ASTM F3510-21
(Guide)Standard Guide for Characterizing Fiber-Based Constructs for Tissue-Engineered Medical Products
Standard Guide for Characterizing Fiber-Based Constructs for Tissue-Engineered Medical Products
SIGNIFICANCE AND USE
5.1 The test methods contained herein guide characterization of the structural, physical, chemical, mechanical, and biological properties of a fiber-based construct. Such properties may be important for the success of a TEMP, especially if they affect cell retention; activity and organization; tensile strength; the delivery of bioactive agents; or the biocompatibility and bioactivity of the construct.
5.2 Tests described herein may be used for quality control during manufacturing or to assess how the product may perform its intended clinical function.
5.3 Plans for product development, product characterization, and the regulatory pathway should be discussed with the appropriate regulatory body.
SCOPE
1.1 This guide is a resource for the characterization of fiber-based constructs intended for use in a tissue-engineered medical product (TEMP). There are existing standards that broadly cover scaffolds in a more generalized fashion (Guides F2150, F2450, F2900, F2902, ISO 21560). This guide focuses specifically on fiber-based constructs.
1.2 Fiber-based constructs may be fabricated by many different methods including, but not limited to the following: electrospinning, forcespinning, meltspinning, pneumatospinning, blowspinning, melt-electrowriting, melt extrusion, wet extrusion, fused deposition, liquid crystal deposition, electrochemical alignment, drawing, spinning, knitting, weaving, braiding, powder bed fusion (laser sintering), vat photopolymerization (stereolithography), binder jetting, directed energy deposition, self-assembly (for example, fibrillogenesis), and hybrid approaches. This document is intended to address fibers made by any of these methods, although electrospun fibers are addressed in greater detail in some sections.
1.3 This guide will focus on constructs made of fibers wherein the average fiber diameter is within the range of approximately 100 nm to 100 µm.
1.4 For the purposes of this standard, a “fiber-based construct” is defined as a construct composed of slender, elongated filaments.
1.5 Units—The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard.
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, health, and environmental practices and determine the applicability of regulatory limitations 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.
General Information
- Status
- Published
- Publication Date
- 31-Mar-2021
- Technical Committee
- F04 - Medical and Surgical Materials and Devices
- Drafting Committee
- F04.42 - Biomaterials and Biomolecules for TEMPs
Relations
- Effective Date
- 15-Mar-2024
- Effective Date
- 01-Mar-2024
- Effective Date
- 15-Nov-2023
- Effective Date
- 01-Nov-2023
- Effective Date
- 01-Aug-2020
- Effective Date
- 01-Jul-2020
- Effective Date
- 01-Jun-2020
- Effective Date
- 01-Jun-2020
- Refers
ASTM F2475-20 - Standard Guide for Biocompatibility Evaluation of Medical Device Packaging Materials - Effective Date
- 01-Jan-2020
- Effective Date
- 01-Nov-2019
- Effective Date
- 01-Nov-2019
- Effective Date
- 01-Oct-2019
- Effective Date
- 01-Oct-2019
- Effective Date
- 01-Sep-2019
- Effective Date
- 01-Jul-2019
Overview
ASTM F3510-21 is the official standard guide developed by ASTM International for the characterization of fiber-based constructs intended for use in tissue-engineered medical products (TEMPs). Fiber-based constructs are widely used as scaffolds in regenerative medicine, providing essential support for cell growth and tissue development. This standard specifically addresses the unique aspects and requirements of fiber-based constructs, distinguishing itself from more general scaffold standards. The guide details recommended test methods and key attributes to evaluate these constructs, ensuring consistency, quality, and informed product development for medical applications.
Key Topics
Characterization Methods:
The guide provides approaches to assess the structural, physical, chemical, mechanical, and biological properties of fiber-based constructs. These properties are essential as they influence cell retention, activity, organization, tensile strength, delivery of bioactive agents, and overall biocompatibility.Structural Attributes:
- Porosity: Critical for nutrient and fluid flow, influencing cell migration and construct performance.
- Pore Structure: Focuses on how "pore size" or void volumes within fiber-based constructs should be defined and measured.
- Fiber Diameter & Orientation: Measurement of fiber diameter distribution and orientation impacts mechanical properties and biological function.
- Mat Thickness: Consistency in thickness supports reproducibility in scaffold performance.
Fabrication Methods:
Fiber-based constructs can be produced through electrospinning, meltspinning, wet extrusion, weaving, knitting, braiding, self-assembly, additive manufacturing, and more. The guide covers constructs made by any of these methods, with special attention to electrospun fibers.Quality Control and Regulatory Considerations:
The standard emphasizes the role of characterization in quality control during manufacturing as well as in evaluating clinical function. It highlights the necessity of consulting with regulatory bodies during product development.
Applications
ASTM F3510-21 is crucial for the following tissue engineering and regenerative medicine applications:
Preclinical and Clinical Scaffold Evaluation:
By standardizing the assessment of fiber-based constructs, it ensures products meet necessary benchmarks for structural integrity, biocompatibility, and consistent performance.Quality Control in Manufacturing:
Manufacturers use the guide to verify product properties batch-to-batch, improving reproducibility and safety.Product Development and Regulatory Submissions:
Developers follow the characterization requirements to facilitate smoother regulatory approvals for TEMPs, ensuring that the constructs meet both function and safety standards.Research and Development:
Academic and industry researchers rely on the guide for reproducible scaffold characterization in developing new regenerative therapies.
Related Standards
Several standards complement ASTM F3510-21 for the characterization and testing of fiber-based and general scaffold constructs:
- ASTM F2150: Guide for characterization and testing of biomaterial scaffolds for TEMPs
- ASTM F2450: Guide for assessing microstructure of polymeric scaffolds for tissue-engineered products
- ASTM F2900 & F2902: Guides for characterization of hydrogels and absorbable polymeric implants
- ASTM F2603, F2664, F2739: Guides for evaluating cell attachment, cell viability, and surface texture
- ISO 21560: General requirements for tissue-engineered medical products
- ISO 10993 Series: Biological evaluation of medical devices, including cytotoxicity, irritation, and systemic toxicity
Practical Value
Adherence to ASTM F3510-21 enhances comparability between products, improves quality assurance, and supports regulatory compliance. By following the guide’s framework and characterization protocols, stakeholders in tissue engineering - including manufacturers, researchers, and regulators - can ensure that fiber-based medical scaffolds are safe, effective, and reliable for clinical use. Employing this standard also facilitates innovation and technology transfer in the growing field of tissue-engineered medical products.
