CHAPTER FIVE

BIOLOGICAL TESTING OF
BIOMATERIALS
 INTRODUCTION

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.
 INTRODUCTION
• 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.
 INTRODUCTION

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.
• After the experiment statistics will help to extract maximum
useful information.
 INTRODUCTION
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)
 INTRODUCTION
IN VITRO(cell cultures in glass)

• Rapid

• Inexpensive

• Poor representation of physiological conditions

• Good as the first step

 INTRODUCTION

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

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.).
 INVITRO ASSESSMENT OF TISSUE
COMPATIBILITY
• It is evaluation of biomaterials by methods that use isolated,
adherent cells in culture to measure cytotoxicity and biological
compatibility.
 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.
• 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.
 Cell Culture Assay Methods

Three primary cell culture assays are used for evaluating

biocompatibility
1. Direct contact
2. Agar diffusion
3. 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.
 Cell Culture Assay Methods

• 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.
 Cell Culture Assay Methods

The choice of method varies with

• The characteristics of the test material
• The rationale for doing the test
• The application of the data for evaluating biocompatibility.
 Cell Culture Assay Methods
To standardize the methods and compare the results of these
assays, the following criteria of test sample must be carefully
controlled.
• 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)
• Surface area
Cell Culture Assay Methods
 Basics of cell culture
 Cells grown in a cell media on petri dish or in
flask
Cell media – solution containing nutrients for cell
growth and phenol red to indicate change in pH
Need to change media to keep cells nourished
 Most cells are adherent ; grow on surface of
petri dish ,Grow in monolayer
As cells replicate, they need to be transferred to
new petri dish
 Cells grow in an incubator at conditions that
replicate those in the human body
37°C with 5% CO for mammalian cells
 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.
• Specimens are placed in humidified incubator for 24 hours at
37 +1oC fix.
• The culture medium and specimens are removed and the cells
are fixed and stained with a cytochemical stain such as
hematoxylin blue.
DIRECT CONTACT TEST
 DIRECT CONTACT TEST
• 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.
 AGAR DIFFUSION TEST

• A near-confluent monolayer of L-929 mammalian fibroblast 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)

 AGAR DIFFUSION TEST
• Place on the surface of plate and the cultures incubated for at

least 24 hours at 37+1oC in a humidified incubator.

• 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.

AGAR DIFFUSION TEST
 ELUTION TEST
• An extract of the material is prepared by using 0.9% sodium
chloride or serum-free culture medium.
• The extract is placed on prepared near-confluent monolayer of
L-929 mammalian fibroblast cells.
• Toxicity is evaluated after 48 hours.
• Live or dead cells may be distinguished by the use of
histochemical or vital stain as agar diffusion test method.
ELUTION TEST
ADVANTAGES AND DISADVANTAGES OF CELL CULTURE
METHODS

Advantages Disadvantages
Direct Eliminate extraction preparation Cellular trauma if material
contact Zone of diffusion moves
Target cell contact with material Cellular trauma with high
Mimic physiological conditions density materials
Standardize amount of test material or test Decreased cell population
indeterminate shapes with highly soluble
toxicants
Can extend exposure time by adding fresh
media
Agar Eliminate extraction preparation Requires flat surface
Diffusion Zone of diffusion Solubility of toxicant in
Better concentration gradient of toxicant agar
Can test one side of a material Limited exposure time
Independent of material density Risk of absorbing water
form agar
 ADVANTAGES AND DISADVANTAGES OF CELL CULTURE
METHODS

Advantages Disadvantages
Elution Separate extraction from testing Additional time and step
Dose response effect
Extend exposure time
 Positive and negative control
Positive Control
 Culture cells in known condition to show that cells will grow normally
 Ensure that there is an effect when there should be an effect
 Used to asses test validity
 If the positive control does not produce expected result, there may be
something wrong with the experimental procedure
Negative Control
 Culture cells in condition with variable being tested as lacking, preventing
growth of cells
 No phenomenon is expected
 Ensure that there is no effect when there should be no effect
 In Vivo Assessment of Tissue Compatibility
 Goal is to determine biocompatibility of device/biomaterial in specific
environment
 Many potential hazards: short-term effects, long-term effects, specific
toxic effects
 Need to consider all materials in the device:
Main material of manufacture
Additives, process contaminants, residues
Leachable substances
Degradation products
Other components and interaction with final product
Properties and characteristics of final product
 Not always practical to test for all potential hazards
 Main Categories Of In vivo Testing
Preliminary in vivo tests:
• To identify any unknown chemical elements that cause
adverse reaction
• Further research and development may be required
In vivo tests of final product:
• To prove effectiveness and biocompatibility in final stages
• To gain regulatory acceptance
• May have to make final adjustments to biomaterial at this
stage
 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 the one.

