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Osteoarthritis is a common chronic degenerative disease that causes pain and disability with increasing incidence worldwide. The osteochondral junction is a dynamic region of the joint that is associated with the early development and progression of osteoarthritis. Despite the substantial advances achieved in the imaging of cartilage and application to osteoarthritis in recent years, the osteochondral junction has received limited attention. This is primarily related to technical limitations encountered with conventional MR sequences that are relatively insensitive to short T2 tissues and the rapid signal decay that characterizes these tissues. MR sequences with ultrashort echo time (UTE) are of great interest because they can provide images of high resolution and contrast in this region. Here, we briefly review the anatomy and function of cartilage, focusing on the osteochondral junction. We also review basic concepts and recent applications of UTE MR sequences focusing on the osteochondral junction.

Ultrashort echo time (UTE) sequences can image tissues with transverse T 2 /T 2 * relaxations too short to be efficiently observed on routine clinical MRI sequences, such as the vertebral body cartilaginous endplate (CEP). Here, we describe a 3D adiabatic inversion-recovery-prepared fat-saturated ultrashort echo time (3D IR-FS-UTE) sequence to highlight the CEP of vertebral bodies in comparison to the intervertebral disc (IVD) and bone marrow fat (BF) at 3 T. The IR-FS-UTE sequence used a 3D UTE sequence combined with an adiabatic IR preparation pulse centered in the middle of the water and fat peaks, while a fat saturation module was used to suppress the signal from fat. A slab-selective half pulse was used for signal excitation, and a 3D center-out cones trajectory was used for more efficient data sampling. The 3D IR-FS-UTE sequence was applied to an ex vivo human spine sample, as well as the spines of six healthy volunteers and of three patients with back pain. Bright continuous lines representing signal from CEP were found in healthy IVDs. The measured contrast-to-noise ratio was 18.5 ± 4.9 between the CEP and BF, and 20.3 ± 4.15 between the CEP and IVD for the six volunteers. Abnormal IVDs showed CEP discontinuity or irregularity in the sample and patient studies. In conclusion, the proposed 3D IR-FS-UTE sequence is feasible for imaging the vertebral body's CEP in vivo with high contrast.

The purpose of this study is to develop a 3D adiabatic inversion recovery prepared ultrashort echo time Cones (3D IR-UTE-Cones) sequence for high resolution and contrast imaging of the region of osteochondral junction (OCJ) of human knee joint using a clinical 3 T scanner. A feasibility study on direct imaging of the OCJ region was performed on a human patellar cartilage sample and on eight cadaveric knee joints using T1 -weighted, proton density (PD)-weighted and short-T2 -weighted 3D IR-UTE-Cones sequences. Contrast to noise ratio was measured to evaluate the effectiveness of the 3D IR-UTE-Cones sequences for selective imaging of the OCJ region. Computed tomography imaging was performed in parallel for the cadaveric knee joints. The optimized T1 -weighted 3D IR-UTE-Cones sequence was used to image the knee joints of eight healthy volunteers and six patients with osteoarthritis (OA) to evaluate morphological changes in the OCJ region. Clinical PD- and T2 -weighted FSE sequences were also performed for comparison. The T1 -weighted 3D IR-UTE-Cones sequence showed high resolution and contrast bright band of the normal OCJ region in the cadaveric joints. Normal OCJ appearances were also seen in healthy volunteers. Abnormal OCJ regions, manifested as ill-defined, focal loss or non-visualization of the high intensity band adjacent to the subchondral bone plate, were observed in the knee joints of both ex vivo and in vivo OA patients. The 3D IR-UTE-Cones sequence can image OCJ regions ex vivo and in vivo, with abnormalities depicted with high resolution and contrast. The technique may be useful for demonstrating involvement of OCJ regions in early OA.

PurposeIn ultrashort echo time (UTE) imaging, fat suppression can improve short T2 * contrast but can also reduce short T2 * signals. The conventional two-point Dixon (2p-Dixon) method does not perform well due to short T2 * decay. In this study, we propose a new method to suppress fat for high contrast UTE imaging of short T2 tissues, utilizing a single-point Dixon (1p-Dixon) method.MethodsThe proposed method utilizes dual-echo UTE imaging, where UTE is followed by the second TE, chosen flexibly. Fat is estimated by applying a 1p-Dixon method to the non-UTE image after correction of phase errors, which is used to suppress fat in the UTE image. In vivo ankle and knee imaging were performed at 3 T to evaluate the proposed method.ResultIt was observed that fat and water signals in tendons were misestimated by the 2p-Dixon method due to signal decay, while the 1p-Dixon method showed reliable fat and water separation not affected by the short T2 * signal decay. Compared with the conventional chemical shift based fat saturation technique, the 1p-Dixon based approach showed much stronger signal intensities in the Achilles, quadriceps, and patellar tendons, with significantly improved contrast to noise ratios (CNRs) of 11.8 ± 2.2, 16.0 ± 1.6, and 26.8 ± 1.3 with the 1p-Dixon method and 0.6 ± 0.2, 4.6 ± 1.0, and 17.5 ± 1.4 with regular fat saturation, respectively.ConclusionThe proposed 1p-Dixon based fat suppression allows more flexible selection of imaging parameters and more accurate fat and water separation over the conventional 2p-Dixon in UTE imaging. Moreover, the proposed method provides much improved CNR for short T2 tissues over the conventional fat saturation method.

Intracortical bone porosity is a key microstructural parameter that determines bone mechanical properties. While clinical MRI visualizes the cortical bone with a signal void, ultrashort echo time (UTE) MRI can acquire high signal from cortical bone, thus enabling quantitative assessments. Magnetization transfer (MT) imaging combined with UTE-MRI can indirectly assess protons in the bone collagenous matrix, which are inversely related to porosity. This study aimed to examine UTE-MT MRI techniques to evaluate intracortical bone porosity. Eighteen human cortical bone specimens from the tibial and fibular midshafts were scanned using UTE-MT sequences on a clinical 3 T MRI scanner and on a high-resolution micro-computed tomography (μCT) scanner. A series of MT pulse saturation powers (500°, 1000°, 1500°) and frequency offsets (2, 5, 10, 20, 50 kHz) were used to measure the macromolecular fraction (MMF) and macromolecular T2 (T2MM ) using a two-pool MT model. The measurements were made on 136 different regions of interest (ROIs). ROIs were selected at three cortical bone layers (from endosteum to periosteum) and four anatomical sites (anterior, mid-medial, mid-lateral, and posterior) to provide a wide range of porosity. MMF showed moderate to strong correlations with intracortical bone porosity (R = -0.67 to -0.73, p

