Methods for evaluation of

BIOCOMPATIBILITY

BIOMATERIALS
Biomaterials are defined as:

any substance (different from drugs) or combination of substances,

with synthetic or natural origin, which can be used for any period of
time, as a whole or as a part of a system which treats, improves, or
replaces any tissue, organ, or function of the body
Biomaterials Science is:

The study and knowledge of interactions between non-living and living

materials
2nd Consensus Conference on Biomaterials Chester (UK), 7-8
September 1991

BIOMATERIALS SCIENCE

BiomaterialTissue
Interactions
Biocompatibility

Biocompatibility is the ability of a material to

perform with an appropriate host response in a specific application
The Williams Dictionary of Biomaterials
Liverpool University Press, 1999

Host responce: The local and/or systemic response of the host

organism to the implanted material or device.
Material responce: The response of the material to a living system.

Host Responces
Material Responces

Short term

Sweeling and Leaching

Acute toxicity

Corrosion and Dissolution

Irritation

Friction and Wear

Sensitization

Deformation and Failure

Hemolysis

Effect of surface morphology

Thrombogenicity

Effect of degradation products

Long term
Subchronic and Chronic toxicity
Genotoxicity
Carcinogenicity
Effect on reproduction including
teratogenicity

The Reality
The host response, involving both humoral and cellular components is
extremely complex
Several of these components involve amplification or cascade events

There is often a two-way relationship between the material variable and the
host response e.g. a degradation process is pro-inflammatory and the products
of inflammation enhance the degradation process

Mechanical stability influences the host response, and in many situations the
host response determines the stability (e.g. osseointegration)
The host response is time dependent

The host response is patient specific, depending on age, sex, health status /
concomitant disease, pharmacological status, lifestyle, etc.
Biocompatibility is species specific - testing materials in young rats in

Liverpool may be of no relevance to senior citizens in Sydney.

The evaluation of the biocompatibility of materials,

i. e. the evaluation of the suitability of materials for
use in implantable medical devices, has evolved over
approximately the last 50 years.
1989: International Standards Organisation concerning biological
evaluation of medical devices (ISO 10993) and currently operating

The ISO 10993 set entails a series of standards for

evaluating the biocompatibility of a medical device prior to
a clinical study.
List of the standards in the Biological evaluetion of medical devices
ISO 10993 (EN 30993)
ISO 10993-1:2009 Evaluation and testing
ISO 10993-2:2006 Animal welfare requirements
ISO 10993-3:2003 Tests for genotoxicity, carcinogenicity and
reproductive toxicity
ISO 10993-4:2002/Amd 1:2006 Selection of tests for interactions
with blood
ISO 10993-5:2009 Tests for in vitro cytotoxicity
ISO 10993-6:2007 Tests for local effects after implantation
ISO 10993-7:2008 Ethylene oxide sterilization residuals
ISO 10993-8:2001 Selection of reference materials
ISO 10993-9:1999 Framework for identification and quantification of
potential degradation products

ISO 10993-10:2010 Tests for irritation and delayed-type

hypersensitivity
ISO 10993-11:2006 Tests for systemic toxicity
ISO 10993-12:2007 Sample preparation and reference materials
(available in English only)
ISO 10993-13:1998 Identification and quantification of degradation
products from polymeric medical devices
ISO Identification and quantification of degradation products from
ceramics
ISO 10993-15:2000 Identification and quantification of degradation
products from metals and alloys
ISO 10993-16:1997 Biological evaluation of medical devices Part 16:
Toxico-kinetic study design for degradation products and leachables
ISO 10993-17:2002 Establishment of allowable limits for leachable
substances
ISO 10993-18:2005 Chemical characterization of materials
ISO/TS 10993-19:2006 Physico-chemical, morphological and
topographical characterization of materials
ISO/TS 10993-20:2006 Principles and methods for immunotoxicology
testing of medical devices

ISO 10993-5
Tests for Cytotoxicity-In vitro Methods
In standard cytotoxicity test methods, cell monolayers are grown to
near confluence in flasks and are then exposed to test or control
materials directly or indirectly by means of fluid extracts.

Indirect Method: Elution test

Direct Method: Contact test

 Indirect Method: Elution test

In the elution test method, extracts are obtained by placing the test
and control materials in separate cell culture media under standard
conditions. Each fluid extract obtained is then applied to a culturedcell monolayer, replacing the medium that had nourished the cells to
that point. In this way, test cells are supplied with a fresh nutrient
medium containing extracts derived from the test material or control.

