Achilles tendon and enthesis assessment using ultrashort echo time magnetic resonance imaging (UTE-MRI) T1 and magnetization transfer (MT) modeling in psoriatic arthritis.

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.

Correlations between elastic modulus and ultrashort echo time (UTE) adiabatic T1ρ relaxation time (UTE-Adiab-T1ρ) in Achilles tendons and entheses.

Patients with psoriatic arthritis commonly have abnormalities of their entheses, which are the connections between tendons and bone. There are shortcomings with the use of conventional magnetic resonance imaging (MRI) sequences for the evaluation of entheses and tendons, whereas ultrashort echo time (UTE) sequences are superior for the detection of high signals, and can also be used for non-invasive quantitative assessments of these structures. The combination of UTE-MRI with an adiabatic-T1ρ preparation (UTE-Adiab-T1ρ) allows for reliable assessment of entheses and tendons with decreased susceptibility to detrimental magic angle effects. This study aimed to investigate the relationship between quantitative UTE-MRI measures and the biomechanical properties of Achilles tendons and entheses. In total, 28 tendon-enthesis sections were harvested from 11 fresh-frozen human cadaveric foot-ankle specimens (52 ± years old). Tendon-enthesis sections were scanned using the UTE-Adiab-T1ρ and UTE-T1 sequences on a clinical 3 T scanner. MRI-based measures and indentation tests were performed on the enthesis, transitional, and tensile tendon zones of the specimens. Hayes' elastic modulus showed significant inverse correlations (Spearman's) with UTE-Adiab-T1ρ in all zones (R= - 0.46, - 0.54, and - 0.61 in enthesis, transition, and tensile tendon zones, respectively). Oliver-Pharr's elastic modulus showed significant inverse correlations with UTE-Adiab-T1ρ in transition (R= - 0.52) and tensile tendon zone (R=- 0.60). UTE-T1 did not show significant correlations with the elastic modulus. UTE-MRI and elastic modulus were significantly lower in the tensile tendon compared with the enthesis regions This study highlights the potential of the UTE-Adiab-T1ρ technique for the non-invasive evaluation of tendons and enthuses.

Correction: Lombardi et al. AcidoCEST-UTE MRI Reveals an Acidic Microenvironment in Knee Osteoarthritis. Int. J. Mol. Sci. 2022, 23, 4466.

In the original publication, there was a mistake in Figure 1 as published [...].

Correction of B0 and linear eddy currents: Impact on morphological and quantitative ultrashort echo time double echo steady state (UTE-DESS) imaging.

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.

Synthetic CT in Musculoskeletal Disorders: A Systematic Review.

ABSTRACT: Repeated computed tomography (CT) examinations increase patients' ionizing radiation exposure and health costs, making an alternative method desirable. Cortical and trabecular bone, however, have short T2 relaxation times, causing low signal intensity on conventional magnetic resonance (MR) sequences. Different techniques are available to create a "CT-like" contrast of bone, such as ultrashort echo time, zero echo time, gradient-echo, and susceptibility-weighted image MR sequences, and artificial intelligence. This systematic review summarizes the essential technical background and developments of ultrashort echo time, zero echo time, gradient-echo, susceptibility-weighted image MR imaging sequences and artificial intelligence; presents studies on research and clinical applications of "CT-like" MR imaging; and describes their main advantages and limitations. We also discuss future opportunities in research, which patients would benefit the most, the most appropriate situations for using the technique, and the potential to replace CT in the clinical workflow.

Ultrashort echo time magnetic resonance imaging of the osteochondral junction.

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.

Accelerated Quantitative 3D UTE-Cones Imaging Using Compressed Sensing.

