not biologically inert: Practitioners should understand that there are no “inert” materials. When a material is placed into living tissue, interactions with the complex biologic systems around it occur, and those interactions result in some sort of biologic
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INTRODUCTION:
Although a wide spectrum of materials, devices exists in diverse
surgical disciplines as ophthalmology, cardiology, neurology, orthopedics,
and dentistry they all have one thing in common. They must have intimate
contact with human tissues, providing real, classical physical interface. The
term biocompatibility has come into existence with reference to the study of
interaction of various materials with human tissues.
DEFINITION:
“It is the ability of a material to elicit an appropriate biological
response in a specific application”.
William D,F 1987
KEY CONCEPTS OF BIOCOMPATIBILITY
Biomaterials are not biologically inert:
Practitioners should understand that there are no “inert” materials.
When a material is placed into living tissue, interactions with the complex
biologic systems around it occur, and those interactions result in some sort
of biologic response. The interactions depend on the material, the host, and
the forces and conditions placed on the material (its function). Regardless,
the material affects the host and the host affects the material. Inertness of
materials implies an absence of such interactions. Most scientists today
agree that no material is truly inert in the body.
Biocompatibility is a dynamic process:
Biocompatibility is a dynamic, ongoing process, not a static one. A
dental implant that is osseointegrated today may or may not be
osseointegrated in the future. The response of the body to a material is
dynamic because the body may change through disease or aging, the
material may change through corrosion or fatigue, or the loads placed on the
material may change through changes in the occlusion or diet. Any of these
changes may alter the conditions that initially promoted an appropriate and
desired biologic response. The interactions among material, host, and
function continue over time; therefore, the biologic response to a material is
an ongoing process.
Biocompatibility is a property of a material and its environment:
Biocompatibility is a property not only of a material, but also of a
material interacting with its environment. In this sense, biocompatibility is
like color. We often ascribe color to a material, but color is a property of
both the material and the material’s interaction with light (its environment).
Without the light interaction, there is no color. Ultimately, the color of the
material depends on the light source, how the light interacts with the
material, and the bias of the observer.
Consider an example of this concept with dental implants. Under the
proper conditions, titanium alloy implants will osseointegrate with the bone
over time. This means that the bone will approximate to within 100 of the
implant with no intervening fibrous tissue. If a cobalt-chromium alloy is
placed into the same situation – same host, same placement technique,
same load – no osseointegration will occur. Conversely, if a titanium alloy
is used as the ball portion of a femoral hip joint, it will wear against the
acetabulum into small particles that ultimately will cause the hip to fail. Yet
the cobalt-chromium alloy which wears less, will do better. Because
biocompatibility depends on the interaction of the material with its
environment, it is inappropriate to label the titanium alloy a “biocompatible
material” and the cobalt-chromium alloy an “incompatible material”. One
cannot define the biocompatibility of a material without defining the
location and function of the material.
The complexities mentioned in the previous paragraphs may leave the
practitioner wondering about the relevance of the definition of
biocompatibility to dental practice. Yet there are profound consequences of
this definition for practitioners. For example, the practitioner must always
consider the health and habits of the patient when assessing the biologic
response, to materials. Is the patient diabetic? Does the patient smoke? If so,
the response of the gingiva to placement of a crown may be affected. Does
the patient drink many acidic liquids? The corrosion properties of partial
denture alloys and the tissue response may be different. The practitioner
must consider what he or she is asking the material to do and must not
assume that, because a material is biologically acceptable in role, it will be
acceptable in a different role. Result materials that are biologically
acceptable as resin-based cements. Finally, the practitioner must monitor the
patient over time. A patient who is not allergic to today may become
allergic in the future, from oral exposure or exposure through other sources
(jewelry, for example).
HOW IS BIOCOMPATIBILITY RELEVANT TO DENTISTS?
Dentists’ potential concerns about biocompatibility can be organized
into 4 areas; safety of the patient safety of the dental staff, regulatory
compliance issues and legal liability.
Safety of the patient:
One of the primary concerns of any dental practitioner is to avoid
harming the patient. Evidence has shown that, although adverse reactions to
dental materials are not common, they can occur for many type of materials,
including alloys, resins, and cements. National registry in Norway, where
there are lion people and 3800 dentists, reported 674 adverse reactions to
dental materials from 1993 to 1997. These adverse events occurred locally
and systemically and involved all classes of dental materials. A 1 in 260
frequency of problems has been reported for dental casting alloys, including
nickel-based alloys. Yet the number of adverse reactions may be
underreported, and existing reports may not be accurate. Better
documentation of the extent of these reactions is needed. One
biocompatibility / patient safety issue that has been prominent in dentistry in
recent years is the hypersensitivity of patients to dental biomaterials.