 CLINICAL USE
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)

 CLINICAL USE

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

 Popup quiz
1, what are the advantages and dis advantages of Evaluations
under in vitro and in vivo conditions? list at least 2
advantages and dis advantages in each conditions.
2, list the three cell culture assay methods?
3, what are Main Categories Of In vivo Testing?
 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.

WHY MEASURE BLOOD COMPATIBILITY?

 THROMBOGENICITY
• Thrombogenic device may cause the accumulation of various blood elements
that are preferentially concentrated locally relative to their concentrations in
circulating blood (thrombus formation).

• 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,..).
 THROMBOGENICITY
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.

Therefore it is difficult and expensive to measure device thrombogenicity in an

extensive and systematic way (experiment. animals or humans).
• Alternative interpretations can be applied to data from ‘’blood compatibility’’
tests.
 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
SCENARIOS FOR BLOOD-MATERIAL
INTERACTIONS
 SCENARIOS FOR BLOOD-MATERIAL
INTERACTIONS

 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.
 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)
 INTERPRETING BLOOD-COMPATIBILITY
TESTS

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
ALTERNATE SCENARIOS
 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.
 BLOOD
• The source and methods for handling blood can have

important effects on blood material interactions.

• The Initial adhesiveness of blood platelets for artificial

surfaces appears to be

• Low in man and some primates

• High in the dog, rat and rabbit

 BLOOD
• 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)
 FLOW
• 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
 FLOW
• 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

 SURFACE
• 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:

• Protein–surface reactions involve complex, dynamic processes of competitive adsorption,

denaturation, and activation

• Cell–surface interactions may modify the protein layer, i.e., cells may deposit lipid and

protein “footprints” derived from the cell membrane

• The importance of specific adsorbed proteins for subsequent cell interactions is not well

defined

• There have been few relevant tests in which both protein adsorption and later thrombus

formation have been assessed

 BLOOD INTERACTION TIMES 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.
DEGRADATION OF MATERIALS IN THE
BIOLOGICAL ENVIRONMENT
 EFFECT OF BIOLOGICAL ENVIRONMENT

• Biological environment surprisingly harsh

– Many specialized mechanisms attempt to break down biomaterials

• 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.
 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.
 BIOLOGICAL DEGRADATION

• 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

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.

 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

 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.
 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, and sulfones.
POLYMER DEGRADATION BY HYDROLYSIS

Hydrolytic degradation mechanism of (A)polyesters,

(B)poly(urethane) and (C)poly(ureas)
 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.

 POLYMER DEGRADATION BY OXIDATION

• 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

 POLYMER DEGRADATION BY OXIDATION

Mechanism of oxidative degradation by H2O2 in

(A)poly(ether urethanes), (B)poly(carbonate urethanes) and
(C)aromatic polyurethanes
 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 generally susceptible to corrosion.

• Susceptibility of ceramics to corrosion varies with solubility

 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 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
 METALLIC CORROSION
• For all corrosion processes, rate of
the anodic or oxidation reaction must Metal Potential (V)
equal the rate of the cathodic or Gold 1.43
reduction reaction.
Platinum 1.2
– Can stop corrosion by inhibiting either
process Mercury .8
• Can measure the standard electrode Silver .79
potential for a metal Copper .34
– Gives general guide to reactivity in
Hydrogen 0
aqueous solutions
• Metals at the top are noble, relatively Lead -0.13
unreactive metals, and those at the Cobalt -0.28
bottom are the more reactive Iron -0.44
• This is first guide to corrosion Titanium -1.63
resistance but it is much more Lithium -3.05
complicated
 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 from

surface and disrupts equilibrium

 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.
 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.

 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
 CERAMIC DEGRADATION
• Degradation depends on chemical composition and microstructure

• 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.

 THANK YOU !!!

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