1、Designation: F3224 17Standard Test Method forEvaluating Growth of Engineered Cartilage Tissue usingMagnetic Resonance Imaging1This standard is issued under the fixed designation F3224; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision
2、, 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 standard is intended as a standard test method forengineered cartilage tissue growth evaluation usi
3、ng MRI.1.2 This standard is intended for use in the development oftissue engineering regenerative medical products for cartilagedamages, such as in knee, hip, or shoulder joints.1.3 This standard has been prepared for evaluation ofengineered cartilage tissue growth at the preclinical stage andsummar
4、izes results from tissue growth evaluation of tissue-engineered cartilage in a few notable cases using waterspin-spin relaxation time, T2, in vitro and in vivo in smallanimal models.1.4 This standard uses the change in mean T2values as afunction of growth time to evaluate the tissue growth ofenginee
5、red cartilage.1.5 This standard provides a method to remove the scaffoldcontribution to the tissue growth evaluation.1.6 Information in this standard is intended to be applicableto most porous natural and synthetic polymers used as ascaffold in engineered cartilage, such as alginate, agarose,collage
6、n, chitosan, and poly-lactic-co-glycolic acid (PLGA).However, some materials (both synthetic and natural) mayrequire unique or varied methods of MRI evaluation that arenot covered in this test method.1.7 This standard does not purport to address all of thesafety concerns, if any, associated with its
7、 use. It is theresponsibility of the user of this standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.8 This international standard was developed in accor-dance with internationally recognized princi
8、ples on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 The following referenced documents are indispensa
9、blefor the application of this document. For dated references, onlythe edition cited applies. For undated references, the latestedition of the referenced document applies.2.2 ASTM Standards:2F2312 Terminology Relating to Tissue Engineered MedicalProductsF2529 Guide for in vivo Evaluation of Osteoind
10、uctive Po-tential for Materials Containing Demineralized Bone(DBM)F2603 Guide for Interpreting Images of Polymeric TissueScaffoldsF2664 Guide for Assessing the Attachment of Cells toBiomaterial Surfaces by Physical MethodsF2978 Guide to Optimize Scan Sequences for Clinical Di-agnostic Evaluation of
11、Metal-on-Metal Hip ArthroplastyDevices using Magnetic Resonance Imaging2.3 ISO Standard:3ISO/TR 16379-2014 Tissue-engineered medical products Evaluation of anisotropic structure of articular cartilageusing DT (Diffusion Tensor)-MR Imaging3. Terminology3.1 Definitions of Terms Specific to This Standa
12、rd:3.1.1 biomaterial, nany substance (other than a drug),synthetic or natural, that can be used as a system or part of asystem that treats, augments, or replaces any tissue, organ, orfunction of the body. F26643.1.2 chondrocyte, na cell that has secreted the matrix ofcartilage and becomes embedded i
13、n it.3.1.3 chondrogenic differentiation, nthe biological pro-cess of stem cells changing their lineage into chondrocytes. Ifthe starting cells are chondrocytes, this term refers to differen-tiation of cells into the same phenotype.1This test method is under the jurisdiction ofASTM Committee F04 on M
14、edicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.44 on Assessment for TEMPs.Current edition approved Nov. 1, 2017. Published February 2018. DOI: 10.1520/F3224-17.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer
15、Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.Copyright ASTM International
16、, 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, Guides a
17、nd Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.1.4 chondrogenic extracellular matirx (chondrogenicECM), nan extracellular matrix containing cartilaginousmatrix proteins such as proteoglycan, collagen type II, collagentype X and other matrix p
18、roteins found in cartilage.3.1.5 echo time (TE), ntime after 90 pulse in an MRIpulse sequence until an echo signal is formed.3.1.6 fast low angle shot (FLASH) MRI, na gradient echoMRI acquisition technique with low flip angle radiofrequencypulse excitation and short repletion time for fast imageacqu
19、isition.3.1.7 field of view (FOV), nMR image acquisition param-eter that defines the dimensions of the imaging plane (ex-pressed in cm cm or mm mm).3.1.8 histological assessment of engineered cartilage tissuegrowth, nhistological assessment is used to assess the pres-ence of cartilage extracellular
