ASTM F2603-06
(Guide)Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
SIGNIFICANCE AND USE
This document provides guidance for users who wish to obtain quantifiable data from images of tissue scaffolds manufactured from polymers that include both high water content gels and woven textiles.
Information derived from tissue scaffold images can be used to optimize the structural characteristics of the matrix for a particular application, to develop better manufacturing procedures or to provide a measure of quality assurance and product traceability. Fig. 1 provides a summary of the key stages of image capture and analysis.
There is a synergy between the analysis of pores in tissue scaffolds and that of particles that is reflected in standards cited and in the analysis described in Section 9. Guide E 1919 provides a compendium of standards for particle analysis that includes measurement techniques, data analytical and sampling methodologies.
FIG. 1 Key Stages in Image Capture, Storage, and Analysis
SCOPE
1.1 This guide covers the factors that need to be considered in obtaining and interpreting images of tissue scaffolds including technique selection, instrument resolution and image quality, quantification and sample preparation.
1.2 The information in this guide is intended to be applicable to porous polymer-based tissue scaffolds, including naturally derived materials such as collagen. However, some materials (both synthetic and natural) may require unique or varied sample preparation methods that are not specifically covered in this guide.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatory limitations prior to use.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F2603 − 06
StandardGuide for
Interpreting Images of Polymeric Tissue Scaffolds
This standard is issued under the fixed designation F2603; 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 3.1.1 aliasing, n—artifactual data that originates from an
insufficient sampling rate.
1.1 This guide covers the factors that need to be considered
3.1.2 biomaterial, n—a natural or synthetic material that is
in obtaining and interpreting images of tissue scaffolds includ-
ing technique selection, instrument resolution and image suitable for introduction into living tissue especially as part of
a medical device (as an artificial heart valve or joint).
quality, quantification and sample preparation.
3.1.3 blind (end) pore, n—a pore that is in contact with an
1.2 The information in this guide is intended to be appli-
exposedinternalwallorsurfacethroughasingleorificesmaller
cable to porous polymer-based tissue scaffolds, including
than the pore’s depth.
naturally derived materials such as collagen. However, some
materials (both synthetic and natural) may require unique or
3.1.4 closed cell, n—void within a solid, lacking any con-
varied sample preparation methods that are not specifically
nectivity with an external surface. Synonym: closed pore.
covered in this guide.
3.1.5 feret diameter, n—the mean value of the distance
1.3 This standard does not purport to address all of the
between pairs of parallel tangents to the periphery of a pore
safety concerns, if any, associated with its use. It is the
(adapted from Practice F1877).
responsibility of the user of this standard to establish appro-
3.1.6 hydrogel, n—a water-based open network of polymer
priate safety and health practices and to determine the
chains that are cross-linked either chemically or through
applicability of regulatory limitations prior to use.
crystalline junctions or by specific ionic interactions.
3.1.7 irregular, adj—an irregular pore that cannot be de-
2. Referenced Documents
scribed as round or spherical. A set of reference figures that
2.1 ASTM Standards:
define the nomenclature are given in Appendix X2. (Adapted
E1919 GuideforWorldwidePublishedStandardsRelatingto
from Practice F1877).
Particle and Spray Characterization
3.1.8 Nyquist criterion—states that a signal must be
E2245 Test Method for Residual Strain Measurements of
sampled at a rate greater than or equal to twice its highest
Thin, Reflecting Films Using an Optical Interferometer
frequency component to avoid aliasing.
F1854 Test Method for Stereological Evaluation of Porous
Coatings on Medical Implants
3.1.9 permeability, n—a measure of fluid, particle, or gas
F1877 Practice for Characterization of Particles flow through an open pore structure.
F2150 Guide for Characterization and Testing of Biomate-
3.1.10 pixel, n—two-dimensional picture element.
rial Scaffolds Used in Tissue-Engineered Medical Prod-
3.1.11 polymer, n—a long chain molecule composed of
ucts
monomers.
F2450 Guide for Assessing Microstructure of Polymeric
3.1.11.1 Discussion—A polymer may be a natural or syn-
Scaffolds for Use in Tissue-Engineered Medical Products
thetic material.
3.1.11.2 Discussion—Examples of polymers include colla-
3. Terminology
gen and polycaprolactone.
3.1 Definitions:
3.1.12 pore, n—a liquid (fluid or gas) filled externally
connecting channel, void, or open space within an otherwise
This guide is under the jurisdiction of ASTM Committee F04 on Medical and solid or gelatinous material (for example, textile meshes
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
composed of many or single fibers (textile based scaffolds),
F04.42 on Biomaterials and Biomolecules for TEMPs.
open cell foams, (hydrogels). Synonyms: open pore, through
Current edition approved Dec. 1, 2006. Published February 2007. DOI: 10.1520/
pore.