Bone stress injury (BSI) incidents have been increasing amongst athletes in recent years as a result of more intense sporting activities. Cortical bone in the tibia and fibula is one of the most common BSI sites. Nowadays, clinical magnetic resonance imaging (MRI) is the recommended technique for BSI diagnosis at an early stage. However, clinical MRI focuses on edema observations in surrounding soft tissues, rather than the injured components of the bone. Specifically, both normal and injured bone are invisible in conventional clinical MRI. In contrast, ultrashort echo time (UTE)-MRI is able to detect the rapidly decaying signal from the bone. This study aimed to employ UTE-MRI for fatigue fracture detection in fibula cortical bone through an ex vivo investigation. Fourteen human fibular samples (47 ± 20 years old, four women) were subjected to cyclic loading on a four-point bending setup. The loading was displacement controlled to induce -5000 ± 1500 μ-strain at 4 Hz. Loading was stopped when bone stiffness was reduced by 20%. Fibula samples were imaged twice, using UTE-MRI and micro-computed tomography (μCT), first pre-loading and second post-loading. After loading, the macromolecular fraction (MMF) from UTE-MT modeling demonstrated a significant decrease (12% ± 20%, P = 0.02) on average. Single-component T2 * also decreased significantly by BSI (12% ± 11%, P = 0.01) on average. MMF reduction is hypothesized to be a result of collagenous matrix rupture and water increase. However, faster T2 * decay might be a result of water shifts towards newly developed microcracks with higher susceptibility. Despite this good sensitivity level of the UTE-MRI technique, the μCT-based porosity at a voxel size of 9 μm was not affected by loading. UTE-MRI shows promise as a new quantitative technique to detect BSI.

Peripheral nerves are a composite tissue consisting of neurovascular elements packaged within a well-organized extracellular matrix. Their composition, size, and anatomy render nerves a challenging medical imaging target. In contrast to morphological MRI, which represents the predominant approach to nerve imaging, quantitative MRI sequences can provide information regarding tissue composition. Here, we applied standard clinical Carr-Purcell-Meiboom-Gill (CPMG) and experimental three-dimensional (3D) ultrashort echo time (UTE) Cones sequences for quantitative nerve imaging including T2 measurement with single-component analysis, T2 * measurement with single-component and bi-component analyses, and magnetization transfer ratio (MTR) analysis. We demonstrated the feasibility and the high quality of single-component T2 *, bi-component T2 *, and MTR approaches to analyze nerves imaged with clinically deployed 3D UTE Cones pulse sequences. For 24 single fascicles from eight nerves, we measured a mean single-component T2 * of 22.6 ±8.9 ms, and a short T2 * component (STC) with a mean T2 * of 1.7 ±1.0 ms and a mean fraction of (6.74 ±4.31)% in bi-component analysis. For eight whole nerves, we measured a mean single-component T2 * of 16.7 ±2.2 ms, and an STC with a mean T2 * of 3.0 ±1.0 ms and a mean fraction of (15.56 ±7.07)% in bi-component analysis. For nine fascicles from three healthy nerves, we measured a mean MTR of (25.2 ±1.9)% for single fascicles and a mean MTR of (23.6 ±0.9)% for whole nerves. No statistically significant correlation was observed between any MRI parameter and routine histological outcomes, perhaps due to the small sample size and lack of apparent sample pathology. Overall, we have successfully demonstrated the feasibility of measuring quantitative MR outcomes ex vivo, which might reflect features of nerve structure and macromolecular content. These methods should be validated comprehensively on a larger and more diverse set of nerve samples, towards the interpretation of in vivo outcomes. These approaches have new and broad implications for the management of nerve disease, injury, and repair.

Inversion recovery ultrashort echo time (IR-UTE) imaging holds the potential to directly characterize MR signals from ultrashort T2 tissue components (STCs), such as collagen in cartilage and myelin in brain. The application of IR-UTE for myelin imaging has been challenging because of the high water content in brain and the possibility that the ultrashort T2 * signals are contaminated by water protons, including those associated with myelin sheaths. This study investigated such a possibility in an ovine brain D2 O exchange model and explored the potential of IR-UTE imaging for the quantification of ultrashort T2 * signals in both white and gray matter at 3 T. Six specimens were examined before and after sequential immersion in 99.9% D2 O. Long T2 MR signals were measured using a clinical proton density-weighted fast spin echo (PD-FSE) sequence. IR-UTE images were first acquired with different inversion times to determine the optimal inversion time to null the long T2 signals (TInull ). Then, at this TInull , images with echo times (TEs) of 0.01-4 ms were acquired to measure the T2 * values of STCs. The PD-FSE signal dropped to near zero after 24 h of immersion in D2 O. A wide range of TInull values were used at different time points (240-330 ms for white matter and 320-350 ms for gray matter at TR = 1000 ms) because the T1 values of the long T2 tissue components changed significantly. The T2 * values of STCs were 200-300 μs in both white and gray matter (comparable with the values obtained from myelin powder and its mixture with D2 O or H2 O), and showed minimal changes after sequential immersion. The ultrashort T2 * signals seen on IR-UTE images are unlikely to be from water protons as they are exchangeable with deuterons in D2 O. The source is more likely to be myelin itself in white matter, and might also be associated with other membranous structures in gray matter.

Inversion recovery ultrashort echo time (IR-UTE) imaging holds the potential to directly characterize MR signals from ultrashort T2 tissue components (STCs), such as collagen in cartilage and myelin in brain. The application of IR-UTE for myelin imaging has been challenging because of the high water content in brain and the possibility that the ultrashort T2 * signals are contaminated by water protons, including those associated with myelin sheaths. This study investigated such a possibility in an ovine brain D2 O exchange model and explored the potential of IR-UTE imaging for the quantification of ultrashort T2 * signals in both white and gray matter at 3 T. Six specimens were examined before and after sequential immersion in 99.9% D2 O. Long T2 MR signals were measured using a clinical proton density-weighted fast spin echo (PD-FSE) sequence. IR-UTE images were first acquired with different inversion times to determine the optimal inversion time to null the long T2 signals (TInull ). Then, at this TInull , images with echo times (TEs) of 0.01-4 ms were acquired to measure the T2 * values of STCs. The PD-FSE signal dropped to near zero after 24 h of immersion in D2 O. A wide range of TInull values were used at different time points (240-330 ms for white matter and 320-350 ms for gray matter at TR = 1000 ms) because the T1 values of the long T2 tissue components changed significantly. The T2 * values of STCs were 200-300 μs in both white and gray matter (comparable with the values obtained from myelin powder and its mixture with D2 O or H2 O), and showed minimal changes after sequential immersion. The ultrashort T2 * signals seen on IR-UTE images are unlikely to be from water protons as they are exchangeable with deuterons in D2 O. The source is more likely to be myelin itself in white matter, and might also be associated with other membranous structures in gray matter.

