Chapter Five

Biological Testing of Biomaterials

Biomaterial & Implant 1

 INTRODUCTION TO TESTING
BIOMATERIALS
• How can biomaterials be evaluated to determine if they are
biocompatible and will function in a biologically appropriate
manner in the in vivo environment?
• Evaluation under in vitro (literally “in glass”) conditions can
provide rapid and inexpensive data on biological interaction.

Biomaterial & Implant 2

 Cont’d
Testing always leads to experimental variability, particularly tests in

living systems.

 The more complex the system (e.g. Humans vs. cultured cells) the

larger the variability that might be expected.

The Statistics should be used at two steps in testing biomaterials.

– Before an experiment is performed, statistical experimental design

will indicate the minimum number of samples that must be evaluated

to yield meaningful results.

Biomaterial & Implant 3
 Cont’d
– After the experiment statistics will help to extract maximum useful

information.
The Detailed protocols are provided by:
– ASTM (American Society for Testing and Materials)
– ISO (International Standards Organization)
– FDA (Food and Drug Administration)
– NIH (National Institute of Health)

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IN VITRO(cell cultures in glass)
– rapid
– inexpensive
– poor representation of physiological conditions
– good as the first step
IN VIVO(animal experiments)
– better approximation to human environment
– demanding protocols (Animal Welfare Act)
– right animal model approximate human environment
– second step prior to clinical use

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 INVITRO ASSESSMENT OF TISSUE
COMPATIBILITY
• The term “cytotoxicity” means to cause toxic effects
(death,alterations in cellular membrane permeability,
enzymatic inhibition, etc.) at the cellular level.
• It is distinctly different from physical factors that affect
cellular adhesion (surface charge of a material,
hydrophobicity, hydrophilicity, etc.).

Biomaterial & Implant 6

 Cont’d
• Evaluation of biomaterials by methods that use isolated,
adherent cells in culture to measure cytotoxicity and biological
compatibility.
• Cell culture methods have been used to evaluate the biological
compatibility of materials for more than two decades

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 BACKGROUND CONCEPTS

Toxicity
• A toxic material is defined as a material that releases a
chemical in sufficient quantities to kill cells either directly or

indirectly through inhibition of key metabolic pathways.

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• Variety of factors affect the toxicity of a chemical (e.g.,
compound, temperature, test system), the most important is the
dose or amount of chemical delivered to the individual cell.

Biomaterial & Implant 9

 ASSAY METHODS
• Three primary cell culture assays are used for evaluating
biocompatibility: direct contact, agar diffusion, and elution
(also known as extract dilution).
• These are morphological assays, meaning that the outcome is
measured by observations of changes in the morphology of the
cells

Biomaterial & Implant 10

 Cont’d
• The three assays differ in the manner in which the test material
is exposed to the cells.
• Test material may be placed directly on the cells or extracted
in an appropriate solution that is subsequently placed on the
cells.

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 Cont’d
• The choice of method varies with the characteristics of the test
material, the rationale for doing the test, and the application of the
data for evaluating biocompatibility.
• To standardize the methods and compare the results of these assays,
the variables of number of cells, growth phase of the cells (period of
frequent cell replication), cell type, duration of exposure, test sample
size (e.g., geometry, density, shape, thickness), and surface area of
test sample must be carefully controlled.

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 Direct Contact Test
• A near-confluent monolayer of L-929 mammalian fibroblast
cells is prepared in a 35-mm-diameter cell culture plate.
• The culture medium is removed and replaced with 0.8 ml of
fresh culture medium.
• Place in humidified incubator for 24 hours at 37 +1oC fix and
stained with cytochemicals Haematoxylin and &Eosin.

Biomaterial & Implant 13

 Cont’d
• Dead cells lose their adherence to the culture plate and live
cells adhere to the culture plate and are stained by the
cytochemical stain.
• Examine cells for morphological changes.
• How do you measure toxicity?
– Toxicity is evaluated by the absence of stained cells under
and around the periphery of the specimen.

