1、Designation: F2450 10Standard Guide forAssessing Microstructure of Polymeric Scaffolds for Use inTissue-Engineered Medical Products1This standard is issued under the fixed designation F2450; the number immediately following the designation indicates the year oforiginal adoption or, in the case of re
2、vision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers an overview of test methods that maybe used to obtain information relating to th
3、e dimensions ofpores, the pore size distribution, the degree of porosity,interconnectivity, and measures of permeability for porousmaterials used as polymeric scaffolds in the development andmanufacture of tissue-engineered medical products (TEMPs).This information is key to optimizing the structure
4、 for aparticular application, developing robust manufacturing routes,and providing reliable quality control data.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This guide does not purport to address all of the safetycon
5、cerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety andhealth practices and to determine the applicability of regula-tory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D2873 Test Method for Interior Porosi
6、ty of Poly(VinylChloride) (PVC) Resins by Mercury Intrusion Porosim-etry3D4404 Test Method for Determination of Pore Volume andPore Volume Distribution of Soil and Rock by MercuryIntrusion PorosimetryE128 Test Method for Maximum Pore Diameter and Per-meability of Rigid Porous Filters for Laboratory
7、UseE1294 Test Method for Pore Size Characteristics of Mem-brane Filters Using Automated Liquid Porosimeter3E1441 Guide for Computed Tomography (CT) ImagingF316 Test Methods for Pore Size Characteristics of Mem-brane Filters by Bubble Point and Mean Flow Pore TestF2150 Guide for Characterization and
8、Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical ProductsF2603 Guide for Interpreting Images of Polymeric TissueScaffolds3. Terminology3.1 Definitions:3.1.1 bioactive agent, nany molecular component in, on,or within the interstices of a device that is intended to elicit adesired t
9、issue or cell response.3.1.1.1 DiscussionGrowth factors and antibiotics are typi-cal examples of bioactive agents. Device structural compo-nents or degradation byproducts that evoke limited localizedbioactivity are not bioactive agents.3.1.2 blind (end)-pore, na pore that is in contact with anexpose
10、d internal or external surface through a single orificesmaller than the pores depth.3.1.3 closed cell, na void isolated within a solid, lackingany connectivity with an external surface. Synonym: closedpore3.1.4 hydrogel, na water-based open network of polymerchains that are cross-linked either chemi
11、cally or throughcrystalline junctions or by specific ionic interactions.3.1.5 macropore/macroporosity (life sciences), na struc-ture (including void spaces) sized to allow substantially unre-stricted passage of chemicals, biomolecules, viruses, bacteria,and mammalian cells. In implants with intercon
12、necting pores,macroporosity provides dimensions that allow for ready tissuepenetration and microvascularization after implantation. In-cludes materials that contain voids with the potential to beobservable to the naked eye (100 m).3.1.6 micropore/microporosity (life sciences), na struc-ture (includi
13、ng void spaces) sized to allow substantially unre-stricted passage of chemicals, biomolecules, and viruses whilesized to control or moderate the passage of bacteria, mamma-lian cells, and/or tissue. Includes materials with typical pore1This guide is under the jurisdiction of ASTM Committee F04 on Me
14、dical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved March 1, 2010. Published April 2010. Originallyapproved in 2004. Last previous edition approved in 2009 as F2450 09. DOI:10.1520/F2450-10
15、.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn. The last approved version of this historical sta
16、ndard is referencedon www.astm.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.sizes of greater than 0.1 m (100 nm) and less than about 100m (100 000 nm), with a common microporous context en-compassing the range of 20 m or less
17、for the filtration of cellsranging from bacteria to common mammalian cells and above30 m for the ingrowth of tissue. Objects in this size rangetypically can be observed by conventional light microscopy.3.1.7 nanopore/nanoporosity (life sciences), na structureinclusive of void spaces sized to control
18、 or moderate thepassage of chemicals, biomolecules, and viruses while sized tosubstantially exclude most bacteria and all mammalian cells.Includes materials with typical pore sizes of less than 100 nm(0.1 m), with common nanoporous context in the range of 20nm or less for the filtration of viruses.3
