ASTM F2721-09(2023)
(Guide)Standard Guide for Preclinical in vivo Evaluation in Critical-Size Segmental Bone Defects
Standard Guide for Preclinical <emph type="ital">in vivo</emph> Evaluation in Critical-Size Segmental Bone Defects
SIGNIFICANCE AND USE
4.1 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.
4.2 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.
4.3 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.
4.4 It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
4.5 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.
4.6 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 preclinical 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F2721 − 09 (Reapproved 2023)
Standard Guide for
Preclinical in vivo Evaluation in Critical-Size Segmental Bone
Defects
This standard is issued under the fixed designation F2721; 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 responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
1.1 This guide covers general guidelines for the in vivo
mine the applicability of regulatory limitations prior to use.
assessment of tissue-engineered medical products (TEMPs)
1.8 This international standard was developed in accor-
intended to repair or regenerate bone. TEMPs included in this
dance with internationally recognized principles on standard-
guide may be composed of natural or synthetic biomaterials
ization established in the Decision on Principles for the
(biocompatible and biodegradable) or composites thereof, and
Development of International Standards, Guides and Recom-
may contain cells or biologically active agents such as growth
mendations issued by the World Trade Organization Technical
factors, synthetic peptides, plasmids, or cDNA. The models
Barriers to Trade (TBT) Committee.
described in this guide are segmental critical size defects
which, by definition, will not fill with viable tissue without
2. Referenced Documents
treatment. Thus, these models represent a stringent test of a
material’s ability to induce or augment bone growth. 2.1 ASTM Standards:
F561 Practice for Retrieval and Analysis of Medical
1.2 Guidelines include a description and rationale of various
Devices, and Associated Tissues and Fluids
animal models including rat (murine), rabbit (leporine), dog
F565 Practice for Care and Handling of Orthopedic Implants
(canine), goat (caprine), and sheep (ovine). Outcome measures
and Instruments
based on radiographic, histologic, and mechanical analyses are
F895 Test Method for Agar Diffusion Cell Culture Screening
described briefly and referenced. The user should refer to
for Cytotoxicity
specific test methods for additional detail.
F981 Practice for Assessment of Compatibility of Biomate-
1.3 This guide is not intended to include the testing of raw
rials for Surgical Implants with Respect to Effect of
materials, preparation of biomaterials, sterilization, or packag-
Materials on Muscle and Insertion into Bone
ing of the product. ASTM standards for these steps are
F1983 Practice for Assessment of Selected Tissue Effects of
available in the Referenced Documents (Section 2).
Absorbable Biomaterials for Implant Applications
1.4 The use of any of the methods included in this guide F2150 Guide for Characterization and Testing of Biomate-
rial Scaffolds Used in Regenerative Medicine and Tissue-
may not produce a result that is consistent with clinical
performance in one or more specific applications. Engineered Medical Products
2.2 Other Documents:
1.5 Other preclinical methods may also be appropriate and
21 CFR Part 58 Good Laboratory Practice for Nonclinical
this guide is not meant to exclude such methods. The material
Laboratory Studies
must be suitable for its intended purpose. Additional biological
21 CFR 610.12 General Biological Products Standards—
testing in this regard would be required.
Sterility
1.6 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3. Terminology
standard.
3.1 Definitions:
1.7 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This guide is under the jurisdiction of ASTM Committee F04 on Medical and contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Surgical Materials and Devices and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
F04.44 on Assessment for TEMPs. the ASTM website.
Current edition approved March 1, 2023. Published March 2023. Originally Available from U.S. Government Printing Office Superintendent of Documents,
approved in 2008. Last previous edition approved in 2014 as F2721 – 09 (2014). 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
DOI: 10.1520/F2721-09R23. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2721 − 09 (2023)
3.1.1 bone regeneration—the formation of bone that has 3.1.15 skeletal maturity—the age at which the epiphyseal
histologic, biochemical, and mechanical properties similar to plates are fused.
that of native bone. 3.1.15.1 Discussion—In rodents, skeletally mature animals
are characterized by defined gonads.