F2603-06.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
3.1.13 porosity, n—property of a solid which contains an
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
inherent or induced network of channels and open spaces.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. Porosity can be determined by measuring the ratio of pore
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2603 − 06
(void) volume to the apparent (total) volume of a porous
material and is commonly expressed as a percentage (Guide
F2150).
3.1.14 rectangular, adj—A pore that approximates a square
or rectangle in shape (derived from Practice F1877).
3.1.15 roundness (R), n—a measure of how closely an
object represents a circle (Practice F1877).
3.1.16 scaffold, n—a support, delivery vehicle, or matrix for
facilitating the migration, binding, or transport of cells or
bioactive molecules used to replace, repair, or regenerate
tissues. (Guide F2150).
3.1.17 segmentation, n—a methodology for distinguishing
different regions (for example, pores and walls) within a tissue
scaffold image.
3.1.18 sphericalpore,adj—aporewithagenerallyspherical
shape.
3.1.18.1 Discussion—A spherical pore appears round in a
photograph (Practice F1877).
3.1.19 threshold, n—isolation of a range of grayscale values
exhibited by one constituent within an image.
3.1.20 through pores, n—an inherent or induced network of
FIG. 1 Key Stages in Image Capture, Storage, and Analysis
voids or channels that permit flow of fluid from one side of the
structure to the other.
micrometres, and (3) the degree of interconnectivity and
3.1.21 tortuosity, n—a measure of the mean free path length
tortuosity of the pores.
of through pores relative to the sample thickness. Alternative
definition: The squared ratio of the mean free path to the
6. Imaging Methods and Conditions
minimum possible path length.
6.1 There are many experimental ways of obtaining key
3.1.22 voxel, n—three-dimensional picture element.
scaffold physical parameters as described in Guide F2450.
4. Significance and Use
When imaging and subsequent quantitative analysis is chosen
as the method for determining these parameters, it is critical
4.1 This document provides guidance for users who wish to
that any image under consideration be a true representation of
obtain quantifiable data from images of tissue scaffolds manu-
the scaffold of interest. Some imaging methods require sample
factured from polymers that include both high water content
preparation.Somedonot.Whensamplepreparationisrequired
gels and woven textiles.
prior to imaging, care must be taken that the procedures do not
4.2 Information derived from tissue scaffold images can be
significantly alter the morphology of the scaffold. See Appen-
used to optimize the structural characteristics of the matrix for
dix X1 for further information on sample preparation.
a particular application, to develop better manufacturing pro-
6.2 Images obtained using techniques such as light
cedures or to provide a measure of quality assurance and
microscopy, electron microscopy, and magnetic resonance
product traceability. Fig. 1 provides a summary of the key
imaging are two-dimensional (2-D) representations of a three-
stages of image capture and analysis.
dimensional (3-D) structure. These can be a planar or cross-
4.3 There is a synergy between the analysis of pores in
sectional view with a relatively large depth of field or a series
tissue scaffolds and that of particles that is reflected in
of physical or virtual 2-D slices, each with a small depth of
standards cited and in the analysis described in Section 9.
field, that can be reassembled in a virtual environment to
Guide E1919 provides a compendium of standards for particle
produce a 3-D mesostructure.
analysis that includes measurement techniques, data analytical
6.3 There are limits to the extent an image (2-D or 3-D) can
and sampling methodologies.
faithfully represent the physical artifacts that are influenced by
5. Measurement Objectives
factors germane to the imaging method, such as spatial
5.1 Much of the research activity in tissue engineering is resolution and dynamic range, image contrast, and the signal-
focusedonthedevelopmentofsuitablematerialsandstructures to-noiseratio.Table1listssomeofthetechniquesavailablefor
for optimal growth of a range of tissue types including producing images of porous structures, along with their con-
cartilage, bone, and nerve. This requires a quantitative assess- trast source, maximum demonstrated spatial resolution, and
ment of the scaffold structure. typical dynamic range. Proper technique selection depends
The key parameters that need to be determined are (1) the both on the material properties of the scaffold (that is, optical
overall level of porosity, (2) the pore size distribution, which methods cannot be used with opaque materials) the contrast
can range from tens of nanometers to several hundred available, and the target pore size range.
F2603 − 06
TABLE 1 Sources of Contrast and Techniques to Generate
in the reassembly process. However, the techniques used to
Images of Tissue Scaffolds
generate virtual 2-D images typically have limited penetration
Generic method Contrast source Maximum resolution Physical slicing
depth.
(lateral/axial) required for 3D
imaging?