19、.1.8 permeability, na measure of fluid, particle, or gasflow through an open pore structure.3.1.9 polymer, na long chain molecule composed ofmonomers including both natural and synthetic materials.Examples include collagen and polycaprolactone.3.1.10 pore, na fluid (liquid or gas) filled externallyc
20、onnecting channel, void, or open space within an otherwisesolid or gelatinous material (for example, textile meshescomposed of many or single fibers (textile based scaffolds),open cell foams, (hydrogels). Synonyms: open-pore, through-pore.3.1.11 porogen, na material used to create pores within aninh
21、erently solid material.3.1.11.1 DiscussionFor example, a polymer dissolved inan organic solvent is poured over a water-soluble powder.Afterevaporation of the solvent, the porogen is leached out, usuallyby water, to leave a porous structure. The percentage ofporogen needs to be high enough to ensure
22、that all the pores areinterconnected.3.1.12 porometry, nthe determination of the distributionof open pore diameters relative to the direction of fluid flow bythe displacement of a non-volatile wetting fluid as a function ofpressure.3.1.13 porosimetry, nthe determination of the pore vol-ume and pore
23、size distribution through the use of a non-wettingliquid (typically mercury) intrusion into a porous material as afunction of pressure.3.1.14 porosity, nproperty of a solid which contains aninherent or induced network of channels and open spaces.Porosity can be determined by measuring the ratio of p
24、ore(void) volume to the apparent (total) volume of a porousmaterial and is commonly expressed as a percentage.3.1.15 scaffold, na support, delivery vehicle, or matrix forfacilitating the migration, binding, or transport of cells orbioactive molecules used to replace, repair, or regeneratetissues.3.1
25、.16 through-pores, nan inherent or induced network ofvoids or channels that permit flow of fluid (liquid or gas) fromone side of the structure to the other.3.1.17 tortuosity, na measure of the mean free path lengthof through-pores relative to the sample thickness. Alternativedefinition: The squared
26、ratio of the mean free path to theminimum possible path length.4. Summary of Guide4.1 The microstructure, surface chemistry, and surface mor-phology of polymer-based tissue scaffolds plays a key role inencouraging cell adhesion, migration, growth, and prolifera-tion. The intention of this guide is t
27、o provide a compendium oftechniques for characterizing this microstructure. The breadthof the techniques described reflects the practical difficulties ofquantifying pore sizes and pore size distributions over lengthscales ranging from nanometres to sub-millimetres and theporosity of materials that d
28、iffer widely in terms of theirmechanical properties.4.2 These microstructural data when used in conjunctionwith other characterization methods, for example, chemicalanalysis of the polymer (to determine parameters such as themolecular weight and its distribution), will aid in the optimi-zation of sc
29、affolds for tissue-engineered medical products(TEMPs). Adequate characterization is also critical to ensurethe batch-to-batch consistency of scaffolds; either to assessbase materials supplied by different suppliers or to developrobust manufacturing procedures for commercial production.4.3 Applicatio
30、n of the techniques described in this guidewill not guarantee that the scaffold will perform the functionsfor which it is being developed but they may help to identifythe reasons for success or failure.4.4 This guide does not suggest that all listed tests beconducted. The choice of technique will de
31、pend on theinformation that is required and on the scaffolds physicalproperties; for example, mercury porosimetry will not yieldTABLE 1 A Guide to the Physical Characterization of Tissue ScaffoldsGeneric Technique Information Available SectionMicroscopy Pore shape, size and size distribution; porosi
32、ty. 6.1 (Electron microscopy)6.2 (Optical microscopy)6.2.3 (Confocal microscopy)6.2.4 (Optical coherence tomography)6.2.5 (Optical coherence microscopy)X-Ray micro-computed Tomography(MicroCT)Pore shape, size and size distribution; porosity. 6.3Magnetic Resonance Imaging Pore shape, size and size di
33、stribution; porosity. 6.4Measurement of density Porosity, pore volume 7.2Porosimetry Porosity, total pore surface area, pore diameter, pore size distribution 7.3Porometry Median pore diameter (assuming cylindrical geometry), through-poresize distribution7.4Diffusion of markers Permeability 8.2NMR Po
34、re size and distribution 7.5F2450 102meaningful data if used to characterize soft materials thatdeform during the test and cannot be used for hydratedscaffolds.4.5 Table 1 provides guidance for users of this guide byproviding a brief overview of the applicability of a range ofdifferent measurement t
