1、Designation: F3259 17Standard Guide forMicro-computed Tomography of Tissue EngineeredScaffolds1This standard is issued under the fixed designation F3259; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A
2、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 is a resource for conducting micro-computedtomography (microCT) imaging and analysis of porous scaf-folds for tissue enginee
3、ring applications. Considerations areprovided for sample preparation, image acquisition parameterselection, post-processing, and data interpretation.1.2 The information in this guide is intended to be appli-cable to products that include a porous scaffold component andare designed for tissue enginee
4、ring repair strategies. Thescaffolds may be fabricated from synthetic polymers (e.g.,absorbable polyesters) or natural materials (e.g., calciumphosphates), mammalian or human derived materials (e.g.,demineralized bone) or combinations of these. While someconsiderations are provided for imaging of ma
5、terials that are ofmoderate to high radiodensity, specific guidelines are notprovided for imaging metallic scaffolds.1.3 Applicability of the guidelines herein will depend onscaffold material type and the users application (e.g., experi-mental design, as manufactured characterization) as appropri-at
6、e.1.4 The guidelines for microCT discussed herein are mostsuitable for specimen scanning in vitro. Specific guidelinesrelevant to direct in vivo imaging of scaffolds are not includedbecause the imaging parameters will be dependent on theimplantation site, animal size, breathing etc. In addition,cons
7、ensus recommendations for in vivo imaging are providedin Bouxsein et al 2010 (1).2While the specific imagingparameters and processing recommendations discussed inBouxsein et al are specific to bone imaging, many of theconsiderations and precautions are also applicable for in vivoscaffold imaging.1.5
8、 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 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-p
9、riate safety and health practices and determine the applica-bility of regulatory limitations prior to use.1.7 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of Intern
10、ational Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:3F2450 Guide for Assessing Microstructure of PolymericScaffolds for Use in Tissue-Engineered Medical ProductsF2603 Guide for Inte
11、rpreting Images of Polymeric TissueScaffolds3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 microarchitecture, nthe set of structural features ofan object defined at the microscale.3.1.2 volume of interest (VOI), na 3D sub-volume insidean image that contains the features to be
12、 analyzed.4. Significance and Use4.1 X-ray microcomputed tomography (microCT) is a non-destructive three-dimensional imaging method that can be usedto reconstruct the microarchitecture of a tissue engineeredmedical product (TEMP) scaffold that may or may not containingrown tissue. MicroCT was first
13、developed to study ceramicsfor the auto-industry and adapted for bone morphology at themicroscale (Feldkamp et al 1989) (2). More recently, theimaging method has been adapted for in vivo applications andstudies of multiple natural and synthetic materials.1This guide is under the jurisdiction of ASTM
14、 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 May 1, 2017. Published September 2017. DOI:10.1520/F3259-17.2The boldface numbers in parentheses refer to the list of
15、 references at the end ofthis standard.3For 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.Copyright ASTM Interna
16、tional, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Gu
17、ides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.14.2 Alternate characterization methods for assessing scaf-fold microarchitecture and tissue ingrowth are limited by theirtwo dimensional nature (e.g., microscopy) and low depth ofpenetration
18、(e.g., optical coherence tomography), even thoughtheir resolution may be increased over microCT. However,microCT is an ideal imaging choice for studying scaffoldmicroarchitecture and tissue ingrowth because it is non-destructive, provides scaffold assessments based on directmeasurements rather than
19、stereological methods, offers theability to perform longitudinal imaging, and can be conductedat length scales relevant to cells and cell attachment (i.e., 1micron to hundreds of microns).4.3 The microarchitecture of tissue engineered scaffoldsplays a critical role in providing structural support an
