3. HISTORY
• Hippocrates (460-377B.C)-developed the concept
of ethical treatment of patients
• Concept of protecting patient is only 30-40 years
old
• IRB oversee testing of new materials in humans.
• The oversight of testing materials in a systematic
way now rests largely with FDA
• Also regulated by ANSI ,ADA and ISO
4. • HIPPOCRATIC OATH: MODERN VERSION
• I swear to fulfill, to the best of my ability and judgment, this covenant:
• I will respect the hard-won scientific gains of those physicians in whose
steps I walk, and gladly share such knowledge as is mine with those who
are to follow.
• I will apply, for the benefit of the sick, all measures [that] are required,
avoiding those twin traps of overtreatment and therapeutic nihilism.
• I will remember that there is art to medicine as well as science, and that
warmth, sympathy, and understanding may outweigh the surgeon's knife
or the chemist's drug.
• I will not be ashamed to say "I know not," nor will I fail to call in my
colleagues when the skills of another are needed for a patient's recovery.
• I will respect the privacy of my patients, for their problems are not
disclosed to me that the world may know. Most especially must I tread
with care in matters of life and death. If it is given me to save a life, all
thanks. But it may also be within my power to take a life; this awesome
responsibility must be faced with great humbleness and awareness of my
own frailty. Above all, I must not play at God.
• I will remember that I do not treat a fever chart, a cancerous growth, but a
sick human being, whose illness may affect the person's family and
economic stability. My responsibility includes these related problems, if I
am to care adequately for the sick.
• I will prevent disease whenever I can, for prevention is preferable to cure.
• I will remember that I remain a member of society, with special
obligations to all my fellow human beings, those sound of mind and body
as well as the infirm.
• If I do not violate this oath, may I enjoy life and art, respected while I live
and remembered with affection thereafter. May I always act so as to
preserve the finest traditions of my calling and may I long experience the
joy of healing those who seek my help.
• —Written in 1964 by Louis Lasagna, Academic Dean of the School of
Medicine at Tufts University, and used in many medical schools today.
6. Definition
• Biocompatibility is formally defined as the ability of a material to
elicit an appropriate biological response in a given application
in the body.
• Implicit in this definition is that biocompatibility is an interaction
between body and material –depends on condition of host,
properties of material and context of use
• Inherent in this definition also is the idea that a single material
may not be biologically acceptable in all applications.
7.
8. Anatomical and pathological aspects of
oral tissues
ENAMEL
• Mature human enamel is highly mineralized (96% by weight),
with only 1% of its weight being organic molecules and 3%
being water.
• The permeability of enamel to most oral molecules is quite low,
and in this sense enamel "seals" the tooth to outside agents.
However, recent evidence indicates that enamel is not
impermeable. Peroxides in bleaching agents have been shown
to permeate intact enamel in just a few seconds.
9. DENTIN AND PULP
• Because of their close anatomical relationship, most researchers
consider the dentin and pulp to be a single organ.
• The dentinal matrix (both calcified and uncalcified) forms the greatest
bulk of the tooth. Calcified dentin is about 20% organic, 70%
inorganic, and 10% aqueous by weight. Collagen constitutes
approximately 85% of the organic portion of dentin, and
hydroxyapatite is the main inorganic compound.
• The dentinal matrix surrounds dentinal tubules that are filled with the
odontoblastic processes.
• A serum-like fluid fills the dentinal tubules. This fluid has continuity
with the extracellular fluid of the pulp tissue. The pulpal circulation
maintains an intercellular hydraulic pressure of about 24 mm Hg (32.5
cm H20)
10.
11. • Moderately deep cavity preparation will damage odontoblasts
by severing the odontoblastic processes. Deep cavity
preparation may destroy most of the dentin and kill the primary
odontoblasts.
• A number of investigators think that the extracellular matrix
(ECM) of dentin and pulp is largely responsible for the
differentiation of secondary odontoblasts that form reparative
dentin.
• The source of secondary odontoblasts is not known, but much
of the proliferative activity of granulation tissue following pulpal
insult is found in perivascular areas proximal to the core of the
pulp.