Classically, this concern has focused on allergy to materials such as nickel
or methacrylates. The incidence of nickel allergy in the general population
ranges between 10% and 20% and is far more common in females than
males. In patients sensitive to nickel, oral exposure to nickel may or may not
elicit an allergic response, but these responses may be spectacular. There is
also crowing concern about the hypersensitivity of patients to resin-based
materials and to latex. Although uncommon, patients can have severe or
even fatal (anaphylactic) reactions to these materials. There have been
recent reports of a growing incidence of contact sensitivity in children to a
variety of substances, including dental materials. One report found that 49%
of children and sensitivity to some type of material or food. The growing
allergic history in children may portend an increasing need for dentists to be
more aware of material allergies in adults in the future.
Thus, there is evidence that the biomaterials used by dental
practitioners can pose safety risks to patients. However, the evidence about
harmful effects from materials is, more often than not, equivocal or
incomplete. It therefore is every practitioner’s responsibility to decide
whether the existing evidence has merit and to assess the risks of these
issues in his or her own practice, taking into account each patient’s unique
history.
Safety of the dental staff:
In many situations, the risk of adverse effects of biomaterials is much
higher for the dental staff than for the patient. The staff may be chronically
exposed to material when they are being manipulated or setting. The classic
example of this problem is dental amalgam because the release of mercury
vapor from amalgam during placement or removal is substantially higher
than when it is undisturbed in the mouth. However, these types of issues
also are relevant to casting alloys, resins, and other dental materials used in
prosthodontics. For example, risks for dental staff appear to result from
chronic contact with latex – and resin-based materials. Adverse effects from
these materials range from cumulative irritation to allergenic responses.
Ironically, gloves do not protect against contact with some materials
because the materials are capable of moving through gloves. Furthermore,
the gloves themselves may be a source of the problem. One study in Sweden
reported that 15% of dentists (vs 9% in the general population) experienced
itching on the hands in response to gloves, particularly latex gloves. Another
study reported that 6.2% of dental staff were allergic to latex.
The mechanisms by which materials cause problems through chronic
exposure are not known, but there is evidence that some resin components
such as 2-hydroxyethyl methacrylate (HEMA), tetraethylene glycol
dimethacrylate (TEGDMA), and camphoroquinone are capable of directly
activating immune cells. If the dental team also grinds laboratory materials
such as acrylic resin, metals, or gypsum, then there is a risk from inhalation
of particulates. Because particles less than 10 m in diameter cannot be
filtered by the respiratory system, it is prudent to wear a mask when
grinding or polishing any material. If laboratory personnel cast alloys
containing beryllium, precautions must be taken to limit exposure to vapor
from the alloy, as the vapor can cause berylliosis in the lungs. Reactions to
many types of prosthodontic materials can be severe, career-threatening, and
even life-threatening in rare instances.
Regulatory compliance issues:
Biocompatibility issues are closely linked to regulations that affect
dental practice. An example of this link is related to dental amalgam.
Because of the biologic concerns about mercury, regulators have considered
monitoring and restricting the amount of mercury in waste water from
dental practices. This debate has spurred considerable research on methods
to eliminate particulate and elemental mercury from dental waste. Another
example is the use of later. Because of allergic reactions to latex (discussed
previously), several US state legislatures have debated but not passed bans
on latex gloves in dental and medical practice.
Legal liability:
Biocompatibility issues also influence liability issues that affect
dental practitioners. Because dental materials can affect the well-being of
patients and dental auxiliaries, practitioners assume a legal risk when using
these materials. Litigation as a result of biomaterials causing harm to a
patient is probably rare. Nevertheless, when these problems occur, they are
(at best) emotionally and financially stressful to the practitioner.
MEASURING BIOCOMPATIBILITY:
Measuring the biocompatibility of a material is not simple, and the
methods of measurement are evolving rapidly as more is known about the
interactions between dental materials and oral tissues and as technologies
for testing improve. Historically, new materials were simply tried in humans
to see if they were biocompatible. However, this practice has not been
acceptable for many years, and current materials must be extensively
screened for biocompatibility before they are ever used in humans. Several
varieties of tests are currently used to try to ensure that new materials are
biologically acceptable. These tests are classified as in vitro, animal, and
usage tests. These three testing types include the clinical trial, which is
really a special case of a usage test in humans. The remainder of this section
will discuss several of each type of test, their advantages and disadvantages,
how the tests are used together, and standards that rely on these tests to
regulate the use of materials in dentistry.
