ASTM F2721-09(2014)

Standard Guide for Pre-clinical <emph type="bdit">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 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.

General Information

• Status: Historical
• Publication Date: 31-Oct-2014
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.44 - Assessment for TEMPs

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F2721 − 09 (Reapproved 2014)
Standard Guide for
Pre-clinical 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 and health practices and determine the applica-
1.1 This guide covers general guidelines for the in vivo
bility of regulatory limitations prior to use.
assessment of tissue-engineered medical products (TEMPs)
intended to repair or regenerate bone. TEMPs included in this
2. Referenced Documents
guide may be composed of natural or synthetic biomaterials
2.1 ASTM Standards:
(biocompatible and biodegradable) or composites thereof, and
may contain cells or biologically active agents such as growth F561 Practice for Retrieval and Analysis of Medical
Devices, and Associated Tissues and Fluids
factors, synthetic peptides, plasmids, or cDNA. The models
described in this guide are segmental critical size defects F565 Practice for Care and Handling of Orthopedic Implants
and Instruments
which, by definition, will not fill with viable tissue without
treatment. Thus, these models represent a stringent test of a F895 Test Method for Agar Diffusion Cell Culture Screening
for Cytotoxicity
material’s ability to induce or augment bone growth.
F981 Practice for Assessment of Compatibility of Biomate-
1.2 Guidelines include a description and rationale of various
rials for Surgical Implants with Respect to Effect of
animal models including rat (murine), rabbit (leporine), dog
Materials on Muscle and Bone
(canine), goat (caprine), and sheep (ovine). Outcome measures
F1983 Practice for Assessment of Selected Tissue Effects of
based on radiographic, histologic, and mechanical analyses are
Absorbable Biomaterials for Implant Applications
described briefly and referenced. The user should refer to
F2150 Guide for Characterization and Testing of Biomate-
specific test methods for additional detail.
rial Scaffolds Used in Tissue-Engineered Medical Prod-
1.3 This guide is not intended to include the testing of raw
ucts
materials, preparation of biomaterials, sterilization, or packag-
2.2 Other Documents:
ing of the product. ASTM standards for these steps are
21 CFR Part 58 Good Laboratory Practice for Nonclinical
available in the Referenced Documents (Section 2).
Laboratory Studies
1.4 The use of any of the methods included in this guide
21 CFR 610.12 General Biological Products Standards—
may not produce a result that is consistent with clinical
Sterility
performance in one or more specific applications.
3. Terminology
1.5 Other pre-clinical methods may also be appropriate and
this guide is not meant to exclude such methods. The material
3.1 Definitions:
must be suitable for its intended purpose. Additional biological
3.1.1 bone regeneration—the formation of bone that has
testing in this regard would be required.
histologic, biochemical, and mechanical properties similar to
that of native bone.
1.6 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this 3.1.2 bone repair—the process of healing injured bone
standard. through cell proliferation and synthesis of new extracellular
matrix.
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 [e-mail]. 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 Nov. 1, 2014. Published March 2015. Originally Available from U.S. Government Printing Office Superintendent of Documents,
approved in 2008. Last previous version approved in 2009 as F2721 – 09. DOI: 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
10.1520/F2721-09R14. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2721 − 09 (2014)
3.1.3 compact bone—classification of ossified bony connec- 3.1.17 weight-bearing versus non-weight bearing models—
tive tissue characterized by the presence of osteon-containing weight bearing is the amount of weight a patient or experimen-
lamellar bone. Lamellar bone is highly organized in concentric tal animal puts on the leg on which surgery has been
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 6 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
3.1.7 endochondral ossification—one of the two main types
clinical repair or regeneration of bone.
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;
occurs mainly in the formation of flat bones (skull).
4.4 It is recommended that safety testing be in accordance
with the provisions of the FDA Good Laboratory Practices
3.1.8 growth plate—the anatomic location within the
Regulations 21 CFR 58.
epiphyseal region of long bones corresponding to the site of
growth of bone through endochondral ossification.
4.5 Safety and effectiveness studies to support regulatory
3.1.8.1 Discussion—The growth plate in skeletally mature
submissions (for example, Investigational Device Exemption
animals is fused.
