ASTM F2150-19

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: F2150 − 13 F2150 − 19
Standard Guide for
Characterization and Testing of Biomaterial Scaffolds Used
in Regenerative Medicine and Tissue-Engineered Medical
Products
This standard is issued under the fixed designation F2150; 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 is a resource of currently available test methods for the characterization of the compositional and structural
aspects of biomaterial scaffolds used to develop in the development and manufacture of regenerative medicine and tissue-
engineered medical products (TEMPs).
1.2 The test methods contained herein guide characterization of the bulk physical, chemical, mechanical, and surface properties
of a scaffold construct. Such properties may be important for the success of a TEMP, especially if they affect the property affects
cell retention, activity and organization, the delivery of bioactive agents, or the biocompatibility and bioactivity within the final
product.
1.3 This guide may be used in the selection of appropriate test methods for the generation of an original equipment manufacture
(OEM) specification. This guide also may be used to characterize the scaffold component of a finished medical product.
1.4 This guide is intended to be utilizedused in conjunction with appropriate characterization(s) and evaluation(s) of any raw
or starting material(s) utilizedused in the fabrication of the scaffold, such as described in Guide F2027.
1.5 This guide addresses natural, synthetic, or combination scaffold materials with or without bioactive agents or biological
activity. This guide does not address the characterization or release profiles of any biomolecules, cells, drugs, or bioactive agents
that are used in combination with the scaffold. scaffold, but may be used to address the effects on other (e.g., structural) properties
as a result of such release. A determination of the suitability of a particular starting material and/or finished scaffold structure to
a specific cell type and/or tissue engineering application is essential, but will require additional in vitro and/or in vivo evaluations
considered to be outside the scope of this guide.
1.6 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory requirementslimitations prior to use.
1.7 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.
2. Referenced Documents
2.1 ASTM Standards:
D412 Test Methods for Vulcanized Rubber and Thermoplastic Elastomers—Tension
D570 Test Method for Water Absorption of Plastics
D638 Test Method for Tensile Properties of Plastics
D648 Test Method for Deflection Temperature of Plastics Under Flexural Load in the Edgewise Position
D695 Test Method for Compressive Properties of Rigid Plastics
D747 Test Method for Apparent Bending Modulus of Plastics by Means of a Cantilever Beam (Withdrawn 2019)
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.42
on Biomaterials and Biomolecules for TEMPs.
Current edition approved Oct. 1, 2013Oct. 1, 2019. Published December 2013October 2019. Originally approved in 2002. Last previous edition approved in 20072013
as F2150 – 07.F2150–13. DOI: 10.1520/F2150-13.10.1520/F2150-19.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2150 − 19
D790 Test Methods for Flexural Properties of Unreinforced and Reinforced Plastics and Electrical Insulating Materials
D792 Test Methods for Density and Specific Gravity (Relative Density) of Plastics by Displacement
D882 Test Method for Tensile Properties of Thin Plastic Sheeting
D1042 Test Method for Linear Dimensional Changes of Plastics Caused by Exposure to Heat and Moisture
D1238 Test Method for Melt Flow Rates of Thermoplastics by Extrusion Plastometer
D1388 Test Method for Stiffness of Fabrics
D1621 Test Method for Compressive Properties of Rigid Cellular Plastics
D1623 Test Method for Tensile and Tensile Adhesion Properties of Rigid Cellular Plastics
D1708 Test Method for Tensile Properties of Plastics by Use of Microtensile Specimens
D2857 Practice for Dilute Solution Viscosity of Polymers
D2990 Test Methods for Tensile, Compressive, and Flexural Creep and Creep-Rupture of Plastics
D3016 Practice for Use of Liquid Exclusion Chromatography Terms and Relationships
D3039/D3039M Test Method for Tensile Properties of Polymer Matrix Composite Materials
D3418 Test Method for Transition Temperatures and Enthalpies of Fusion and Crystallization of Polymers by Differential
Scanning Calorimetry
D4001 Test Method for Determination of Weight-Average Molecular Weight of Polymers By Light Scattering
D4404 Test Method for Determination of Pore Volume and Pore Volume Distribution of Soil and Rock by Mercury Intrusion
Porosimetry
D4603 Test Method for Determining Inherent Viscosity of Poly(Ethylene Terephthalate) (PET) by Glass Capillary Viscometer
