ASTM F2529-13(2021)
(Guide)Standard Guide for in vivo Evaluation of Osteoinductive Potential for Materials Containing Demineralized Bone (DBM)
Standard Guide for <emph type="ital"> in vivo</emph> Evaluation of Osteoinductive Potential for Materials Containing Demineralized Bone (DBM)
SIGNIFICANCE AND USE
4.1 This guide covers animal implantation methods and analysis of the explanted DBM-containing material to determine whether a material or substance possesses osteoinductive potential, as defined by its ability to cause bone to form in vivo at a site that would otherwise not support bone formation, that is, heterotopically in a skeletal muscle implant site. For in vitro evaluation see Test Method F2131 for in vitro assessment of rhBMP 2.
4.2 The test methods described here may be suitable for defining product specifications, cGMP lot release testing, research evaluation, regulatory submission, and so forth, but a positive outcome should not be presumed to indicate that the product will be osteoinductive in a human clinical application. At present, the only direct assays to assess new bone formation are in vivo, since the property of bone conduction or induction can only be assessed in a heterotopic or orthotopic site in a living animal. When these products are implanted in an orthotopic site, osteogenic factors already present at the implantation site may contribute to and enhance bone formation in conjunction with the osteoconductive nature of the product. Thus, orthotopic implantation of products may result in bone formation by acting on existing bone-forming cells and not by causing mesenchymal stem cells to become osteochondroprogenitor cells. In contrast, when these products are implanted in a heterotopic site, no native osteogenic factors are present to contribute to or enhance bone formation. Thus, heterotopic implantation of products will only result in new bone formation by causing mesenchymal stem cells to become osteochondroprogenitor cells. In vitro assays have been described and some believe they may correlate to the results obtained from in vivo assays. However such in vitro assays measure only some of the biochemical marker(s) associated with in vivo bone formation and are therefore only indirect assays for osteoinductive activity or the capacity t...
SCOPE
1.1 This guide covers general guidelines to evaluate the effectiveness of DBM-containing products intended to cause and/or promote bone formation when implanted or injected in vivo. This guide is applicable to products that may be composed of one or more of the following components: natural biomaterials (such as demineralized bone), and synthetic biomaterials (such as calcium sulfate, glycerol, and reverse phase polymeric compounds) that act as additives, fillers, and/or excipients (radioprotective agents, preservatives, and/or handling agents) to make the demineralized bone easier to manipulate. It should not be assumed that products evaluated favorably using this guidance will form bone when used in a clinical setting. The primary purpose of this guide is to facilitate the equitable comparison of unique bone-forming products in in vivo heterotopic models of osteoinductivity. The purpose of this guide is not to exclude other established methods.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with the use of DBM-containing bone-forming/promoting products. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices involved in the development of said products in accordance with applicable regulatory guidance documents and in implementing this guide to evaluate the bone-forming/promoting capabilities of the product.
1.4 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: F2529 − 13 (Reapproved 2021)
Standard Guide for
in vivo Evaluation of Osteoinductive Potential for Materials
Containing Demineralized Bone (DBM)
This standard is issued under the fixed designation F2529; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This guide covers general guidelines to evaluate the
effectiveness of DBM-containing products intended to cause
2. Referenced Documents
and/or promote bone formation when implanted or injected in
2.1 ASTM Standards:
vivo. This guide is applicable to products that may be com-
D1193Specification for Reagent Water
posed of one or more of the following components: natural
D5056Test Method for Trace Metals in Petroleum Coke by
biomaterials (such as demineralized bone), and synthetic bio-
Atomic Absorption
materials (such as calcium sulfate, glycerol, and reverse phase
E508Test Method for Determination of Calcium and Mag-
polymeric compounds) that act as additives, fillers, and/or
nesium in Iron Ores by Flame Atomic Absorption Spec-
excipients (radioprotective agents, preservatives, and/or han-
trometry
dling agents) to make the demineralized bone easier to ma-
F565PracticeforCareandHandlingofOrthopedicImplants
nipulate. It should not be assumed that products evaluated
and Instruments
favorably using this guidance will form bone when used in a
F895TestMethodforAgarDiffusionCellCultureScreening
clinical setting. The primary purpose of this guide is to
for Cytotoxicity
facilitate the equitable comparison of unique bone-forming
F981Practice for Assessment of Compatibility of Biomate-
products in in vivo heterotopic models of osteoinductivity.The
rials for Surgical Implants with Respect to Effect of
purpose of this guide is not to exclude other established
Materials on Muscle and Insertion into Bone
methods.
