Biocompatibility of Dental Materials

METHODS OF TESTING
BIOCOMPATIBILITY OF
DENTAL MATERIALS
PADMAPRIYA RAMANUJAM
I YEAR POSTGRADUATE
DEPARTMENT OF CONSERVATIVE DENTISTRY
AND ENDODONTICS
TABLE OF CONTENTS
 DEFINITION
 INTRODUCTION
 CHARACTERISTICS OF A BIOCOMPATIBLE MATERIAL
 MEASURING BIOCOMPATIBILITY
 REQUIREMENTS OF A BIOCOMPATIBLE TEST
 INVITRO, ANIMAL AND USAGE TESTS
 ETHICAL ISSUES WITH ANIMAL AND USAGE TESTS
 EARLIER STRATERGIES AND CURRENT TRENDS IN MODELS
 STANDARDS THAT REGULATE BIOCOMPATIBILITY TESTING
 ADVERSE EFFECTS ON EXPOSURE TO DENTAL MATERIALS
 BIOCOMPATIBILITY OF FREQUENTLY USED DENTAL MATERIALS IN CONSERVATIVE
DENTISTRY AND ENDODONTICS
DEFINITION
 The ability of a biomaterial to perform its desired function with respect to a medical (or dental) therapy,
without eliciting any undesirable local or systemic effect in the recipient or beneficiary of that therapy, but
generating the most appropriate beneficial cellular or tissue response in that specific situation, and
optimizing the clinically relevant performance of that therapy (Williams, 2008)
 Biocompatibility is defined as the ability of a material to elicit an appropriate biological response in a given
application in the body (Craig)
 ADA Specification No. 41
 ISO Specification No. 7405
INTRODUCTION
 Biocompatibility is a property of a material interacting with its environment
 The biological response will change with change in the
Host
Material itself Application of the material

 Whether or not a material is biocompatible therefore depends on the
Types and locations
Physical and chemical Surface Physical function for of patient tissues that
nature of its characteristics of the which the material will be exposed to the
components material will be used material
Amount and nature of Biological response

Duration of exposure substances eluted that will be required
from the material from it
PHILLIPS’ SCIENCE OF DENTAL MATERIALS, 12th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION
CHARACTERISTICS OF A BIOCOMPATIBLE MATERIAL
- They should not sensitize and produce allergic reactions
- They should not undergo biodegradation
- They should not contain any toxic diffusible substances which may get released and
enter into the circulatory system
- They should not cause adverse effects to soft and hard tissues of the oral cavity in
particular and the whole body in general
- They should not be carcinogenic/ mutagenic/ teratogenic
MEASURING BIOCOMPATIBILTY
 Historically, new materials- simply tested in humans- to assess their biocompatibility- no longer accepted
 Currently, the usefulness for predicting biocompatibility is directly proportional to the fidelity with which
the test mimics the clinical use of the material in every regard, including time, location, environment, and
placement technique
IN
ANIMAL USAGE
VITRO

REQUIREMENTS OF A BIOCOMPATIBILITY TEST
 The test should be performed under conditions that simulate the actual use of
the material in the body
 The test conditions should reflect the effects of the material’s time in the body
on the biological response
 The stresses induced in the material under its intended function should be
considered in the interpretation of the biological response

IN VITRO TESTS
Done outside a living organism, require placement of a material or a component of a material in contact with a cell, enzyme, or some
other isolated biological system
CONTACT CYTOTOXICITY MUTAGENESIS