D5226 Practice for Dissolving Polymer Materials
D5296 Test Method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance
Size-Exclusion Chromatography
D6420 Test Method for Determination of Gaseous Organic Compounds by Direct Interface Gas Chromatography-Mass
Spectrometry
D6474 Test Method for Determining Molecular Weight Distribution and Molecular Weight Averages of Polyolefins by High
Temperature Gel Permeation Chromatography
D6539 Test Method for Measurement of the Permeability of Unsaturated Porous Materials by Flowing Air
D6579 Practice for Molecular Weight Averages and Molecular Weight Distribution of Hydrocarbon, Rosin and Terpene Resins
by Size-Exclusion Chromatography
E128 Test Method for Maximum Pore Diameter and Permeability of Rigid Porous Filters for Laboratory Use
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E384 Test Method for Microindentation Hardness of Materials
E456 Terminology Relating to Quality and Statistics
E473 Terminology Relating to Thermal Analysis and Rheology
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E793 Test Method for Enthalpies of Fusion and Crystallization by Differential Scanning Calorimetry
E794 Test Method for Melting And Crystallization Temperatures By Thermal Analysis
E967 Test Method for Temperature Calibration of Differential Scanning Calorimeters and Differential Thermal Analyzers
E968 Practice for Heat Flow Calibration of Differential Scanning Calorimeters
E996 Practice for Reporting Data in Auger Electron Spectroscopy and X-ray Photoelectron Spectroscopy
E1078 Guide for Specimen Preparation and Mounting in Surface Analysis
E1142 Terminology Relating to Thermophysical Properties
E1294 Test Method for Pore Size Characteristics of Membrane Filters Using Automated Liquid Porosimeter (Withdrawn 2008)
E1298 Guide for Determination of Purity, Impurities, and Contaminants in Biological Drug Products (Withdrawn 2014)
E1356 Test Method for Assignment of the Glass Transition Temperatures by Differential Scanning Calorimetry
E1642 Practice for General Techniques of Gas Chromatography Infrared (GC/IR) Analysis
E1829 Guide for Handling Specimens Prior to Surface Analysis
E1994 Practice for Use of Process Oriented AOQL and LTPD Sampling Plans
F316 Test Methods for Pore Size Characteristics of Membrane Filters by Bubble Point and Mean Flow Pore Test
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F1249 Test Method for Water Vapor Transmission Rate Through Plastic Film and Sheeting Using a Modulated Infrared Sensor
F1634 Practice forIn-Vitro Environmental Conditioning of Polymer Matrix Composite Materials and Implant Devices
(Withdrawn 2017)
F1635 Test Method for in vitro Degradation Testing of Hydrolytically Degradable Polymer Resins and Fabricated Forms for
Surgical Implants
F1884 Test Methods for Determining Residual Solvents in Packaging Materials
F1980 Guide for Accelerated Aging of Sterile Barrier Systems for Medical Devices
F1983 Practice for Assessment of Selected Tissue Effects of Absorbable Biomaterials for Implant Applications
F2150 − 19
F2025 Practice for Gravimetric Measurement of Polymeric Components for Wear Assessment
F2027 Guide for Characterization and Testing of Raw or Starting Materials for Tissue-Engineered Medical Products
F2212 Guide for Characterization of Type I Collagen as Starting Material for Surgical Implants and Substrates for Tissue
Engineered Medical Products (TEMPs)
F2312 Terminology Relating to Tissue Engineered Medical Products
F2450 Guide for Assessing Microstructure of Polymeric Scaffolds for Use in Tissue-Engineered Medical Products
F2603 Guide for Interpreting Images of Polymeric Tissue Scaffolds
F2791 Guide for Assessment of Surface Texture of Non-Porous Biomaterials in Two Dimensions
F2809 Terminology Relating to Medical and Surgical Materials and Devices (Withdrawn 2019)
F2883 Guide for Characterization of Ceramic and Mineral Based Scaffolds used for Tissue-Engineered Medical Products
(TEMPs) and as Device for Surgical Implant Applications
F2900 Guide for Characterization of Hydrogels used in Regenerative Medicine
F2902 Guide for Assessment of Absorbable Polymeric Implants
G120 Practice for Determination of Soluble Residual Contamination by Soxhlet Extraction
2.2 AAMI Standards:
AAMI STBK-1 Sterilization—Part 1: Sterilization in Health Care Facilities
AAMI STBK-2 Sterilization—Part 2: Sterilization Equipment
AAMI STBK-3 Sterilization—Part 3: Industrial Process Control
2.3 ANSI Standards:
ANSI/ISO/ASQ Q9000: Quality Management Systems—Fundamentals and Vocabulary
ANSI/ISO/ASQ Q9001: Quality Management Systems: Requirements
2.4 British Standards Institute:
BSI BS EN 12441–1 British Standard—Animal Tissues and Their Derivatives Utilized in the Manufacture of Medical
Devices—Part 1: Analysis and Management of Risk