12. • Nerves and blood vessels, which arborize from the core of the pulp
as they approach the odontoblastic layer,may influence the extent
of the inflammatory response and the amount of new dentinal
matrix formed during dentin repair.
• When toxic bacterial or chemical products traverse the dentin,
odontoblasts and the pulpal connective tissue usually respond first
by focal necrosis (0 to 12 hours), which may be followed by an
acute, but more widespread, pulpitis (12 hours to several days).
This response may resolve naturally if the injurious agent is
removed or the tubules are blocked. If the pulpitis does not
resolve, it may spread to more completely involve the pulp in
liquefaction necrosis (especially if the pulpitis results from bacterial
products) or in chronic inflammation
13. DENTIN PERMEABILITY
• Two types of dentin permeability occur-Convection and
diffusion
• Convection of fluids across dentin varies with the fourth power
of the radius of the dentinal tubule(r4), and thus very sensitive
to the diameter of the tubules.
• Diffusion is proportional to the length of the dentinal tubules
and thus ,roughly, to the thickness of the dentin between cavity
preparation and the pulp
14. • Smear layers created in cavity preparation are better than
cavity liners and sealers at limiting diffusional permeability
.
• the capillary beds and vascular dynamics in most healthy pulps
are probably capable of removing relatively large amounts of
cytotoxic chemicals and bacterial products once they diffuse
through the dentin. However, if the pulp is already damaged
(inflamed because of caries or trauma), edema and sluggish
circulation probably compromise the removal of these
materials.
15. BONE
• Bone is an extracellular matrix (ECM) with accompanying cells
and tissue. The ECM of bone is a mineralized tissue composed
of about 23% organic substances and about 77%
hydroxyapatite.
• Osteoblasts die if surgical manipulation destroys the vascular
supply or heats the bone above 45' C for more than a few
minutes
16. Osseointegration and
Biointegration
• Osseointegration is defined as the close approximation
of bone to an implant material
• To achieve osseointegration, the bone must be viable, the
space between the bone and implant must be less than 100 A
and contain no fibrous tissue, and the bone-implant interface
must be able to survive loading by a dental prosthesis. In
current practice, osseointegration is an absolute requirement
for the successful implant-supported dental prosthesis.
17. • In recent years there has been a trend to coat titanium alloys
with a calcium-phosphate ceramic to better promote an implant
bone connection. If successful, the ceramic coating becomes
completely fused with the surrounding bone. In this case, the
interface is called biointegration rather than osseointegration
and there is no intervening space between the bone and the
implant .
• A number of ceramic coatings have been used in this manner,
including tricalcium phosphate, hydroxyapatite, and Bioglass.
The long-term integrity of the ceramic coating in vivo is not
known, but some evidence indicates that these coatings will
resorb over time.
18.
19. PERIODONTIUM
• The periodontium is a combination of tissues, including the
periodontal ligament (PDL), cementum, and alveolar bone. The
cementum and alveolar bone are mineralized extracellular
matrices with the associated cells that are responsible for
generating and maintaining them.
20. GINGIVA AND MUCOSA
• The gingiva is a connective tissue with an epithelial surface that covers the
alveolar ridge, surrounds the cervices of the teeth, and fills interproximal
spaces between teeth.
• The free gingiva fuses with the attachment epithelium, which surrounds the
tooth at its cervix in the young, healthy tooth. Some of the crevicular
epithelium and all the attachment epithelium, at least in the young individual,
are derived embryologically from reduced enamel epithelium.
• The gingival response to injury may be complicated by its association with the
tooth. Calculus deposition on the tooth, malocclusion, and faulty restorations
may enhance the destructive effects of microorganisms. The crevicular
epithelium then becomes vulnerable to endotoxin and various exogenous and
endogenous chemicals.
21. • The resulting breakdown of tissue leads to an acute
inflammatory response by the gingival connective tissue called
acute gingivitis. This condition is often reversible if the injurious
agent is removed and the reaction is limited to the connective
tissue above the alveolar bony crest.
• If the insult continues, the inflammatory infiltrate becomes
mixed and then predominantly mononuclear. Inflamed
epithelial-lined granulation tissue gradually spreads apically to
and below the alveolar bony crest. At this point the condition is
described as chronic periodontal disease, a progressive
disease process that is probably reinforced by immune
mechanisms. The relationship between periodontal disease
and dental materials that are in close contact with the gingiva is
not known but is an active area of research.
