The document discusses various tests used to evaluate the biocompatibility of dental materials. It describes in vitro tests like cytotoxicity tests, cell function tests, and indirect tests that are conducted outside of a living organism using cell cultures. Animal tests involve implanting materials in animal tissues and observing inflammatory responses. Usage tests are considered the gold standard as they involve placing materials in animals or humans in situations replicating clinical use conditions. The tests aim to measure biological responses like toxicity, tissue irritation, and inflammatory reactions to determine if a material can perform appropriately in the body.

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2. Presented by,
SACHIN SUNNY OTTA
4rd year KUHS
St.Gregorios Dental College
Submitted to,
Dept. of Conservative
Dentistry
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3. Ability Of a Material To Perform With
Appropriate Host Response In a Specific
Application {IUPAC} .
•Capacity of the material implanted in
the body to exist in harmony with
tissue without causing damage.
•Material that is acceptable for one
application may not be acceptable for
another performance!
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4. Measuring biocompatibility
 Location of the material
 Duration of material exposure
 Stresses placed on the material
(physical,chemical,biological)
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5. biocompatibility TESTS
1. IN VITRO TESTS
2. ANIMAL TESTS
3. USAGE TESTS
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6. IN VITRO TESTS
 Done in test tubes, cell culture dish (outside of living
organism)
 PRINCIPLE: the material is placed in contact with the
cell; in vitro, and analyzed for cellular changes after a
few days
 Broadly divided into:
I. Direct tests
II. Indirect tests
 Direct-material directly contact the cell system
 Indirect-barrier present between cell and material
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7.  TYPES OF CELL USED:
a) PRIMARY CELL
 directly from animal into culture
 grow only for limited time
 retain many characteristics of cell in vivo
 more relevant
Viral/bacterial agents may alter its behavior
b) CONTINOUS CELL
 Primary cells allowed to grow indefinitely
 Do not retain many characteristics of cell in vivo
 Consistently exhibit any feature that the pertain
 Genetic & metabolic stability
 INFERENCES:
The features that are to be checked to define a material..
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8. 1.CYTOTOXICITY TESTS
 Assess the cell number or growth after exposure to the
material
 If cells remain attached to the well & proliferate with
time – material is not cytotoxic
 If cells stop growth, exhibit cytopathic features, & get
detached from well – material is cytotoxic
 RING OF INHIBITION / ZONE OF INHIBITED CELL
GROWTH : zone of inhibited growth around the
cytotoxic material.It assess the cell density
 Teflon used as negative control & plasticized PVC as
positive control.
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9.  Assess the Membrane Permeability :loss of membrane
permeability is equivalent to death!
 Allows the cell to be in contact with dye
 Identifies the cells that are dead or alive
 Two types of dye used:
a) Vital dyes
Actively transported to viable cells (neutral red, Na51CrO4)
Taken by vital cells & drained if cells injured
b) Non vital dyes
Transported if membrane permeability is
compromised(trypan blue, propidium iodine)
Taken by injured cells
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10. 5/21/2018 10smile always
51Cr
TB
NR
51Cr
NR
TBHEALTHY
CELL
INJURED
CELL

11. 2.CELL METABOLISM / CELL FUNCTION TESTS
 Measurement of enzymatic activity of the cell
 Assess DNA & protein synthesis
 MTT test (quantitative cytotoxicity)
MTT FORMAZAN
(yellow (blue
soluble) insoluble)
 If cellular dehydrogenase are not active (coz of
cytotoxicity) formazan will not be formed.Amount of
formazan can be quantified by dissolving it &
measuring optical density of solution.
 Measure gene activation
 Measure specific cell function
Cell.dehydrogenase
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12.  3.INDIRECT TEST
 Test that use barriers between cell & material
 No direct contact
 Agar overlay method : agar act as a barrier which is jammed
between NR stained cell layer & material.
©
 INFERENCE: I f cells are injured, NR released leaving a zone
of inhibition .
 variability of agars diffusion properties
+ control sample -control
1% agar(b)
Neutral red stained cell layer (a)
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13.  MILLIPORE FILTER ASSAY:
a. Monolayer of cells on filters made of cellulose ester
b. Medium containing 1% agar allowed to gel over the cells
c. Sample material
Monolayer – gel is detached & turned upside down so that
filter is on top for placement of sample material
 INFERENCE: if sample is cytotoxic, width of cytotoxic
zone is assessed.
 May be influenced by leaching of toxic products from test
material
 DENTIN BARRIER TEST :thickness of dentin act as barrier
to protect the pulp.
 Dentin disks are placed between cell & material.
 Effect of medium on cell metabolism measured
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A : TEST MATERIAL PLACED
B : DEVICE TO HOLD DENTIN DISK
C :CELL CULTURE MEDIUM , Renewed via inlet & outlet.