BSI BS EN 12442–2 British Standard—Animal Tissues and Their Derivatives Utilized in the Manufacture of Medical
Devices—Part 2: Controls on Sourcing, Collection, and Handling
BSI BS EN 12442–3 British Standard—Animal Tissues and Their Derivatives Utilized in the Manufacture of Medical
Devices—Part 3: Validation of the Elimination and/or Inactivation of Viruses and Transmissible Agents
2.4 ISO Standards:
ISO 1133–1 Determination of the Melt-Mass Flow Rate (MFR) and the Melt Volume-Flow Rate (MVR) of Thermoplastics
ISO 10993-1 Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing within a Risk Management Process
ISO 10993-9 Biological Evaluation of Medical Devices—Part 9: Degradation of Materials Related to Biological Testing
ISO 10993-13 Biological Evaluation of Medical Devices—Part 13: Identification and Quantification of Degradation Products
from Polymers
ISO 10993-14 Biological Evaluation of Medical Devices—Part 14: Identification and Quantification of Degradation Products
from Ceramics
ISO 10993-15 Biological Evaluation of Medical Devices—Part 15: Identification and Quantification of Degradation Products
from Coated and Uncoated Metals and Alloys
ISO 10993-18 Biological Evaluation of Medical Devices — Part 18: Chemical Characterization of Medical Device Materials
within a Risk Management Process
ISO 11357-1 Plastics—Differential Scanning Calorimetry (DSC)—Part 1: General Principles
ISO 11357-2 Plastics—Differential Scanning Calorimetry (DSC)—Part 2: Determination of Glass Transition Temperature and
Glass Transition Step Height
ISO 13781 Implants for Surgery—Homopolymers, Copolymers and Blends on Poly(Lactide)—In Vitro Degradation
Testing—Second Edition
ISO 22442-1 Medical Devices Utilizing Animal Tissues and Their Derivatives—Part 1: Application of Risk Management—
Second Edition
ISO 22442-2 Medical Devices Utilizing Animal Tissues and Their Derivatives—Part 2: Controls on Sourcing, Collection and
Handling—Second Edition
ISO 22442-3 Medical Devices Utilizing Animal Tissues and Their Derivatives—Part 3: Validation of the Elimination and/or
Inactivation of Viruses and Transmissible Spongiform Encephalopathy (TSE) Agents—First Edition
Available from the Association for the Advancement of Medical Instrumentation, 1110901 N. Glebe Rd., Suite 220,300, Arlington, VA 22201-4795.22203,
http://www.aami.org.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
Switzerland, http://www.iso.org.
F2150 − 19
ISO TR 22442-4 Medical Devices Utilizing Animal Tissues and Their Derivatives—Part 4: Principles for Elimination and/or
Inactivation of Transmissible Spongiform Encephalopathy (TSE) Agents and Validation Assays for Those Processes—First
Edition
ISO 80000–9 Quantities and Units—Part 9: Physical Chemistry and Molecular Physics
2.5 U.S. Code of Federal Regulations:
21 CFR Part 58 Title 21—Food And Drug Administration, Part 58—Good Laboratory Practice For Nonclinical Laboratory
Studies
21 CFR Part 820 Title 21—Food and Drugs Services, Part 820—Quality System Regulation
2.6 U.S. Pharmacopeia (USP) Standards:
<51> Antimicrobial Effectiveness Testing
<71> Sterility Tests
<87> Biological Reactivity Tests, in vitro
<88> Biological Reactivity Tests, in vivo
<151> Pyrogen Test
<197> Spectrophotometric Identification Test
<231> Heavy Metals
<232> Elemental Impurities—Limits
<233> Elemental Impurities—Procedures
<381> Elastomeric Closures for Injections
<616> Bulk Density and Tapped Density
<661> Containers—Plastics
<699> Density of Solids
<701> Disintegration
<731> Loss on Drying
<736> Mass Spectrometry
<741> Melting Range or Temperature
<761> Nuclear Magnetic Resonance
<776> Optical Microscopy
<786> Particle Size Distribution Estimation by Analytical Sieving
<846> Specific Surface Area
<851> Spectrophotometry and Light-Scattering
<881> Tensile Strength
<891> Thermal Analysis
<911> Viscosity
<921> Water Determination
<941> X-Ray Diffraction
<1031> Biocompatibility of Materials Used in Drug Containers, Medical Devices, and Implants
<1045> Biotechnology Derived Biotechnology-Derived Articles
<1050> Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin
<1074> Excipient Biological Safety Evaluation Guidelines
<1078> Good Manufacturing Practices for Bulk Pharmaceutical Excipients
<1181> Scanning Electron Microscopy
<1211> Sterilization and Sterility Assurance of Compendial Articles
<1225> Validation of Compendial Procedures
2.7 NIST Document:
NIST SP811 Special Publication SP811: Guide for the Use of the International System of Units (SI)
2.8 ICH Guideline:
ICH Q3D Guideline for Elemental Impurities
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.
Available from U.S. Pharmacopeia, 12601 Twinbrook Pkwy., Rockville, MD 20852, or through http://www.usp.org/products/USPNF/. The standards are listed by
appropriate USP citation number.