The cultures are then returned to the 37C incubator and periodically
removed for microscopic examination at designated times for as long
as three days.

 Direct Method: Contact test

In the contact test method, samples of test and control material can be
applied directly to monolayers of cells covered with nutrient medium.
During the subsequent incubation period, extracts from the samples will
migrate into the nutrient medium or through the nutrient agar overlay
to the underlying cells. After incubation, the monolayers are evaluated

in terms of the presence or absence of a zone of cellular effects

beneath and surrounding the sample. Extraction conditions in the direct
contact methods are less rigorous than in the elution test. However,

this method is particularly useful if only very small quantities of samples

are available or when only one surface of a material needs to be
evaluated.

 Indirect Method: Elution test

Direct Method: Contact test

Cytotoxicity Evaluation
Quantitative evaluation
Test for determination of cell viability:

Trypan blue dye exclusion assay

MTT or WST1 test
Test for determination of enzymatic release:
LDH assay

Trypan blue dye exclusion test

Cell viability is determined by staining
the cells with trypan blue. Trypan blue
is a vital stain used to selectively
colour dead tissue or cells in blue. Live
cells or tissues with intact cell
membrane are not coloured. They are
excluded from staining while dead
cells are shown as a distinctive blue
colour under a microscope.

Ex: 6/9=0,666
0.66*100=66,6%
Percentage of viable cells

Percentage % of viable cells =

N of unstained cells/total N of cells x 100

MTT or WST1 assay

The MTT assay is a colorimetric
assay for measuring the activity
of enzymes that reduce MTT or
close dyes (WSTs) to formazan,
giving a purple color.
A microtiter plate after an MTT
assay. Increasing amounts of
cells resulted in increased
purple colouring.

A main application of this assay allows to assess the

viability and the proliferation of cells. It can also be used
to determine cytotoxicity of potential medicinal agents
and toxic materials, since those agents would stimulate or
inhibit cell viability and growth.

LDH assay
LDH is a cytoplasmic enzyme that is released into the
cytoplasm upon cell lysis. The LDH assay, therefore, is a
measure of membrane integrity and of cell viability
because LDH release into the media is a marker of cell
dead.

(1) LDH oxidizes

lactate to pyruvate

(2) Pyruvate reacts with the tetrazolium salt

to form formazan

(3) the water-soluble formazan dye is

detected spectrophotometrically at a
wavelength of 340 nm

Cytotoxicity Evaluation
Qualitative evaluation
Microscopy examination:
Cells are observed for visible signs of toxicity (such as a change in the
size or appearance of cellular components or a disruption in their
configuration) in response to the test and control materials (Figures 1
and 2).
Figure 1. A confluent
monolayer (100 x
magnification) of L929
mouse fibroblast cells.
This appearance is
indicative of a
noncytotoxic response in
the elution test method.

Figure 2. Cytotoxic reaction

in the elution test method.
The L929 mouse fibroblast
cells cells (100 x
magnification) are grainy
and lack normal cytoplasmic
space; the considerable open
areas between cells indicate
that extensive cell lysis
(disintegration) has
occurred.

Morphological Evaluation of Cell Death

Etiology of Cell Death

Necrosis vs Apoptosis

Major Factors

Accidental

Genetic

NECROSIS

APOPTOSIS

Necrosis:
A pathological response to cellular injury. It is the sum of the
morphologic changes that follow cell death in a living tissue or organ
Apoptosis:
a physiological process that includes specific suicide signals leading to
cell death

Necrosis
Consequences of cell injury
NORMAL
REVERSIBLE CHANGES
Disaggregated polysomes
Focal chromatin margination
Mild mitochondria swelling
IRREVERSIBLE CHANGES
High amplitude mitochondria swelling
Mitochondria matrix densities
Progressive dilatation of endoplasmic reticulum
Lysosomal rupture
Plasma membrane rupture
Nuclear dissolution
Loss of recognisable organelles

Apoptosis
Morphological changes that occur during the apoptosis:
I) The normal cell
II) Shrink and the condensed
chromatin collapses into
crescents around the nuclear
envelope
III)The membrane begins to bulge
and bleb
IV) The blebing increases and the
cell finally breaks into a number
of apoptotic bodies
V) Which lyse in vitro
VI) And phagocytoses in vivo

Necrosis: a pathological response

to cellular injury

Apoptosis: a physiological response to

specific suicide signals, or lack of survival
signals
Chromatin condenses and migrates to
nuclear
membrane.
Internucleosomal
cleavage leads to laddering of DNA at the
nucleosomal repeat length, ca. 200 bp.