IN VITRO TESTS:
In vitro tests for biocompatibility are done in a test tube, cell-culture
dish, or otherwise outside of 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, where the material contacts the cell system without barriers, or
indirect, where there is a barrier of some sort between the material and the
cell system. Direct tests can be further subdivided into those in which the
material is physically present with the cells and those in which some extract
from the material contacts the cell system. In vitro tests can be roughly
subdivided into those that measure cytotoxicity or cell growth, those that
measure some metabolic or other cell function, and those that measure the
effect on the genetic material in a cell (mutagenesis assays). Often there is
some overlap in what a test measures. In vitro tests have a number of
significant advantages over other types of biocompatibility tests. They are
relatively quick to perform, generally cost much less than animal or usage
tests, can be standardized, are well suited to large scale screening, and can
be tightly controlled to address specific scientific questions. The overriding
disadvantage of in vitro tests is their questionable relevance to the final in
vivo use of the material (see later section on correlation between tests).
Other disadvantages include the lack of inflammatory and other tissue-
protective mechanisms in the in vitro environment. It should be emphasized
that in vitro tests alone cannot usually predict the overall biocompatibility of
a material.
Standardization of in vitro tests is a primary concern of those trying to
evaluate materials. Two types of cells can be used for in vitro assays.
Primary cells are cells taken directly from an animal into culture. These
cells will grow for only a limited time in culture but may retain many of the
characteristics of cells in vivo. Continuous cells are primary cells that have
been transformed to allow them to grow more or less indefinitely in culture.
Because of their transformation, these cells may not retain all in vivo
characteristics, but they consistently exhibit any features that they do retain.
Primary cell cultures would seemingly be more relevant than continuous
cell lines for measuring cytotoxicity of materials. However, primary cells
have the problems of being from a single individual, possibly harboring
viral or bacterial agents that alter their behavior, and often rapidly losing
their in vivo functionality once placed in a cell culture. Furthermore, the
genetic and metabolic stability of continuous cells lines contributes
significantly toward standardizing assay methods. In the end, both primary
and continuous cells play an important role in invitro testing; both should be
used to assess a material.
Cytotoxicity tests:
Cytotoxicity tests assess the cytotoxicity of a material by measuring
cell number of growth after exposure to a material. Cells are plated in a well
of a cell-culture dish where they attach. The material is then placed in the
test system. If the material is not cytotoxic, the cells will remain attached to
the well and will proliferate with time. If the material is cytotoxic, the cells
may stop growing, exhibit cytotoxic, the cells may stop growing, exhibit
cytopathic features or detach from the well. If the material is a solid, then
the density (number of cells per unit area) of cells 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, semi-
quantitatively, or quantitatively. Substances such as Teflon can be used as
negative (non-cytotoxic) controls, whereas materials such as plasticized
polyvinyl chloride can be used as positive (cytotoxic) controls. Control
materials should be well defined and commercially available to facilitate
comparisons among testing laboratories.
Another group of tests is used to measure cytotoxicity by a change in
membrane permeability. Membrane permeability is the ease with which a
dye can pass through a cell membrane. This test is used on the basis that a
loss in membrane permeability is equivalent to or very nearly equivalent to
cell death. The advantage of the membrane permeability test is that it
identifies cells that are alive (or dead) under the microscope. This feature is
important because it is possible for cells to be physically present, but dead
(when materials fix the cells). There are two basic types of dyes used. Vital
dyes are actively transported into viable cells, where they are retained unless
cytotoxic effects increase the permeability of the membrane. It is important
to establish that the dye itself does not exhibit cytotoxicity during the time
frame of the test. Nonvital dyes are not actively transported, and are only
taken up if membrane permeability has been compromised by cytotoxicity.
Many types of vital dyes have been used, including neutral red and Na2
CrO4. The use of neutral red and Na2 CrO4 are particularly advantageous
because they are neither synthesized nor metabolized by the cell examples
of nonvital dyes include try pan blue and propodium iodide.
Tests for Cell Metabolism or Cell Function:
Some in vitro tests for biocompatibility use the biosynthetic or
enzymatic activity of cells to assess cytotoxic response. Tests that measure
deoxyribonucleic acid (DNA) synthesis or protein synthesis are common
examples of this type of test. The synthesis of DNA or protein by cells is
usually analyzed by adding radioisotope-labeled precursors to the medium
and quantifying the radioisotope (e.g., H-thymidine or H-leucine)
incorporated into DNA or protein. A commonly used enzymatic test for
cytotoxicity is the MTT test. This test measures the activity of cellular
dehydrogenases, which convert a chemical called MTT, via several cellular
reducing agents, to a blue, insoluble formazan compound. If the
dehydrogenases are not active because of cytotoxic effects, the formazan
will not form. The production of formazan can be quantified by dissolving it
and measuring the optical density of the resulting solution. Alternatively,
the formazan can be localized around the test sample by light or electron
microscopy. Other formazan-generating chemicals have been used,
including NBT, XTT, and WST. Furthermore, many other activities of cells
can be followed qualitatively or quantitatively in vitro. Recently, in vitro
tests to measure gene activation, gene expression, cellular oxidative stress,
and other specific cell functions have been proposed. However, these types
of tests are not yet routinely used to assess the biocompatibility of materials.
