Biomaterials
Eng.
BIOMEDICAL ENGINEERING
BIOMATERIALS
Biological Analyses & Sterilization
of Biomaterials
SECTION 3
1
Biomaterials
Eng.
Biological analyses
For Bachelor Science(B.Sc.) Students
Esmaeil Biazar
Ph.D in
Biomedical Engineering
Material attributes for biomedical applications
Property
Desirables
Biocompatibility
Noncarcinogenic, nonpyrogenic,
nontoxic, nonallergenic, blood
compatible, non-inflammatory
Sterilizability
Not destroyed by typical sterilizing
techniques such as autoclaving, dry heat,
radiation, ethylene oxide
Physical characteristics
Strength, elasticity, durability
Manufacturability
Machinable, moldable, extrudable
2
Adverse
Effects Of Biomaterial/Bio-device
• B = f (X1,X2......Xn)
• Where X: Material, Design, Application etc.
3
Adverse
Effects Of Biomaterial/Bio-device
Reliability
r = 1-f
rt = r1 × r2 …… rn
Example
r ? For Knee replacement after 1 years
Fi = 5% , Fw = 3% , Fl= 2% , Fsur= 1% and Ffr=4%
r = (1-0.05).(1-0.03).(1-0.02).(1-0.04) = 0.89
4
Human Biomedical Devices
Based on the duration of the device use, invasiveness and risk to the user.
 Class I: crutches, bedpans, tongue depressors,
adhesive bandages etc. minimal invasiveness,
does not contact the user internally.
 Class II: hearing aids, blood pumps, catheters,
contact lens, electrodes etc. higher degree of
invasiveness and risk, but relatively short
duration.
 Class III: cardiac pacemakers, intrauterine
devices, intraocular lenses, heart valves,
orthopedic implants, etc. considerably more
invasive and can pose immense risk to the
user-implantables.
5
Human Biomaterials Devices
Materials
FIRST GENERATION IMPLANTS
• “ad hoc” implants.
• Specified by physicians using common and borrowed materials.
• Most successes were accidental rather than by design.
Examples
•
•
•
•
Gold fillings, wooden teeth, dental prosthesis.
Steel, gold, ivory, etc., bone plates.
Glass eyes and other body parts.
Dacron and parachute cloth vascular implants.
6
SECOND GENERATION IMPLANTS
•
•
•
•
Engineered implants using common and borrowed materials.
Developed through collaborations of physicians and engineers.
Built on first generation experiences.
Used advances in materials science (from other fields).
Examples
• Titanium alloy dental and orthopaedic implants.
• Cobalt-chromium-molybdinum orthopaedic implants.
• UHMW polyethylene bearing surfaces for total joint
replacements.
• Heart valves and pacemakers.
7
Third generation implants
•
•
•
•
Bioengineered implants using bioengineered materials.
Few examples on the market.
Some modified and new polymeric devices.
Many under development.
Example
• Tissue engineered implants designed to regrow rather than
replace tissues.
• Integra LifeSciences artificial skin.
• Genzyme cartilage cell procedure.
• Some resorbable bone repair cements.
• Genetically engineered “biological” components (Genetics
Institute and Creative Biomolecules BMPs).
Biological Analyses
Objective is to minimize inflammatory responses and toxic effects.
•
Biocompatibility has been defined as the ability of a medical device to perform
with an appropriate host response in a specific application, and biocompatibility
assessment is considered to be a measurement of the magnitude and duration of the
adverse alterations in homeostatic mechanisms that determine the host response.
• Bioactivity
– The characteristic that allows the material to form a bond with living tissue
(Hench, 1971)
– The ability of a material to stimulate healing and trick the tissue system
into responding as if it were a natural tissue (Hench 2002).
– Advantages: Bone tissue – implant interface, enhanced healing response,
extends implant life
• Biodegradability
– Breakdown of implant due to chemical or cellular actions
– If timed to rate of tissue healing transforms implant to scaffold for tissue
regeneration
– Negates issues of stress shielding, implant loosening, long term stability
Biological Analyses
• Biology is a science of surfaces and interfaces
• Understanding and controlling performance
 Physical
 Chemical
 Biological
• Relevant material performance is under biological conditions
– 37 ºC, aqueous, saline, Extra Cellular Matrix (ECM)
– Material properties as a function of time
• Initial negative biological response - toxicity
• Long term biological response – rejection
– Seldom (never) at equilibrium
Host can affect the implant
Physically
 Abrasive, adhesive, delamination wear
 Fatigue and Fracture
 Stress Corrosion cracking
 General corrosion.
Biologically
 Absorption of substances from the tissues
 Enzymatic degradation
 Calcification
11
Host can affect the implant
Host Factors:
Age and health status
 Immunological/metabolic status
 Choice of surgeon: minimize tissue damage and contamination,
proper implantation.