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 Agar Diffusion Test
• A near-confluent monolayer of L-929 is prepared in a 60-mm-diameter plate .
The culture medium is removed and replaced with a culture medium
containing 2% agar.
• Overlay monolayer of L-929 fibroblasts with thin layer of agar containing a
vital dye (neutral red to ready visualise live cells).
• Place on the surface of plate and the cultures incubated for at least 24 hours at
37+1oC in a humidified incubator.
• Fix and analyze.
• Vital stains, such as neutral red, are taken up and retained by healthy, viable
cells. Dead or injured cells do not retain neutral red and remain colourless.
• Toxicity is evaluated by the loss of the vital stain under and around the
periphery of the specimens

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Biomaterial & Implant 16
 Elution test

• Incubate material in growth media at body temperature,

• Remove aliquot after some time;
• Add to confluent L929 culture
• Fix and analyse after 24-48 hr.

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Cont’d

Biomaterial & Implant 18

Cont’d

Biomaterial & Implant 19

 CLINICAL USE

• Products for in vitro fertilization procedures would be tested for adverse

effects on a very low cell population.

– A new material for culturing cells would be assayed by comparing growth

rates of cells in contact with the new material with those of currently marketed

materials.

– Current experience: a material non-toxic in vitro will be non- toxic in in

vivo assays.

– But: the clinical acceptability of a material depends on many different

factors; target cell toxicity is but one.

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 Cont’d

• In vivo testing: critical for development of clinical devices

– In vitro tests cannot replace in vivo tests:

• no inflammation
• no immune response
• single cell type
• no tissue remodeling
• No acquired toxicity through processing (eg the liver modifies many
foreign compounds)
Biomaterial & Implant 21
 Cont’d

– In vivo tests provide:

• Interactions of different cell types

• Effects of hormonal factors
• Interactions with extracellular matrix
• Interactions with blood-borne cells, proteins and molecules
• Overall determination of: wether the device performs as intended and
provides no significant harm to the patient or user

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 Blood Compatibility Tests

• Blood compatibility” can be defined as the property of a

material or device that permits it to function in contact with
blood without inducing adverse reactions.

Biomaterial & Implant 23

 Cont’d
• Devices may be modified to improve characteristics other than
BMI.
• However, since these changes may also affect blood responses,
and since BMIs are not entirely predictable based on
knowledge of device composition and configuration, blood
compatibility testing is nearly always required to document
safety.

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 Thrombogenicity
The local blood reaction may produce systemic effects.
• Thrombi may detach (embolize) and impair blood flow in
peripheral vessels.
• Chronic devices may “consume” circulating blood elements.
-Mechanical destruction of red blood cells by heart prostheses or
dialyzers.
-Removal of platelets as a result of continuing thrombus
formation.
- Mediators of inflammatory responses and vessel tone may be
produced or released from cells (platelets, white cells,..)

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 Cont’d
• The types of devices used are:
– Numerous, exhibit complex flow geometries, and are
continuously evolving.
• The possible blood responses are:
– numerous, complex, dynamic, and not fully understood.
It is difficult and expensive to measure device thrombogenicity in
an
• Extensive and systematic way (experiment. animals or
humans).
• The Alternative interpretations can be applied to data from
blood compatibility“ tests.

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Scenarios for blood-material interactions

A) Device remains free of thrombus

B) Large thrombus forms and remains attached
C) Large thrombus forms but detaches (embolizes)
D) Surface is highly reactive toward blood but deposited
material is quickly removed through microembolism and or
lysis.
 Inspection of devices C and D could lead to the incorrect
conclusion that these surfaces are blood compatible
 Overall it can be difficult to interpret results of blood
compatibility tests to know whether a surface is compatible or
not.
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Cont’d

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 Cont’d
• In vitro tests:
– Usually of short duration
– Strongly influenced by the blood source, handling methods, the
use of anticoagulants
– Can not predict longer term BMI and in vivo outcome events
– Useful in screening materials.
• The In vivo tests:
– Insertion for short/long periods into the arteries or veins of
experimental animals.
• Arteriovenous (AV) or
• Arterioarterial (AA) shunt

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Cont’d

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 Interpreting blood-compatibility tests
 Depending on evaluation method, biomaterials scientist must relate
significance of the events being observed to blood compatibility of the device.
 To do this, need to have good understanding of the physical and biological
mechanisms of blood–materials interactions.
 Tests are interaction between device and solutes, proteins and cells in blood
under defined conditions (exposure time, blood composition, and blood flow)
 Therefore, a researcher cannot:
1) Extrapolate results obtained under one set of test conditions to another set
of conditions
2) Use short-term testing to predict long-term results
3) Predict in vivo device performance based on BMIs testing of materials in
vitro