Tests that use barriers (Indirect Tests):
Most of the cytotoxicity tests presented thus far are performed with
the material in direct contact with the cell culture. 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. One such test is the agar
overlay method in which a monolayer of cultured cells is established before
adding 1% agar or agarose (low melting temperature) plus a vital stain, such
as neutral red, to fresh culture media. The agar forms a barrier between the
cells and the materials, which is placed on top of the agar. Nutrients, gas,
and soluble toxic substances can diffuse through the agar. Solid test samples
or liquid samples adsorbed onto filter paper can be tested with this assay for
up to 24 hours. This assay correlated positively with the direct-contact assay
correlates positively with the direct-contact assays described above and the
intramuscular implantation test in rabbits. However, the agar may not
adequately represent barriers that occur in vivo. Furthermore, because of
variability of the agar’s diffusion properties, it is difficult to correlate the
intensity of color or width of the zone around a material with the
concentration of leachable toxic products.
A second barrier assay is the Millipore filter assay. 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 monolyer-
gel is detached and turned over so that the filler is on top for placement of
solid or soluble test samples for 2 or more hours. After exposure to the test
samples, the filter is removed and an assay is used to determine the effect of
the sample on a cellular metabolic activity. The succinyl dehydrogenase
assay described previously can be used with this test. Like the agar overlay
test and the cell contact tests, toxicity in the Millipore filter test is assessed
by the width of the cytotoxic zone around each test sample. This test also
has the drawback of arbitrarily influencing the diffusion of leachable
products from the test material. The agar diffusion and Millipore filter tests
can provide, at best, a cytotoxic ranking among materials.
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. A number of studies have shown
that dentin forms a barrier through which toxic materials must diffuse to
reach pulpal tissue. Thus pulpal reaction to zinc oxide-eugenol is relatively
mild as compared with the more severe reactions to the same material in
direct contact with cells in in vitro assays and tissue in implantation tests.
The thickness of the dentin correlates directly with the protection offered to
the pulp. Thus assay have been developed that incorporate dentin disks
between the test sample and the cell assay system. The use of dentin disks
offers the added advantage of directional diffusion between the restorative
material and the culture medium.
Other Assays for Cell Function:
In vitro assays to measure immune function or other tissue reactions
have also been used. The in vivo significance of these assays is yet to be
ascertained, but many show promise for being able to reduce the number of
animal tests required to assess the biocompatibility of a material. These
assays measure 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 cycle or
activate complement. The activation of complement is of particular concern
to researchers working on artificial or “engineered” 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 complement activation by
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.
Mutagenesis assays :
Mutagenesis assay assess the effect of materials on a cell’s genetic
material. There is a wide range of mechanisms by which materials can affect
the genetic material of the cell. Genotoxic mutagens directly alter the DNA
of the cell through various types of mutations. Each chemical may be
associated with a specific type of DNA mutation. Genotoxic chemicals may
be mutagens in their native states, or may require activation or
biotransformation to be mutagens, in which case they are called
promutagens. Epigenetic mutagens do not alter the DNA themselves, but
support tumor growth by altering the cell’s biochemistry, altering the
immune system, acting as hormones, or other mechanisms. Carcinogenesis
is the ability to cause cancer in vivo. Mutagens may or may not be
carcinogens, and carcinogens may or may not be mutagens. Thus the
quantification and relevance of tests that attempt to measure mutagenesis
and carcinogenesis are extremely complex. A number of government-
sponsored programs evaluate the ability of in vitro mutagenesis assays to
predict carcinogenicity.
The Ames’ test is the most widely used short-term mutagenesis test
and the only short-term test that is considered thoroughly validated. It uses
mutant stocks of Salmonella typbimurium that require exogenous histidine.
Native stocks of bacteria do not require exogenous histidine. Exclusion of
histidine from the culture medium allows a chemical to be tested for its
ability to convert the mutant strain to a native strain. Chemicals that
significantly increase the frequency of reversion back to the native state
have a reportedly high probability of being carcinogenic material.
Performance of this test requires experience in the field and special strains
of salmonella to produce meaningful results. Several stains of salmonella
are used, each to detect a different type of mutation transformation.
Furthermore, chemicals can be “metabolized” in vitro using homogenates of
liver enzymes to simulate the body’s action on chemicals before testing for
mutagenicity.
A second test for mutagenesis is the Styles’ Cell Transformation test.
This test on mammalian cells was developed to offer an alternative to
bacterial tests (Ames test), which may not be relevant to mammalian
systems. This assay quantifies the ability of potential carcinogens to
transform standardized cell lines so they will grow in soft agar.
