ASTM F2884-21

Standard Guide for Pre-clinical <emph type="bdit">in vivo</emph> Evaluation of Spinal Fusion

SIGNIFICANCE AND USE
4.1 This guide is aimed at providing a range of in vivo models to aid in pre-clinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone in the spine.  
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 utilize 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.  
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 pre-clinical in vivo assessment of tissue-engineered medical products (TEMPs) intended to repair or regenerate bone in an interbody and/or posterolateral spinal environment. TEMPs included in this guide may be composed of, but are not limited to, natural or synthetic biomaterials 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 document represent a stringent test of a material’s ability to induce and/or augment bone growth in the spinal environment.  
1.2 While clinically TEMPs may be combined with hardware for initial stabilization or other purposes, the focus of this guide is on the appropriateness of the animal model chosen and evaluation of the TEMP-induced repair and as such does not focus on issues of components or constructs.  
1.3 Guidelines include a description and rationale of various animal models for the in vivo assessment of the TEMP. The animal models utilize a range of species including rat (murine), rabbit (lapine), dog (canine), goat (caprine), pig (porcine), sheep (ovine), and non-human primate (primates). Outcome measures include in vivo assessments based on radiographic, histologic, and CT imaging as well as subsequent in vitro assessments of the repair, including histologic analyses and mechanical testing. All methods are described briefly and referenced. The user should refer to specific test methods for additional detail.  
1.4 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 Referenced Documents (Section 2).  
1.5 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.  
1.6 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.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard.  
1.9 This international standard was developed in accordance with internationally recognized prin...

General Information

• Status: Published
• Publication Date: 14-Dec-2021
• ICS: 11.100.99 - Other standards related to laboratory medicine
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.44 - Assessment for TEMPs

Relations

• Refers: ASTM F2150-19 - Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Regenerative Medicine and Tissue-Engineered Medical Products
• Effective Date: 01-Oct-2019
• Refers: ASTM F561-19 - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Jan-2019
• Refers: ASTM F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Refers: ASTM F2150-13 - Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
• Effective Date: 01-Oct-2013
• Refers: ASTM F565-04(2013) - Standard Practice for Care and Handling of Orthopedic Implants and Instruments
• Effective Date: 01-Oct-2013
• Refers: ASTM F561-13 - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Sep-2013
• Refers: ASTM F895-11 - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Oct-2011
• Refers: ASTM F561-05a(2010) - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Sep-2010
• Refers: ASTM F981-04(2010) - Standard Practice for Assessment of Compatibility of Biomaterials for Surgical Implants with Respect to Effect of Materials on Muscle and Bone
• Effective Date: 01-Jun-2010
• Refers: ASTM F565-04(2009) - Standard Practice for Care and Handling of Orthopedic Implants and Instruments
• Effective Date: 01-Apr-2009
• Refers: ASTM F565-04(2009)e1 - Standard Practice for Care and Handling of Orthopedic Implants and Instruments
• Effective Date: 01-Apr-2009
• Refers: ASTM F1983-99(2008) - Standard Practice for Assessment of Compatibility of Absorbable/Resorbable Biomaterials for Implant Applications
• Effective Date: 01-Aug-2008
• Refers: ASTM F2150-07 - Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
• Effective Date: 01-Dec-2007
• Refers: ASTM F895-84(2006) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Mar-2006
• Refers: ASTM F561-05a - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Oct-2005

Overview

ASTM F2884-21 is the Standard Guide for Pre-clinical in vivo Evaluation of Spinal Fusion, developed by ASTM International. This standard provides comprehensive guidelines for the pre-clinical assessment of tissue-engineered medical products (TEMPs) intended to repair or regenerate bone in the spine. The guide outlines the selection and use of animal models, surgical methods, tissue handling, and various analysis techniques essential for evaluating the efficacy and safety of TEMPs prior to clinical use.

Adopting ASTM F2884-21 supports robust research and development practices, ensuring that new spinal fusion medical products undergo thorough pre-clinical testing to meet global regulatory standards.

Key Topics

• Animal Models: Covers the selection and use of appropriate animal species such as rats, rabbits, dogs, goats, pigs, sheep, and non-human primates to evaluate spinal bone growth and regeneration.
• Surgical Considerations: Includes guidance for performing surgical procedures in both interbody and posterolateral spinal environments, with attention to defect creation, fixation options, and minimizing animal distress.
• Specimen Processing: Provides protocols for collecting, handling, and processing spinal tissue samples, detailing both qualitative and quantitative assessments through radiographic imaging, histology, and mechanical testing.
• Safety and Biocompatibility: Recommends compliance with relevant ASTM and ISO standards for cytotoxicity and biocompatibility testing of materials. Encourages conformity with FDA Good Laboratory Practices and international regulatory requirements for investigational submissions (e.g., IDE, PMA, 510K).
• Interpretation of Results: Advises on the limitations of animal model outcomes, emphasizing careful interpretation before extrapolating results to human clinical scenarios.

Applications

Implementing ASTM F2884-21 is essential for:

• Medical Device Developers: Provides guidance for pre-clinical validation of spinal implants, bone graft substitutes, and biologically active scaffolds, ensuring suitability for regulatory submission.
• Tissue Engineering Research: Offers structured methodologies for pre-clinical evaluation of new materials and products for bone repair and regeneration in the spine.
• Regulatory Compliance: Supports preparation of robust data packages for regulatory authorities in applications such as Investigational Device Exemptions (IDE), Premarket Approvals (PMA), 510K submissions, and Biologics License Applications (BLA).
• Academic and Contract Research: Assists laboratories in designing scientifically valid and reproducible animal studies that conform to international best practices.
• Quality Assurance: Promotes consistent safety testing and assessment, helping manufacturers meet both U.S. FDA and international standards.