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Alternate scenarios

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 KEY CONSIDERATIONS FOR BMI
ASSESSMENT
• Virchow’s triad three factors that contribute to blood
coagulation and influence the results of blood-compatibility
testing
blood

Surface flow

Interaction time of blood with materials (seconds to years)

influences three components of the triad

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 Blood: Factors Affecting Its Properties
• The source and methods for handling blood can have important effects on
blood material interactions.
• The Initial adhesiviness of blood platelets for artificial surfaces appears to be
– low in man and some primates and
– high in the dog, rat and rabbit.
• The Animal blood donors are relatively homogenous:
– Age, health status, blood response
• The In vitro testing generally requires anticoagulation of the blood (can have
profound effects).
• The In vivo testing and the use of extracorporeal circuits are also commonly
performed with anticoagulats:
– Sodium citrate (chelates Ca2+)
– Heparin (used to block thrombin)

Biomaterial & Implant 34

 Flow factors that affect testing
 Blood flow controls rate of transport of cells and proteins in
area of biomaterial surfaces
 Initial attachment of platelets to artificial surfaces can depend
on the rate of blood flow.
 After initial platelets have adhered, platelet aggregation and in
vivo thrombus formation (over minutes to hours) may be
reaction controlled.
 Depends on substrate reactivity and material properties
 Generally interested in measuring two rates:
 r1 - rate of platelet transport from blood to the surface
 r2 - rate of reaction of a platelet with the surface

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 Surface factors that affect testing
 Known that surface physicochemical properties of materials and
devices have important effects on early events
 E.g. protein adsorption and platelet adhesion
 Do not know how these effects relate to subsequent thrombus
formation because:
1) Protein–surface reactions involve complex, dynamic processes of
competitive adsorption, denaturation, and activation
2) Cell–surface interactions may modify the protein layer, i.e., cells
may deposit lipid and protein “footprints” derived from the cell
membrane
3) The importance of specific adsorbed proteins for subsequent cell
interactions is not well defined
4) There have been few relevant tests in which both protein
adsorption and later thrombus formation have been assessed
Biomaterial & Implant 36
 Blood Interaction Time with Materials
and Devices
 Different events may occur over short and long periods of
biomaterial interaction with blood
 Reactions can change over entire period of device
exposure
 May see completely different result when blood contacts a
material for a few minutes, hours or days.
 Maximum number of platelets thought to adhere in a few
hours
 Short tests may be adequate for this
 But platelet adhesion alone not an adequate measure of
thrombogenicity.

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Tests to evaluate biomaterial interaction
with blood
 Different methods to assess changes in blood and blood
interaction with biomaterial surface
 Can put biomaterials in a flow chamber with circulating blood
 Materials contact blood under conditions to simulate
human physiology
 Many flow geometries have been tested
 This test helped us to understand how proteins and
platelets are transported to, and react with, artificial
surfaces
 This test of short duration and influenced by blood source
 Short test not used to predict results in vivo.

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Degradation of Materials in the
Biological Environment

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 Effect of Biological environment
 Biological environment surprisingly harsh
 Many specialized mechanisms attempt to break down
biomaterials.
 Evolved over millions of years to protect animals from foreign
substances
 Biomaterial exposed to continuous cyclic stress, abrasion and
flexure in an aqueous, ionic environment that is
electrochemically active to metals and breaks down polymers.
 Specific biological mechanisms cause proteins to adsorb, this
can enhance rate of biomaterial corrosion
 Cells secrete oxidizing agents that digest biomaterials
 Degradative agents are concentrated between the cell and the
material

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 Biological Degradation
 To understand biological degradation of implant materials must
consider other pathways that contribute
 E.g. Cracks open fresh surface area for degradation
 Swelling increases surface area
 Degradation products can change local pH, stimulating further
reaction
 Biodegradation can occur over minutes and years
 Can be engineered to happen at specific time after implantation, or can
be long-term unexpected consequence of severe biological
environment
 Implanted biomaterials can solubilize, crumble, become
rubbery, or rigid over time
 Degradation products can be toxic or have pharmacological
effects
 Degradation seen in metals, polymers, ceramics and composites
Biomaterial & Implant 41
 Biological Degradation
 Biodegradation = The chemical breakdown of materials by
action of living organisms  leads to changes in physical
properties
 Ranges from decomposition of environmental waste involving
microorganisms to host-induced deterioration of biomaterials
in implanted medical devices
 Specific biological processes required.