F1854Test Method for Stereological Evaluation of Porous
1.2 The values stated in SI units are to be regarded as
Coatings on Medical Implants
standard. No other units of measurement are included in this
F2131Test Method forIn Vitro Biological Activity of Re-
standard.
combinant Human Bone Morphogenetic Protein-2
(rhBMP-2) Using the W-20 Mouse Stromal Cell Line
1.3 This standard does not purport to address all of the
F2721Guide for Pre-clinical in vivo Evaluation in Critical
safety concerns, if any, associated with the use of DBM-
Size Segmental Bone Defects
containing bone-forming/promoting products. It is the respon-
sibility of the user of this standard to establish appropriate 2.2 Federal Documents:
safety, health, and environmental practices involved in the 21 CFR 58Good Laboratory Practice for Nonclinical Labo-
development of said products in accordance with applicable ratory Studies
regulatory guidance documents and in implementing this guide 21 CFR 820Quality System Regulation
to evaluate the bone-forming/promoting capabilities of the 21 CFR 1270Human Tissue Intended for Transplantation
product. 21 CFR 1271Human Cells, Tissues, and Cellular and
Tissue-Based Products
1.4 This international standard was developed in accor-
21 CFR 610.12General Biological Products Standards—
dance with internationally recognized principles on standard-
General Provisions—Sterility
ization established in the Decision on Principles for the
Q1E Evaluation of Stability DataFDAGuidance for Indus-
Development of International Standards, Guides and Recom-
try
1 2
This guide is under the jurisdiction ofASTM 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 service@astm.org. For Annual Book of ASTM
F04.44 on Assessment for TEMPs. Standardsvolumeinformation,refertothestandard’sDocumentSummarypageon
Current edition approved Aug. 1, 2021. Published August 2021. Originally the ASTM website.
approved in 2013. Last previous edition approved in 2013 as F2529–13. DOI: Available from Food and DrugAdministration (FDA), 10903 New Hampshire
10.1520/F2529-13R21. Ave., Silver Spring, MD 20993-0002, http://www.fda.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2529 − 13 (2021)
Container and Closure Integrity Testing in Lieu of Sterility the physical or chemical properties/characteristics of that
Testing as a Component of the Stability Protocol for bone-forming product.
Sterile ProductsFDA Guidance Document
3.1.3 bone—hardconnectivetissueoftheskeletalsystemin
Eligibility Determination for Donors of Human Cells,
vertebrates comprised of collagen, growth factors, and an
Tissues, and Cellular and Tissue-Based ProductsGuid-
inorganicrigidmatrixcontainingcalcium,phosphateandother
ance for Industry
minerals, and various cellular elements, including osteoblasts,
Guidance for the Preparation of a Premarket Notification
osteocytes, osteoclasts, and hematopoietic cells.
Application for a Surgical MeshGuidance for Industry
3.1.3.1 Discussion—Bone is made up of cortical and can-
and/or for FDA Reviewers/Staff and/or Compliance
cellous bone tissue. It serves as the point of attachment for
2.3 AAMI/ISO Documents:
muscles and tendons and is load-bearing.
AAMI TIR 17Compatibility of Materials Subject to Steril-
3.1.4 bone-forming product (containing DBM)—as used in
ization
this guide, a DBM-containing bone-forming product may be
AAMI/ISO 22442-01Medical Devices Utilizing Animal
comprisedofmultiplecomponentsincluding,butnotrestricted
Tissues and Their Derivatives—Part 1: Application of
to, demineralized bone, growth factors, differentiation factors,
Risk Management
osteoprogenitor cells, mesenchymal stem cells, biologically
AAMI/ISO 22442-03Medical Devices Utilizing Animal
active carrier(s), and/or non-biologically active carrier(s).
