Ligamentization of the reconstructed ACL differs between the intraarticular and intraosseous regions: A quantitative assessment using UTE-T2* mapping.

BACKGROUND: The purpose of this study was to prospectively observe the trends of ultrashort echo time (UTE)-T2* values for the intraarticular and intraosseous regions of reconstructed anterior cruciate ligaments from 6 to 12 months after anterior cruciate ligament reconstruction by using UTE-T2* mapping, and to investigate the changes and differences over time in each region.
METHODS: Ten patients underwent UTE-T2* mapping of the operated knee at 6, 9, and 12 months after anterior cruciate ligament reconstruction. The UTE-T2* values of intraarticular and intraosseous regions of reconstructed anterior cruciate ligaments at 6, 9, and 12 months postoperatively were statistically compared.
RESULTS: The UTE-T2* values of the intraarticular region at 6 months postoperatively were significantly higher than those at 9 and 12 months. There were no significant differences in the UTE-T2* values at 6, 9, and 12 months postoperatively in the intraosseous region. At 6 months postoperatively, the UTE-T2* values of the intraarticular region were significantly higher than those of the intraosseous region. The UTE-T2* values of the intraosseous region at the tibia were significantly lower than those of the other sites at any postoperative time point.
CONCLUSIONS: According to UTE-T2*mapping-based findings, histological maturation of reconstructed ACLs is faster in the intraosseous region than in the intraarticular region. In particular, the intraarticular region is still undergoing rapid histologic changes at 6 months postoperatively, and its tissue structure is less substantial than normal. The findings of this study may provide clues to determine the optimal timing for safe return to sports in terms of ligamentaization of reconstructed ACLs.

Introduction

Patients who undergo primary anterior cruciate ligament (ACL) reconstruction (ACLR) are at a high risk of ipsilateral retear in the first 12 months, and early return to sport (RTS) after ACLR is one of the most important risk factors [1-4]. The ligamentization status of the reconstructed ACL is an important factor when considering RTS [3-5]. Reconstructed ACLs undergo cytological rearrangement and adapt to their biological and mechanical environment over time after ACLR [6]. These findings are based on samples obtained from animals and humans, but not all results from studies with animals can be correlated with physiological changes in humans [6-8]. Histological monitoring of the reconstructed ACL may be ideal, but ethical considerations limit the possibility of a second look and tissue harvesting in patients showing good progress, and the evaluations are limited to the collection site [3,4]. Magnetic resonance imaging (MRI) has been used to evaluate the ligamentization process of the reconstructed ACL to compensate for these limitations.

MRI signals, such as the signal-to-noise quotient (SNQ) and median signal intensity (SI), reflect the biological processes underlying cell proliferation and extracellular matrix remodeling, and have been considered useful for assessing the ligamentization process of reconstructed ACL [5,9-11]. However, these MRI signals show problems related to accuracy and quantification. A previous review has shown that the SNQ and SI of reconstructed ACLs vary significantly even in studies with similar imaging acquisition protocols and postoperative time points [9]. This can be attributed to the fact that the conventional MRI signal intensity is affected by the image sequence and scanner hardware. Moreover, Tendon and ligaments normally have short T2 relaxation times leading to low MRI signal in conventional MRI protocols [3-5,9].

Relaxation time T2* reflects the intrinsic property of tissue and should be independent on image sequence and acquisition parameters [12]. These variables reflects the T2* relaxation of bounded water with collagen of tendons and ligaments, and an ideal to capture the changes in tissue structure and organization during ligamentization of reconstructed ACL [3,4,13]. The ultrashort echo time (UTE) pulse sequence is a method of acquiring data immediately after excitation by using short radiofrequency pulses. By acquiring multiple echoes, the UTE-T2* relaxation time of tendons and ligaments, which cannot be assessed in conventional gradient-echo based MRI assessment due to sub-ms T2* values of collagen-bound water [13-15]. Thus, the UTE T2* technique is an excellent tool for observing the ligamentization process of a reconstructed ACL [3,4].

The critical time for RTS after ACLR is 6–12 months postoperatively, but data evaluating the ligamentization process of the reconstructed ACL by using the UTE-T2* technique are extremely limited [3,4]. Although the intraarticular and intraosseous regions of reconstructed ACLs undergo different maturation processes, the difficulties in biopsy have limited the available knowledge of the intraosseous region of reconstructed ACLs. Although the influence of the differences in each process in the images is important, no study has evaluated the maturation process of the intraosseous region of reconstructed ACLs by using the UTE-T2* technique.

To address these aspects, the purpose of this study was to prospectively observe the trends in UTE T2* values for the intraarticular and intraosseous regions of reconstructed ACLs from 6 to 12 months after ACLR by using UTE-T2* mapping, and to investigate the changes and differences over time in each region. We hypothesized that the UTE T2* values and their trends would differ for the intraarticular and intraosseous regions of reconstructed ACLs at each postoperative time point.