In this study, the feasibility of accelerated quantitative Ultrashort Echo Time Cones (qUTE-Cones) imaging with compressed sensing (CS) reconstruction is investigated. qUTE-Cones sequences for variable flip angle-based UTE T1 mapping, UTE adiabatic T1ρ mapping, and UTE quantitative magnetization transfer modeling of macromolecular fraction (MMF) were implemented on a clinical 3T MR system. Twenty healthy volunteers were recruited and underwent whole-knee MRI using qUTE-Cones sequences. The k-space data were retrospectively undersampled with different undersampling rates. The undersampled qUTE-Cones data were reconstructed using both zero-filling and CS reconstruction. Using CS-reconstructed UTE images, various parameters were estimated in 10 different regions of interests (ROIs) in tendons, ligaments, menisci, and cartilage. Structural similarity, percentage error, and Pearson's correlation were calculated to assess the performance. Dramatically reduced streaking artifacts and improved SSIM were observed in UTE images from CS reconstruction. A mean SSIM of ~0.90 was achieved for all CS-reconstructed images. Percentage errors between fully sampled and undersampled CS-reconstructed images were below 5% for up to 50% undersampling (i.e., 2× acceleration). High linear correlation was observed (>0.95) for all qUTE parameters estimated in all subjects. CS-based reconstruction combined with efficient Cones trajectory is expected to achieve a clinically feasible scan time for qUTE imaging.

Imaging of Paget's Disease of Bone.

Paget's disease is a metabolic bone disorder affecting the elderly and characterized by bone resorption followed by compensatory bone formation. Radiography is the imaging modality of choice for the diagnosis whereas bone scintigraphy helps stage the extent of the disease and assess response to treatment. MRI and CT are important imaging methods in the assessment of complications and surgical planning. Osteolytic lesions of Paget's first phase present with well-defined margins on radiographs, most commonly in the femur, pelvis, and skull. Cortical thickening, trabecular coarsening, bone marrow sclerosis, and deformities of long bones are present in the mixed- and late-sclerotic phases.

Lower Macromolecular Content in Tendons of Female Patients with Osteoporosis versus Patients with Osteopenia Detected by Ultrashort Echo Time (UTE) MRI.

Tendons and bones comprise a special interacting unit where mechanical, biochemical, and metabolic interplays are continuously in effect. Bone loss in osteoporosis (OPo) and its earlier stage disease, osteopenia (OPe), may be coupled with a reduction in tendon quality. Noninvasive means for quantitatively evaluating tendon quality during disease progression may be critically important for the improvement of characterization and treatment optimization in patients with bone mineral density disorders. Though clinical magnetic resonance imaging (MRI) sequences are not typically capable of directly visualizing tendons, ultrashort echo time MRI (UTE-MRI) is able to acquire a high signal from tendons. Magnetization transfer (MT) modeling combined with UTE-MRI (i.e., UTE-MT-modeling) can indirectly assess macromolecular proton content in tendons. This study aimed to determine whether UTE-MT-modeling could detect differences in tendon quality across a spectrum of bone health. The lower legs of 14 OPe (72 ± 6 years) and 31 OPo (73 ± 6 years) female patients, as well as 30 female participants with normal bone (Normal-Bone, 36 ± 19 years), are imaged using UTE sequences on a 3T MRI scanner. Institutional review board approval is obtained for the study, and all recruited subjects provided written informed consent. A T1 measurement and UTE-MT-modeling are performed on the anterior tibialis tendon (ATT), posterior tibialis tendon (PTT), and the proximal Achilles tendon (PAT) of all subjects. The macromolecular fraction (MMF) is estimated as the main measure from UTE-MT-modeling. The mean MMF in all the investigated tendons was significantly lower in OPo patients compared with the Normal-Bone cohort (mean difference of 24.2%, p < 0.01), with the largest Normal-Bone vs. OPo difference observed in the ATT (mean difference of 32.1%, p < 0.01). Average MMF values of all the studied tendons are significantly lower in the OPo cohort compared with the OPe cohort (mean difference 16.8%, p = 0.02). Only the PPT shows significantly higher T1 values in OPo patients compared with the Normal-Bone cohort (mean difference 17.6%, p < 0.01). Considering the differences between OPo and OPe groups with similar age ranges, tendon deterioration associated with declining bone health was found to be larger than a priori detected differences caused purely by aging, highlighting UTE-MT MRI techniques as useful methods in assessing tendon quality over the course of progressive bone weakening.

Myelin water imaging using a short-TR adiabatic inversion-recovery (STAIR) sequence.