Related Standards

Organizations working in spinal fusion research and development should also reference:

• ASTM F561 - Practice for Retrieval and Analysis of Medical Devices and Associated Tissues and Fluids
• ASTM F565 - Practice for Care and Handling of Orthopedic Implants and Instruments
• ASTM F895 - Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• ASTM F981 - Practice for Assessment of Compatibility of Biomaterials for Surgical Implants
• ASTM F1983 - Practice for Assessment of Selected Tissue Effects of Absorbable Biomaterials
• ASTM F2150 - Guide for Characterization and Testing of Biomaterial Scaffolds
• ISO 10993-5 - Biological Evaluation of Medical Devices: Tests for In Vitro Cytotoxicity
• 21 CFR Part 58 - Good Laboratory Practice for Nonclinical Laboratory Studies

By utilizing ASTM F2884-21 in conjunction with these related standards, organizations can enhance the reliability, reproducibility, and regulatory compliance of their pre-clinical spinal fusion evaluations. This promotes the safe and effective development of innovative tissue-engineered products and medical devices for spinal surgery.

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: F2884 − 21
Standard Guide for
Pre-clinical in vivo Evaluation of Spinal Fusion
This standard is issued under the fixed designation F2884; 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 1.6 Other pre-clinical methods may also be appropriate, and
this guide is not meant to exclude such methods. The material
1.1 This guide covers general guidelines for the pre-clinical
must be suitable for its intended purpose.Additional biological
in vivo assessment of tissue-engineered medical products
testing in this regard would be required.
(TEMPs) intended to repair or regenerate bone in an interbody
1.7 The values stated in SI units are to be regarded as
and/or posterolateral spinal environment. TEMPs included in
standard. No other units of measurement are included in this
this guide may be composed of, but are not limited to, natural
or synthetic biomaterials or composites thereof, and may standard.
contain cells or biologically active agents such as growth
1.8 The values stated in inch-pound units are to be regarded
factors, synthetic peptides, plasmids, or cDNA. The models
as standard. The values given in parentheses are mathematical
described in this document represent a stringent test of a
conversions to SI units that are provided for information only
material’s ability to induce and/or augment bone growth in the
and are not considered standard.
spinal environment.
1.9 This international standard was developed in accor-
1.2 While clinically TEMPs may be combined with hard-
dance with internationally recognized principles on standard-
ware for initial stabilization or other purposes, the focus of this
ization established in the Decision on Principles for the
guideisontheappropriatenessoftheanimalmodelchosenand
Development of International Standards, Guides and Recom-
evaluation of the TEMP-induced repair and as such does not
mendations issued by the World Trade Organization Technical
focus on issues of components or constructs.
Barriers to Trade (TBT) Committee.
1.3 Guidelinesincludeadescriptionandrationaleofvarious
2. Referenced Documents
animal models for the in vivo assessment of the TEMP. The
2.1 ASTM Standards:
animalmodelsutilizearangeofspeciesincludingrat(murine),
F561 Practice for Retrieval and Analysis of Medical
rabbit (lapine), dog (canine), goat (caprine), pig (porcine),
Devices, and Associated Tissues and Fluids
sheep (ovine), and non-human primate (primates). Outcome
F565 PracticeforCareandHandlingofOrthopedicImplants
measures include in vivo assessments based on radiographic,
and Instruments
histologic, and CT imaging as well as subsequent in vitro
F895 TestMethodforAgarDiffusionCellCultureScreening
assessments of the repair, including histologic analyses and
for Cytotoxicity
mechanical testing. All methods are described briefly and
F981 Practice for Assessment of Compatibility of Biomate-
referenced. The user should refer to specific test methods for
rials for Surgical Implants with Respect to Effect of
additional detail.
Materials on Muscle and Insertion into Bone
1.4 This guide is not intended to include the testing of raw
F1983 Practice forAssessment of Selected Tissue Effects of
materials, preparation of biomaterials, sterilization, or packag-
Absorbable Biomaterials for Implant Applications
ing of the product. ASTM standards for these steps are
F2150 Guide for Characterization and Testing of Biomate-
available in Referenced Documents (Section 2).
rial Scaffolds Used in Regenerative Medicine and Tissue-
1.5 The use of any of the methods included in this guide Engineered Medical Products
may not produce a result that is consistent with clinical 2.2 Other Standards:
performance in one or more specific applications. ISO 10993 Biological Evaluation of Medical TEMPs—Part
5: Tests for in vitro Cytotoxicity
1 2
This guide is under the jurisdiction of ASTM Committee F04 on Medical and For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
F04.44 on Assessment for TEMPs. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Dec. 15, 2021. Published January 2022. Originally the ASTM website.
approvedin2012.Lastpreviouseditionapprovedin2012asF2884 – 12,whichwas Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
withdrawn July 2021 and reinstated in December 2021. DOI: 10.1520/F2884-21. 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
F2884 − 21
21 CFR Part 58 Good Laboratory Practice for Nonclinical 3.1.9 interbody spine fusion—a method of obtaining spinal
Laboratory Studies fusion that involves placing bone graft between adjacent
21 CFR 610.12 General Biological Product Standards— vertebrae in the area usually occupied by the intervertebral
Sterility disc.
3.1.10 marrow—soft, gelatinous tissue that fills the cavities
3. Terminology
of the bones. It is either red or yellow, depending upon the
3.1 Definitions:
preponderance of hematopoietic (red) or fatty (yellow) tissue.
3.1.1 bone regeneration—the formation of bone that has
3.1.10.1 Discussion—Red marrow is also called myeloid
histologic, biochemical, and mechanical properties similar to
tissue.
that of native bone.
3.1.11 matrix—a term applied to either the exogenous im-
3.1.2 bone remodeling—a lifelong process where old bone