Untransformed fibroblasts normally will not grow within an agar gel,
whereas genetically transformed cells will grow below the gel surface. This
characteristic of transformed fibroblasts is the only characteristic that
correlates with the ability of cells to produce tumors in vivo. at least four
different continuous cell lines have been used. In 1978, Styles claimed 94%
“accuracy in determining carcinogenic or noncarcinogenic activity” when
testing 120 compounds in two cell lines. However, there has been some
difficulty in reproducing these results.
In a recent report, four short-term tests (STTs) for gene toxicity were
compared. The Ames test was the most specific (86% of non-carcinogens
yielding a negative result). The Ames test also had the highest positive
predictability (83% of positives were actually carcinogens) and displayed
negative predictability equal to that of other STTs (i.e. 51% of all Ames test
negatives were noncarcinogenic). However, the results were in agreement
(concordance) with rodent carcinogenicity tests for only 62% of the
chemicals. Also, the Ames test was sensitive to only 4% of the carcinogens;
that is it, missed over half of the known carcinogens. The other three STTs
were assays for chromosomal aberration, sister chromatid exchanges in
CHO cells, and the mouse lymphoma 1.5178Y cell mutagenesis assay. The
sister chromatid exchange method, the mouse lymphoma mutagenesis assay,
and the Ames test had 73%, 70%, and 45% sensitivity, respectively.
However, because the Ames test is widely used, extensively described in the
literature, and technically easier to conduct in a testing laboratory than the
other tests, it is most often conducted in a screening program. These studies
suggest that not all carcinogens are genotoxic (mutagenic) and not all
mutagnes are carcinogenic. Thus, although STTs for mutagenesis are
helpful for predicting some carcinogens, STTs cannot predict all of them.
ANIMAL TESTS :
Animal tests for biocompatibility are usually used in mammals such
as mice, rats, hamsters, or guinea pigs, although many types of 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. The use of an animal allows many complex interactions
between the material and a functioning, complete biological system to
occur. For example, an immune response may occur or complement may be
activated in an animal system in a way that would be difficult to mimic in a
cell-culture system. Thus the biological responses in animal tests are more
comprehensive and may be more relevant than in vitro tests, and these are
the major advantages of these tests. The main disadvantages of animal tests
are that they can be difficult to interpret and control, are expensive, may be
time consuming, and often involve significant ethical concerns and paper
work. 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. A variety of animal tests have been
used to assess biocompatibility, and a few are discussed in detail below.
The mucous membrane irritation test determines if a material causes
inflammation to mucous membranes or abraded skin. This test is conducted
by placing the test materials and positive and negative controls 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.
In the skin sensitization test in guinea pigs (guinea pig maximization
test), the materials are injected intradermally to test for development of skin
hypersensitivity reactions. Freund’s adjuvant can be used to augment the
reaction. This injection is followed by secondary treatment with adhesive
patches containing the test substance. If hypersensitivity developed from the
initial injection, the patch will elicit an inflammatory response. The skin-
patch test can result in a spectrum from no reaction to intense redness and
swelling. The degree of reaction in the patch test and the percentage of
animals that show a reaction are the bases for estimating the allergenicity of
the material.
Animal tests that measure the mutagenic and carcinogenic properties
of materials have been developed by toxicologists. These (tests are
employed with a strategy called the decision-point approach. Using this
strategy, tests are applied in a specific order, and testing is stopped when
any one indicates mutagenic potential of the material or chemical. The
validity of any of these tests may be affected by issues of species, tissue,
gender, and other factors. Tests are generally divided into limited-term in
vivo tests and long-term or lifetime tests. Limited-term in vivo tests measure
altered liver function or increased tumor induction when animals are
exposed to the chemicals for a fraction of their lifetimes. Long-term in vivo
tests are performed by keeping the chemical in contact with the animal over
the majority of its lifetime.
Implantation tests are used to evaluate materials that will contact
subcutaneous tissue or bone. The location of the implant site is determined
by the use of the material, and may include connective tissue, bone or
muscle. Although amalgams and alloys are tested because the margins or
the restorative materials contact the gingival, most subcutaneous tests are
used for materials that will directly contact soft tissue during implantation,
endodontic, or periodontal treatment. Short-term implantation 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 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.
Implantation tests of longer duration, for identification of either chronic
duration, 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.
USAGE TESTS :
Usage tests may be done in animals or in human volunteers. 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 of a
usage test 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. 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 tests is identical to a clinical trial. The overwhelming
advantage for a usage test is its relevance. These tests are the gold standard
of tests in that they give the ultimate answer to whether a material will be
biocompatible. One might ask, then why bother with in vitro or animal tests
at all. The answer is in the significant disadvantages of the usage test. These
tests are extremely expensive, last for long periods, involve many ethical
and often legal concerns, and are exceptionally difficult to control and
interpret accurately. The statistical analysis of these tests is often a daunting
process. In dentistry, dental pulp, periodontium, and gingival or mucosal
tissues are generally the targets of usage tests.