12
Biological Analyses
Material
Contact time
syringe needle
1-2 s
tongue depressor
10 s
contact lens
12 hr - 30 days
bone screw / plate
3-12 months
total hip replacement
10-15 yrs
intraocular lens
30 + yrs
13
Biological Analyses
Biocompatibility factors
 Components and natural properties of materials.
 Tissue type

Implantation time
 Limited X ≤ 24 hrs.
 Prolonged 24 hrs. < X < 30 days.
 Permanent X > 30 days.

14
Biological Analyses
Screening tests












Cytotoxicity
Sensitization
Irritation or intracutaneous reactivity
Acute systemic toxicity
Subacute toxicity
Genotoxicity
Implantation effect
Hemocompatibility
Chronic toxicity
Carcinogenocity
Reproductive and developmental toxicity
Biodegradation
15
Standards
• Assistance in the design of many biomaterials tests is available
through national and international standards-organizations.
• Thus, the American Society for Testing Materials (ASTM) and
the International Standards Organization (IS0) can often provide
detailed protocols for widely accepted, carefully thought out
testing procedures.
•
Other testing protocols are available through government
agencies (e. g., the FDA) and through commercial testing
laboratories.
16
Standards
USP (U.S. Pharmacopeia).
 ISO (International Standard Organization), ISO 10993.
 FDA ( Food and Drug Administration), Blue Book.
 AAMI (The Association for the Advancement of Medical
Instrumentation).
 ANSI (American National Standards Institute).
 BSI (British Standard Institute).
 NHLBI (National Heart , Lung and Blood Institute).
 UIPAC (Union for Pure and Applied Chemistry).
 IACUC (Institutional Animal Care and Use Committee).

17
Standards
List of the standards in the 10993 series
• ISO 10993-1:2003 Biological evaluation of medical devices
Part 1: Evaluation and testing.
• ISO 10993-2:2006
Part 2: Animal welfare requirements.
• ISO 10993-3:2003
Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity.
• ISO 10993-4:2002/Amd 1:2006
Part 4: Selection of tests for interactions with blood.
• ISO 10993-5:1999
Part 5: Tests for in vitro cytotoxicity.
18
Standards
• ISO 10993-6:1994
Part 6: Tests for local effects after implantation.
• ISO 10993-7:1995
Part 7: Ethylene oxide sterilization residuals.
• ISO 10993-8:2001
Part 8: Selection and qualification of reference materials for biological
tests.
• ISO 10993-9:1999
Part 9: Framework for identification and quantification of potential
degradation products.
• ISO 10993-10:2002/Amd 1:2006
Part 10: Tests for irritation and delayed-type hypersensitivity.
19
Standards
• ISO 10993-11:2006
Part 11: Tests for systemic toxicity.
• ISO 10993-12:2002
Part 12: Sample preparation and reference materials.
• ISO 10993-13:1998
Part 13: Identification and quantification of degradation products from polymeric medical
devices.
• ISO 10993-14:2001
Part 14: Identification and quantification of degradation products from ceramics.
• ISO 10993-15:2000
Part 15: Identification and quantification of degradation products from metals and alloys.
20
Standards
• ISO 10993-16:1997
Part 16: Toxicokinetic study for degradation products and leachable.
• ISO 10993-17:2002
Part 17: Establishment of allowable limits for leachable substances.
• ISO 10993-18:2005
Part 18: Chemical characterization of materials.
• ISO 10993-19:2006
Part 19: Physico-chemical, morphological and topographical characterization of
materials.
• ISO 10993-20:2006
21
Part 20: Principles and methods for immunotoxicology testing of medical devices.
Standards
22
Biological Analyses
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.
 The number of cells that are affected is an indication of the dose and potency of the
chemical.
 Although a variety of factors affect the toxicity of a chemical (e.g., compound,
23
temperature, test system), the most important is the dose or amount of chemical
delivered to the individual cell.
Biological Analyses
BACKGROUND CONCEPTS
Delivered and Exposure Doses
 Delivered dose: the dose that is actually absorbed by the cell.
 Exposure dose: the amount applied to a test system.
• For example, if an animal is exposed to an atmosphere containing a
noxious substance (exposure dose), only a small portion of the inhaled
substance will be absorbed and delivered to the internal organs and
cells (delivered dose).
24
Biological Analyses
BACKGROUND CONCEPTS
DELIVERED AND EXPOSURE DOSES
o The cells that are most sensitive are referred to as the target cells.
o Cell culture methods: evaluate target cell toxicity by using
delivered doses of the test substance.
 This distinguishes cell culture methods from whole animal
studies, which evaluate the exposure dose and do not determine
the target cell dose of the test substance.
25
Biological Analyses
BACKGROUND CONCEPTS
SAFETY FACTORS
• This practice requires being able to exaggerate the anticipated human clinical
dosage in the nonhuman test system.
• Local toxicity model in animals: there is ample opportunity for reducing the
target cell dose by distribution, diffusion, metabolism, and changes in the
number of exposed cells (because of the inflammatory response).
• Cell culture models: the variables of metabolism, distribution, and absorption
are minimized, the dosage per cell is maximized to produce a highly sensitive
26
test system.
Biological Analyses
BACKGROUND CONCEPTS
 How can biomaterials be evaluated to determine if they
are biocompatible and will function in a biologically
appropriate manner in the in vivo environment?
 This introduction: common to all biomaterials biological
testing.
27
Biological Analyses
BACKGROUND CONCEPTS
Tests

