1、Designation: F 2450 04Standard Guide forAssessing Microstructure of Polymeric Scaffolds for Use inTissue Engineered Medical Products1This standard is issued under the fixed designation F 2450; the number immediately following the designation indicates the year oforiginal adoption or, in the case of
2、revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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
3、 the 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 struct
4、ure for aparticular application, developing robust manufacturing routes,and for providing reliable quality control data.1.2 This guide does not purport to address all of the safetyconcerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate
5、 safety andhealth practices and to determine the applicability of regula-tory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 2873 Test Method for Interior Porosity of Poly(VinylChloride) (PVC) Resins by Mercury Intrusion Porosim-etry3D 4404 Test Method for Determination of Por
6、e Volume andPore Volume Distribution of Soil and Rock by MercuryIntrusion PorosimetryE 128 Test Method for Maximum Pore Diameter and Per-meability of Rigid Porous Filters for Laboratory UseE 1294 Test Method for Pore Size Characteristics of Mem-brane Filters Using Automated Liquid PorosimeterF 316 T
7、est Method for Pore Size Characteristics of Mem-brane Filters by Bubble Point and Mean Flow Pore TestF 2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical Products2.2 ISO Standard:4ISO 845 Cellular Plastics and RubbersDetermination ofApparent (Bulk
8、) Density3. 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 tissue or cell response.3.1.1.1 DiscussionGrowth factors and antibiotics are typi-cal examples of bioactive agents. Device structura
9、l compo-nents or degradation byproducts that evoke limited localizedbioactivity are not included.3.1.2 blind (end)-pore, na pore that is in contact with anexposed internal or external surface through a single orificesmaller than the pores depth.3.1.3 closed cell, na void isolated within a solid, lac
10、kingany connectivity with an external surface. Synonym: closedpore3.1.4 hydrogel, na water-based open network of polymerchains that are cross-linked either chemically or throughcrystalline junctions or by specific ionic interactions.3.1.5 permeability, na measure of fluid, particle, or gasflow throu
11、gh an open pore structure.3.1.6 polymer, na long chain molecule composed ofmonomers including both natural and synthetic materials, forexample, collagen, polycaprolactone.3.1.7 pore, na liquid (fluid or gas) filled externally con-necting channel, void, or open space within an otherwise solidor gelat
12、inous material (for example, textile meshes composedof many or single fibers (textile based scaffolds), open cellfoams, (hydrogels). Synonyms: open-pore, through-pore.3.1.8 porogen, na material used to create pores within aninherently solid material.3.1.8.1 DiscussionFor example, a polymer dissolved
13、 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 that all the pores areinterconnected.1This guide is under the jurisdiction
14、 of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Nov. 1, 2004. Published December 2004.2For referenced ASTM standards, visit the ASTM website, www.astm.org, o
15、rcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.4Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.1Copyright AST
16、M International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.9 porometry, nthe determination of the distribution ofopen pore diameters relative to the direction of fluid flow bythe displacement of a non-volatile wetting fluid as a function ofpressure.3.1.1
17、0 porosimetry, nthe determination of the pore vol-ume and pore size distribution through the use of a non-wettingliquid (typically mercury) intrusion into a porous material as afunction of pressure.3.1.11 porosity, nproperty of a solid which contains aninherent or induced network of channels and ope
18、n spaces.Porosity can be determined by measuring the ratio of pore(void) volume to the apparent (total) volume of a porousmaterial and is commonly expressed as a percentage.3.1.12 scaffold, na support, delivery vehicle, or matrix forfacilitating the migration, binding, or transport of cells orbioact
19、ive molecules used to replace, repair, or regeneratetissues.3.1.13 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.3.1.14 tortuosity, na measure of the mean free path lengthof through-pores relati
20、ve to the sample thickness. Alternativedefinition: The squared 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, migratio
21、n, growth, and prolifera-tion. The intention of this guide is to 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 na
22、nometres to sub-millimetres and theporosity of materials that differ 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 we
23、ight and its distribution), will aid in the optimi-zation of scaffolds 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 ma
24、nufacturing procedures for commercial production.4.3 Application 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 tha
25、t all listed tests beconducted. The choice of technique will depend on theinformation that is required and on the scaffolds physicalproperties; for example, mercury porosimetry will not yieldmeaningful data if used to characterize soft materials thatdeform during the test and cannot be used for high
26、ly hydratedscaffolds.4.5 Table 1 provides guidance for users of this guide byproviding a brief overview of the applicability of a range ofdifferent measurement techniques that can be used to physi-cally characterize tissue scaffolds. This list of techniques is notdefinitive.5. Significance and Use5.