22. • The oral mucosa is composed of a loose fibro elastic
connective tissue with a well-vascularized and innervated
lamina propria and submucosa and is covered mainly by a
parakeratinized stratified squamous epithelium.
• The oral mucous membrane can be injured chemically or
physically by dental materials. If the injury is short-term (acute)
and leads to loss of tissue but does not involve infection by
pathogenic microorganisms, the connective tissue defect is
filled in with granulation tissue within 3 to 4 days and epithelium
regenerates over the surface within a week. The tissue is
remodeled to nearly normal by the end of 2 to 3 weeks.
24. MEASURING BIOCOMPATIILITY
• Historically, new materials were simply tried in humans to see if
they were biocompatible
• Current materials must be extensively screened for
biocompatibility before they are ever used in humans.
• 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.
25. 1)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.
26. 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).
28. • 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.
29. • 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
cell lines contributes significantly toward standardizing assay
methods.
• In the end, both primary and continuous cells play an important
role in in vitro testing; both should be used to assess a material
30. A)Cytotoxicity Tests
• Cytotoxicity tests assess the cytotoxicity of a material by
measuring cell number or 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 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
either qualitatively, semi-quantitatively, or quantitatively.
31. 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.
32. • 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)
33. • 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 cytoxicity 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
51CrO4.The use of neutral red andNa2
51CrO4 are particularly
advantageous because they are neither synthesized nor metabolized
by the cell.
• Examples of nonvital dyes include trypan blue and propidium iodide.
34.
35. B)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., 3thymidine or 3leucine) incorporated
into DNA or protein.
• A commonly used enzymatic test for cytotoxicity is the
MTT test
• Recently in vitro tests to measure gene activation, gene
expression, cellular oxidative stress, and other specific
cell functions have been proposed.
36. 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.
37. C)Tests That Use Barriers (Indirect
Tests)
• 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.
• Several in vitro barrier tests have been
developed to mimic in vivo conditions. One
such test is the agar overlay method
38. This assay correlates positively with the direct-contact assays 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.
39. • A second barrier assay is the Millipore filter assay.
• 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
40. D)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
• 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 cell
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.
41. E)Mutagenesis Assays
• Mutagenesis assays assess the effect of materials on a
cell's genetic material.
• 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.
• .
42. • 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
• The Ames' test is the most widely used mutagenesis test and the only
short-term test that is considered thoroughly validated.
• It uses mutant stocks of Salmonella typhimurium 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
44. • 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 (Chang,
BHK, HeLa, WI-38) have been used.
45. COMPARISION OF MUTAGENIC TESTS
These studies suggest that not all carcinogens are genotoxic
(mutagenic) and not all mutagens are carcinogenic.
46. 11)ANIMAL TESTS
• Animal tests for biocompatibility are usually used in
mammals such as mice, rats, hamsters, or guinea
pigs
• 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
47. • 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.
48. • 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.
49. • 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.
50. • 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 of the restorative materials contact the gingiva, most
subcutaneous tests are used for materials that will directly
contact soft tissue during implantation, endodontic, or
periodontal treatment.
• Implantation tests of longer 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.
51. 111)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 test is identical to a clinical trial.
• These tests are the gold standard of tests in that
they give the ultimate answer to whether a
material will be biocompatible
52. 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.
• 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.
53. • 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 on its appearance after treatment.
• 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.
• Usage tests that study teeth with induced pulpitis allows
evaluation of types and amount of reparative dentin formed.
54. 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,
(3) radiographs indicating either osseous integration or
radiolucency around the implant
55. 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.
56. • 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.
58. CORRELATION AMONG IN
VITRO, ANIMAL, AND
USAGE 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.
• Barriers between the material and tissues may exist in
usage tests or clinical use that may not exist in in vitro or
animal tests.
• 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.
59. • The best example of a barrier that occurs in use but not in vitro
is the dentin barrier.
