Transcript Biological Testing of Biomaterials From

BME Experiment
2010 Summer
Ming-Long Yeh
8/25/2010
Biological Testing of Biomaterials
From: Biomaterials Science by Ratner
1
5. Biological Testing of Biomaterials
5.1 INTRODUCTION TO TESTING BIOMATERIALS
5.2 IN VITRO ASSESSMENT OF TISSUE COMPATIBILITY
5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY
5.4 EVALUATION OF BLOOD-MATERIALS
INTERACTIONS
5.5 LARGE ANIMAL MODELS IN CARDIAC AND
VASCULAR BIOMATERIALS RESEARCH AND
TESTING
5.6 MICROSCOPY FOR BIOMATERIALS SCIENCE
2
5.1 INTRODUCTION TO
TESTING BIOMATERIALS
3
5.1 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?
 This introduction: common to all
biomaterials biological testing.

4
5.1 INTRODUCTION TO TESTING
BIOMATERIALS
Evaluation under in vitro (literally "in glass")
conditions: rapid and inexpensive data on
biological interaction (Chapter 5. 2).
 The question: will the in vitro test measure
parameters relevant to what will occur in
vivo environment?

5
5.1 INTRODUCTION TO TESTING
BIOMATERIALS




For example, tissue culture polystyrene, a
surface modified polymer, will readily attach and
grow most cells in culture.
Untreated polystyrene will neither attach nor
grow mammalian cells.
Yet when implanted in vivo, both materials heal
almost indistinguishably with a thin foreign body
capsule.
Thus, the results of the in vitro test do not
provide information relevant to the implant
situation.
6
5.1 INTRODUCTION TO TESTING
BIOMATERIALS



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.
7
5.1 INTRODUCTION TO TESTING
BIOMATERIALS




Animals are used for testing biomaterials to
model the environment that might be
encountered in humans (Chapter 5. 3).
However, there is great range in animal anatomy,
physiology, and biochemistry.
Will the animal model provide data useful for
predicting how a device performs in humans?
Without validation to human clinical studies, it is
often difficult to draw strong conclusions from
performance in animals.
8
5.1 INTRODUCTION TO TESTING
BIOMATERIALS


The first step in designing animal testing
procedures is to choose an animal model that
offers a reasonable parallel anatomically or
biochemically to the situation in humans.
Experiments designed



to minimize the number of animals needed,
ensure that the animals are treated humanely (e. g.,
NIH guidelines for the use of laboratory animals), and
maximize the relevant information generated by the
testing procedure are essential.
9
5.1 INTRODUCTION TO TESTING
BIOMATERIALS


Some biomaterials fulfill their intended function in
seconds.
Others are implanted for a lifetime (10 years ? 70
years?).

Are 6-month implantation times useful to learn about a
device intended for 3-minute insertion?

Will six months implantation in a test model provide
adequate information to draw conclusions about the
performance of a device intended for lifetime
implantation?
10
5.1 INTRODUCTION TO TESTING
BIOMATERIALS



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.
11
5.2 IN VITRO ASSESSMENT
OF TISSUE COMPATIBILITY
12
5.2 IN VITRO ASSESSMENT OF
TISSUE COMPATIBILITY



“Cytotoxicity”: 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.).
Evaluation of biomaterials by methods that use
isolated, adherent cells in culture to measure
cytotoxicity and biological compatibility.
13
HISTORICAL REVIEW
Cell culture methods: more than two
decades (Northup, 1986).
 Most often today the cells used for culture
are from established cell lines purchased
from biological suppliers or cell banks.
 Primary cells are seldom used


less assay repeatability, reproducibility,
efficiency, and, in some cases, availability.
14
BACKGROUND CONCEPT
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效力 of the chemical.
Although a 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.
15
BACKGROUND CONCEPT
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有毒的 substance (exposure dose),
only a small portion of the inhaled substance will be
absorbed and delivered to the internal organs and cells
(delivered dose).
16
Delivered and Exposure Doses



The cells that are most sensitive are referred to
as the target cells.
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.
17
Safety Factors



A highly sensitive test system is desirable for
evaluating the potential hazards of biomaterials
because the inherent characteristics of the
materials often do not allow the dose to be
exaggerated.
There is a great deal of uncertainty in
extrapolating from one test system to another,
such as from animals to humans.
To allow for this, toxicologists have used the
concept of safety factors to take into account
intra- and interspecies variation.
18
Safety Factors



This practice requires being able to exaggerate
the anticipated預期 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 test system.
19
ASSAY METHODS
20
ASSAY METHODS