Dental pulp irritation tests :
Generally, materials to be tested on the dental pulp are placed in
class-5 cavity preparations in intact, noncarious teeth of monkeys or other
suitable animals. Care is taken to prepare uniformly sized cavities. After
anesthesia and a thorough prophylaxis of teeth, cavities are prepared under
sterile conditions with an efficient water-spray coolant to ensure minimal
trauma to the pulp. The compounds are placed in an equal number of
anterior and posterior teeth of the maxilla and mandible to ensure uniform
distribution in all types of teeth. The materials are left in place from 1 to 8
weeks. Zinc oxide – eugenol and silicate cement have been used as negative
and positive control materials, respectively.
At the conclusion of the study, the teeth are removed and sectioned
for microscopic examination. The tissue sections are evaluated by the
investigators without knowledge of the identity of the materials, and
necrotic and inflammatory reactions are classified according to the intensity
of the response. The thicknesses of the remaining dentin and reparative
dentin for each histological specimen is measured with a photomicrometer
and recorded. The response of the pulp is evaluated based n its appearance
after treatment. The severity of the lesions is based on disruption of the
structure of the tissue and the number of inflammatory cells (usually both
acute and chronic) present. Pulpal response is classified as either slight
(mild hyperemia, few inflammatory cells, slight hemorrhage in
odontoblastic zone), moderate (definite increase in number of inflammatory
cells, hyperemia, and slight disruption of odontoblastic zone), or severe
(decided inflammatory infiltrate, hyperemia, total disruption of
odontoblastic layer in the zone of cavity preparation, reduction or absence
of predentin, and perhaps even localized abscesses). As with dental caries,
the mononuclear cells are usually most prominent in the inflammatory
response. If neutrophils are present, the presence of bacteria or bacterial
products must be suspecteds. Some investigators now use zinc oxide-
eugenol (ZOE) cements to “surface-seal” the restorations to eliminate the
effects of microleakage on the pulp.
Until recently, most dental-pulp irritation tests have involved intact,
noncarious teeth, without inflamed pulps. There has been increased concern
that inflamed dental pulp tissue may respond differently than normal pulps
to liners, cements, and restorative agents. Efforts have been made to develop
techniques that identify bacterial insults to the pulp. Usage tests that study
teeth with induced pulpitis allows evaluation of types and amount of
reparative dentin formed and will probably continue to be developed.
Dental Implants into Bone :
At present, the best estimations of the success and failure of implants
are gained from three tests: (1) penetration of a periodontal probe along the
side of the implant, (2) mobility of the implant, and (3) radiographs
indicating either osseous integration or radiolucency around the implant.
Currently, an implant is considered successful if it exhibits no mobility, no
radiographic evidence of peri-implant radiolucency, minimal vertical bone
loss, and absence of persistent peri-implant soft tissue complications.
Previously, investigators argued that formation of a fibrous connective
tissue capsule around a subperiosteal implant or root cylinder was the
natural reaction of the body to a material. They argued that this was actually
an attachment similar to the periodontal ligament and should be considered
a sign of an acceptable material. However, in most cases it resembled the
wall of a cyst, which is the body’s attempt to isolate the implanted material
as the material slowly degrades and leaches its components into tissue.
Currently, for implants in bone, implants should be completely encased in
bone, the most differentiated state of that tissue. Fibrous capsule formation
is a sign of irritation and chronic inflammation.
Mucosa and Gingival Usage Tests :
Because various dental materials contact gingival and mucosal
tissues, the tissue response to these materials must be measured. Materials
are placed in cavity preparations with subgingival extensions. The
materials’ effects on gingival tissues are observed at 7 days and again after
30 days. Responses are categorized as slight, moderate, or severe. A slight
response is characterized by a few mononuclear inflammatory cells (mainly
lymphocytes) in the epithelium and adjacent connective tissue. A moderate
response is indicated by numerous mononuclear cells in the connective
tissue and a few neutrophils in the epithelium. A severe reaction evokes a
significant mononuclear and neutrophilic infiltrate and thinned or absent
epithelium.
A difficulty with this type of study is the frequent presence of some
degree of preexisting inflammation in gingival tissue. Bacterial plaque is the
most important factor in causing this inflammation. Secondary factors are
the surface roughness of the restorative material, open or overhanging
margins, and overcontouring or undercontouring of the restoration. One way
to reduce the interference of inflammation caused by plaque is to perform
dental prophylaxis before preparing the cavity and placing the material.