Available from National Institute of Standards and Technology (NIST), 100 Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://www.nist.gov.
Available from International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH Secretariat, 9,
chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland, http://www.ich.org.
F2150 − 19
2.9 Other Documents/Web Sites:
U.S. Food &and Drug Administration (FDA) Center for Devices &and Radiologic Health (CDRH), Consensus Standards
Database
FDA-CDRH Guidance Documents Database
FDA-CDRH Premarket Approval (PMA) Database
FDA-CDRH 510(k) (Premarket Notification) Database
3. Terminology
3.1 Unless provided otherwise in 3.2, terminology shall be in conformance with Terminologiesthe F2809 and TEMPs
terminology standard, Terminology F2312.
3.2 Definitions:
3.2.1 bioactive agents, n—any molecular component in, on, or within the interstices of a device that elicits a desired tissue or
cell response. Growth factors, antibiotics, and antimicrobials are typical examples of bioactive agents. Device structural
components or degradation byproducts that evoke limited localized bioactivity are not included.
3.2.2 pores, n—an inherent or induced network of channels and open spaces within an otherwise solid structure.
3.2.3 porometry, n—the determination of the distribution of pore diameters relative to the direction of fluid flow by the
displacement of a wetting liquid as a function of pressure.
3.2.4 porosimetry, n—the determination of pore volume and pore size distribution through the use of a nonwettingnon-wetting
liquid (typically mercury) intrusion into a porous material as a function of pressure.
3.2.5 porosity, n—property of a solid which contains an inherent or induced network of channels and open spaces. Porosity can
be measured by the ratio of pore (void) volume to the apparent (total) volume of a porous material and is commonly expressed
as a percentage.
4. Summary of Guide
4.1 The physicochemical and three-dimensional characteristics of the scaffold material are expected to influence the properties
of TEMPs. It is the intent of this guide to provide a compendium of materials characterization techniques for properties that may
be related directly to the functionality of scaffolds for TEMPs.
4.2 Other characterizations for scaffolds utilizedused in TEMPs may include compositional identity, physical and chemical
properties or characteristics, viable sterilization techniques, degradability/resorbability,degradability/absorbability, and mechanical
properties.
4.3 Application of the test methods contained within this guide does not guarantee clinical success of a finished product but will
help to ensure consistency in the properties and characterization of a given scaffold material.
4.4 This guide does not suggest that all of the listed tests be conducted. The decision regarding applicability or suitability of
any particular test method remains the responsibility of the supplier, user, or regulator of the scaffold material based on applicable
regulations, characterizations, and preclinical/clinical testing.
5. Significance and Use
5.1 Scaffolds potentially may be metallic, ceramic, polymeric, natural, or composite materials. Scaffolds are usually porous to
some degree, but may be solid. Scaffolds can range from mechanically rigid to gelatinous and can be either absorbable/degradable
or nonresorbable/nondegradable.non-absorbable/non-degradable. The scaffold may or may not have a surface treatment. Because
of this large breadth of possible starting materials and scaffold constructions, this guide cannot be considered as exhaustive in its
listing of potentially applicable tests. A voluntary guidance for the development of tissue-engineered products can be found in
Omstead, et al (1). Guide F2027 contains a listing of potentially applicable test methods specific to various starting materials.
Guidance regarding the evaluation of absorbable polymeric materials and constructs can be found in Guide F2902. Guidance
regarding the evaluation of collagen-based materials can be found in Guide F2212. Guidance regarding the evaluation of scaffolds
composed of ceramic or mineral based mineral-based material is available in Guide F2883. Similarly, guidance for the assessment
of unique aspects of scaffolds based on hydrogels (for example, gel kinetics, mechanical stability, and mass transport properties)
may be found in Guide F2900.
5.2 Each TEMP scaffold product is unique and may require testing not within the scope of this guide or other guidance
documents. Users of this guide are encouraged to examine the references listed herein and pertinent FDA or other regulatory
Available from http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm.
Available from http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfggp/search.cfm.
Available from http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMA/pma.cfm.
Available from http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm
The boldface numbers in parentheses refer to the list of references at the end of this standard.
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guidelines or practices, and conduct a literature search to identify other procedures particularly pertinent for evaluation of their
specific scaffold material (2,3,4). It is the ultimate responsibility of the TEMP scaffold designer to determine the appropriate
testing, whether or not it is described in this guide.
5.3 A listing of potentially applicable tests for characterizing and analyzing the materials utilizedused to fabricate the scaffold
may be found in Guide F2027. However, conformance of a raw material to this and/or any other compendial standard(s) does not,
in itself, ensure that the selected material is suitable or that the provided quality is adequate to meet the needs of a particular
application. Thus, other characterization procedures may also be relevant and not covered by this guide.