We report a novel three-dimensional (3D) ultrashort echo time (UTE) sequence employing Cones trajectory and T1ρ preparation (UTE-Cones-T1ρ ) for quantitative T1ρ assessment of short T2 tissues in the musculoskeletal system. A basic 3D UTE-Cones sequence was combined with a spin-locking preparation pulse for T1ρ contrast. A relatively short TR was used to decrease the scan time, which required T1 measurement and compensation using 3D UTE-Cones data acquisitions with variable TRs. Another strategy to reduce the total scan time was to acquire multiple Cones spokes (Nsp ) after each T1ρ preparation and fat saturation. Four spin-locking times (TSL = 0-20 ms) were acquired over 12 min, plus another 7 min for T1 measurement. The 3D UTE-Cones-T1ρ sequence was compared with a two-dimensional (2D) spiral-T1ρ sequence for the imaging of a spherical CuSO4 phantom and ex vivo meniscus and tendon specimens, as well as the knee and ankle joints of healthy volunteers, using a clinical 3-T scanner. The CuSO4 phantom showed a T1ρ value of 76.5 ± 1.6 ms with the 2D spiral-T1ρ sequence, as well as 85.7 ± 3.6 and 89.2 ± 1.4 ms for the 3D UTE-Cones-T1ρ sequences with Nsp of 1 and 5, respectively. The 3D UTE-Cones-T1ρ sequence provided shorter T1ρ values for the bovine meniscus sample relative to the 2D spiral-T1ρ sequence (10-12 ms versus 16 ms, respectively). The cadaveric human Achilles tendon sample could only be imaged with the 3D UTE-Cones-T1ρ sequence (T1ρ  = 4.0 ± 0.9 ms), with the 2D spiral-T1ρ sequence demonstrating near-zero signal intensity. Human studies yielded T1ρ values of 36.1 ± 2.9, 18.3 ± 3.9 and 3.1 ± 0.4 ms for articular cartilage, meniscus and the Achilles tendon, respectively. The 3D UTE-Cones-T1ρ sequence allows volumetric T1ρ measurement of short T2 tissues in vivo.

MRI biomarkers such as T2 , T2 * and T1rho have been widely used, but are confounded by the magic angle effect. The purpose of this study is to investigate the use of the two-dimensional ultrashort echo time magnetization transfer (UTE-MT) sequence for potential magic angle independent MR biomarkers. Magnetization transfer was investigated in cadaveric Achilles tendon samples using the UTE-MT sequence at five MT powers and five frequency offsets ranging from 2 to 50 kHz. The protocol was applied at five sample orientations ranging from 0 to 90° relative to the B0 field. The results were analyzed with a two-pool quantitative MT model. Multiple TE data were also acquired and mono-exponential T2 * was calculated for each orientation. Macromolecular proton fractions and exchange rates derived from UTE-MT modeling did not appreciably change between the various orientations, whereas the T2 * relaxation time demonstrated up to a sixfold increase from 0° to 55°. The UTE-MT technique with two-pool modeling shows promise as a clinically compatible technique that is resistant to the magic angle effect. This method provides information on the macromolecular proton pool that cannot be directly obtained by other methods, including regular UTE techniques.

Ultrashort-TE (UTE) sequences can obtain signal directly from short-T2 , collagen-rich tissues. It is generally accepted that bound and free water can be detected with UTE techniques, but the ability to detect protons directly on the collagen molecule remains controversial. In this study, we investigated the potential of UTE sequences on a 3-T clinical scanner to detect collagen protons via freeze-drying and D2 O-H2 O exchange studies. Experiments were performed on bovine cortical bone and human Achilles tendon specimens, which were either subject to freeze-drying for over 66 h or D2 O-H2 O exchange for 6 days. Specimens were imaged using two- and three-dimensional UTE with Cones trajectory techniques with a minimum TE of 8 μs at 3 T. UTE images before treatment showed high signal from all specimens with bi-component T2 * behavior. Bovine cortical bone showed a shorter T2 * component of 0.36 ms and a longer T2 * component of 2.30 ms with fractions of 78.2% and 21.8% by volume, respectively. Achilles tendon showed a shorter T2 * component of 1.22 ms and a longer T2 * component of 15.1 ms with fractions of 81.1% and 18.9% by volume, respectively. Imaging after freeze-drying or D2 O-H2 O exchange resulted in either the absence or near-absence of signal. These results indicate that bound and free water are the sole sources of UTE signal in bovine cortical bone and human Achilles tendon samples on a clinical 3-T scanner. Protons on the native collagen molecule are not directly visible when imaged using UTE sequences. Copyright © 2016 John Wiley & Sons, Ltd.

Ultrashort-TE (UTE) sequences can obtain signal directly from short-T2 , collagen-rich tissues. It is generally accepted that bound and free water can be detected with UTE techniques, but the ability to detect protons directly on the collagen molecule remains controversial. In this study, we investigated the potential of UTE sequences on a 3-T clinical scanner to detect collagen protons via freeze-drying and D2 O-H2 O exchange studies. Experiments were performed on bovine cortical bone and human Achilles tendon specimens, which were either subject to freeze-drying for over 66 h or D2 O-H2 O exchange for 6 days. Specimens were imaged using two- and three-dimensional UTE with Cones trajectory techniques with a minimum TE of 8 μs at 3 T. UTE images before treatment showed high signal from all specimens with bi-component T2 * behavior. Bovine cortical bone showed a shorter T2 * component of 0.36 ms and a longer T2 * component of 2.30 ms with fractions of 78.2% and 21.8% by volume, respectively. Achilles tendon showed a shorter T2 * component of 1.22 ms and a longer T2 * component of 15.1 ms with fractions of 81.1% and 18.9% by volume, respectively. Imaging after freeze-drying or D2 O-H2 O exchange resulted in either the absence or near-absence of signal. These results indicate that bound and free water are the sole sources of UTE signal in bovine cortical bone and human Achilles tendon samples on a clinical 3-T scanner. Protons on the native collagen molecule are not directly visible when imaged using UTE sequences. Copyright © 2016 John Wiley & Sons, Ltd.

