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European Journal of Radiology 83 (2014) 349– 353

Contents lists available at ScienceDirect

European Journal of Radiology

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ffects of repetitive freeze–thawing cycles on T2 and T2*f the Achilles tendon

ric Y. Changa,b,∗, Won C. Baeb, Sheronda Statumb, Jiang Dub, Christine B. Chungb,a

Department of Radiology, VA San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161, United StatesDepartment of Radiology, University of California, 200 West Arbor St., San Diego, CA 92103, United States

r t i c l e i n f o

rticle history:eceived 18 April 2013eceived in revised form 24 August 2013ccepted 12 October 2013

eywords:chilles tendonreeze–thaw22*

a b s t r a c t

Introduction: In this study we sought to evaluate the effects of multiple freezing and thawing cycles ontwo MR parameters to study Achilles tendon, T2 and T2*.Materials and methods: Four fresh Achilles tendons were imaged on a 3T clinical scanner and again after 1,2, 4, and 5 freeze–thaw cycles with spin-echo (SE) and ultrashort echo time (UTE) sequences. Regions ofinterest were manually drawn over the entire Achilles tendon and mono-exponential curves were usedto determine T2 and T2* relaxation times.Results: There was no statistically significant difference in mean T2 or T2* values between the freshspecimens and after subsequent cycles of freeze–thaw treatment (p > 0.1). Linear regression betweenSE T2 values at baseline and after successive freeze–thaw cycles demonstrated moderate agreement

TE (r = 0.60) whereas UTE T2* values at baseline and after successive-freeze thaw cycles demonstrated strongagreement (r = 0.92).Conclusion: These findings suggest that changes between specimens seen in vitro are due to factors otherthan frozen storage. Furthermore, our results suggest that there is stronger agreement between baseline(fresh) and successive freeze–thaw T2* values of tendon obtained with the UTE technique in comparison

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. Introduction

For clinical and research use, cadaveric tendons often undergoultiple freeze–thaw cycles. Clinically, this is most often seenith tendon allografts, such as those used for anterior cruciate

igament reconstruction. In the research arena, it is often difficulto obtain and process material immediately after death and in vitroissue is frequently subject to at least one freeze–thaw cycle foronvenience.

The effects of successive freeze–thaw cycles on material proper-ies of tendon have been studied, and most investigators have foundegligible or no decline in biomechanical parameters with one [1,2]r two cycles [3]. This is not invariable, however, with some authorsoting biomechanical change after a single freeze thaw cycle [4–6].

s the number of cycles approaches four [7] or five [8], the generalonsensus is that detrimental biomechanical changes will be seen.

∗ Corresponding author at: VA San Diego Healthcare System, 3350 La Jolla Villagerive, San Diego, CA 92161, United States. Tel.: +1 858 552 8585x7656;

ax: +1 619 471 0503.E-mail addresses: ericchangmd@gmail.com (E.Y. Chang), wbae@ucsd.edu

W.C. Bae), sherondastatum@msn.com (S. Statum), jiangdu@ucsd.edu (J. Du),bchung@ucsd.edu (C.B. Chung).

720-048X/$ – see front matter. Published by Elsevier Ireland Ltd.ttp://dx.doi.org/10.1016/j.ejrad.2013.10.014

nventional clinical CPMG technique.Published by Elsevier Ireland Ltd.

Multi-parametric quantitative magnetic resonance imaging(qMRI) has shown promise as a surrogate for the structural andmaterial properties of biologic tissue [9], including tendon [10].

The effects of freeze–thawing on cartilage has been studiedby multiple authors and it has been demonstrated that a singlefreeze–thaw cycle increases mono-exponential T2 values [11,12]with a corresponding increase with the total number of cycles[12]. Studies on other tissues have been performed, such as withfish muscle, and it has been shown that T2 is less sensitive tofreeze–thaw changes than T1 and magnetization transfer rate [13].

Discrepancies between qMRI values obtained on tendon in vivoand in vitro have been noted and some have suggested thesechanges are, in part, due to freezing and thawing [10,14–16]. How-ever, we know of no studies that systematically evaluate the effectsof successive freeze–thawing on qMRI values of human tendon. Inthis study we sought to evaluate the effects of multiple freezingand thawing cycles on two MR parameters used to study Achillestendon, T2 and T2*.

2. Methods

2.1. Sample preparation

This anonymized cadaveric study was exempted by the Institu-tional review board. Four fresh human ankles from three donors (3

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emales, mean age 78 years old, range 69–82) were harvested belowhe knee within 6 h of death. The Achilles tendons were imagedithin the intact ankles in three specimens and in one specimen,

he tendon was imaged after it was carefully dissected free fromurrounding tissues. In particular, the tendon was not mechanicallyamaged during removal.