Patients and methods

Patient selection

The study design was approved by the Ethical Committee of the Graduate School of Medical Sciences, Kanazawa University (#2936), and conducted in accordance with the principles expressed in the Declaration of Helsinki. Patients who underwent initial ACLR with hamstring tendons during 2018–2020 were eligible for inclusion. The purpose of this study was explained to the participants, and written informed consent was obtained from the participants or their parents. Patients with a history of ipsilateral or contralateral knee injury or surgery and those who were unable to attend the hospital or undergo MRI were excluded.

Ten female patients were enrolled, and they underwent UTE-T2*mapping of the operated knee at 6, 9, and 12 months postoperatively. The mean age of participants at the start of the study was 18.4 ± 4.3 years. The mean body mass index (BMI) was 21.7±2.1 kg/m2 and the mean time to surgery was 42.1 ± 11.3 days. Five patients had right knee injuries and five had left knee injuries, and all were non-contact injuries.

Surgical procedure

All patients were treated by a single orthopedic surgeon specializing in arthroscopy. In all cases, the transplanted tendon was created with a single bundle of semitendinosus or semitendinosus and gracilis tendons. The femoral tunnel was created in the middle of the anatomical ACL footprint by the inside-out method using a rounded rectangular dilator [16]. The tunnel on the tibial side was created in a circular shape in the middle of the anatomical ACL footprint. The femoral side of the graft tendon was fixed using a cortical device (Tight Rope; Arthrex, USA). After the graft was pretensioned several times, the tibial side was fixed using a tibial fixation implant (Tension-Loc; Arthrex, USA). All patients underwent rehabilitation using a standardized postoperative protocol.

Imaging procedure

A clinical 1.5-T MRI scanner (Ingenia 1.5 T CX; Philips Healthcare, Best, The Netherlands) and an eight-channel receiver knee coil were used for all patients. The UTE-T2*maps were calculated via monoexponential fitting of a series of T2*-weighted MR images, which were acquired using the 3D fast-field echo technique. The typical acquisition parameters were as follows: slice thickness, 3 mm; number of slices = 45; field of view = 16 cm; echo time (TE)/repetition time (TR) = 0.14, 4.74, 9.34, and 13.94 ms/29 ms; flip angle = 25°; acquisition matrix = 272 × 272; bandwidth = 522 Hz; and scan time = 9 min 31 s. Four sets of images were obtained using a single four-echo UTE acquisition. Images were obtained from a picture archiving and communication system. T2* maps were directly calculated on a pixel-by-pixel basis by using a monoexponential fitting algorithm available on the scanner. The equation is expressed as follows: SI(TE) = S0*exp(-TE/T2*), where SI(TE) is the single intensity at each TE, and S0 is the equilibrium magnetization.

The slices in the UTE-T2* map for measuring T2* values were selected by referring to the slice in which the reconstructed ACL was more distinct in the oblique sagittal T2-weighted image. The UTE-T2* values for the intraarticular region of the reconstructed ACLs were measured at three sites based on the method previously reported by Okuda et al [15]: proximal, middle, and distal. Values for the intraosseous regions of the reconstructed ACLs were measured at one site each in the tibia and femur. One orthopedic surgeon (RY, Observer 1) used a 5–10 mm2 circle to manually segment the regions of interest (ROIs) within areas unaffected by artifacts (Fig 1). All measurements were taken three times, and the average value was used as the UTE-T2* value for each region. The UTE-T2* values of the intraarticular region were calculated by further averaging those of the proximal, middle, and distal sites. To assess interobserver reliability, another orthopedic surgeon (YY, Observer 2) independently performed measurements using the same method.

Fig 1

Measurement of the UTE T2* values.

The UTE-T2* values for the intraarticular region of reconstructed ACLs were measured at three sites, and those for the intraosseous region of reconstructed ACLs were measured at one site each. The regions of interest (ROIs) for each site were segmented at the areas unaffected by artifacts by using a 5–10 mm2 circle.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics for Windows, version 27.0. The UTE-T2* values for the intraarticular and intraosseous regions of reconstructed ACLs at 6, 9, and 12 months postoperatively were compared using one-way analysis of variance (ANOVA). The UTE-T2* values for the intraarticular and intraosseous regions of reconstructed ACLs at each postoperative month were also compared using ANOVA. Statistical significance was set at P < 0.05. The intra- and interobserver reliabilities (intraclass correlation coefficient [ICC]) of the UTE-T2* values at 6 months after ACLR were calculated, and the measured values were rated as follows; 0.00–0.40, poor; 0.41–0.75, fair to good; and 0.76–1.00, good to excellent. Sample size was calculated using G-power 3.1 (effect size, 1.3; α-error, 0.05; and target power, 0.8); a minimum of nine participants was recommended on the basis of a previous study [4].