(IDE)), Premarket Approval (PMA), 510K, Investigational
New Drug (IND), or Biologics License Application (BLA)
3.1.9 long bone—bone that is longer than it is wide, and
submissions in the U.S.) should conform to appropriate guide-
grows primarily by elongation of the diaphysis. The long bones
lines of the regulatory bodies for development of medical
include the femurs, tibias, and fibulas of the legs, the humeri,
devices, biologics, or drugs, respectively.
radii, and ulnas of the arms, the metacarpals and metatarsals of
the hands and feet, and the phalanges of the fingers and toes.
4.6 Animal model outcomes are not necessarily predictive
3.1.10 marrow—soft, gelatinous tissue that fills the cavities of human results and should, therefore, be interpreted cau-
of the bones. It is either red or yellow, depending upon the tiously with respect to potential applicability to human condi-
preponderance of hematopoietic (red) or fatty (yellow) tissue. tions.
3.1.10.1 Discussion—Red marrow is also called myeloid
tissue. 5. Animal Models
NOTE 1—This section provides a description of the options to consider
3.1.11 matrix—either the exogenous implanted scaffold or
in determining the appropriate animal model and bone defect size and
the endogenous extracelluar substance (otherwise known as
location.
extracellular matrix) derived from the host.
NOTE 2—Research using these models needs to be conducted in
accordance with governmental regulations and guidelines appropriate to
3.1.12 metaphyseal—pertaining to the dense end-section of
the locale for the care and use of laboratory animals. Study protocols
long bones.
should be developed after consultation with the institutional attending
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.
3.1.15 skeletal maturity—the age at which the epiphyseal
5.1.2 In principle, critical-size defects may be achieved in
plates are fused.
both metaphyseal and diaphyseal locations. For the purpose of
3.1.15.1 Discussion—In rodents, skeletally mature animals
this guide, only defects created in the diaphyseal section of
are characterized by defined gonads.
long bones will be described.
3.1.16 trabecular bone—ossified bony connective tissue 5.1.3 Significant variability exists between animal species
characterized by spicules surrounded by marrow space. with respect to the size and weight of the animal, anatomy, and
F2721 − 09 (2014)
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
5.2.2 Potential differences in outcome when using weight-
in modulating bone development and maintenance, as well as
bearing versus non-weight bearing models should be carefully
bone degeneration The impact of mechanical load extent or
considered.
duration on the implanted TEMP, and surrounding native bone,
5.3 Chromosomal Sex:
varies depending on the anatomic site. The defect site chosen
5.3.1 Due to the impact of circulating steroids on cartilage
to evaluate implants should, therefore, factor the impact of
and bone metabolism and regeneration, the choice of chromo-
mechanical load on the performance of the implant.
somal sex should be considered. Animals in lactation should
5.1.5 It is recommended that an appropriate species and
not be used. For some purposes, the use of aged or ovariecto-
anatomic site be chosen, that have dimensions sufficiently large
mized females (especially rats) may be indicated to simulate
to adequately investigate and optimize the formulation, design,
osteoporotic conditions.
dimensions, and associated instrumentation envisaged for hu-
5.3.2 It is recommended that the chromosomal sex be the
man use, especially in late stages of development.
same within the cohort, and that needs to be reported. The
5.1.6 Larger animals may be more appropriate for studying
investigator should be aware that variances can occur between
repair in defects and locations that more closely approximate
sexes and that appropriate statistical power needs to be
those in humans.
instituted.
5.1.7 Larger defect dimensions generally require a method
of fixation to secure the implant and thereby reduce implant
5.4 Age:
dislocation. The method of implant immobilization can nega-
5.4.1 Bone undergoes dynamic changes in metabolism and
tively impact both the surrounding host tissue and repair tissue.
remodeling during growth. Due to the impact of these physi-
Accordingly, the difference in the design of the test TEMP in
ologic processes on tissue repair, skeletally mature animals
models which generally do not require fixation should be
should be used. The cohorts should have fused epiphyseal
factored into the interpretation of results with respect to growth plates. Skeletal maturity varies between species and
predictability of outcomes in larger animal models and humans
can be determined radiographically if necessary.
requiring fixation.
5.4.2 Older animals have a greater propensity for osteopenia
5.1.8 For each species, a critical size defect is defined as the
and have a decreased capacity to repair bone defects. If specific
minimum defect dimension that the animal is incapable of
conditions are considered important for the intended TEMP
repairing without intervention. The dimensions of critical
assessment, then an appropriate model should be used.
defects generally differ for each species and should be consid-
5.4.3 The mesenchymal stem cell pool, growth factor
ered carefully when designing the implant dimensions and
responsiveness, and metabolic activity of cells generally de-
method of fixation. As an empirical rule, the length of the
creases with age. Thus, reparative processes that are dependent
defect (created by ostectomy) should at least be equal to 1.5
on the number and activity of native cells may be partially
times the diameter of the selected bone (1, 2). Some authors
compromised in older animals.
recommend at least 2 times the diameter of the selected bone
5.5 Diet or Concurrent Pathology:
(3).