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Frequently Asked Questions
ASTM F3510-21 is a guide published by ASTM International. Its full title is "Standard Guide for Characterizing Fiber-Based Constructs for Tissue-Engineered Medical Products". This standard covers: SIGNIFICANCE AND USE 5.1 The test methods contained herein guide characterization of the structural, physical, chemical, mechanical, and biological properties of a fiber-based construct. Such properties may be important for the success of a TEMP, especially if they affect cell retention; activity and organization; tensile strength; the delivery of bioactive agents; or the biocompatibility and bioactivity of the construct. 5.2 Tests described herein may be used for quality control during manufacturing or to assess how the product may perform its intended clinical function. 5.3 Plans for product development, product characterization, and the regulatory pathway should be discussed with the appropriate regulatory body. SCOPE 1.1 This guide is a resource for the characterization of fiber-based constructs intended for use in a tissue-engineered medical product (TEMP). There are existing standards that broadly cover scaffolds in a more generalized fashion (Guides F2150, F2450, F2900, F2902, ISO 21560). This guide focuses specifically on fiber-based constructs. 1.2 Fiber-based constructs may be fabricated by many different methods including, but not limited to the following: electrospinning, forcespinning, meltspinning, pneumatospinning, blowspinning, melt-electrowriting, melt extrusion, wet extrusion, fused deposition, liquid crystal deposition, electrochemical alignment, drawing, spinning, knitting, weaving, braiding, powder bed fusion (laser sintering), vat photopolymerization (stereolithography), binder jetting, directed energy deposition, self-assembly (for example, fibrillogenesis), and hybrid approaches. This document is intended to address fibers made by any of these methods, although electrospun fibers are addressed in greater detail in some sections. 1.3 This guide will focus on constructs made of fibers wherein the average fiber diameter is within the range of approximately 100 nm to 100 µm. 1.4 For the purposes of this standard, a “fiber-based construct” is defined as a construct composed of slender, elongated filaments. 1.5 Units—The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard. 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, health, and environmental practices and determine the applicability of regulatory limitations 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.
SIGNIFICANCE AND USE 5.1 The test methods contained herein guide characterization of the structural, physical, chemical, mechanical, and biological properties of a fiber-based construct. Such properties may be important for the success of a TEMP, especially if they affect cell retention; activity and organization; tensile strength; the delivery of bioactive agents; or the biocompatibility and bioactivity of the construct. 5.2 Tests described herein may be used for quality control during manufacturing or to assess how the product may perform its intended clinical function. 5.3 Plans for product development, product characterization, and the regulatory pathway should be discussed with the appropriate regulatory body. SCOPE 1.1 This guide is a resource for the characterization of fiber-based constructs intended for use in a tissue-engineered medical product (TEMP). There are existing standards that broadly cover scaffolds in a more generalized fashion (Guides F2150, F2450, F2900, F2902, ISO 21560). This guide focuses specifically on fiber-based constructs. 1.2 Fiber-based constructs may be fabricated by many different methods including, but not limited to the following: electrospinning, forcespinning, meltspinning, pneumatospinning, blowspinning, melt-electrowriting, melt extrusion, wet extrusion, fused deposition, liquid crystal deposition, electrochemical alignment, drawing, spinning, knitting, weaving, braiding, powder bed fusion (laser sintering), vat photopolymerization (stereolithography), binder jetting, directed energy deposition, self-assembly (for example, fibrillogenesis), and hybrid approaches. This document is intended to address fibers made by any of these methods, although electrospun fibers are addressed in greater detail in some sections. 1.3 This guide will focus on constructs made of fibers wherein the average fiber diameter is within the range of approximately 100 nm to 100 µm. 1.4 For the purposes of this standard, a “fiber-based construct” is defined as a construct composed of slender, elongated filaments. 1.5 Units—The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard. 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, health, and environmental practices and determine the applicability of regulatory limitations 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.
ASTM F3510-21 is classified under the following ICS (International Classification for Standards) categories: 11.040.40 - Implants for surgery, prosthetics and orthotics. The ICS classification helps identify the subject area and facilitates finding related standards.
ASTM F3510-21 has the following relationships with other standards: It is inter standard links to ASTM F2791-24, ASTM E96/E96M-24, ASTM E96/E96M-23, ASTM D854-23, ASTM F2603-06(2020), ASTM D3787-16(2020), ASTM F1249-20, ASTM D4833/D4833M-07(2020), ASTM F2475-20, ASTM F316-03(2019), ASTM E128-99(2019), ASTM F2150-19, ASTM F2664-19, ASTM F2739-19, ASTM D1777-96(2019). Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
ASTM F3510-21 is available in PDF format for immediate download after purchase. The document can be added to your cart and obtained through the secure checkout process. Digital delivery ensures instant access to the complete standard document.
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: F3510 − 21
Standard Guide for
Characterizing Fiber-Based Constructs for Tissue-
Engineered Medical Products
This standard is issued under the fixed designation F3510; 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 1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This guide is a resource for the characterization of
ization established in the Decision on Principles for the
fiber-based constructs intended for use in a tissue-engineered
Development of International Standards, Guides and Recom-
medical product (TEMP). There are existing standards that
mendations issued by the World Trade Organization Technical
broadly cover scaffolds in a more generalized fashion (Guides
Barriers to Trade (TBT) Committee.
F2150, F2450, F2900, F2902, ISO 21560). This guide focuses
specifically on fiber-based constructs.
2. Referenced Documents
1.2 Fiber-based constructs may be fabricated by many
2.1 ASTM Standards:
different methods including, but not limited to the following:
C1559 Test Method for Determining Wicking of Fibrous
electrospinning, forcespinning, meltspinning,
Glass Blanket Insulation (Aircraft Type)
pneumatospinning, blowspinning, melt-electrowriting, melt
D257 Test Methods for DC Resistance or Conductance of
extrusion, wet extrusion, fused deposition, liquid crystal
Insulating Materials
deposition, electrochemical alignment, drawing, spinning,
D412 Test Methods forVulcanized Rubber andThermoplas-
knitting, weaving, braiding, powder bed fusion (laser
tic Elastomers—Tension
sintering), vat photopolymerization (stereolithography), binder
D638 Test Method for Tensile Properties of Plastics
jetting,directedenergydeposition,self-assembly(forexample,
D648 Test Method for Deflection Temperature of Plastics
fibrillogenesis), and hybrid approaches. This document is
Under Flexural Load in the Edgewise Position
intended to address fibers made by any of these methods,
D695 Test Method for Compressive Properties of Rigid
although electrospun fibers are addressed in greater detail in
Plastics
some sections.
D790 Test Methods for Flexural Properties of Unreinforced
1.3 This guide will focus on constructs made of fibers
and Reinforced Plastics and Electrical Insulating Materi-
wherein the average fiber diameter is within the range of
als
approximately 100 nm to 100 µm.