Three primary cell culture assays are
used for evaluating biocompatibility:
 direct
contact,
 agar diffusion,
 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.
21
cell culture assays

http://en.wikipedia.org/wiki/Cell_Culture_Assays
 direct
contact,
 agar diffusion,
 elution
22
Direct contact method
1.
2.
3.
4.
5.
6.
7.
8.
A near confluent layer of fibroblasts are prepared in a
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
The material is removed
The culture media is removed
The remaining cells are fixed and stained, 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 the material
23
Agar diffusion method
1.
2.
3.
4.
5.
6.
7.
8.
A near confluent layer of fibroblasts 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
When the agar solidifies the cells will have dispersed
throughout its volume
The material is then placed on the surface of the agar
and incubated for 24 hours at 37 degrees Celsius
Live cells take up the vital stain and retain it, dead cells
do not
The toxicity of the material is evaluated by the loss of
vital stain under and around the material
Surface microscopy is also needed to evaluate the
material-cell interfacae
24
Elution method
1.
2.
3.
4.
5.
A near confluent layer of fibroblasts are prepared in a
culture plate
An extract of the material which is being tested is
prepared using physiological saline or serum free media
(the latter is generally preferred)
Extraction conditions are used which are appropriate for
the type of exposure which the cells would receive in
the in vivo environment if the material were to be
implanted
The extract is placed on the cells and incubated for 48
hours at 37 degrees Celsius
After 48 hours the toxicity is evaluated using either a
histochemical or vital stain
25
ASSAY METHODS

To standardize the methods and compare the
results of these assays, the variables
 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
26
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近親交配的 animal strains used for in vivo
studies.
Cell lines maintain their genetic and
morphological characteristics throughout a long
(sometimes called infinite) life span.
27
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.
28
ASSAY METHODS

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.
The positive controls: low-molecular-weight
organotin-stabilized poly (vinyl chloride), gum
rubber, and dilute solutions of toxic chemicals,
such as phenol and benzalkonium chloride.

29
30
CLINICAL USE
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.

31
CLINICAL USE




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.
32
5.3 IN VIVO ASSESSMENT
OF TISSUE
COMPATIBILITY
33
5.3 IN VIVO ASSESSMENT OF
TISSUE COMPATIBILITY
INTRODUCTION
 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.
34
INTRODUCTION
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.

35
INTRODUCTION


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.
36
INTRODUCTION


Recently, extensive efforts have been made by
government agencies, i. e., FDA, and regulatory
bodies, i.e., ASTM, IS0, and USP, to provide
procedures, protocols, guidelines, and standards
that may be used in the in vivo assessment of
the tissue compatibility of medical devices.
This chapter draws heavily on the IS0 10993
standard, Biological Evaluation of Medical
Devices, in presenting a systematic approach to
the in vivo assessment of tissue compatibility of
medical devices.
37
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 Biological evaluation of
medical devices Part 2: Animal welfare
requirements
 ISO 10993-3:2003 Biological evaluation of
medical devices Part 3: Tests for genotoxicity,
carcinogenicity and reproductive toxicity
 ISO 10993-4:2002/Amd 1:2006 Biological
evaluation of medical devices Part 4: Selection
of tests for interactions with blood
 ISO 10993-5:1999 Biological evaluation of
medical devices Part 5: Tests for in vitro
cytotoxicity
38





ISO 10993-6:1994 Biological evaluation of medical
devices Part 6: Tests for local effects after
implantation
ISO 10993-7:1995 Biological evaluation of medical
devices Part 7: Ethylene oxide sterilization residuals
ISO 10993-8:2001 Biological evaluation of medical
devices. Part 8: Selection and qualification of
reference materials for biological tests
ISO 10993-9:1999 Biological evaluation of medical
devices Part 9: Framework for identification and
quantification of potential degradation products
ISO 10993-10:2002/Amd 1:2006 Biological evaluation
of medical devices Part 10: Tests for irritation and
delayed-type hypersensitivity
39





ISO 10993-11:2006 Biological evaluation of medical
devices Part 11: Tests for systemic toxicity
ISO 10993-12:2002 Biological evaluation of medical
devices Part 12: Sample preparation and reference
materials (available in English only)
ISO 10993-13:1998 Biological evaluation of medical
devices Part 13: Identification and quantification of
degradation products from polymeric medical
devices
ISO 10993-14:2001 Biological evaluation of medical
devices Part 14: Identification and quantification of
degradation products from ceramics
ISO 10993-15:2000 Biological evaluation of medical
devices Part 15: Identification and quantification of
degradation products from metals and alloys
40





ISO 10993-16:1997 Biological evaluation of medical
devices Part 16: Toxicokinetic study design for
degradation products and leachables
ISO 10993-17:2002 Biological evaluation of medical
devices Part 17: Establishment of allowable limits
for leachable substances
ISO 10993-18:2005 Biological evaluation of medical
devices Part 18: Chemical characterization of
materials
ISO/TS 10993-19:2006 Biological evaluation of
medical devices Part 19: Physico-chemical,
morphological and topographical characterization of
materials
ISO/TS 10993-20:2006 Biological evaluation of
medical devices Part 20: Principles and methods for
immunotoxicology testing of medical device
41
INTRODUCTION
42
BIOMATERIALS AND DEVICE
PERSPECTIVES IN IN VIVO TESTINGS
43
BIOMATERIALS AND DEVICE
PERSPECTIVES IN IN VIVO TESTINGS
44
Sensitization, irritation刺激, 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.
45
Sensitization, irritation, and intracutaneous
(intradermal) Reactivity