5.4 The following provides a listing of links to U.S. Food & Drug Administration (FDA)—Center for Devices & Radiologic
Health (CDRH) web sites that may potentially contain additional guidance relevant to biomaterial scaffolds covered within this
document.
5.4.1 Recognized FDA-CDRH Consensus Standards Database:
5.4.1.1 http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm
5.4.1.2 This database provides a resource for locating FDA-recognized consensus standards for medical products.
5.4.2 FDA-CDRH Good Guidance Practice (GGP) Database:
5.4.2.1 http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfggp/search.cfm
5.4.2.2 This database provides a resource for locating non-binding FDA guidance documents intended for CDRH staff,
regulated industry and the public that relate to the processing, content, and evaluation of regulatory submissions, the design,
production, manufacturing, and testing of regulated products, and FDA inspection and enforcement procedures.
5.4.2.3 A document within this database possessing content that warrants particular consideration for its potential applicability
for tissue engineering tissue-engineering scaffolds is Guidance for the Preparation of a Premarket Notification Application for a
Surgical Mesh; Final.
5.4.3 FDA-CDRH Premarket Approval (PMA) Database:
5.4.3.1 http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMA/pma.cfm
5.4.4 FDA-CDRH 510(k) (Premarket Notification) Database:
5.4.4.1 http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm
6. Chemical Properties and Tests
NOTE 1—Chemical properties are the chemical composition characteristics of a compound. Chemical tests provide information about the identity or
nature of the chemical components of a scaffold. Chemical tests include those that provide information about the nature or size of constituent molecules,
the product’s purity, and/or the chemical nature of the scaffold surface.
6.1 Identification of Impurities:
6.1.1 Chemical impurities are expected and unexpected materials that are not part of the intended design of the scaffold.
Acceptable levels are a function of the nature of the impurity and the scaffold’s intended in vitro or in vivo application, and may
be evaluated by appropriate qualification studies. A more precise definition of both contaminants and impurities and guidance
regarding their significance may be found in Guide E1298.
6.1.2 Expected impurities of potential biological significance should be monitored through appropriate analytic means.
Impurities can occur in both synthetic and natural based materials (for example, proteins, such as collagen and elastin;
polysaccharides, such as cellulose, alginate, hyaluronan, and chitin based derivatives) and may include, but are not limited to,
processing aids or solvents, unreacted cross-linking agents, residual monomers, endotoxins, sterilization residuals, and residual
solutions that, by their chemical nature or relative concentrations, carry potential for influencing cell or tissue response.
6.1.3 Impurities may be identified or quantitatively determined by infrared (IR) spectroscopy, nuclear magnetic resonance
(NMR), combined gas chromatography/mass spectrometry (GC/MS), or other analytic methods as appropriate. Polyacrylamide gel
electrophoresis is a possible method for assessing the presence of impurities in biologically derived scaffold materials (for example,
collagen, hyaluronic acid). Impurities separated within such gels can be detected with Coomassie Blue (as a general protein stain)
or silver (as a general protein and carbohydrate stain), and characterized further by immunonblot analysis and/or protein
sequencing to identify specific impurities that may possess critical biological activities (for example, elastin immunogenicity,
cytokines and growth factors). Once characterized, such impurities can be assessed by other robust and sensitive methods well
suited to a manufacturing environment (for example, ELISA the enzyme-linked immunosorbent assay (ELISA) test for specific
substances identified by immunoblot analysis or protein sequencing.)
6.1.4 Generally, impurities are isolated more readily when the scaffold in its entirety can be solvated along with possible
contaminants. If the scaffold cannot be dissolved, exhaustive extraction with one or more solvents appropriate to the suspected
impurity is necessary.
6.1.4.1 Solvation/Dissolution—In the absence of known or established dissolution solvents for a particular material, Practice
D5226 may provide added guidance in identifying suitable potential solvents for dissolving a scaffold material. Samples should
not be dissolved in analytic solvents that can be considered as potential contaminants or create analytic interferences.
6.1.4.2 Extraction of residuals may be undertaken by methods such as Practice G120. The extract may then be concentrated and
analyzed by appropriate chromatographic analysis.
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6.1.5 The amount of any expected impurity should be quantified and the analytic detection limit reported. Both solvated and
extracted samples should provide results that specify the amount of expected impurity per mass of test sample in either percentage,
ppm (μg/g;mg/kg), ppb (ng/g;μg/kg), or other appropriate units.
6.1.6 The following analytic methods may be applicable in the determination and quantification of potential impurities:
6.1.6.1 Gas chromatography (GC) may be used for the routine detection of volatile relatively low molecular mass (formerly
known as molecular weight) impurities or contaminants. Some methods that may prove suitable include Test MethodF1884.