6.7 Confocal microscopy (OCT), for example, has a pen-
Widefield Optical Refractive index 1 µm/(10 µm) Y etration depth of approximately 100 µm, a value that depends
Microscopy Fluorescence
on the wavelength of the light used and the amount of
Absorbance
scattering that occurs within the sample. Scanning acoustic
Confocal Optical Refractive index 0.5 µm/1 µm N
Microscopy Fluorescence
microscopy (SAM) can extend the penetration depth to ap-
Absorbance
proximately 1 mm in polymer scaffolds albeit with a reduction
Optical Coherence Refractive index 1 µm/1 µm N
in image resolution.
Tomography (or
Microscopy)
6.8 In general, using longer wavelength radiation to im-
Scanning Acoustic Acoustic 0.1 µm/0.1 µm N
Microscopy (SAM) impedance (depending on the prove penetration of the radiation is accompanied by a reduc-
wavelength chosen)
tion in resolution.
Magnetic Nuclear spin 10 µm/10 µm N
Resonance
Imaging (MRI)
7. Image Capture and Storage
X-ray Micro- Electron density 10 µm/10 µm N
Computed 7.1 Image acquisition in this guide refers to the process of
Tomography
capturing an image through digitization that is then stored for
(µ-CT)
subsequent analysis. Care should be taken during this stage to
Transmission Electron density Approximately 0.2 Y
Electron nm in plane avoid loss of fidelity by controllable factors that are not related
Microscopy (TEM)
to the methodology used to produce the image. These factors
Scanning Electron Electron density Approximately 10 nm N
include the spatial sampling frequency of the detector system,
Microscopy (SEM)
the dynamic range of analogue to digital (A/D) conversion,
segmenting (thresholding) operations (discussed in Section 9),
and both image compression and decompression.
7.2 Spatial sampling frequency and appropriate A/D con-
6.4 The images generated by the techniques shown in Table
version are straightforward issues; the sampling frequency
1 cannot reproduce features smaller than the spatial resolution
should be at least twice the inverse spatial resolution, so as to
of the method. Features that are faint, that is, those that do not
fulfill the Nyquist criterion. Sampling at frequencies below this
have significant contrast, or signal significantly above
will lead to the display of artifacts, most image processing
background, will be resolved at length scales larger than the
systems have anti-aliasing filters that remove frequencies
maximum resolution. Excessive contrast can also limit the
greater than F /2 Hz, where F is the digital sampling rate. The
s s
penetration depth due to scattering effects. This is particularly
A/D conversion should utilize a sufficient number of bits to
true of optical microscopies using differences in refractive
cover the dynamic range of the imaging / detector system.
index as the contrast mechanism. An appropriate level of
Eight-bit conversion and recording is used for most common
contrast that can be established by experimentation is therefore
imaging applications, resulting in images with 256 grayscale
critical to high quality imaging.
levels, where 0 corresponds to pure black and 255 to pure
6.5 Contrast can be enhanced by using exogenous agents,
white respectively. If 8-bit conversion is used in a color (RGB)
such as florescence tags in optical microscopy and stains
image there are 256 possible color combinations.
containing heavy metal complexes in electron microscopies.
NOTE 1—The gamut, or range of the grayscale reflects the image
Excessive contrast can be ameliorated in optical microscopies
contrast.
by imbibing the structure with a fluid that has an index of
7.3 It is important to record the minimum measurement
refraction similar to that of the solid making up the structure
value (that is, the dimensions of a single pixel) when using
(this is termed “index-matching”). There are many excellent
digital capture or digitizing film-based images at all magnifi-
resources describing factors influencing widefield and confocal
3 cations used in measurements (Test Method F1854).
optical microscopy (1-3), optical coherence microscopy (4),
MRI (5), and electron microscopies (6).
7.4 Image compression is used to facilitate rapid display of
data and easy file transmission. However, many compression
6.6 The reconstruction of the mesostructure in 3-D from a
methods (JPEG, PNG, and GIF) cause a loss of data. This loss
series of 2-D images obtained from a sample that has been
generally occurs in the high-frequency components of the
physically sectioned requires considerably more effort than
spatial Fourier spectrum of the image, leading to an oscillating,
assembly of virtual sections produced by techniques that are
smearedgrayscale,orcolorintensityprofileattheobjectedges.
able to focus on a plane within the sample. The virtual
Some proprietary compression methods are purported to in-
approach is also less prone to sample distortion since it
volve no loss of information and thus be completely reversible
obviatestheneedforphysicalsectioningandregistrationerrors
(7). There are excellent internet resources describing compres-
sion and decompression techniques (7).
7.5 After capture, an image can be manipulated to facilitate
The boldface numbers in parentheses refe
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