3.1.2 bone repair—the process of healing injured bone
3.1.16 trabecular bone—ossified bony connective tissue
through cell proliferation and synthesis of new extracellular
matrix. characterized by spicules surrounded by marrow space.
3.1.17 weight-bearing versus non-weight bearing models—
3.1.3 compact bone—classification of ossified bony connec-
tive tissue characterized by the presence of osteon-containing weight bearing is the amount of weight a patient or experimen-
tal animal puts on the leg on which surgery has been
lamellar bone. Lamellar bone is highly organized in concentric
sheets. performed, generally described as a percentage of the body
weight.
3.1.4 cortical bone—one of the two main types of osseous
3.1.17.1 Discussion—Non-weight bearing means the leg
tissue. Cortical bone is dense and forms the surface of bones.
must not touch the floor (i.e., supports 0 % of the body weight).
3.1.5 critical size defect—a bone defect, either naturally
3.1.17.2 Discussion—Full weight bearing means the leg can
occurring or artificially created, which will not heal without
carry 100 % of the body weight on a step.
intervention. In the clinical setting, this term applies to
exceeding a healing period of approximately six months (in
4. Significance and Use
otherwise healthy adults).
4.1 This guide is aimed at providing a range of in vivo
3.1.6 diaphyseal—pertaining to the mid-section of long
models to aid in preclinical research and development of
bones.
tissue-engineered medical products (TEMPs) intended for the
clinical repair or regeneration of bone.
3.1.7 endochondral ossification—one of the two main types
of bone formation, where a cartilaginous matrix forms first and
4.2 This guide includes a description of the animal models,
is subsequently replaced by osseous tissue.
surgical considerations, and tissue processing as well as the
3.1.7.1 Discussion—Endochondral ossification is respon-
qualitative and quantitative analysis of tissue specimens.
sible for much of the bone growth in vertebrate skeletons,
4.3 The user is encouraged to use appropriate ASTM and
especially in long bones.
other guidelines to conduct cytotoxicity and biocompatibility
3.1.7.2 Discussion—The other main mechanism for bone
tests on materials, TEMPs, or both, prior to assessment of the
formation is intramembraneous ossification, where osseous
in vivo models described herein.
tissue is formed directly, without cartilaginous precursor;
4.4 It is recommended that safety testing be in accordance
occurs mainly in the formation of flat bones (skull).
with the provisions of the FDA Good Laboratory Practices
3.1.8 growth plate—the anatomic location within the epi-
Regulations 21 CFR 58.
physeal region of long bones corresponding to the site of
4.5 Safety and effectiveness studies to support regulatory
growth of bone through endochondral ossification.
submissions (for example, Investigational Device Exemption
3.1.8.1 Discussion—The growth plate in skeletally mature
(IDE), Premarket Approval (PMA), 510K, Investigational New
animals is fused.
Drug (IND), or Biologics License Application (BLA) submis-
3.1.9 long bone—bone that is longer than it is wide, and
sions in the U.S.) should conform to appropriate guidelines of
grows primarily by elongation of the diaphysis. The long bones
the regulatory bodies for development of medical devices,
include the femurs, tibias, and fibulas of the legs; the humeri,
biologics, or drugs, respectively.
radii, and ulnas of the arms; the metacarpals and metatarsals of
4.6 Animal model outcomes are not necessarily predictive
the hands and feet; and the phalanges of the fingers and toes.
of human results and should, therefore, be interpreted cau-
3.1.10 marrow—soft, gelatinous tissue that fills the cavities
tiously with respect to potential applicability to human condi-
of the bones. It is either red or yellow, depending upon the
tions.
preponderance of hematopoietic (red) or fatty (yellow) tissue.
3.1.10.1 Discussion—Red marrow is also called myeloid
5. Animal Models
tissue.