.2. MR imaging acquisition at baseline and after successivereeze–thaw cycles

Specimens were placed parallel to the B0 field and imaging inhe anatomically axial plane was performed on a clinical 3T MRcanner (Signa HDx, GE Healthcare Technologies, Milwaukee, WI)hich had gradients capable of a slew rate of 150 T/m/s and ampli-

ude of 40 mT/m on each axis. Hardware modifications includedhe addition of a custom transmit-receive switch to the receiverreamplifiers for rapid switching at the end of a radiofrequencyxcitation pulse to allow for ultrashort echo time (UTE) imagingith a nominal TE of 8 �s. A 2 in. receive-only surface coil was used

or the whole ankles and a 1-in. diameter birdcage transmit-receiveoil was used for the dissected tendon.

The quantitative imaging protocol is shown in Table 1. T2 and2* was measured for each tendon. Carr–Purcell–Meiboom–GillCPMG) acquisitions were acquired for T2 quantification, wherepin echo (SE) signal from eight echoes (TE = 10, 20, 30, 40, 50,0, 70 and 80 ms, TR = 2000 ms) was subject to a simple expo-ential signal decay model to calculate T2, similar to what haseen previously used on Achilles tendon [16]. 2D UTE acquisitionsere used for T2* quantification, where UTE signal acquired after

series of TEs (TE = 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, 2, 4, 10, 15, 20,nd 30 ms, TR = 100 ms) was subject to a simple exponential sig-al decay model to calculate T2*. For each of the sequences used

n this study, a field of view (FOV) of 5 cm and a slice thickness of mm were prescribed.

Imaging was performed at the same location within the mid-ensile portion of the tendon in the fresh specimens and after 1,, 4, and 5 freeze–thaw cycles. Each cycle consisted of a 24–36 hreeze period at −70 ◦C followed by a 24 h thaw period at roomemperature. Each specimen was wrapped in moist gauze duringreezing to prevent dehydration.

.3. Image analysis

T2 and T2* values were obtained using Levenberg–Marquardttting algorithm developed in-house. The analysis algorithm was

ritten in MATLAB (The Mathworks Inc., Natick, MA, USA) andas executed offline on axial images obtained with the protocolsescribed above. Regions of interest were manually drawn overhe entire Achilles tendon and a mono-exponential curve was used

ig. 1. UTE source images (A) show abundant signal in the Achilles tendon on many imagetting. Echo times are in milliseconds (ms).

Radiology 83 (2014) 349– 353

to determine T2 and T2* relaxation times. T2 was derived throughexponential fitting of the equation: S(TE) ∝ exp(−TE/T2) + constant.T2* was derived through exponential fitting of the equation:S(TE) ∝ exp(−TE/T2*) + constant. Constant refers to backgroundnoise, a separate fitting parameter.

2.4. Statistical analysis

Statistical analyses were performed with Excel (version 2011,Microsoft Corporation, Redmond, Washington) and R software, ver-sion 2.10.1 (2009) (R Foundation for Statistical Computing, Vienna,Austria). First, data was summarized for each solution and eachimaging parameter. To determine effects of freeze–thaw cycles onSE T2 and UTE T2* values, repeated measures ANOVA was used.Next, linear regression between T2 and T2* values of baselineand successive freeze–thaw cycles was performed to determinestrength of correlation as well as intraclass correlation coefficients[17]. To determine the agreement between the baseline and suc-cessive freeze–thaw T2 and T2* values, an analysis similar toBland–Altman analysis [18] was performed and the bias and limitsof agreement were calculated. For all statistical analyses, p-valuesless than 0.05 were considered significant.

3. Results

The Achilles tendon demonstrated very low signal intensity onall CPMG source images whereas source images acquired with theUTE technique showed abundant intratendinous signal on manyimages (Fig. 1). No tendon demonstrated tendinosis or tendon tear-ing on CPMG sources images. However, excellent curve fitting wasable to be performed on all data. Mean SE T2 and UTE T2* valuesfor all four specimens at baseline (fresh) and after five freeze–thawcycles are shown in Table 2. Mean SE T2 value at baseline for all fourspecimens was 17.23 ± 6.49 ms and after five freeze–thaw cycleswas 17.09 ± 1.93 ms. Mean UTE T2* value at baseline for all fourspecimens was 1.18 ± 0.45 ms and after five freeze–thaw cycles was1.11 ± 0.37 ms. Repeated measures ANOVA did not find a statisti-cally significant difference in mean SE T2 values (p = 0.59) or UTET2* values (p = 0.14) (Fig. 2) after freeze–thaw cycles.