Results

The UTE-T2* values of the intraarticular region of reconstructed ACLs were 13.1 ± 1.9 ms, 11.7 ± 1.5 ms, and 11.1 ± 1.3 ms, respectively, at 6, 9, and 12 months postoperatively, and the UTE-T2* value at 6 months postoperatively was significantly higher than those at 9 and 12 months (P < 0.01 vs. 9 months; P < 0.01 vs. 12 months). Compared to the UTE-T2* value of the normal ACL reported previously, the T2* value at 6 months postoperatively was significantly higher (P<0.01) (Fig 2). In the intraosseous region of reconstructed ACLs, the UTE-T2* values at the tibial site were 7.4 ± 1.2 ms, 7.1 ± 1.0 ms, and 6.9 ± 1.2 ms, respectively, at 6, 9, and 12 months postoperatively, with no significant difference between the values at 6 and 9 months (P = 0.44), 6 and 12 months (P = 0.20), and 9 and 12 months (P = 0.85). The UTE-T2* values at the femoral site were 11.5 ± 2.4 ms, 11.0 ± 1.7 ms, and 11.1 ± 1.5 ms at 6, 9, and 12 months postoperatively, also showing no significant difference between the values 6 and 9 months (P = 0.56), 6 and 12 months (P = 0.72), and 9 and 12 months (P = 0.97). At 6 months postoperatively, the UTE-T2* values were significantly higher for the intraarticular region of reconstructed ACLs than for the intraosseous region of reconstructed ACLs (P < 0.01 vs. tibial site; P < 0.01 vs. femoral site). The UTE-T2* values at the tibial site for the intraosseous region were significantly lower than those at the other sites at all time points (9 months: P < 0.01 vs. femoral site, P < 0.01 vs. intraarticular; 12 months: P < 0.01 vs. femoral site, P < 0.01 vs. intraarticular) (Table 1, Fig 3). Interobserver reliability was good to excellent for both intraarticular and intraosseous regions in the segmentation and registration process at 6 months after ACLR (ICC, 0.84; 95% CI, 0.57–0.93; tibial site: ICC, 0.94; 95% CI, 0.88–0.97; femoral site: ICC, 0.76; 95% CI, 0.50–0.89). Intraobserver reliability at 6 months after ACLR was also good to excellent in all regions (intraarticular: ICC, 0.97; 95% CI, 0.93–0.99; tibial site: ICC, 0.85; 95% CI, 0.65–0.96; femoral site: ICC, 0.93; 95% CI, 0.83–0.98).

Fig 2

Boxplot showing the UTE-T2* values for the intraarticular region of reconstructed ACLs between 6 to 12 months after ACLR, relative to the values for the normal ACL.

The UTE-T2 values for the intraarticular region were comparable to those for the normal ACL from 9 months postoperatively. UTE, ultrashort echo time; ACL, anterior cruciate ligament; ACLR, anterior cruciate ligament reconstruction.

Table 1

UTE-T2* values (ms) for each region at 6–12 months after ACLR.

	6 months	9 months	12 months
Intraarticular region of reconstructed ACL
Distal	12.3±1.4	10.6±1.6	10.3±1.5
Middle	13.1±2.2	12.1±1.7	11.4±1.7
Proximal	13.8±2.6	12.6±1.8	11.7±1.5
Mean	13.1±1.8	11.7±1.5*	11.1±1.3*
Intraosseous region of reconstructed ACL
Tibia site	7.4±1.2	7.1±1.0	6.9±1.2
Femoral site	11.5±2.3	11.0±1.6	11.1±1.5

Values are presented as mean ± SD. UTE, ultrashort echo time; ACL, anterior cruciate ligament.

Fig 3

Boxplot of changes in UTE-T2* values for the intraarticular and intraosseous regions of reconstructed ACLs between 6 to 12 months after ACLR.

The UTE-T2* values for the intraarticular and intraosseous regions show different transitions. UTE, ultrashort echo time; ACL, anterior cruciate ligament; ACLR, anterior cruciate ligament reconstruction.

Discussion

In this study, we investigated the changes in T2* values for the reconstructed ACLs at 6, 9, and 12 months after ACLR by using UTE-T2*mapping separately for the intraarticular and intraosseous regions. The most important finding of this study was that the intraarticular region of reconstructed ACLs showed significantly lower UTE-T2* values from 6 to 9 months postoperatively, while the values for the intraosseous region of reconstructed ACLs did not change significantly. The UTE-T2* values at the tibial site in the intraosseous region of reconstructed ACLs were significantly lower than those at the femoral sites and the intraarticular regions at all time points.

Previous studies using UTE-T2* techniques in patients after ACLR have focused on assessment of changes in the knee cartilage and meniscus over time [17-19]. UTE-T2* mapping is suitable for assessing the ligamentization process of the reconstructed ACL because it can image organized collagen structures and capture microscopic changes [3,4,13-15]. However, there are limited data regarding UTE-T2* assessments of the reconstructed ACL, and the results of this study may provide an insight into the ligamentization process of the reconstructed ACL.

The signal intensities acquired from long-TE sequences used in conventional MRI vary depending on the acquisition protocol, software, and inherent parameters [3-5,9]. In contrast, UTE-T2*mapping is an innovative tool for evaluating the ligamentization process of the reconstructed ACL because it shows no such limitations and can provide objective data [3,4].

