1、Designation: F2603 06 (Reapproved 2012)Standard Guide forInterpreting Images of Polymeric Tissue Scaffolds1This standard is issued under the fixed designation F2603; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last
2、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 the factors that need to be consideredin obtaining and interpreting images of tissue scaffolds includ-ing
3、 technique selection, instrument resolution and imagequality, quantification and sample preparation.1.2 The information in this guide is intended to be appli-cable to porous polymer-based tissue scaffolds, includingnaturally derived materials such as collagen. However, somematerials (both synthetic
4、and natural) may require unique orvaried sample preparation methods that are not specificallycovered in this guide.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafet
5、y concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and to determine theapplicability of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1919 Guide for Worldwide Publis
6、hed Standards Relating toParticle and Spray CharacterizationE2245 Test Method for Residual Strain Measurements ofThin, Reflecting Films Using an Optical InterferometerF1854 Test Method for Stereological Evaluation of PorousCoatings on Medical ImplantsF1877 Practice for Characterization of ParticlesF
7、2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical Prod-uctsF2450 Guide for Assessing Microstructure of PolymericScaffolds for Use in Tissue-Engineered Medical Products3. Terminology3.1 Definitions:3.1.1 aliasing, nartifactual data that originates
8、 from aninsufficient sampling rate.3.1.2 biomaterial, na natural or synthetic material that issuitable for introduction into living tissue especially as part ofa medical device, such as an artificial heart valve or joint.3.1.3 blind (end) pore, na pore that is in contact with anexposed internal wall
9、 or surface through a single orifice smallerthan the pores depth.3.1.4 closed cell, nvoid within a solid, lacking any con-nectivity with an external surface. Synonym: closed pore.3.1.5 feret diameter, nthe mean value of the distancebetween pairs of parallel tangents to the periphery of a pore(adapte
10、d from Practice F1877).3.1.6 hydrogel, na water-based open network of polymerchains that are cross-linked either chemically or throughcrystalline junctions or by specific ionic interactions.3.1.7 irregular, adjan irregular pore that cannot be de-scribed as round or spherical. A set of reference figu
11、res thatdefine the nomenclature are given in Appendix X2. (Adaptedfrom Practice F1877).3.1.8 Nyquist criteriona criterion that states that a signalmust be sampled at a rate greater than or equal to twice itshighest frequency component to avoid aliasing.3.1.9 permeability, na measure of fluid, partic
12、le, or gasflow through an open pore structure.3.1.10 pixel, ntwo-dimensional picture element.3.1.11 polymer, na long chain molecule composed ofmonomers.3.1.11.1 DiscussionA polymer may be a natural or syn-thetic material.3.1.11.2 DiscussionExamples of polymers include colla-gen and polycaprolactone.
13、3.1.12 pore, na liquid, fluid, or gas-filled externally con-necting channel, void, or open space within an otherwise solidor gelatinous material (for example, textile meshes composedof many or single fibers (textile-based scaffolds), open cellfoams, (hydrogels). Synonyms: open pore, through pore.1Th
14、is guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devicesand is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Oct. 1, 2012. Published February 2007. DOI: 10.1520/F2603-06.2For referenced
15、 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Consh
16、ohocken, PA 19428-2959. United States13.1.13 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
17、a percentage (GuideF2150).3.1.14 rectangular, adjA pore that approximates a squareor rectangle in shape (derived from Practice F1877).3.1.15 roundness (R), na measure of how closely anobject represents a circle (Practice F1877).3.1.16 scaffold, na support, delivery vehicle, or matrix forfacilitating
18、 the migration, binding, or transport of cells orbioactive molecules used to replace, repair, or regeneratetissues. (Guide F2150).3.1.17 segmentation, na methodology for distinguishingdifferent regions (for example, pores and walls) within a tissuescaffold image.3.1.18 spherical pore, na pore with a
19、 generally sphericalshape.3.1.18.1 DiscussionA spherical pore appears round in aphotograph (Practice F1877).3.1.19 threshold, nisolation of a range of grayscale valuesexhibited by one constituent within an image.3.1.20 through pores, nan inherent or induced network ofvoids or channels that permit fl