planted scaffold or the endogenous extracelluar substance
is removed from the skeleton (a sub-process called bone
(otherwise known as extracellular matrix) derived from the
resorption) and new bone is added (a sub-process called bone
host.
formation).
3.1.12 posterolateral spine fusion—a method of obtaining
3.1.2.1 Discussion—These processes also control the re-
spinal fusion that involves placing bone graft in the “gutter” in
shaping or replacement of bone during growth and following
the posterolateral portion of the spine between the transverse
injuries. Remodeling responds to functional demands and
process and the spinous process.
muscle attachments. As a result, bone is added where needed
3.1.12.1 Discussion—Posterolateral spine fusion is also
and removed where it is not required.
known as posterolateral gutter spine fusion.
3.1.3 bone repair—process of healing injured bone through
3.1.13 remodeling—a lifelong process where old bone is
cell proliferation and synthesis of new extracellular matrix.
removed from the skeleton (bone resorption) and new bone is
3.1.4 cancellous bone—(also known as trabecular, or
added (bone formation).
spongy, bone), a type of osseous tissue with a low apparent
3.1.14 residence time—time at which an implanted material
density and strength but very high surface area, that fills the
(synthetic or natural) can no longer be detected in the host
inner cavity of long bones.
tissue.
3.1.4.1 Discussion—The orientation of the trabecular bone
is such that the trabecular “struts” tend to follow the lines of 3.1.15 skeletal maturity—the age at which the epiphyseal
stress to which the bones are normally subjected. The external plates are fused.
layer of cancellous bone contains red bone marrow where the
3.1.15.1 Discussion—In rodents, skeletally mature animals
production of blood cellular components (known as he- are characterized by defined gonads.
matopoiesis) takes place. Cancellous bone is also where most
3.1.16 spinal fusion—also known as spondylosyndesis, is a
of the arteries and veins of bone organs are found.
surgical technique used to combine two or more vertebrae.
3.1.5 compact bone—classification of ossified bony connec-
3.1.16.1 Discussion—Supplementary bone tissue (either au-
tive tissue characterized by the presence of osteon-containing
tograftorallograft)isoftenusedinconjunctionwiththebody’s
lamellar bone; lamellar bone is highly organized in concentric
naturalosteoblasticprocesses.Thisprocedureisusedprimarily
sheets.
to eliminate the pain caused by abnormal motion of the
vertebrae by immobilizing the vertebrae themselves. Spinal
3.1.6 cortical bone—one of the two main types of osseous
fusion is done most commonly in the lumbar region of the
tissue; cortical bone is dense and forms the surface of bones.
spine,butitisalsousedtotreatcervicalandthoracicproblems.
3.1.7 endochondral ossification—one of the two main types
3.1.17 trabecular bone—bony connective tissue character-
of bone formation, where a cartilaginous matrix forms first and
ized by spicules surrounded by marrow space.
is subsequently replaced by osseous tissue.
3.1.7.1 Discussion—Endochondral ossification is respon-
3.1.18 vertebra (plural: vertebrae)—the vertebral column is
sible for much of the bone growth in vertebrate skeletons,
the individual irregular bones that make up the spinal column
especially in long bones.
(also known as ischis)—a flexuous and flexible column.
3.1.7.2 Discussion—The other main mechanism for bone
3.1.18.1 Discussion—There are normally thirty-three (33)
formation is intramembraneous ossification, where osseous
vertebrae in humans, including the five that are fused to form
tissue is formed directly, without cartilaginous precursor; it
the sacrum (the others are separated by intervertebral discs)
occurs mainly in the formation of flat bones (skull).
and the four coccygeal bones which form the tailbone. The
upperthreeregionscomprisetheremaining24andaregrouped
3.1.8 growth plate—the anatomic location within the
epiphyseal region of long bones corresponding to the site of under the names cervical (seven vertebrae), thoracic (twelve
vertebrae), and lumbar (five vertebrae), according to the
growth through endochondral bone formation.
3.1.8.1 Discussion—The growth plate in skeletally mature regionstheyoccupy.Thisnumberissometimesincreasedbyan
additional vertebra in one region, or it may be diminished in
animals is fused.
oneregion,thedeficiencyoftenbeingsuppliedbyanadditional
vertebra in another. The number of cervical vertebrae is,
AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
however, very rarely increased or diminished. Each vertebra is
732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov. composed of a body anteriorly and a neural arch posteriorly.
F2884 − 21
The arch encloses an opening, the vertebral foramen, which 4.6 Animal model outcomes are not necessarily predictive
helps to form a canal in which the spinal cord is housed. of human results and should, therefore, be interpreted cau-
tiously with respect to potential applicability to human condi-
Protruding from the posterior extreme of each neural arch is a
tions.
spinous process and extending from the lateral edges of each
arch are transverse processes. These bony elements serve as
5. Animal Models
important sites of attachment of deep back muscles.The neural
NOTE 1—This section provides a description of the options to consider
arch of each vertebrae is divided into component parts by these
in determining the appropriate animal model and fusion location.
processes.Thepartsoftheneuralarchbetweenthespinousand
NOTE 2—Research using these models needs to be conducted in
transverse processes are known as the laminae and the parts of accordance with governmental regulations appropriate to the locale and
guidelines for the care and use of laboratory animals. Study protocols
the arch between the transverse processes and the body are the
should be developed after consultation with the institutional attending
pedicles. At the point where the laminae and pedicles meet,
veterinarian,andneedappropriatereviewandapprovalbytheinstitutional
each vertebra contains two superior articular facets and two
animal care and use committee prior to study initiation.
inferior articular facets. The former pair of facets form
5.1 Defect Considerations:
articulations, which are synovial joints, with the two inferior
