Scholarly article on topic 'Morphological and compositional monitoring of a new cell-free cartilage repair hydrogel technology – GelrinC by MR using semi-quantitative MOCART scoring and quantitative T2 index and new zonal T2 index calculation'

Morphological and compositional monitoring of a new cell-free cartilage repair hydrogel technology – GelrinC by MR using semi-quantitative MOCART scoring and quantitative T2 index and new zonal T2 index calculation Academic research paper on "Medical engineering"

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Osteoarthritis and Cartilage
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{"MR imaging" / "Articular cartilage" / "Cartilage repair tissue" / "T2 mapping" / "Global and zonal T2 index" / GelrinC}

Abstract of research paper on Medical engineering, author of scientific article — S. Trattnig, K. Ohel, V. Mlynarik, V. Juras, S. Zbyn, et al.

Summary Objective To evaluate cartilage repair tissue (RT) using MOCART scoring for morphological and T2 mapping for biochemical assessment following implantation of GelrinC, a biosynthetic, biodegradable hydrogel implant. Design MR imaging (1.5/3T) was performed on 21 patients at six sites. Standard protocols were used for MOCART evaluation at 1 week (baseline) 1, 3, 6, 12, 18 and 24 months. Multi-echo SE was used for T2 mapping. Global (T2 in RT divided by T2 in normal cartilage) and zonal T2 index (deep T2 divided by superficial T2) of RT were calculated. Results Average MOCART score was 71.8 (95% CI 62.2 to 81.3) at six, 75.2 (95% CI 62.8 to 87.5) at twelve, 71.8 (95% CI 55.4 to 88.2) at eighteen and 84.4 (95% CI 77.7 to 91.0) at twenty-four months. The global T2 index ranged between 0.8 and 1.2 (normal healthy cartilage) in 1/11 (9%) patients at baseline, 8/12 (67%) at 12 months, 11/13 (85%) at 18 months and 13/16 (81%) at 24 months. The zonal T2 index for RT was <20% difference to the zonal T2 index for normal cartilage in: 6/12 patients (50%) at 12 months, 7/13 (53.8%) at 18 months and 10/16 (63.5%) at 24 months. The standard deviation for T2 showed a significant decrease over the study. Conclusions The increase of MOCART scores over follow-up indicates improving cartilage repair tissue. Global and zonal T2 repair values at 24 months reached normal cartilage in 81% and 63.5% of the patients respectively, reflecting collagen organization similar to hyaline cartilage.

Academic research paper on topic "Morphological and compositional monitoring of a new cell-free cartilage repair hydrogel technology – GelrinC by MR using semi-quantitative MOCART scoring and quantitative T2 index and new zonal T2 index calculation"

Morphological and compositional monitoring of a new cell-free cartilage repair hydrogel technology - GelrinC by MR using semi-quantitative MOCART scoring and quantitative T2 index and new zonal T2 index calculation

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S. Trattnig t§ *, K. Ohel t, V. Mlynarik y, V. Juras y, S. Zbyn y, A. Korner t

y High Field MR Center, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Waehringer Guertel 18-20, Vienna, Austria

t Regentis Biomaterials Ltd., 12 Ha'ilan Street, Northern Industrial Zone, P.O.B. 260, Or Akiva, 3060000, Israel § CD Laboratory for Clinical Molecular MR Imaging, Austria

ARTICLE INFO

Article history:

Received 9 December 2014

Accepted 7 July 2015

Keywords: MR imaging Articular cartilage Cartilage repair tissue T2 mapping

Global and zonal T2 index GelrinC

SUMMARY

Objective: To evaluate cartilage repair tissue (RT) using MOCART scoring for morphological and T2 mapping for biochemical assessment following implantation of GelrinC, a biosynthetic, biodegradable hydrogel implant.

Design: MR imaging (1.5/3T) was performed on 21 patients at six sites. Standard protocols were used for MOCART evaluation at 1 week (baseline) 1, 3, 6, 12, 18 and 24 months. Multi-echo SE was used for T2 mapping. Global (T2 in RT divided by T2 in normal cartilage) and zonal T2 index (deep T2 divided by superficial T2) of RT were calculated.

Results: Average MOCART score was 71.8 (95% CI 62.2 to 81.3) at six, 75.2 (95% CI 62.8 to 87.5) at twelve, 71.8 (95% CI 55.4 to 88.2) at eighteen and 84.4 (95% CI 77.7 to 91.0) at twenty-four months. The global T2 index ranged between 0.8 and 1.2 (normal healthy cartilage) in 1/11 (9%) patients at baseline, 8/12 (67%) at 12 months, 11/13 (85%) at 18 months and 13/16 (81%) at 24 months. The zonal T2 index for RT was <20% difference to the zonal T2 index for normal cartilage in: 6/12 patients (50%) at 12 months, 7/13 (53.8%) at 18 months and 10/16 (63.5%) at 24 months. The standard deviation forT2 showed a significant decrease over the study.

Conclusions: The increase of MOCART scores over follow-up indicates improving cartilage repair tissue. Global and zonal T2 repair values at 24 months reached normal cartilage in 81% and 63.5% of the patients respectively, reflecting collagen organization similar to hyaline cartilage.

© 2015 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Magnetic resonance imaging (MRI) techniques have been widely used for the evaluation of the morphological status as well

* Address correspondence and reprint requests to: S. Trattnig, High Field MR Center, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Waehringer Guertel 18-20, Vienna, Austria. Tel: 431-40-40064600; Fax: 431-40-400-64750.