15. 4)OTHER ASSAYS FOR CELL FUNCTION
a. Immune function
b. CK production by lymphocyte & macrophages
c. Chemotaxis
d. Complement activation
e. T-cell rosetting to sheep RBC
Their significance is to reduce animal tests
 Activation of complement by resin or their corrosion
products may prolong inflammation in the gingiva or
pulp
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16. INFERENCES
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17. CELLULAR CHANGES
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NON CYTOTOXIC
INTERACTION
ALIVE FIBROBLAST
CYTOTOXIC
INTERACTION
DEAD / DYING
FIBROBLAST
DETACHED &
ROUNDENED
FIBROBLAST

18. CELLULAR CHANGES DUE
TO COMPROMISED
BIOCOMPATABILITY
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19. 5)MUTAGENESIS ASSAY
 Assess the effect of material on cell`s genetic material
a) Genotoxic mutagens : directly alter DNA of cell.Each
chemical has specific DNA mutation. They mostly
require activation or biotransformation
(promutagens)
b) Epigenetic mutagens : support tumor growth by
altering the cell`s biochemistry. Donot directly alter
DNA.
 Mutagens may or may not be carcinogens & vice versa.
 Ames` test : short term mutagenesis test
 Mutant strains of Salmonella typhimurium require
exogenous histidine
 These strains were grown on histidine free culture
media
 Check whether mutant strains were converted to
native strains5/21/2018 19smile always

20. INFERENCE: chemicals that significantly increase the
frequency of reversion back to native state have high
probability of being carcinogenic.5/21/2018 20smile always

21.  STYLES` CELL TRANSFORMATION TEST: alternative to
bacterial test
 Quantifies ability of potential carcinogens to transform
a standardized cell
 Transformed cells only grow within an agar gel.
 SISTER CHROMATID EXCHANGE TEST (SCE)
 MOUSE LYMPHOMA ASSAY (MOLY)
 CHROMOSOME ABBERATION TEST (ABS)
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22. ANIMAL TESTS
 Mammals viz, mice, rats , hamsters, guinea pigs are
used.
1)MUCOUS MEMBRANE IRRITATION TEST
 Place the test material , positive & negative control in
contact with hamster cheek pouch tissue or rabbit oral
tissue.
 After several weeks, tissue examined & photographed.
 Animals are then sacrificed & biopsy specimen are
prepared for histological examination.
 INFERENCE : inflammation to mucous membrane
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23. 2)SKIN SENSITIZATION TEST (guinea pig
maximization test)
 Material injected intradermally into guinea pigs
 Assess skin hypersensitivity reactions
 Followed by secondary treatment with adhesive patches
containing test material
 INFERENCE : if hypersensitivity develop from initial
treatment, patch test will elicit an inflammmatory
response (no reaction to intense redness).
3)DECISION POINT APPROACH :measure mutagenic &
carcinogenic properties.
 Set of tests broadly divided as limited term in vivo test &
long term tests.
 They are applied in specific order & testing is stopped
when any one indicate mutagenic potential.
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24. 4)IMPLANTATION TESTS : for materials in contact with
subcutaneous tissue or bone.
 Amalgams & alloys (contact with gingiva)
 Aseptically place the compound in small, open ended
polyethylene tubes & then placed into the tissue.
 Allowed to remain for 1-11 weeks(short term) or for 1-2
years(long term test for identification of chronic
inflammation or tumor formation)
 INFERENCE : tissue response evaluated by histological,
or histochemical methods
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25. USAGE TESTS
 GOLD STANDARD OF TESTS.
 DONE IN ANIMALS OR HUMAN VOLUNTEERS
 Situation identical to its clinical use (distinct from animal
tests)
 Dogs or monkeys are used (similar oral environment to
humans)
 If humans are used, its CLINICAL TRIAL.
1)DENTAL PULP IRRITATION TESTS :
 Sample placed in class5 cavity preparation in intact non
carious tooth
 Allowed to remain for 1-8 weeks
 ZnOE –negative control & silicate cement-positive control
 Note: ZnOE causes irritation if placed in direct contact wih
the pulp!
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26.  INFERENCE: teeth removed & sectioned for
microscopic examination shows necrotic &
inflammatory reactions of pulp.
 Types of pulpal response:
a) Slight
Mild hyperemia,few inflammatory cells, slight
hemorrhage in odontoblastic zone
b) Moderate
Increase in number of inflammatory
cell,hyperemia,slight disruption of odontoblastic
zone.
c) Severe
Increased inflammatory infiltrate,hyperemia,total
disruption of odontoblastic layer in the zone of cavity
preparation,reduction or absence of
predentin,localized abscesses5/21/2018 26smile always

