Biocompatibility of Dental Materials and Related Researches

Biocompatibility of Dental Materials
PRESENTED BY-
DR. SOUMIK KARMAKAR
1st YEAR PGT
DEPT. OF PROSTHODONTICS AND CROWN AND BRIDGE
GURU NANAK INSTITUTE OF DENTAL SCIENCES AND RESEARCH
UNDER THE ABLE GUIDANCE OF:

Prof. Dr. JAYANTA BHATTACHARYYA (HOD & PRINCIPAL)
Prof. Dr. SAMIRAN DAS
Prof. Dr. SOUMITRA GHOSH
Prof. Dr. PREETI GOEL
Dr. SAYAN MAJUMDAR
CONTENTS
 Introduction
 Definition
 Evolution
 Importance
 Biological interface
 Adverse effects of dental materials
 Biocompatibility of common dental materials
 Tests for evaluation of biocompatibility
 Strategies for evaluating biocompatibility
 Standards of measurement of biocompatibility
 Conclusion
 References
INTRODUCTION
 Biocompatibility refers to the study of interaction of various materials
with human tissues.
 Materials used in dentistry come into direct contact with the hard tissues
of the teeth, the oral mucosa, the pulp & the periapical tissues.
 Due to this intimate, long term contact, the materials should exhibit a
high degree of biocompatibility.
 For the biocompatibility of a biomaterial ,it is not only important that
minimal diffusible substances are released when it is in body contact, but
the material must also solve the purpose for which it has been designed.
“Practitioners should understand that there are no inert materials. When material is
placed into living tissue, interaction with the complex biologic systems around it occur,
and those interactions result in some sort of biologic response.
Wataha J.C., 2001
biocompatibility
Biocompatibility of
Restorative Dental Materials
and Related Researches
DRES 407 725, 407 723
27 June and 4 July 2013
Dr. Sitthikorn Kunawarote
DEFINITION
 BIOCOMPATIBILITY IS FORMALLY DEFINED AS THE
ABILITY OF A MATERIAL TO ELICIT AN APPROPRIATE
BIOLOGICAL RESPONSE IN A GIVEN APPLICATION IN
THE BODY. (CRAIG)
 If examined closely, this definition implies an interaction among
a host, a material and an expected function of the material.
 All three factors must be in harmony before the material can be
considered biocompatible.
Saigal A, Sharma AK. A comparative study between criteria for selection, evaluation and collection of e-resources in IIMS Libraries. Library Progress
(International). 2017;37(2):269-84.
EVOLUTION
 Before 400BC, the Etruscans fabricated bridges and partial dentures using gold
combined with animal or extracted human teeth.
 Autian proposed a structured approach for evaluating tissue response to dental
materials consisting of three levels namely nonspecific toxicity (cell cultures or
small laboratory animals); specific toxicity (usage tests, e.g. in subhuman
primates) and clinical testing in humans.
 The ISO (1984) in Technical Report 7405 adopted the following sequence of
tests for evaluation of dental materials i.e initial tests (cytotoxicity,
mutagenicity); secondary tests (sensitization, implantation tests, mucosal
irritation) and usage tests.
 In both concepts, newly developed materials should be subjected to the three
steps in the given sequence from the simple to the complicated test method,
from in vitro to animal tests and from preclinical to clinical testing on humans.
(International). 2017;37(2):269-84.
Ganapathy D. BIOCOMPATIBILITY OF DENTAL RESTORATIVE MATERIALS. European Journal of Molecular & Clinical Medicine. 2021 Jan 10;8(1):504-
12.
IMPORTANCE
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
The primary purpose of biocompatibility tests is to protect dental
patients who will be treated with the materials and the office
staff and lab technicians who will be handling these materials.
Since no dental biomaterial is absolutely free from the potential
risk of adverse reactions, the testing of biocompatibility is
related to risk assessment. Thus, the challenge is to select those
products for which the known benefits far outweigh the known
risks.
(International). 2017;37(2):269-84. Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD
BIOLOGICAL INTERFACE
 As the definition of biocompatibility suggests an
interaction between the body & the material.
 Placement of a material in the body creates an interface
that is normally not present.
 This interface is not static, rather it is the site of many
dynamic interactions between the material & the body
through which the body may alter the material or the
material may alter the body.
Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD
 The activity of this interface depends on:
 1. The location of material.
 2. Its duration in the body.
 3. The properties of the material.
 4. Health of the host.
 4 types of interaction can take place:

 1. Between the material and oral cavity.
 2. Between the material and the pulp (Via the
dentinal tubules).
 3. Between the material and periodontium.
 4. Between the material and the periapical bone.
ADVERSE EFFECTS OF DENTAL MATERIALS
 A critical adverse effect is the first event that is observed at the lowest exposure level. The
location of this effect is called the critical tissue, or critical organ, and the concentration of
a substance that produces this effect is the critical concentration.
 Figure shows the critical tissue and organ sites that can be affected by exposure to dental
restorative materials. Material components can be released during melting and casting of
metals, fabrication of prostheses, grinding and polishing procedures, adhesive bonding, or
cementing to prepared teeth.
Phillips’ Science of Dental Materials

KENNETH J. ANUSAVICE, PhD, DM
 The biocompatibility of a material depends on several factors:
1. The chemical nature of its components
2. The physical nature of the components
3. The types and locations of patient tissues that will be exposed to
the device
4. The duration of the exposure
5. The surface characteristics of the material
6. The amount and nature of substances eluted from the material

 There are a number of possible biological reactions to materials.
 Classically, these reactions have been separated into toxic, inflammatory,
allergic, and mutagenic reactions.

TOXICITY
 TOXICITY OF A MATERIAL DESCRIBES THE ABILITY TO DAMAGE
A BIOLOGICAL SYSTEM BY CHEMICAL MEANS.
 Of the biological responses to materials, the first screening test used for almost
all materials is a toxicity test
 Materials may be capable of releasing substances into a patient's body, and the
release of certain substances in adequate amounts can cause overt toxicity.
 For example, early dental materials containing lead posed real risk to the patient
because of the toxic properties of the lead that leached into the patient's body.
 Fortunately, most materials capable of causing overt toxicity are no longer used
in dentistry

 IMMUNOTOXICITY
• SUBSTANCES LEACHED FROM MATERIALS
CAN ALTER IMMUNE SYSTEM CELLS,
RESULTING IN ENORMOUS BIOLOGICAL
CONSEQUENCES BECAUSE OF THE
AMPLIFYING NATURE OF IMMUNE CELLS.
• THIS MAY LEAD TO:
1. IMPAIRED HOST DEFENCE.
2. TISSUE DAMAGE.
• GLUTATHIONE CONTENT OF HUMAN

MONOCYTES WHICH HELPS IN MAINTAINING
OXIDATIVE STRESS IN CELLS. MERCURY
IONS ARE KNOWN TO INCREASE WHEREAS
PALLADIUM IONS DECREASE THE CELLS’
GLUTATHIONE CONTENT. Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD
 SYSTEMIC TOXICITY
 Here site of application & site of adverse reactions are different.
 Almost all dental materials release substances into the oral cavity, from where they
may enter the systemic circulation via different routes.
According to the time frame:
1. Acute (up to an exposure period of 24 h)
2. Subacute (up to 3 months)
3. Chronic toxicity are differentiated
Substances that have a long life in the body, such as mercury, may accumulate and
reach critical levels more easily than other substances that are readily excreted.
Furthermore, not all tissues react equally. Systemic reactions may also be
influenced by the liver that alter substances in an attempt to digest or excrete them.
 LOCAL TOXICITY
 Inflammation of the gingiva in contact with a
porcelain- fused-to-metal crown
 Pulp necrosis after application of resin fillings
•Substances released from dental materials may
generate a reaction (e.g., inflammation or necrosis)
in adjacent tissues such as oral mucosa/ gingiva,
pulp or alveolar bone.
• There may be other factors like:
1. Bacterial accumulation on the surface, at the
margin, or under a material.
2. Mechanical/physical irritation, such as pressure
caused by dentures.
INFLAMMATION
 Inflammation is a fundamental type of biological
response to a material.
 The inflammatory response is complex & it occurs to
ward off some threat.
 Histologically, the inflammatory response is characterised
by 1. Edema of the tissue. 2. Inflammatory cells
infiltration such as neutrophills (in the short term) or
monocytes and other lymphocytic cells(in the long term).
The contribution of dental materials to inflammatory
reactions is especially important because pulpal and
periodontal diseases are largely chronic inlflammatory
responses toPhillips’
long-term infections.
Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD
ALLERGY
 Allergy is an abnormal antigen – antibody reaction to a substance that is
harmless to most individuals.
 It is not dose dependent.
 These allergic reactions are also called Hypersensitivity reactions & are
mediated by IgE.
ALLERGIC REACTIONS ELICITED BY DENTAL MATERIALS CAN