E-mail addresses: siegfried.trattnig@meduniwien.ac.at (S. Trattnig), Kitty@ regentis.co.il (K. Ohel), vladimir.mlynarik@meduniwien.ac.at (V. Mlynarik), vladimir.juras@meduniwien.ac.at (V. Juras), stefan.zbyn@meduniwien.ac.at (S. Zbyn), Amit@regentis.co.il (A. Korner).

as for the quality of regenerated cartilage during postoperative follow-up. However, the visualization and quantification of the ultrastructural composition of cartilage repair tissue (RT), specifically comparing its hyaline-like vs fibrous characteristics, requires specialized MR imaging techniques.

In addition to the evaluation of gross cartilage morphology of the RT, MR sequences such as delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and the mapping of the transverse relaxation time (T2 mapping) permit the assessment of key matrix components of the tissue, specifically, dGEMRIC for glycosaminoglycan (GAG) content and T2 mapping for water concentration and collagen architecture and orientation1-3. While dGEMRIC requires injection of a gadolinium contrast agent, T2 mapping is a "natural"

http://dx.doi.org/10.1016/j.joca.2015.07.007

1063-4584/© 2015 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

MRI evaluation tool. Cartilage T2 values reflect the interaction of free water molecules with the extracellular matrix, in particular collagen fibers and can detect differences in zonal collagen fibers orientation between healthy and damaged cartilage.

T2 relaxation time measurements define different structural tissue characteristics of cartilage RT and enable monitoring the maturation process4-9. In general it could be demonstrated that in the early stages after cartilage repair surgery elevated T2 values were reported up to 1 year, while at 1 year and later after surgery T2 values decreased to values close to normal cartilage. This was found in patients after microfracture as well as after matrix assisted autologous chondrocyte transplantation (MACT)8,10—15. Since fibrous tissue reduces T2 relaxation times compared to hyaline cartilage, lower T2 values were observed in RT after microfracture as compared to MACT RT in studies with a follow-up of more than 2 years after surgery4,10,16. In addition most studies reported that normal zonal variation of hyaline cartilage with lower T2 relaxation times in the deep zone of cartilage and higher T2 relaxation times in the superficial zone due to the collagen fiber network with more anisotropic collagen fibers in the deep zone, was lacking in RT after microfracture10,13,17-19.

Fibrous tissue has generally disorganized collagen fibers orientation with lack of zonal variation as well as reduced mobility of water molecules leading to a decrease in T2 values. Hyaline cartilage is characterized by zonal organization of articular cartilage with collagen fibers running perpendicular to cortical bone in the deep zone of cartilage. Anisotropic orientation of the collagen fibers in the deep zone affects the mobility of water molecules and thus reduce the water proton T2 relaxation time compared to the superficial zone. In the superficial zone, the collagen fibers are oriented more randomly resulting in less restricted mobility of water molecules and thus longer T2 relaxation times. This so called zonal variation has been reported to be a marker for collagen fiber organization and network in healthy hyaline cartilage10.

Surgical techniques for cartilage repair vary from tissue debridement and bone marrow stimulation such as drilling and microfracture (MFX), to more sophisticated cell based transplantation such as autologous chondrocyte implantation (ACI) and MACT. Despite success with cell based treatments, there is no consensus as to the quality of the regenerated cartilage and long term outcome of the RT20,21. These technologies are associated with higher cost compared to MFX.

Newly available, cell-free scaffolds have the advantage of requiring only a single surgical procedure and are typically made from biodegradable synthetic or natural polymers. These scaffolds provide a matrix onto which mesenchymal stem cells (MSCs) originating from the microfractured subchondral bone attach, differentiate and develop into RT. GelrinC (GelrinC, Regentis Biomaterials, Or Akiva, Israel) is a new cell-free biosynthetic hydrogel implant requiring a single procedure. GelrinC was developed to support consistent and effective cartilage regeneration in a simple, single-step procedure without the need for either autologous or allogeneic cells.

GelrinC solution is composed of synthetic component - polyethylene glycol diacrylate (PEG-DA), and a natural component — denatured human fibrinogen (DHF). The cured implant consists of a continuous network of cross-linked PEG-DA that is covalently conjugated to a backbone of denatured disulfide-reduced fibrin-ogen chains via its reduced thiol group.

GelrinC is provided to the surgeon as a liquid solution. Following preparation of the defect using a standard debridement and microfracture procedure22, GelrinC is applied to the defect in liquid form so that it completely fills the lesion. Following 90-sec exposure to UVA light, GelrinC is converted into a soft, elastomeric implant. GelrinC implant acts as a scaffold for tissue repair by

gradually eroding over time, allowing for new cartilage to take its place and it is completely resorbed in-vivo within 6-12 months.

GelrinC is ready-to-use, cell-free implant, provided to the surgeon as an off-the-shelf product.

GelrinC procedure is simple, one-step - It adds about 15 min to a standard microfracture. It is applied to the defect as liquid and photo-cured in situ. This allows for perfect filling of the defect regardless of its geometry, shape and depth. The resultant implant is optimally integrated with surrounding cartilage and bone tissue.

Clinical study demonstrated that GelrinC has outstanding safety and efficacy outcome and new hyaline-like cartilage is formed from the periphery of the defect towards the center. GelrinC, by presenting a gradually receding surface to the surrounding tissue, allows incoming cells to adhere to the implant matrix and for the regeneration process to proceed. As a result, as the implant degrades on its surface, primarily through the action of proteases secreted by attached cells, new hyaline-like cartilage is formed from the periphery of the defect towards the center.

The aim of this multi-center single arm study was to assess the quality of RT of patients after GelrinC implantation in the femoral condyle. Quality was evaluated using clinical outcome, morphological appearance and quantitative T2 values using MRI.