5.1.1 Spinal fusion is typically performed on a patient who
articular facets of the vertebra immediately above (or the skull,
has sustained trauma in order to stabilize the spine, to relieve
in the case of the first cervical vertebra). The pedicle of each
aneuraldeficitrelatedtobonystenosis,ortotreatdegenerative
vertebra is notched at its superior and inferior edges. Together
disc disease. A high proportion of injuries in humans occur in
the notches from two contiguous vertebra form an opening, the
the spine. Accordingly, defects created in the spine are com-
intervertebral foramen, through which spinal nerves pass.
monly used for assessing spinal bone repair/regeneration in
animal models.
3.1.19 vertebral body—the largest part of a vertebra and is
5.1.2 Defects may be created surgically in both the inter-
approximately cylindrical in shape.
bodyandposterolateralspinallocations.Forthepurposeofthis
3.1.19.1 Discussion—Its upper and lower surfaces are flat-
guide, defects created in both spinal regions will be described.
tened and rough, and give attachment to the intervertebral
5.1.3 Significant variability exists between animal species
fibrocartilages, and each presents a rim around its circumfer-
with respect to the size and weight of the animal, anatomy, and
ence.Infront,thebodyisconvexfromsidetosideandconcave
gait thereby influencing kinetics, range of motion, and me-
from above downward. Behind, it is flat from above downward
chanical forces on defects. These factors influence bone
and slightly concave from side to side. Its anterior surface
architecture and structure. These factors play a significant role
presents a few small apertures, for the passage of nutrient
in the response to injury or disease of bone. The user should
vessels. On the posterior surface is a single large, irregular
consider carefully the animal model that is appropriate for the
aperture, or occasionally more than one, for the exit of the
stage of investigation of an implanted TEMPs. Table X1.1 is
basi-vertebral veins from the body of the vertebrae.
provided to give guidance for the selection of animal models
and the relevancy of their results.
4. Significance and Use
5.1.4 Mechanical load has been shown to affect bone repair.
The intermittent hydrostatic pressure and load-bearing stresses
4.1 This guide is aimed at providing a range of in vivo
play an important role in modulating bone development and
models to aid in pre-clinical research and development of
maintenance as well as bone degeneration. The impact of the
tissue-engineered medical products (TEMPs) intended for the
amount and duration of the mechanical load on the implanted
clinical repair or regeneration of bone in the spine.
TEMPs, and surrounding native bone, varies depending on the
4.2 This guide includes a description of the animal models, anatomic site.
5.1.5 It is recommended that an appropriate species and
surgical considerations, and tissue processing as well as the
anatomic site having dimensions sufficiently large to ad-
qualitative and quantitative analysis of tissue specimens.
equately investigate and optimize the formulation, design,
4.3 The user is encouraged to utilize appropriateASTM and
dimensions, and associated instrumentation envisaged for hu-
other guidelines to conduct cytotoxicity and biocompatibility
man use be chosen, especially in late stages of development.
tests on materials, TEMPs, or both, prior to assessment of the
5.1.6 Spinal interbody surgical procedures generally require
in vivo models described herein.
a method of stabilization, typically some sort of load-bearing
interbodyimplant.Largeranimalsmaybemoreappropriatefor
4.4 It is recommended that safety testing be in accordance
studying repair in the interbody location due to size constraints
with the provisions of the FDA Good Laboratory Practices
associated with applying spinal interbody fusion devices used
Regulations 21 CFR 58.
to provide load support, as well as sizing of appropriate
4.5 Safety and effectiveness studies to support regulatory
stabilization components or constructs such as spinal rods,
submissions (for example, Investigational Device Exemption
plates, and/or screws.
(IDE),PremarketApproval(PMA),510K,InvestigationalNew
5.1.7 The use of pedicle screw and rod constructs varies in
Drug (IND), or Biologics License Application (BLA) submis-
the literature and is dependent upon several factors, including
sions in the U.S.) should conform to appropriate guidelines of the amount of instability created by the surgery as well as how
the regulatory bodies for development of medical devices,
closely researchers may wish to mimic the human clinical
biologics, or drugs. scenario. Accordingly, the difference in the design of the test
F2884 − 21
TEMP in models which generally do not require fixation mans must be considered. In some instances of new intended
shouldbefactoredintotheinterpretationofresultswithrespect use and/or new materials, human clinical data may still be
necessary.
to predictability of outcomes in larger animal models and
humans.
5.2 Handling:
5.1.8 In regard to instrumentation, both interbody fusion
5.2.1 Exposure of implants to extreme and highly variable
devices and pedicle screws, there are pros and cons. Pros
mechanical forces as a result of jumping and running can lead
include the fact that the surgical intervention more closely
to increased variability in outcome measures.
mimics that of human clinical surgeries. Cons include in-
5.2.2 Potential differences in outcome when using instru-
creased study cost, animal intervention, and surgical time. The mented versus non-instrumented models should be carefully
use of instrumentation must be balanced against the desired
considered.
outcomes of the study and the frequency of healing in the
5.3 Chromosomal Sex:
particular animal model compared to the human.
5.3.1 Due to the impact of circulating steroids on cartilage
5.1.9 Each study should include a control group containing
and bone metabolism and regeneration, the choice of chromo-
an acceptable standard of care, usually autograft for positive
somal sex should be considered. Animals in lactation should
controls or shams for negative controls, to confirm that the
not be used. For some purposes, the use of aged or ovariecto-
modelresultsdemonstrateconsistencywithacceptedvaluesfor
mized females (especially rats) may be indicated to simulate