OCCUR INTRAORALLY OR AS REMOTE REACTIONS
EXTRAORALLY.
 Dental materials may cause allergies of type I (immediate reaction) and
type IV (delayed reaction).
 Examples 1. Immediate reaction is Contact urticaria following
occupational exposure to latex proteins in disposable gloves.
2. Delayed reaction : Allergic reaction to components of the root
canal sealer AH26, Pronounced gingivitis of an orthodontic patient (nickel-
containing device)
CONCURRENT- ALLERGY CROSS-ALLERGY
It is generated by 2 allergen that are If an individual is allergic to a particular

frequently present at same time within element, then it may be assumed that he
a material oral environment. will also allergic to chemically similar
elements.
e.g.–Nickel and palladium.
e.g.–Ethylene glycol dimethacrylate
(EGDMA) Hydroxyethyl
Methacrylate (HEMA)

OTHER REACTIONS
1. Genotoxicity : Refers to the ability of substance released
from materials to cause alterations of the genome DNA.
2. Mutagenicity : It is the ability of a substance to pass genetic
damage on the next generation. e.g. – Ni, Cu and Be are
known mutagens.
3. Carcinogenicity: It is the ability of a material or substance
released from it to induce malignant tumors.
4. Teratogenicity: It is the ability of certain substance to cause
malformation during embryonic development.
BIOCOMPATILITY OF COMMON DENTAL MATERIALS
 Amalgam
 The mercury used in the reaction may leach out of the restoration and may lead to
corrosion reactions.
 It can be very toxic when it occurs as methyl mercury.
 It can enter into human body through the skin, by ingestion, or by inhalation of
mercury vapors. Mercury in vapour form is easily taken up by the body.
 The mercury present in an amalgam reaction exists in the metallic form and is not
easily absorbed from the digestive system if swallowed. It is completely bound up
with the other metals present in the amalgam because the reaction is a chemical
one that combines it with the metals to form an alloy.
 Amalgam has been considered to be either inert or only mildly irritating to the pulp
or body tissues in dogs, rats and humans. Any pulpal response to amalgam is
related mainly to the pressure of condensation
 Safe level of mercury exposure in the dental office is 50 µg Hg / Cubic Meter of
air /day.
 Allergic reaction Type 1V – Delayed allergic type reaction may be seen in the oral
mucosa in contact with the restorations. They manifest as Oral Lichenoid
BIOCOMPATIBILITY OF DENTAL MATERIALS: A COMPREHENSIVE REVIEW
Reactions, which resolve on removal of the restorations.
 Composites and polymeric materials
 Unpolymerized monomers can leach into saliva
and cause adverse reactions
 The main problem occurring with the use of
these materials is allergy i.e Type IV
hypersensitivity reaction.
 Contact with acrylic resin was the main cause of
hand dermatitis in dental technicians, and more
than 12% of adverse reactions in patients were
associated with resin-based dental materials.
 Pronounced inflammation of the palatal mucosa
beneath a PMMA denture may occur. OF DENTAL MATERIALS: A COMPREHENSIVE REVIEW
BIOCOMPATIBILITY
 Metallic materials
 Certain noble metals, pure metals and alloys used in dentistry derive
biocompatibility from the formation of a protective layer on the surface called a
passive film, which is an oxide of one or more of the components of the alloy.
These films are products of an oxidative (corrosive) reaction that reduces the
corrosion rate by several orders of magnitude and essentially prevents further
corrosion once the passive layer is formed.
 Contact between two different metals in the mouth or changes in the temperature
or pH in the mouth can cause breakdown in protection and can, in the case of
dissimilar metals, lead to galvanic corrosion.
 Although uncommon, the metal most frequently responsible for an allergic
response is nickel which is present in most stainless steels, most cobalt/chromium
alloys, nickel-titanium alloys, and nickel-chromium alloys. If an adverse allergic
response occurs, little can be done other than to exchange the metal component
for one that does not contain nickel
 Zinc phosphate cement:
Strong to moderate cytotoxic reactions that
decrease with time after setting. Leaching of
zinc ions and a low pH is cause of these effects
initial pH on setting is 4.2 at 3 minutes. Best
protection against phosphoric acid penetration
is provided by coating the dentin with two
coats of varnish, dentin bonding agents or liner.
If left over sulcular region after cementation may
cause periodontal destruction and bone loss.
 Zinc-oxide eugenol:
 Eugenol from ZOE fixes cells, depresses
cell respiration and reduces nerve
transmission with direct contact.
 ZOE may form a temporary seal against
bacterial invasion.
 It inhibits the synthesis of prostaglandin
and leukotriens (anti-inflammatory).
 In usage tests: The response is slight-
moderate within first week and mild in
 Glass Ionomer cement:
 Initially low pH may produce chemically irritating conditions for
the dental pulp. The actual pH depends importantly on
manipulation procedures, such as the mixing ratio of components
 In screening and usage tests – pulp reaction to GIC was found to
be mild. Weak nature of polyacrylic acid, unable to diffuse
through dentin because of its high molecular weight.
 As with other materials, irritation of the pulp may be caused by
hydraulic pressure and etching during placement of the
restoration.
 Calcium hydroxide:
Calcium hydroxide is alkaline in nature and is biocompatible.
The high pH is due to presence of free hydroxyl ions in the
set cement
 Latex:
6-7% of surgical allergic reactions to latex were
reported in 1991. Hypersensitivity to latex-
containing gloves is mainly due to hydrolysis
of ammonia which degrades sap proteins to
produce allergens