6.1.6.2 Gas chromatography can be coupled with both quantitative and qualitative analytic methods such as IR or MS to provide
compositional identification while quantitatively detecting low molecular mass volatile impurities or contaminants. Some
particular methods that may prove useful include Test Method D6420 and Practice E1642.
6.2 Molar Mass Determination:
NOTE 2—The term molecular weight (abbreviated MW) is obsolete and should be replaced by the SI (Système Internationale) equivalent of either
relative molecular mass (Mr), which reflects the dimensionless ratio of the mass of a single molecule to an atomic mass unit (see ISO 80000–9), or molar
mass (M), which refers to the mass of a mole of a substance and is typically expressed as grams/mole. For polymers and other macromolecules, use of
the symbols Mw, Mn, and Mz continue, referring to mass-average molar mass, number-average molar mass, and z-average molar mass, respectively. For
more information regarding proper utilization of SI units, see NIST SP811.
6.2.1 For polymeric materials (synthetic or natural), the molar mass and molar mass distribution may be determined through
size exclusion chromatography (SEC) or gel permeation chromatography (GPC). Other procedures such as inherent or intrinsic
viscosity (both abbreviated with the acronym “IV”), light scattering, or membrane osmometry may be used. For protein impurities,
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) has proven robust and generally applicable. In specific instances, mass
spectrometry can provide highly accurate mass determinations as well.
6.2.2 In any of the preceding tests, the solvent solubility characteristics of the scaffold will be highly significant in allowing
determination of suitable molar mass test methods. In the absence of known or established dissolution solvents for a particular
scaffold construct, Practice D5226 provides added guidance in identifying suitable potential solvents for dissolving a particular
material.
6.2.3 The following test methods may be applicable in the determining the molar mass of the fabricated scaffold.
NOTE 3—The following GPC/SEC and IV methods are considered to be suitable for use on linear polymer systems only. Branched polymer systems
should use light-scattering techniques.
6.2.3.1 Gel Permeation Chromatography (GPC), Also Known as Size Exclusion Chromatography (SEC)—See Test Methods
D5296 and D6474 and Practices D3016 and D6579.
NOTE 4—The SEC solvent system and calibration standard polymer type should be specified with any obtained result.
6.2.3.2 Inherent Viscosity—See Practice D2857 and Test Method D4603.
NOTE 5—The test temperature, solvent system, and sample concentration should be included with any reported result.
6.2.3.3 Light Scattering—See Test Method D4001.
NOTE 6—This test method is suitable for both linear and branched polymer systems.
TABLE 1 USP Chemical Tests
USP
Test Description
Test No.
<197> Spectrophotometric identification
<231> Heavy metals
<232> Elemental Impurities—Limits
<233> Elemental Impurities—Procedures
<381> Elastomeric closures for injections—physicochemical
test procedures
<731> Loss in drying (water content)
<736> Mass spectroscopy-purity or elemental analysis
<761> Nuclear magnetic resonance-purity or component
analysis
(for example, copolymers)
<851> Spectrophotometry and light scattering (molar mass
information)
<891> Thermal analysis (purity)
<911> Viscosity (molar mass)
<921> Water determination
6.2.3.4 Melt Flow—If a scaffold or starting material is found to be insoluble after utilizing the guidance contained within
Practice D5226, melt rheology (melt flow rate) may replace the measurements of solution properties to obtain an indication of the
material’s molar mass and molar mass distributions. Potentially useful methods include Test Method D1238 and ISO
1133–1991.1133–1.
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6.3 USP Chemical Tests—See Table 1.
7. Physical Properties and Tests
NOTE 7—Physical properties are those of a compound that can change without involving a change in chemical composition (5). Physical testing
determines the physical properties of materials based on observation and measurement. Such tests include those that provide information about the
porosity, density, crystallinity, or physical surface properties of a scaffold material.
7.1 Visual Image Interpretation—Guide F2603 covers considerations needed when interpreting visual images of three-
dimensional polymeric (including collagen-based) and hydrogel structures.
7.2 Porosity Characterization—The porous macrostructure and microstructure of a scaffold exertsexert a strong influence on
both the elicited cell response and the tissue-engineered result. Guide F2450 provides an overview of available pore
characterization methods and their respective range of applicability with respect to pore sizes and material characteristics. While
Guide F2450 may indicate more suitable method(s) for a specific scaffold structure, the following test methodologies are
recommended for consideration in the evaluation and characterization of the porosity of scaffolds possessing the 50 to 500 μm pore
sizes most typical for the encouragement of cell growth within TEMPs (see X1.2 of this guide for further discussion on the nature,
significance, and potential applicability of these test methods):
7.2.1 Porosimetry (Liquid Intrusion)—Methodologies suitable for the mercury intrusion measurement of porosity include Test
Method D4404.