IN VITRO

EX VIVO

IN VIVO
28
Biological Analyses
BACKGROUND CONCEPTS
IN VITRO
• Evaluation under in vitro (literally "in glass") conditions: rapid and
inexpensive data on biological interaction .
 No one material will be appropriate for all medical device application.
 The material, its composition and degradation products may affect host cells
and tissues.
 The host environment may also affect material properties and device
performance.
• The question: will the in vitro test measure parameters relevant to what will
29
occur in vivo environment?
Biological Analyses
BACKGROUND CONCEPTS
IN VITRO TESTING
Types of in - Vitro tests for estimating biocompatibility :
 Cytotoxicity – Elution or extract test (MEM Elution)
Direct contact test
Agar or agarose overlay test
 Hemocompatibility - Hemolysis assay
Clotting and complement activation
 Mutagenecity – Ames test
 Hypersensitivity – Lymphocyte transformation test
Leukocyte migration & inhibition test
30
Biological Analyses
IN VITRO
Cytotoxicity
• Three primary cell culture assays are used for evaluating
biocompatibility (Cytotoxicity):
o Direct contact
o Agar diffusion
o 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.
31
Biological Analyses
IN VITRO
DIRECT CONTACT METHOD




In this method, a piece of test material is placed directly onto cells growing
on culture medium.
Cell cultures are grown to a standard monolayer.
During incubation, leachable chemicals in the test material can diffuse into
the culture medium and contact the cell layer.
Subsequently, the monolayers are examined microscopically for the presence
of morphological changes, reduction in cell density or lysis of cells around
the test material.
32
Biological Analyses
IN VITRO
DIRECT CONTACT METHOD
•
A near-confluent monolayer of L-929 mammalian fibroblast cells is prepared in a
35 mm diameter cell culture plate.
• Old cell culture media (agar generally) is removed .
• Fresh media is added .
• Material being tested is placed onto the cultures, which are incubated for 24 hours
at 37 degrees Celsius (Incubator in 37 degrees Celsius, 5% CO2 , Humidity 9095%).
(Specimen of negative or positive controls and the test article are carefully placed in
prepare culture and incubated for 24 hour).
• The material is removed .
• The culture media is removed .
• The remaining cells are fixed and stained by the cytochemical stain.
• Dead cells are lost during fixation and only the live cells are stained .
• The toxicity of the material is indicated by the absence of stained cells around 33
the
material .
Biological Analyses
IN VITRO
AGAR DIFFUSION METHOD
In this method, a thin layer of nutrient-supplemented agar is placed over the
cultured cells (L-929 mouse fibroblast cells ).
The test material (or an extract of the test material dried on filter
paper/sample) is placed on top of the agar layer, and the cells are incubated
for 24 hours.
Cytotoxic leachates diffuse into the cell layer via the agar, and a zone of
malformed, degenerative or lysed cells under and around the test material
indicates cytotoxicity.
34
Biological Analyses
IN VITRO
AGAR DIFFUSION METHOD
•
•
•
•
•
•
•
•
A near confluent layer of L-929 mammalian fibroblast cells are prepared in a
culture plate .
Old cell culture media is removed .
The cells are covered with a solution of 2% agar, which often contains red vital
stain.
After the agar has solidified, specimen of negative and positive controls and the test
article are placed on the surface of the same prepared plate and the culture
incubated for at least 24 hours (Incubator in 37 degrees Celsius, 5% CO2 ,
Humidity 90-95%).
This assay also contain red stain in the agar mixture, which allows ready
visualization of live cells.
Healthy cells retain red stain.