 Ceramic materials
 The tissue response to ceramic materials are mainly due to i.e porcelains and other
hard ceramics used in crowns, inlays and onlays and ceramics that are intended to
react with surrounding tissues.
 In cell culture experiments, some ceramics were found to cause little suppression of
mitochondria activity, some were found to be initially toxic, but that toxicity
declined after an artificial aging process.
 The aging process was removing cytotoxic chemicals from the ceramics but that the
leaching was only from the surface in some cases while being from the bulk of the
material in others.
 The sintering process would have bound up the toxic species into the bulk and
prevented a tissue response.
 The other class of tissue responses to ceramics that are intended to interact with the
surrounding tissues at least a small amount of dissolution at the surface that contain
calcium and phosphorus.
 Bioglass and calcium phosphate ceramics, interact with surrounding
bone to form a bond between the material and the tissue that can be
stronger than either the bone or the material itself.
 In a process called osteointegration, the materials become integrated
into the bone as it repairs itself and may reside permanently within the
healed bone, although some are dissolved or resorbed and replaced by
new bone.
 Wu et al concluded that oxygen plasma treatment was certified to be
effective in modifying the surface state of Zirconia ceramic and has
potential in the creation and maintenance of hydrophilic surfaces and
the enhancement of cell proliferation and differentiation
 The relative incidence of biological side effects of dental ceramics
compared with other restorative materials is considered to be low. In
general, conventional dental ceramics are considered to be the most
 Chlorhexidine
 The toxicity of chlorhexidine to gingival cells is due to the toxic potency of
chlorhexidine is dependent on the length of exposure and the composition of the
exposure medium.
 Boyce et al. (1995) found 0.05% chlorhexidine is uniformly toxic to both
cultured human cells and microorganisms.
 Agarwal et al. In 1997 found that the chlorhexidine rapidly disrupts the cell
membrane of both crevicular and peripheral blood neutrophils at concentrations
above 0.005% in 5 min, indicating that its inhibitory effect on neutrophil function
is mostly due to its lytic properties.
 Yesilsoy et al. (1995) assessed the short-term toxic effects of chlorhexidine in the
subcutaneous tissue of guinea pigs and found a moderate inflammation present
after 2 days, followed by a foreignbody granuloma formation at 2 weeks.
Ganapathy D. BIOCOMPATIBILITY OF DENTAL RESTORATIVE MATERIALS. European Journal of Molecular & Clinical Medicine. 2021 Jan
10;8(1):504-12.
 Sodium Hypochlorite
 Complications of the use of sodium hypochlorite is due to accidental
injection beyond the apex which can cause violent tissue reactions
characterized by pain, swelling, haemorrhage, and in some cases the
development of secondary infection and paraesthesia (Reeh&
Messer 1989, Becking 1991, Ehrich et al.1993).
 Hypersensitivity reactions to sodium hypochlorite have also been
reported. A great deal of care should be taken when using sodium
hypochlorite during endodontic irrigation.
 Ehrich et al. (1993) suggested that a clinician should check, both
clinically and radiographically, for immature apices, root resorption,
apical perforations or any other conditions that may result in larger
than normal volumes of irrigant to be extruded from the root-canal
system into the surrounding tissue.(Ribeiro, Marques and Salvadori,
2006)
Ganapathy D. BIOCOMPATIBILITY OF DENTAL RESTORATIVE MATERIALS. European Journal of Molecular & Clinical Medicine. 2021
Jan 10;8(1):504-12.
 ETDA
 The leakage of EDTA to periapical tissues during
root-canal preparation may inhibit macrophage
function, and thus alter the inflammatory response
in periapical lesions.
 EDTA has been shown to have weak antibacterial