Chromatin clumps
Mitochondria swell and rupture
Plasma membrane lyses
Cell contents spill out
General inflammatory response is
triggered

Cytoplasm
rupture

shrinks

without

membrane

Blebbing of plasma and nuclear membranes

Cell contents are packaged in membrane
bounded bodies, internal organelles still
functioning, to be engulfed by neighbours
Epitopes appear on plasma membrane
marking cell as a phagocytic target.
No spillage, no inflammation

ISO 10933-4
Selection of tests for interactions with
blood-Hemocompatibility
Blood represents one of the most complex biochemical systems in living
organisms, and its various components play integral roles in several life
functions. Because these functions are critical, medical devices that
contact blood during routine use must be hemocompatible, therefore,
they must not adversely interact with any blood components so as to
cause their inappropriate activation or even destruction.
Blood/device interaction:
any interaction between blood or any component of blood and a device
resulting in effects on the blood, or any organ or tissue or on the
device. Such effects may or may not have clinically significant or
undesiderable consequences.

Blood is a specialized fluid of body that delivers necessary substances

to the body's cells such as nutrients and oxigen and transports
waste products away from those same cells.

It is composed of a multitude
of cell types, suspended in a
liquid called blood plasma.
The blood cells are mainly:
Erythrocites-they transport oxigen
Lynphocytes-cells that destroy invading pathogens
Platelets-important cells in clotting of blood

The types of tests required by ISO depend on the blood

contact category and on the time of contact of the
device or material (< 24h, 24h > t < 30 days, > 30 days):
- Device that hasnt contact with circulating blood
(blood cell counter, etc..)
- Device that has contact with circulating blood
indirectly
system to collect blood (blood bag, etc..)
directly
external communicating devices (dreinage
catherters, buttarfly needles, etc..)
implant devices (stents, cardiac valve, etc..)

Test

Methods

Comments

Thrombosis

Light microscopy (adhered

platelets, leukocytes,
aggregates, erythrocytes,
fibrin, etc.)

Light microscopy can be replaced by

scanning electron microscopy if the
nature of the material presents technical
problems for light microscopy.

Coagulation

Partial thromboplastin
time (nonactivated)

Platelets

Platelet count

Hematology

Leukocyte count and

differential; hemolysis
(plasma hemoglobin)

Hemolysis is regarded as an especially

significant screening test to perform in
this category because of its measurement
of red blood cell membrane fragility in
contact with materials and devices. The
method used should be one of the
normative standard test methods for
hemolysis.

Immunology

C3a, C5a, TCC, Bb, iC3b,

C4d, SC5b-9

A panel including the last four tests

encompasses the various complement
activation pathways.

Some examples of Tests

Hemolysis (ASTM F 756)
ASTM F 756 is a standard test method to evaluate whether direct contact with
the matherial or an extract of the material would cause in vitro red blood cell
hemolysis. This test involves a quantitative measurement of plasma hemoglobin
by using a hemoglobin reagent and spettrophotometric measurement at
wavelenght of 540 nm. An increase in plasma hemoglobin correlates with lysis of
red blood cells, thereby indicating hemolytic activity of the material exposed to
the cells.
Coagulation (determination of the rate of clot formation)
A device's effects on blood coagulation may be measured in vitro by determining
the rate of clot formation or the partial thromboplastin time (PTT) of plasma
exposed to the biomaterial or device during an incubation period.
Thrombosis
Thrombosis may be addressed by performing either an in vivo or ex vivo test. An
evaluation of the thrombogenic potential of a device typically involves placing
the device in a simulated clinical setting for a period of time, then removing the
device and evaluating the extent of thrombus formation on or in it.