Dead or injured cells do not retain neutral red and remain colorless.
Toxicity is evaluated by the loss of the stain under and around the periphery of the
35
specimens.
Biological Analyses
IN VITRO
ELUTION METHOD
• A near confluent layer of L-929 mammalian fibroblast cells are prepared in a
culture plate .
• An extract of the material which is being tested is prepared using
physiological saline (0.9% sodium chloride) or serum-free culture medium
(the latter is generally preferred) .
• The extract is placed on the cells and incubated for 48 hours at 37 degrees
Celsius (Incubator in 37 degrees Celsius, 5% CO2 , Humidity 90-95%).
• After 48 hours the toxicity is evaluated using either a histochemical or vital
stain.
36
Biological Analyses
IN VITRO
37
Biological Analyses
IN VITRO
ASSAY METHODS
• To standardize the methods and compare the results of these assays, the
variables:
–
–
–
–
–
–
Number of cells.
Growth phase of the cells.
Cell type.
Duration of exposure.
Test sample size (e.g., geometry, density, shape, thickness).
Surface area of test sample.
– Cell morphology
Trypan blue (enters dead cells), neutral red (actively taken up by living cells)
38
Biological Analyses
IN VITRO
39
Biological Analyses
IN VITRO
ASSAY METHODS
• In general, cell lines that have been developed for growth in vitro are
preferred to primary cells that are freshly harvested from live
organisms because the cell lines improve the reproducibility of the
assays and reduce the variability among laboratories.
• That is, a cell line is the in vitro counterpart of inbred animal strains
used for in vivo studies.
• Cell lines maintain their genetic and morphological characteristics
throughout a long (sometimes called infinite) life span.
40
Biological Analyses
IN VITRO
ASSAY METHODS
• Cell lines from other tissues or species may also be used.
• Selection of a cell line is based upon the type of assay, the
investigator’s experience, measurement endpoints (viability,
enzymatic activity, species specific receptors, etc.), and various
other factors.
• It is not necessary to use human cell lines for this testing
because, by definition, these cells have undergone some
dedifferentiation and lost receptors and metabolic pathways in
the process of becoming cell lines.
41
Biological Analyses
IN VITRO
Organ of origin
Primary cultures or isolated
cells
Cell lines
Nervous system
Chick embryo ganglia;
chick embryo brain cells;
mouse and rat cerebellum cells
C 1300 (mouse);
C 6 (rat)
Lung
Human, rabbit and rat alveolar
macrophages
P 388Dl (mouse);
A 549 (human)
Reticuloendothelial
system
Human, mouse lymphocytes and
erythrocytes;
rat and mouse peritoneal
macrophages
------
Liver
Rat and chick embryo hepatocytes
Chang (human);
CC1144 (rat);
ARL (rat);
RLC-GA (rat)
42
Biological Analyses
IN VITRO
SPECIAL CELLS
Chondrocyte (Cartilage)
 Epithelial (Skin)
 Hepatocyte (Liver)
 Endothelial (Vascular)
 Osteoblast (Bone)
 Fibroblast (Connective tissue)
 Schwann cell (Nerve)

43
Biological Analyses
IN VITRO
CELL TOXICITY

MTT
Neutral red assay
Calcein assay
Alamar Blue
Radioisotope 3-H-Leucine assessment
Bromodeoxyuridine (BrdU) assessment
Lactate dehydrogenase (LDH) assessment

MTT (Quantitative cytotoxic assays)






Dehydrogenase enzyme: conversion of the water-soluble yellow dye MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan by the
action of mitochondrial reductase (ELISA).
Dead cells do not have active mitochondrial reductases (as the cellular reduction is only
44
catalyzed by living cells), MTT is not reduced and the purple formazan is not formed.
Biological Analyses
IN VITRO

Neutral Red (NR) assay


Lysosomal activity
Membrane permeability

The
neutral
red
(NR
(3-amino-7-dimethyl-2-methylphenazine
hydrochloride)) assay is a cell survival chemo sensitivity assay.