Tissues and Their Derivatives—Part 3: Validation of the
3.1.5 bone marrow—tissuelocatedinthecancellousportion
Elimination and/or Inactivation of Viruses and Transmis-
and cavities (medullary canal) of most bones.
sible Spongiform Encephalopathy (TSE) Agents
3.1.5.1 Discussion—Bone marrow is highly vascular and
2.4 ANSI/AAMI/ISO Documents:
occurs in two forms: white/yellow marrow, comprised mostly
ANSI/AAMI/ISO 11137Parts 1–3 Sterilization of Health
of adipose cells located primarily in the long bones, and red
Care Products—Radiation
marrow, which primarily produces and contains pluripotent
ANSI/AAMI/ISO 10993Biological Evaluation of Medical
stemscellsandredbloodcells,platelets,andwhitebloodcells
Products
derived from them. In adults, red marrow is located primarily
2.5 ICH Documents:
in the flat bones.
ICH Harmonised Tripartite Guideline Q5CQuality of Bio-
3.1.6 bone tissue—the tissue component of a bone com-
technological Products—Stability Testing of
prised of a mineralized collagenous matrix formed and main-
Biotechnological/Biological Products
tained through the action of osteoblastic cells and osteocytes
ICHHarmonisedTripartiteGuidelineQ5A(R1)ViralSafety
remodeled through the action of osteoclasts.
Evaluation of Biotechnology Products Derived from Cell
Lines of Human or Animal Origin
3.1.7 cartilage—connective tissue that is a major constitu-
2.6 USP Document: ent of the embryonic and young vertebrate skeleton, largely
United States Pharmacopeia Chapter <71>
replaced with bone and bone marrow bone with maturation.
3.1.7.1 Discussion—It is comprised mostly of Type II col-
3. Terminology
lagen and proteoglycans and found in joints, the outer ear,
bronchi, and larynx. There are three major types of cartilage:
3.1 Definitions:
hyaline cartilage, which is adapted for joint surfaces by virtue
3.1.1 additive—ingredientsthatmaybeusedtopreservethe
of its smoothness and ability to withstand compression;
product, provide radioprotection, and/or act as bulk filler
fibrocartilage, found in the outer ear, nose, and meniscus; and
and/or binding agent.
elasticcartilage,foundintheouterearandepiglottis.Cartilage
3.1.1.1 Discussion—Ithasnointendedmodeofaction,such
is also formed by the action of bone morphogenetic protein(s)
as causing cells to transform lineage, once implanted.
(BMPs) in concert with other peptide factors on mesenchymal
3.1.2 biologically active carrier—a component added to a
stem cells.
DBM-containing bone-forming product that results in a physi-
3.1.8 cortical bone—thin superficial layer of compact bone
ological and/or biochemical transformation in the implant site
independentofotherconstituentsinthebone-formingproduct. tissuethatconstitutestheprimaryload-bearingcomponentofa
bone.
3.1.2.1 Discussion—Thetransformationmaybedesirableor
untoward. A biologically active carrier may also contribute to
3.1.9 demineralized bone—bone tissue wherein the average
mineral content, typically measured as calcium, is less than or
equal to 8% by dry weight.
Available from Association for the Advancement of Medical Instrumentation
3.1.9.1 Discussion—As used in this guide, dry weight
(AAMI), 4301 N. Fairfax Dr., Suite 301, Arlington, VA 22203-1633, http://
means lacking significant or measurable residual moisture
www.aami.org.
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
(<5%).
4th Floor, New York, NY 10036, http://www.ansi.org.
3.1.10 differentiation factors—proteins with the ability to
Available from International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH
induce or alter cell differentiation and/or proliferation with
Secretariat, c/o IFPMA, 15 ch. Louis-Dunant, P.O. Box 195, 1211 Geneva 20,
subsequent tissue morphogenesis.
Switzerland, http://www.ich.org.