20、matrix proteins in the engineeredcartilage to evaluate the tissue growth (e.g. Safranin O stainingfor proteoglycan assessment).3.1.9 hydrogel, na water-based open network of polymerchains that are cross-linked either chemically or throughcrystalline junctions or by specific ionic interactions. F2603
21、3.1.10 in-plane resolution, nthe spatial resolution of animage (typically expressed in mm mm or m m). It isgiven by = FOV/acquired matrix size.3.1.11 magnetic resonance imaging (MRI), nan imagingtechnique that uses static and time-varying magnetic fields toprovide tomographic images of tissue by the
22、 magnetic reso-nance of nuclei. F29783.1.12 matrix size, nthe number of pixels in each imagedimension of FOV.3.1.13 mesenchymal stem cell (MSC), na multipotent cellderived from mesenchyme that is capable of proliferating anddifferentiating in chondrogenic lineage and can produce acartilage extracell
23、ular matrix.3.1.14 multi slice multi echo (MSME) MRI, nan MRIpulse sequence for the measurement of T2where a series of180 RF pulses (number of echoes) is followed by a 90 RFpulse in a multi-slice MRI pulse sequence. This pulse sequenceis the MRI extension of similar nuclear magnetic resonance(NMR) s
24、pectroscopy sequence named Carr-Purcell-Meiboom-Gill (CPMG) echo train pulse sequence for T2measurement.3.1.15 number of averages (NA), nthe number of times anidentical MRI experiment is repeated to improve the SNR.3.1.16 pulse sequence, nprogrammed train of RF andgradient pulses. In MRI, it is a ti
25、me protocol for obtainingimages.3.1.17 quantitative real-time polymerase chain reaction(qRPCR), na laboratory technique for the detection,selection, and amplification of specific gene transcripts basedon their genetic sequence. Commonly, it is used to assess thepresence of chondrogenic markers such
26、as Sox9, RUNX2,ECM proteins, etc. in a tissue-engineered cartilage.3.1.18 radiofrequency pulse (RF pulse), na short durationradiofrequency electromagnetic pulse used for changing thedirection of magnetization vector.3.1.19 rapid acquisition with refocused echoes (RARE)MRI, nan MRI pulse sequence for
27、 fast image acquisition.This MRI pulse sequence is characterized by a series of 180RF rephasing pulses followed by a 90 RF pulse, with eachecho is individually phase-encoded for fast image acquisition.3.1.20 region of interest (ROI), na user-defined area of animage in which parameter of interest is
28、calculated.3.1.21 relaxation rate (R2), ninverse of spin-spin relax-ation time (R2= 1/T2).3.1.22 repetition time (TR), ntime interval between con-secutive 90 RF pulses or the time interval when the basic unitof MRI pulse sequence is repeated. ISO/TR 16379-20143.1.23 scaffold, nthree-dimensional natu
29、ral or syntheticbiomaterial typically made out of one or more polymers(natural or synthetic) and used as a skeleton for cell seeding.F26033.1.24 signal to noise ratio (SNR), nthe ratio of theamplitude of any signal of interest to the amplitude of theaverage background noise which includes both coher
30、ent andnon-coherent types of noise.3.1.25 slice thickness, nthe thickness of the 2D imagingplane in an MRI image. ISO/TR 16379-20143.1.26 spin echo (SE) MRI, na method for acquiring MRimages based on the spin-echo pulse sequence.3.1.27 spin-spin relaxation time (T2), nT2refers to thecharacteristic e
31、xponential time constant of the transversemagnetization. This is typically the time taken for the trans-verse magnetization to decrease to 37% of the initial value It istypically depicted in milliseconds (ms).3.1.28 stem cell, nan undifferentiated cell that is capableof developing into many differen
32、t cell types.3.1.29 voxel, nthe minimum unit volume of a three-dimensional MRI image. ISO/TR 16379-20144. Significance and Use4.1 Tissue-engineered cartilage is prepared by seeding stemcells or chondrocytes in a three-dimensional biodegradablescaffold under controlled growth conditions. It is expect
33、ed thatthe cells will differentiate towards chondrogenic lineage andproduce an ample amount of cartilage extracellular matrixproteins, proteoglycans, and collagen type-II. Longitudinalassessment is needed weekly for the first few weeks in vitroand monthly at a later stage in vivo to determine the gr
34、owth rateof tissue-engineered cartilage. Traditional testing methods suchas histological staining, mechanical testing, and qPCR areinvasive, destructive, and cannot be performed in vivo after thetransplantation of engineered tissue as a regenerative treat-ment. In the regenerative medicine of cartil