Method

Institutional review board approval was obtained from all participating medical centres. The study included screening evaluation for eligibility, diagnostic arthroscopy and surgery as well as follow-up of clinical and radiological (MRI) evaluations, rehabilitation program, adverse events, concomitant medication and subsequent interventions. All patients signed an informed consent before participating in the study.

Patient population

Twenty-one (5 females and 16 males, mean age 36 ± 10 years; age range 18^51) patients were treated with GelrinC in the period between 2009 and 2012. All patients were treated for a single full thickness cartilage defect.

Inclusion and exclusion criteria

Inclusion criteria included patients with one or two contained symptomatic lesion/s to the femoral condyle, lesion size between 1 and 6 cm2 with a maximal diameter of 2.5 cm and with less than 6 mm into the subchondral bone. Body mass index (BMI) <32, knee should be stable and previous or concurrent meniscus resection of no more than 50%. Excluded were patients younger than 18 years or older than 65 years, with lesions greater than 6 cm2 or smaller than 1 cm2, lesions to the patella or trochlea, patients with diffuse degenerative joint disease and those who had contraindications to perform MRI (i.e., patients with pacemakers, claustrophobia, etc).

Procedure

GelrinC was implanted in a single procedure following standard MFX treatment. The patient was prepared for surgery, anaesthetized and a tourniquet applied. The lesion was debrided of any cartilage fragments and the calcified layer was completely removed before standard MFX was performed. The lesion was then evaluated for eligibility. If eligible, GelrinC liquid was applied to completely fill the defect. A GelrinC accessory kit was used to seal the defect and facilitate injection of GelrinC in non-horizontal positioned lesions. Using a light guide, the implant was exposed to ultraviolet light for 90 s to cure the GelrinC hydrogel into a soft, elastomeric implant.

After curing, the accessories were removed and the implant inspected for completeness. The joint was then flexed to ensure that the implant was secured in the lesion. To complete the procedure, the joint capsule and surrounding tissue were closed in standard fashion. The GelrinC implant naturally degrades in synchronization with the growth of cartilage RT within 6—12 months.

Clinical evaluation

For assessment of the clinical outcome, Overall KOOS, individual KOOS subscales and International Knee Documentation Committee (IKDC) scores were used.

Image acquisition

Each subject underwent several MR examinations in pre-defined intervals after surgery: 1 week (baseline), 1, 3, 6, 12, 18 and 24 months. According to protocol design, MRI scan for 1 and 3 months were performed only on the first 6 patients. All MR examinations were performed at six sites in Europe and Israel using a standardized examination protocol on a 1.5 or 3T MR scanner (Siemens Healthcare, Philips or GE Healthcare) using a gradient strength of 40 mT/m and dedicated knee coils (mostly using an 8-channel phased array knee coil). All MRIs acquired on the same individual were performed on the same scanner, using the same coil. Special attention was paid to ensure that the patients were positioned consistently with the knee extended and minimally externally rotated in the coil to minimize potential magic angle effects. For morphologic evaluation, the following sequences were performed at all sites and with all MR scanners to enable a semi-quantitative morphological scoring based on the MOCART scoring system23.

Sagittal T1-SE for scoring of bone changes in MOCART score, sagittal proton density (PD) FSE, coronal PD-FSE with fat-saturation, 3D gradient recalled echo (GRE) sequences (not available at all sites) with parameters adapted according to MR vendor, scanner type and coil used. The basic measurement parameters are listed in Table I.

The T2 relaxation times were obtained from T2 maps reconstructed using a CPMG multi-echo spin-echo technique with a repetition time (TR) of 2640 ms. Eight echo times (TE) in the range of 12.5—87.5 ms were collected. Images were acquired in the sagittal plane for the femoro-tibial compartments. Main measurement parameters were as follows: slice thickness of 3 mm, number of slices 15, FOV of 160 x 160 mm2, matrix size 256 x 225 pixels, fat suppression was not applied, time of acquisition was 5 m 10 s.

MRI evaluation

The MOCART scoring system23 was used for MRI morphological evaluation. This point-scoring system was designed to

systematically record the constitution of the area of cartilage repair and surrounding tissues, and has been shown to be reliable and reproducible and can be applied to different surgical cartilage repair techniques24,25. The maximum score achievable in the evaluation of nine variables is 100, except in cases when GRE images were not provided resulting in a maximum possible MOCART score of 85. In such cases, a correction factor of a 100/ 85 = 1.176 was applied. In practice, if the total MOCART score without GRE was evaluated for example as 85, a correction was performed by multiplying 85 by the correction factor 1.176 for a total of 99.96 (rounded to 100.0).

Special emphasis was placed on the variables "degree of defect repair and filling of the defect" and "signal intensity" of the MOCART score, which represents degree of maturation of the RTover time23,26.

The baseline score (1 week post-surgery) on the point scale of the MOCART score was judged to be "0". This was based on the fact that at 1 week post-surgery, GelrinC implant displays fluid-like signal intensity on MR indicating that the lesion is completely filled and presenting "false" high scores. In addition, 1 week after surgery the MFX procedure displays subchondral bone changes, the subchondral lamina is perforated and effusion is present.

All MRI images were evaluated for total MOCART scoring separately by two experienced senior musculoskeletal radiologists (one with 24 and the other with 6 years of experience in musculoskeletal MR). Any disagreements were discussed and a consensus was reached. Reviewers were blinded to the patient's clinical history and the location of the lesion (medial or lateral femoral condyle).