This assay is based on the incorporation of NR into the lysosomes of
viable cells after being incubation with test agents.

Therefore, it is possible to distinguish between viable, damaged or
dead cells as viable cells take up the NR dye, damaged or dead cells
do not.
45
Biological Analyses
IN VITRO
• Positive and negative controls are often included in the assays to
ensure the operation and suitability of the test system.
• The negative control of choice: high-density polyethylene
material or TCPS (Tissue Culture Poly Styrene).
• The positive controls: low-molecular-weight organo tin-stabilized
poly (vinyl chloride), gum rubber, and dilute solutions of toxic
chemicals, such as phenol and benzalkonium chloride.
46
Biological Analyses
IN VITRO
 The in vitro cytotoxicity assays are the primary biocompatibility
screening tests for a wide variety of elastomeric, polymeric, and
other materials used in medical devices.
 After the cytotoxicity profile of a material has been determined,
then more application specific tests are performed to assess the
biocompatibility of the material.
47
Biological Analyses
IN VITRO
HEMOCOMPATIBILITY TESTS
 Are used to evaluate the effect of a material on blood coagulation processes,
thrombus formation and hemolysis (destruction of red blood cells).
 Materials or theirs extracts are incubated with red blood cells, isolated from
rabbits, mice, or rats for three hours with intermittent shaking to keep
samples mixed and in contact with blood.
 The amount of hemoglobin released into the supernatant from the cells is
determined spectrophotometrically and reported as percent hemolysis with
respect to negative controls.
 To evaluate the effect of materials and their surfaces on blood clotting,
materials are exposed to whole blood serum.
 Turbulent flow of blood may increase hemolysis and or clotting – the
number of adherent platelets may be determined per unit area after exposure
48
to whole blood.
Biological Analyses
IN VITRO
MUTAGENECITY AND GENOTOXICITY
 Mutagenes – modify the genome of a host, so materials may be classified as
genotoxic. It is widely accepted that carcinogenic behavior proceeds via a
mutation in the genome.
Ames test – uses a mutant bacterial cell line (Salmonella typhimurium or
Escherichia coli) that must be supplied with histidine to growth. The cells are
cultured in histidine-free environment and only those material that mutate the
cells back to state of histidine indipendence will allow the cells back to grow.
Styles test (Fibroblast cell deformation , Agar gel)
 Hypersensitivity Tests – the leukocyte migration inhibition and lyphocyte
transformation tests have been used as in-vitro models to estimate delayed
hypersensitivity reaction to implant materials and their released components.
49
Biological Analyses
IN VITRO
CONSIDERATIONS
Choice of cells
 Conformity of Biomaterials to tissue
 Repetition (Number of replicates)
 Statically evaluations (P-value)
CAS (Cell Analysis System) or Image Pro Plus