healing. Allograft may also be an option for use in animal
osteoporotic conditions (1-24).
models where donor material is from animals of genetically 5.3.2 It is recommended that the chromosomal sex be the
identical strains, for example, athymic (rnu/rnu) rats. In cases
same within the cohort, and be reported. The investigator
where the product being tested consists of a combination of should be aware that variances can occur between sexes, and
agents (for example, cells and a matrix), each separate com- that appropriate statistical power needs to be instituted.
ponent of the combination product should be tested individu-
5.4 Age:
ally as controls, where possible or appropriate. If/once the
5.4.1 Bone undergoes dynamic changes in metabolism and
model is very well characterized and considered “validated,”
remodeling during growth. Due to the impact of these physi-
the use of historical data (from published literature or lab
ologic processes on tissue repair, skeletally mature animals
studies using an identical “validated” model) instead of actual
should be used. The cohorts should have fused epiphyseal
control animals should be considered, in order to save on
growth plates. Skeletal maturity varies between species and
animal numbers, unless this would compromise the objectives
can be determined radiographically if necessary.
of the study. For example, in pivotal pre-clinical proof-of-
5.4.2 Older animals have a greater propensity for osteope-
concept studies, concurrent controls are likely to be appropri-
nia and have a decreased capacity to repair bone defects. If
ate.
specific conditions are considered important for the intended
5.1.10 For screening materials, small animals (rats, rabbits) TEMP assessment, then an appropriate model should be used.
are best due to relative cost and a sizeable amount of literature 5.4.3 The mesenchymal stem cell pool, growth factor
responsiveness, and metabolic activity of cells generally de-
to support their use in posterolateral spine material evaluations
crease with age. Thus, reparative processes that are dependent
for bone fusion.
on the number and activity of native cells may be partially
5.1.11 Largeranimalsmaybemoreappropriateforstudying
compromised in older animals.
repair in the interbody location due to size constraints associ-
ated with applying interbody spinal fusion devices used to
5.5 Diet or Concurrent Pathology—In general, studies are
provide load support, as well as sizing of appropriate stabili- performed with healthy animals under normal diet conditions.
zation components or constructs such as spinal rods, plates,
However, the addition of fluoride, as well as deprivation of
and/or screws. Vitamin D and/or calcium to mimic specific bone disease
states, has been reported (13, 21, 25, 26). In situations where
5.1.12 In TEMPs which use components that depend on a
treatment of patients with systemic conditions that may affect
particular dose range in order to function appropriately, the
bone repair are contemplated, non-clinical models that mimic
dose ranges should be appropriate for the animal model used.
the disease or condition under consideration may be appropri-
In general, larger animals require doses of material scaled
ate.
appropriately. Non-human primates are likely the best choice
when targeting doses which may potentially approach the
5.6 Study Duration:
ranges of human clinical dose ranges.
5.6.1 The length of the study depends on the stage ofTEMP
5.1.13 Regardless, all animal models contain inherent limi- development, the species used, the size of the defect, and
tations and these limitations should be noted where possible. composition and design of the implant.
5.6.2 Short-terminsmallanimals(rats,rabbits)canbetaken
Drawbacks may include factors such as more rapid bone
healing than observed in humans, relatively small amounts of to mean less than twelve weeks in life, long-term is twelve to
24 weeks or greater. In large animals (dogs, pigs, sheep, goats,
material that can be implanted, and these models do not reflect
the range of pathology (age, osteoporosis, soft tissue injury) or
deleterious systemic agents (steroids, malnutrition, smoking)
that may be present in humans. Also, differences in loading 5
The boldface numbers in parentheses refer to a list of references at the end of
environments between quadripedal animals and bipedal hu- this standard.
F2884 − 21
non-human primates) short-term can be considered to mean 5.9.2 Adult rabbits with closed growth plates are preferred
less than six months in life, and long-term six months or (more than approximately 20 weeks old).
greater.
5.9.3 Surgical defects are typically performed at the L4-L5
5.6.3 In small animals, small defects implanted for five to or L5-L6 lumbar levels.
twelve weeks provide information regarding residence time of
5.9.4 For more details, see Appendix X2, Table X2.2.
implant and fixation of the TEMPas well as the type of repair.
5.10 Dog Posterolateral Spine Model:
5.6.4 Using larger animals, study periods of eight to twelve
5.10.1 Caninessuchasmedium-sizemongrels(forexample,
weeks are limited to providing information regarding the
mean 10 to 20 kg) and hounds have been utilized in postero-
biocompatibility, early cellular responsiveness, and the persis-
lateral spinal models (144-153).
tence and condition of the implant within the defect.
5.10.2 Surgical defects are typically performed at one or
5.6.5 Periods of more than three months for mid-size to
more of the L2-L3, L3-L4, L4-L5, or L5-L6 lumbar levels.
largeranimalsaregenerallynecessarytogainconfidenceinthe
5.10.3 An average of approximately 2 to 3 g (145, 149) or
extent of success in the repair or regeneration of bone based on
15 cc (150) of the desired graft material is placed at the
histologic and biochemical outcome measures.
operative site bilaterally.
5.6.6 Depending on the study objective, it might be advis-
5.10.4 For more details, see Appendix X2, Table X2.3.
able to evaluate one or more cohorts in the study before full
healing occurs. This may be of interest when comparing a new
5.11 Dog Interbody Spine Model:
material with a standard material like autograft, where the
5.11.1 Caninessuchasmedium-sizemongrels(forexample,
difference between treatment groups may reach a transient
mean 10 to 15 kg) and hounds have been utilized in interbody
maximum and then diminish over time. In general, it is