The assessment of the T2 values, based on a region-of-interest (ROI) evaluation, was performed by a single reviewer, which has an experience of 24 years in MSK MR imaging. The average number of pixels on the RT and on the reference normal tissue was 14 ± 5 and 13 ± 4, respectively. On the same condyle, an area of RT and an area of healthy cartilage at least 1 cm in distance from the RT (as an internal control) were selected using the morphologic MRI dataset. The selection of the ROIs was made on 1 to 4 consecutive slices depending on the size of cartilage repair. In cases where there was more than one slice used, the T2 values were averaged. The ROIs for cartilage RTand healthy hyaline cartilage were selected to cover the full thickness of the cartilage layer. For the purpose of zonal variation assessment, these ROIs were divided equally into a deep and a superficial half. T2 maps were calculated using a pixel-wise, mono-exponential, non-negative least-squares (NNLS) fit analysis (IDL 6.3, Interactive Data Language, RSI, Inc., Boulder, CO, USA). The individual T2 index, which is a dimensionless coefficient, was calculated by expressing global mean T2 of the RT relative to global mean T2 of healthy cartilage. For the zonal variation, a "zonal T2 index" was calculated as a T2 relaxation times of the RT in the deep zone divided by the T2 relaxation times of the RT in the superficial zone. To our best knowledge, this is first time when the T2 zonal variation index was calculated, in order to eliminate differences in

Table I

Basic imaging parameters of all sequences are listed

Orient. Contrast Sl.thick [mm] Fatsat TR [ms] TE [ms] No. of slices FOV [mm] Matrix Phase- res. (%) Scan time (min:sec)

sag PD + T2 2 no 3050 11 + 80 19 160 448 80 3:17

sag PD 2 mm 2 no 2000 37 19 120 384 85 3:20

cor PD fs 3 yes 2970 27 25 160 448 80 3:29

sag T1 se 2 no 680 12 19 160 384 100 2:50

sag T2 map 3 no 2640 12.5-87.5 15 160 256 88 5:10

sag PD + T2 2 no 3480 13 + 94 19 160 384 90 4:16

sag PD 2 mm 2 no 2000 27 19 120 320 90 4:24

cor PD fs 3 yes 3430 31 25 160 384 100 4:36

sag T1 se 2 no 600 13 19 160 384 100 4:09

PD fs, proton density fat suppressed; T1 se, T1 spin echo.

the T2 relaxation time values due to multicenter and multivendor character of our study. The same calculation of the zonal T2 index was performed for normal healthy cartilage. The absolute global T2 values were only used for the analysis of longitudinal development of the standard deviation of T2 relaxation times.

Data and statistical analysis

Comparison of data from each visit to 6 months time point was performed using a paired t-test for MOCART score as well as T2 index at the different time points. Furthermore, Spearman correlation coefficient was used for evaluating possible correlation between each of the different calculated parameters (MOCART morphological scores, the biochemical zonal T2 index values for both RT and reference native cartilage) and clinical outcome with time after implantation, with the age of the patient and with each other. Correlation of the above parameters with the lesion size (pre-and post-debridement) was also performed. All statistical calculations were performed by using statistical software (SPSS version 21, SPSS, Chicago, Ill, USA).

Results

Patient follow-up, implant size and location

Nineteen patients completed 12 months follow-up, 19 patients completed 18 months follow-up and 18 patients completed 24 months follow-up. Analysis was performed on 17 patients at 24 months, 14 patients at 18 months and 19 patients at 12 months where MRI images were available. The GelrinC implant was located on the medial femoral condyle in 17 patients (81%) and on the lateral femoral condyle in 4 patients (19%). The mean implant area was 2.54 ± 1.12 cm2 (range: 1^5 cm2). Six patients had an implant area smaller than 2 cm2 (mean: 1.41 ± 0.34 cm2, range: 1^1.8 cm2) and 13 patients had an implant area of 2 cm2 and above (mean: 2.99 ± 0.99 cm2, range: 2^5 cm2). Two patients did not have documentation of the implanted area.

Morphological evaluation

The mean global MOCART scoring improvement from baseline of the RT is shown in Table II with an increase of the total MOCART at all time points and maximum mean score up to 84.4 points (95% CI 77.7 to 91.0) at 24 months. From 6 months to 12 months, though there was an increase of MOCART score from 71.8 (95% CI 62.2 to 81.3) to 75.2 (95% CI 62.8 to 87.5) it was not statistically significant (N = 18, P = 0.308), however, changes from 6 to 24 months were statistically significant with an increase of the MOCART score from 71.8 (95% CI 62.2 to 81.3) at 6 months to 84.4 (95% CI 77.7. to 91.0) (N = 17, P < 0.005). Fig. 1 shows an example of follow-up of a patient at 1 week, 6,12 and 24 months post-surgery. In six analyses of the MOCART score a disagreement between readers was discussed and a consensus was reached.

The variable "signal intensity" of the MOCART score showed an increase from 9.3 points at 6 month (N = 20, 95% CI 6.7 to 11.8) to 14.4 points (maximum 15 points) at 24 months (N = 17, 95% CI 13.2 to 15.7) and thus nearly reaching the maximum value compared to normal, healthy cartilage (Table III).

Evaluation of the global T2 index

The mean T2 index showed a decrease over time for global cartilage of 2.5 (95% CI 1.8 to 3.3, N = 11); superficial cartilage zone: 2.1 (95% CI 1.6 to 2.7, N = 13); deep cartilage zone: 2.8 (95% CI 2.2 to 3.4, N = 13) at baseline (1 week after surgery) and mean T2 index for global cartilage of 1.3 (95% CI 1.1 to 1.5, N = 16); superficial cartilage zone: 1.2 (95% CI 1.0 to 1.4, N = 16); deep cartilage zone: 1.3 (95% CI 1.1 to 1.5, N = 16) at 24 months follow-up (Table IV).