Magnetization transfer (MT) imaging is one way to indirectly assess pools of protons with fast transverse relaxation. However, conventional MT imaging sequences are not applicable to short T2 tissues such as cortical bone. Ultrashort echo time (UTE) sequences with TE values as low as 8 µs can detect signals from different water components in cortical bone. In this study we aim to evaluate two-dimensional UTE-MT imaging of cortical bone and its application in assessing cortical bone porosity as measured by micro-computed tomography (μCT) and biomechanical properties. In total, 38 human cadaveric distal femur and proximal tibia bones were sectioned to produce 122 rectangular pieces of cortical bone for quantitative UTE-MT MR imaging, μCT, and biomechanical testing. Off-resonance saturation ratios (OSRs) with a series of MT pulse frequency offsets (Δf) were calculated and compared with porosity assessed with μCT, as well as elastic (modulus, yield stress, and strain) and failure (ultimate stress, failure strain, and energy) properties, using Pearson correlation and linear regression. A moderately strong negative correlation was observed between OSR and μCT porosity (R(2)  = 0.46-0.51), while a moderate positive correlation was observed between OSR and yield stress (R(2)  = 0.25-0.30) and failure stress (R(2)  = 0.31-0.35), and a weak positive correlation (R(2)  = 0.09-0.12) between OSR and Young's modulus at all off-resonance saturation frequencies. OSR determined with the UTE-MT sequence provides quantitative information on cortical bone and is sensitive to μCT porosity and biomechanical function.

Water is present in cortical bone in different binding states. In this study we aimed to investigate the effects of inversion time (TI) on the signal from bound and pore water in cortical bone using an adiabatic inversion recovery prepared ultrashort echo time (IR-UTE) sequence on a clinical 3 T scanner. In total ten bovine midshaft samples and four human tibial midshaft samples were harvested for this study. Each cortical sample was imaged with the UTE and IR-UTE sequences with a TR of 300 ms and a series of TI values ranging from 10 to 240 ms. Five healthy volunteers were also imaged with the same sequence. Single- and bi-component models were utilized to calculate the T2 * and relative fractions of short and long T2 * components. Bi-component behavior of the signal from cortical bone was seen with the IR-UTE sequence, except with a TI of around 80 ms, where the short T2 * component alone were seen and a mono-exponential decay pattern was observed. In vivo imaging with the IR-UTE sequence provided high contrast-to-noise images with direct visualization of bound water and reduced signal from long T2 muscle and fat. Our preliminary results demonstrate that selective nulling of the pore water component can be achieved with the IR-UTE sequence with an appropriate TI, allowing selective imaging of the bound water component in cortical bone in vivo using clinical MR scanners.

Water is present in cortical bone in different binding states. In this study we aimed to investigate the effects of inversion time (TI) on the signal from bound and pore water in cortical bone using an adiabatic inversion recovery prepared ultrashort echo time (IR-UTE) sequence on a clinical 3 T scanner. In total ten bovine midshaft samples and four human tibial midshaft samples were harvested for this study. Each cortical sample was imaged with the UTE and IR-UTE sequences with a TR of 300 ms and a series of TI values ranging from 10 to 240 ms. Five healthy volunteers were also imaged with the same sequence. Single- and bi-component models were utilized to calculate the T2 * and relative fractions of short and long T2 * components. Bi-component behavior of the signal from cortical bone was seen with the IR-UTE sequence, except with a TI of around 80 ms, where the short T2 * component alone were seen and a mono-exponential decay pattern was observed. In vivo imaging with the IR-UTE sequence provided high contrast-to-noise images with direct visualization of bound water and reduced signal from long T2 muscle and fat. Our preliminary results demonstrate that selective nulling of the pore water component can be achieved with the IR-UTE sequence with an appropriate TI, allowing selective imaging of the bound water component in cortical bone in vivo using clinical MR scanners.

Muscle degeneration following rotator cuff tendon tearing is characterized by fatty infiltration and fibrosis. While tools exist for the characterization of fat, the ability to noninvasively assess muscle fibrosis is limited. The purpose of this study was to evaluate the capability of quantitative ultrashort echo time T1 (UTE‐T1) and UTE magnetization transfer (UTE‐MT) mapping with and without fat suppression (FS) for the differentiation of injured and control rotator cuff muscles and for the detection of fibrosis. A rat model of chronic massive rotator cuff tearing (n = 12) was used with tenotomy of the right supraspinatus and infraspinatus tendons and silicone implants to prevent healing. Imaging was performed on a 3‐T scanner, and UTE‐T1 mapping with and without FS and UTE‐MT with and without FS for macromolecular fraction (MMF) mapping was performed. At 20 weeks postinjury, T1 and MMF were measured in the supraspinatus and infraspinatus muscles of the injured and contralateral, internal control sides. Histology was performed and connective tissue fraction (CTF) was measured, defined as the area of collagen‐rich extracellular matrix divided by the total muscle area. Paired t‐tests and correlation analyses were performed. Significant differences between injured and control sides were found for CTF in the supraspinatus (mean ± SD, 14.5% ± 3.9% vs. 11.3% ± 3.7%, p = 0.01) and infraspinatus (17.0% ± 5.4% vs. 12.5% ± 4.6%, p < 0.01) muscles, as well as for MMF using UTE‐MT FS in the supraspinatus (9.7% ± 0.3% vs. 9.5% ± 0.2%, p = 0.04) and infraspinatus (10.9% ± 0.8% vs. 10.1% ± 0.5%, p < 0.01) muscles. No significant differences between sides were evident for T1 without or with FS or for MMF using UTE‐MT. Only MMF using UTE‐MT FS was significantly correlated with CTF for both supraspinatus (r = 0.46, p = 0.03) and infraspinatus (r = 0.51, p = 0.01) muscles. Fibrosis occurs in rotator cuff muscle degeneration, and the UTE‐MT FS technique may be helpful to evaluate the fibrosis component, independent from the fatty infiltration process. [ABSTRACT FROM AUTHOR]