spinal models, mostly in the location of the cervical spine
necessary to match the claim and study end, taking into
(154-165).
consideration the statistical power.
5.11.2 Surgical defects are typically performed at one or
more of the C3-C4 and C5-C6 cervical levels.
5.7 Number of Animals—A statistically significant number
5.11.3 The discs of the chosen levels are excised leaving the
of animals per group is recommended to be used, if possible.
posterior longitudinal ligaments intact.
The required number depends on the intrinsic variability
5.11.4 Opposing vertebral cartilaginous endplates are
among the animals being used, the consistency of the surgical
procedure which will be performed, the accuracy of the scraped clean with a curette and a high-speed burr.
evaluation methods, anticipated attrition rate of animals during
5.11.5 Care should be taken to produce a flat surface for
the study, and the statistical techniques which will be used to implant insertion and seating (assuming an impacted-type
analyze the data (27). Another important factor may be the
implant).
objective of the study (for example, general feasibility/efficacy
5.11.6 The interbody fusion device is packed with the
compared to an empty defect, or comparability of different
desired TEMP.
constructs) and the variability of the treatment (for example,
5.11.7 Usingfingerpressureorgentleimpaction,thedesired
load of cells/factors, implant dimensions). The group size can
interbody fusion device is inserted.
bedeterminedfromexistingdataiftherespectivemodeliswell
5.11.8 The interbody fusion device is placed such that it is
established (literature or results from preliminary studies). For
in contact with the anterior cortices.
a pilot study, a group size of six to eight is likely appropriate
5.11.9 For more details, see Appendix X2, Table X2.4.
for histologic and mechanical testing as evaluation methods
5.12 Sheep Posterolateral Spine Model:
(27). For group sizes reported in the literature, see the
5.12.1 Sheep are commonly used for posterolateral spinal
appendix.
fusion studies in large species animals (166-182).
5.8 Rat Posterolateral Spine Model:
5.12.2 Surgical defects are typically performed at one or
5.8.1 Rats are amongst the most commonly used species for
more of the L2-L3, L3-L4, L4-L5, or L5-L6 lumbar levels.
early-phase development, due to relatively low cost, housing
5.12.3 Ten (10) cc of autogenous cancellous bone may be
space, and ease of maintenance (28-44). Often, Sprague-
harvested, if used as a control, per side.
Dawley or athymic rats are used to assess results because the
5.12.4 The transverse processes of the operative levels are
fusions involve human-derived materials (such as demineral-
decorticated bilaterally.
ized bone products). In cases where autograft or synthetic
5.12.5 Treatment or control materials are placed along the
biomaterials are used, normothymic Sprague-Dawley rats may
“gutters” between the transverse processes.
be used (32).
5.12.6 Optionally, transpedicular screw fixation using
5.8.2 Surgical defects are typically performed at the L4-L5
screws and rods may be used for fixation.
lumbar level.
5.12.7 For more details, see Appendix X2, Table X2.5.
5.8.3 For more details, see Appendix X2, Table X2.1.
5.13 Sheep Interbody Spine Model:
5.9 Rabbit Posterolateral Spine Model:
5.13.1 Sheeparecommonlyusedforinterbodyspinalfusion
5.9.1 Rabbitsarethemostcommonlyusedanimalmodelfor
studies in large species animals (166, 176, 181, 183-213).
spinal posterolateral fusion (39, 45-142) assessment due to a
variety of factors (cost, model validation work, and so on) and 5.13.2 Surgical defects are typically performed at one or
nonunions spontaneously occur at a similar rate as in human more of the L2-L3 or L4-L5 lumbar levels or the C2-C3,
(55, 143). C3-C4, C4-C5, or C5-C6 cervical levels.
F2884 − 21
5.13.3 An interbody fusion device is filled with an appro- 5.18.3 Approximately4gof autogenous cancellous bone
priate bone graft material and implanted at each disc space. may be harvested, if used as a control, per side.
5.13.4 Optionally, the lumbar fusion sites may be stabilized 5.18.4 The transverse processes of the operative levels are
with unilaterally placed pedicle screws and a connecting rod. decorticated bilaterally.
5.13.5 For more details, see Appendix X2, Table X2.6. 5.18.5 Treatment or control materials are placed along the
“gutters” between the transverse processes.
5.14 Goat Posterolateral Spine Model—Goats have not
5.18.6 For more details, see Appendix X2, Table X2.10.
typically been used for posterolateral spinal fusion studies in
large species animals. They have been used to evaluate a 5.19 Non-Human Primate Interbody Model:
variety of bone graft materials using cassettes containing
5.19.1 Non-human primates have been utilized in interbody
multiple materials for evaluation at a single transverse process spinal models (287-294).
site or for studying posterior construct mechanics (214-218).
5.19.2 Surgical defects are typically performed at one or
more of the L2-L3, L3-L4, L5-L6, or L7-S1 lumbar levels.
5.15 Goat Interbody Spine Model:
5.19.3 An interbody fusion device is filled with an appro-
5.15.1 Goats are commonly used for interbody spinal fusion
priate bone graft material and implanted at each disc space.
studies in large species animals (219-247).
5.19.4 For more details, see Appendix X2, Table X2.11.
5.15.2 In comparison to sheep, goats are generally less
adversetohumaninteractionandarethereforeeasiertohandle.
6. Considerations for the Spinal Fusion Site
5.15.3 Goats should be screened by blood test for caprine
encephalitis prior to inclusion in cohort group.
6.1 The focus of this guide is on interbody and posterolat-
5.15.4 Surgical defects are typically performed at one or
eral fusion sites in the spine. Not all sites have been reported
more of the L2-L3, L3-L4, L4-L5, or L5-L6 lumbar levels or
for all species.
the C2-C3, C3-C4, C4-C5, or C5-C6 cervical levels.
6.2 Considerationsshouldalsoincludethelevelofdifficulty
5.15.5 An interbody fusion device is filled with an appro-
ofperformingthesurgicalprocedureinregardstobothsurgical
priate bone graft material and implanted at each disc space.
access and implant fixation.
5.15.6 Optionally, the lumbar fusion sites may be stabilized
6.3 Consideration should be given to the level of translat-
with unilaterally placed pedicle screws and a connecting rod.