TESTS TESTS FOR CELL ASSAYS
Measure METABOLISM OR Measure the effect
DIRECT INDIRECT
Advantages cytotoxicity or cell CELLDisadvantages
FUNCTION on the genetic
When the material When there is a
growth material in a cell
Quick
contacts cellperform barrier of some sort
the to Relevance to in vivo is questionable
systemLeast expensive
without between the
Can be standardized material and the
barriers
Large-scale screening cell system TYPES OF CELL LINES
Good experimental control
Excellence for mechanisms of interactions
Some
The material PRIMARY CELLS CONTINUOUS CELL LINES
extract from - Cells taken directly from - Cells that have been transformed
is physically
the material an animal and cultured previously to allow them to grow
present with
contacts the - Grow for only a limited more or less indefinitely in culture
the cells
cell system time in culture but usually - Because of this transformation,
retain many of the these cells do not retain all in vivo
characteristics of cells in characteristics, but they do
vivo consistently exhibit those features
that they do retain
CYTOTOXICITY TESTS
Assess cell death caused by a material by measuring cell number or growth
before and after exposure to that material
 MEMBRANE PERMEABILITY tests are used to measure cytotoxicity by the ease with which a dye can
pass through a cell membrane, because membrane permeability is equivalent to or very nearly equivalent to
cell death
Noncytotoxic interaction with Cytotoxic interaction with

calcium hydroxide pulp-capping agent a type of calcium hydroxide pulp-capping agent
TESTS FOR CELL METABOLISM/ CELL FUNCTION
Use the biosynthetic or enzymatic activity of cells to assess cytotoxic response
 Common examples- tests that measure deoxyribonucleic acid (DNA) synthesis or protein synthesis
 A commonly used enzymatic test for cytotoxicity- MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] test
Colorimetric assays based on - NBT (nitroblue tetrazolium)
different tetrazolium salts - XTT [2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide salt]
- WST (a water-soluble tetrazolium)
 Alamar Blue tests- quantitatively measure cell proliferation using a fluorescent indicator that allows
continuous monitoring of cells over time

TESTS THAT USE BARRIERS
(INDIRECT TESTS)
 Since direct contact often does not exist between cells and materials during in vivo use, several in vitro
barrier tests have been developed to mimic in vivo conditions
AGAR OVERLAY METHOD DENTIN DISK BARRIER TEST METHOD

OTHER ASSAYS FOR CELL FUNCTION
 Includes in vitro assays to measure immune function or other tissue reactions
 Measure cytokine production by lymphocytes and macrophages, lymphocyte proliferation, chemotaxis, or
T-cell rosetting to sheep red blood cells and the ability of a material to alter the cell cycle or activate
complement

MUTAGENESIS ASSAYS
Assess the effect of a biomaterial on a cell’s genetic material
 Genotoxic mutagens- Directly alter cell DNA through various types of mutations
- Some 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, immune system, acting on hormones, or by other mechanisms
 The Ames test- most widely used, only validated short term mutagenesis test
- considers the conversion of a mutant stock of Salmonella typhimurium back to a native
strain, because chemicals that increase the frequency of reversion back to the native
state have a high probability of being carcinogenic in mammals
 Styles’ cell transformation test- Alternative, but may not be very relevant to mammalian systems

ANIMAL TESTS
 Involve mammals such as mice, rats, hamsters, or guinea pigs, where the material is not placed in the animal
with regard to its final use
 As use of an animal allows for the complex interactions between the material and a functioning, complete
biological system to occur, these biological responses in animal tests are more comprehensive and relevant
Advantages Disadvantages
than in vitro test
Allows complex systemic interactions Relevance to use of material is questionable
 The mucousmore
Response membrane irritation
comprehensive test-
than determines
in vitro tests whether a material causes inflammation to mucous
Expensive
More relevant
membranes than in vitro
or abraded skintests Time consuming
Legal/ethical concerns
 In a skin sensitization test, materials are injected intradermally
Difficult toto test for development of skin
control
hypersensitivity reactions, followed by secondary treatment with
Difficult adhesive
to interpret patches
and quantifycontaining the test
substance
 Implantation tests are used to evaluate materials that will contact subcutaneous tissue or bone. The location of
the implantation site is determined by the use of the material and may include connective tissue, bone, or
muscle