• The effect of the dentin barrier is illustrated by the following
classic study
Comparision reactions of 3 materials by screening and usage tests
60. USING IN VITRO, ANIMAL, AND
USAGE TESTS TOGETHER
• 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
61.
62. • Two newer testing schemes have evolved in the past few years 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 market 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.
63.
64. STANDARDS THAT REGULATE THE
MEASUREMENT OF BlOCOMPATlBlLlTY
• The first efforts 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.
• 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.
65. • 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
66. ANSI/ADA Document 41
• Three categories of tests are described in the 1982 ANSI/ ADA document:
initial, secondary, and usage tests.
• 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
67. IS0 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 (IS0 10993) was published in 1992 and is the
most recent standard available for biological testing. IS0 10993
contains 12 parts, each dealing with a different aspect of biological
testing.
68. • 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.
• Unlike the IS0 standard, which covers all biomedical devices, the
ANSI/ADA document is limited to dental devices
69. BlOCOMPATlBlLlTY OF DENTAL
MATERIALS
• REACTIONS OF PULP
• Microleakage
• If a bond does not form or debonding occurs, bacteria, food
debris, or saliva may be drawn into the gap between the
restoration and the tooth by capillary action. This effect has been
termed microleakage
70. • Recently, the new concept of nanoleakage hasbeen put
forward.
• Nanoleakage refers to the leakage of saliva, bacteria, or
material components through the interface between a material
and tooth structure. However, nanoleakage refers specifically
to dentin bonding, and may occur between mineralized dentin
and a bonded material in the very small spaces of
demineralized collagen matrix into which the bonded material
did not penetrate.
• it is thought to play at least some role and it is suspected of
contributing to the hydrolytic degradation of the dentin-material
bond, leading ultimately much more serious microleakage.
71. Dentin Bonding
• Because the dentinal tubules and their resident
odontoblasts are extensions of the pulp,
bonding to dentin also involves biocompatibility
issues.
• From the standpoint of biocompatibility, the
removal of the smear layer may pose a threat
to the pulpal tissues for three reasons.
• First, its removal juxtaposes resin materials
and dentin without a barrier.
• Second, the removal of the smear layer makes
any microleakage more significant because a
significant barrier to the diffusion of bacteria or
bacterial products toward the pulp is removed.
• Third, the acids used to remove the smear
layer are a potential source of irritation
themselves.
72. • The biocompatibility of acids used to remove the smear layer has been
extensively studied. Numerous acids have been used to remove the
smear layer, including phosphoric, hydrochloric, citric, and lactic acids.
The effect of the acids on pulpal tissues depends on a number of factors,
including the thickness of dentin between the restoration and the pulp, the
strength of the acid, and the degree of etching. Most studies have shown
that dentin is a very efficient buffer of protons, and most of the acid may
never reach the pulp if sufficient dentin remains.
• A dentin thickness of 0.5 mm has proven adequate in this regard. Citric or
lactic acids are less well buffered, probably because these weaker acids
do not dissociate as efficiently.
• Usage tests that have studied the effects of acids have shown that
phosphoric, pyruvic, and citric acids produce moderate pulpal
inflammatory responses, which resolve after 8 weeks. Recent research
has shown that in most cases the penetration of acids into the dentin is
probably less than 100 µm.
73. • Dentin Bonding Agents
• Many of these reagents are cytotoxic to cells in vitro if tested
alone.
• However, when placed on dentin and rinsed with tap water
between applications of subsequent reagents as prescribed,
cytotoxicity is often reduced.
• Longer-term in vitro studies suggest, however, that sufficient
components of many bonding agents permeate up to0.5 mm of
dentin to cause significant suppression of cellular metabolism
for up to 4 weeks after their application. This suggests that
residual unbound reagents may cause adverse reactions.