ability of the surgical procedure to human clinical patients.
5.15.7 For more details, see Appendix X2, Table X2.7.
5.16 Pig Posterolateral Spine Model:
7. Test Procedures
5.16.1 Pigs have been utilized in posterolateral spinal
7.1 Implant Preparation:
models, although not as frequently in literature as other large
7.1.1 All materials to be implanted into animals should be
animal models (248-251).
verified to be non-cytotoxic and biocompatible. Implant com-
5.16.2 Surgical defects are typically performed at one or
ponents can be sterilized and prepared aseptically or endpoint
more of the L2-L3, L3-L4, L4-L5, or L5-L6 lumbar levels.
sterilized by methods known to be acceptable to the implant
5.16.3 Approximately 4 to8gof autogenous cancellous
composition and function.
bone may be harvested, if used as a control, per side.
7.1.2 Bioburden or sterility testing, as appropriate, should
5.16.4 The transverse processes of the operative levels are
be completed on representative test articles. Note that for
decorticated bilaterally.
TEMPS regulated as biologics in the United States, each lot
5.16.5 Treatment or control materials are placed along the
must be tested for sterility in accordance with 21 CFR 610.12.
“gutters” between the transverse processes.
7.1.3 See Guide F2150, Practices F1983, F981, F565, and
5.16.6 Optionally, transpedicular screw fixation using
Test Method F895. See also ISO 10993 and 21 CFR Part 58.
screws and rods may be used for fixation.
Practice F1983 covers the assessment of compatibility of
5.16.7 For more details, see Appendix X2, Table X2.8.
absorbable biomaterials for implant applications.
5.17 Pig Interbody Spine Model:
7.2 Defect Generation:
5.17.1 Pigs have been utilized in interbody spinal models,
7.2.1 The defect should be created in a standard and
although not as frequently in literature as other large animal
reproducible manner.
models (252-273).
7.2.2 Templates or other sizing tools should be considered,
5.17.2 Surgical defects are typically performed at one or
where feasible, for preparation of consistently sized defects.
more of the L2-L3, L3-L4, L4-L5, or L6-L7 lumbar levels.
7.2.3 Defectsinallanimalswithinastudyshouldbecreated
5.17.3 An interbody fusion device is filled with an appro-
with the same type of tools and instruments.
priate bone graft material and implanted at each disc space.
5.17.4 Optionally, the lumbar fusion sites may be stabilized
7.3 Test TEMP Implantation and Fixation:
with unilaterally placed pedicle screws and a connecting rod.
7.3.1 The test TEMP should be implanted in a standard and
5.17.5 For more details, see Appendix X2, Table X2.9.
reproducible manner.
5.18 Non-Human Primate Posterolateral Spine Model: 7.3.2 Care should be exercised to ensure that the surround-
5.18.1 Non-human primates have been utilized in postero- ing bone is not excessively damaged and that the TEMP is in
lateral spinal models (274-286). contact with as much of the area of the defect as possible.
5.18.2 Surgical defects are typically performed at the L4-L5 7.3.3 The defect should be fixed in a standard and repro-
lumbar level. ducible manner, if fixation is required.
F2884 − 21
7.4 Recovery and Husbandry: nonmineralized) in the defect, resorption of bone graft, cortex
7.4.1 Recovery conditions should be designed to reduce remodeling, marrow changes, and spinal fusion. In addition,
potential for stress and excessive motion. For goats and sheep, fibrous connective tissue should be evaluated with regard to
recovery pens that are sized to reduce excessive range of inflammation.
mobility for a period of two to three days are recommended. 8.1.1.3 Histomorphometric analyses can be utilized to mea-
7.4.2 All housing conditions should be approved by the sure histological parameters, including (but not limited to)
United States Department of Agriculture (USDA), or the tissue volume, lamellar bone (area, %), periosteal fibrosis
respectivegovernmentalagencyofthecountrywherethestudy (area, %), marrow fibrosis (area, %), and cellularity (number,
is conducted. mean/field).
7.4.3 Animals should be monitored frequently and observa- 8.1.1.4 Histological sectioning should ensure that the entire
tions recorded to ascertain appropriate health and physical defect site, as well as some additional surrounding tissue, is
condition. encompassed and assessed.
7.4.4 A veterinarian should approve the health condition of 8.1.1.5 Note that time points of less than six months for
animals prior to returning them to larger groups or herds. large animals and less than twelve weeks in small animals do
not necessarily reflect the long-term outcome due to the
7.5 In-Life Period:
potential for changes in the biochemical composition and
7.5.1 Radiographs should be used as appropriate for a given
organization of repair tissue over time.
study to assess placement of the implants.
8.1.1.6 Short-term histologic evaluation can be used for
7.5.2 Followingrecovery,largeanimalsshouldbecontained
screening and optimization. Long-term assessment should be
within protected stalls for a minimum of nine days. After this
based on histologic and mechanical measures.
period the animals can either remain in protected stalls or be
allowed to roam freely in group herds. 8.2 Radiography:
7.5.3 A qualified veterinarian should examine animals rou-
8.2.1 Radiographs are important to evaluate the amount and
tinely for any gross abnormalities and for signs of discomfort. quality of the new bone forming during the in-life portion of
7.5.4 Survival time should be designated based on the
the study, as well as at the endpoint.
objective of the study. Typically, an early time point (for 8.2.2 Typically,radiographsshouldbetakenintwoorthogo-
example, to examine the effect on early healing, including, for
nal planes to allow assessment of proper alignment and a
example, acceleration of healing), and one or two later time quasi-three-dimensional view (for example, anterior-posterior
point(s) (for example, when full or nearly full healing is
and lateral).
anticipated) are chosen. Historically used in-life periods are 8.2.3 Radiographic healing may be one of the decisive
listed in the tables in Appendix X2.
factors used to terminate a study.
8.2.4 Various radiographic scoring systems have been pub-
7.6 Necropsy:
lished. The scoring system should be specified for the species.
7.6.1 Animals should be euthanized in a humane manner