35、echniques that can be used to physi-cally characterize tissue scaffolds. This list of techniques is notdefinitive.5. Significance and Use5.1 The ability to culture functional tissue to repair damagedor diseased tissues within the body offers a viable alternative toxenografts or heterografts. Using t
36、he patients own cells toproduce the new tissue offers significant benefits by limitingrejection by the immune system. Typically, cells harvestedfrom the intended recipient are cultured in vitro using atemporary housing or scaffold. The microstructure of thescaffold, that is, its porosity, the mean s
37、ize, and size distribu-tion of pores and their interconnectivity is critical for cellmigration, growth and proliferation (Appendix X1). Optimiz-ing the design of tissue scaffolds is a complex task, given therange of available materials, different manufacturing routes,and processing conditions. All o
38、f these factors can, and will,affect the surface texture, surface chemistry, and microstructureof the resultant scaffolds. Factors that may or may not besignificant variables depend on the characteristics of a givencell type at any given time (that is, changes in cell behavior dueto the number of pa
39、ssages, mechanical stimulation, and cultureconditions).5.2 Tissue scaffolds are typically assessed using an overallvalue for scaffold porosity and a range of pore sizes, though thedistribution of sizes is rarely quantified. Published mean poresizes and distributions are usually obtained from electro
40、nmicroscopy images and quoted in the micrometer range. Tissuescaffolds are generally complex structures that are not easilyinterpreted in terms of pore shape and size, especially in threedimensions. Therefore, it is difficult to quantifiably assess thebatch-to-batch variance in microstructure or to
41、make a system-atic investigation of the role that the mean pore size and poresize distribution has on influencing cell behavior based solelyon electron micrographs (Tomlins et al, (1).45.2.1 Fig. 1 gives an indication of potential techniques thatcan be used to characterize the structure of porous ti
42、ssuescaffolds and the length scale that they can measure. Clearly arange of techniques must be utilized if the scaffold is to becharacterized in detail.5.2.2 The classification and terminology of pore sizes, suchas those given in Table 2, has yet to be standardized, with4The boldface numbers in pare
43、ntheses refer to the list of references at the end ofthis standard.FIG. 1 A Range of Techniques is Required to Fully Characterize Porous Materials(NoteFigure redrawn from Meyer (2).)F2450 103definitions of terms varying widely (as much as three orders ofmagnitude) between differing applications and
44、industries. BothTable 2 and the supporting detailed discussion included withinAppendix X2 describe differences that exist between IUPAC(International Union of Pure and Applied Chemistry) defini-tions and the common terminology currently utilized withinmost life science applications, which include bo
45、th implant andtissue engineering applications.5.2.2.1 Since the literature contains many other terms fordefining pores (Perret et al (3), it is recommended that theterms used by authors to describe pores be defined in order toavoid potential confusion. Additionally, since any of thedefinitions in Ta
46、ble 2 can shift, dependending on the pore sizedetermination method (see Table 1 and Fig. 1), an accompa-nying statement describing the utilized assessment technique isessential.5.2.3 All the techniques listed in Table 1 have limitations forassessing complex porous structures. Fig. 2a and Fig. 2b sho
47、wa through- and a blind-end pore respectively. Porometrymeasurements (see 7.4) are only sensitive to the narrowestpoint along a variable diameter through-pore and therefore cangive a lower measure of the pore diameter than other investi-gative techniques, such as scanning electron microscope(SEM), w
48、hich may sample at a different point along the pore.The physical basis of porometry depends on the passage of gasthrough the material. Therefore, the technique is not sensitiveto blind-end or closed pores. Therefore, estimates of porositybased on porometry data will be different from those obtainedf
49、rom, for example, porosimetry (see 7.3), which is sensitive toboth through- and blind-pores or density determinations thatcan also account for through-, blind-end, and closed pores. Thesignificance of these differences will depend on factors such asthe percentage of the different pore types and their dimensions.Further research will enable improved guidance to be devel-oped.5.2.4 Polymer scaffolds range from mechanically rigidstructures to soft hydrogels. The methods currently used tomanufacture these structures include, but are not limited to:5.2.
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