35、age, it is importantto evaluate whether the implanted tissue regenerates as anarticular cartilage over time. MRI is the only available non-invasive imaging modality that is utilized for post-operativemonitoring and assessment of cartilage regeneration in clinics.Therefore, it is important to evaluat
36、e tissue-engineered carti-lage using MRI at the preclinical stage as well.4.2 Preclinical in vivo assessment of tissue-engineered car-tilage is performed in small animal models such as mice, ratsF3224 172or rabbits, and in large animal models such as goats, pigs, andhorses. It is possible to evaluat
37、e engineered cartilage tissuegrowth at each stage of development non-invasively usingMRI. This may reduce the number of animals needed for theassessment and will provide a good estimate of cartilageregeneration.4.3 Parametric MRI technique allows non-invasive quanti-tative assessment of tissue growt
38、h in vitro and in vivo. Whenthe amount of extracellular matrix increases over time, theinteraction of the water molecule with its surroundingschanges, and this creates a change in T2. The amount of changein T2is directly correlated with the amount of matrix generatedwith high sensitivity and specifi
39、city. The T2MRI is thus usedto observe tissue growth for use commonly in longitudinaldiagnosis following cell seeding in a scaffold in vitro orfollowing tissue implantation in vivo.4.4 The T2MRI for preclinical evaluation of engineeredcartilage takes into account the presence of a scaffold in thedev
40、eloping tissue-engineered cartilage. These data are pub-lished in refereed journals and book chapters, and includedhere as a guide for preclinical quantitative evaluation forengineered cartilage tissue growth (2-12).4Additional datautilizing T2MRI for tissue growth evaluation of engineeredcartilage
41、can be found in the references (13-15).4.5 Magnetic resonance parameters of water protons intissue are sensitive to the tissue microstructure. In cartilagetissue engineering, cells produce primarily cartilage extracel-lular matrix proteins, proteoglycans, and collagen, type-II. Astissue matures with
42、 the production of ECM, the matrix changesthe environment around water molecules. The water nuclearspins find several new pathways for relaxation and the T2generally is lower from the original value. Fig. 1 shows theeffect of water tumbling rate and magnetic field strength on T2.As shown by the blue
43、 arrow, when the engineered cartilagetissue matures, the tumbling rate of the water molecule is lowerand as a result, the T2is lower. The reduction of T2as afunction of tissue growth is the basis of engineered cartilageassessment using MRI. Fig. 1 also shows that this principleholds true from low to
44、 high magnetic field strengths (1.5 T 11.7 T) that are commonly used in MRI assessment.4.6 As shown in Fig. 1, the change in T2is dependent on themagnetic field strength and initial tumbling rate of the watermolecule that signifies its surrounding.4.7 The principle of reducedT2with increased tissue
45、growthgenerally holds true for scaffold-free cartilage tissue engineer-ing. However, in scaffold-based cartilage tissue engineering,the following relationship should be used to assess the tissuegrowth (3, 6):R2ECM! 5 R2TEC!2R2Control! (1)4The boldface numbers in parentheses refer to the list of refe
46、rences at the end ofthis standard.FIG. 1 The change in the water relaxation time T2as a function of the magnetic field and the tumbling rate of the water molecule usingBPP theory of relaxation (1). Note that the tumbling rate of the water molecule decreases with increasing tissue growth. The blue ar
47、rowshows the direction of change of the relaxation time, T2, as a function of the tissue growth.F3224 173where:R2= 1/T2,R2(ECM) = the calculated relaxation rate arising fromcartilage extracellular matrix,R2(TEC) = the measured experimental relaxation rate ofthe tissue-engineered cartilage graft, and
48、R2(Control) = the relaxation rate of the scaffold withoutcells.4.7.1 The change in calculated relaxation rate, R2(ECM),using Eq 1 have been found to be positively correlated withtissue growth (3, 6).5. MRI Assessment of Engineering Cartilage TissueGrowth5.1 Sample Preparation:5.1.1 In Vitro MRI Asse
49、ssmentTypically, MRI tissue as-sessment of early stage in vitro samples is performed usingvertical bore high field MRI scanner and a small diameterradiofrequency probe ( 5-10 mm) that is equipped withgradients in the x-, y-, and z-directions and relevant acquisitionsoftware for pulse sequence generation and data acquisition.The samples that are small ( 3-5 mm in diameter) can bepacked using the technique shown in Fig. 2. As shown in thefigure, samples are placed in an MRI-compatible tube on top ofa susceptibility matched plug (or
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