Linear regression between SE T2 values at baseline and aftersuccessive freeze–thaw cycles demonstrated moderate agreement(r = 0.60, Fig. 3A) whereas UTE T2* values at baseline and aftersuccessive-freeze thaw cycles demonstrated a strong agreement(r = 0.92, (Fig. 3C). Comparison between SE T2 values at base-line and after successive freeze–thaw cycles showed a small bias

of 1.5 ms with a moderate limit of agreement (LoA) of ±4.6 ms(Fig. 3B) whereas UTE T2* values at baseline and after succes-sive freeze–thaw cycles showed a negligible bias of 0.09 with asmall LoA of ±0.18 ms (Fig. 3D). These findings suggest stronger

s. Mono-exponential decay curve fitting for determination of T2* (B) show accurate

E.Y. Chang et al. / European Journal of Radiology 83 (2014) 349– 353 351

Table 1Imaging parameters for CPMG T2 and UTE T2* sequences.

Sequence TR [ms] TE [ms] FOV [cm] Matrix

T2 2000 10, 20, 30, 40, 50, 60, 70, 80 5 320 × 256UTE T2* 100 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, 2, 4, 10, 15, 20, 30 5 256 × 256

Table 2Results.

Specimen 1 (whole)mean ± SD

Specimen 2 (whole)mean ± SD

Specimen 3 (whole)mean ± SD

Specimen 4 (dissectedtendon) mean ± SD

Overall (4 specimens)mean ± SD

T2 (ME-SE)Fresh 21.04 ± 3.72 10.09 ± 1.95 13.64 ± 1.50 24.15 ± 6.64 17.23 ± 6.49Post 1 cycle 18.69 ± 2.01 11.52 ± 1.55 19.12 ± 3.35 11.02 ± 5.83 15.09 ± 4.42Post 2 cycles 22.91 ± 5.68 14.10 ± 2.38 14.21 ± 0.65 16.71 ± 1.79 16.98 ± 4.13Post 4 cycles 20.98 ± 4.95 14.36 ± 1.37 18.57 ± 1.50 22.10 ± 3.45 19.00 ± 3.43Post 5 cycles 18.92 ± 3.51 14.38 ± 2.36 17.76 ± 1.42 17.31 ± 2.05 17.09 ± 1.93

T2* (UTE)Fresh 1.04 ± 0.07 1.78 ± 0.17 1.17 ± 0.11 0.71 ± 0.04 1.18 ± 0.45Post 1 cycle 0.98 ± 0.08 1.41 ± 0.16 1.12 ± 0.14 0.69 ± 0.05 1.05 ± 0.30

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Post 2 cycles 1.05 ± 0.08 1.67 ± 0.17

Post 4 cycles 0.94 ± 0.06 1.69 ± 0.16

Post 5 cycles 0.88 ± 0.08 1.57 ± 0.12

greement and higher reproducibility of UTE T2* values after mul-iple freeze–thaw cycles in comparison to SE T2 values.

. Discussion

The purpose of this study was to evaluate the change of suc-essive freezing and thawing cycles on qMRI parameters used totudy tendon. In this pilot study, we have found that MR imaging ofadaveric tendon in the fresh state and again after five successivereeze–thaw cycles does not significantly alter T2 and T2* mea-urements. These findings are important because they suggest thatrozen storage can be continued to be used in the study of the MRmaging characteristics of tendon ex vivo.

Histologic studies have shown that freezing tissue results

n changes at the cellular level, including dysfunction of cell

etabolism, ice crystal formation and cell death [19,20]. In fact,uthors have noted that a single freeze–thaw cycle completely

ig. 2. Variations in (A) conventional spin echo T2 and (B) UTE T2* values in tendonamples with freeze/thaw cycles. Mean ± Standard Error of the Mean, n = 4.

8 ± 0.20 0.74 ± 0.05 1.26 ± 0.445 ± 0.14 0.64 ± 0.06 1.11 ± 0.443 ± 0.16 0.76 ± 0.05 1.11 ± 0.37

kills the scant tendon cells that are present [21]. On electronmicroscopy, changes in the extra-cellular matrix have also beennoted with an increase in the mean diameter of collagen fibrils anda decrease in the mean number of fibrils [6]. However, authors havefound that there are no gross macroscopic changes even after eightfreeze–thaw cycles [22]. Despite presumed microscopic changes,our findings are in keeping with the lack of macroscopic change inthat T2 and T2* were not sensitive up to five cycles of freeze–thawtreatment. A possible explanation for this is that the total watercontent and orientation of collagen fibrils remains the same.