USAGE TESTS
 Done in animals or in human study participants where the material is placed in a situation identical to its
intended clinical use Advantages Disadvantages
 Relevance
Usually to use
employ of material
larger animals is that
assured Very expensive
have similar oral environments to humans, such as dogs, mini-swine or
Very time consuming
monkeys as the usefulness of biocompatibility tests is directly proportional
Major legal to the fidelity with which the test
/ ethical issues
mimics the clinical use of the material Can be difficult to control
Difficult to interpret and quantify
 When humans are used, the usage test is termed a clinical trial- ultimate golden standard
 In dentistry, dental pulp, periodontium, and gingival or mucosal tissues are the main targets of usage tests

DENTAL PULP IRRITATION DENTAL IMPLANTS IN MUCOSA AND GINGIVAL ENDODONTIC USAGE
TESTS BONE USAGE TESTS TESTS
Material- placed in class 5 - Assessed by placement in cavity
cavity preparations in preparations with subgingival
- Material to be
intact, noncarious teeth Best predictors for success of extensions
tested is placed in
implants are- - Responses are categorized as
the root canals of
At conclusion the teeth are  Careful patient selection slight/moderate/severe, depending
animal models
removed and sectioned for  Ideal clinical conditions on the number of mononuclear
(Primates and dogs)
microscopic examination Three commonly used tests inflammatory cells (mainly
after root canal
to predict implant success- lymphocytes and neutrophils) in the
preparation
Classified according to the (1) Penetration of epithelium and adjacent connective
- Sometimes, pulpal
intensity of the necrotic/ a periodontal probe along the tissues
infection is
inflammatory response side of the implant - Bias- frequent presence of some
provoked
 Inflammed pulp Normal (2) Mobility of the implant degree of preexisting inflammation
experimentally
pulp (3) Radiographs indicating in gingival tissue due to bacterial
- Histological
 Usage tests with induced either osseous integration or plaque, surface roughness of the
evaluation of the
pulpitis, which allow radiolucency around the restorative material, open or
apical tissues is
evaluation of the type and implant overhanging margins, and
done
amount of reparative dentin over/under-contouring of the
formed are more futuristic restoration

CLINICAL TRIALS
Most ideal method- test on human subjects
After FDA approval  3 stages of clinical trails

Once a material passes all 3 previous
stages- an IND (Notice of Claimed Phase I trials Phase III trials
Investigational Exemption for a New - Material is tested in a few - Test material is subjected to a large-
healthy individuals after Phase II trials scale study with sufficient number of
Drug) application with following details obtaining informed consent - Material is tested in a small subjects and unbiased allotment of
is submitted to the FDA for approval: - Administered in increasing group of patients
these subjects to the control and test
groups
1. Novelty of the test material, its increments until the required - Helps determine the - Test groups must comprise of even
result is achieved /till an “Therapeutic dose” of the other materials with which the test
composition and manufacture adverse effect manifests material can be compared
material
2. Results of all the in vitro and animal - Thus, the “Safe dose” of the - Helps prove that the test material is
“Safe and Effective”
tests conducted on the test material material is determined
3. Proposed application of the material,
- Data obtained from these trials Phase IV trials
availability & method of use  submitted to the FDA in NDA - Reports on any allergies,
4. Methods to determine the safety of the (New Drug Application) interaction with other materials, and
the amount of material
drug and its outcome in humans - Complete  Permitted for circulated/distributed is submitted to
marketing the FDA at timely intervals
- Incomplete  Subjected to more - Aid in recognizing any delayed
tests adverse effects of the material
MATERIALS USED IN DENTISTRY, S. MAHALAKSHMI

ETHICAL ISSUES REGARDING ANIMAL AND HUMAN USAGE IN
BIOCOMPATIBILITY
Latest ISO 7405 guidelines  recommended the evaluation of alternative nonpatient/nonanimal testing strategies
2 experimental in vitro strategies  Most relevant to clinical situations
TOOTH SLICE CULTURE TEST

