ASTM F2603-06(2012)
(Guide)Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
SIGNIFICANCE AND USE
4.1 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.
4.2 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.
FIG. 1 Key Stages in Image Capture, Storage, and Analysis
4.3 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 E1919 provides a compendium of standards for particle analysis that includes measurement techniques, data analytical and sampling methodologies.
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.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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
Relations
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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 (Reapproved 2012)
Standard Guide 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 F2450 Guide for Assessing Microstructure of Polymeric
Scaffolds for Use in Tissue-Engineered Medical Products
1.1 This guide covers the factors that need to be considered
in obtaining and interpreting images of tissue scaffolds includ-
3. Terminology
ing technique selection, instrument resolution and image
quality, quantification and sample preparation. 3.1 Definitions:
3.1.1 aliasing, n—artifactual data that originates from an
1.2 The information in this guide is intended to be appli-
insufficient sampling rate.
cable to porous polymer-based tissue scaffolds, including
naturally derived materials such as collagen. However, some
3.1.2 biomaterial, n—a natural or synthetic material that is
materials (both synthetic and natural) may require unique or
suitable for introduction into living tissue especially as part of
varied sample preparation methods that are not specifically
a medical device, such as an artificial heart valve or joint.
covered in this guide.
3.1.3 blind (end) pore, n—a pore that is in contact with an
1.3 The values stated in SI units are to be regarded as
exposedinternalwallorsurfacethroughasingleorificesmaller
standard. No other units of measurement are included in this
than the pore’s depth.
standard.
3.1.4 closed cell, n—void within a solid, lacking any con-
1.4 This standard does not purport to address all of the
nectivity with an external surface. Synonym: closed pore.
safety concerns, if any, associated with its use. It is the
3.1.5 feret diameter, n—the mean value of the distance
responsibility of the user of this standard to establish appro-
between pairs of parallel tangents to the periphery of a pore
priate safety and health practices and to determine the
(adapted from Practice F1877).
applicability of regulatory limitations prior to use.
3.1.6 hydrogel, n—a water-based open network of polymer
2. Referenced Documents chains that are cross-linked either chemically or through
crystalline junctions or by specific ionic interactions.
2.1 ASTM Standards:
3.1.7 irregular, adj—an irregular pore that cannot be de-
E1919 GuideforWorldwidePublishedStandardsRelatingto
scribed as round or spherical. A set of reference figures that
Particle and Spray Characterization (Withdrawn 2014)
define the nomenclature are given in Appendix X2. (Adapted
E2245 Test Method for Residual Strain Measurements of
from Practice F1877).
Thin, Reflecting Films Using an Optical Interferometer
F1854 Test Method for Stereological Evaluation of Porous
3.1.8 Nyquist criterion—a criterion that states that a signal
Coatings on Medical Implants
must be sampled at a rate greater than or equal to twice its
F1877 Practice for Characterization of Particles
highest frequency component to avoid aliasing.
F2150 Guide for Characterization and Testing of Biomate-
3.1.9 permeability, n—a measure of fluid, particle, or gas
rial Scaffolds Used in Tissue-Engineered Medical Prod-
flow through an open pore structure.
ucts
3.1.10 pixel, n—two-dimensional picture element.
3.1.11 polymer, n—a long chain molecule composed of
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
monomers.
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
3.1.11.1 Discussion—A polymer may be a natural or syn-
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Oct. 1, 2012. Published February 2007. DOI: 10.1520/
thetic material.
F2603-06.
3.1.11.2 Discussion—Examples of polymers include colla-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
gen and polycaprolactone.
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
3.1.12 pore, n—a liquid, fluid, or gas-filled externally con-
the ASTM website.
necting channel, void, or open space within an otherwise solid
The last approved version of this historical standard is referenced on
www.astm.org. or gelatinous material (for example, textile meshes composed
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2603 − 06 (2012)
of many or single fibers (textile-based scaffolds), open cell
foams, (hydrogels). Synonyms: open pore, through pore.
3.1.13 porosity, n—property of a solid which contains an
inherent or induced network of channels and open spaces.
Porosity can be determined by measuring the ratio of pore
(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 spherical pore, n—a pore with a generally spherical
shape.
FIG. 1 Key Stages in Image Capture, Storage, and Analysis
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
for optimal growth of a range of tissue types including
exhibited by one constituent within an image.
cartilage, bone, and nerve. This requires a quantitative assess-
ment of the scaffold structure.
3.1.20 through pores, n—an inherent or induced network of
The key parameters that need to be determined are (1) the
voids or channels that permit flow of fluid from one side of the
overall level of porosity, (2) the pore size distribution, which
structure to the other.
can range from tens of nanometers to several hundred
3.1.21 tortuosity, n—a measure of the mean free path length
micrometres, and (3) the degree of interconnectivity and
of through pores relative to the sample thickness. Alternative
tortuosity of the pores.
definition: The squared ratio of the mean free path to the
minimum possible path length.
6. Imaging Methods and Conditions
3.1.22 voxel, n—three-dimensional picture element.
6.1 There are many experimental ways of obtaining key
scaffold physical parameters as described in Guide F2450.