The global T2 index at baseline were found to be between 0.8 and 1.2 (what is considered normal healthy cartilage) in 1/11 (9%) of patient at baseline, in 8/12 (67%) of patients at 12 months, in 11/13 (85%) of patients at 18 months and in 13/16 (81%) of patients at 24 months (Fig. 2).

Evaluation of the zonal T2 index

The zonal T2 index for the RT was found to be close (less than 20% difference) to the zonal T2 index of normal cartilage in 6/12 patients (50%) at 12 months, in 7/13 (53.8%) patients at 18 months and in 10/16 (63.5%) patients at 24 months, with 7 of 10 patients showing a difference of the zonal T2 index of less than 10% at 24 months. An example of T2 zonal evaluation is shown on Fig. 3. Superficial and deep ROIs for cartilage transplant tissue and normal healthy cartilage are shown.

Longitudinal development of standard deviation of global T2 in RT and normal cartilage

The standard deviation of global T2 among pixels in the evaluated ROIs in RT was decreasing over time (Fig. 4). An initial (early post-surgery) standard deviation of global T2 showed an average of 49.4. Later post-surgery standard deviations of global T2 in evaluated ROIs at 12,18 and 24 months reached in average 17.3,15.6 and 17.0, respectively. For a reference, the standard deviation of global T2 in ROIs drawn in the normal hyaline cartilage was evaluated to be 12.9.

Correlation between T2 mapping and the MOCART score

Considering different variables of the MOCART score, a negative correlation was found between T2 values and the degree of defect repair (r = -0.62, P = 0.01) at the 24 months follow-up. A high negative correlation was also found between T2 values and the integration with the border zone (r = -0.75, P < 0.01) and the surface of the RT (r = -0.70, P < 0.01) at 24 months. Total MOCART score also showed negative correlation with T2 values (r = -0.62,

Table II

Descriptive Statistics for MOCART, changes from baseline

Visit Difference from baseline

Mean Min Median Max 95% Lower CL 95% Upper CL N

1 Month Follow-Up 61.8 47.0 65.0 76.0 49.0 74.6 6

3 Months Follow-Up 63.7 41.0 65.0 76.0 49.5 77.9 6

6 Months Follow-Up 71.8 6.0 73.5 94.0 62.2 81.3 20

12 Months Follow-Up 75.2 6.0 82.0 100.0 62.8 87.5 19

18 Months Follow-Up 71.8 6.0 79.0 100.0 55.4 88.2 14

24 Months Follow-Up 84.4 53.0 88.0 100.0 77.7 91.0 17

Fig. 1. Series of proton density fat suppressed images in coronal plane show development of cartilage transplant in follow-up examinations. Arrows and circle delineate RT area.

Table III

Descriptive Statistics of signal intensity of the repair tissue (Dual FSE) in MOCART

Visit Signal intensity of the repair tissue

Mean Min Median Max 95% Lower CL 95% Upper CL N

1 Month Follow-Up 1.7 0.0 0.0 5.0 0.0 4.4 6

3 Months Follow-Up 3.3 0.0 5.0 5.0 0.6 6.0 6

6 Months Follow-Up 9.3 0.0 5.0 15.0 6.7 11.8 20

12 Months Follow-Up 11.1 0.0 15.0 15.0 8.1 14.0 19

18 Months Follow-Up 12.1 0.0 15.0 15.0 8.8 15.5 14

24 Months Follow-Up 14.4 5.0 15.0 15.0 13.2 15.7 17

Table IV

Descriptive Statistics of T2 mapping — effectiveness population

T2 Parameter/Visit Mean Min Median Max 95% Lower CL 95% Upper CL N

Deep 1 Week Follow-Up 2.8 1.2 2.9 4.4 2.2 3.4 13

12 Months Follow-Up 1.1 0.8 1.1 1.6 0.9 1.3 12

18 Months Follow-Up 1.3 0.8 1.0 4.9 1.1 1.5 13

24 Months Follow-Up 1.3 0.6 1.0 5.4 1.1 1.5 16

Global 1 Week Follow-Up 2.5 0.9 2.3 4.9 1.8 3.3 11

12 Months Follow-Up 1.1 0.9 1.1 1.3 1 1.2 12

18 Months Follow-Up 1.2 0.8 0.9 4.2 1.1 1.4 13

24 Months Follow-Up 1.3 0.7 1.0 5.1 1.1 1.5 16

Superficial 1 Week Follow-Up 2.1 1.0 1.9 4.5 1.6 2.7 13

12 Months Follow-Up 1.2 0.8 1.2 1.5 1.1 1.3 12

18 Months Follow-Up 1.2 0.8 1.0 3.5 0.9 1.5 13

24 Months Follow-Up 1.2 0.6 1.0 4.0 1 1.4 16

Fig. 2. T2 index development at 12,18 and 24 months after surgery using an early post-surgery (1 week) measurement as a reference. A 95% CI as a measure of uncertainty is indicated.

Fig. 3. An example of T2 zonal evaluation. Superficial and deep ROIs for cartilage transplant tissue (posterior aspect on the image) and normal healthy cartilage (anterior aspect of the image) are shown.

P = 0.01) at 24 months. Image examples of morphological and T2 mapping of the post-surgery period are shown in Fig. 5. Cartilage in T2 maps is pseudo-colored for better visualization.

Discussion

In this prospective, single arm, longitudinal multicentre study the safety and efficacy of a new GelrinC cartilage repair procedure in patients at different time intervals post-surgery (1 week, 1,3,6,12,18 and 24 months) were evaluated. Patients were evaluated for clinical outcome, morphological and biochemical properties using MRI.

