ASTM F2450-04

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Designation:F2450–04
Standard Guide for
Assessing Microstructure of Polymeric Scaffolds for Use in
Tissue Engineered Medical Products
This standard is issued under the fixed designation F 2450; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 ISO Standard:
ISO 845 Cellular Plastics and Rubbers—Determination of
1.1 This guide covers an overview of test methods that may
Apparent (Bulk) Density
be used to obtain information relating to the dimensions of
pores, the pore size distribution, the degree of porosity,
3. Terminology
interconnectivity, and measures of permeability for porous
3.1 Definitions:
materials used as polymeric scaffolds in the development and
3.1.1 bioactive agent, n—any molecular component in, on,
manufacture of tissue engineered medical products (TEMPs).
or within the interstices of a device that is intended to elicit a
This information is key to optimizing the structure for a
desired tissue or cell response.
particular application, developing robust manufacturing routes,
3.1.1.1 Discussion—Growthfactorsandantibioticsaretypi-
and for providing reliable quality control data.
cal examples of bioactive agents. Device structural compo-
1.2 This guide does not purport to address all of the safety
nents or degradation byproducts that evoke limited localized
concerns, if any, associated with its use. It is the responsibility
bioactivity are not included.
of the user of this standard to establish appropriate safety and
3.1.2 blind (end)-pore, n—a pore that is in contact with an
health practices and to determine the applicability of regula-
exposed internal or external surface through a single orifice
tory limitations prior to use.
smaller than the pore’s depth.
2. Referenced Documents 3.1.3 closed cell, n—a void isolated within a solid, lacking
any connectivity with an external surface. Synonym: closed
2.1 ASTM Standards:
pore
D 2873 Test Method for Interior Porosity of Poly(Vinyl
3.1.4 hydrogel, n—a water-based open network of polymer
Chloride) (PVC) Resins by Mercury Intrusion Porosim-
chains that are cross-linked either chemically or through
etry
crystalline junctions or by specific ionic interactions.
D 4404 Test Method for Determination of Pore Volume and
3.1.5 permeability, n—a measure of fluid, particle, or gas
Pore Volume Distribution of Soil and Rock by Mercury
flow through an open pore structure.
Intrusion Porosimetry
3.1.6 polymer, n—a long chain molecule composed of
E 128 Test Method for Maximum Pore Diameter and Per-
monomers including both natural and synthetic materials, for
meability of Rigid Porous Filters for Laboratory Use
example, collagen, polycaprolactone.
E 1294 Test Method for Pore Size Characteristics of Mem-
3.1.7 pore, n—a liquid (fluid or gas) filled externally con-
brane Filters Using Automated Liquid Porosimeter
necting channel, void, or open space within an otherwise solid
F 316 Test Method for Pore Size Characteristics of Mem-
or gelatinous material (for example, textile meshes composed
brane Filters by Bubble Point and Mean Flow Pore Test
of many or single fibers (textile based scaffolds), open cell
F 2150 Guide for Characterization and Testing of Biomate-
foams, (hydrogels). Synonyms: open-pore, through-pore.
rial Scaffolds Used inTissue-Engineered Medical Products
3.1.8 porogen, n—a material used to create pores within an
inherently solid material.
3.1.8.1 Discussion—For example, a polymer dissolved in
anorganicsolventispouredoverawater-solublepowder.After
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
evaporation of the solvent, the porogen is leached out, usually
Surgical Materials and Devices and is the direct responsibility of Subcommittee
by water, to leave a porous structure. The percentage of
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Nov. 1, 2004. Published December 2004. porogenneedstobehighenoughtoensurethatalltheporesare
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
interconnected.
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F2450–04
3.1.9 porometry, n—the determination of the distribution of 4.3 Application of the techniques described in this guide
open pore diameters relative to the direction of fluid flow by will not guarantee that the scaffold will perform the functions
thedisplacementofanon-volatilewettingfluidasafunctionof for which it is being developed but they may help to identify
pressure. the reasons for success or failure.