The purpose of this study is to investigate the use of ultrashort echo time (UTE) magnetic resonance imaging (MRI) techniques (T1 and magnetization transfer [MT] modeling) for imaging of the Achilles tendons and entheses in patients with psoriatic arthritis (PsA) compared with asymptomatic volunteers. The heels of twenty‐six PsA patients (age 59 ± 15 years, 41% female) and twenty‐seven asymptomatic volunteers (age 33 ± 11 years, 47% female) were scanned in the sagittal plane with UTE‐T1 and UTE‐MT modeling sequences on a 3‐T clinical scanner. UTE‐T1 and macromolecular proton fraction (MMF; the main outcome of MT modeling) were calculated in the tensile portions of the Achilles tendon and at the enthesis (close to the calcaneus bone). Mann–Whitney‐U tests were used to examine statistically significant differences between the two cohorts. UTE‐T1 in the entheses was significantly higher for the PsA group compared with the asymptomatic group (967 ± 145 vs. 872 ± 133 ms, p < 0.01). UTE‐T1 in the tendons was also significantly higher for the PsA group (950 ± 145 vs. 850 ± 138 ms, p < 0.01). MMF in the entheses was significantly lower in the PsA group compared with the asymptomatic group (15% ± 3% vs. 18% ± 3%, p < 0.01). MMF in the tendons was also significantly lower in the PsA group compared with the asymptomatic group (17% ± 4% vs. 20% ± 5%, p < 0.01). Percentage differences in MMF between the asymptomatic and PsA groups (−16.6% and −15.0% for the enthesis and tendon, respectively) were higher than the T1 differences (10.8% and 11.7% for the enthesis and tendon, respectively). The results suggest higher T1 and lower MMF in the Achilles tendons and entheses in PsA patients compared with the asymptomatic group. This study highlights the potential of UTE‐T1 and UTE‐MT modeling for quantitative evaluation of entheses and tendons in PsA patients. [ABSTRACT FROM AUTHOR]

The purpose of the current study was to investigate the effects of B0 and linear eddy currents on ultrashort echo time double echo steady state (UTE‐DESS) imaging and to determine whether eddy current correction (ECC) effectively resolves imaging artifacts caused by eddy currents. 3D UTE‐DESS sequences based on either projection radial or spiral cones trajectories were implemented on a 3‐T clinical MR scanner. An off‐isocentered thin‐slice excitation approach was used to measure eddy currents. The measurements were repeated four times using two sets of tested gradient waveforms with opposite polarities and two different slice locations to measure B0 and linear eddy currents simultaneously. Computer simulation was performed to investigate the eddy current effect. Finally, a phantom experiment, an ex vivo experiment with human synovium and ankle samples, and an in vivo experiment with human knee joints, were performed to demonstrate the effects of eddy currents and ECC in UTE‐DESS imaging. In a computer simulation, the two echoes (S+ and S‐) in UTE‐DESS imaging exhibited strong distortion at different orientations in the presence of B0 and linear eddy currents, resulting in both image degradation as well as misalignment of pixel location between the two echoes. The same phenomenon was observed in the phantom, ex vivo, and in vivo experiments, where the presence of eddy currents degraded S+, S‐, echo subtraction images, and T2 maps. The implementation of ECC dramatically improved both the image quality and image registration between the S+ and S‐ echoes. It was concluded that ECC is crucial for reliable morphological and quantitative UTE‐DESS imaging. [ABSTRACT FROM AUTHOR]

The objective of the current study was to investigate the feasibility of quantitative 3D ultrashort echo time (UTE)‐based biomarkers in detecting proteoglycan (PG) loss and collagen degradation in human cartilage. A total of 104 cartilage samples were harvested for a trypsin digestion study (n = 44), and a sequential trypsin and collagenase digestion study (n = 60), respectively. Forty‐four cartilage samples were randomly divided into a trypsin digestion group (tryp group) and a control group (phosphate‐buffered saline [PBS] group) (n = 22 for each group) for the trypsin digestion experiment. The remaining 60 cartilage samples were divided equally into four groups (n = 15 for each group) for sequential trypsin and collagenase digestion, including PBS + Tris (incubated in PBS, then Tris buffer solution), PBS + 30 U col (incubated in PBS, then 30 U/ml collagenase [30 U col] with Tris buffer solution), tryp + 30 U col (incubated in trypsin solution, then 30 U/ml collagenase with Tris buffer solution), and tryp + Tris (incubated in trypsin solution, then Tris buffer solution). The 3D UTE‐based MRI biomarkers included T1, multiecho T2*, adiabatic T1ρ (AdiabT1ρ), magnetization transfer ratio (MTR), and modeling of macromolecular proton fraction (MMF). For each cartilage sample, UTE‐based biomarkers (T1, T2*, AdiabT1ρ, MTR, and MMF) and sample weight were evaluated before and after treatment. PG and hydroxyproline assays were performed. Differences between groups and correlations were assessed. All the evaluated biomarkers were able to differentiate between healthy and degenerated cartilage in the trypsin digestion experiment, but only T1 and AdiabT1ρ were significantly correlated with the PG concentration in the digestion solution (p = 0.004 and p = 0.0001, respectively). In the sequential digestion experiment, no significant differences were found for T1 and AdiabT1ρ values between the PBS + Tris and PBS + 30 U col groups (p = 0.627 and p = 0.877, respectively), but T1 and AdiabT1ρ values increased significantly in the tryp + Tris (p = 0.031 and p = 0.024, respectively) and tryp + 30 U col groups (both p < 0.0001). Significant decreases in MMF and MTR were found in the tryp + 30 U col group compared with the PBS + Tris group (p = 0.002 and p = 0.001, respectively). It was concluded that AdiabT1ρ and T1 have the potential for detecting PG loss, while MMF and MTR are promising for the detection of collagen degradation in articular cartilage, which could facilitate earlier, noninvasive diagnosis of osteoarthritis. [ABSTRACT FROM AUTHOR]