20、d/orfacilitating cell adhesion, proliferation, and phenotype as wellas matrix deposition. These parameters are essential elementsof the tissue engineering strategy. During scaffold degradation,either in vitro or in vivo, changes to the microarchitecturecontinue to influence the eventual tissue repai
21、r. Therefore, it iscritical to characterize the microarchitecture over time. Suchcharacterization can aid the optimal design of TEMP scaffolds,establishment of manufacturing consistency, and monitoring ofscaffold structure and/or tissue response.4.4 This guide provides a compendium of informationrel
22、ated to the use of microCT for the structural assessment ofscaffold microarchitecture and tissue ingrowth. While themicroarchitecture of tissue engineered scaffolds, as well aschanges to it over time, can be assessed using multiplemethods, (e.g., such as those described in Guide F2450), thisguide fo
23、cuses on unique considerations for conducting themicroCT analyses.4.5 The user of this guide is provided with considerationsfor each aspect of a complete microCT study including samplepreparation, image acquisition, assessing image quality andartifacts, post-processing, and image interpretation base
24、d onthe specific application.4.6 This standard provides imaging and analysis consider-ations for the following broad types of applications: (a)scaffold microarchitecture analysis in vitro either before orafter different stages of degradation, (b) ex vivo analysis ofscaffold microarchitecture followi
25、ng partial degradation in anin vivo animal model, (c) deriving microarchitectural informa-tion when multiple materials are used in the scaffold, and (d)differentiating between scaffold microarchitectural changesand new tissue ingrowth.4.7 The information provided in this standard guide is notintende
26、d as a test method for microCTcharacterization becausethe users specific application and experimental design willsignificantly influence the imaging methodology and interpre-tation.5. MicroCT Characterization Objectives5.1 A significant amount of tissue engineering research isfocused on developing o
27、ptimal scaffold microstructure tofacilitate tissue ingrowth, modulate cell phenotype, and controlthe repair response. Due to the non-destructive nature ofmicroCT, many investigators have utilized this imaging tech-nique as a way to measure numerous architectural parametersquantitatively and to track
28、 them at progressive time points.Specific indices and types of architectural indices that can betracked for scaffolds are discussed in Section 10.5.2 The objective of a microCT assessment of tissue engi-neered scaffolds is dependent on numerous factors and con-trolled by the investigators. Some cons
29、iderations for definingthe objective of the study include the need for point-in-time vslongitudinal assessments, quality assurance, and monitoringtissue growth vs scaffold degradation. This guide is suitable forthe following experimental objectives when performing mi-croCT assessment of a tissue eng
30、ineered scaffold:5.2.1 Quantification of microstructural features (e.g., strutthickness) in the scaffold. This type of assessment may beperformed as part of a quality assurance characterization, (e.g.to test the degree of agreement between design andproduction), or to characterize the microstructura
31、l features.5.2.1.1 These analyses are most typically performed onscaffolds that have not been exposed to an in vivo environment.5.2.1.2 The same set of analyses can be applied to scaffoldsthat have undergone simulated degradation in vitro. MicroCTprovides a simple and non-destructive way to track mi
32、crostruc-tural and physical changes to the scaffold during degradation.Since the technique provides a three-dimensional image of thescaffold, it can be used to determine potential areas ofnon-uniform degradation or other structural features.5.2.1.3 While assessment of these indices is most com-monly
33、 completed following all manufacturing processes, simi-lar considerations would apply if assessing the scaffold atinterim points in the manufacturing process or after exposure toshelf life aging.5.2.1.4 Examples of scaffolds imaged with microCT appearin Fig. 1 to illustrate the type of information t
34、hat may begathered and the heterogeneity of visual representation forTEMP scaffolds.5.2.2 Ex vivo characterization of changes to the scaffoldmicroarchitecture following degradation and tissue ingrowth invivo. This type of assessment includes removal of the scaffoldfrom the animal model prior to imag
35、ing. This characterizationwould provide the user with information on geometric altera-tions to the structure of the scaffold over time followingimplantation in an animal model.5.2.2.1 Performing microCT of the scaffold after implanta-tion in an animal model has unique challenges as illustrated bythe