74. • In 1996 Olea et al claimed that dental sealants
released estrogenic substances
• The concern about estrogen centred around a
chemical called bisphenol A(BPA)
• One common test used to assess
xenoestrogenic activity is called E Screen
Assay
• This in vitro test relies on growth response of
breast cancer cells that are estrogen sensitive
to purported estrogenic compounds
75. Resin-Based Materials
• In vitro, freshly set chemically cured and light-cured resins often cause
moderate cytotoxic reactions in cultured cells over 24 to 72 hours of
exposure
• The cytotoxicity is significantly reduced 24 to 48 hours after setting and by
the presence of a dentin barrier
• In all cases, cytotoxicity is thought to be mediated by resin components
released from the materials
• The primary risks for these materials appear to be allergy
• The allergenicity of methylmethacrylate is well documented and the use of
gloves is not effective in preventing contact because most monomers pass
easily through gloves
• The allergic reactions are primarily contact dermatitis
76. Amalgams
• The biocompatibility of amalgams is thought determined largely by
corrosion products released while in service. Corrosion depends
on the type of amalgam, whether it contains the γ2, phase, and the
composition of the amalgam.
• In usage tests, the response of the pulp to amalgam in shallow
cavities or in deeper but lined cavities is minimal. In deep cavities
(0.5 mm or less of remaining dentin), pain results from using
amalgams in unlined cavity preparations.
• An inflammatory response is seen after 3 days and after 5 weeks.
In cavities with 0.5 to 1.0 mm of dentin relining in the floor, the
cavity preparation should be lined for two other reasons.
77. • First, thermal conductivity with amalgam is significant and can be a
problem clinically.
• Second, margins of newly placed amalgam restorations show significant
microleakage. Marginal leakage of corrosion and microbial products is
probably enhanced by the daily natural thermal cycle in the oral cavity.
The short-term pulpal response is significantly reduced when the cavity is
lined, and amalgam rarely causes irreversible damage to the pulp. Long-
term sealing of the margins occurs through the buildup of corrosion
• Amalgams based on gallium rather than mercury have been developed
to provide direct restorative materials that are free of mercury
• Clinically, these materials show much higher corrosion rates than
standard amalgams, leading to roughness and discoloration. There are
few reports of pulpal responses to these materials
78. Glass Ionomers
• Light-cured ionomer systems have also been introduced; these systems
use Bis-GMA or other oligomers as pendant chains on the polyacrylate
main chain. In screening tests, freshly prepared ionomer is mildly
cytotoxic, but this effect is reduced with increased times after setting.
• The fluoride release from these materials, which is probably of some
therapeutic value, may cause cytotoxicity in in vitro tests
• The overall pulpal biocompatibility of these materials has been attributed
to the weak nature of the polyacrylic acid, which is unable to diffuse
through dentin because of its high molecular weight. In usage tests the
pulp reaction to glass ionomer cements is mild.
79. Liners, Varnishes, and Nonresin Cements
• Calcium hydroxide cavity liners come in many forms, ranging from
saline suspensions with a very alkaline pH (above 12) to modified
forms containing zinc oxide, titanium dioxide, and resins.
• The high pH of calcium hydroxide in suspension leads to extreme
cytotoxicity in screening tests
• Calcium hydroxide cements containing resins cause mild-to-
moderate cytotoxic effects in tissue culture in both the freshly set
and long-term set conditions. The inhibition of cell metabolism is
reversible in tissue culture by high levels of serum proteins,
suggesting that protein binding or buffering in inflamed pulpal
tissue may play an important role in detoxifying these materials in
vivo.
80. • The initial response of exposed pulpal tissue to the highly
alkaline aqueous pulp-capping agents is necrosis to a depth of
1 mm or more. The alkaline pH also helps to coagulate any
hemorrhagic exudate of the superficial pulp. Shortly after
necrosis occurs, neutrophils infiltrate into the subnecrotic zone.
Eventually, after 5 to 8 weeks, only a slight inflammatory
response remains.
• Within weeks to months, the necrotic zone undergoes
dystrophic calcification, which appears to be a stimulus for
dentin bridge formation.
82. Conclusion
• The biocompatibility of a dental material depends on its
composition, location, and interactions with the oral cavity.
Metal, ceramic, and polymer materials elicit different
biological responses because of their differences in
composition.
• Furthermore, diverse biological responses to these
materials depend on whether they release their
components and whether those components are toxic,
immunogenic, or mutagenic at the released
concentrations.
• The paradigm of evaluating material biocompatibility
needs to be meticulously assessed and informed consent
and continual evaluation and reevaluation of materials
must be done
