ASTM F2721-08
(Guide)Standard Guide for Pre-clinical in vivo Evaluation in Critical Size Segmental Bone Defects
Standard Guide for Pre-clinical <span class="bdit">in vivo</span> Evaluation in Critical Size Segmental Bone Defects
SIGNIFICANCE AND USE
This guide is aimed at providing a range of in vivo models to aid in preclinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone.
This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.
The user is encouraged to use appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.
It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE)), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs, respectively.
Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the in vivo assessment of tissue engineered medical products (TEMPs) intended to repair or regenerate bone. TEMPs included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this guide are segmental critical size defects which, by definition, will not fill with viable tissue without treatment. Thus, these models represent a stringent test of a material’s ability to induce or augment bone growth.
1.2 Guidelines include a description and rationale of various animal models including rat (murine), rabbit (leporine), dog (canine), goat (caprine), and sheep (ovine). Outcome measures based on radiographic, histologic, and mechanical analyses are described briefly and referenced. The user should refer to specific test methods for additional detail.
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in the Referenced Documents (Section 2).
1.4 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.
1.5 Other pre-clinical methods may also be appropriate and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:F2721–08
Standard Guide for
Pre-clinical in vivo Evaluation in Critical Size Segmental
Bone Defects
This standard is issued under the fixed designation F 2721; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
1.1 This guide covers general guidelines for the in vivo
assessment of tissue engineered medical products (TEMPs)
2. Referenced Documents
intended to repair or regenerate bone. TEMPs included in this
2.1 ASTM Standards:
guide may be composed of natural or synthetic biomaterials
F 561 Practice for Retrieval and Analysis of Medical De-
(biocompatible and biodegradable) or composites thereof, and
vices, and Associated Tissues and Fluids
may contain cells or biologically active agents such as growth
F 565 Practice for Care and Handling of Orthopedic Im-
factors, synthetic peptides, plasmids, or cDNA. The models
plants and Instruments
described in this guide are segmental critical size defects
F 895 Test Method forAgar Diffusion Cell Culture Screen-
which, by definition, will not fill with viable tissue without
ing for Cytotoxicity
treatment. Thus, these models represent a stringent test of a
F 981 Practice for Assessment of Compatibility of Bioma-
material’s ability to induce or augment bone growth.
terials for Surgical Implants with Respect to Effect of
1.2 Guidelinesincludeadescriptionandrationaleofvarious
Materials on Muscle and Bone
animal models including rat (murine), rabbit (leporine), dog
F 1983 Practice for Assessment of Compatibility of
(canine), goat (caprine), and sheep (ovine). Outcome measures
Absorbable/Resorbable Biomaterials for Implant Applica-
based on radiographic, histologic, and mechanical analyses are
tions
described briefly and referenced. The user should refer to
F 2150 Guide for Characterization and Testing of Biomate-
specific test methods for additional detail.
rial Scaffolds Used inTissue-Engineered Medical Products
1.3 This guide is not intended to include the testing of raw
F 2451 Guide for in vivo Assessment of Implantable De-
materials, preparation of biomaterials, sterilization, or packag-
vices Intended to Repair or Regenerate Articular Cartilage
ing of the product. ASTM standards for these steps are
2.2 Other Documents:
available in the Referenced Documents (Section 2).
ISO10993 BiologicalEvaluationofMedicalDevices—Part
1.4 The use of any of the methods included in this guide
5: Tests for in vitro Cytotoxicity
may not produce a result that is consistent with clinical
21 CFR Part 58 Good Laboratory Practice for Nonclinical
performance in one or more specific applications.
Laboratory Studies
1.5 Other pre-clinical methods may also be appropriate and
21 CFR 610.12 General Biological Products Standards—
this guide is not meant to exclude such methods. The material
Sterility
must be suitable for its intended purpose.Additional biological
testing in this regard would be required.
3. Terminology
1.6 The values stated in SI units are to be regarded as
3.1 Definitions:
standard. No other units of measurement are included in this
standard.
1.7 This standard does not purport to address all of the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
safety concerns, if any, associated with its use. It is the
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
responsibility of the user of this standard to establish appro-
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
This guide is under the jurisdiction of ASTM Committee F04 on Medical and 4th Floor, New York, NY 10036, http://www.ansi.org.