Morphological MRI evaluation

Twenty-four months after the surgery, a MOCART score of 84.4 was achieved. Significant differences in the MOCART scores in the entire cohort were found when comparing cartilage implant between 6 and 24 months. These findings are different from results seen in other studies following MACT procedure at early postoperative follow-up (3 months) and at 12 months follow-up period4,11 and in patients after MFX procedure27 in which the MOCART score did not significantly change at different time intervals. For the "degree of defect repair and filling of the defect", the values achieved already after 6 months post-surgery did not significantly change over the next 18 months, which may be explained by the nature of the GelrinC procedure with a tightly fixed soft, elastomeric implant filling the defect completely starting at the surgery. A steady improvement in "signal intensity" was observed from 6 to 24 months and a near complete normalization of the RT signal intensity is reported at 24 months. This corresponds to a morphologically excellent maturation process of the tissue. Only low positive correlation was found between the MOCART score and the clinical outcome KOOS Sport Score at 24 months follow-up. This is consistent with a similar finding reported by Buda et al.28 where a high correlation between KOOS and the MOCART "signal intensity" score was found.

T2 mapping results

It has been previously demonstrated that fibrous RT shows lower T2 relaxation times (which corresponds to T2 index < 1) than normal hyaline cartilage16. Correspondingly, lower T2 values were observed in RT after MFX as compared to MACT RT, while no differences in Lysholm or MOCART scores were detected10,27,29. Oneto et al. initially found elevated T2 values (corresponding to a T2

Fig. 4. Change of T2-standard deviations among the pixels of the ROIs drawn on the RT in post-surgery period. The numbers in brackets indicate number of ROIs included. As a reference a standard deviation of healthy tissue was used.

Fig. 5. Image examples of morphological (left column) and T2 mapping (right column) of the post-surgery period are shown. Cartilage in T2 maps is pseudo-colored for better visualization. Color-bar is added for T2 map quantification.

index > 1) in MFX RT, which came close to normal cartilage values after 2 years in cases of good graft maturation and lower T2 values for therapy failure12. Theologis et al. reported that 3—6 months after surgery MFX RT had significantly higher Tlrho and T2 values relative to normal cartilage9. At 1 year, T2 values of RT decreased to reach values comparable to normal cartilage (Tlrho remained significantly different). Welsh et al. detected in two different studies reduced T2 values in cartilage repair area after MFX, whereas, after MACT, T2 similar to normal cartilage was observed between 2.3 and 2.7 years after surgery10,27. Most studies however

reported that normal zonal variation was lacking after MFX10,13,16,18,19. Tissue maturation processes can also be depicted after ACI16. Kurkijarvi et al. showed that T2 values for RT after ACI were higher and more heterogeneous than T2 of normal control cartilage about 1 year after surgery with a lack of zonal organization30. T2 relaxation times were higher for RT than for normal cartilage at 3—13 months after ACI, but no significant difference was detected at later time points in several studies (after 19—42,12—59, and 20 months)4,10,13,14. However, T2 relaxation times of RT after MACT generally decreased during longer postoperative in-tervals10,13,31. According to Salzmann et al. RT had significantly lower T2 values than normal cartilage about 3.5 years after MACT15. In an international, multicentre, randomized controlled trial, BST-CarGel treatment was evaluated and compared with MFX alone in the repair of cartilage lesions in the knee joint32. The repair cartilage T2 relaxation times for the BST-CarGel treatment group were significantly different and lower than that of the MFX treatment group, albeit not yet at the level of native cartilage after only 12 months and no information on zonal variation of T2 mapping within the RT was given.

In the GelrinC study, the T2 index results are in accordance with these published findings of absolute T2 relaxation times mentioned above and show T2 index values from 0.8 to 1.2 for the majority of RT after 12 months. At 24 months, values are similar to healthy, native hyaline cartilage.

Using the T2 index calculation, Domayer et al.,29 found a correlation between the Lysholm score and the IKDC subjective knee evaluation form and the T2 index in patients after MFX and Salzmann et al.15 found a correlation between the Lysholm score and T2 mapping in patients after MACT.

Using the zonal T2 index in the current study, zonal variation in the implanted area was clearly detected at 12—24 months after surgery with T2 index of RT close to T2 index of normal healthy cartilage in the majority of patients. Our new approach of calculating the zonal T2 index allows us also to avoid the problems mentioned above regarding absolute T2 values. Zonal differences between the different cartilage layers have previously been re-ported10,16,33,34. However, these zonal differences seem to vary during the maturation process after ACI. Whereas after 12 months, no zonal stratification was detected30, in this study zonal variation was obvious already at 12 months in the majority of our patients. At later time points, during the second year after MACT, T2 zonal organization approached that of control healthy cartilage and persisted during the period, which nicely corresponds with our results as opposed to the lack of zonal variation described after MFX4,10,13,14,35.

The new calculation of the mean standard deviation of T2 relaxation time over time provides additional information on the tissue organization, since higher standard deviation means higher heterogeneity of the RT and vice versa. In our study, the standard deviation of the T2 relaxation time measurements decreased with the longer follow-up periods up to 24 months significantly and came close to values of normal hyaline cartilage.

Overall, these findings for T2 relaxation time measurements of cartilage RT may correspond to the described histological differences of the tissue and indicate maturation of both layers with a decrease in water content and an increase of collagen content and

orientation2'.