The most severely irritating chemical leachables 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
leachables.
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.
46
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.
Peritoneal 腹膜的
47
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.
Sub-chronic toxicity is considered to be the adverse
effects occurring after administration of a single dose or
multiple doses of a test sample per day given during a
part of the life span, usually 90 days but not exceeding
10 % of the life span of the animal.
48
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

Pyrogenicity: producing or produced by heat or fever
49
Genotoxicity



In vivo genotoxicity tests are carried out if indicated by
the chemistry and/or composition of the biomaterial (see
Table 1) 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, and
 chromosomal aberrations .
50
Implantation
Dystrophic: 營養不良的
51
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.

52
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.

53
Hemocompatibility



Hemocompatibility tests evaluate effects on
blood and/or blood components by bloodcontacting 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血栓, coagulation凝結,
platelets, hematology血液學, and immunology
(complement and leukocytes).
54
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.
55
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.
56
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 (see Chapter 4. 7) 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.
57
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產前的
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.
58
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), and
the qualitative and quantitative assessment of
degradation products and leachables in adjacent
tissues and in distant organs.
59
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.
60
過敏的
61
SELECTION OF ANIMAL MODELS FOR IN
VIVO TESTS
蕁麻疹
62
Immune Response



Direct measures of immune system activity by
functional assays are the most important types
of tests for immunotoxicity.
Functional assays are generally more important
than tests for soluble mediators, which are more
important than phenotyping.
Signs of illness may be important in in vivo
experiments but symptoms may also have a
significant role in studies of immune function in
clinical trials and postmarket studies.
63
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 (Table7).
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
(also see Chapter 5. 5).
64
SELECTION OF ANIMAL MODELS FOR IN
VIVO TESTS
65
SELECTION OF ANIMAL MODELS
FOR IN VIVO TESTS
Thus, the choice of this animal model for
bioprosthetic 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,

66
SELECTION OF ANIMAL MODELS
FOR IN VIVO TESTS
The in vivo assessment of tissue
responses to vascular graft materials is
an example in which animal models
presents particularly misleading
picture of what generally occurs in
humans.
 Virtually all animal models, including
nonhuman primates, heal rapidly and
completely with an endothelial bloodcontacting surface.

67
SELECTION OF ANIMAL MODELS
FOR IN VIVO TESTS


Humans, on the other hand, do not show
extensive endothelialization of vascular graft
materials and the resultant pseudointima from the
healing response in humans has potential
thrombogenicity.
Consequently, despite favorable results in animals,
small-diameter vascular grafts (less than 4mm in
internal diameter) yield early thrombosis in humans,
the mayor mechanism of failure, which is secondary
to the lack of endothelialization in the luminal
surface healing response.
intima 內膜
68
STANDARDS
69
70
71
SELECTION OF ANIMAL MODELS FOR IN
VIVO TESTS
72
73
Preoperative Care



Anesthesia Dogs should be fasted for 12
hours prior to their operation to reduce the risk
of aspiration肺內異物的吸入 during anesthetic
induction and endotracheal intubation氣管內插管.
Preparation of the animal involves administration
of a mild intramuscular (IM) sedative such as
acepromazine, a phenothiazine tranquillizer.
Peripheral intravenous (IV) accesses can then
be established.
74
Preoperative Care Anesthesia



Prior to intubation, 0.05 mg/kg of atropine阿托品,
an anticholinergic抗副交感神經藥., may be
administered to reduce the amount of oral
secretions and ease endotracheal (ET)
intubation.
Once a secure airway has been obtained,
anesthesia can be fully induced with a
barbiturate such as thiopental sodium (12.5 mg /
kg IV).
Table 3 lists several of the recommended drugs
and doses for sedation and anesthesia in dogs.
75
76
Preoperative Care



Anesthesia Dogs should be fasted for 12
hours prior to their operation to reduce the risk
of aspiration肺內異物的吸入 during anesthetic
induction and endotracheal intubation氣管內插管.
Preparation of the animal involves administration
of a mild intramuscular (IM) sedative such as
acepromazine, a phenothiazine tranquillizer.
Peripheral intravenous (IV) accesses can then
be established.
77
Preoperative Care Anesthesia



Prior to intubation, 0.05 mg/kg of atropine阿托品,
an anticholinergic抗副交感神經藥., may be
administered to reduce the amount of oral
secretions and ease endotracheal (ET)
intubation.
Once a secure airway has been obtained,
anesthesia can be fully induced with a
barbiturate such as thiopental sodium (12.5 mg /
kg IV).
Table 3 lists several of the recommended drugs
and doses for sedation and anesthesia in dogs.
78
79
Swine vascular graft
80
81
82
83
84
85
86
87
88
89
90
Thanks!
91
92