50
Biological Analyses
IN VITRO
• In vitro tests minimize the use of animals in research, a desirable goal.
• Also, in vitro testing is required by most regulatory agencies in the device
approval process for clinical application.
• When appropriately used, in vitro testing provides useful insights that can
dictate whether a device need be further evaluated in expensive in vivo
experimental models.
51
Biological Analyses
IN VITRO
•
•
•
•
•
•
In vitro tests minimize the use of animals in research, a desirable goal.
Also, in vitro testing is required by most regulatory agencies in the device approval
process for clinical application.
When appropriately used, in vitro testing provides useful insights that can dictate
whether a device need be further evaluated in expensive in vivo experimental models.
Current experience indicates that a material that is judged to be nontoxic in vitro will be
nontoxic in in vivo assays.
This does not necessarily mean that materials that are toxic in vitro could not be used in
a given clinical application.
The clinical acceptability of a material depends on many different factors, of which
target cell toxicity is but one.
 For example, glutaraldehyde-fixed porcine valves produce adverse effects in vitro
owing to low residues of glutaraldehyde; however, this material has the greatest clinical
efficacy for its unique application.
52
Biological Analyses
IN VITRO
Advantages
 Low
expense
 Low work
 High speed
 High reproducibility
 Cell and tissue (biopsy) access
 High Performability
53
Biological Analyses
IN VIVO
The goal of in vivo assessment of tissue compatibility of a
biomaterial, prosthesis, or medical device is to determine the
biocompatibility or safety of the biomaterial, prosthesis, or medical
device in a biological environment.
• From a practical perspective, the in vivo assessment of tissue
compatibility of medical devices is carried out to determine that
the device performs as intended and presents no significant harm
to the patient or user.
• Thus, the goal of the in vivo assessment of tissue compatibility is
to predict whether a medical device presents potential harm to
the patient or user by evaluations under conditions simulating
clinical use.
54
Biological Analyses
IN VIVO
55
Biological Analyses
IN VIVO
SENSITIZATION, IRRITATION, AND INTRACUTANEOUS
(INTRADERMAL) REACTIVITY
• Exposure to or contact with even minute amounts of potential
leachables from medical devices or biomaterials can result in
allergic or sensitization reactions.
• Sensitization tests estimate the potential for contact sensitization
to medical devices, materials, and/or their extracts.
• Symptoms of sensitization are often seen in skin and tests are
often carried out topically in guinea pigs.
56
Biological Analyses
IN VIVO
SENSITIZATION, IRRITATION, AND INTRACUTANEOUS
(INTRADERMAL) REACTIVITY
• The most severely irritating chemical leachable may be discovered prior to in
vivo studies by careful material characterization and in vitro cytotoxicity tests.
• Irritant tests emphasize utilization of extracts of the biomaterials to determine
the irritant effects of potential leachable.
• Intracutaneous (intradermal) reactivity tests determine the localized reaction of
tissue to intracutaneous injection of extracts of medical devices, biomaterials,
or prostheses in the final product form.
• Intracutaneous tests may be applicable where determination of irritation by
dermal or mucosal tests are not appropriate.
• Albino rabbits are most commonly used.
57
Biological Analyses
IN VIVO
Systemic Toxicity
Acute, Subacute, and Subchronic Toxicity
Mice, rats, or rabbits are the usual animals of choice for the conduct
of these tests and oral, dermal, inhalation, intravenous,
intraperitoneal, or subcutaneous application of the test substance
may be used, depending on the intended application of the
biomaterial.
58
Biological Analyses
IN VIVO
SYSTEMIC TOXICITY
ACUTE, SUBACUTE, AND SUBCHRONIC TOXICITY
• Acute toxicity is considered to be the adverse effects that occur
after administration of a single dose or multiple doses of a test
sample given within 24 hours.
• Subacute toxicity (repeat-dose toxicity) focuses on adverse effects
occurring after administration of a single dose or multiple doses
of a test sample per day during a period of from 14 to 28 days.
59
Biological Analyses
IN VIVO
SYSTEMIC TOXICITY
ACUTE, SUBACUTE, AND SUBCHRONIC TOXICITY
• Pyrogenicity tests are also included in the systemic toxicity
category to detect material-mediated fever-causing reactions to
extracts of medical devices or materials.
• It is noteworthy that no single test can differentiate pyrogenic
reactions that are material-mediated from those due to endotoxin
contamination.
60
Biological Analyses
IN VIVO
GENOTOXICITY
• In vivo genotoxicity tests are carried out if indicated by the
chemistry and/or composition of the biomaterial or if in vitro test
results
indicate
potential
genotoxicity
[changes
in
deoxyribonucleic acid (DNA)].
• Initially, at least three in vitro assays should be used and two of
these assays should utilize mammalian cells.
• The initial in vitro assays should cover the three levels of
genotoxic effects:
– DNA destruction.
– Gene mutations.
– Chromosomal aberrations.
61
Biological Analyses
IN VIVO
IMPLANTATION
62
Biological Analyses
IN VIVO
IMPLANTATION