5.5.1 In general, studies are perfor
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F2721 − 09 F2721 − 09 (Reapproved 2014)
Standard Guide for
Pre-clinical 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
1.1 This guide covers general guidelines for the in vivo assessment of tissue engineered 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.
2. Referenced Documents
2.1 ASTM Standards:
F561 Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
F565 Practice for Care and Handling of Orthopedic Implants and Instruments
F895 Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
F981 Practice for Assessment of Compatibility of Biomaterials for Surgical Implants with Respect to Effect of Materials on
Muscle and Bone
F1983 Practice for Assessment of Selected Tissue Effects of Absorbable Biomaterials for Implant Applications
F2150 Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
F2451 Guide forin vivo Assessment of Implantable Devices Intended to Repair or Regenerate Articular Cartilage
2.2 Other Documents:
ISO 10993 Biological Evaluation of Medical Devices—Part 5: Tests for in vitro Cytotoxicity
21 CFR Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.44
on Assessment for TEMPs.
Current edition approved June 1, 2009Nov. 1, 2014. Published June 2009March 2015. Originally approved in 2008. Last previous version approved in 20082009 as
F2721 – 08.F2721 – 09. DOI: 10.1520/F2721-09.10.1520/F2721-09R14.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2721 − 09 (2014)
21 CFR 610.12 General Biological Products Standards—Sterility
3. Terminology
3.1 Definitions:
3.1.1 bone regeneration—the formation of bone that has histologic, biochemical, and mechanical properties similar to that of
native bone.
3.1.2 bone repair—the process of healing injured bone through cell proliferation and synthesis of new extracellular matrix.
3.1.3 compact bone—classification of ossified bony connective tissue characterized by the presence of osteon-containing
lamellar bone. Lamellar bone is highly organized in concentric sheets.
3.1.4 cortical bone—one of the two main types of osseous tissue. Cortical bone is dense and forms the surface of bones.
3.1.5 critical size defect—a bone defect, either naturally occurring or artificially created, which will not heal without
intervention. In the clinical setting, this term applies to exceeding a healing period of approximately 6 months (in otherwise healthy
adults).
3.1.6 diaphyseal—pertaining to the mid-section of long bones.
3.1.7 endochondral ossification—one of the two main types of bone formation, where a cartilaginous matrix forms first and is
subsequently replaced by osseous tissue.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
3.1.7.1 Discussion—
Endochondral ossification is responsible for much of the bone growth in vertebrate skeletons, especially in long bones.
3.1.7.2 Discussion—
The other main mechanism for bone formation is intramembraneous ossification, where osseous tissue is formed directly, without
cartilaginous precursor; occurs mainly in the formation of flat bones (skull).
3.1.8 growth plate—the anatomic location within the epiphyseal region of long bones corresponding to the site of growth of
bone through endochondral ossification.
3.1.8.1 Discussion—
The growth plate in skeletally mature animals is fused.
3.1.9 long bone—bone that is longer than it is wide, and grows primarily by elongation of the diaphysis. The long bones include
the femurs, tibias, and fibulas of the legs, the humeri, radii, and ulnas of the arms, the metacarpals and metatarsals of the hands
and feet, and the phalanges of the fingers and toes.
3.1.10 marrow—soft, gelatinous tissue that fills the cavities of the bones. It is either red or yellow, depending upon the
preponderance of hematopoietic (red) or fatty (yellow) tissue.
3.1.10.1 Discussion—
Red marrow is also called myeloid tissue.
3.1.11 matrix—a term applied to either the exogenous implanted scaffold or the endogenous extracelluar substance (otherwise
known as extracellular matrix) derived from the host.
3.1.12 metaphyseal—pertaining to the dense end-section of long bones.
3.1.13 remodeling—a life long process where old bone is removed from the skeleton (bone resorption) and new bone is added
(bone formation).
3.1.14 residence time—the time at which an implanted material (synthetic or natural) can no longer be detected in the host
tissue.
3.1.15 skeletal maturity—the age at which the epiphyseal plates are fused.
3.1.15.1 Discussion—
F2721 − 09 (2014)
In rodents, skeletally mature animals are characterized by defined gonads.