and antifungal properties.
Jan 10;8(1):504-12.
 Casting Alloys
 The biocompatibility of cast metallic restorations is primarily
determined by the amount and nature of released cations. The
biological effects of these metal ions are significantly different.
 Many investigators have reported that Cu, Ni, and Be have
pronounced cytotoxic potency.
 There is also evidence from in vitro investigations that various
metallic elements, like Ni, Co, and Cr, can modulate the
immune response.
 Local and systemic allergic reactions to many metals have been
observed, with Ni being the most frequent allergenic element.
 Additionally, various other factors could contribute to
biological interactions of metallic restorations, such as
physicochemical surface parameters (atomic ratio of noble to
Ganapathy non noble metals,
D. BIOCOMPATIBILITY etc.),RESTORATIVE
OF DENTAL phase formation, wear,Journal
MATERIALS. European andofthe quality
Molecular & Clinical
of the manufacturing process itself. Medicine. 2021 Jan 10;8(1):504-12.
 Dental Implants
 Although titanium is the preferred choice for dental implants as it is
an inert material, it may encourage toxic or allergic type I or IV
reactions.
 Allergy due to titanium might be accountable for the failure of
implants in some cases. The risk of titanium allergy is more
prevalent in patients having sensitivity to other metals.
 Titanium and zirconium are highly reactive metals, and when
exposed to fluid media or air they quickly develop a layer of
titanium dioxide (TiO2) or zirconium dioxide (ZrO2). This layer of
metal dioxide forms a boundary at the interface between the
biological medium and the metal structure and prevents further
deterioration of materials. It produces passivation of the metal,
determining the degree of biocompatibility and the biological
response to the implant. Any rupture of the oxide layer may
produce corrosion of these metals and affect compatibility .
(SoheiliMajd, Goldberg and Stanislawski, 2003) Ganapathy D. BIOCOMPATIBILITY OF DENTAL RESTORATIVE MATERIALS.
European Journal of Molecular & Clinical Medicine. 2021 Jan 10;8(1):504-12.
 Impression Materials
 Allergic reactions are reported to polyether impression materials which
manifest as swelling, itching and redness. It was seen on patch testing that
a component of the catalyst paste caused the allergy and on replacement of
this component, no allergic reactions were observed.
 There is only a single allergic case reported in which a patient developed
hypersensitivity reaction to polysulfide material in the form of redness,
itching and oedema following secondary impression for upper and lower
complete dentures and on treatment with topical corticosteroids
(Betamethasone valerate ointment 0.1%) she recovered.
 A retrospective case report of fatal anaphylactic shock to alginate
impression material has also been documented.(Schmalz and Arenholt-
Bindslev, 2009) Jan 10;8(1):504-12.
 Resin-based dental materials
 Composite resins and denture-base materials come into direct
contact with oral mucosa and can cause adverse reactions on oral
mucosa such as mucosal irritation, epithelial proliferation and oral
lichenoid reactions
 Restorative materials and dentine bonding agents can also affect the
pulp due to release of leachable components through the permeable
dentin.
Moharamzadeh K, Brook IM, Van Noort R. Biocompatibility of resin-based dental materials. Materials. 2009 Jun;2(2):514-48.
PRECAUTIONS
Beneath a composite resin restoration, a suitable base should be
placed to protect the pulp
Rubber dam should be used always
Dental personnel should always avoid any contact of skin or
even gloves with resin-based composites .
Biocompatibility of dental materials- kelly
TESTS FOR EVALUATION OF
BIOCOMPATIBILITY
Measuring the biocompatibility of a material is not simple.
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.
Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.
Clinical Use
Autian, 1970
New Materials Number of Materials