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 Polymer degradation
 Polymeric components of implantable devices are
generally reliable for their intended lifetimes
 But no polymer completely resistant to chemical
processes and mechanical actions of the body.
 Polymer biomaterials generally degrade because they are
attacked by body constituents
 Many operations performed on a polymer from synthesis
to its use in the body.
 E.g. Extrusion, pelletizing, drying, packaging, molding,
cleaning… etc.
 Physical and chemical deterioration can occur at any time
in these stages
 E.g. Gamma irradiation sterilization of polyethylene
causes free radicals Biomaterial & Implant 43
 Polymer degradation
 Polymeric surfaces in contact with body fluids immediately
adsorb proteins, water, ions and lipids.
 Cellular elements attach to surfaces and initiate chemical
processes
 Most polymers can withstand attack by cells and many
chemical agents, including oxidants and enzymes
 Fibrous capsule will probably form  rate of release of
powerful chemicals from activated cells will decrease
 Hydrolysis and oxidation almost always cause of chemically
degraded polymers Biomaterial & Implant 44
 Polymer degradation by Hydrolysis
 Hydrolysis - breakage of molecular functional groups by
reaction with water
 Polymer’s susceptibility to hydrolysis is result of chemical
structure, morphology, dimensions, and body’s
environment
 Catalyzed by acids, bases, salts, or enzymes.
 Functional groups susceptible to hydrolysis have different
degradation rates.
 Polymers that degrade by hydrolysis at a higher rate: have
more hydrolyzable groups, high hydrophilicity, low
crystallinity, low crosslink density and high ratio of
exposed surface area to volume.
 Polymers more resistant to degradation by hydrolysis:
have hydrocarbons, dimethylsiloxanes,
Biomaterial & Implant and sulfones 45
 Polymer degradation by Hydrolysis
• Hydrolytic degradation mechanism of (A) polyesters, (B)
poly(urethane) and (C) poly(ureas)

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 Polymer degradation by Oxidation
 Functional groups that prevent hydrolysis can also prevent
degradation by oxidation
 Site for oxidative attack allow abstraction of an atom or ion and
provide resonance stabilization of the resultant radical or ion
 Hosts can generate molecular species that encourage oxidative
processes
 Reactive molecules can come from activated macrophages
responding to injury at implant site.
 Polyurethanes are resistant to hydrolysis but susceptible to stress
cracking by oxidation, this material use in pacemaker leads.
 In some circumstances body can transmit electromagnetic
radiation that affects polymer structure.
 E.g. Cornea and skin can absorb UV rays

Biomaterial & Implant 47

 Polymer degradation by Oxidation
• Mechanism of oxidative degradation by H2O2 in poly(ether
urethanes) (A), poly(carbonate urethanes) (B) and aromatic
polyurethanes (C)