NOTE 1—This section provides a description of the options to consider
in determining the appropriate animal model and bone defect size and
3.1.11 matrix—either the exogenous implanted scaffold or
location.
the endogenous extracelluar substance (otherwise known as
NOTE 2—Research using these models needs to be conducted in
extracellular matrix) derived from the host.
accordance with governmental regulations and guidelines appropriate to
the locale for the care and use of laboratory animals. Study protocols
3.1.12 metaphyseal—pertaining to the dense end-section of
should be developed after consultation with the institutional attending
long bones.
veterinarian, and need appropriate review and approval by the institutional
3.1.13 remodeling—a life-long process where old bone is
animal care and use committee prior to study initiation.
removed from the skeleton (bone resorption) and new bone is
5.1 Defect Size:
added (bone formation).
5.1.1 A high proportion of fracture injuries in humans occur
3.1.14 residence time—the time at which an implanted in long bones. Accordingly, defects created in long bones are
material (synthetic or natural) can no longer be detected in the commonly used for assessing bone repair/regeneration in
host tissue. animal models.
F2721 − 09 (2023)
5.1.2 In principle, critical-size defects may be achieved in the model is very well characterized, the use of historical data
both metaphyseal and diaphyseal locations. For the purpose of instead of actual control animals should be considered, in order
this guide, only defects created in the diaphyseal section of to save on animal numbers, unless this would compromise the
long bones will be described. objectives of the study. For example, in pivotal preclinical
5.1.3 Significant variability exists between animal species proof-of-concept studies, concurrent controls are likely to be
with respect to the size and weight of the animal, anatomy, and appropriate.
gait thereby influencing kinetics, range of motion, and me-
5.1.11 The use of unilateral defect models is generally
chanical forces on defects. These factors influence bone
recommended. This is especially true for weight-bearing loca-
architecture and structure. These factors play a significant role
tions in animals that use all four limbs for weight bearing
in the response to injury or disease of bone. The user should
(especially goats, sheep, and horses).
consider carefully the animal model that is appropriate for the
5.2 Handling:
stage of investigation of an implanted TEMP.
5.2.1 Exposure of implants to extreme and highly variable
5.1.4 Mechanical load has been shown to affect bone repair.
mechanical forces as a result of jumping and running can lead
Amongst the mechanobiological factors, intermittent hydro-
to increased variability in outcome measures.
static pressure and load-bearing stresses play an important role
in modulating bone development and maintenance, as well as 5.2.2 Potential differences in outcome when using weight-
bearing versus non-weight bearing models should be carefully
bone degeneration. The impact of mechanical load extent or
duration on the implanted TEMP, and surrounding native bone, considered.
varies depending on the anatomic site. The defect site chosen
5.3 Chromosomal Sex:
to evaluate implants should, therefore, factor the impact of
5.3.1 Due to the impact of circulating steroids on cartilage
mechanical load on the performance of the implant.
and bone metabolism and regeneration, the choice of chromo-
5.1.5 It is recommended that an appropriate species and
somal sex should be considered. Animals in lactation should
anatomic site be chosen, that have dimensions sufficiently large
not be used. For some purposes, the use of aged or ovariecto-
to adequately investigate and optimize the formulation, design,
mized females (especially rats) may be indicated to simulate
dimensions, and associated instrumentation envisaged for hu-
osteoporotic conditions.
man use, especially in late stages of development.
5.3.2 It is recommended that the chromosomal sex be the
5.1.6 Larger animals may be more appropriate for studying
same within the cohort, and that needs to be reported. The
repair in defects and locations that more closely approximate
investigator should be aware that variances can occur between
those in humans.
sexes and that appropriate statistical power needs to be
5.1.7 Larger defect dimensions generally require a method
instituted.
of fixation to secure the implant and thereby reduce implant
dislocation. The method of implant immobilization can nega-
5.4 Age:
tively impact both the surrounding host tissue and repair tissue.
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 Older animals have a greater propensity for osteopenia
minimum defect dimension that the animal is incapable of
and have a decreased capacity to repair bone defects. If specific
repairing without intervention. Th
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