NOTE 8—An alternative porosimetry suitable non-wetting liquid may be utilizedused instead of mercury, provided that the resulting maximum pore
size limitation is acceptable based on scaffold design and both recognized and accounted for within the results interpretation.
7.2.1.1 The sample data recommended to be obtained and reported are as follows:
Median pore diameter and standard deviation
(based on volume)—in μm
Pore diameter range or distribution—in μm
Total intrusion (void) volume—in cm /g
Bulk density—in g/cm
Total percentage porosity
Total intrusion (void) volume (in cm /g)
= —————————————————
1 / [bulk density (in g/cm )]
7.2.2 Porometry—Methodology suitable for the capillary flow measurement of pore size and its distribution include Test
Methods E128, E1294, and F316.
7.2.2.1 The sample data recommended to be obtained and reported are maximum or bubble point pore diameter (in
micrometres); mean flow pore diameter (in micrometres); and pore size range or distribution, or both (in micrometres).
7.2.3 Pneumatic Permeability—The methodology suitable for measurement of the pneumatic permeability of a scaffold
structure includes Test Method D6539.
7.2.3.1 The sample data recommended to be obtained and reported is as follows:
Average coefficient of pneumatic permeability—report in Darcy
2 2
(0.99 μm ) or millidarcy (0.000 99 μm )
NOTE 9—In each of the aforementioned porosity, porometry, and permeability tests, bulkier samples may require modification into a thinner profile
to allow proper specimen placement into the apparatus (for example, microtome or other sectioning techniques). In such situations, the specimen thickness
should be adjusted to be as thick as practical and the test thickness as tested reported with the result. If the sample is anisotropic in nature, separate
porometry or permeability sampling profiles for each orientation is recommended.
NOTE 10—If evidence of collapse or distortion of the scaffold’s porous structure is observed as a result of the application of analytic test pressures (that
is, induced reversible or non-reversible distortions not reasonably expected under in vivo or in vitro service conditions), either method modifications (for
example, use of an alternative fluid or reduced test pressure range) or alternative pore characterization methodologies should be employed. If significant
distortion or other analytic interferences are suspected, utilization of one or more alternative characterization methods may be needed to either corroborate
or discard the obtained results.
NOTE 11—If scaffold construction can be reasonably expected to possess either bimodal (for example, both macroporosity and microporosity) or
multi-modal distribution of pore sizes, such characteristics should be both quantified and reported and, dependent on actual pore size, may require
utilization of multiple pore characterization methodologies.
7.3 Glass transition temperatures, melting temperatures, and crystallinity may have an effect on the mechanical properties of
polymer-based scaffolds. Measurement of these properties may be appropriate to ensure consistency in mechanical properties and
to identify lot-to-lot variations of scaffold materials.
7.3.1 Methodologies that may be suitable for differential scanning calorimetry (DSC) measurement of glass transition and
melting temperatures, or crystallinity of scaffolds include Test Methods D3418, E793, E794, E1356; Terminologies E473 and
E1142; and Practices E967 and E968. Other potentially relevant standards include ISO 11357–1 and ISO 11357–2.
NOTE 12—Crystallinity also may be determined by X-ray diffraction.
7.4 USP Physical Tests—See Table 2.
7.5 Other Physical Tests:
7.5.1 Water absorption characteristics may be ascertained using Test Method D570.
F2150 − 19
TABLE 2 USP Physical Tests
USP
Test Description
Test No.
<616> Bulk density and tapped density
<661> Containers—biological tests (PET, PE and
Ophthalmic polymers)
<699> Density of solids
<701> Disintegration
<741> Melting range or temperature
<776> Optical microscopy
<786> Particle size distribution by analytical
sieving
<846> Specific surface area
<941> X-ray diffraction—crystallinity
<1045> Biotechnology derived articles (may be
useful for natural materials)
<1181> Scanning electron microscopy
(characterization of surfaces)
7.5.2 Density may be assessed using Test Methods D792 if not evaluated within a porosimetry method as described in 7.2.1.
7.5.3 Surface Properties—The extent of surface characterization of a scaffold will depend on the nature of the scaffold material
and its particular use. Users are encouraged to consider Ratner, et al (6,7) for guidance about the methods of surface
characterization of scaffold starting materials, which includes determination of the surface free energy. A guide for the assessment
of the surface texture of non-porous materials is available in Guide F2791. Other methods that may be pertinent include Guides
E1078 and E1829, and Practice E996.
7.5.4 Vapor Permeability of Films—In the event the scaffold contains a film-like component, vapor permeability may be
determined using Test Method F1249. Ref (8) also contains methods potentially useful in determining film permeability.
8. Mechanical Properties and Tests
NOTE 13—Mechanical properties are those which involve a relationship between stress and strain or provide a reaction to an applied physical force
(5).