36、 images in Fig. 2.5.2.2.2 Specific considerations for each aspect of perform-ing a microCT study of a scaffold while implanted in an in vivoanimal model, however, are beyond the scope of this guide andare covered in Bouxsein et al 2010 (1).5.2.3 Ex vivo characterization of tissue ingrowth. Inadditio
37、n, to understanding how the scaffold degrades and isaltered following implantation, microCT can be used to quan-tify the extent of tissue ingrowth and provide some basicinformation on the type of tissue regenerated.5.2.3.1 While in theory, using microCT to quantify theamount of tissue ingrowth in th
38、e presence of a tissue engi-neered scaffold is feasible, it is limited by the ability of themicroCT to differentiate the radiodensity of scaffold materialF3259 172as compared to tissue. In practice, this has been most readilyachieved by quantifying the production (Peyrin 2011) (3) ofbone since this
39、tissue type has a much higher density than thatof many synthetic absorbable polymeric scaffolds.5.3 MicroCT characterizations of tissue engineered scaf-folds may be completed on structures that are fabricated fromone or multiple materials. The ability to differentiate multiplematerials within a scaf
40、fold will be dependent on the composi-tion of those materials and their radiodensity.5.4 Some applications may necessitate designing the experi-ment in order to include various types of controls. Examples ofcontrols which may be used to facilitate microCT imageanalysis and/or interpretation may incl
41、ude the following:5.4.1 Blank scaffolds that are stable and do not change theirarchitecture (i.e., without any cells or degradation).5.4.2 For applications where the tissue engineered scaffoldof interest is designed from multiple materials, the microCTexperiment may necessitate imaging of different
42、scaffolds,each manufactured with only one of the pure materials. Theseadditional images may be used to aid threshold selection.6. Sample Preparation6.1 Scaffold dimensions and/or design are important whenpreparing TEMPs scaffolds for microCT imaging. Duringsample preparation, it is recommended that
43、the key features ofthe scaffold (e.g. pore size, strut thickness, density, etc) whichFIG. 1 Examples of TEMP Scaffolds Scanned Alone in vitro by X-ray MicroCTF3259 173need to be resolved and quantified be identified in order toprepare the sample appropriately. In particular, the desiredvoxel size fo
44、r the scaffold should be considered when prepar-ing samples for microCT imaging.6.1.1 The voxel size is of critical importance to the microCTusers and their ability to extract quantitative information. Voxelsize is dependent on many aspects of the microCT experiment,including the field of view (see
45、section 6.3), scanning param-eters (see Section 7), and reconstruction (see Section 8).6.1.2 When selecting the size of the specimen holder/ fieldof view, microCT scan parameters and reconstruction, thevoxel size will be calculated and presented to the user. The usershould ensure that the voxel size
46、 is appropriate for imagingstructures of interest. It is recommended to image scaffoldswith voxel dimensions that are at least one third and moreoptimally one tenth of the size of the relevant scaffold features(e.g., strut size).6.1.3 It should be noted that voxel size is not the same asspatial reso
47、lution of the microCT image and microCT manu-facturers may report this information differently. A discussionof the difference between voxel size and spatial resolution canbe found in Bousxein et al., 2010 (1).6.2 The microCT scan resolution will be determined by thescaffold size and structures of in
48、terest within the scaffold. Ingeneral, higher resolution microCT scans are obtained whenusing a smaller sized specimen holder/ field of view (FOV). Ifvery small structures need to be resolved and analyzed, theFOV should be small enough to achieve a resolution sufficientto resolve all the small struc
49、tures of interest.6.2.1 Typically, there is no optimal size to address allresearch questions for a scaffold. In this case, the scaffold mayhave to be cut into different size specimens to facilitatemeasurements at different resolutions.NOTE 1These examples illustrate some of the challenges associated with differentiating the scaffold from surrounding tissue if the radiodensity issimilar. In (A), a grayscale image of a polycaprolactone (PCL) scaffold impregnated with bone morphogenetic protein (BMP) and implanted in rat muscletissue. Voxel size 6.8 microns. The PCL scaffold itsel