Previous UTE-T2* studies have reported that UTE-T2* values for the intraarticular region of reconstructed ACLs increase rapidly until about 6 months and then slowly decrease, which is consistent with the findings of conventional MRI studies [3,4,9-11]. In the early postoperative period, the reconstructed ACLs undergo a stepwise and combined process of increasing fibroblast number, intense revascularization, and disintegration of collagen fibrils and their orientation. In particular, it has been shown that the regular collagen orientation and crimp pattern of reconstructed ACLs are lost in the early postoperative period and slowly restored only during the remodeling phase [6]. In other words, the increase in UTE-T2* values up to 6 months postoperatively may reflect the disruption of the collagen matrix in the reconstructed ACL in the early postoperative period, and the subsequent changes in the UTE-T2* values may be due to the transition to the main phase of remodeling [3,4,6-8]. The key question is how long the significant histological changes in the reconstructed ACL, that is, the rapid changes in UTE-T2* values, will continue. In this study, the UTE-T2* values for the intraarticular region of reconstructed ACLs decreased significantly from 6 to 9 months after surgery, during which time the remodeling changes in the reconstructed ACLs may occur rapidly. Warth et al. showed that the UTE-T2* values decreased significantly from 6 to 9 months in 10 patients after ACLR with hamstring or patellar tendons [4]. This is the only study that observed changes in the UTE-T2* values for reconstructed ACLs at 6, 9, and 12 months postoperatively, which supports the findings of the previous study. Chu et al. observed the evolution of UTE-T2* values for the intraarticular region of reconstructed ACLs and reported a significant decrease from 1 year to 2 years postoperatively, but the change was clearly slower than from 6 months to 1 year postoperatively [3]. Histologically, the reconstructed ACL undergoes structural changes up to 2 years postoperatively, and the structural changes stop at a stage of microstructure that is strictly different from that of the normal ACL [20]. In summary, the reconstructed ACL at 6 to 9 months postoperatively continues to undergo rapid tissue changes during the remodeling phase, and the tissue structure is unstable. After that time, the speed of histological changes declines, and the tissue structure becomes relatively stable.

As mentioned earlier, we had previously investigated the UTE-T2* values of the normal ACL in 12 healthy knees by using the same measurement methods used in this study [15]. When the results of this study were compared with those obtained for a normal ACL by ANOVA, the UTE-T2* value of the normal ACL was 11.9 ± 2.4 ms, which differed significantly from the value obtained 6 months postoperatively in the intraarticular region of the reconstructed ACL but not from the values obtained at 9 and 12 months postoperatively (Fig 3). This result suggests that the tissue structure of the reconstructed ACL is less substantial than that of the normal ACL at 6 months postoperatively. Previous studies comparing the UTE-T2* values for the intact ACL of the contralateral knee and reconstructed ACL showed no difference in the UTE-T2* values at 6 months postoperatively, but they did not consider influences of ACL injury and ACLR on the intact ACL of the contralateral knee [4]. The results of this study suggest that the histological structure of a reconstructed ACL is different from that of a normal ACL, but it is possible to reach a similar histological structure 9 months postoperatively.

The maturation processes of the intraarticular and intraosseous regions of reconstructed ACLs differ in relation to the biological processes at the early stages after ACLR [6-8,21,22]. Specifically, the intraarticular region of a reconstructed ACL undergoes revascularization from synovial fluid, and the intraosseous region of the reconstructed ACL undergoes revascularization from the adjacent cancellous bone. In a recent review, these processes were completed by 3 to 6 months postoperatively, with no significant difference in the rate of progression [6-8,21,22]. In this study, the UTE-T2* values of the intraosseous region of reconstructed ACLs remained unchanged from 6 to 12 months postoperatively and were significantly lower than those for the intraarticular region of reconstructed ACLs, especially at 6 months postoperatively. This may indicate that histological maturation of the reconstructed ACL is faster in the intraosseous region than in the intraarticular region. In the intraosseous region, a fibrous interface in continuous contact with the bone tunnel was formed and stress-shielded by 6 months postoperatively, and this stress-shielding may have provided an advantage in histological maturation [21,22].

Furthermore, the UTE-T2* values were significantly lower at the tibial site in the intraosseous region of reconstructed ACLs than at the femoral site at all time points. Ahn et al. investigated the maturation in the intraosseous region of reconstructed ACLs using conventional MRI with SNQ, which also showed significant maturation of the tibial site [11]. Microvessels derived from the fat pad and posterior synovial tissue were considered to form a rich vascular envelope that contributes to the numerous intra-ligamentous branches for perfusion at the graft site. This may also reflect the mechanical environment at each site. Rodeo et al. investigated the relationship between tunnel motion in the reconstructed ACL and histological maturation of the femoral and tibial sites [23]. They concluded that the histological maturation of the reconstructed ACL in the femoral tunnel is inversely proportional to the magnitude of the graft tunnel motion, which affects local histological maturation.