ODONTOBLAST CELL CULTURE TEST - Uses cultured slices of teeth that have retained pulp
- Use primary cell culture of odontoblasts or vitality
pulp fibroblasts that simulate human in vivo tissue - Advantages- it permits the cytotoxicity
responses better screening of test materials on pulpal tissues,
- Attempts are being made to culture odontoblasts, allows the evaluation of restorative therapies,
but cannot be readily grown when separated from growth factor, stem cell and gene therapy, without
the dentin matrix presenting a risk to animal or human health
- Pulp fibroblasts can be readily cultured but their - It is also reproducible because the teeth are
growth characteristics can slightly vary from culture maintained in identical experimental conditions
to culture - Allows the role of cavity preparation and
restoration variables to be measured (can only be
measured in animals and human clinical trials)

EARLY AND CONTEMPORARY STRATEGIES FOR THE USE OF
BIOCOMPATIBILITY TESTS TO ASSESS THE SAFETY OF
MATERIALS

TWO SUGGESTED FUTURE STRATEGIES FOR
BIOCOMPATIBILITY TESTING OF MATERIALS

STANDARDS
Final THAT REGULATE MEASUREMENT
tests-
OF BIOCOMPATIBILITY
ANSI/ADA Specification
one/mor 41- 2005 ISO 7405
e Revision of the dental components of ISO 10993, published in 1992
in resulted in ISO 7405:2008 “Preclinical evaluation of biocompatibility
vivo of medical devices used in dentistry—Test methods for dental
usage materials.” the most recent ISO standard available for
tests, biocompatibility testing of dental materials
first in
larger
animals
Secondary tests in small
animals(primate
(in vivo) for- Initial tests
s)
inflammatory/&immunogenic Supplementary tests
- Cytotoxicity, sensitization
potential finally - Chronic toxicity,
(e.g., dermal
with irritation,
& systemic toxicity
carcinogenicity,
subcutaneousFDAand bony - In vitro/ in animals tests in
implantation, hypersensitivity
approva biodegradation
tests) non-usage situations
l, in - Done in animal systems
humans
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
organism

ADVERSE EFFECTS FROM EXPOSURE TO DENTAL
MATERIALS
TYPES OF TOXIC AGENTS
 Local agents- producing local tissue reaction
 Systemic toxins- act on target organs or organ systems
 Asphyxiants- deprive the tissues of oxygen (anoxia)
 Irritants- cause inflammation of tissues by direct contact
 Allergic sensitizers- activate the production of antibodies- in turn, react with the antigen to
make it a latent complex
 Carcinogens- cause cancer in an organism
 Reproductive toxins- mutagens or teratogens

PATHWAYS TO LOCAL AND SYSTEMIC EFFECTS

INFLAMMATORY AND ALLERGIC REACTIONS

PATCH TEST PRICK TEST
(DELAYED HYPERSENSITIVITY) (IMMEDIATE HYPERSENSITIVITY)
- Questions about existing allergies should always be part

of the first medical examination of the patient
- Patient should also be asked at regular intervals on newly
- Adhesive tapes with potential developed
allergens atallergies and general medical history should
concentrations that are just highperiodically
enough to trigger the
be updated - Allergen is applied as a drop to the skin, and
allergic reaction are adhered to the clinically sound skin
then the skin is “pricked” through the drop
of the patient’s back - After 5–30 min, the skin reaction is assessed
- Patient should avoid excessive sweating, exposure
(redness, formation of weals)
to sun, scratching of the back and bathing
- Skin reactions (redness, itching, blisters) are assessed
after 2 , 3, 5, 7 days since immunocompetent T
lymphocytes require several days before they cause a
visible allergic reaction
BIOCOMPATIBILITY OF DENTAL MATERIALS, SCHMALTZ AND BINDSLEV