D792 Test Methods for Density and Specific Gravity (Rela-
tive Density) of Plastics by Displacement
1.4 For the purposes of this standard, a “fiber-based con-
D854 Test Methods for Specific Gravity of Soil Solids by
struct”isdefinedasaconstructcomposedofslender,elongated
Water Pycnometer
filaments.
D882 Test Method for Tensile Properties of Thin Plastic
1.5 Units—The values stated in SI units are to be regarded
Sheeting
as the standard. No other units of measurement are included in
D1388 Test Method for Stiffness of Fabrics
this standard.
D1621 Test Method for Compressive Properties of Rigid
1.6 This standard does not purport to address all of the Cellular Plastics
D1623 Test Method for Tensile and Tensile Adhesion Prop-
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- erties of Rigid Cellular Plastics
D1708 Test Method forTensile Properties of Plastics by Use
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use. of Microtensile Specimens
D1777 Test Method for Thickness of Textile Materials
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee For referenced ASTM standards, visit the ASTM website, www.astm.org, or
F04.42 on Biomaterials and Biomolecules for TEMPs. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved April 1, 2021. Published April 2021. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
F3510-21. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3510 − 21
D1876 Test Method for Peel Resistance of Adhesives (T- F2027 Guide for Characterization and Testing of Raw or
Peel Test) Starting Materials for Tissue-Engineered Medical Prod-
D1894 Test Method for Static and Kinetic Coefficients of ucts
F2150 Guide for Characterization and Testing of Biomate-
Friction of Plastic Film and Sheeting
rial Scaffolds Used in Regenerative Medicine and Tissue-
D2256/D2256M Test Method forTensile Properties ofYarns
Engineered Medical Products
by the Single-Strand Method
F2212 Guide for Characterization of Type I Collagen as
D2990 Test Methods for Tensile, Compressive, and Flexural
Starting Material for Surgical Implants and Substrates for
Creep and Creep-Rupture of Plastics
Tissue Engineered Medical Products (TEMPs)
D3039/D3039M Test Method forTensile Properties of Poly-
F2256 Test Method for Strength Properties of Tissue Adhe-
mer Matrix Composite Materials
sives in T-Peel by Tension Loading
D3418 Test Method for Transition Temperatures and En-
F2450 Guide for Assessing Microstructure of Polymeric
thalpies of Fusion and Crystallization of Polymers by
Scaffolds for Use in Tissue-Engineered Medical Products
Differential Scanning Calorimetry
F2475 Guide for Biocompatibility Evaluation of Medical
D3786/D3786M Test Method for Bursting Strength of Tex-
Device Packaging Materials
tile Fabrics—Diaphragm Bursting StrengthTester Method
F2477 Test Methods for in vitro Pulsatile Durability Testing
D3787 Test Method for Bursting Strength of Textiles—
of Vascular Stents
Constant-Rate-of-Traverse (CRT) Ball Burst Test
F2529 Guide for in vivo Evaluation of Osteoinductive Po-
D4404 Test Method for Determination of Pore Volume and
tential for Materials Containing Demineralized Bone
Pore Volume Distribution of Soil and Rock by Mercury
(DBM)
Intrusion Porosimetry
F2603 Guide for Interpreting Images of Polymeric Tissue
D4496 Test Method for D-C Resistance or Conductance of
Scaffolds
Moderately Conductive Materials
F2606 Guide for Three-Point Bending of Balloon Expand-
D4833/D4833M Test Method for Index Puncture Resistance
able Vascular Stents and Stent Systems
of Geomembranes and Related Products
F2664 Guide for Assessing the Attachment of Cells to
D6420 Test Method for Determination of Gaseous Organic
Biomaterial Surfaces by Physical Methods
Compounds by Direct Interface Gas Chromatography-
F2739 Guide for Quantifying Cell Viability and Related
Mass Spectrometry
Attributes within Biomaterial Scaffolds
D6539 Test Method for Measurement of the Permeability of
F2791 Guide for Assessment of Surface Texture of Non-
Unsaturated Porous Materials by Flowing Air
Porous Biomaterials in Two Dimensions
D6701 Test Method for Determining Water Vapor Transmis-
F2900 Guide for Characterization of Hydrogels used in
sion Rates Through Nonwoven and Plastic Barriers
Regenerative Medicine (Withdrawn 2020)
D6797 Test Method for Bursting Strength of Fabrics
F2902 Guide for Assessment of Absorbable Polymeric Im-
Constant-Rate-of-Extension (CRE) Ball Burst Test
plants
D7264 Test Method for Flexural Properties of Polymer
F2952 Guide for Determining the Mean Darcy Permeability
Matrix Composite Materials
Coefficient for a Porous Tissue Scaffold
E96/E96M Test Methods for Water Vapor Transmission of
F2997 Practice for Quantification of Calcium Deposits in
Materials
Osteogenic Culture of Progenitor Cells Using Fluorescent
E128 Test Method for Maximum Pore Diameter and Perme-
Image Analysis
ability of Rigid Porous Filters for Laboratory Use
F3036 Guide for Testing Absorbable Stents
E793 Test Method for Enthalpies of Fusion and Crystalliza-
F3089 Guide for Characterization and Standardization of
tion by Differential Scanning Calorimetry
Polymerizable Collagen-Based Products and Associated
E1868 Test Methods for Loss-On-Drying by Thermogravi-
Collagen-Cell Interactions
metry
F3106 Guide for in vitro Osteoblast Differentiation Assays
F316 Test Methods for Pore Size Characteristics of Mem-
F3142 Guide for Evaluation of in vitro Release of Biomol-
brane Filters by Bubble Point and Mean Flow Pore Test
ecules from Biomaterials Scaffolds for TEMPs
F748 PracticeforSelectingGenericBiologicalTestMethods F3224 Test Method for Evaluating Growth of Engineered
for Materials and Devices
Cartilage Tissue using Magnetic Resonance Imaging
F1249 Test Method for Water Vapor Transmission Rate F3259 Guide for Micro-computed Tomography of Tissue
Through Plastic Film and Sheeting Using a Modulated Engineered Scaffolds
Infrared Sensor F3294 Guide for Performing Quantitative Fluorescence In-
F1306 Test Method for Slow Rate Penetration Resistance of tensity Measurements in Cell-based Assays with Wide-
Flexible Barrier Films and Laminates field Epifluorescence Microscopy
F3369 GuideforAssessingtheSkeletalMyoblastPhenotype
F1635 Test Method for in vitro Degradation Testing of
HydrolyticallyDegradablePolymerResinsandFabricated
Forms for Surgical Implants
F1983 Practice forAssessment of Selected Tissue Effects of 3
The last approved version of this historical standard is referenced on
Absorbable Biomaterials for Implant Applications www.astm.org.