20、ow of fluid from one side of thestructure to the other.3.1.21 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.3.1.22 voxel, nthree-dimensional pictu
21、re element.4. Significance and Use4.1 This document provides guidance for users who wish toobtain quantifiable data from images of tissue scaffolds manu-factured from polymers that include both high water contentgels and woven textiles.4.2 Information derived from tissue scaffold images can beused t
22、o optimize the structural characteristics of the matrix fora particular application, to develop better manufacturing pro-cedures or to provide a measure of quality assurance andproduct traceability. Fig. 1 provides a summary of the keystages of image capture and analysis.4.3 There is a synergy betwe
23、en the analysis of pores intissue scaffolds and that of particles that is reflected instandards cited and in the analysis described in Section 9.Guide E1919 provides a compendium of standards for particleanalysis that includes measurement techniques, data analyticaland sampling methodologies.5. Meas
24、urement Objectives5.1 Much of the research activity in tissue engineering isfocused on the development of suitable materials and structuresfor optimal growth of a range of tissue types includingcartilage, bone, and nerve. This requires a quantitative assess-ment of the scaffold structure.The key par
25、ameters that need to be determined are (1) theoverall level of porosity, (2) the pore size distribution, whichcan range from tens of nanometers to several hundredmicrometres, and (3) the degree of interconnectivity andtortuosity of the pores.6. Imaging Methods and Conditions6.1 There are many experi
26、mental ways of obtaining keyscaffold physical parameters as described in Guide F2450.When imaging and subsequent quantitative analysis is chosenas the method for determining these parameters, it is criticalthat any image under consideration be a true representation ofthe scaffold of interest. Some i
27、maging methods require samplepreparation. Some do not. When sample preparation is requiredprior to imaging, care must be taken that the procedures do notsignificantly alter the morphology of the scaffold. See Appen-dix X1 for further information on sample preparation.6.2 Images obtained using techni
28、ques such as lightmicroscopy, electron microscopy, and magnetic resonanceimaging are two-dimensional (2-D) representations of a three-dimensional (3-D) structure. These can be a planar or cross-sectional view with a relatively large depth of field or a seriesof physical or virtual 2-D slices, each w
29、ith a small depth offield, that can be reassembled in a virtual environment toproduce a 3-D mesostructure.6.3 There are limits to the extent an image (2-D or 3-D) canfaithfully represent the physical artifacts that are influenced byfactors germane to the imaging method, such as spatialresolution and
30、 dynamic range, image contrast, and the signal-to-noise ratio. Table 1 lists some of the techniques available forproducing images of porous structures, along with their con-trast source, maximum demonstrated spatial resolution, andtypical dynamic range. Proper technique selection dependsFIG. 1 Key S
31、tages in Image Capture, Storage, and AnalysisF2603 06 (2012)2both on the material properties of the scaffold (that is, opticalmethods cannot be used with opaque materials) the contrastavailable, and the target pore size range.6.4 The images generated by the techniques shown in Table1 cannot reproduc
32、e features smaller than the spatial resolutionof the method. Features that are faint, that is, those that do nothave significant contrast, or signal significantly abovebackground, will be resolved at length scales larger than themaximum resolution. Excessive contrast can also limit thepenetration de
33、pth due to scattering effects. This is particularlytrue of optical microscopies using differences in refractiveindex as the contrast mechanism. An appropriate level ofcontrast that can be established by experimentation is thereforecritical to high quality imaging.6.5 Contrast can be enhanced by usin
34、g exogenous agents,such as florescence tags in optical microscopy and stainscontaining heavy metal complexes in electron microscopies.Excessive contrast can be ameliorated in optical microscopiesby imbibing the structure with a fluid that has an index ofrefraction similar to that of the solid making
35、 up the structure(this is termed “index-matching”). There are many excellentresources describing factors influencing widefield and confocaloptical microscopy (1-3),3optical coherence microscopy (4),MRI (5), and electron microscopies (6).6.6 The reconstruction of the mesostructure in 3-D from aseries