Our results also show stronger agreement and higher repro-ducibility between baseline (fresh) and successive freeze–thaw T2*values of tendon obtained with the UTE technique in comparisonto T2 values obtained with a conventional clinical CPMG technique.One possible explanation for this is that tendon demonstrates amajority of “short” T2/T2* components [15] and the signal fromthe majority of the tendon has decayed by the time the first echois acquired on the clinical CPMG technique. Hence there are moreuncertainties in plotting only the “long” T2 components as there isfar less signal compared with UTE techniques.

Quantitative T2 values obtained in our in vitro study are com-parable to those recently reported by Juras et al. who utilized asimilar multi-echo spin-echo technique, which were reported torange from 8.3 to 25.6 at 7 T [16]. Our mono-exponential T2* val-ues are comparable to a prior in vivo study performed by Gold et al.which averaged 1.2 ± 0.2 ms [23,24]. In a prior study, Juras et al.also measured T2* of in vivo tendon at both 3 T and 7 T, utilizing bi-exponential fitting [15]. Linear combination of the short and longcomponents which were reported in their paper results in mono-exponential T2* values of 2.7–9.6 ms. Previously we have shownthat histologically normal areas of in vitro Achilles tendon demon-strated a global mean T2* value of 2.18 ms (range 1.76–2.6 ms)[10,25]. The fact that the T2* values obtained in this current studyare shorter than those by Juras et al. [15] and our previouslyreported results [10,25] are likely due to specimen variation.

Our study has a number of limitations. First, the sample size issmall, limiting the generalizability of these results. However, stud-ies that involve repeated measurements are lengthy, and our studyinvolved 20 successive scanning sessions. Regardless, future stud-

ies that include a larger sample of normal and abnormal tendons arenecessary. Second, these results should not be generalized to otherMR parameters which have been used to study tendon, including T1and T1 rho. In fact, other authors have found T1 and magnetization

352 E.Y. Chang et al. / European Journal of Radiology 83 (2014) 349– 353

Fig. 3. Linear regression of post-freeze/thaw vs. baseline (A) SE T2 and (C) UTE T2* values show a moderate agreement for SE T2 with r = 0.60 and strong agreement for UTET valuel a nem

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2* with r = 0.92. Plots of post-pre difference vs. baseline (B) SE T2 and (D) UTE T2*imit of agreement (LoA) of ±4.6 ms (27% of the mean). (D) UTE T2* values showed

ean).

ransfer rate to be sensitive to the effects of frozen storage onsh muscle [13], but these parameters were not included in thistudy. Additionally, other physical properties of tendon such aslasticity and echotexture which could be measured on ultrasoundould be interesting to evaluate in future studies. Third, we didot perform histological or biomechanical evaluation to correlateith the degree of cellular or material change. Although most prior

tudies have shown that detrimental biomechanical changes areeen as the number of freeze–thaw cycles approaches four or five7,8], we cannot assume that the few specimens used in this studyould have demonstrated this. Fourth, mono-exponential analysesere primarily utilized in this study although tendon demonstratesulti-exponential decay [15,26,27]. When we retrospectively re-

nalyzed our data using a bi-component fitting model [28], we werenable to reliably detect the longer component (consistently lesshan 5% for all samples and time points) likely due to uncorrectedusceptibility causing rapid decay at longer echo times. This is toe expected since our T2* values ranged from 0.64–1.78 ms andhe best fit curve was, in fact, mono-exponential. In comparison tohe gradient echo UTE T2* acquisition, the longer components ofendon were able to be detected with the CPMG technique.

In conclusion, in our pilot study, we have not found a signifi-ant difference in mono-exponential T2 or T2* values on Achillesendons imaged fresh and up to five freeze–thaw cycles. These find-ngs suggest that changes between specimens seen in vitro are dueo factors other than frozen storage. Furthermore, our results sug-est that there is stronger agreement and less variability betweenaseline (fresh) and successive freeze–thaw T2* values of ten-on obtained with the UTE technique in comparison to T2 valuesbtained with a conventional clinical CPMG technique.

onflict of interest

None declared.

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s. (B) SE T2 values showed a small bias of 1.5 ms (9% of the mean) with a moderategligible bias of 0.09 ms (8% of the mean) with a small LoA of ±0.18 ms (15% of the

Acknowledgement

The authors thank grant support from the Veterans AffairsClinical Science Research and Development Service (Career Devel-opment Award 1IK2CX000749-01).

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