This study had some limitations. First, although we limited the measurement of UTE-T2* values to the part where the influence of artifacts was suppressed, the influence of joint edema and magic angle effects was unavoidable. In particular, in the reconstructed ACL on the femoral side, the magic angle effect may be a factor responsible for overestimation of the UTE-T2* values due to the large bending angle. Second, we used only mono exponential UTE-T2* mapping techniques. Using mono exponential T2* fitting, the estimated apparent T2* values as reported in this study may include the effect from changes of bound/unbound tissue water fraction during ACL ligamentization. The fraction of bound water is quite high in both reconstructed and normal ACLs and include a first echo (TE = 0.1 ms), which may introduce errors in estimation of the mono-exponential map. In this study, bi-exponential UTE-T2* mapping was deemed inappropriate for routine clinical MRI because of its long acquisition time, although partial acquisition could have reduced the time [24,25]. Third, factors affecting the UTE-T2* values of reconstructed ACLs and their correlations with clinical and functional outcomes were not evaluated. We could not control for potential factors that may have influenced the results of this study. Although the participants enrolled in this study were women who underwent single-bundle ACLR with hamstring tendons, sex and the surgical technique including graft selection, may have influenced the results of this study. Future studies involving larger sample sizes and incorporating covariates that may affect the maturity of reconstructed ACLs are warranted. Investigation of the relationship of UTE-T2* values with clinical and functional outcomes is also important to provide ideas on safe return to sports in terms of maturation of reconstructed ACLs.

Despite these limitations, this is one of the few studies to investigate the graft maturation process in both intraarticular and intraosseous regions of reconstructed ACLs using UTE-T2* mapping. The objective data obtained in this study will play an important role in understanding the overview of the ligamentization process in a reconstructed ACL.

Conclusions

According to UTE-T2*mapping-based findings, histologic maturation of reconstructed ACLs is faster in the intraosseous region than in the intraarticular region. In particular, the intraarticular region of ACLs is still undergoing rapid histological changes at 6 months postoperatively, and its tissue structure is less substantial than normal. The findings of this study may provide clues to determine the optimal timing for safe return to sports in terms of ligamentaization of reconstructed ACLs.

Measurement data.

(XLSX)

Click here for additional data file.

25 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Markus Geßlein, Ph.D.

Section Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments:

Dear Authors,

thank you very much for submitting your well-performed study to PlosOne. After review by two experienced colleagues in this field I would kindly ask your for some improvments in your manuscript. You will find the detailled reviewers comments below.

Furthermore, I would kindly ask you to adress the additional issues below. I think this will make your manuscript more interesting for a broader readership.

Abstract

Conclusion

- Is a bit of repetition according to the abstracts result section? Please provide any potential clinical benefit of your study or describe why this finding could be important for future research (see comment on discussion below).

- Conclusion in the abstract and the manuscript should be same.

Material & Methods

-Please provide more information about the female patients included e.g. BMI, Height, side of injury, time from injury to surgery, maybe injury mechanism etc..

Discussion

-The first paragraph of the discussion should contain the most important findings of your study. I would suggest using the actual conclusion as first paragraph for the discussion. Then rewriting the conclusion with reference to the clinical importance or future research topics you would suggest.

-I would like to make the manuscript more interesting to read for orthopedic surgeons having to deal with the RTA/RTS issues in daily routine.

-I would like to ask you to move figure 3 to the results section. Further, please explain in the methods section how “normal ACL” values were aquired.

Limitations

-You did not provide data about the stability of knee after surgery. Orthopedic Surgeons well know that technical errors during surgery may result in decreased stability and that might affect the biological graft transformation. If you could provide data about the knee stability (clinical like Lachmann or KT 1000) that would improve the findings. If you don not have that data please mention this fact in the limitations.

-Please add information how the choice of enrolled participants could have influenced the results (only females..time between injury and surgery etc..)

-Did metal remnants from tunnel drilling interfere with the scan /measures?

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, authors investigated the maturation of reconstructed ACL using UTE-T2* imaging. This is a well-written manuscript. I do have some minor concerns:

1. Statistical Analysis: Only pairwise comparison of T2* values was conducted at three time points. As demonstrated in Figure 2, there are some clear trends of T2* over time, especially in intraarticular region. Correlation T2* values with recovery time would be interesting.

2. In Discussion, authors claimed that this is the first study to investigate regional graft maturation with UTE. There is at least one recent publication in this topic: Fukuda T, et al, Abbreviated quantitative UTE imaging in anterior cruciate ligament reconstruction, BMC Musculoskeletal Disords. 2019;20(1):426;

3. In Discussion, authors claimed the long acquisition duration needed for bi-exponential UTE study. It would be true if using fully-sampled acquisition. As described in Fukuda et al paper, it is possible to reduce the time by partial acquisition.

4. The confounds of mono-exponential instead of bi-exponential should be discussed. The fraction of bound water is quite high in both reconstructed and healthy ACLs. The inclusion of first echo (TE=0.1 ms) may led to errors in mono-exponential T2* estimation. A comparison of results between using all 4 TEs vs. using only 3 TEs (excluding first TE) would be meaningful.

5. As indicated in literature, there are significant changes during the first 6 months post-op. Some discussion/clarification are needed.