F3510 − 21
2.2 ISO Standards: ISO 10993-20 Biological evaluation of medical devices—
ISO 2758 Paper—Determination of bursting strength Part 20: Principles and methods for immunotoxicology
ISO 2759 Board—Determination of bursting strength
testing of medical devices
ISO 7198 Cardiovascular implants and extracorporeal
ISO 10993-22 Biological evaluation of medical devices—
systems—Vascular prostheses—Tubular vascular grafts
Part 22: Guidance on nanomaterials
and vascular patches
ISO 11137-1 Sterilization of health care products—
ISO 9000 Quality management systems—Fundamentals and
Radiation—Part 1: Requirements for development, vali-
vocabulary
dation and routine control of a sterilization process for
ISO 9001 Quality management systems—Requirements
medical devices
ISO 9073-6 Textiles—Test methods for nonwovens Part 6:
ISO 11607-1 Packaging for terminally sterilized medical
Absorption
devices Part 1: Requirements for materials, sterile barrier
ISO9277 Determinationofthespecificsurfaceareaofsolids
systems and packaging systems
by gas adsorption—BET method
ISO 11607-2 Packaging for terminally sterilized medical
ISO10993-1 Biologicalevaluationofmedicaldevices—Part
devices Part 2: Validation requirements for forming,
1: Evaluation and testing within a risk management
sealing and assembly processes
process
ISO 11737-1 Sterilization of health care products—
ISO10993-2 Biologicalevaluationofmedicaldevices—Part
Microbiological methods—Part 1: Determination of a
2: Animal welfare requirements
population of microorganisms on products
ISO10993-3 Biologicalevaluationofmedicaldevices—Part
ISO 13019 Tissue-engineered medical products—
3:Tests for genotoxicity, carcinogenicity and reproductive
Quantification of sulfated glycosaminoglycans (sGAG)
toxicity
for evaluation of chondrogenesis
ISO10993-4 Biologicalevaluationofmedicaldevices—Part
ISO 13408-1 Aseptic processing of health care products—
4: Selection of tests for interactions with blood
Part 1: General requirements
ISO10993-5 Biologicalevaluationofmedicaldevices—Part
ISO 13408-2 Aseptic processing of health care products—
5: Tests for in vitro cytotoxicity
Part 2: Sterilizing filtration
ISO10993-6 Biologicalevaluationofmedicaldevices—Part
ISO 13408-3 Aseptic processing of health care products—
6: Tests for local effects after implantation
Part 3: Lyophilization
ISO10993-7 Biologicalevaluationofmedicaldevices—Part
ISO 13408-4 Aseptic processing of health care products—
7: Ethylene oxide sterilization residuals
ISO10993-9 Biologicalevaluationofmedicaldevices—Part Part 4: Clean-in-place technologies
9: Framework for identification and quantification of ISO 13408-5 Aseptic processing of health care products—
potential degradation products Part 5: Sterilization in place
ISO 10993-10 Biological evaluation of medical devices— ISO 13408-6 Aseptic processing of health care products—
Part 10: Tests for irritation and skin sensitization
Part 6: Isolator systems
ISO 10993-11 Biological evaluation of medical devices—
ISO 13408-7 Aseptic processing of health care products—
Part 11: Tests for systemic toxicity
Part 7: Alternative processes for medical devices and
ISO 10993-12 Biological evaluation of medical devices—
combination products
Part 12: Sample preparation and reference materials
ISO 13485 Medical devices—Quality management
ISO 10993-13 Biological evaluation of medical devices—
systems—Requirements for regulatory purposes
Part 13: Identification and quantification of degradation
ISO 14644-1 Cleanrooms and associated controlled
products from polymeric medical devices
environments—Part 1: Classification of air cleanliness by
ISO 10993-14 Biological evaluation of medical devices—
particle concentration
Part 14: Identification and quantification of degradation
ISO 14644-2 Cleanrooms and associated controlled
products from ceramics
environments—Part 2: Monitoring to provide evidence of
ISO 10993-15 Biological evaluation of medical devices—
cleanroom performance related to air cleanliness by par-
Part 15: Identification and quantification of degradation
ticle concentration
products from metals and alloys
ISO 14644-3 Cleanrooms and associated controlled
ISO 10993-17 Biological evaluation of medical devices—
environments—Part 3: Test methods
Part 17: Establishment of allowable limits for leachable
ISO 14644-4 Cleanrooms and associated controlled
substances
environments—Part 4: Design, construction and start-up
ISO 10993-18 Biological evaluation of medical devices—
ISO 14644-5 Cleanrooms and associated controlled
Part 18: Chemical characterization of materials
environments—Part 5: Operations
ISO 10993-19 Biological evaluation of medical devices—
ISO 14644-7 Cleanrooms and associated controlled
Part19:Physico-chemical,morphologicalandtopographi-
environments—Part 7: Separative devices (clean air
cal characterization of materials
hoods, gloveboxes, isolators and mini-environments)
ISO 14644-8 Cleanrooms and associated controlled
4 environments—Part 8: Classification of air cleanliness by
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
4th Floor, New York, NY 10036, http://www.ansi.org. chemical concentration (ACC)
F3510 − 21
ISO 14644-9 Cleanrooms and associated controlled 21 CFR 820 Quality System Regulation
environments—Part 9: Classification of surface cleanli- 21 CFR 1271 Human Cells, Tissues, and Cellular and
ness by particle concentration Tissue-Based Products
ISO 14644-10 Cleanrooms and associated controlled 21 CFR 1271.210 Human Cells, Tissues, and Cellular and
environments—Part 10: Classification of surface cleanli- Tissue-Based Products; Supplies and Reagents
ness by chemical concentration BS 3424-18 Testing Coated Fabrics—Part 18: Methods 21A
ISO 14644-13 Cleanrooms and associated controlled and 21B: Methods for Determination of Resistance to
environments—Part 13: Cleaning of surfaces to achieve Wicking and Lateral Leakage to Air
defined levels of cleanliness in terms of particle and FDAGuidance on 10993-1 Guidance for Industry and Food
chemical classifications and DrugAdministration Staff: Use of International Stan-
ISO 14644-14 Cleanrooms and associated controlled dard ISO 10993-1, “Biological evaluation of medical
