1、Designation: F 2603 06Standard Guide forInterpreting Images of Polymeric Tissue Scaffolds1This standard is issued under the fixed designation F 2603; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A numb
2、er 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 the factors that need to be consideredin obtaining and interpreting images of tissue scaffolds includ-ing technique sele
3、ction, instrument resolution and image qual-ity, 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 and natural)
4、may require unique orvaried sample preparation methods that are not specificallycovered in this guide.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and he
5、alth practices and to determine theapplicability of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E 1919 Guide for Worldwide Published Standards Relatingto Particle and Spray CharacterizationE 2245 Test Method for Residual Strain Measurements ofThin, Reflecting Films
6、 Using an Optical InterferometerF 1854 Test Method for Stereological Evaluation of PorousCoatings on Medical ImplantsF 1877 Practice for Characterization of ParticlesF 2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical ProductsF 2450 Guide for Ass
7、essing Microstructure of PolymericScaffolds for Use in Tissue Engineered Medical Products3. Terminology3.1 Definitions:3.1.1 aliasing, nartifactual data that originates from aninsufficient sampling rate.3.1.2 biomaterial, na natural or synthetic material that issuitable for introduction into living
8、tissue especially as part ofa medical device (as an artificial heart valve or joint).3.1.3 blind (end) pore, na pore that is in contact with anexposed internal wall or surface through a single orifice smallerthan the pores depth.3.1.4 closed cell, nvoid within a solid, lacking anyconnectivity with a
9、n 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(adapted from Practice F 1877).3.1.6 hydrogel, na water-based open network of polymerchains that are cross-linked either chemically or throughcry
10、stalline 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 figures thatdefine the nomenclature are given in Appendix X2. (Adaptedfrom Practice F 1877).3.1.8 Nyquist criterionstates that a signal must
11、besampled at a rate greater than or equal to twice its highestfrequency component to avoid aliasing.3.1.9 permeability, na measure of fluid, particle, 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
12、.1.11.1 DiscussionA polymer may be a natural or syn-thetic material.3.1.11.2 DiscussionExamples of polymers include col-lagen and polycaprolactone.3.1.12 pore, na liquid (fluid or gas) filled externallyconnecting channel, void, or open space within an otherwisesolid or gelatinous material (for examp
13、le, textile meshescomposed of many or single fibers (textile based scaffolds),open cell foams, (hydrogels). Synonyms: open pore, throughpore.3.1.13 porosity, nproperty of a solid which contains aninherent or induced network of channels and open spaces.Porosity can be determined by measuring the rati
14、o of pore1This guide is under the jurisdiction 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 Dec. 1, 2006. Published February 2007.2For referenced ASTM stan
15、dards, 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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
16、PA 19428-2959, United States.(void) volume to the apparent (total) volume of a porousmaterial and is commonly expressed as a percentage (GuideF 2150).3.1.14 rectangular, adjA pore that approximates a squareor rectangle in shape (derived from Practice F 1877).3.1.15 roundness (R), na measure of how c
17、losely anobject represents a circle (Practice F 1877).3.1.16 scaffold, na support, delivery vehicle, or matrix forfacilitating the migration, binding, or transport of cells orbioactive molecules used to replace, repair, or regeneratetissues. (Guide F 2150).3.1.17 segmentation, na methodology for dis
18、tinguishingdifferent regions (for example, pores and walls) within a tissuescaffold image.3.1.18 spherical pore, adja pore with a generally spheri-cal shape.3.1.18.1 DiscussionA spherical pore appears round in aphotograph (Practice F 1877).3.1.19 threshold, nisolation of a range of grayscale valuese
19、xhibited by one constituent within an image.3.1.20 through pores, nan inherent or induced network ofvoids or channels that permit flow 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. Alte
20、rnativedefinition: The squared ratio of the mean free path to theminimum possible path length.3.1.22 voxel, nthree-dimensional picture 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 p
21、olymers that include both high water contentgels and woven textiles.4.2 Information derived from tissue scaffold images can beused to optimize the structural characteristics of the matrix fora particular application, to develop better manufacturing pro-cedures or to provide a measure of quality assu
22、rance andproduct traceability. Fig. 1 provides a summary of the keystages of image capture and analysis.4.3 There is a synergy between the analysis of pores intissue scaffolds and that of particles that is reflected instandards cited and in the analysis described in Section 9.Guide E 1919 provides a
23、 compendium of standards for particleanalysis that includes measurement techniques, data analyticaland sampling methodologies.5. Measurement Objectives5.1 Much of the research activity in tissue engineering isfocused on the development of suitable materials and structuresfor optimal growth of a rang
24、e of tissue types includingcartilage, bone, and nerve. This requires a quantitative assess-ment of the scaffold structure.The key parameters that need to be determined are (1) theoverall level of porosity, (2) the pore size distribution, whichcan range from tens of nanometers to several hundred mi-c
25、rometres, and (3) the degree of interconnectivity and tortuos-ity of the pores.6. Imaging Methods and Conditions6.1 There are many experimental ways of obtaining keyscaffold physical parameters as described in Guide F 2450.When imaging and subsequent quantitative analysis is chosenas the method for
26、determining these parameters, it is criticalthat any image under consideration be a true representation ofthe scaffold of interest. Some imaging methods require samplepreparation. Some do not. When sample preparation is requiredprior to imaging, care must be taken that the procedures do notsignifica