Reviewer #2: In the present prospective study, the authors Rikuto Y et al. use a UTE T2* mapping technique on a defined patient collective (10 female patients, 18 +/- 4 years of age) to assess the UTE T2* value changes within the tendon graft in the first postoperative year after ACL reconstruction with regards to the histological process of ligamentisation. The progressing ligamentisation has been shown to correlate with an increasing stability of the ACL reconstruction (although mainly in animal trials), which is an important factor with regards to return to physical activity as well as to avoiding the risk of early graft re-tear.

In correlation with other recent studies by Chu CR et al (Orthop J Sports Med. 2019) and Warth RJ and al. (Am J Sports Med. 2020), the presented UTE T2* values of the intraarticular segment of the ACL grafts drop significantly between 6 to 9 months postoperatively, without another significant change in the last quarter of the first postoperative year. The study also assesses the UTE T2* values of the femoral and tibial intraosseous segments of the ACL grafts, which are significantly lower than the intraarticular UTE T2* values, especially 6 months postoperatively. Compared to previously assessed UTE T2* values in uninjured ACL (see Okuda M et al., Acta Radiol. 2021), the intraosseous graft segments present similar values. In addition, a significant difference of UTE T2* values between the femoral and the tibial intraosseous segments of the ACL grafts is noted.

The authors conclude that the ligamentisation processes in the intraarticular and intraosseous segments of the ACL reconstructions differ significantly within the first 6 months postoperatively with a slowly increasing convergence after 9 to 12 months, which points towards a significant tendency for faster maturation intraosseously. Moreover, a significant difference is described in ligamentisation of the intraosseous segments in the first postoperative year, which is discussed to possibly be in relation to tunnel motion.

The authors present their findings and conclusions, as well as the study’s limitations, in a clear and concise manner. The correlating histological and imaging findings by other studies, added in the introduction and the discussion, give weight to the study’s overall conclusion.

The patient collective is explained to have been selected prospectively and includes patients operated on by one orthopaedic surgeon with a precisely explained surgical method. While the patient collective is small, consisting of just one gender and limited to a certain age, it is within the limit of the predetermined, calculated sample size.

The technique used for imaging acquisition, including its limitations (e.g. metal artefacts, magic angle effect), is explained in detail and is easily understandable, both in the introduction and in the method section. The measurement method as well as the statistical evaluation are well described and not too technical. As a radiologist, I especially appreciate the precise explanations with regards to the differences in MRI imaging and MRI imaging acquisition concerning this kind of tissue material.

In the last few years, mapping techniques in MR imaging have gained importance in the diagnostic assessment of tissue characteristics, and in particular of tissue pathologies (e.g. T1/T2 mapping of myocardium in cardiac imaging). UTE sequences have been increasingly applied in the diagnostics of the musculoskeletal system, given that they can reveal alterations in tissues with short to ultra-short transverse relaxation times, such as bone, ligaments or tendons. Tissues with moderate to long transverse relaxation times, e.g. cartilage, can already be reliably assessed with conventional MRI sequences (see also Chang E. et al, Journal of Magnetic Resonance Imaging 41:870–883 (2015)).

From a radiologist’s point of view, studies like the present authored by Rikuto Y et al. may give rise to more detailed studies, which will hopefully contribute to establishing these type of measuring methods in the daily routine of MRI imaging. The aforementioned is subject to the condition that the correlation between measured values and biomechanical changes prove to be significant to the clinical outcome of patients.

No major issues can be observed in the manuscript.

However, following minor issues could be considered:

- Page 12, Line 193: The measurements were taken by one orthopaedic surgeon as defined in the methodology. I found no mention of other evaluators in the text. It is not completely clear to me how the interobserver reliability could be assessed precisely in the results.

- Page 10 Line 156-160, Page 13/14 Line 207-211, Page 18 Line 281-284: The same, detailed passages of measurement and result explanations, which are included already in the adjacent text, can be found underneath the figures’ titles. They read repetitive, especially since the figures aren’t shown within the text in the downloaded PDF version. In my opinion, captions of figures should be kept succinct.

- Page 16 Line 248-252 / Page 17 Line 267-270: Another exact text passage repetition. While both passages fit within the context of the discussion, their proximity within the text impacts the fluency of the narrative. Paraphrasing of one passage may be better.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Xiang He

Reviewer #2: Yes: Dr. Maria Elena Misu

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

4 Jun 2022

Abstract

Conclusion

- Is a bit of repetition according to the abstracts result section? Please provide any potential clinical benefit of your study or describe why this finding could be important for future research (see comment on discussion below).

- Conclusion in the abstract and the manuscript should be same.

Response: Thank you for pointing this out. We have revised the conclusions to highlight the potential clinical benefits of the study in the Abstract (Page 3, Lines 39) as well as main text (Page 21, Lines 377). We have also ensured consistency between the conclusions in the Abstract and those in the main text.

Material & Methods

-Please provide more information about the female patients included e.g. BMI, Height, side of injury, time from injury to surgery, maybe injury mechanism etc..

Response: Thank you for the recommendations. We have added the required information in the Patients and Methods section (Page 8, Lines 119).