environments—Part 14: Assessment of suitability for use devices—Part 1: Evaluation and testing within a risk
of equipment by airborne particle concentration management process” https://www.fda.gov/media/85865/
ISO 14644-15 Cleanrooms and associated controlled download
environments—Part 15: Assessment of suitability for use FDAGuidanceonGMPforCombinationProducts Guidance
of equipment and materials by airborne chemical concen- for Industry and FDAStaff: Current Good Manufacturing
tration Practice Requirements for Combination Products, https://
ISO 14698-1 Cleanrooms and associated controlled www.fda.gov/media/90425/download
environments—Biocontamination control Part 1: General FDA Guidance on Validation Guidance for Industry: Ana-
principles and methods lytical Procedures and Methods Validation for Drugs and
ISO 14698-2 Cleanrooms and associated controlled Biologics, https://www.fda.gov/media/87801/download
environments—Biocontamination control Part 2: Evalua- FDA Guidance on Surgical Meshes Guidance for Industry
tion and interpretation of biocontamination data and/or for FDA Reviewers/Staff and/or Compliance:
ISO 14971 Medical devices—Application of risk manage- Guidance for the Preparation of a Premarket Notification
ment to medical devices Application for a Surgical Mesh, https://www.fda.gov/
ISO 16379 Tissue-engineered medical products— media/71828/download
Evaluation of anisotropic structure of articular cartilage ICH Q2(R1) International Conference on Harmonisation,
using DT (Diffusion Tensor)-MR Imaging Validation of Analytical Procedures: Text and Methodol-
ISO 19074 Leather—Physical and mechanical tests— ogy Q2(R1), https://www.ich.org/page/quality-guidelines
Determination of water absorption by capillary action ICH Q7 International Conference on Harmonisation, Good
(wicking) Manufacturing Practice for Active Pharmaceutical Ingre-
ISO 19090 Tissue-engineered medical products—Bioactive dients Q7, https://www.ich.org/page/quality-guidelines
ceramics—Method to measure cell migration in porous NIST Special Publication 960-17 Porosity and Specific Sur-
materials face Area Measurements for Solid Materials
ISO 19997 Guidelines for good practices in zeta-potential NIST SRM 1898 Titanium Dioxide Nanomaterial, Certifi-
measurement cate of Analysis, https://www.nist.gov/srm
ISO 20399-1 Biotechnology—Ancillary materials present NIST SRM 1900 Silicon Nitride Powder-Specific Surface
during the production of cellular therapeutic products— Area Standard, Certificate of Analysis, https://
Part 1: General requirements www.nist.gov/srm
ISO 20399-2 Biotechnology—Ancillary materials present NIST SRM 1917 Mercury Porosimetry Standard, https://
during the production of cellular therapeutic products— www.nist.gov/srm
Part 2: Best practice guidance for ancillary material NIST SRM 2206 Controlled Pore Glass—BET Specific
suppliers Surface Area (Nominal Pore Diameter 300 nm), Certifi-
ISO 20399-3 Biotechnology—Ancillary materials present cate of Analysis, https://www.nist.gov/srm
during the production of cellular therapeutic products— NIST SRM 2207 Controlled Pore Glass—BET Specific
Part 3: Best practice guidance for ancillary material users SurfaceArea (Nominal Pore Diameter 18 nm), Certificate
ISO 21560 General requirements of tissue-engineered medi- of Analysis, https://www.nist.gov/srm
cal products NIST SRM 2696 Silica Fume (Powder Form), Certificate of
Analysis, https://www.nist.gov/srm
2.3 Other Documents:
PDA Technical Report 13-2 Fundamentals of an Environ-
21 CFR 210 Current Good Manufacturing Practice in
mental Monitoring Program Annex 1: Environmental
Manufacturing,Processing,Packing,orHoldingofDrugs;
Monitoring of Facilities Manufacturing Low Bioburden
General
21 CFR 211 Current Good Manufacturing Practice for Fin-
ished Pharmaceuticals
Available from British Standards Institution (BSI), 389 Chiswick High Rd.,
London W4 4AL, U.K., http://www.bsigroup.com.
Available from U.S. Food and Drug Administration (FDA), 10903 New
Available from U.S. Government Printing Office, Superintendent of Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http:// Available from National Institute of Standards and Technology (NIST), 100
www.access.gpo.gov. Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://www.nist.gov.
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Products, https://webstore.ansi.org/standards/pda/ 5.2 Tests described herein may be used for quality control
pdatr132020 during manufacturing or to assess how the product may
USP <71> Sterility Tests perform its intended clinical function.
USP <85> Bacterial Endotoxins Test
5.3 Plans for product development, product
USP <161> Medical Devices—Bacterial Endotoxin and Py-
characterization, and the regulatory pathway should be dis-
rogen Tests
cussed with the appropriate regulatory body.
USP <467> Residual Solvents
USP <861> Sutures—Diameter
6. Structural Characterization
USP <881> Tensile Strength
6.1 General Considerations:
USP<1043> Ancillary Materials for Cell, Gene, andTissue-
6.1.1 Structure may be the most important attribute of
Engineered Products
fiber-based constructs. It is the fiber-based structure that makes
these constructs attractive for biomedical applications. The
3. Terminology
fiber structure can mimic that of a native extracellular matrix
3.1 Definitions of Terms Specific to This Standard:
that provides a supportive niche for cells. The gross geometry
3.1.1 fiber-based construct, n—a construct composed of
of a fiber-based construct is often planar, which makes them
slender, elongated filaments intended for use in biological
useful as barrier membranes and as scaffolds for epithelial
applications, such as a tissue-engineering scaffold.
tissues (guided tissue regeneration, dental, dura mater), tubular
3.1.2 nonwoven fiber mat, n—a textile structure held to-
structures, or filamentous structures (urethra, bladder,
getherbyinterlockingoffibersinarandomweb,accomplished
esophagus, tendon, ligament). Fiber-based constructs typically
by mechanical, chemical, thermal, or solvent means. (http://
haveasignificantvoidvolumewhichmakesthempermeableto
www.fabriclink.com/dictionaries/textile.cfm#N)
biological fluids, cell culture medium, nutrients, ions, small
molecules, and proteins.