3.1.10 porosimetry, n—the determination of the pore vol- 4.4 This guide does not suggest that all listed tests be
umeandporesizedistributionthroughtheuseofanon-wetting conducted. The choice of technique will depend on the
liquid (typically mercury) intrusion into a porous material as a information that is required and on the scaffold’s physical
function of pressure. properties; for example, mercury porosimetry will not yield
3.1.11 porosity, n—property of a solid which contains an meaningful data if used to characterize soft materials that
inherent or induced network of channels and open spaces. deform during the test and cannot be used for highly hydrated
Porosity can be determined by measuring the ratio of pore scaffolds.
(void) volume to the apparent (total) volume of a porous 4.5 Table 1 provides guidance for users of this guide by
material and is commonly expressed as a percentage. providing a brief overview of the applicability of a range of
3.1.12 scaffold, n—asupport,deliveryvehicle,ormatrixfor different measurement techniques that can be used to physi-
facilitating the migration, binding, or transport of cells or cally characterize tissue scaffolds.This list of techniques is not
bioactive molecules used to replace, repair, or regenerate definitive.
tissues.
5. Significance and Use
3.1.13 through-pores, n—an inherent or induced network of
voids or channels that permit flow of fluid (liquid or gas) from 5.1 Theabilitytoculturefunctionaltissuetorepairdamaged
one side of the structure to the other.
ordiseasedtissueswithinthebodyoffersaviablealternativeto
3.1.14 tortuosity, n—ameasureofthemeanfreepathlength xenografts or heterografts. Using the patient’s own cells to
of through-pores relative to the sample thickness. Alternative
produce the new tissue offers significant benefits by limiting
definition: The squared ratio of the mean free path to the rejection by the immune system. Typically, cells harvested
minimum possible path length.
from the intended recipient are cultured in vitro using a
temporary housing or scaffold. The microstructure of the
4. Summary of Guide
scaffold, that is, its porosity, the mean size, and size distribu-
4.1 The microstructure, surface chemistry, and surface mor- tion of pores and their interconnectivity is critical for cell
phology of polymer-based tissue scaffolds plays a key role in migration, growth and proliferation (Appendix X1). Optimiz-
encouraging cell adhesion, migration, growth, and prolifera- ing the design of tissue scaffolds is a complex task, given the
tion.The intention of this guide is to provide a compendium of range of available materials, different manufacturing routes,
techniques for characterizing this microstructure. The breadth and processing conditions. All of these factors can, and will,
of the techniques described reflects the practical difficulties of affect the surface roughness, surface chemistry, and micro-
quantifying pore sizes and pore size distributions over length structure of the resultant scaffolds. Factors that may or may not
scales ranging from nanometres to sub-millimetres and the besignificantvariablesdependonthecharacteristicsofagiven
porosity of materials that differ widely in terms of their cell type at any given time that is, changes in cell behavior due
mechanical properties. to the number of passages, mechanical stimulation, and culture
4.2 These microstructural data when used in conjunction conditions.
with other characterization methods, for example, chemical 5.2 Tissue scaffolds are typically assessed using an overall
analysis of the polymer (to determine parameters such as the valueforscaffoldporosityandarangeofporesizes,thoughthe
molecular weight and its distribution), will aid in the optimi- distribution of sizes is rarely quantified. Published mean pore
zation of scaffolds for tissue engineered medical products sizes and distributions are usually obtained from electron
(TEMPs). Adequate characterization is also critical to ensure microscopy images and quoted in the micron range. Tissue
the batch-to-batch consistency of scaffolds; either to assess scaffolds are generally complex structures that are not easily
base materials supplied by different suppliers or to develop interpreted in terms of pore shape and size, especially in
robust manufacturing procedures for commercial production. three-dimensions. Therefore it is difficult to quantifiably assess
TABLE 1 A Guide to the Physical Characterization of Tissue Scaffolds
Generic Technique Information Available Section
Microscopy Pore shape, size and size distribution, porosity. 6.1 (Electron microscopy)
6.2 (Optical microscopy)
6.3 (Confocal microscopy)
6.4 (Optical coherence tomography)
6.5 (Optical coherence microscopy)
Micro X-ray computer tomography Pore shape, size and size distribution, porosity. 6.6
Magnetic Resonance Imaging Pore shape, size and size distribution, porosity. 6.7
Measurement of density Porosity, pore volume 7.2
Porosimetry Porosity, total pore surface area, pore diameter, pore size distribution 7.3
Porometry Median pore diameter (assuming cylindrical geometry), through-pore 7.4
size distribution
Diffusion of markers Permeability 8.2
NMR Pore size and distribution 8.3
F2450–04
the batch-to-batch variance in microstructure or to enable a gative techniques, such as SEM, which may sample at a
systematic investigation to be made of the role that the mean different point along the pore. The physical basis of porometry
pore size and pore size distribution has on influencing cell
depends on the passage of gas through the material. Therefore,
behavior based solely on electron micrographs (Tomlins et al, the technique is not sensitive to blind-end or enclosed pores.