36、 of 2-D images obtained from a sample that has beenphysically sectioned requires considerably more effort thanassembly of virtual sections produced by techniques that areable to focus on a plane within the sample. The virtualapproach is also less prone to sample distortion since itobviates the need
37、for physical sectioning and registration errorsin the reassembly process. However, the techniques used togenerate virtual 2-D images typically have limited penetrationdepth.6.7 Confocal microscopy (OCT), for example, has a pen-etration depth of approximately 100 m, a value that dependson the wavelen
38、gth of the light used and the amount ofscattering that occurs within the sample. Scanning acousticmicroscopy (SAM) can extend the penetration depth to ap-proximately 1 mm in polymer scaffolds albeit with a reductionin image resolution.6.8 In general, using longer wavelength radiation to im-prove pen
39、etration of the radiation is accompanied by a reduc-tion in resolution.7. Image Capture and Storage7.1 Image acquisition in this guide refers to the process ofcapturing an image through digitization that is then stored forsubsequent analysis. Care should be taken during this stage toavoid loss of fi
40、delity by controllable factors that are not relatedto the methodology used to produce the image. These factorsinclude the spatial sampling frequency of the detector system,the dynamic range of analogue to digital (A/D) conversion,segmenting (thresholding) operations (discussed in Section 9),and both
41、 image compression and decompression.7.2 Spatial sampling frequency and appropriate A/D con-version are straightforward issues; the sampling frequencyshould be at least twice the inverse spatial resolution, so as tofulfill the Nyquist criterion. Sampling at frequencies below thiswill lead to the dis
42、play of artifacts. Most image processingsystems have anti-aliasing filters that remove frequenciesgreater than Fs/2 Hz, where Fsis the digital sampling rate. TheA/D conversion should utilize a sufficient number of bits tocover the dynamic range of the imaging / detector system.Eight-bit conversion a
43、nd recording is used for most commonimaging applications, resulting in images with 256 grayscalelevels, where 0 corresponds to pure black and 255 to purewhite respectively. If 8-bit conversion is used in a color (RGB)image there are 256 possible color combinations.NOTE 1The gamut, or range of the gr
44、ayscale reflects the imagecontrast.7.3 It is important to record the minimum measurementvalue (that is, the dimensions of a single pixel) when usingdigital capture or digitizing film-based images at all magnifi-cations used in measurements (Test Method F1854).7.4 Image compression is used to facilit
45、ate rapid display ofdata and easy file transmission. However, many compressionmethods (JPEG, PNG, and GIF) cause a loss of data. This lossgenerally occurs in the high-frequency components of thespatial Fourier spectrum of the image, leading to an oscillating,smeared grayscale, or color intensity pro
46、file at the object edges.Some proprietary compression methods are purported to in-volve no loss of information and thus be completely reversible(7). There are excellent internet resources describing compres-sion and decompression techniques (7).7.5 After capture, an image can be manipulated to facil
47、itatesubsequent analysis. Such transformations include noise3The boldface numbers in parentheses refer to a list of references at the end ofthis standard.TABLE 1 Sources of Contrast and Techniques to GenerateImages of Tissue ScaffoldsGeneric method Contrast source Maximum resolution(lateral/axial)Ph
48、ysical slicingrequired for 3Dimaging?Widefield OpticalMicroscopyRefractive indexFluorescenceAbsorbance1 m/(10 m) YConfocal OpticalMicroscopyRefractive indexFluorescenceAbsorbance0.5 m/1 m NOptical CoherenceTomography (orMicroscopy)Refractive index 1 m/1 m NScanning AcousticMicroscopy (SAM)Acousticim
49、pedance0.1 m/0.1 m(depending on thewavelength chosen)NMagneticResonanceImaging (MRI)Nuclear spin 10 m/10 m NX-ray Micro-ComputedTomography(-CT)Electron density 10 m/10 m NTransmissionElectronMicroscopy (TEM)Electron density Approximately 0.2nm in planeYScanning ElectronMicroscopy (SEM)Electron density Approximately 10 nm NF2603 06 (2012)3suppression, enhancement of regions that are of particularinterest, and corrections that compensate for instrument orexperimental limitations.7.6 Noise SuppressionThe minor random fluctuations insignal inten