However, the prophylaxis and cavity preparation will themselves cause
some inflammation of the soft tissues. Thus , if margins are placed
subgingivally, time for healing (typically 8 to 14 days) must be allowed
before assessing the effects of the restorative agents.
CORRELATION AMONG IN VITRO, ANIMAL AND USAGE
TESTS :
In the filed 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,
the lack of correlation is not surprising in light of the differences among
these tests. In vitro and animal tests often measure aspects of the biological
response that are more subtle or less prominent than in a material’s clinical
usage. Furthermore, barriers between the material and tissues may exist in
usage tests or clinical use that may not exist in in vitro or animal tests. Thus
it is important to remember that each type of test has been designed to
measure different aspects of the biological response to materials, and
correlation may not always be expected.
The best example of a barrier that occurs in use but not in vitro is the
dentin barrier. When restorative materials are placed in teeth, dentin will
generally be interposed between the material and the pulp. The dentin
barrier, although possibly only a fraction of a millimeter thick, is effective
in modulating the effects of dental materials. The effect of the dentin barrier
is illustrated by the following classic study. three methods were used to
evaluate the following materials: a ZOE cement, a composite material, and a
silicate cement. The evaluation methods included (1) four different cell
culture tests, (2) an implantation test, and (3) a usage test in class 5 cavity
preparations in monkey teeth. The results of the four cells culture tests were
relatively consistent, with silicate having only a slight effect and ZOE a
severe effect. These three materials were also embedded subcutaneously in
connective tissue in polyethylene tubes (secondary test), and observations
were made at 7, 30, and 90 days. Reactions at 7 days could not be
determined because of inflammation caused by the operative procedure. At
30 days, ZOE appeared to cause a more severe reaction than silicate cement.
The inflammatory reactions at 90 days caused by ZOE and silicate were
slight, and the reaction to composite materials was moderate. When the
three materials were evaluated in class 5 cavity preparations under
prescribed conditions of cavity size and depth (usage test), the results were
quite different from those obtained by the screening methods. The silicate
was found to have the most severe inflammatory reaction the composite had
a moderate to slight reaction, and the ZOE had little or no effect.
The apparent contraindictions in this study may be explained by
considering the components that were released from the materials and the
environments into which they were released. The silicate cement released
hydrogen ions that were probably buffered in the cell culture and
implantation tests but may not have been adequately buffered by the dentin
in the usage tests. Microleakage of bacteria or bacterial products may have
added to the inflammatory reaction in the usage test. Thus this material
appeared most toxic in the usage test. The composites released low-
molecular-weight resins, and the ZOE released eugenol and zinc ions. In the
cell-culture tests, these compounds had direct access to cells and probably
caused the moderate to severe cytotoxicity. In the implantation tests, the
released components may have caused some cytotoxicity, but the severity
may have been reduced because of the capacity of the surrounding tissue to
disperse the toxins. In usage tests, these materials probably were less toxic
because the diffusion gradient of the dentin barrier reduced concentrations
of the released molecules to low levels. The slight reaction observed with
the composites also may also have been caused in part by microleakage
around these restorations. The ZOE did not show this reaction, however,
because the eugenol and zinc probably killed bacteria in the cavity, and the
ZOE may have somewhat reduced microleakage.
Another example of the lack of correlation of usage tests with
implantation tests is the inflammatory response of the gingiva at the
gingival and interproximal margins of restorations that accumulate bacterial
plaque and calculus. Plaque and calculus cannot accumulate on implanted
materials and therefore the implantation test cannot hope to duplicate the
usage test. However, connective tissue implantation tests are of great value
in demonstrating the cytotoxic effects of materials and evaluating materials
that will be used ion contact with alveolar bone and apical periodontal
connective tissues. In these cases, the implant site and the usage sites are
sufficiently similar to compare the test results of the two sites.
USING IN VITRO, ANIMAL AND USAGE TESTS TOGETHER
(SCHEME) :
For about 20 years, scientists, industry, and the government have
recognized that the most accurate and cost-effective means to assess the
biocompatibility of a new material is a combination of in vitro, animal, and
usage tests. Implicit in this philosophy is the idea that no single test will be
adequate to completely characterize the biocompatibility of a material. The
ways by which these tests are used together, however, are controversial and
have evolved over the years as knowledge has increased and new
technologies developed. This evolution can be expected to continue as we
ask materials to perform more-sophisticated functions for longer periods.
Early combination schemes proposed a pyramid testing protocol, I
which all materials were tested at the bottom of the pyramid and materials
were “weeded out” as the testing continued toward the top of the pyramid.
Tests at the bottom of the pyramid were “unspecific toxicity” tests of any
type (in vitro or animal) with conditions that did not necessarily reflect
those of the material’s use. The next tier shows specific toxicity tests that
presumably dealt with conditions more relevant to the use of the material.