Biocompatibility of
Restorative Dental Materials
and Related Researches
 In vitro tests
 Cytotoxicity Tests
 Tests for Cell Metabolism or Cell Function
 Tests that Use Barriers (Indirect tests)
 Other Assays for Cell Function
 Mutagenesis Assays
 Animal Tests
 The mucous membrane irritation tests
 Implantation tests
 Usage Tests
 Dental Pulp Irritation Tests
 Dental Implants into Bone
 Mucosa and Gingival Usage Tests
In vitro tests
 Cytotoxicity Tests/ Cell Culture Test
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 Culture: Ring of Inhibition

 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.
NR = Nutral Red
TB= Trypan Blue
51Cr=Chromium51
a change in membrane permeability

 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.
 The advantages of the membrane permeability test is that it identifies cells

that are alive (or dead) under the microscope.

 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 tests.
 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

 Tests that Use Barriers (Indirect tests)
 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.
Agar layer is placed over the cells on which the test material is
incubated for 24 hrs. Agar forms a barrier between the cells and the
material.
If the test material is cytotoxic, it will lead to loss of dye within cells
as lysis occurs. Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.
 Dentin Barrier tests have shown improved correlation
with the cytotoxicity of dental materials
 A number of studies have shown that dentin forms a
barrier through which toxic materials must diffuse to
reach pulpal tissue.
 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 vitro assays and
tissue in implantation tests.
 The thickness of the dentin correlates directly with the
protection offered to the pulp.
 Thus assays 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
Kunawarote material
S. Biocompatibility and Dental Materials and Related Researches.
of Restorative
 Other Assays for Cell Function
 In vitro assays to measure immune function or other
tissue reactions have also been used.
 These assays measure cytokine production by
lymphocytes and macrophages, lymphocyte
proliferation in sheep red blood cells.
 Other tests measure the ability of a material to alter the
cell cycle or activate compliment.

 Mutagenesis Assays
 Mutagenesis assays assess the effect of materials on a
cell’s genetic materials.
 Genotoxic mutagens directly alter the DNA of the cell
through various types of mutations. Each chemical may be
associated with a specific type 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.

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.
 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
paperwork.
 The relevance of the test to the in vivo use of a
material can be quite unclear, especially in
estimating theKunawarote
appropriateness
S. Biocompatibility of Restorativeof
Dentalan animal
Materials and Related Researches.
 The mucous membrane irritation tests
 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.
 The skin sensitization tests
 In the skin sensitization test in guinea pigs, the
materials are injected intradermally to test for
development of skin hypersensitivity reactions.
 This injection is followed by secondary treatment with
adhesive patches containing the test substance.
 If hypersensitivity developed from the initial injection,
the patch will 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.
 Implantation tests
 To evaluate materials that will contact subcutaneous tissue or bone
 The location of the implant site is determined by the use of 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.
 Short-term implantation is studied by aseptically placing the compounds in small, open-
ended, polyethylene tubes into the tissue.
 The test samples and control are placed at separate sites, and allowed to remain for 1 to 11
weeks.
 The tissue response can be evaluated by normal histological, histochemical, or
immunohistochemical methods.
 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.
Advantages of animal tests:
 The ability to assess the biological response that cannot
modeled by in vitro test, including blood interaction,
wound healing, infection, hypersensitivity response,
carcinogenesis and chronic inflammation, etc.
 Generally less expensive than human clinical trials.
 The ability to completed more quickly and can be
controlled to a grater degree.
 Animals may be tested in many stages of life
(embryo, children) in manner that is not possible in humans.
. Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.
 Disadvantages of animal tests:
 Due to the species differences, the congruity of animal response to human
response cannot be assumed, and may be, at worst, misleading.
 Limitation of an animal tests to mimic the human material interface, for
example occlusal force and food, etc.
 Interpretation of response is complex
in animal tests because many
overlapping complex events are
occurring simultaneously.
 Ethical and cost considerations