PURPOSE: To develop a new myelin water imaging (MWI) technique using a short-TR adiabatic inversion-recovery (STAIR) sequence on a clinical 3T MR scanner. METHODS: Myelin water (MW) in the brain has both a much shorter T1 and a much shorter T2 * than intracellular/extracellular water. A STAIR sequence with a short TR was designed to efficiently suppress long T1 signals from intracellular/extracellular water, and therefore allow selective imaging of MW, which has a much shorter T1 . Numerical simulation and phantom studies were performed to investigate the effectiveness of long T1 signal suppression. TheT2 * in white matter (WM) was measured with STAIR and compared with T2 * measured with a conventional gradient recall echo in in vivo study. Four healthy volunteers and 4 patients with multiple sclerosis were recruited for qualitative and quantitative MWI. Apparent MW fraction was generated to compare MW in normal WM in volunteers to MW in lesions in patients with multiple sclerosis. RESULTS: Both simulation and phantom studies showed that when TR was sufficiently short (eg, 250 ms), the STAIR sequence effectively suppressed long T1 signals from tissues with a broad range of T1 s using a single TR/TI combination. The volunteer study showed a short T2 * of 9.5 ± 1.7 ms in WM, which is similar to reported values for MW. Lesions in patients with multiple sclerosis showed a significantly lower apparent MW fraction (4.5% ± 1.0%) compared with that of normal WM (9.2% ± 1.5%) in healthy volunteers (p < 0.05). CONCLUSIONS: The STAIR sequence provides selective MWI in brain and can quantify reductions in MW content in patients with multiple sclerosis.

AcidoCEST-UTE MRI Reveals an Acidic Microenvironment in Knee Osteoarthritis.

A relationship between an acidic pH in the joints, osteoarthritis (OA), and pain has been previously demonstrated. Acidosis Chemical Exchange Saturation Transfer (acidoCEST) indirectly measures the extracellular pH through the assessment of the exchange of protons between amide groups on iodinated contrast agents and bulk water. It is possible to estimate the extracellular pH in the osteoarthritic joint using acidoCEST MRI. However, conventional MR sequences cannot image deep layers of cartilage, meniscus, ligaments, and other musculoskeletal tissues that present with short echo time and fast signal decay. Ultrashort echo time (UTE) MRI, on the other hand, has been used successfully to image those joint tissues. Here, our goal is to compare the pH measured in the knee joints of volunteers without OA and patients with severe OA using acidoCEST-UTE MRI. Patients without knee OA and patients with severe OA were examined using acidoCEST-UTE MRI and the mean pH of cartilage, meniscus, and fluid was calculated. Additionally, the relationship between the pH measurements and the Knee Injury and Osteoarthritis Outcome Score (KOOS) was investigated. AcidoCEST-UTE MRI can detect significant differences in the pH of knee cartilage, meniscus, and fluid between joints without and with OA, with OA showing lower pH values. In addition, symptoms and knee-joint function become worse at lower pH measurements.

Comprehensive assessment of in vivo lumbar spine intervertebral discs using a 3D adiabatic T1ρ prepared ultrashort echo time (UTE-Adiab-T1ρ) pulse sequence.

BACKGROUND: T1ρ has been extensively reported as a sensitive biomarker of biochemical changes in the nucleus pulposus (NP) and annulus fibrosis of intervertebral discs (IVDs). However, no T1ρ study of cartilaginous endplates (CEPs) has yet been reported because the relatively long echo times (TEs) of conventional clinical T1ρ sequences cannot effectively capture the fast-decaying magnetic resonance signals of CEPs, which have very short T2/T2*s. This can be overcome by using ultrashort echo time (UTE) T1ρ acquisitions. METHODS: Seventeen subjects underwent UTE with adiabatic T1ρ preparation (UTE-Adiab-T1ρ) and T2-weighted fast spin echo imaging of their lumbar spines. Each IVD was manually segmented into seven regions (i.e., outer anterior annulus fibrosis, inner anterior annulus fibrosis, outer posterior annulus fibrosis, inner posterior annulus fibrosis, superior CEP, inferior CEP, and NP). T1ρ values of these sub-regions were correlated with IVD modified Pfirrmann grades and subjects' ages. In addition, T1ρ values were compared in subjects with and without low back pain (LBP). RESULTS: Correlations of T1ρ values of the outer posterior annulus fibrosis, superior CEP, inferior CEP, and NP with modified Pfirrmann grades were significant (P<0.05) with R values of 0.51, 0.36, 0.38, and -0.94, respectively. Correlations of T1ρ values of the outer anterior annulus fibrosis, outer posterior annulus fibrosis, and NP with ages were significant with R equal to 0.52, 0.71, and -0.76, respectively. T1ρ differences of the outer posterior annulus fibrosis, inferior CEP, and NP between the subjects with and without LBP were significant (P=0.005, 0.020, and 0.000, respectively). CONCLUSIONS: The UTE-Adiab-T1ρ sequence can quantify T1ρ of whole IVDs including CEPs. This is an advance, and of value for comprehensive assessment of IVD degeneration.