3.1.10.1 Discussion—For example, BMP-2, BMP-4, and
AvailablefromU.S.Pharmacopeia(USP),12601TwinbrookPkwy.,Rockville,
MD 20852-1790, http://www.usp.org. BMP-7(OP-1) are differentiation factors with the ability to
F2529 − 13 (2021)
induce bone and cartilage formation in tissues or implantation 3.1.23.1 Discussion—A carrier may also contribute to the
sites that would otherwise not form bone or cartilage by physical or chemical properties/characteristics of that bone-
causing mesenchymal progenitor cells to differentiate into forming product.
chondrocytes and/or osteoblasts.
3.1.24 nude mouse—a mouse with a genetically deficient
3.1.11 endochondral ossification—process by which bones immune system as the result of a dysfunctional rudimentary
in embryonic and juvenile animals grow while maintaining thymus and thus is deficient in T-cells and incapable of
functional proportions of the skeleton and joints.
producing cytotoxic effector cells and a graft-versus-host
3.1.11.1 Discussion—Bone-forming products considered in response.
this guide mimic this process: post-implantation inflammation
3.1.24.1 Discussion—ThereforetheT-cellresponsetoxeno-
leads to recruitment of mesenchymal stem cells, and the
genic material is abrogated. However, the animals are able to
BMPS/growth factors cause the mesenchymal stems cells to
mount an innate immune response (macrophages, giant cells,
differentiate into chondroblasts, which synthesize cartilage
and granulocytes) and thus are capable of mounting an
matrix. The chondrocytes undergo hypertrophy and calcify
immunological response to non-biocompatible products.
their extracellular matrix. This matrix is then resorbed by
3.1.25 nude rat—a rat with a genetically deficient immune
osteoclasts, angiogenesis occurs, and osteoprogenitor cells
systemastheresultofadysfunctionalrudimentarythymusand
migrate to the calcified cartilage by means of the neovascula-
thus deficient in T-cells and incapable of producing cytotoxic
ture. Osteoblasts synthesize bone on the cartilage scaffold and
effector cells and a graft-versus-host response.
new bone marrow forms, and is incorporated into the new
3.1.25.1 Discussion—ThereforetheT-cellresponsetoxeno-
tissue and ultimately is remodeled.
genic material is abrogated. However, the animals are able to
3.1.12 evidence of endochondral bone formation—
mount an innate immune response (macrophages, giant cells,
chondroblasts,chondrocytes,cartilage,osteoblasts,osteocytes,
and granulocytes) and thus are capable of mounting an
new bone, and bone marrow.
immunological response to non-biocompatible products.
3.1.13 excipient—constituents of a DBM-containing bone-
3.1.26 orthotopic—in the context of this guide, an
forming product that are not the active substance.
implantation/transplantation site within or in direct contact
3.1.14 fascia—fibrous connective tissue between two layers with bone tissue.
of tissues such as muscle, or an internal organ and the
3.1.27 ossicle—a spherical piece of bone consisting of an
abdominal wall.
outer core of cortical bone and an inner region of trabecular
3.1.15 fibrous connective tissue—connective tissue pro- bone and bone marrow.
duced by fibroblasts.
3.1.28 osteoblasts—cells exhibiting the appropriate protein
3.1.16 fillers—an ingredient that may used to expand the
andgeneexpressioncharacteristicsnecessaryfortheformation
volume of the implant material and may possess osteoconduc- and maintenance.
tive properties but does not cause cells to transform lineage
3.1.29 osteoconduction—the ability of a material/bony im-
once implanted.
plant site to support new bone formation by providing an
3.1.17 gelatin capsule—capsule made of gelatin used to environment in which pre-existing osteoid cells capable of
contain materials for implantation.
synthesizing and secreting components essential to the forma-
tion lead to growth of new host bone into/onto the material.
3.1.18 growth factors—proteins produced by cells that
stimulate or inhibit cell proliferation and may affect differen-
3.1.30 osteogenic—indicating the presence of cells capable
tiation.
of synthesizing and secreting components essential to the
formation of bone at some implant site as well as the
3.1.19 heterotopic—in the context of this guide, an
osteoconductive capacity necessary for causing/allowing bone
implantation/transplantation site that is not in direct contact
formation in a tissue or implantation site.
with bone, such as skeletal muscle.
3.1.30.1 Discussion—Additionally, BMPs would also be
3.1.20 intermuscular—within that space occurring between
considered osteogenic as these are so
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.