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 Degradation of Metals and ceramics
 Biomaterials for prolonged use are exposed to various
anions, cations, organic substances, and dissolved oxygen
 Main cations: Na+, K+, Ca2+, and Mg2+
 Dissolved oxygen influences aggressive nature of
environment.
 Proteins have a significant influence on the corrosive
nature of body fluids.
 PH normally 7.4, but for short periods following surgery
can drop as low as 4 or 5 due to inflammation
 Metals are generally susceptible to corrosion.
 Susceptibility of ceramics to corrosion varies with
solubility
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 Corrosion of metals and ceramics
 Even corrosion-resistant metals suffer in vivo.
 Some ceramics with combination of very strong partially
ionic, partially covalent bonds can resist breakdown.
 Must consider the following questions in relation to the
corrosion and degradation of metals and ceramics:
1) How does the susceptibility to corrosion and degradation
vary for one material type; what mechanisms cause
interfacial reactions; how can we use this knowledge to
select an appropriate material (and treatment)?
2) Are there variables within this biological environment other
than those described above that can influence these
processes?
3) What are the consequences of corrosion and degradation
phenomena? Biomaterial & Implant 50
 Metallic Corrosion
 Most pertinent form of corrosion related to metallic biomaterials
is aqueous corrosion.
 Electrochemical reactions take place on a metallic surface in
an aqueous electrolyte.
 Always two reactions that occur:
1) Anodic reaction, which yields metallic ions
 E.g. Oxidation of metal to its salt:
M  M(n+) +n(electrons)
2) Cathodic reaction, where the electrons are consumed
 E.g. Reduction of hydrogen
2H+  2E- + H2
 For all corrosion processes, rate of the anodic or oxidation
reaction must equal the rate of the cathodic or reduction reaction
 Can stop corrosion by inhibiting either process
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 Metallic Corrosion
Metal Potential (V)
 Can measure the standard electrode Gold 1.43
potential for a metal Platinum 1.2
Mercury .8
 Gives general guide to reactivity
Silver .79
in aqueous solutions
Copper .34
 Metals at the top are noble, relatively
Hydrogen 0
unreactive metals, and those at the
Lead -0.13
bottom are the more reactive.
Cobalt -0.28
 This is first guide to corrosion Iron -0.44
resistance but it is much more Titanium -1.63
complicated Lithium -3.05

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 Metallic Corrosion
 In vivo metal is positive ions placed in solution of free electrons.
 Metals reach a charge transfer equilibrium in vivo
 Rate of anodic/oxidation reaction equals rate of
cathodic/reduction reaction
 Corrosion occurs
 Can predict rate of corrosion for a metal in homogeneous
constant environment
 In vivo environment constantly changing, difficult to predict
 If accumulating positive metal ions in surrounding media or
accumulating electrons in metal are removed, rate of corrosion
will change
 This happens due to interaction of proteins with metal ion
 Metal ions can form complexes with proteins  transports
metals ions away fromBiomaterial
surface& Implant
and disrupts equilibrium 53
 Metallic Corrosion
1. In theory, corrosion resistance can be predicted from standard
electrode potentials
 Explains nobility and reactivity of some metals
 Not useful for predicting occurrence of corrosion of most
alloy systems in vivo
2. Corrosion resistance of many materials determined by ability
to become passivated by oxide layer that protects underlying
metal
3. Corrosion processes in practice influenced by variations in
surface microstructural features and environment that disrupts
charge transfer equilibrium

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 Ceramic Degradation
 Rate of degradation of ceramics usually highly corrosion-
resistant or highly soluble.
 Generally expect high degree of resistance to degradation with
ceramics and glasses.
 Corrosion in metals is conversion from metal to ceramic
structure (e.g. metal to metal oxide)
 Ceramic is lower energy state  less driving force for
degradation
 Interatomic bonds in ceramics are largely ionic but partly
covalent
 Large amounts of energy required for their disruption
 Ceramics such as Al2O3, ZrO2, TiO2, SiO2, and TiN are
generally stable under normal conditions
Biomaterial & Implant 55
 Ceramic Degradation
• Generally classified as either:
1. Inert, or “nearly inert” ceramics
2. Resorbable ceramics
3. Ceramics of controlled surface reactivity
• Many ceramic structures that, although stable in the air, will
dissolve in aqueous environments
– E.g. NaCl – Classic ionic ceramic structure that degrades in
water
• Based on chemical structure possible to predict which ceramics
will degrade in body (can control degradation).
• Need to select material where degradation products (anions and
cations) are harmless.
– This is why calcium (calcium phosphates and carbonates) and
sodium are popular. Biomaterial & Implant 56
 Ceramic Degradation
 Degradation depends on chemical composition and microstructure
 E.g. tricalcium phosphate [Ca3(PO4)2] degrades fairly rapidly,
calcium hydroxyapatite [Ca10(PO4)6(OH)2] relatively stable
 Porosity also influences rate of degradation
 Dense materials degrade more slowly, microporous materials more
rapidly
 Degradation rates in vivo can generally be predicted from behavior in
simple aqueous solution
 Some variations, especially at different implantation sites
 Can be due to action of cells
 Small group of materials where there is selective degradation at
surface (Ca and P released) then degradation reaction stops because
of formation of stable SiO2 layer on surface – useful for bonding to
bone
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