Discussion

-The first paragraph of the discussion should contain the most important findings of your study. I would suggest using the actual conclusion as first paragraph for the discussion. Then rewriting the conclusion with reference to the clinical importance or future research topics you would suggest.

Response: Thank you for pointing this out. We have now revised the first paragraph of the Discussion section (Page 15, Lines 226) and the Conclusions section (Page 21, Lines 343) in accordance with your suggestions.

-I would like to make the manuscript more interesting to read for orthopedic surgeons having to deal with the RTA/RTS issues in daily routine.

-I would like to ask you to move figure 3 to the results section. Further, please explain in the methods section how “normal ACL” values were aquired.

Response: Thank you for your suggestions. We have moved Figure 3 to the Results section (Page 12, Lines 184) and added a reference citation pertaining to the measurement of normal ACL values in the Methods section (Page 10, Lines 148).

Limitations

-You did not provide data about the stability of knee after surgery. Orthopedic Surgeons well know that technical errors during surgery may result in decreased stability and that might affect the biological graft transformation. If you could provide data about the knee stability (clinical like Lachmann or KT 1000) that would improve the findings. If you don not have that data please mention this fact in the limitations.

Response: Thank you for the valuable insights. We are currently investigating the association of UTE-T2* values with clinical and functional outcomes (IKDC score and KOOS at 1 year postoperatively) of patients, including the subjects of the present study. Data regarding knee stability after surgery were insufficient in the present study. In the limitations section of the current manuscript, we have mentioned that investigation of the relationship of UTE-T2* values with clinical and functional outcomes is also important to provide ideas on safe return to sports in terms of maturation of reconstructed ACLs (Page 20, Lines 327).

-Please add information how the choice of enrolled participants could have influenced the results (only females..time between injury and surgery etc..)

Response: Thank you for pointing this out. We agree that selection bias is a concern in this study. We have mentioned this in the limitations paragraph and highlighted that future studies involving larger sample sizes and incorporating covariates that may affect the maturity of reconstructed ACLs are warranted (Page 20, Lines 328).

-Did metal remnants from tunnel drilling interfere with the scan /measures?

Response: Thank you for the pertinent question. If metal remnants were present, they would have a significant impact on the measurement as large artifacts. There were no such cases in the present study.

Reviewer #1: In this manuscript, authors investigated the maturation of reconstructed ACL using UTE-T2* imaging. This is a well-written manuscript. I do have some minor concerns:

1. Statistical Analysis: Only pairwise comparison of T2* values was conducted at three time points. As demonstrated in Figure 2, there are some clear trends of T2* over time, especially in intraarticular region. Correlation T2* values with recovery time would be interesting.

2. In Discussion, authors claimed that this is the first study to investigate regional graft maturation with UTE. There is at least one recent publication in this topic: Fukuda T, et al, Abbreviated quantitative UTE imaging in anterior cruciate ligament reconstruction, BMC Musculoskeletal Disords. 2019;20(1):426;

Response: Thank you for pointing that out. As you pointed out, previous studies have measured the T2* values of reconstructed ACLs separately in the joint and in the bony foramen. We have deleted the word “first” and rephrased the sentence in the revised manuscript (Page 15, Lines 226 and Page21, Lines 337).

3. In Discussion, authors claimed the long acquisition duration needed for bi-exponential UTE study. It would be true if using fully-sampled acquisition. As described in Fukuda et al paper, it is possible to reduce the time by partial acquisition.

4. The confounds of mono-exponential instead of bi-exponential should be discussed. The fraction of bound water is quite high in both reconstructed and healthy ACLs. The inclusion of first echo (TE=0.1 ms) may led to errors in mono-exponential T2* estimation. A comparison of results between using all 4 TEs vs. using only 3 TEs (excluding first TE) would be meaningful.

Response: Thank you for your valuable comments and suggestions. We have mentioned in the limitations that biexponential UTE-T2* mapping was deemed inappropriate for routine clinical MRI in this study because of its long acquisition time, although partial acquisition could have reduced the time. We have also discussed the confounds of using mono exponential mapping instead of biexponential mapping in the limitations (Page 20, Lines 321).

5. As indicated in literature, there are significant changes during the first 6 months post-op. Some discussion/clarification are needed.

Response: Thank you for the helpful comments. We have discussed the main histological findings pertaining to the changes during the first 6 months after surgery in the Discussion section (Page 16, Lines 249).

Reviewer #2

From a radiologist’s point of view, studies like the present authored by Rikuto Y et al. may give rise to more detailed studies, which will hopefully contribute to establishing these type of measuring methods in the daily routine of MRI imaging. The aforementioned is subject to the condition that the correlation between measured values and biomechanical changes prove to be significant to the clinical outcome of patients.

Response: Thank you for your detailed review and positive feedback for our study. We are currently investigating the association of UTE-T2* values with clinical and functional outcomes (IKDC score and KOOS at 1 year postoperatively) of patients, including the subjects of the present study. We will analyze and present the results once a sufficient sample is collected.