3.1.3 yarn, n—a continuous strand of textile fibers created
6.1.2 Many fiber-based constructs, such as those made by
when a cluster of individual fibers are twisted together.
electrospinning, are nonwoven. The fibers may lay down upon
(http://www.fabriclink.com/dictionaries/textile.cfm#N)
one another creating a structure that resembles a bowl of
4. Summary of Guide
noodles (Fig. 1(a)). Fiber-based constructs often have an
irregularly shaped void volume that does not have a typical
4.1 The structural, mechanical, physical, chemical, and
“pore” with a repeating structure. Nonwovens are often
biological properties of the fiber-based constructs will influ-
anisotropic, since the long axes of the fibers typically extend
ence their function in tissue-engineered medical products
the in the X- and Y-direction with fibers stacking upon one
(TEMPs).Itistheintentofthisguidetoprovideacompendium
another in the Z-direction.
of techniques for characterizing fiber-based constructs for use
6.1.3 Fiber-based constructs, such as electrospun mats, may
in TEMPs. Application of the test methods contained within
have the consistency of fabrics, whereby they are thin and
this guide does not guarantee clinical success of a finished
pliable.
product but will help to ensure consistency in the properties,
6.1.4 Fiber-based constructs must be handled carefully and
characterization of a given construct, and meaningful compari-
consistently. The constructs are often delicate and their prop-
son between constructs using consistent test methodologies.
erties may be affected by their handling. They can be suscep-
This guide does not suggest that all of the listed tests be
tibletoperturbationsbyfingersortweezersduringsimpletasks
conducted.The decision regarding applicability of any particu-
such as transfering from one container to another. This minute
lar test method is the responsibility of the developer and will
damage may manifest during imaging, structural
depend on the intended use.
measurements, or mechanical tests. Cells may respond to
4.2 The reader should be aware of a guidance document
surface features caused by handling perturbations.
issued by the U.S. Food and Drug Administration (FDA) for
6.1.5 When fiber-based constructs are used for their in-
surgical meshes that may apply to some fiber-based constructs
tended clinical indication, they are likely to experience me-
(FDA Guidance on Surgical Meshes).
chanical forces. This ought to be considered when planning
how to characterize their structure. Porosity assessed under
5. Significance and Use
zero load may be higher than when a clinically relevant load is
5.1 The test methods contained herein guide characteriza-
applied. It may be helpful to assess structural attributes when
tion of the structural, physical, chemical, mechanical, and
the construct is under a clinically relevant load.Application of
biologicalpropertiesofafiber-basedconstruct.Suchproperties
aclinicallyrelevantloadcouldaffectporosityandtheabilityof
may be important for the success of a TEMP, especially if they
cells or solutes to penetrate the construct.
affect cell retention; activity and organization; tensile strength;
6.2 Key Structural Attributes:
the delivery of bioactive agents; or the biocompatibility and
6.2.1 Porosity—Porosity is the fraction of the total scaffold
bioactivity of the construct.
volume that is void space. It is defined as follows: Porosity =
(V / V )=[V /(V + V )]. Porosity is calculated by dividing
V T V V F
Available from Parenteral Drug Association (PDA), 4350 East West Highway,
the void volume (V ) by the total scaffold volume (V ), where
V T
Suite 600, Bethesda, MD 20814, http://www.pda.org.
V is the sum of the void volume (V ) and the volume of the
Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook T V
Pkwy., Rockville, MD 20852-1790, http://www.usp.org. fibers (V ). Porosity is important for function since it will
F
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(a)SEMofamatofelectrospunpolymerfibers(PhotoCredit:NathanHotaling).Therearenothrough-holesvisibleintheimage.Determiningtheporesizebymeasuring
the distance between two arbitrarily chosen fibers, as shown in the image, may not be meaningful. (b) SEM of a woven mesh of fiber bundles. The mesh has onlyafew
layers of fibers and through-holes are present (indicated by asterisk). The dimensions of the through-holes (short axis 203 µm; long axis 476 µm) represent more
meaningful measurements of a pore size. Image is used with permission from Xu and Simon (1). (c) SEM of a collagen braid. Collagen fibers were manufactured by
microfluidic wet extrusion into a continuous yarn that was subsequently braided (Photo Credit: Michael Francis).
FIG. 1 Defining “Pore Size” for Fiber-Based Constructs
influence the flow of liquid and nutrients through the 6.2.1.2 Defining Porosity—For fiber-based constructs, the
constructs, cell migration into the construct, and mechanical determined porosity value is dependent upon how the user
properties. defines porosity. The user must think carefully about how to
6.2.1.1 Void Volume—Thevoidvolume(V )orvoidfraction define porosity for their construct. Since fiber mats are typi-
V
of a fiber-based construct is the empty regions within a cally thin in the Z-direction (like a fabric), small deviations in
construct that occupy the spaces between the fibers. For a defining the location of the top and bottom surfaces of the
nonwoven structure, the void volume is an irregular and scaffold may have large effects on the volume calculation.This
continuous volume that is not broken into discrete pores. concept is illustrated in Fig. 2, which shows an example of a
The edge of the fiber mat was created by immersing in liquid nitrogen and slicing with a razor blade. The same micrograph is shown in all panels. (a) The yellow
arrowheadindicatespinchingoftheedgeoffibermatthatoccursduringslicing.(b)Thefibermatsdonothaveaconsistentthickness.Themeasuredthicknesswilldepend
upon the location at which the thickness is measured. The double-sided arrows show the thickness range. (c) The thickness could be approximated as indicated by the
red lines. (d) The thickness could be determined at multiple positions and averaged. If image analysis routines were used for the analysis, then thickness determination
would depend on the algorithm (Photo Credit: Wojtek Tutak).
FIG. 2 Determining Thickness of a Mat of Airbrushed Poly(D,L-Lactic Acid) Fibers By Observing the Edge of the Mat By Scanning
Electron Microscopy (SEM)
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fiber mat and the variability in the different ways that the morphology must be considered when measuring fiber diam-
scaffold thickness could be defined. eter. Fibers are typically cylindrical, but elliptical, ribbon-
6.2.2 Pore Structure:
shaped, and irregularly shaped fibers have been fabricated.The
6.2.2.1 The term “pore size” may be confusing when
consistency of the fiber diameter may be a measure of
applied to fiber-based constructs. The term “pore size” implies
consistent manufacturing. Fiber diameter variation across
that a construct has voids or pockets, with a characteristic and
batches may be an indicator that the raw materials are not
repeating size and shape. Constructs made by electrospinning
homogeneous or that instabilities are present at the spinneret.
arenonwovensanddonothaveporesinthetraditionalsenseof
6.2.4 Fiber Orientation—Fibers can be randomly dispersed
the term, but instead have a continuous void volume which
or aligned to varying degrees. Electrospun fibers are often
surrounds the fibers (Fig. 1(a)). The void volume of nonwoven
deposited into a nonwoven mat in a random alignment.