27. 2)DENTAL IMPLANTS INTO BONE :
 Success of implants assessed by:
a. Penetration of periodontal probe along side of implant
b. Mobility of implant absent
c. Radiograph indicating either osseous integration or
absence of radiolucency around implant
d. Minimal vertical bone loss
 Failure of implant was denoted by formation of wall of
a cyst around the implant caused by slow degradation
of implant material into tissue
 Fibrous capsule formation was a sign of irritation &
chronic inflammation.
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28. 3)MUCOSA & GINGIVAL USAGE TESTS :
 Materials place in cavity with subgingival extensions
 Observed at 7th day & 30th day.
 Response graded as:
a. Slight
Few lymphocytes in epith. & adjacent CT
b. Moderate
Numerous lymphocytes in CT & few neutrophils in epith.
c. Severe
Significant mononuclear & neutrophil infiltation &
thinned or absent epith.
Perform oral prophylaxis before the test as bacterial
plaque initiate inflammation in gingiva that may
hinder the response.
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29. Correlation of tests
 Each type of test are designed to measure different
aspects of biological response to materials
 No single test would be adequate to completely
characterize the biocompatibility of a material.
 Method of diagnosis : PYRAMID TESTING
PROTOCOL.
 All materials were tested at the bottom of the pyramid
& materials were webbed out as the testing continued
to the top of the pyramid.
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30. EARLY STRATEGY
CLINICAL
TRIALS
SPECIFIC
TOXICITY
UNSPECIFIC TOXICITY
No. of tests
•Number of materials tested is
represented by width of the
triangle.
•Unspecific toxicity :condition
that did not necessarily reflect
those of materials use (in vitro
or animal tests)
•Specific toxicity : condition
more relevant to materials
clinical use.
•Clinical trial : usage tests
Progress of
testing
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31. PRIMARY
SECONDARY
USAGE
No.of tests
Progress of
testing
CONTEMPORARY STRATEGY
•Primary tests : in vitro & in
vivo tests
•Secondary tests: advanced
biological tests
•Usage tests :clinical trials
•The concept was similar to
early scheme except types of
tests were extended to
include other than toxicity,
such as immunogenicity &
mutagenicity.
•Only material that passed
the first tier of tests were
graduated to second tier , &
only those that passed second
tier were graduated to clinical
trial.5/21/2018 31smile always

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33. FUTURE SRTATEGIES
 All three test done initially, but as testing
progresses,usage test predominates. Primary & secondary
tests play a continuing but a decreased role as test
continues
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34. The most common progression is from primary to
secondary to usage test.
Tests may be performed at any time depending upon
the problems encountered.
Tests accurately screen in or out a material
Assessing biocompatibility is an ongoing process.
Who regulate the measurement of
biocompatibility?
ANSI/ADA Document 41
ISO 10993
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36. Why not in vivo tests??
 Contact between cells & material is of question
 Separation of cells & materials may occur from
keratinized epithelium, dentin & cell matrix
 Reduce other systemic toxicity
 Thorough monitoring required.
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37. EFFECTS OF NON
BIOCOMPATIBLE MATERIALS??
1. TOXICITY
2. INFLAMMATION
3. ALLERGY
4. MUTAGENICITY
5. IMMUNOTOXICITY
6. ESTROGENICITY :bisphenol A
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38. BIBLIOGRAPHY
Restorative Dental Materials-Craig (11th
edition)
Phillips Science of Dental
Materials(11th edition)
www.ncbi.gov
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