(1)).
Therefore, estimates of porosity based on porometry data will
5.2.1 Fig. 1 gives an indication of potential techniques that
be different to those obtained from, for example, porosimetry
can be used to characterize porous tissue scaffolds and the
(see7.3),whichissensitivetoboththrough-andblind-poresor
length scale that they can measure. Clearly a range of tech-
density determinations that can also account for through-,
niquesmustbeutilizedifthescaffoldistobeassessedindetail.
blind-end, and enclosed pores. The significance of these
5.2.2 The classification of pore sizes given in Fig. 1 has yet
differenceswilldependonfactorssuchasthepercentageofthe
to be standardized.Typical size ranges for pores are, according
different pore types and on their dimensions. Further research
to their dimensions:
will enable improved guidance to be developed.
5.2.2.1 Micropores (;0.5 to 2 nm)
5.2.5 Polymer scaffolds range from being mechanically
5.2.2.2 Mesopores (;2to50nm)
rigid to those that are soft hydrogels. The methods currently
5.2.2.3 Macropores (;50 to 2000 nm)
used to manufacture these structures include, but are not
5.2.2.4 Capillaries (;2000 nm to 0.8 mm)
limited to:
5.2.2.5 Macrocapillaries (>0.8 µm)
5.2.5.1 Casting a polymer, dissolved in an organic solvent,
5.2.3 This list is by no means complete; the literature
overawater-solubleparticulateporogen,followedbyleaching.
contains many other terms for defining pores (Perret et al (3)).
5.2.5.2 Melt mixing of immiscible polymers followed by
It is recommended that the terms used by authors to describe
leaching of the water-soluble component.
pores are defined in order to avoid potential confusion.
5.2.4 All the techniques listed in Table 1 have their limita-
5.2.5.3 Dissolution of supercritical carbon dioxide under
tions for assessing complex porous structures. Fig. 2a and Fig. pressure into an effectively molten polymer, a phenomenon
2b show a through- and a blind-end pore respectively. Porom-
attributed to the dramatic reduction in the glass transition
etry measurements (see 7.4) are only sensitive to the narrowest
temperature which occurs, followed by a reduction in pressure
point along a variable diameter through-pore and therefore can
that leads to the formation of gas bubbles and solidification.
give a lower measure of the pore diameter than other investi-
5.2.5.4 Controlled deposition of molten polymer to produce
a well-defined three-dimensional lattice.
5 5.2.5.5 The manufacture of three-dimensional fibrous
The boldface numbers in parentheses refer to the list of references at the end of
this standard. weaves, knits, or non-woven structures.
NOTE—(Redrawn from Meyer (2))
FIG. 1 A Range of Techniques is Required to Fully Characterize Porous Materials
F2450–04
FIG. 2 A through-pore showing a variation of pore diameter, D (a); and an example of a blind-pore (b).
5.2.5.6 Chemical or ionic cross-linking of a polymeric microscope. This method is less appropriate for investigating
matrix. hydrogels that can be gradually dehydrated using a series of
5.2.6 Considerations have been given to the limitations of alcohol solutions, following standardized procedures, before
these methods in Appendix X1. embedding.However,thisproceduretendstoreducethesizeof
5.2.7 This guide focuses on the specific area of character- the water-filled pores within the sample. The quantifiable pore
ization of polymer-based porous scaffolds and is an extension size data subsequently obtained are of value if microstructural
of an earlier ASTM guide, Guide F 2150. comparisons between different samples are required. Conse-
quently, these data are likely to be inappropriate for character-
6. Imaging
izing the microstructure of samples per se due to the artifacts.
6.1 Electron Microscopy—Both transmission and scanning 6.2 Optical Microscopy— Images of the surfaces of tissue
electron micr
...