fiber-based constructs is irregular and lacks a repeating struc-
Electrospun fibers can be aligned through deposition onto a
ture. In addition, the void volume of fiber-based constructs is
spinning mandrel during the electrospinning process. Melt-
anisotropic whereby the distances between fibers in the X- and
extruded fibers can be deposited into structured constructs
Y-directions are typically larger than the distances between
through additive manufacturing mechanisms. Yarns of com-
fibers in the Z-direction.The term “void structure” might make
posite fibers may be plied or twisted together, while fibers and
more sense when discussing fiber-based constructs. However,
yarns may be braided with discrete fiber alignments. Fiber
the term “pore size” is embedded in the lexicon and is difficult
alignment can be advantageous for a given indication. For
to avoid, especially when discussing test methods for assessing
example, aligned fibers have anisotropic mechanical properties
structure.
that may be useful for tendon, while randomly oriented fibers
6.2.2.2 If a fiber-based construct has only a few layers, then
willhaveisotropicmechanicalpropertiesthatmightbesuitable
through-holes may be present. The through-holes may have a
for planar tissues such as epithelium or bladder.
repeating shape with a characteristic size and may be more
6.2.5 Mat Thickness—Fiber-based constructs are often pla-
appropriately described as pores (Fig. 1(b)). Electrospun fiber
nar. The mat thickness is the thickness along the short axis
mats may not have through-holes.
perpendicular to the plane of the mat. Consistent mat thickness
6.2.2.3 Whenreportingporestructure,itiscriticaltoclearly
is an indicator of consistent manufacturing. In addition, con-
describehowporestructureisbeingdefinedandhowitisbeing
struct permeability, construct degradation, and cell infiltration
measured. Many test methods report a pore size based on
into constructs may depend upon mat thickness. Mat thickness
volume, pressure, or flow measurements for liquids and gases
is also a key input value for determination of porosity by
thatareusedtofillorflowthroughthevoidsofaconstruct.The
gravimetric methods. See Table 1.
user must consider what these “pore size” values mean for a
fiber-based construct that has an ill-defined void structure that
6.2.6 Attributes of Individual Fibers—The properties of the
lacks pores with a repeating, uniform structure.
individual fibers themselves may be important. There may be
6.2.2.4 Pore structure and pore size are important since they
pores within the fibers, the fibers may have their own surface
affect diffusion of solutes in a construct and the ability of cells
texture, and fibers may have a core sheath morphology, as can
to penetrate a construct.
be obtained from coaxial electrospinning designs.
6.2.3 Fiber Diameter—Fiber diameter is the cross-sectional
6.3 Structural Measurements:
thickness of a fiber. This attribute of fiber-based constructs is
6.3.1 Scanning Electron Microscopy—Scanning electron
probably the easiest to quantify and the most commonly
quantified. It is important to measure the diameter of many microscopy (SEM) is probably the most commonly used
method to assess the structure of fiber-based constructs. Dry
fibersinaconstructtoprovideanestimateofthefiberdiameter
distribution, since the diameter of fibers in fiber-based con- constructs may be sputter-coated with a thin layer of gold or
structs often have a wide range of diameters. The fiber other material to improve SEM contrast. SEM images can
A,B,C
TABLE 1 Common Measurement Methods for Assessing Key Structural Attributes of Fiber-Based Constructs
Fiber Diameter Fiber Pore Structure Mat
Measurement Methods Porosity
(size range) Orientation (size range) Thickness
D D
Scanning electron microscopy 1 nm to 1 mm yes n/a n/a yes
Gravimetry n/a n/a yes n/a n/a
Mercury intrusion porosimetry n/a n/a yes 4 nm to 60 µm n/a
Brunauer-Emmett-Teller (BET) gas adsorption n/a n/a yes 2 nm to 300 nm n/a
Liquid extrusion porosimetry n/a n/a yes 1 µm to 1 mm n/a
Porometry/bubble point test n/a n/a n/a 100 nm to 100 µm n/a
X-ray microcomputed tomography 5 µm to 1 mm yes yes 5 µm to 1 mm yes
Confocal microscopy 1 µm to 1 mm yes yes 1 µm to 1 mm yes
Atomic force microscopy 1 nm to 1 mm yes n/a n/a yes
X-ray microscopy 1 nm to 1 mm yes yes 1 nm to 1 mm yes
Calipers n/a n/a n/a n/a yes
Laser displacement profiler n/a n/a n/a n/a yes
A
The table lists common methods, but other suitable methods for measuring the given attributes may exist.
B
Values given in the table are approximate and will depend on the characteristics of a given specimen.
C
n/a = not applicable.
D
Since SEM is a 2D imaging modality, porosity and pore structure measurements made with SEM are 2D approximations.
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achieve several pixels per nm and provide the highest resolu- diameter of 17 µm, which is large enough to be verified by
tion 2D images of fibers. orthogonal methods, including calipers and light microscopy
6.3.1.1 SEM Measurement of Fiber Diameter—SEM is well
(Fig. 3).
suited for determining fiber diameter. Image captures of fibers
6.3.1.2 The fiber samples can be tilted in the SEM to assess
can be assessed manually using a line tool in an image analysis
whether fibers have a cylindrical or elliptical morphology,
program. ImageJ and Fiji are open-source image analysis
contain a solid core, or are hollow and tube-like. This can be
software that may be used (2-4). Fiber diameter can also be
achieved by imaging the same field of view of fibers at 0° tilt
determined with automated methods which are less biased and
and 60° tilt to see if there is a difference in fiber diameter.
faster (5, 6). Automated methods can rapidly generate a lot of
6.3.1.3 SEM Measurement of Porosity or Pore Structure—
data so that histograms of fiber diameter that can be used to
SEM is often used to assess porosity and pore structure by
assess the uniformity of fiber diameter can be generated. A
measuring distances between fibers in micrographs. However,
training website is available for one of the algorithms (7).In
this approach may not be meaningful. SEM micrographs
addition, reference images of highly uniform fibers with a
present a two-dimensional view of a 3D structure and may not
known diameter are publicly available for users to ensure the
beappropriatefordetermining3Dattributessuchasporosityor
performance of analytical routines (8). The reference images
pore structure.
were generated by taking SEMs of steel wire that has a
6.3.1.4 When SEM is used to ascribe a pore size to a
nonwoven mat of fibers, what is typically being measured is
the interfiber spacing along the long axes of the fiber mat (X-
and Y-direction). If the interfiber spacing is measured by
The boldface numbers in parentheses refer to a list of references at the end of
this standard.
(a) Optical image and (b) SEM image of reference wire with a fiber diameter of 16.7 µm. (c) Histogram of fiber diameter measurements determined using algorithm #1
to analyze SEM images of the reference wire. (d) Graph comparing measurements of the fiber diameter of the reference wire. “Manufacturer’s Diameter” was reported
by the manufacturer from caliper measurements. “Light Microscope” = human analysis of bright-field images of the reference wire by manually using a line tool in analysis
software (ImageJ) (2). “Operator #1 Manual” and “Operator #2 Manual”: Human manual analysis (by two different people) of SEM images of the reference wire using
ImageJ line tool. “Algorithm #1” and “Algorithm #2”: automated analysis of SEM images of the reference wire using two different image analysis algorithms. Data are
adapted from Hotaling et al. (6). Image is used with permission from Garcia et al. (9).