The final tier was a clinical trial of the material. Later, another pyramid
scheme was proposed that divided tests into initial, secondary, and usage
tests. The philosophy was similar to the first scheme, except the types of
tests were broadened to encompass biological reactions other than toxicity,
such as immunogenicity and mutagenicity. The concept of a usage test in an
animal was also added (vs. a clinical trial in a human). There are several
important features of these early schemes. First, only materials that “passed”
the first tier of tests were graduated to the second tier, and only those that
passed the second tier were graduated to the clinical trials.
Presumably, then this scheme fed safer materials into the clinical
trials area and eliminated unsafe materials. This strategy was welcomed
because clinical trials are the most expensive and time-consuming aspect of
biocompatibility tests. Second, any material that survived all three tiers of
tests were deemed acceptable for clinical use. Third, each tier of the system
put a great deal of onus on the tests use to accurately screen in or out a
material. Although still used in principle today, the inability of in vitro and
animal tests to unequivocally screen materials in or out has led to the
development of newer schemes in biocompatibility testing.
Two newer testing schemes have evolved in the past 5 year s with
regard to using combinations of biocompatibility tests to evaluate materials.
Both of these newer schemes accommodate several important ideas. First,
all tests (in vitro, animal, and usage) continue to be of value in assessing the
biocompatibility of a material during its development and even in its clinical
service. For example, tests in animals for inflammation may be useful
during the development of a material, but may also be useful after a problem
is noted with the material after it has been on the marker for a time. Second,
the newer schemes recognize the inability of current testing methods to
accurately and absolutely screen in or out a material. Third, these newer
schemes incorporate the philosophy that assessing the biocompatibility of a
material is an ongoing process. Undoubtedly, we will see still newer
strategies in the use of combinations of biocompatibility tests as the roles of
materials change and the technologies for testing improve.
STANDARDS THAT REGULATE THE MEASUREMENT OF
BIOCOMPATIBILITY :
The first effort of the ADA to establish guidelines for dental materials
came in 1926 when scientists at the National Bureau of Standards, now the
National Institute of Science and Technology, developed specifications for
dental amalgam. Unfortunately, recommendations on materials and
conditions for biological compatibility have not kept pace with the
technological development of dental materials. Reasons for this are (1) the
fast advance of cellular and molecular biology, (2) the variety of tests
available for assessing biocompatibility of materials, and (3) the lack of
standardization of these tests.
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 the
study 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. Small, standardized pieces of gold,
amalgam, gutta-percha, silicates, and copper amalgam were sterilized and
placed in uniformly sized pockets within skeletal muscle tissue. Biopsy
specimens were evaluated microscopically after 6 months. Other early
attempts to standardize techniques were carried out by Mitchell (1959) on
connective tissue and by Massler (1958) on tooth pulp. Not until the passage
of the Medical Device bill by Congress in 1976 was biological testing for all
medical devices (including dental materials) given a high priority. In 1972
the Council on Dental Materials, Instruments, and Equipment of
ANSI/ADA approved Document 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. In 1982, an addendum
was made to this document, including an update of the Ames test for
mutagenic activity.
ANSI / ADA DOCUMENT 41 :
Three categories of tests are described in the 1982 ANSI/ADA
document: initial, secondary, and usage tests. This document uses the
testing scheme. The initial tests include in vitro assays for cytotoxicity, red
blood cell membrane lysis (hemolysis), mutagenesis and carcinogenesis at
the cellular level, and in vivo acute physiological distress and death at the
level of the whole 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 (placement of the materials in
their intended contexts, first in larger animals, often primates, and finally,
with Food and Drug administration approval, in humans). The ANSI/ADA
Doc. 41, 1982 Addendum, has two assays for mutagenesis, the Ames test
and the Styles cell transformation test.
Iso 10993 :
In the past decade, an international effort was initiated by several
standards organizations to develop international standards for biomedical,
materials and devices. Several multinational working groups, including
scientists from ANSI and the international Standards Organization (ISO)
were formed to develop these standards. The final document (ISO 10993)
was published in 1992 and is the most recent standard available for
biological testing. ISO 10993 contains 12 parts, each dealing with a
different aspect of biological testing. For example, part 2 addressed animal
welfare requirements, part 3 addresses tests for genotoxicity
carcinogenicity, and reproductive toxicity, and part 4deals with tests for
interactions with blood. The standard divides 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 nonusage situations.
Supplementary tests are tests such as chronic toxicity, carcinogenicity, and
biodegradation. Most of the supplementary tests are done in animal systems,
many in usage situations. The selection of tests for a specific material is left
up to the manufacturer, who must present and defend the testing results.
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 current working version of the ISO standard is available from the
International Organization for Standardization (www.iso.ch), Case Postale