Limitations of the study include a rather small number of patients. However, to recruit patients for a longitudinal study which comprises at least 4 follow-up examinations and considers all inclusion and exclusion criteria severely reduced the number of available patients. Another limitation of this longitudinal study is the restriction of time interval after surgery to 24 months. Further development of the RT after GelrinC beyond 24 months will be

reported in next study. This was a single arm study and comparison with a second arm, such as microfracture or other cartilage repair procedures would be beneficial in future studies. The main purpose of this study was evaluation of radiological outcome.

The in-plane resolution used in this study was a limitation for T2 mapping. However, it was given by the selected matrix size. In case a higher matrix size would have been selected the SNR would drop and consequently the uncertainty of T2 values would increase. The overall quality of T2 evaluation depends on the experience of person drawing ROIs. Unfortunately, there is not an alternative so far and evaluator experience is crucial in study like this one. The lack of histological control data in this study is another limitation, however the dedicated histological large animal model study to evaluate safety, performance and degradation profile of GelrinC in-vivo was already performed (not published yet - currently under the revisions).

In conclusion, significant improvement can be expected already 1 year after GelrinC implantation. This is supported by a relatively high mean MOCART score and the results of T2 relaxation times calculation with T2 index, new zonal T2 index and standard deviation of T2 over time, which in most cases, were comparable to values from normal hyaline cartilage.

Author contributions

The authors declare the following contributions to the preparation of the manuscript:

• study conception and design (Trattnig, Korner, Ohel, Mlynarik,

Zbyn, Juras);

• analysis and interpretation of data (Trattnig, Juras);

• drafting of the manuscript (Trattnig, Mlynarik, Zbyn, Juras);

• critical revision of the manuscript for important intellectual

content (Korner, Ohel);

• final approval of the article (Trattnig, Korner, Ohel, Mlynarik,

Zbyn, Juras);

• collection and assembly of data (Korner, Ohel).

All authors take responsibility for the integrity of the work. Conflict of interest

None of the authors have any conflict of interest relating to the submitted work.

Acknowledgments - Role of funding source

Funding for this study was provided by the Vienna Spots of Excellence of the Vienna Science and Technology Fund (WWTF): Vienna Advanced Imaging Center-VIACLIC FA102A0017 and FWF-DACH Programme 1652-B19 "Ultrahigh-field (7T) MR of early cartilage degeneration". The financial support by the Austrian Federal Ministry of Science, Research and Economy and the National Foundation for Research, Technology and Development is gratefully acknowledged. The study sponsors did not have a role in study design, the collection and analysis of data, interpretation of results and decision to publish.

References

1. Glaser C. New techniques for cartilage imaging: T2 relaxation time and diffusion-weighted MR imaging. Radiol Clin North Am 2005;43:641-53. vii.

2. Recht MP, Goodwin DW, Winalski CS, White LM. MRI of articular cartilage: revisiting current status and future directions. AJR Am J Roentgenol 2005;185:899-914.

3. Lusse S, Claassen H, Gehrke T, Hassenpflug J, Schunke M, Heller M, et al. Evaluation of water content by spatially resolved transverse relaxation times of human articular cartilage. Magn Reson Imaging 2000;18:423-30.

4. Welsch GH, Mamisch TC, Marlovits S, Glaser C, Friedrich K, Hennig FF, et al. Quantitative T2 mapping during follow-up after matrix-associated autologous chondrocyte transplantation (MACT): full-thickness and zonal evaluation to visualize the maturation of cartilage repair tissue. J Orthop Res 2009;27:957-63.

5. Gilbert JE. Current treatment options for the restoration of articular cartilage. Am J Knee Surg 1998;11:42-6.

6. Crema MD, Roemer FW, Marra MD, Burstein D, Gold GE, Eckstein F, et al. Articular cartilage in the knee: current MR imaging techniques and applications in clinical practice and research. Radiographics 2011;31:37-61.

7. Brown WE, Potter HG, Marx RG, Wickiewicz TL, Warren RF. Magnetic resonance imaging appearance of cartilage repair in the knee. Clin Orthop Relat Res 2004:214-23.

8. Potter HG, Black BR, Chong le R. New techniques in articular cartilage imaging. Clin Sports Med 2009;28:77-94.

9. Theologis AA, Schairer WW, Carballido-Gamio J, Majumdar S, Li X, Ma CB. Longitudinal analysis of T1rho and T2 quantitative MRI of knee cartilage laminar organization following microfracture surgery. Knee 2012;19:652-7.

10. Welsch GH, Mamisch TC, Domayer SE, Dorotka R, Kutscha-Lissberg F, Marlovits S, etal. Cartilage T2 assessment at 3-T MR imaging: in vivo differentiation of normal hyaline cartilage from reparative tissue after two cartilage repair procedures-initial experience. Radiology 2008;247:154-61.

11. Dhollander AA, Huysse WC, Verdonk PC, Verstraete KL, Verdonk R, Verbruggen G, et al. MRI evaluation of a new scaffold-based allogenic chondrocyte implantation for cartilage repair. Eur J Radiol 2010;75:72-81.

12. Oneto JM, Ellermann J, LaPrade RF. Longitudinal evaluation of cartilage repair tissue after microfracture using T2-mapping: a case report with arthroscopic and MRI correlation. Knee Surg Sports Traumatol Arthrosc 2010;18:1545-50.

13. Welsch GH, Trattnig S, Scheffler K, Szomonanyi P, Quirbach S, Marlovits S, et al. Magnetization transfer contrast and T2 mapping in the evaluation of cartilage repair tissue with 3T MRI. J Magn Reson Imaging 2008;28:979-86.

14. Trattnig S, Mamisch TC, Welsch GH, Glaser C, Szomolanyi P, Gebetsroither S, et al. Quantitative T2 mapping of matrix-associated autologous chondrocyte transplantation at 3 Tesla: an in vivo cross-sectional study. Invest Radiol 2007;42: 442-8.