Bleeding with iron deposition and vascular remodelling in subchondral cysts: A newly discovered feature unique to haemophilic arthropathy.

INTRODUCTION: Joint iron accumulation is the incendiary factor triggering osteochondral destruction, synovial hypertrophy, inflammation, and vascular remodelling in haemophilic arthropathy (HA). Hemosiderin depositions have been described in synovium and, more recently, in cartilage. Clinical observations also suggest hemosiderin accumulation in subchondral cysts, implying cyst bleeding. AIM: We explored associations between cystic iron accumulation, vascular remodelling and HA status to determine if cystic bleeding may contribute to HA progression. METHODS: Thirty-six haemophilic joints (16 knees, 10 ankles, and 10 elbows; 31 adult patients with haemophilia A/B) were evaluated by magnetic resonance imaging (MRI) for subchondral cysts and hemosiderin. Cyst score (WORMS) and hemosiderin presence were compared between haemophilic and osteoarthritic knees, matched for the degree of arthritis (Kellgren-Lawrence score). Cystic iron accumulation, vascular remodelling and macrophage cell counts were also compared by immunohistochemistry in explanted joint tissues. In haemophilic knees, cyst number and extent of hemosiderin deposition were correlated with haemophilia joint health scores (HJHS). RESULTS: Cystic hemosiderin was detected in 78% of haemophilic joints. Cyst score and presence of hemosiderin were significantly higher in haemophilic compared to osteoarthritic knees. Cyst score and presence of hemosiderin strongly correlated with HJHS. Moreover, iron deposition and vascular remodelling were significantly more pronounced within cysts in haemophilic compared to osteoarthritic knees, with similar total cell and macrophage count. CONCLUSION: These findings suggest the presence of subchondral bleeding in haemophilia, contributing to poor joint health outcomes. Observations of bleeding into osseous structures are novel and should inform investigations of new therapies.

Knee Cartilage Imaging.

Articular cartilage injury and degeneration represent common causes of knee pain, which can be evaluated accurately and noninvasively using MRI. This review describes the structure of cartilage focusing on its histologic appearance to emphasize that structure will dictate patterns of tissue failure as well as MR appearance. In addition to identifying cartilage loss, MRI can demonstrate signal changes that correspond to intrinsic structural abnormalities which place the cartilage at risk for subsequent more serious injury or premature degeneration, allowing for earlier intervention and treatment of important causes of pain and morbidity.

High contrast cartilaginous endplate imaging using a 3D adiabatic inversion-recovery-prepared fat-saturated ultrashort echo time (3D IR-FS-UTE) sequence.

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.

High-contrast osteochondral junction imaging using a 3D dual adiabatic inversion recovery-prepared ultrashort echo time cones sequence.

While conventional MRI sequences cannot visualize tissues from the osteochondral junction (OCJ) due to these tissues' short transverse T2 /T2 * relaxations, ultrashort echo time (UTE) sequences can overcome this limitation. A 2D UTE sequence with a dual adiabatic inversion recovery preparation (DIR-UTE) for selective imaging of short T2 tissues with high contrast has previously been developed, but high sensitivity to eddy currents and aliased out-of-slice excitation make it difficult to image the thin layer of the OCJ in vivo. Here, we combine the DIR scheme with a 3D UTE cones sequence for volumetric imaging of OCJ tissues in vivo, aiming to generate higher OCJ contrast compared with a recently developed single IR-prepared UTE sequence with a fat saturation module (IR-FS-UTE). All sequences were implemented on a 3-T clinical scanner. The DIR-UTE cones sequence combined a 3D UTE cones sequence with two narrow-band adiabatic IR preparation pulses centered on water and fat spectrum frequencies, respectively. The 3D DIR-UTE cones sequence was first applied to a phantom, then to the knees of four healthy volunteers and four patients diagnosed with osteoarthritis and compared with the IR-FS-UTE sequence. In both phantom and volunteer studies, the proposed DIR-UTE cones sequence showed much higher contrast for OCJ imaging than the IR-FS-UTE sequence did. The 3D DIR-UTE cones sequence showed a significantly higher contrast-to-noise ratio between the OCJ and subchondral bone fat (mean, standard deviation [SD]: 25.7 ± 2.3) and between the OCJ and superficial layers of cartilage (mean, SD: 22.2 ± 3.5) compared with the IR-FS-UTE sequence (mean, SD: 10.8 ± 2.5 and 16.3 ± 2.6, respectively). The 3D DIR-UTE cones sequence is feasible for imaging of the OCJ region of the knee in vivo and produces both high resolution and high contrast.