 Platelets (platelets count, adhesion

and aggregation)
PLATELETS COUNT
Manually platelets count
Automatic cell counter
ADHESION and AGGREGATION
SEM analysis

ISO 10993-3
Tests for genotoxicity, carcinogenicity and
reproductive toxicity
Three major type of
genotoxic effect:

Three major type of tests for

genotoxicity, carcinogenicity and
reproductive toxicity :

Gene Mutations

Gene Mutation Tests

Chromosomal Aberrations

Chromosomal Aberration Tests

DNA Effects

DNA Effect Tests

The Ames bacterial reverse mutation

assay is most commonly used to detect
gene mutations and utilizes histidinedependent Salmonella typhimurium strains
as the test organisms. Rat liver extract
are incorporated into a portion of the test
organisms
to
simulate
whole-animal
exposure. Following exposure to the fluid
extract from the test material, the
organisms are plated in triplicate onto
histidine-free growth nutrient agar and
incubated for a specified period. The
colonies are then enumerated and these
data are compared to counts obtained for
negative control conditions. Since the
unreverted test strains will not grow
without histidine, any further growth
indicates that exposure to a genotoxic
agent has caused point mutations that
have produced bacterial strains that no
longer require histidine.

Gene Mutation test:

Ames Test

Ames test
Mutagen

His
bacteria

Medium
containing
histidine

Test system auxotrophic strain of

Salmonella typhimurium survives only in
medium with histidine (dies in normal
medium without histidine)
After treatment with mutagen some
auxotrophic cells are turned into normal
ones that synthesize histidine and survive in
a normal medium.
These cells are called revertants (due to
reverse mutation).

Reverse mutation

Dies in a
normal
medium

His+
bacteria

Normal
medium

Result of Ames test

Negative
control

A dish with a
compound to
be tested

Positive
control

SPONTANEOUS
REVERTANTS

GENOTOXICITY
CONFIRMED

IS USED FOR
CONTROL OF
THE TEST

Chromosomal aberration testing detect chromosomal

damage induced after one cellular division; structural
changes in the chromosomes are evaluated while cells
are in the metaphase stage of division. The in vitro
model employs Chinese hamster ovary cells. Gaps,
breaks, and exchanges are other examples of
observable aberrations (Figure).

Chromosomal
Aberration Tests

The mouse bone marrow micronucleus test is an in vivo assay that detect
damage to the chromosomes or the mitotic apparatus of immature red blood
cells found in bone marrow.
DNA Effects Tests
During cell division, if the chromosomes are broken or the
mitotic apparatus of the cell is damaged, chromosome
fragments may be incorporated in secondary nuclei
instead of into the main nucleus. Secondary nuclei are
much smaller than the main nucleus and are referred to
as micronuclei.
When erythroblasts develop into polychromatic erythrocytes (PCEs), the main
nucleus is extruded but any micronuclei that are present remain behind. Thus,
an increase in the number of micronucleated PCEs in animals treated with the
test article extract is an indication of the presence of a genotoxin (Figure).

Possible results of
chromosomal analysis
Percentage of cells with
chromosomal aberrations

Result

Less then 2%

Normal finding, spontaneous

level of aberrations

24%

A border result mutagenic

effect is neither confirmed
nor excluded

More than 4%

Mutagenic effect confirmed

with high probability

Result of micronucleus test

Carcinogenicity Test
Carcinogenicity:
The potential of a device material that comes in contact with a patient
to cause or incite the growth of malignant cells.

The ISO 10993 standards, which covers genotoxicity, carcinogenicity,

and reproductive toxicity, describes carcinogenicity testing as the
means "to determine the tumorigenic potential of devices, materials,
and/or extracts to either a single or multiple exposures over a period
of the total life-span of the test animal. Specifically, such testing
should be considered for a device that will have permanent contact
(longer than 30 days) with tissues. The standard further indicates
that "carcinogenicity tests should be conducted only if there are
suggestive data from other sources." Thus, not every device needs to
be subjected to this time-consuming (total life-span) and expensive
testing (in vivo test).

In vivo Test
In vivo is experimentation using a whole, living organism as opposed to
an in vitro (i.e., in a test tube or petri dish) controlled environment.
Animal tests and clinical trials are two forms of in vivo research.

Supporters of the use of animals in experiments argue that virtually

every medical achievement in the 20th century relied on the use of
animals in some way, arguing that even sophisticated computers are
unable to model interactions between molecules, cells, tissues, organs,
organisms, and the environment, making animal research necessary in
many areas.
Other scientists arguing that it is cruel, poorly regulated, that medical
progress is being held back by misleading animal models, that some of
the tests are outdated, that it cannot reliably predict effects in
humans, that the costs outweigh the benefits, or that animals have an
intrinsic right not to be used for experimentation.