USAGE TEST
 LARGER ANIMALS or IN HUMAN VOLUNTEERS
 Requires that material be placed in a situation identical to its intended clinical use
DENTAL PULP IRRITATION TEST

TEST MATERIAL PLACED IN CLASS V CAVITY PREPARED IN INTACT, NON
CARIOUS TEETH
TEETH REMOVED, SECTIONED FOR MICROSCOPIC EXAMINATION
TISSUE NECROSIS AND INFLAMMATION GRADED ACCORDING TO

INTENSITY
INTRAOSSEOUS IMPLANT TEST
MATERIALS USED FOR DENTAL IMPLANTS ARE INSERTED
INTO THE JAW.
Criteria for implant success
• Early implant success 1-3 years
• Intermediate implant success 3-7 years
• Long term implant success >7 years
Tests used
 Passage of periodontal probe along side of the implant
 Mobility of the implant
 Radiographic evidence
Available data from these studies show that implants based on titanium
or ceramics, are generally well tolerated by the surrounding tissue.

 MUCOSA AND GINGIVAL USAGE TESTS
 Materials are placed in cavity preparations with sub gingival extensions.
 Slight, moderate or severe based on amount of inflammatory cells
depending on the number of mononuclear inflammatory cells (mainly
lymphocytes and neutrophils) in the epithelium and adjacent connective
tissues
 Disadvantages: • Presence of plaque • Preexisting inflammation in the
gingival tissue • Surface roughness of the restorative material • Over
contouring and under contouring of the restoration

Clinical tests ( in human)
Cross-sectional test
Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches .

 Retrospective test
“Reviewing of the patient records after the fact to assess material
performance.”
Strengths:
 Simplest and least expensive
 Do not require direct patient
examination
Weakness:
 Heavily depend on the quality
of information that recorded.
 The risk of selection bias, due to
the data quality and past practitioners.
 Cross-sectional test
“A patient cohort examined at one point in
time.”
Strengths:
 Ability to define exclusion and inclusion
criteria.
 Collect specific data in standardized condition.
Weakness
 Lack of control of how material was used.
 The variables that may have been important
but were unrecorded.
 Skills and limitation of examiner.
 Prospective/Longitudinal test
Controlled clinical trials/Randomized control trials
Strengths:
 Assure blinding and randomization.s
 The most reliable and interpretable information
Weakness:
 Skill of the operator may not represent the ability of average
practitioner.
 The disease stage treated may not be relevant to clinical practice.
 Expensive and Kunawarote
time consuming
S. Biocompatibility of Restorative Dental Materials and Related Researches.
 Advantages and Disadvantages of Biocompatibility Tests
Test Advantages Disadvantages
In vitro tests Quick to perform
Least expensive
Relevance to in vivo is questionable
Can be standardized
Large-scale screening
Good experimental control
Excellence for mechanisms of interactions
In vivo tests Allows complex systemic interactions

Response more comprehensive than in vitro
Relevance to use of material
questionable
tests Expensive
More relevant than in vitro tests Time consuming
Legal/ethical concerns
Difficult to control
Difficult to interpret and quantify
Usage tests Relevance to use of material is assured Very expensive

Very time consuming
Major legal/ethical issues
Con be difficult to control
Difficult to interpret and quantify
Wataha JC; Biocompatibility of Dental Materials; Chapter 5 from Craig RG & Powers JM, Restorative
Dental Materials, 11th Edition, 2002 Mosby, Inc.
STRATEGIES FOR EVALUATING BIOCOMPATIBILITY
 A. the earliest strategy, in which the testing strategy is focused on toxicity only. Unspecific
toxicity refers to tests not necessarily related to the use of the material, whereas tests under
specific toxicity are more relevant. Clinical trials are equivalent to usage tests in this
scheme.
 B. the contemporary strategy used in most standards documents. All of the products are
screened initially using primary tests. Those have favourable test results are then subjected
to secondary tests. Best materials are used for clinical trials.

NEWER SCHEMES FOR THE PROGRESSION
A. the pyramid scheme of is retained, but it is acknowledged that primary and secondary tests
will play a continuing (but decreased) role as the progress of the testing continues.
B. the usage test has the most stature and the most common progression of tests is from primary
to secondary to usage, but the need to go through several interactions between testing types
is acknowledged.