The protons in collagen‐rich musculoskeletal (MSK) tissues such as the Achilles tendon are subject to strong dipolar interactions which are modulated by the term (3cos2θ‐1) where θ is the angle between the fiber orientation and the static magnetic field B0. The purpose of this study was to investigate the magic angle effect in three‐dimensional ultrashort echo time Cones Adiabatic T1ρ (3D UTE Cones‐AdiabT1ρ) imaging of the Achilles tendon using a clinical 3 T scanner. The magic angle effect was investigated by Cones‐AdiabT1ρ imaging of five cadaveric human Achilles tendon samples at five angular orientations ranging from 0° to 90° relative to the B0 field. Conventional Cones continuous wave T1ρ (Cones‐CW‐T1ρ) and Cones T2* (Cones‐T2*) sequences were also applied for comparison. On average, Cones‐AdiabT1ρ increased 3.6‐fold from 13.6 ± 1.5 ms at 0° to 48.4 ± 5.4 ms at 55°, Cones‐CW‐T1ρ increased 6.1‐fold from 7.0 ± 1.1 ms at 0° to 42.6 ± 5.2 ms at 55°, and Cones‐T2* increased 12.3‐fold from 2.9 ± 0.5 ms at 0° to 35.8 ± 6.4 ms at 55°. Although Cones‐AdiabT1ρ is still subject to significant angular dependence, it shows a much‐reduced magic angle effect compared to Cones‐CW‐T1ρ and Cones‐T2*, and may be used as a novel and potentially more effective approach for quantitative evaluation of the Achilles tendon and other MSK tissues. [ABSTRACT FROM AUTHOR]

Inversion recovery ultrashort echo time (IR-UTE) imaging holds the potential to directly characterize MR signals from ultrashort T2 tissue components (STCs), such as collagen in cartilage and myelin in brain. The application of IR-UTE for myelin imaging has been challenging because of the high water content in brain and the possibility that the ultrashort T2* signals are contaminated by water protons, including those associated with myelin sheaths. This study investigated such a possibility in an ovine brain D2O exchange model and explored the potential of IR-UTE imaging for the quantification of ultrashort T2* signals in both white and gray matter at 3 T. Six specimens were examined before and after sequential immersion in 99.9% D2O. Long T2 MR signals were measured using a clinical proton density-weighted fast spin echo (PD-FSE) sequence. IR-UTE images were first acquired with different inversion times to determine the optimal inversion time to null the long T2 signals (TInull). Then, at this TInull, images with echo times (TEs) of 0.01-4 ms were acquired to measure the T2* values of STCs. The PD-FSE signal dropped to near zero after 24 h of immersion in D2O. A wide range of TInull values were used at different time points (240-330 ms for white matter and 320-350 ms for gray matter at TR = 1000 ms) because the T1 values of the long T2 tissue components changed significantly. The T2* values of STCs were 200-300 μs in both white and gray matter (comparable with the values obtained from myelin powder and its mixture with D2O or H2O), and showed minimal changes after sequential immersion. The ultrashort T2* signals seen on IR-UTE images are unlikely to be from water protons as they are exchangeable with deuterons in D2O. The source is more likely to be myelin itself in white matter, and might also be associated with other membranous structures in gray matter. [ABSTRACT FROM AUTHOR]

We report a novel three-dimensional (3D) ultrashort echo time (UTE) sequence employing Cones trajectory and T1ρ preparation (UTE-Cones- T1ρ) for quantitative T1ρ assessment of short T2 tissues in the musculoskeletal system. A basic 3D UTE-Cones sequence was combined with a spin-locking preparation pulse for T1ρ contrast. A relatively short TR was used to decrease the scan time, which required T1 measurement and compensation using 3D UTE-Cones data acquisitions with variable TRs. Another strategy to reduce the total scan time was to acquire multiple Cones spokes ( Nsp) after each T1ρ preparation and fat saturation. Four spin-locking times (TSL = 0-20 ms) were acquired over 12 min, plus another 7 min for T1 measurement. The 3D UTE-Cones- T1ρ sequence was compared with a two-dimensional (2D) spiral- T1ρ sequence for the imaging of a spherical CuSO4 phantom and ex vivo meniscus and tendon specimens, as well as the knee and ankle joints of healthy volunteers, using a clinical 3-T scanner. The CuSO4 phantom showed a T1ρ value of 76.5 ± 1.6 ms with the 2D spiral- T1ρ sequence, as well as 85.7 ± 3.6 and 89.2 ± 1.4 ms for the 3D UTE-Cones- T1ρ sequences with Nsp of 1 and 5, respectively. The 3D UTE-Cones- T1ρ sequence provided shorter T1ρ values for the bovine meniscus sample relative to the 2D spiral- T1ρ sequence (10-12 ms versus 16 ms, respectively). The cadaveric human Achilles tendon sample could only be imaged with the 3D UTE-Cones- T1ρ sequence ( T1ρ = 4.0 ± 0.9 ms), with the 2D spiral- T1ρ sequence demonstrating near-zero signal intensity. Human studies yielded T1ρ values of 36.1 ± 2.9, 18.3 ± 3.9 and 3.1 ± 0.4 ms for articular cartilage, meniscus and the Achilles tendon, respectively. The 3D UTE-Cones- T1ρ sequence allows volumetric T1ρ measurement of short T2 tissues in vivo. [ABSTRACT FROM AUTHOR]