27、ntly alter the morphology of the scaffold. See Appen-dix X1 for further information on sample preparation.6.2 Images obtained using techniques such as light micros-copy, electron microscopy, and magnetic resonance imagingare two-dimensional (2-D) representations of a three-dimensional (3-D) structur
28、e. 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 with 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-
29、D) canfaithfully represent the physical artifacts that are influenced byfactors germane to the imaging method, such as spatialresolution and dynamic range, image contrast, and the signal-to-noise ratio. Table 1 lists some of the techniques available forproducing images of porous structures, along wi
30、th their con-trast source, maximum demonstrated spatial resolution, andtypical dynamic range. Proper technique selection dependsboth 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
31、 images generated by the techniques shown in Table1 cannot reproduce features smaller than the spatial resolutionof the method. Features that are faint, that is, those that do notFIG. 1 Key Stages in Image Capture, Storage, and AnalysisF2603062have significant contrast, or signal significantly above
32、 back-ground, will be resolved at length scales larger than themaximum resolution. Excessive contrast can also limit thepenetration depth due to scattering effects. This is particularlytrue of optical microscopies using differences in refractiveindex as the contrast mechanism. An appropriate level o
33、fcontrast that can be established by experimentation is thereforecritical to high quality imaging.6.5 Contrast can be enhanced by using exogenous agents,such as florescence tags in optical microscopy and stainscontaining heavy metal complexes in electron microscopies.Excessive contrast can be amelio
34、rated in optical microscopiesby imbibing the structure with a fluid that has an index ofrefraction similar to that of the solid making up the structure(this is termed “index-matching”). There are many excellentresources describing factors influencing widefield and confocaloptical microscopy (1-3),3o
35、ptical coherence microscopy (4),MRI (5), and electron microscopies (6).6.6 The reconstruction of the mesostructure in 3-D from aseries of 2-D images obtained from a sample that has beenphysically sectioned requires considerably more effort thanassembly of virtual sections produced by techniques that
36、 areable to focus on a plane within the sample. The virtualapproach is also less prone to sample distortion since itobviates the need for physical sectioning and registration errorsin the reassembly process. However, the techniques used togenerate virtual 2-D images typically have limited penetratio
37、ndepth.6.7 Confocal microscopy (OCT), for example, has a pen-etration depth of approximately 100 m, a value that dependson the wavelength of the light used and the amount ofscattering that occurs within the sample. Scanning acousticmicroscopy (SAM) can extend the penetration depth to ap-proximately
38、1 mm in polymer scaffolds albeit with a reductionin image resolution.6.8 In general, using longer wavelength radiation to im-prove penetration 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 ofcapturi
39、ng an image through digitization that is then stored forsubsequent analysis. Care should be taken during this stage toavoid loss of fidelity by controllable factors that are not relatedto the methodology used to produce the image. These factorsinclude the spatial sampling frequency of the detector s
40、ystem,the dynamic range of analogue to digital (A/D) conversion,segmenting (thresholding) operations (discussed in Section 9),and both image compression and decompression.7.2 Spatial sampling frequency and appropriate A/D con-version are straightforward issues; the sampling frequencyshould be at lea
41、st twice the inverse spatial resolution, so as tofulfill the Nyquist criterion. Sampling at frequencies below thiswill lead to the display of artifacts, most image processingsystems have anti-aliasing filters that remove frequenciesgreater than Fs/2 Hz, where Fsis the digital sampling rate. TheA/D c
42、onversion should utilize a sufficient number of bits tocover the dynamic range of the imaging / detector system.Eight-bit conversion and recording is used for most commonimaging applications, resulting in images with 256 grayscalelevels, where 0 corresponds to pure black and 255 to purewhite respect
43、ively. If 8-bit conversion is used in a color (RGB)image there are 256 possible color combinations.NOTE 1The gamut, or range of the grayscale 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
44、 digitizing film-based images at all magnifi-cations used in measurements (Test Method F 1854).7.4 Image compression is used to facilitate rapid display ofdata and easy file transmission. However, many compressionmethods (JPEG, PNG, and GIF) cause a loss of data. This lossgenerally occurs in the hig
45、h-frequency components of thespatial Fourier spectrum of the image, leading to an oscillating,smeared grayscale, or color intensity profile 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 excell
46、ent internet resources describing compres-sion and decompression techniques (7).7.5 After capture, an image can be manipulated to facilitatesubsequent analysis. Such transformations include noise sup-pression, enhancement of regions that are of particular interest,and corrections that compensate for
47、 instrument or experimentallimitations.7.6 Noise SuppressionThe minor random fluctuations insignal intensity or noise that are present in digitized images candegrade the quality of the image if the contrast between thematerial and background is low.Averaging a number of frames,3The boldface numbers
48、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)Physical slicingrequired for 3Dimaging?Widefield OpticalMicroscopyRefractive indexFl
49、uorescenceAbsorbance1 m/(10 m) YConfocal OpticalMicroscopyRefractive indexFluorescenceAbsorbance0.5 m/1 m NOptical CoherenceTomography (orMicroscopy)Refractive index 1 m/1 m NScanning AcousticMicroscopy (SAM)Acousticimpedance0.1 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 NF2603063