OTHER REACTIONS
Mutagenicity
Genotoxicity
- Ability to pass genetic damages on to
- Ability to cause alterations of the the next generation
genome DNA - When the components of a material alter
- A transfer of these genetic damages the base-pair sequences of the DNA in
to subsequent generations of cells the cells, mutagenic reactions (mutations)
result
can be avoided by programmed cell
death - Example- metals such as Ni, Cu, and Be
are known mutagens
Carcinogenicity Teratogenicity
Ability to induce malignant Ability to cause malformations
tumors during embryonic development

RESTORATIVE MATERIALS
 SILVER AMALGAM
 GLASS IONOMER CEMENT
 ZINC OXIDE EUGENOL
 ZINC PHOSPHATE
 ZINC POLYCARBOXYLATE
 ZINC POLYACRYLATE
 RESIN BASED CEMENTS
SILVER AMALGAM
STURDEVANT’S ART AND SCIENCE OF OPERATIVE DENTISTRY, A SOUTH ASIAN EDITION

GLASS IONOMER CEMENT

ZINC OXIDE EUGENOL

ZINC PHOSPHATE

ZINC POLYCARBOXYLATE

ZINC POLYACRYLATE
- Evoke a pulpal response similar to ZOE, with a slight-to-moderate response

after 3 days, and only mild chronic inflammation after 5 weeks
- Reparative dentine formation is minimal with these cements and thus
recommended only for use in the floor of cavity preparations with intact
dentin
MALLINENI et al, BIOCOMPATIBILITY OF VARIOUS DENTAL MATERIALS IN CONTEMPORARY DENTISTRY: A

NARRATIVE INSIGHT, JOURNAL OF INVESTIGATIVE AND CLINICAL DENTISTRY 2013
RESIN BASED CEMENTS

PRE TREATMENT AND POST OPERATIVE MATERIALS
 ETCHING AGENTS
 BONDING AGENTS
 FISSURE SEALANTS
 BLEACHING AGENTS
 LATEX
ETCHING AGENTS
 Phosphoric acid is a strongly corrosive, mineral acid that, if allowed to come into contact with
the gingiva or the lip, might result in severe burning of the soft tissues.
 Thus, etchants that run off the teeth onto tissues should be rinsed off immediately with copious
amounts of water
 Hydrofluoric acid should only be used extra-orally, due to its toxicity and extremely strong
corrosive effect on living tissues

BONDING AGENTS
 Components of the bonding agents might penetrate up to 0.5 mm into dentin and cause
significant suppression of cellular metabolism for up to 4 weeks after application
 HEMA is less cytotoxic in tissue culture than bis-GMA
 If the dentine in the floor of the cavity preparation is thin (i.e. <0.1 mm), HEMA might be
cytotoxic in vivo

FISSURE SEALANTS
 Leaching of monomers (bis- DMA and bis- GMA) from resins can occur during the initial
setting period in conjunction with fluid sorption and desorption over time  bioactive
 Potential estrogenecity with BPA

BLEACHING AGENTS
 Predominantly contain contain some form of carbamide peroxide or hydrogen peroxide in a gel
matrix- rapidly traverse in vitro the dentine in sufficient concentrations to be cytotoxic
 The occurrence of tooth sensitivity is very common with the use of these agents
 They might chemically burn the gingival and other soft tissues if not sequestered adequately in
the bleaching tray

LATEX

ENDODONTIC MATERIALS
 OBTURATING MATERIALS
 ROOT CANAL SEALERS
 IRRIGANTS
 CaOH
 BIODENTIN
OBTURATING MATERIALS

ROOT CANAL SEALERS

IRRIGANTS
NaOCl
- Highly toxic at high
concentrations and tends to CHLORHEXIDINE
induce tissue irritation upon EDTA - Chlorhexidine is stable, minimally
contact - Leakage into periapical absorbed by the mucosa and skin
- Most complications tissues during RCT might inhibit and is well tolerated in animals
accidental injection beyond the macrophage function and alter the - Concentrations of chlorhexidine
root apex pain, swelling and inflammatory response of periapical clinically used have acceptable
hemorrhaging, secondary lesions biocompatibility with
infection, paraesthesia and relative absence of cytotoxicity
hypersensitive reactions are
noted