3.1.16 trabecular bone—ossified bony connective tissue characterized by spicules surrounded by marrow space.
3.1.17 weight-bearing versus non-weight bearing models—weight bearing is the amount of weight a patient or experimental
animal puts on the leg on which surgery has been performed, generally described as a percentage of the body weight.
3.1.17.1 Discussion—
Non weight Non-weight bearing means the leg must not touch the floor (i.e., supports 0 % of the body weight).
3.1.17.2 Discussion—
Full weight bearing means the leg can carry 100 % of the body weight on a step.
4. 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.
5. Animal Models
NOTE 1—This section provides a description of the options to consider in determining the appropriate animal model and bone defect size and location.
NOTE 2—Research using these models needs to be conducted in accordance with governmental regulations and guidelines appropriate to the locale for
the care and use of laboratory animals. Study protocols should be developed after consultation with the institutional attending veterinarian, and need
appropriate review and approval by the institutional animal care and use committee prior to study initiation.
5.1 Defect Size:
5.1.1 A high proportion of fracture injuries in humans occur in long bones. Accordingly, defects created in long bones are
commonly used for assessing bone repair/regeneration in animal models.
5.1.2 In principle, critical-size defects may be achieved in both metaphyseal and diaphyseal locations. For the purpose of this
guide, only defects created in the diaphyseal section of long bones will be described.
5.1.3 Significant variability exists between animal species with respect to the size and weight of the animal, anatomy, and gait
thereby influencing kinetics, range of motion, and mechanical forces on defects. These factors influence bone architecture and
structure. These factors play a significant role in the response to injury or disease of bone. The user should consider carefully the
animal model that is appropriate for the stage of investigation of an implanted TEMP.
5.1.4 Mechanical load has been shown to affect bone repair. Amongst the mechanobiological factors, intermittent hydrostatic
pressure and load-bearing stresses play an important role in modulating bone development and maintenance, as well as bone
degeneration The impact of mechanical load extent or duration on the implanted TEMP, and surrounding native bone, varies
depending on the anatomic site. The defect site chosen to evaluate implants should, therefore, factor the impact of mechanical load
on the performance of the implant.
5.1.5 It is recommended that an appropriate species and anatomic site be chosen, that have dimensions sufficiently large to
adequately investigate and optimize the formulation, design, dimensions, and associated instrumentation envisaged for human use,
especially in late stages of development.
5.1.6 Larger animals may be more appropriate for studying repair in defects and locations that more closely approximate those
in humans.
5.1.7 Larger defect dimensions generally require a method of fixation to secure the implant and thereby reduce implant
dislocation. The method of implant immobilization can negatively impact both the surrounding host tissue and repair tissue.
F2721 − 09 (2014)
Accordingly, the difference in the design of the test TEMP in models which generally do not require fixation should be factored
into the interpretation of results with respect to predictability of outcomes in larger animal models and humans requiring fixation.
5.1.8 For each species, a critical size defect is defined as the minimum defect dimension that the animal is incapable of repairing
without intervention. The dimensions of critical defects generally differ for each species and should be considered carefully when
designing the implant dimensions and method of fixation. As an empirical rule, the length of the defect (created by ostectomy)
should at least be equal to 1.5 times the diameter of the selected bone (1, 2). Some authors recommend at least 2 times the diameter
of the selected bone (3).
5.1.9 Whether or not the periosteum from the resected segment of bone is still present can influence healing within the bone
defect. The periosteum is typically removed in most studies of segmental critical-size defects. Whether or not the periosteum has
been removed should be stated when reporting results.
5.1.10 Each study should include an empty-defect control group to confirm that the model is a critical-size defect. If/once the
model is very well characterized, the use of historical data instead of actual control animals should be considered, in order to save
on animal numbers, unless this would compromise the objectives of the study. For example, in pivotal preclinical proof-of-concept
studies, concurrent controls are likely to be appropriate.
5.1.11 The use of unilateral defect models is generally recommended. This is especially true for weight-bearing locations in
animals that use all four limbs for weight bearing (especially goats, sheep, and horses).
5.2 Handling:
5.2.1 Exposure of implants to extreme and highly variable mechanical forces as a result of jumping and running can lead to
increased variability in outcome measures.
5.2.2 Potential differences in outcome when using weight-bearing versus non-weight bearing models should be carefully
considered.
5.3 Chromosomal Sex:
5.3.1 D
...