STANDARDS OF MEASUREMENT OF BIOCOMPATIBILITY
In an effort to make the testing of dental materials for biocompatibility more
uniform within the world, standard organizations have issued documents
specifying or recommending the testing that should be performed to
determine the suitability of new and existing materials in the oral and
maxillofacial environment.
 American National Standard Institute(ANSI) via ADA
 American Society of Testing and Materials(ASTM)
 The Committee on European Normalization(CEN)
 The International Organization of Standardization(ISO)
 Nordic institute of Dental Materials(NIOM)
 The European Union(EN) Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.
John KR. Biocompatibility of dental materials. Dental Clinics of North America. 2007 Jul 1;51(3):747-60.
CONCLUSION
• DUE TO RISE IN NUMBER OF PATIENTS WITH ALLERGIES FROM
DIFFERENT MATERIALS, THE PRACTICING DENTISTS SHOULD BE
AWARE ABOUT THE ALLERGIES DOCUMENTED TO KNOWN
MATERIALS
• FOR ESTABLISHING DIAGNOSIS, IT IS ESSENTIAL TO OBTAIN
PROPER HISTORY RELATED TO ALLERGY, CLINICAL EXAMINATION
AND CONFIRMATORY TESTS.
• IT IS MANDATORY FOR THE CLINICIAN TO KNOW AND
UNDERSTAND THE BIOCOMPATIBILITY OF THE DENTAL
MATERIALS, SO AS TO PROVIDE MAXIMUM ADVANTAGE &
MINIMUM RISK TO THE PATIENT.
REFERENCES
 Biocompatibility of Restorative Dental Materials and Related Researches DRES 407 725, 407 723 27 June and 4
July 2013 Dr. Sitthikorn Kunawarote
 Saigal A, Sharma AK. A comparative study between criteria for selection, evaluation and collection of e-resources
in IIMS Libraries. Library Progress (International). 2017;37(2):269-84.
 Ganapathy D. BIOCOMPATIBILITY OF DENTAL RESTORATIVE MATERIALS. European Journal of Molecular
& Clinical Medicine. 2021 Jan 10;8(1):504-12.
 Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD
 BIOCOMPATIBILITY OF DENTAL MATERIALS: A COMPREHENSIVE REVIEW
 Ganapathy D. BIOCOMPATIBILITY OF DENTAL RESTORATIVE MATERIALS. European Journal of Molecular
& Clinical Medicine. 2021 Jan 10;8(1):504-12.
 Moharamzadeh K, Brook IM, Van Noort R. Biocompatibility of resin-based dental materials. Materials. 2009
Jun;2(2):514-48.
 Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches
 Biocompatibility of Restorative Dental Materials and Related Researches
 Wataha JC; Biocompatibility of Dental Materials; Chapter 5 from Craig RG & Powers JM, Restorative Dental
Materials, 11th Edition, 2002 Mosby, Inc
 John KR. Biocompatibility of dental materials. Dental Clinics of North America. 2007 Jul 1;51(3):747-60
 Biocompatibility of dental materials- kelly

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Biocompatibility of Dental Materials

UNDER THE ABLE GUIDANCE OF:

 4 types of interaction can take place:

Phillips’ Science of Dental Materials

Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD

Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD

Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD

• GLUTATHIONE CONTENT OF HUMAN

ALLERGIC REACTIONS ELICITED BY DENTAL MATERIALS CAN

It is generated by 2 allergen that are If an individual is allergic to a particular

Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD

BIOCOMPATIBILITY OF DENTAL MATERIALS: A COMPREHENSIVE REVIEW

New Materials Number of Materials

Cell Culture: Ring of Inhibition

a change in membrane permeability

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

 The advantages of the membrane permeability test is that it identifies cells

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

 A commonly used enzymatic test for cytotoxicity

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

DENTAL PULP IRRITATION TEST

TEETH REMOVED, SECTIONED FOR MICROSCOPIC EXAMINATION

TISSUE NECROSIS AND INFLAMMATION GRADED ACCORDING TO

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches.

Kunawarote S. Biocompatibility of Restorative Dental Materials and Related Researches .

In vivo tests Allows complex systemic interactions

Usage tests Relevance to use of material is assured Very expensive

Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD

Phillips’ Science of Dental Materials KENNETH J. ANUSAVICE, PhD, DMD

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