AcidoCEST MRI Evaluates the Bone Microenvironment in Multiple Myeloma.

PURPOSE: Multiple myeloma (MM) is an incurable disease of malignant plasma cells in the bone marrow (BM). Adaptive responses to hypoxia may be an essential element in MM progression and drug resistance. This metabolic adaptation involves a decrease in extracellular pH (pHe), and it depends on the upregulation of glucose transporters (GLUTs) that is common in hypoxia and in cancer cells. CEST MRI is an imaging technique that assesses pHe indirectly by the exchange rate of magnetic saturation transfer between labile protons on a solute and water. Thus, this study aimed to determine the feasibility of acidoCEST MRI for pHe measurement using an orthotopic mouse model of MM compared with GLUT1 immunofluorescence staining as a reference. PROCEDURES: Orthotopic BM engrafted MM xenografts were established in NSG/NOD mice using the human RPMI8226 myeloma cell line. AcidoCEST MRI was performed approximately 6 weeks after intravenous challenge, before and after intravenous administration of iopamidol. BM pHe values were generated via fitting the CEST spectrum with the Bloch-McConnell equations. Samples were decalcified, sectioned, and immunostained for GLUT1 expression. Pearson's correlation was used to assess the relationship between pHe and [H3O+] versus GLUT1 expression. RESULTS: Ten mice underwent acidoCEST MRI followed by immunofluorescent histologic analysis. A strong negative correlation was seen between pHe versus GLUT1 expression (r = - 0.75, p < 0.001). After transformation of pH to [H3O+], a strong positive correlation between [H3O+] and GLUT1 expression was observed (r = 0.8, p < 0.001). CONCLUSIONS: AcidoCEST MRI can measure the extracellular pH of bone marrow affected by multiple myeloma. In this MM orthotopic mouse model, pHe measured by acidoCEST MRI showed strong correlations with the metabolic phenotype of BM tumor assessed by immunofluorescent histological assessment of GLUT1 overexpression.

Novel fluorescent staining protocol for thick sections of human osteochondral tissues to facilitate correlation with MRI and CT.

OBJECTIVE: To describe a novel fluorescent histochemical protocol to visualize osteoclasts, vasculature, and nerves in thick sections of human osteochondral tissues and to demonstrate its feasibility for use in radiologic-pathologic research correlation studies. MATERIALS AND METHODS: Consecutive patients scheduled for total knee arthroplasty surgeries underwent pre-operative MRI. CT imaging was performed after tissue collection, and abnormal osteochondral regions were sectioned to 1-2 mm in thickness and decalcified. Fluorescent labeling of osteoclasts was performed by staining for tartrate-resistant alkaline phosphatase activity with a fluorescent substrate. Vascular structure was visualized with fluorescently labeled lectin Ulex europaeus Agglutinin I (UEA-I). Immunostaining was performed for proteins including smooth muscle actin expressed in smooth muscle cells surrounding arterioles and fibrotic myofibroblasts, as well as for neuropeptide Y expressed in sympathetic nerves. Sections were then recut at 5 µm and stained with hematoxylin and eosin (H&E). RESULTS: Edema-like and cyst-like regions identified with MRI and CT were easily located in fluorescent images and appeared to have increased osteoclast activity. Fibrotic regions were identified with thickened arterioles and increased myofibroblasts. Sympathetic nerve fibers traveled alongside arborizing blood vessels. Stained sections became transparent in a water-based refractive index-matched medium, permitting deep 3D visualization of the elaborate neurovascular network in bone. Sequential staining procedures were successfully performed with the same sections, demonstrating the potential to compare multiple cellular markers from the same locations. Routine H&E staining could be performed after the fluorescent staining protocol. CONCLUSION: We have developed a multimodal framework to facilitate comparisons between histology and clinical MRI and CT.