4. Significance and Use
When imaging and subsequent quantitative analysis is chosen
4.1 This document provides guidance for users who wish to
as the method for determining these parameters, it is critical
obtain quantifiable data from images of tissue scaffolds manu-
that any image under consideration be a true representation of
factured from polymers that include both high water content
the scaffold of interest. Some imaging methods require sample
gels and woven textiles.
preparation.Somedonot.Whensamplepreparationisrequired
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-
cedures or to provide a measure of quality assurance and
6.2 Images obtained using techniques such as light
product traceability. Fig. 1 provides a summary of the key
microscopy, electron microscopy, and magnetic resonance
stages of image capture and analysis.
imaging are two-dimensional (2-D) representations of a three-
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
and sampling methodologies.
6.3 There are limits to the extent an image (2-D or 3-D) can
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
F2603 − 06 (2012)
TABLE 1 Sources of Contrast and Techniques to Generate
assembly of virtual sections produced by techniques that are
Images of Tissue Scaffolds
able to focus on a plane within the sample. The virtual
Generic method Contrast source Maximum resolution Physical slicing
approach is also less prone to sample distortion since it
(lateral/axial) required for 3D
obviatestheneedforphysicalsectioningandregistrationerrors
imaging?
in the reassembly process. However, the techniques used to
Widefield Optical Refractive index 1 µm/(10 µm) Y
Microscopy Fluorescence generate virtual 2-D images typically have limited penetration
Absorbance
depth.
Confocal Optical Refractive index 0.5 µm/1 µm N
Microscopy Fluorescence
6.7 Confocal microscopy (OCT), for example, has a pen-
Absorbance
etration depth of approximately 100 µm, a value that depends
Optical Coherence Refractive index 1 µm/1 µm N
on the wavelength of the light used and the amount of
Tomography (or
Microscopy)
scattering that occurs within the sample. Scanning acoustic
Scanning Acoustic Acoustic 0.1 µm/0.1 µm N
microscopy (SAM) can extend the penetration depth to ap-
Microscopy (SAM) impedance (depending on the
wavelength chosen) proximately 1 mm in polymer scaffolds albeit with a reduction
Magnetic Nuclear spin 10 µm/10 µm N
in image resolution.
Resonance
Imaging (MRI)
6.8 In general, using longer wavelength radiation to im-
X-ray Micro- Electron density 10 µm/10 µm N
prove penetration of the radiation is accompanied by a reduc-
Computed
Tomography tion in resolution.
(µ-CT)
Transmission Electron density Approximately 0.2 Y
7. Image Capture and Storage
Electron nm in plane
Microscopy (TEM)
7.1 Image acquisition in this guide refers to the process of
Scanning Electron Electron density Approximately 10 nm N
Microscopy (SEM) capturing an image through digitization that is then stored for
subsequent analysis. Care should be taken during this stage to
avoid loss of fidelity by controllable factors that are not related
to the methodology used to produce the image. These factors
producing images of porous structures, along with their con-
include the spatial sampling frequency of the detector system,
trast source, maximum demonstrated spatial resolution, and
the dynamic range of analogue to digital (A/D) conversion,
typical dynamic range. Proper technique selection depends
segmenting (thresholding) operations (discussed in Section 9),
both on the material properties of the scaffold (that is, optical
and both image compression and decompression.
methods cannot be used with opaque materials) the contrast
available, and the target pore size range.
7.2 Spatial sampling frequency and appropriate A/D con-
version are straightforward issues; the sampling frequency
6.4 The images generated by the techniques shown in Table
should be at least twice the inverse spatial resolution, so as to
1 cannot reproduce features smaller than the spatial resolution
fulfill the Nyquist criterion. Sampling at frequencies below this
of the method. Features that are faint, that is, those that do not
will lead to the display of artifacts. Most image processing
have significant contrast, or signal significantly above
systems have anti-aliasing filters that remove frequencies
background, will be resolved at length scales larger than the
greater than F /2 Hz, where F is the digital sampling rate. The
maximum resolution. Excessive contrast can also limit the
s s
A/D conversion should utilize a sufficient number of bits to
penetration depth due to scattering effects. This is particularly
cover the dynamic range of the imaging / detector system.
true of optical microscopies using differences in refractive
Eight-bit conversion and recording is used for most common
index as the contrast mechanism. An appropriate level of
imaging applications, resulting in images with 256 grayscale
contrast that can be established by experimentation is therefore
levels, where 0 corresponds to pure black and 255 to pure
critical to high quality imaging.
white respectively. If 8-bit conversion is used in a color (RGB)
6.5 Contrast can be enhanced by using exogenous agents,
image there are 256 possible color combinations.
such as florescence tags in optical microscopy and stains
containing heavy metal complexes in electron microscopies. NOTE 1—The gamut, or range of the grayscale reflects the image
contrast.
Excessive contrast can be ameliorated in optical microscopies
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
cations used in measurements (Test Method F1854).
optical microscopy (1-3), optical coherence microscopy (4),
7.4 Image compression is used to facilitate rapid display of
MRI (5), and electron microscopies (6).
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,
smearedgrayscale,orcolorintensityprofileattheobjectedges.
Some proprietary compression me
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.