15. Salzmann GM, Paul J, Bauer JS, Woertler K, Sauerschnig M, Landwehr S, et al. T2 assessment and clinical outcome following autologous matrix-assisted chondrocyte and osteo-chondral autograft transplantation. Osteoarthritis Cartilage 2009;17:1576-82.

16. Nieminen MT, Nissi MJ, Mattila L, Kiviranta I. Evaluation of chondral repair using quantitative MRI. J Magn Reson Imaging 2012;36:1287-99.

17. McCauley TR, Disler DG. Magnetic resonance imaging of articular cartilage of the knee. J Am Acad Orthop Surg 2001;9: 2-8.

18. Mamisch TC, Hughes T, Mosher TJ, Mueller C, Trattnig S, Boesch C, et al. T2 star relaxation times for assessment of articular cartilage at 3 T: a feasibility study. Skeletal Radiol 2012;41:287-92.

19. White LM, Sussman MS, Hurtig M, Probyn L, Tomlinson G, Kandel R. Cartilage T2 assessment: differentiation of normal hyaline cartilage and reparative tissue after arthroscopic

cartilage repair in equine subjects. Radiology 2006;241: 407—14.

20. Kon E, Delcogliano M, Filardo G, Montaperto C, Marcacci M. Second generation issues in cartilage repair. Sports Med Arthrosc 2008;16:221—9.

21. Niemeyer P, Pestka JM, Kreuz PC, Erggelet C, Schmal H, Suedkamp NP, et al. Characteristic complications after autol-ogous chondrocyte implantation for cartilage defects of the knee joint. AmJ Sports Med 2008;36:2091—9.

22. Steadman JR, Rodkey WG, Rodrigo JJ. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin Orthop Relat Res 2001:S362—9.

23. Marlovits S, Striessnig G, Resinger CT, Aldrian SM, Vecsei V, Imhof H, et al. Definition of pertinent parameters for the evaluation of articular cartilage repair tissue with highresolution magnetic resonance imaging. Eur J Radiol 2004;52:310—9.

24. Marlovits S, Zeller P, Singer P, Resinger C, Vecsei V. Cartilage repair: generations of autologous chondrocyte transplantation. Eur J Radiol 2006;57:24—31.

25. Trattnig S, Millington SA, Szomolanyi P, Marlovits S. MR imaging of osteochondral grafts and autologous chondrocyte implantation. Eur Radiol 2007;17:103—18.

26. Marlovits S, Singer P, Zeller P, Mandl I, Haller J, Trattnig S. Magnetic resonance observation of cartilage repair tissue (MOCART) for the evaluation of autologous chondrocyte transplantation: determination of interobserver variability and correlation to clinical outcome after 2 years. Eur J Radiol 2006;57:16—23.

27. Welsch GH, Trattnig S, Domayer S, Marlovits S, White LM, Mamisch TC. Multimodal approach in the use of clinical scoring, morphological MRI and biochemical T2-mapping and diffusion-weighted imaging in their ability to assess differences between cartilage repair tissue after microfracture therapy and matrix-associated autologous chondrocyte transplantation: a pilot study. Osteoarthritis Cartilage 2009;17:1219—27.

28. Buda R, Vannini F, Cavallo M, Grigolo B, Cenacchi A, Giannini S. Osteochondral lesions of the knee: a new one-step repair technique with bone-marrow-derived cells. J Bone Joint Surg Am 2010;92(Suppl 2):2—11.

29. Domayer SE, Kutscha-Lissberg F, Welsch G, Dorotka R, Nehrer S, Gabler C, et al. T2 mapping in the knee after microfracture at 3.0 T: correlation of global T2 values and clinical outcome — preliminary results. Osteoarthritis Cartilage 2008;16:903-8.

30. Kurkijarvi JE, Mattila L, Ojala RO, Vasara AI, Jurvelin JS, Kiviranta I, et al. Evaluation of cartilage repair in the distal femur after autologous chondrocyte transplantation using T2 relaxation time and dGEMRIC. Osteoarthritis Cartilage 2007;15:372-8.

31. Eshed I, Trattnig S, Sharon M, Arbel R, Nierenberg G, Konen E, et al. Assessment of cartilage repair after chondrocyte transplantation with a fibrin-hyaluronan matrix—correlation of morphological MRI, biochemical T2 mapping and clinical outcome. Eur J Radiol 2012;81:1216-23.

32. Stanish WD, McCormack R, Forriol F, Mohtadi N, Pelet S, Desnoyers J, et al. Novel scaffold-based BST-CarGel treatment results in superior cartilage repair compared with microfracture in a randomized controlled trial. J Bone Joint Surg Am 2013;95:1640-50.

33. Trattnig S, Domayer S, Welsch GW, Mosher T, Eckstein F. MR imaging of cartilage and its repair in the knee-a review. Eur Radiol 2009;19:1582-94.

34. Trattnig S, Mamisch TC, Pinker K, Domayer S, Szomolanyi P, Marlovits S, et al. Differentiating normal hyaline cartilage from post-surgical repair tissue using fast gradient echo imaging in delayed gadolinium-enhanced MRI (dGEMRIC) at 3 Tesla. Eur Radiol 2008;18:1251-9.

35. Domayer SE, Apprich S, Stelzeneder D, Hirschfeld C, Sokolowski M, Kronnerwetter C, et al. Cartilage repair of the ankle: first results of T2 mapping at 7.0 T after microfracture and matrix associated autologous cartilage transplantation. Osteoarthritis Cartilage 2012;20:829-36.