8.1 Mechanical properties that reflect scaffold loading expectations both during manufacturing processes and in the intended
clinical application should be at least understood and preferably characterized. While characterization of the macroscopic
mechanical properties of a scaffold are commonly undertaken and important, such properties can differ significantly when
measured at different length scales. Thus, it is important to understand a scaffold’s mechanical properties on multiple length scales.
–3
For example, an electrospun structure may display very different mechanical properties on a millimeter (10 m) scale when
–6
compared to the same scaffold measured on a micron (10 m) scale, while both length scales could affect and thereby contribute
to individual and collective cell behavior.
8.2 Where possible, mechanical evaluations should occur in an environment similar to the expected service condition or
expected condition of use. Sample preconditioning may be needed and can be conducted as described in PracticeGuide
F1634F2902. in vitro conditioning typically employs buffered saline solutions at 37°C as described in Test Method F1635. or ISO
13781.
8.3 Special mounting of specimens may be necessary, depending on the configuration of the scaffold and measurement
equipment variety and dimensions.
8.4 Compressive Properties—Depending on a scaffold’s physical or dimensional characteristics, its compressive properties may
be evaluated using methodology found in one or more of the following Test Methods: D695 and D1621. For length scales for which
–6 –5 –4
standards do not currently exist, a well-designed bench protocol would reveal the mechanical properties at 10 , 10 , 10 , and
-3
10 m. This may include, for example, microindentation as described in Test Method E384 and other appropriate methods, to be
determined by a justified selection of the relevant length scales.
8.5 Tensile Properties—Depending on a scaffold’s physical or dimensional characteristics, its tensile properties may be
evaluated using methodology found in one or more of the following Test Methods: D412, D638, D882, D1623, D1708,
D3039/D3039Mand, D3039/D3039M.and USP <881>.
8.6 Flexural/Bending Properties—Depending on a scaffold’s physical or dimensional characteristics, its flexural properties may
be evaluated using methodology found in one or more of the following Test Methods: D648, D747, D790, and D1388.
8.7 Creep Characteristics—If a scaffold is to be used in applications in which it is expected to maintain its mechanical properties
while under constant strain, methodology found in Test Methods D2990 may be useful.
8.7 USP Mechanical Tests—See Table 3.
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9. Biological Tests and Evaluations
9.1 For many biomaterials, the in vivo response has been thoroughly characterized by way of both clinical use and long-term
evaluations in laboratory animals. When new applications of a biomaterial or through both extensive preclinical animal evaluation
as well as long-term clinical use. When new biomaterials or substantial modifications to the physical form or performance
requirements/specifications of the biomaterial are being considered, then the recommendations and test methods described within
the following practices should be considered:
9.1.1 Practice F748; and
9.1.2 Practice F1983.
9.1.3 ISO 10993—Biological10993-1—Biological Evaluation of Medical Devices—This standard contains is the first of a series
of parts, each of which can for which ISO 10993-1 provides guidance to assist the user dependent on evaluation needs. Particularly
relevant selections for consideration in the characterization of TEMP scaffolds include the following: on evaluation needs
dependent on the material, duration in the body, and intended application. ISO 10993-1 also integrates a risk assessment approach
for determining the relevant needed testing.
9.1.3.1 Part 1—Evaluation and testing;
9.1.3.2 Part 3—Tests for genotoxicity, carcinogenicity, and reproductive toxicity;
9.1.3.3 Part 5—Tests for cytotoxicity: in vitro methods;
9.1.3.4 Part 6—Tests for local effects after implantation;
9.1.3.5 Part 9—Framework for the identification and quantification of potential degradation products;
9.1.3.6 Part 10—Tests for irritation and sensitization;
9.1.3.7 Part 11—Tests for systemic toxicity;
9.1.3.8 Part 12—Sample preparation and reference materials;
9.1.3.9 Part 13—Identification and quantification of degradation products from polymeric medical devices;
9.1.3.10 Part 16—Toxicokinetic study design for degradation products and leachables;
9.1.3.11 Part 17—Establishment of allowable limits for leachable substances;
9.1.3.12 Part 18—Chemical characterization of materials;
9.1.3.13 Part 19—Physico-chemical, morphological and topographical characterization of materials; and
9.1.3.14 Part 20—Principles and methods for immunotoxicology testing of medical devices.
9.1.4 USP <87>, USP <88>, and USP <1031>—These three USP General Chapters provide guidance regarding in vitro and
in vivo biological evaluations.
9.1.5 USP:USP <1074> and USP <1078>—These two references USP General Chapters offer guidance for safety evaluation
of and good manufacturing practices (GMP) for pharmaceutical excipients. These tests can be generally applied to medical
materials used for TEMP scaffolds.
9.1.6 Further but more specific guidance may be indicated, depending on t
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