Ultrashort-TE (UTE) sequences can obtain signal directly from short- T 2 , collagen-rich tissues. It is generally accepted that bound and free water can be detected with UTE techniques, but the ability to detect protons directly on the collagen molecule remains controversial. In this study, we investigated the potential of UTE sequences on a 3-T clinical scanner to detect collagen protons via freeze-drying and D 2 O-H 2 O exchange studies. Experiments were performed on bovine cortical bone and human Achilles tendon specimens, which were either subject to freeze-drying for over 66 h or D 2 O-H 2 O exchange for 6 days. Specimens were imaged using two- and three-dimensional UTE with Cones trajectory techniques with a minimum TE of 8 μs at 3 T. UTE images before treatment showed high signal from all specimens with bi-component T 2 * behavior. Bovine cortical bone showed a shorter T 2 * component of 0.36 ms and a longer T 2 * component of 2.30 ms with fractions of 78.2% and 21.8% by volume, respectively. Achilles tendon showed a shorter T 2 * component of 1.22 ms and a longer T 2 * component of 15.1 ms with fractions of 81.1% and 18.9% by volume, respectively. Imaging after freeze-drying or D 2 O-H 2 O exchange resulted in either the absence or near-absence of signal. These results indicate that bound and free water are the sole sources of UTE signal in bovine cortical bone and human Achilles tendon samples on a clinical 3-T scanner. Protons on the native collagen molecule are not directly visible when imaged using UTE sequences. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mechanical and microstructural evaluations of cortical bone using ultrashort echo time magnetic resonance imaging (UTE‐MRI) have been performed increasingly in recent years. UTE‐MRI acquires considerable signal from cortical bone and enables quantitative bone evaluations. Fitting bone apparent transverse magnetization (T2*) decay using a bicomponent model has been regularly performed to estimate bound water (BW) and pore water (PW) in the quantification of bone matrix and porosity, respectively. Human cortical bone possesses a considerable amount of fat, which appears as MRI T2* signal oscillation and can subsequently lead to BW overestimation when using a bicomponent model. Tricomponent T2* fitting model has been developed to improve BW and PW estimations by accounting for fat contribution in the MRI signal. This study aimed to investigate the correlations of microstructural and mechanical properties of human cortical bone with water pool fractions obtained from a tricomponent T2* model. 135 cortical bone strips (~4 × 2 × 40 mm3) from tibial and femoral midshafts of 37 donors (61 ± 24 years old) were scanned using ten sets of dual‐echo 3D‐UTE‐Cones sequences (TE = 0.032–24.0 ms) on a 3 T MRI scanner for T2* fitting analyses. Average bone porosity and pore size were measured using microcomputed tomography (μCT) at 9 μm voxel size. Bone mechanical properties were measured using 4‐point bending tests. Using a tricomponent model, bound water fraction (FracBW) showed significant strong (R = 0.70, P < 0.01) and moderate (R = 0.58–0.62, P < 0.01) correlations with porosity and mechanical properties, respectively. Correlations of bone microstructural and mechanical properties with water pool fractions were higher for tricomponent model results compared with the bicomponent model. The tricomponent T2* fitting model is suggested as a useful technique for cortical bone evaluation where the MRI contribution of bone fat is accounted for. [ABSTRACT FROM AUTHOR]

Knee degeneration involves all the major tissues in the joint. However, conventional MRI sequences can only detect signals from long T2 tissues such as the superficial cartilage, with little signal from the deep cartilage, menisci, ligaments, tendons and bone. It is highly desirable to develop new sequences that can detect signal from all major tissues in the knee. We aimed to develop a comprehensive quantitative three‐dimensional ultrashort echo time (3D UTE) cones imaging protocol for a truly "whole joint" evaluation of knee degeneration. The protocol included 3D UTE cones actual flip angle imaging (3D UTE‐Cones‐AFI) for T1 mapping, multiecho UTE‐Cones with fat suppression for T2* mapping, UTE‐Cones with adiabatic T1ρ (AdiabT1ρ) preparation for AdiabT1ρ mapping, and UTE‐Cones magnetization transfer (UTE‐Cones‐MT) for MT ratio (MTR) and modeling of macromolecular proton fraction (f). An elastix registration technique was used to compensate for motion during scans. Quantitative data analyses were performed on the registered data. Three knee specimens and 15 volunteers were evaluated at 3 T. The elastix motion correction algorithm worked well in correcting motion artifacts associated with relatively long scan times. Much improved curve fitting was achieved for all UTE‐Cones biomarkers with greatly reduced root mean square errors. The averaged T1, T2*, AdiabT1ρ, MTR and f for knee joint tissues of 15 healthy volunteers were reported. The 3D UTE‐Cones quantitative imaging techniques (ie, T1, T2*, AdiabT1ρ, MTR and MT modeling) together with elastix motion correction provide robust volumetric measurement of relaxation times, MTR and f of both short and long T2 tissues in the knee joint. [ABSTRACT FROM AUTHOR]

Compositional changes can occur in the osteochondral junction (OCJ) during the early stages and progressive disease evolution of knee osteoarthritis (OA). However, conventional magnetic resonance imaging (MRI) sequences are not able to image these regions efficiently because of the OCJ region's rapid signal decay. The development of new sequences able to image and quantify OCJ region is therefore highly desirable. We developed a comprehensive ultrashort echo time (UTE) MRI protocol for quantitative assessment of OCJ region in the knee joint, including UTE variable flip angle technique for T 1 mapping, UTE magnetization transfer (UTE-MT) modeling for macromolecular proton fraction (MMF) mapping, UTE adiabatic T 1ρ (UTE-AdiabT 1ρ ) sequence for T 1ρ mapping, and multi-echo UTE sequence for T 2 * mapping. B 1 mapping based on the UTE actual flip angle technique was utilized for B 1 correction in T 1 , MMF, and T 1ρ measurements. Ten normal and one abnormal cadaveric human knee joints were scanned on a 3T clinical MRI scanner to investigate the feasibility of OCJ imaging using the proposed protocol. Volumetric T 1 , MMF, T 1ρ , and T 2 * maps of the OCJ, as well as the superficial and full-thickness cartilage regions, were successfully produced using the quantitative UTE imaging protocol. Significantly lower T 1 , T 1ρ , and T 2 * relaxation times were observed in the OCJ region compared with those observed in both the superficial and full-thickness cartilage regions, whereas MMF showed significantly higher values in the OCJ region. In addition, all four UTE biomarkers showed substantial differences in the OCJ region between normal and abnormal knees. These results indicate that the newly developed 3D quantitative UTE imaging techniques are feasible for T 1 , MMF, T 1ρ , and T 2 * mapping of knee OCJ, representative of a promising approach for the evaluation of compositional changes in early knee OA., (© 2024 John Wiley & Sons Ltd.)