CALCIUM HYDROXIDE

BIODENTINE
 Laurent et al (2008) were the first to show the promising biological properties of Biodentine on
human fibroblast cultures.
 Zhou et al (2013) compared biodentine with white MTA and glass ionomer cement using human
fibroblasts, where both white MTA and biodentine were found to be less toxic compared to
glass ionomer during the 1- and 7-day observation period
MALKONDU ET AL, A REVIEW ON BIODENTINE, A CONTEMPORARY DENTINE REPLACEMENT AND REPAIR MATERIAL, BIOMED RES INT 2014
ALLOYS AND METALS
 CAST ALLOYS
 GOLD ALLOYS
CAST ALLOYS

GOLD ALLOYS
 Adverse reactions- burning sensation
- lichenoid lesions
 Pulpal reactions due to- Condensation force
- Thermal conductivity
- Dehydration of the cavity
- Microleakage
 Toxicity- Pulpal inflammation
- Destruction of odontoblasts
- Hemorrhaging

CERAMIC BIOMATERIALS
 DENTAL PORCELAIN
 ALUMINA AND ZIRCONIA
DENTAL PORCELAIN

ALUMINA AND ZIRCONIA
ALUMINA ZIRCONIA
- First introduced as a reinforcing inclusion for - Remarkably dense, inert, and hard surface, and
dental porcelain almost 50 years ago is highly biocompatible
- However, the inherently low tensile strength of - Used as root canal posts, subgingival dental
porcelain does not allow it to be used in areas implant abutments, frameworks (cores) for
of high stress all-ceramic fixed dental prosthesis
- Alternatively used in the production of
orthodontic brackets
Liu et al (2012)-
Contraindications are
unknown/ atleast unreported

- Dental material reported to cause most adverse reactions in patients is
AMALGAM and the incidence of ORAL LICHENOID REACTIONS
adjacent to amalgam restorations occur more often than other dental
materials
- The most common allergic reactions in dental staff are allergies to LATEX,
ACRYLATES AND FORMALDEHYDE
- While polymethylmethacrylates and latex trigger DELAYED hypersensitivity
reactions, sodium metabisulphite and nickel cause IMMEDIATE reactions
TAKE HOME MESSAGE!
Over the last few years, due to the rise in number of patients with allergies from different
materials, practicing dentists should have knowledge about documented allergies to known
materials, helping avoid such allergic manifestations in the dental clinic

Biocompatibility of Dental Materials

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Biocompatibility of Dental Materials

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METHODS OF TESTING

Material itself Application of the material

Amount and nature of Biological response

PHILLIPS’ SCIENCE OF DENTAL MATERIALS, 12th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CONTACT CYTOTOXICITY MUTAGENESIS

Noncytotoxic interaction with Cytotoxic interaction with

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

AGAR OVERLAY METHOD DENTIN DISK BARRIER TEST METHOD

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

After FDA approval  3 stages of clinical trails

MATERIALS USED IN DENTISTRY, S. MAHALAKSHMI

TOOTH SLICE CULTURE TEST

MATERIALS USED IN DENTISTRY, S. MAHALAKSHMI

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

CRAIG’S RESTORATIVE DENTAL MATERIALS, 13th EDITION

PHILLIPS’ SCIENCE OF DENTAL MATERIALS, 12th EDITION

PHILLIPS’ SCIENCE OF DENTAL MATERIALS, 12th EDITION

PHILLIPS’ SCIENCE OF DENTAL MATERIALS, 12th EDITION

- Questions about existing allergies should always be part