- Page 12, Line 193: The measurements were taken by one orthopaedic surgeon as defined in the methodology. I found no mention of other evaluators in the text. It is not completely clear to me how the interobserver reliability could be assessed precisely in the results.

Response: We apologize for the lack of clarity regarding interobserver reliability. We have now mentioned in the Patients and Methods section that another orthopedic surgeon (YY, Observer 2) independently performed measurements using the same method in order to assess interobserver reliability (Page 10, Lines 155).

- Page 10 Line 156-160, Page 13/14 Line 207-211, Page 18 Line 281-284: The same, detailed passages of measurement and result explanations, which are included already in the adjacent text, can be found underneath the figures’ titles. They read repetitive, especially since the figures aren’t shown within the text in the downloaded PDF version. In my opinion, captions of figures should be kept succinct.

Response: Thank you for pointing this out. We have revised each caption to be more concise.

- Page 16 Line 248-252 / Page 17 Line 267-270: Another exact text passage repetition. While both passages fit within the context of the discussion, their proximity within the text impacts the fluency of the narrative. Paraphrasing of one passage may be better.

Response: Thank you for pointing this out. We have now deleted and rearranged some text in the repetitive paragraph (Page 17-18, Lines 266-289).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

11 Jul 2022

Ligamentization of the reconstructed ACL differs between the intraarticular and intraosseous regions: A quantitative assessment using UTE-T2* mapping

PONE-D-22-05680R1

Dear Dr. Nakase,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Markus Geßlein, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Authors,

thank you very much for submitting your fine work to PLOS ONE.

The manuscript is now accepted for publication. I would kindly ask you to further adress some minor changes requested by one of the reviewers boefore we proceed:

In this revised manuscript, authors have adequately addressed all my concerns. There are only some very minor changes:

1. Page 6, Line 78-81: Change into: " ... the conventional MRI signal intensity is affected by the image sequence and scanner hardware. Moreover, Tendon and ligaments normally have short T2 relaxation times leading to low MRI signal in conventional MRI protocols".

2. Page 6, Line 83: Relaxation time T2* reflects the intrinsic property of tissue and should be independent on image sequence and acquisition parameters.

3. Page 6, Line 84-85: Change the original sentence into: "These variables reflects the T2* relaxation of bounded water with collagen of tendons and ligaments, and an ideal to capture the changes in tissue structure and organization during ligamentization of reconstructed ACL".

4. Page 6, Line 88-89: Change the original sentence into: "...., which cannot be assessed in conventional gradient-echo based MRI assessment due to sub-ms T2* values of collagen-bound water, ....".

5. Page 20, Line 326: Adding one sentence: "Using mono-exponential T2* fitting, the estimated apparent T2* values as reported in this study may include the effect from changes of bound/unbound tissue water fraction during ACL ligamentization."

Thank you very much and congratulatuions!

Markus Geßlein, PhD

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this revised manuscript, authors have adequately addressed all my concerns. There are only some very minor changes:

1. Page 6, Line 78-81: Change into: " ... the conventional MRI signal intensity is affected by the image sequence and scanner hardware. Moreover, Tendon and ligaments normally have short T2 relaxation times leading to low MRI signal in conventional MRI protocols".

2. Page 6, Line 83: Relaxation time T2* reflects the intrinsic property of tissue and should be independent on image sequence and acquisition parameters.

3. Page 6, Line 84-85: Change the original sentence into: "These variables reflects the T2* relaxation of bounded water with collagen of tendons and ligaments, and an ideal to capture the changes in tissue structure and organization during ligamentization of reconstructed ACL".

4. Page 6, Line 88-89: Change the original sentence into: "...., which cannot be assessed in conventional gradient-echo based MRI assessment due to sub-ms T2* values of collagen-bound water, ....".

5. Page 20, Line 326: Adding one sentence: "Using mono-exponential T2* fitting, the estimated apparent T2* values as reported in this study may include the effect from changes of bound/unbound tissue water fraction during ACL ligamentization."

Reviewer #2: The reviewed article has addressed all remarks made by both the editor and the reviewers, and the text reads fluently and concisely after the revision.

The patient collective is explained much better now, as is the trauma mechanism leading to the ACL repair in the first place.

The revised article also contains additional information on the histological changes that occur, and the article correlates them well to the signal changes and T2* values acquired in this study. The limitations to the study are also far more precise, and pointing towards the need of future studies with a broader patient collective.

One question that remains is whether the very small patient collective (mainly one gender and one age-range) as well as the choosing of only non-contact trauma mechanisms could potentially limit the concluded results to just one particular group of patients (e.g. young adults), and therefore not make it fully applicable to the range of patients with ACL repairs, esp. when it comes to the ideal time for the return to sports.

No major issues can be observed in the revised manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dr. Maria Elena Misu

**********

14 Jul 2022

PONE-D-22-05680R1

Ligamentization of the reconstructed ACL differs between the intraarticular and intraosseous regions: A quantitative assessment using UTE-T2* mapping

Dear Dr. Nakase:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Markus Geßlein

Section Editor

PLOS ONE