Surgical Materials and Devices and is the direct responsibility of Subcommittee AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
F04.44 on Assessment for TEMPs. 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
Current edition approved Nov. 1, 2008. Published March 2009. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2721–08
3.1.1 bone regeneration—the formation of bone that has 3.1.17 weight-bearing versus non-weight bearing models—
histologic, biochemical, and mechanical properties similar to weight bearing is the amount of weight a patient or experimen-
that of native bone. tal animal puts on the leg on which surgery has been per-
3.1.2 bone repair—the process of healing injured bone formed, generally described as a percentage of the body
weight.
through cell proliferation and synthesis of new extracellular
matrix. 3.1.17.1 Discussion—Non weight bearing means the leg
mustnottouchthefloor(i.e.,supports0 %ofthebodyweight).
3.1.3 compact bone—classificationofossifiedbonyconnec-
3.1.17.2 Discussion—Fullweightbearingmeansthelegcan
tive tissue characterized by the presence of osteon-containing
carry 100 % of the body weight on a step.
lamellar bone. Lamellar bone is highly organized in concentric
sheets.
4. Significance and Use
3.1.4 cortical bone—one of the two main types of osseous
4.1 This guide is aimed at providing a range of in vivo
tissue. Cortical bone is dense and forms the surface of bones.
models to aid in preclinical research and development of
3.1.5 critical size defect—a bone defect, either naturally
tissue-engineered medical products (TEMPs) intended for the
occurring or artificially created, which will not heal without
clinical repair or regeneration of bone.
intervention. In the clinical setting, this term applies to
4.2 This guide includes a description of the animal models,
exceeding a healing period of approximately 6 months (in
surgical considerations, and tissue processing as well as the
otherwise healthy adults).
qualitative and quantitative analysis of tissue specimens.
3.1.6 diaphyseal—pertaining to the mid-section of long
4.3 The user is encouraged to use appropriate ASTM and
bones.
other guidelines to conduct cytotoxicity and biocompatibility
3.1.7 endochondral ossification—one of the two main types
tests on materials, TEMPs, or both, prior to assessment of the
of bone formation, where a cartilaginous matrix forms first and
in vivo models described herein.
is subsequently replaced by osseous tissue.
4.4 It is recommended that safety testing be in accordance
3.1.7.1 Discussion—Endochondral ossification is respon-
with the provisions of the FDA Good Laboratory Practices
sible for much of the bone growth in vertebrate skeletons,
Regulations 21 CFR 58.
especially in long bones.
4.5 Safety and effectiveness studies to support regulatory
3.1.8 growth plate—the anatomic location within the epi-
submissions (for example, Investigational Device Exemption
physeal region of long bones corresponding to the site of
(IDE)), Premarket Approval (PMA), 510K, Investigational
growth of bone through endochondral ossification.
New Drug (IND), or Biologics License Application (BLA)
3.1.8.1 Discussion—The growth plate in skeletally mature
submissions in the U.S.) should conform to appropriate guide-
animals is fused.
lines of the regulatory bodies for development of medical
3.1.9 long bone—bone that is longer than it is wide, and
devices, biologics, or drugs, respectively.
growsprimarilybyelongationofthediaphysis.Thelongbones
4.6 Animal model outcomes are not necessarily predictive
include the femurs, tibias, and fibulas of the legs, the humeri,
of human results and should, therefore, be interpreted cau-
radii, and ulnas of the arms, the metacarpals and metatarsals of
tiously with respect to potential applicability to human condi-
the hands and feet, and the phalanges of the fingers and toes.
tions.
3.1.10 marrow—soft, gelatinous tissue that fills the cavities
5. Animal Models
of the bones. It is either red or yellow, depending upon the
preponderance of hematopoietic (red) or fatty (yellow) tissue.
NOTE 1—This section provides a description of the options to consider
3.1.10.1 Discussion—Red marrow is also called myeloid
in determining the appropriate animal model and bone defect size and
location.
tissue.
NOTE 2—Research using these models needs to be conducted in
3.1.11 matrix—a term applied to either the exogenous
accordance with governmental regulations and guidelines appropriate to
implanted scaffold or the endogenous extracelluar substance
the locale for the care and use of laboratory animals. Study protocols
(otherwise known as extracellular matrix) derived from the
should be developed after consultation with the institutional attending
host.
veterinarian,andneedappropriatereviewandapprovalbytheinstitutional
3.1.12 metaphyseal—pertaining to the dense end-section of
animal care and use committee prior to study initiation.
long bones.
5.1 Defect Size:
3.1.13 remodeling—a life long process where old bone is
5.1.1 Ahigh proportion of fracture injuries in humans occur
removed from the skeleton (bone resorption) and new bone is
in long bones. Accordingly, defects created in long bones are
added (bone formation).
commonly used for assessing bone repair/regeneration in
3.1.14 residence time—the time at which an implanted
animal models.
material (synthetic or natural) can no longer be detected in the
5.1.2 In principle, critical-size defects may be achieved in
host tissue.
both metaphyseal and diaphyseal locations. For the purpose of
3.1.15 skeletal maturity—the age at which the epiphyseal
this guide, only defects created in the diaphyseal section of
plates are fused.
long bones will be described.