FIG. 3 Use of Uniform 53-Gauge Steel Wire as a Reference Material for Validating Automated Measurements of Fiber Diameter in
Scanning Electron Micrographs (SEMs)
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scanning electron microscopy (SEM), then the fibers that are thick, a relatively large specimen should be used to reduce the
closer to the objective are more illuminated while illumination contributions of errors in the mass and dimensional measure-
is decreased for fibers that are further from the objective (Fig. ments. A 2 cm specimen may be appropriate. The length and
1(a)). Eventually, the background in SEM images appears dark width (X- andY-dimensions) of the specimen can be measured
for the fibers that have no illumination. A pore size that is
with a ruler. Since fiber mats are typically planar and thin
calculatedfromthewell-illuminatedfibersintheforegroundof (hundreds of micrometers), determining their volume is depen-
SEM micrographs arbitrarily depends upon the electron illu-
dent upon a reliable measure of the thickness of the mat.
mination depth for the given image and is not a true pore size. However, measuring the thickness of a fiber mat is challenging
and is discussed extensively in several sections below.
6.3.1.5 SEM Measurement of Mat Thickness—Thespecimen
can be immersed in liquid nitrogen to make it brittle and then
6.3.2.1 Liquid Intrusion for Measuring Porosity—Liquid
cut with a razor to expose a cross section for examination by
intrusion is a variation on the gravimetric approach for
SEM. However, polymers often compress and pinch along the
measuring porosity of fiber-based constructs whereby a liquid
cut line, similar to cutting soft bread with a dull knife (Fig. 2).
(instead of air) fills the voids (13).The mass of a dry specimen
This makes it difficult to know if the fibers have been
is determined by weighing on a balance and the specimen is
compressed by the cutting process. Analysis of the SEMs to
immersed in a liquid that doesn’t dissolve or swell the fibers.
determinematthicknessrequiresthoughtsincethethicknessof
The specimens are sonicated to facilitate diffusion of the liquid
the mat may vary. The thickness of the fiber mat shown in Fig.
into the void volume of the construct. The specimen is blotted
2(b) ranges from 107 µm to 191 µm.The top and bottom of the
onanabsorbentmaterialtoremoveexternalliquid,whilemuch
mat could be approximated (Fig. 2(c)) or multiple thickness
of the liquid within the void volume is retained. The specimen
measurements could be averaged (Fig. 2(d)). Note that differ-
isweighedagain.Themassofliquidabsorbedbythespecimen
ent users may place the red dotted lines in Fig. 2(c) and (d) in
isdeterminedbysubtractingthedrymassofthespecimenfrom
different places. A consistent process for determining the top
the mass after liquid absorption. Knowing the mass of the
and bottom of the mat in SEM images is required to get a
absorbed liquid and density of the liquid allows the liquid
reproducible measurement. Fig. 2(b) shows that the thickness
volume (V ) to be calculated. Knowing the mass of the dry
L
of fiber mats can vary locally, but thickness can also vary from
specimen and the density of the material used to make the
region to region within a sample. Multiple regions in a sample
fibersallowsthevolumeofthefibers(V )tobecalculated.The
F
should be examined for thickness to provide a more reliable
following equation can then be used to calculate the percent
fiber mat thickness measurement.
porosity: V /(V + V ).The advantage of this approach is that
L L F
6.3.1.6 SEM Measurement of Fiber Orientation—Image it avoids having to determine the volume of the construct,
analysis of SEM images of fibers can be used to determine the whichisdependentuponanaccuratemeasurementofconstruct
degree of fiber alignment. An open-source algorithm called thickness. However, the variability in the blotting process may
“OrientationJ” (10), which runs in ImageJ (2-4), may be behardtocontrol,leadingtovariabilityinthedeterminationof
effective for this metric. the liquid volume.
6.3.1.7 Environmental Scanning Electron Microscopy 6.3.2.2 Caliper Measurement of Mat Thickness—Digital
(ESEM)—ESEM allows imaging of fibers in a hydrated state. calipers can be used to measure the thickness of fiber mats.
Hydrated imaging fibers may be helpful if the fibers are However, the accuracy and consistency of the results may not
intended for use in a hydrated state, such as implantation into bereliable.Fibermatsmaybesoftandmaycompresswhenthe
a patient. This would be particularly important if the fibers are calipers are closed upon the specimen. It may not be possible
expected to swell in an aqueous medium. to reliably determine when the calipers are contacting the
specimen. There may be significant variability between mea-
6.3.1.8 SEM Data Capture—A consistent method for
surements due to differences in how much compression is
sample preparation, SEM imaging, and image capture should
applied to the specimen during measurements.
beusedforallanalyses.Allsamplesandimagecapturesshould
use the same sample mounting procedure, sputter-coating 6.3.2.3 Force Caliper Measurement of Mat Thickness—
process (if required), working distance, instrument settings Force calipers (also called “low-pressure calipers” or “spring-
(voltage), image size, and magnification. Image data should be loaded micrometers”) have a mechanism to ensure that a
saved in a noncompressed, lossless format such as “tif” (Guide consistent amount of force is applied by the calipers to the
F3294). specimen, which improves the repeatability of thickness mea-
surements. A similar mechanism is used for measuring the
6.3.2 Gravimetric Measurement of Porosity—Gravimetric
thickness of textiles which employs a thickness gauge with
analysis uses the following relationship to determine porosity:
weightstoapplyaconstantforcetothefabricusingaweighted
[(V –(M/D)] / V ; where M is mass of the construct, V is
T T T
presser foot (Test Method D1777).
volume of the construct, and D is density of the material used
to make the fibers. The fiber volume (V ) equals mass divided 6.3.2.4 Impedance Caliper Measurement of Mat
F
by density (M/D). The density of the bulk material used to Thickness—Home-builtsystemsusingdigitalcalipersthathave
makethefiberscanbeobtainedfromtheliteratureormeasured been equipped with impedance-sensing hardware can be used
(Test Methods D792 and D854). Electrospinning processes to assess the thickness of fiber mats. When the surfaces of the
may affect polymer packing and crystallinity (11, 12) which caliper contact the specimen, a change in capacitance is
may affect material density. Specimen mass can be determined detected by the sensor to make possible a repeatable measure-
on a balance. For a fiber mat that is a few hundred microns ment of mat thickness.
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6.3.2.5 Non-Contact Opt
...
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