• For short-term implantation evaluation out to 12 weeks, mice,
rats, guinea pigs, or rabbits are the usual animals utilized in these
studies.
• For longer-term testing in subcutaneous tissue, muscle, or bone,
animals such as rats, guinea pigs, rabbits, dogs, sheep, goats, pigs,
and other animals with relatively long life expectancy are suitable.
63
Biological Analyses
IN VIVO
IMPLANTATION
• If a complete medical device is to be evaluated, larger species
may be utilized so that human-sized devices may be used in the
site of intended application.
 For example
– substitute heart valves are usually tested as heart valve
replacements in sheep,
– whereas calves are usually the animal of choice for ventricular
assist devices and total artificial hearts.
64
Biological Analyses
IN VIVO
IMPLANTATION
Haematoxylin and eosin (H&E) staining
Ziehl-Neelsen stain
Papanicolaou staining
Masson's trichrome
….
Haematoxylin and eosin (H&E) staining
65
Biological Analyses
IN VIVO
HEMOCOMPATIBILITY OR BLOOD COMPATIBILITY
• Hemocompatibility tests evaluate effects on blood and/or blood
components by blood-contacting medical devices or materials.
• In vivo hemocompatibility tests are usually designed to simulate
the geometry, contact conditions, and flow dynamics of the device
or material in its clinical application.
• From the IS0 standards perspective, five test categories are
indicated for hemocompatibility evaluation :
 Thrombosis, coagulation, platelets, hematology, and immunology
(complement and leukocytes).
66
Biological Analyses
IN VIVO
HEMOCOMPATIBILITY
In vivo testing in animals may be convenient, but species'
differences in blood reactivity must be considered and these may
limit the predictability of any given test in the human clinical
situation.
67
Biological Analyses
IN VIVO
CHRONIC TOXICITY
 Chronic toxicity tests determine the effects of either single or
multiple exposures to medical devices, materials, and/or their
extracts during a period of at least 10 % of the lifespan of the test
animal, e.g. over 90 days in rats.
 Chronic toxicity tests may be considered an extension of
subchronic (subacute) toxicity testing and both may be evaluated
in an appropriate experimental protocol or study.
68
Biological Analyses
IN VIVO
CARCINOGENICITY
• Carcinogenicity tests determine the tumorigenic potential of
medical devices, materials, and/or their extracts from either single
or multiple exposures or contacts over a period of the major
portion of the lifespan of the test animal.
• Since tumors associated with medical devices have been rare
carcinogenicity tests should be conducted only if data from other
sources suggest a tendency for tumor induction.
• In addition, both carcinogenicity (tumorigenicity) and chronic
toxicity may be studied in a single experimental study.
69
Biological Analyses
IN VIVO
REPRODUCTIVE AND DEVELOPMENTAL TOXICITY
• These tests evaluate the potential effects of medical devices,
materials, and/or their extracts on reproductive function,
embryonic development (teratogenicity), and prenatal and early
postnatal development.
• The application site of the device must be considered and tests
and/or bioassays should only be conducted when the device has a
potential impact on the reproductive potential of the subject.
70
Biological Analyses
IN VIVO
BIODEGRADATION
 Biodegradation tests determine the effects of a biodegradable
material and its biodegradation products on the tissue response.
• They focus on:
– The amount of degradation during a given period of time (the
kinetics of biodegradation).
– The nature of the degradation products.
– The origin of the degradation products (e. g., impurities,
additives, corrosion products, bulk polymer).
– The qualitative and quantitative assessment of degradation
products and leachable in adjacent tissues and in distant organs.
71
Biological Analyses
IN VIVO
IMMUNE RESPONSE
• Immune response evaluation is not a component of the standards
currently available for in vivo tissue compatibility assessment.
• However, ASTM, IS0, and the FDA currently have working
groups developing guidance documents for immune response
evaluation where pertinent.
• Synthetic materials are not generally immunotoxic.
• However, immune response evaluation is necessary with modified
natural tissue implants such as collagen, which has been utilized
in a number of different types of implants and may elicit
immunological responses.
72
Biological Analyses
IN VIVO
SELECTION OF ANIMAL MODELS FOR IN VIVO TESTS
• Animal models are used to predict the clinical behavior, safety,
and biocompatibility of medical devices in humans.
• The selection of animal models for the in vivo assessment of
tissue compatibility must consider the advantages and
disadvantages of the animal model for human clinical application.
• Several examples follow, which exemplify the advantages and
disadvantages of animal models in predicting clinical behavior in
humans.
73
Biological Analyses
IN VIVO
74
Biological Analyses
IN VIVO
SELECTION OF ANIMAL MODELS FOR IN VIVO TESTS
 Thus, the choice of this animal model for bio prosthetic heart
valve evaluation is made on the basis of accelerated calcification
in rapidly growing animals, which has its clinical correlation in
young and adolescent humans.
 Nevertheless, normal sheep may not provide a sensitive
assessment of the propensity of a valve to thrombosis.
75