ASTM F2450-2009 Standard Guide for Assessing Microstructure of Polymeric Scaffolds for Use in Tissue Engineered Medical Products.pdf

1、Designation: F 2450 09Standard 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 () 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 structu

4、re for aparticular application, developing robust manufacturing routes,and for 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 saf

5、etyconcerns, 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:2D 2873 Test Method for Interior

6、 Porosity of Poly(VinylChloride) (PVC) Resins by Mercury Intrusion Porosim-etry3D 4404 Test Method for Determination of Pore 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 La

7、boratory UseE 1294 Test Method for Pore Size Characteristics of Mem-brane Filters Using Automated Liquid Porosimeter3F 316 Test Methods 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 Us

8、ed in Tissue-Engineered Medical Products3. 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 bio

9、active agents. Device structural 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 vo

10、id 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 chemically or throughcrystalline junctions or by specific ionic interactions.3.1.5 macropore/macroporosity (life

11、sciences), na struc-ture inclusive of void spaces sized to allow substantiallyunrestricted passage of chemicals, biomolecules, viruses, bac-teria, and mammalian cells. In implants with interconnectingpores, provides dimensions that allow for ready tissue penetra-tion and microvascularization after i

12、mplantation. Includesmaterials that contain voids with potential to be observable tothe naked eye (100 m).3.1.6 micropore/microporosity (life sciences), na struc-ture inclusive of void spaces sized to allow substantiallyunrestricted passage of chemicals, biomolecules, and viruseswhile sized to contr

13、ol or moderate the passage of bacteria,mammalian cells, and/or tissue. Includes materials with typicalpore sizes of greater than 0.1 m (100 nm) and less than about100 m (100 000 nm), with a common microporous context1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical M

14、aterials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved June 1, 2009. Published July 2009. Originally approvedin 2004. Last previous edition approved in 2004 as F 2450 04.2For referenced ASTM standards, visit the

15、 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 standard is referencedon www.astm.org.1Copyri

16、ght ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.encompassing the range of 20 m or less for the filtration ofcells ranging from bacteria to common mammalian cells andabove 30 micrometer for the ingrowth of tissue. Objects in thissize range t

17、ypically can be observed by conventional lightmicroscopy.3.1.7 nanopore/nanoporosity (life sciences), na structureinclusive of void spaces sized to control or moderate thepassage of chemicals, biomolecules, and viruses while sized tosubstantially exclude most bacteria and all mammalian cells.Include

18、s materials with typical pore sizes of less than 100 nm(0.1 m), with common nanoporous context in the range ofapproximately 20 nm or less for the filtration of viruses.3.1.8 permeability, na measure of fluid, particle, or gasflow through an open pore structure.3.1.9 polymer, na long chain molecule c

19、omposed ofmonomers including both natural and synthetic materials, forexample, collagen, polycaprolactone.3.1.10 pore, na fluid (liquid or gas) filled externallyconnecting channel, void, or open space within an otherwisesolid or gelatinous material (for example, textile meshescomposed of many or sin

20、gle fibers (textile based scaffolds),open cell foams, (hydrogels). Synonyms: open-pore, through-pore.3.1.11 porogen, na material used to create pores within aninherently solid material.3.1.11.1 DiscussionFor example, a polymer dissolved inan organic solvent is poured over a water-soluble powder.Afte

21、revaporation 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.3.1.12 porometry, nthe determination of the distributionof open pore diameters relative to the direct

22、ion 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 size distribution through the use of a non-wettingliquid (typically mercury) intrusion into a porous material as afunction of pressure.3.1

23、.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 pore(void) volume to the apparent (total) volume of a porousmaterial and is commonly expressed as a percentage.3.1.15 scaffold, na support,

24、 delivery vehicle, or matrix forfacilitating the migration, binding, or transport of cells orbioactive molecules used to replace, repair, or regeneratetissues.3.1.16 through-pores, nan inherent or induced network ofvoids or channels that permit flow of fluid (liquid or gas) fromone side of the struc

25、ture 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 ratio of the mean free path to theminimum possible path length.4. Summary of Guide4.1 The microstructure, surface chemistry, and surface m

26、or-phology of polymer-based tissue scaffolds plays a key role inencouraging cell adhesion, migration, 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

27、difficulties ofquantifying pore sizes and pore size distributions over lengthscales ranging from nanometres 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 met

28、hods, for example, chemicalanalysis of the polymer (to determine parameters such as themolecular weight 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

29、of scaffolds; either to assessbase materials supplied by different suppliers or to developrobust manufacturing 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

30、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 depend on theinformation that is required and on the scaffolds physicalproperties; for example, mercury porosimetry will not yieldmeaningful

31、 data if used to characterize soft materials thatdeform during the test and cannot be used for highly hydratedscaffolds.4.5 Table 1 provides guidance for users of this guide byTABLE 1 A Guide to the Physical Characterization of Tissue ScaffoldsGeneric Technique Information Available SectionMicroscop

32、y Pore shape, size and size distribution, porosity. 6.1 (Electron microscopy)6.2 (Optical microscopy)6.2.2 (Confocal microscopy)6.2.3 (Optical coherence tomography)6.2.4 (Optical coherence microscopy)X-Ray Micro-computed Tomography(MicroCT)Pore shape, size and size distribution, porosity. 6.3Magneti

33、c Resonance Imaging Pore shape, size and size distribution, 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 distributi

34、on7.4Diffusion of markers Permeability 8.2NMR Pore size and distribution 8.3F2450092providing 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 an

35、d Use5.1 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, cel

36、ls harvestedfrom 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 X

37、1). Optimiz-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. Facto

38、rs that 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 sca

39、ffold porosity 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 micrometer range. Tissuescaffolds are generally complex structures that are not easilyinter

40、preted in terms of pore shape and size, especially inthree-dimensions. Therefore, it is difficult to quantifiably assessthe 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 cel

41、lbehavior based solely on 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 tissuescaffolds and the length scale that they can measure. Clearly arange of techniques must be utilized if the scaffol

42、d 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, withdefinitions of terms varying widely (as much as three orders ofmagnitude) between differing applications and industries. BothTable 2 and the suppor

43、ting detailed discussion included withinAppendix X2 describe differences that exist between IUPAC4The boldface numbers in parentheses 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 Mey

44、er (2).)F2450093(International Union of Pure and Applied Chemistry) defini-tions and the common terminology currently utilized withinmost life science applications, which include both implant andtissue engineering applications5.2.2.1 Since the literature contains many other terms fordefining pores (

45、Perret et al (3), it is recommended that theterms used by authors to describe pores are defined in order toavoid potential confusion. Additionally, since any of thedefinitions described within Table 2 can shift dependent on thepore size determination method (see Table 1 and Fig. 1), anaccompanying s

46、tatement describing the utilized assessmenttechnique is essential.5.2.3 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-end pore respectively. Porom-etry measurements (see 7.4) are only sensitive to

47、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. The physical basis of porometrydepends on the passage of gas through the materi

48、al. 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 sensitive to both through- and blind-pores ordensity determinations that can also

49、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 guidance to be developed.5.2.4 Polymer scaffolds range from being mechanicallyrigid to those that are soft hydrogels. The methods currentlyused to manufacture these structures include, but are notlimited to:5.2.4.1 Casting a polymer, dissolved in an organic solvent,over a water-soluble particulate porogen, followed by leaching.5.2.4.