Magnetization transfer (MT) magnetic resonance imaging (MRI) can be used to estimate the fraction of water and macromolecular proton pools in tissues. MT modeling paired with ultrashort echo time acquisition (UTE-MT modeling) has been proposed to improve the evaluation of the myotendinous junction and fibrosis in muscle tissues, which the latter increases with aging. This study aimed to determine if the UTE-MT modeling technique is sensitive to age-related changes in the skeletal muscles of the lower leg. Institutional review board approval was obtained, and all recruited subjects provided written informed consent. The legs of 31 healthy younger (28.1 ± 6.1 years old, BMI = 22.3 ± 3.5) and 20 older (74.7 ± 5.5 years old, BMI = 26.7 ± 5.9) female subjects were imaged using UTE sequences on a 3 T MRI scanner. MT ratio (MTR), macromolecular fraction (MMF), macromolecular T2 (T2-MM), and water T2 (T2-W) were calculated using UTE-MT modeling for the anterior tibialis (ATM), posterior tibialis (PTM), soleus (SM), and combined lateral muscles. Results were compared between groups using the Wilcoxon rank sum test. Three independent observers selected regions of interest (ROIs) and processed UTE-MRI images separately, and the intraclass correlation coefficient (ICC) was calculated for a reproducibility study. Significantly lower mean MTR and MMF values were present in the older compared with the younger group in all studied lower leg muscles. T2-MM showed significantly lower values in the older group only for PTM and SM muscles. In contrast, T2-W showed significantly higher values in the older group. The age-related differences were more pronounced for MMF (-17 to -19%) and T2-W (+20 to 47%) measurements in all muscle groups compared with other investigated MR measures. ICCs were higher than 0.93, indicating excellent consistency between the ROI selection and MRI measurements of independent readers. As demonstrated by significant differences between younger and older groups, this research emphasizes the potential of UTE-MT MRI techniques in evaluating age-related skeletal muscle changes., (© 2024 John Wiley & Sons Ltd.)

The purpose of the current study was to investigate the effects of B 0 and linear eddy currents on ultrashort echo time double echo steady state (UTE-DESS) imaging and to determine whether eddy current correction (ECC) effectively resolves imaging artifacts caused by eddy currents. 3D UTE-DESS sequences based on either projection radial or spiral cones trajectories were implemented on a 3-T clinical MR scanner. An off-isocentered thin-slice excitation approach was used to measure eddy currents. The measurements were repeated four times using two sets of tested gradient waveforms with opposite polarities and two different slice locations to measure B 0 and linear eddy currents simultaneously. Computer simulation was performed to investigate the eddy current effect. Finally, a phantom experiment, an ex vivo experiment with human synovium and ankle samples, and an in vivo experiment with human knee joints, were performed to demonstrate the effects of eddy currents and ECC in UTE-DESS imaging. In a computer simulation, the two echoes (S+ and S-) in UTE-DESS imaging exhibited strong distortion at different orientations in the presence of B 0 and linear eddy currents, resulting in both image degradation as well as misalignment of pixel location between the two echoes. The same phenomenon was observed in the phantom, ex vivo, and in vivo experiments, where the presence of eddy currents degraded S+, S-, echo subtraction images, and T 2 maps. The implementation of ECC dramatically improved both the image quality and image registration between the S+ and S- echoes. It was concluded that ECC is crucial for reliable morphological and quantitative UTE-DESS imaging., (© 2023 John Wiley & Sons Ltd.)

Inversion recovery ultrashort echo time (IR-UTE) imaging holds the potential to directly characterize MR signals from ultrashort T 2 tissue components (STCs), such as collagen in cartilage and myelin in brain. The application of IR-UTE for myelin imaging has been challenging because of the high water content in brain and the possibility that the ultrashort T 2 * signals are contaminated by water protons, including those associated with myelin sheaths. This study investigated such a possibility in an ovine brain D 2 O exchange model and explored the potential of IR-UTE imaging for the quantification of ultrashort T 2 * signals in both white and gray matter at 3 T. Six specimens were examined before and after sequential immersion in 99.9% D 2 O. Long T 2 MR signals were measured using a clinical proton density-weighted fast spin echo (PD-FSE) sequence. IR-UTE images were first acquired with different inversion times to determine the optimal inversion time to null the long T 2 signals (TI null ). Then, at this TI null , images with echo times (TEs) of 0.01-4 ms were acquired to measure the T 2 * values of STCs. The PD-FSE signal dropped to near zero after 24 h of immersion in D 2 O. A wide range of TI null values were used at different time points (240-330 ms for white matter and 320-350 ms for gray matter at TR = 1000 ms) because the T 1 values of the long T 2 tissue components changed significantly. The T 2 * values of STCs were 200-300 μs in both white and gray matter (comparable with the values obtained from myelin powder and its mixture with D 2 O or H 2 O), and showed minimal changes after sequential immersion. The ultrashort T 2 * signals seen on IR-UTE images are unlikely to be from water protons as they are exchangeable with deuterons in D 2 O. The source is more likely to be myelin itself in white matter, and might also be associated with other membranous structures in gray matter., (Copyright © 2017 John Wiley & Sons, Ltd.)

We report a novel three-dimensional (3D) ultrashort echo time (UTE) sequence employing Cones trajectory and T 1ρ preparation (UTE-Cones-T 1ρ ) for quantitative T 1ρ assessment of short T 2 tissues in the musculoskeletal system. A basic 3D UTE-Cones sequence was combined with a spin-locking preparation pulse for T 1ρ contrast. A relatively short TR was used to decrease the scan time, which required T 1 measurement and compensation using 3D UTE-Cones data acquisitions with variable TRs. Another strategy to reduce the total scan time was to acquire multiple Cones spokes (N sp ) after each T 1ρ preparation and fat saturation. Four spin-locking times (TSL = 0-20 ms) were acquired over 12 min, plus another 7 min for T 1 measurement. The 3D UTE-Cones-T 1ρ sequence was compared with a two-dimensional (2D) spiral-T 1ρ sequence for the imaging of a spherical CuSO 4 phantom and ex vivo meniscus and tendon specimens, as well as the knee and ankle joints of healthy volunteers, using a clinical 3-T scanner. The CuSO 4 phantom showed a T 1ρ value of 76.5 ± 1.6 ms with the 2D spiral-T 1ρ sequence, as well as 85.7 ± 3.6 and 89.2 ± 1.4 ms for the 3D UTE-Cones-T 1ρ sequences with N sp of 1 and 5, respectively. The 3D UTE-Cones-T 1ρ sequence provided shorter T 1ρ values for the bovine meniscus sample relative to the 2D spiral-T 1ρ sequence (10-12 ms versus 16 ms, respectively). The cadaveric human Achilles tendon sample could only be imaged with the 3D UTE-Cones-T 1ρ sequence (T 1ρ  = 4.0 ± 0.9 ms), with the 2D spiral-T 1ρ sequence demonstrating near-zero signal intensity. Human studies yielded T 1ρ values of 36.1 ± 2.9, 18.3 ± 3.9 and 3.1 ± 0.4 ms for articular cartilage, meniscus and the Achilles tendon, respectively. The 3D UTE-Cones-T 1ρ sequence allows volumetric T 1ρ measurement of short T 2 tissues in vivo., (Copyright © 2017 John Wiley & Sons, Ltd.)