27、1 The ability to culture functional tissue to repair damagedor diseased tissues within the body offers a viable alternative toxenografts or heterografts. Using the patients own cells toproduce the new tissue offers significant benefits by limitingrejection by the immune system. Typically, cells harv
28、estedfrom the intended recipient are cultured in vitro using atemporary housing or scaffold. The microstructure of thescaffold, that is, its porosity, the mean size, and size distribu-tion of pores and their interconnectivity is critical for cellmigration, growth and proliferation (Appendix X1). Opt
29、imiz-ing the design of tissue scaffolds is a complex task, given therange of available materials, different manufacturing routes,and processing conditions. All of these factors can, and will,affect the surface roughness, surface chemistry, and micro-structure of the resultant scaffolds. Factors that
30、 may or may notbe significant variables depend on the characteristics of a givencell type at any given time that is, changes in cell behavior dueto the number of passages, mechanical stimulation, and cultureconditions.5.2 Tissue scaffolds are typically assessed using an overallvalue for scaffold por
31、osity and a range of pore sizes, though thedistribution of sizes is rarely quantified. Published mean poresizes and distributions are usually obtained from electronmicroscopy images and quoted in the micron range. Tissuescaffolds are generally complex structures that are not easilyinterpreted in ter
32、ms of pore shape and size, especially inthree-dimensions. Therefore it is difficult to quantifiably assessTABLE 1 A Guide to the Physical Characterization of Tissue ScaffoldsGeneric Technique Information Available SectionMicroscopy Pore shape, size and size distribution, porosity. 6.1 (Electron micr
33、oscopy)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. 6.6Magnetic Resonance Imaging Pore shape, size and size distribution, porosity. 6.7Measurement
34、 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 Pore size and distribution 8.3F2450042
35、the batch-to-batch variance in microstructure or to enable asystematic investigation to be made of the role that the meanpore size and pore size distribution has on influencing cellbehavior based solely on electron micrographs (Tomlins et al,(1).55.2.1 Fig. 1 gives an indication of potential techniq
36、ues thatcan be used to characterize porous tissue scaffolds and thelength scale that they can measure. Clearly a range of tech-niques must be utilized if the scaffold is to be assessed in detail.5.2.2 The classification of pore sizes given in Fig. 1 has yetto be standardized. Typical size ranges for
37、 pores are, accordingto their dimensions:5.2.2.1 Micropores (;0.5 to 2 nm)5.2.2.2 Mesopores (;2to50nm)5.2.2.3 Macropores (;50 to 2000 nm)5.2.2.4 Capillaries (;2000 nm to 0.8 mm)5.2.2.5 Macrocapillaries (0.8 m)5.2.3 This list is by no means complete; the literaturecontains many other terms for defini
38、ng pores (Perret et al (3).It is recommended that the terms used by authors to describepores are defined in order to avoid potential confusion.5.2.4 All the techniques listed in Table 1 have their limita-tions for assessing complex porous structures. Fig. 2a and Fig.2b show a through- and a blind-en
39、d pore respectively. Porom-etry measurements (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 SEM, which may sample at adifferent point along the pore. Th
40、e physical basis of porometrydepends on the passage of gas through the material. Therefore,the technique is not sensitive to blind-end or enclosed pores.Therefore, estimates of porosity based on porometry data willbe different to those obtained from, for example, porosimetry(see 7.3), which is sensi
41、tive to both through- and blind-pores ordensity determinations that can also account for through-,blind-end, and enclosed pores. The significance of thesedifferences will depend on factors such as the percentage of thedifferent pore types and on their dimensions. Further researchwill enable improved
42、 guidance to be developed.5.2.5 Polymer scaffolds range from being mechanicallyrigid to those that are soft hydrogels. The methods currentlyused to manufacture these structures include, but are notlimited to:5.2.5.1 Casting a polymer, dissolved in an organic solvent,over a water-soluble particulate
43、porogen, followed by leaching.5.2.5.2 Melt mixing of immiscible polymers followed byleaching of the water-soluble component.5.2.5.3 Dissolution of supercritical carbon dioxide underpressure into an effectively molten polymer, a phenomenonattributed to the dramatic reduction in the glass transitionte
44、mperature which occurs, followed by a reduction in pressurethat leads to the formation of gas bubbles and solidification.5.2.5.4 Controlled deposition of molten polymer to producea well-defined three-dimensional lattice.5.2.5.5 The manufacture of three-dimensional fibrousweaves, knits, or non-woven
45、structures.5The boldface numbers in parentheses refer to the list of references at the end ofthis standard.NOTE(Redrawn from Meyer (2)FIG. 1 A Range of Techniques is Required to Fully Characterize Porous MaterialsF24500435.2.5.6 Chemical or ionic cross-linking of a polymericmatrix.5.2.6 Consideratio
46、ns have been given to the limitations ofthese methods in Appendix X1.5.2.7 This guide focuses on the specific area of character-ization of polymer-based porous scaffolds and is an extensionof an earlier ASTM guide, Guide F 2150.6. Imaging6.1 Electron MicroscopyBoth transmission and scanningelectron
47、microscopy can be used to image intact or fracturedsurfaces or sections cut from tissue scaffolds. The resultantimages can be interpreted using image analysis softwarepackages to generate data concerning the shape of pores withinthe scaffold, their mean size, and distribution. Estimates ofboth perme
48、ability and tortuosity can be made from three-dimensional virtual images generated from transmission elec-tron microscopic images of serially sectioned samples.6.1.1 There is likely to be a high degree of uncertainty in thereliability of quantitative data derived from electron micro-scopic examinati
49、on of soft or especially highly hydrated softpolymer-based scaffolds due to the presence of artifacts createdduring sample preparation. These problems may be overcomeby using environmental scanning electron microscopy. Highlyhydrated scaffolds need to be freeze-dried before examinationunder vacuum in a conventional scanning electron microscope.This process, if carried out in liquid nitrogen, usually results ina significant amount of ice damage due to the relatively slowcooling rates that are encountered due to the thin layer ofinsulating nitrogen gas that form