8.2.5 Inclusion of a metal wedge in the picture may help to
according to accepted practices of the Animal Welfare Act (in
normalize radiographs.
the United States) or other applicable local statutes.
8.2.6 Radiopaque implants and fixation materials may have
7.6.2 The implanted site should be removed along with the
an impact on the ability to assess healing from radiographs.
surrounding cartilage and bone.
8.2.7 Plain-film radiographs are not considered to be suffi-
7.6.3 Retrieved tissue should be placed in a solution con-
ciently discriminating to positively identify fusion or pseudar-
sistent with intended outcome measures such as histology
throsis and should be combined with other methods to verify
(decalcified paraffin versus nondecalcified plastic embedded),
fusion.
biochemistry, or mechanical testing.
8.3 Computer Tomography:
8. Evaluation and Results
8.3.1 Computer tomography (CT) has been evolving in
8.1 Histology—For histological processing procedures, re- recent years as a useful tool, allowing 3D imaging of bone
fer to Practice F561. Histological sections should be used to regeneration in harvested bone, as well as being used for
monitoring bone regeneration in vivo over time.
assesstheamountandqualityoftissueregenerationorrepairof
the fusion mass. Histological sections should be serially cut 8.3.2 CTimages to assess bone (mineralized tissue) area are
and stained in a manner to allow for assessment of the quality also useful for correct calculation and interpretation of me-
of tissue and for detection calcified tissue. Standard stains chanical test results.
include: hemotoxylin/eosin, Toluidine Blue, or Modified 8.3.3 ThebiggestchallengewithCTanalysesistothreshold
Trichrome stain, and others (27). Consideration should be appropriately to exclude the scaffold from newly forming bone
given to using decalcified versus nondecalcified sections, within the defect.
which may require different staining methods. 8.3.4 Appropriate controls, calibrations, and scan param-
8.1.1 Microscopic Analysis and Scoring: eters (energy intensity, integration time, and so on) should be
8.1.1.1 Histological sections should be analyzed for adverse utilized in order to ensure that the results are internally
tissue reactions using typical histopathologic indices. consistent within a study.
8.1.1.2 For assessment of TEMP performance, a scoring 8.3.5 Where fusion versus pseudarthrosis is an outcome
system should be utilized to determine several aspects such as measure, CToutcomes should be verified by histology, manual
the following: new bone formation (mineralized/ manipulation, or mechanical testing.
F2884 − 21
8.4 Microtomography: 8.5.4 Typical nondestructive testing includes protocols to
determine global and localized range of motion (ROM) and
8.4.1 Microtomography, or micro-CT, uses X-rays to create
stiffness.Testing typically occurs under either load or displace-
cross sections of a 3D object that later can be used to recreate
ment control.
a virtual model without destroying the original model. The
8.5.5 Typicaldestructivetestingincludestensiontestingand
term micro is used to indicate that the pixel sizes of the cross
torsional strength testing for posterolateral fusion and dynamic
sections are in the micrometer range. Scanners are much
cyclic load to failure for interbody fusions.
smallerindesigncomparedtothehumanversionsandareused
8.5.6 Due to viscoelastic effects, consideration has to be
to model smaller objects. Micro-CT scanning is more focused
giventothetestspeedutilizedinstatictesting,whichshouldbe
than regular CT scanning, meaning that it brings out details as
lower than an appropriate % length change for the test, for
fine as 1000th of a millimeter. Thus, it has two to three
example 0.5 % strain/min, and reported.
thousand times the resolution of a regular CT scan.
8.5.7 From typical stress-strain curves, the strength (maxi-
8.4.2 Microtomography analysis can be used to assess
mum torque), maximum force, stiffness, and total energy to
volume rendering and for image segmentation. Similar to CT,
failurecanbecalculated.Fromtorsionaltests,itisnecessaryto
micro-CT images to assess bone (mineralized tissue) area are
also report the angle at failure. From cyclic load tests, it is
also useful for correct calculation and interpretation of me-
necessarytoreportthefrequencyandamplitudeoftheloading,
chanical test results.
as well as the cycles to failure.
8.4.3 Appropriate controls, calibrations, and scan param-
8.5.8 It is recommended to monitor and report where the
eters (energy intensity, integration time, and so on) should be
fracture at failure occurs (in or through the newly formed bone
utilized in order to ensure that the results are internally
tissue, or in the original bone outside the defect). Faxitron
consistent within a study.
radiographs may be used as a tool for this purpose.
8.4.4 Where fusion versus pseudarthrosis is an outcome
9. Analysis
measure, CToutcomes should be verified by histology, manual
manipulation, or mechanical testing.
9.1 Statistical Analysis—The mean and standard deviation
should be calculated for the individual categories and the total
8.5 Mechanical Testing of Repair Tissue:
score for each of the graded specimens. Fisher exact test,
8.5.1 Mechanical testing of the fusion usually follows
chi-square test, or Kruskal-Wallis test (a one-way non-
dissection. Care has to be taken when separating the spine
parametric analysis of variance) can be used for analyzing the
sections if fusion is observed. Sample preparation may involve
differences between the scores of different groups.
partial embedding into resin blocks to allow proper mounting
in the fixtures. 10. Keywords
8.5.2 Standard nondestructive testing may include manual
10.1 animal models; biomaterials; bone; bone regeneration;
palpation as an assessment of spinal fusion.
bonerepair;implants;interbodyspinefusion;invivo;mechani-
8.5.3 The specific testing apparatus, load cell resolution, cal testing; pre-clinical; products; posterolateral spine fusion;
loading constraints, loading profile, and other test parameters spinal fusion; spine; synthetic biomaterials; TEMPs (tissue
as required need to be documented. engineered medical products)
APPENDIXES
(Nonmandatory Information)
X1. COMMON ANIMAL MODEL PARAMETERS AND RELEVANCE IN SPINAL FUSION PRE-CLINICAL MODELS
F2884 − 21
A
TABLE X1.1 Co
...