3.1.15.1 Discussion—In rodents, skeletally mature animals
5.1.3 Significant variability exists between animal species
are characterized by defined gonads. with respect to the size and weight of the animal, anatomy, and
3.1.16 trabecular bone—ossified bony connective tissue gait thereby influencing kinetics, range of motion, and me-
characterized by spicules surrounded by marrow space. chanical forces on defects. These factors influence bone
F2721–08
architecture and structure. These factors play a significant role 5.1.11 The use of unilateral defect models is generally
in the response to injury or disease of bone. The user should recommended. This is especially true for weight-bearing loca-
consider carefully the animal model that is appropriate for the tions in animals that use all four limbs for weight bearing
stage of investigation of an implanted TEMP. (especially goats, sheep, and horses).
5.1.4 Mechanical load has been shown to affect bone repair.
5.2 Handling:
Amongst the mechanobiological factors, intermittent hydro-
5.2.1 Exposure of implants to extreme and highly variable
static pressure and load-bearing stresses play an important role
mechanical forces as a result of jumping and running can lead
in modulating bone development and maintenance, as well as
to increased variability in outcome measures.
bone degeneration The impact of mechanical load extent or
5.2.2 Potential differences in outcome when using weight-
durationontheimplantedTEMP,andsurroundingnativebone,
bearing versus non-weight bearing models should be carefully
varies depending on the anatomic site. The defect site chosen
considered.
to evaluate implants should, therefore, factor the impact of
5.3 Chromosomal Sex:
mechanical load on the performance of the implant.
5.3.1 Due to the impact of circulating steroids on cartilage
5.1.5 It is recommended that an appropriate species and
and bone metabolism and regeneration, the choice of chromo-
anatomicsitebechosen,thathavedimensionssufficientlylarge
somal sex should be considered. Animals in lactation should
to adequately investigate and optimize the formulation, design,
not be used. For some purposes, the use of aged or ovariecto-
dimensions, and associated instrumentation envisaged for hu-
mized females (especially rats) may be indicated to simulate
man use, especially in late stages of development.
osteoporotic conditions.
5.1.6 Larger animals may be more appropriate for studying
5.3.2 It is recommended that the chromosomal sex be the
repair in defects and locations that more closely approximate
same within the cohort, and that needs to be reported. The
those in humans.
investigator should be aware that variances can occur between
5.1.7 Larger defect dimensions generally require a method
sexes and that appropriate statistical power needs to be
of fixation to secure the implant and thereby reduce implant
instituted.
dislocation. The method of implant immobilization can nega-
5.4 Age:
tivelyimpactboththesurroundinghosttissueandrepairtissue.
5.4.1 Bone undergoes dynamic changes in metabolism and
Accordingly, the difference in the design of the test TEMP in
remodeling during growth. Due to the impact of these physi-
models which generally do not require fixation should be
ologic processes on tissue repair, skeletally mature animals
factored into the interpretation of results with respect to
should be used. The cohorts should have fused epiphyseal
predictability of outcomes in larger animal models and humans
growth plates. Skeletal maturity varies between species and
requiring fixation.
can be determined radiographically if necessary.
5.1.8 For each species, a critical size defect is defined as the
5.4.2 Olderanimalshaveagreaterpropensityforosteopenia
minimum defect dimension that the animal is incapable of
andhaveadecreasedcapacitytorepairbonedefects.Ifspecific
repairing without intervention. The dimensions of critical
conditions are considered important for the intended TEMP
defects generally differ for each species and should be consid-
assessment, then an appropriate model should be used.
ered carefully when designing the implant dimensions and
5.4.3 The mesenchymal stem cell pool, growth factor re-
method of fixation. As an empirical rule, the length of the
sponsiveness, and metabolic activity of cells generally de-
defect (created by ostectomy) should at least be equal to 1.5
creases with age.Thus, reparative processes that are dependent
times the diameter of the selected bone (1, 2). Some authors
on the number and activity of native cells may be partially
recommend at least 2 times the diameter of the selected bone
compromised in older animals.
(3).
5.5 Diet or Concurrent Pathology:
5.1.9 Whether or not the periosteum from the resected
5.5.1 In general, studies are performed with healthy animals
segmentofboneisstillpresentcaninfluencehealingwithinthe
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