The effect of cartilage dehydration and rehydration on quantitative ultrashort echo time biomarkers.

Background: The effect of dehydration of ex vivo cartilage samples and rehydration with native synovial fluid or normal saline on quantitative ultrashort echo time (UTE) biomarkers are unknown. We aimed to investigate the effect of cartilage dehydration-rehydration on UTE biomarkers and to compare the rehydration capabilities of native synovial fluid and normal saline. Methods: A total of 37 cartilage samples were harvested from patients (n=5) who underwent total knee replacement. Fresh cartilage samples were exposed to air to dehydrate for 2 hours after baseline magnetic resonance (MR) scanning, then randomly divided into two groups: one soaking in native synovial fluid (n=17) and the other in normal saline (n=20) to rehydrate for 4 hours. UTE-based biomarkers [T1, adiabatic T1r (AdiabT1r), macromolecular fraction (MMF), magnetization transfer ratio (MTR), and T2*] and sample weights were evaluated for fresh, dehydrated, and rehydrated cartilage samples. Differences and agreements between groups were assessed using the values of fresh cartilage samples as reference standard. Results: Dehydrating in air for 2 hours resulted in significant weight loss (P=0.000). T1, AdiabT1r, and T2* decreased significantly while MMF and MTR increased significantly (all P<0.02). Non-significant differences were observed in cartilage weights after rehydrating in both synovial fluid and normal saline, with P values being 0.204 and 0.769, respectively. There were no significant differences in T1, AdiabT1r, MMF, and MTR after rehydrating in synovial fluid (P>0.0167, with Bonferroni correction) while T2* (P=0.001) still had significant differences compared with fresh samples. However, no significant differences were detected for any of the evaluated UTE biomarkers after rehydrating in normal saline (all P>0.05). No differences were detected in the agreement of UTE biomarker measurements between fresh samples and samples rehydrated with synovial fluid and normal saline. Conclusions: Cartilage dehydration resulted in significant changes in UTE biomarkers. Rehydrating with synovial fluid or normal saline had non-significant effect on all the evaluated UTE biomarkers except T2* values, which still had significant differences compared with fresh samples after rehydrating with synovial fluid. No significant difference was observed in the rehydration capabilities of native synovial fluid and normal saline.

Correlation between the elastic modulus of anterior cruciate ligament (ACL) and quantitative ultrashort echo time (UTE) magnetic resonance imaging.

Conventional magnetic resonance imaging (MRI) often acquires no signal in anterior cruciate ligament (ACL) due to the short apparent transverse relaxation time of ACL. Ultrashort echo time (UTE) MRI is capable of imaging ACL with high signal which enables quantitative ACL assessment. This study aimed to investigate the correlations of the mechanical and microstructural properties of human ACL specimens with quantitative three-dimensional UTE Cones (3D-UTE-Cones) MRI measures. ACL specimens were harvested from cadaveric knee joints of 13 (50.9 ± 21.1 years old, 11 males and 2 female) donors. Specimens were scanned using a series of quantitative 3D-UTE-Cones T2 * (UTE-T2 *), T1 (UTE-T1 ), Adiabatic T1ρ (UTE-Adiab-T1ρ ), and magnetization transfer (UTE-MT) sequences in a wrist coil on a clinical 3T scanner. ACL elastic modulus was measured using a uniaxial tensile mechanical test. Histomorphometry analysis was performed to measure the average fascicle specific surface, fascicle size, and number of cells per unit area. Spearman's rank correlations of UTE-MRI biomarkers with mechanical and histomorphometry measures were investigated. The elastic modulus of ACL showed significant moderate correlations with UTE-Adiab-T1ρ (R = -0.59, p = 0.01), macromolecular fraction from MT modeling (R = 0.54, p = 0.01), magnetization transfer ratio (R = 0.53, p = 0.01), UTE-T2* (R = -0.53, p = 0.01), and average fascicle specific surface (R = 0.54, p = 0.01). UTE-MRI showed nonsignificant correlations with histomorphometry measures. UTE-MRI biomarkers may be useful noninvasive tools for the ACL mechanical assessment.

Evaluation of enzymatic proteoglycan loss and collagen degradation in human articular cartilage using ultrashort echo time-based biomarkers: A feasibility study.

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.

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.

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.

High-Density Mineralized Protrusions and Central Osteophytes: Associated Osteochondral Junction Abnormalities in Osteoarthritis.

The aim of this study was to determine the association between high-density mineralized protrusions (HDMPs) and central osteophytes (COs), and describe the varying appearance of these lesions using advanced clinical imaging and a novel histological protocol. Seventeen consecutive patients with clinically advanced knee osteoarthritis undergoing knee arthroplasty were included. Surgical tissues containing the osteochondral region were investigated using computed tomography (CT); a subset was evaluated using confocal microscopy with fluorescence. Tissues from seven subjects (41.2%) contained HDMPs, and tissues from seven subjects (41.2%) contained COs. A significant association between HDMPs and COs was present (p = 0.003), with 6 subjects (35.2%) demonstrating both lesions. In total, 30 HDMPs were found, most commonly at the posterior medial femoral condyle (13/30, 43%), and 19 COs were found, most commonly at the trochlea (5/19, 26.3%). The HDMPs had high vascularity at their bases in cartilaginous areas (14/20, 70%), while the surrounding areas had elevated levels of long vascular channels penetrating beyond the zone of calcified cartilage (p = 0.012) compared to HDMP-free areas. Both COs and HDMPs had noticeable bone-resorbing osteoclasts amassing at the osteochondral junction and in vascular channels entering cartilage. In conclusion, HDMPs and COs are associated lesions in patients with advanced knee osteoarthritis, sharing similar histologic features, including increased vascularization and metabolic bone activity at the osteochondral junction. Future studies are needed to determine the relationship of these lesions with osteoarthritis progression and symptomatology.

Pectoralis major tendon and enthesis: anatomic, magnetic resonance imaging, ultrasonographic, and histologic investigation.

BACKGROUND: This study evaluates the pectoralis major (PM) tendon humeral insertion, using imaging and histologic assessment in cadaveric specimens. Current descriptions of the pectoralis major tendon depict a bilaminar enthesis, and clarification of the anatomy is important for diagnostic and surgical considerations. MATERIALS AND METHODS: Fourteen fresh-frozen whole upper extremity specimens were used in this study. Magnetic resonance (MRI) and ultrasonographic (US) imaging of the PM muscles, tendons, and entheses were performed, followed by anatomic dissection and inspection. Morphology of the lateral tendon and entheses were evaluated, focused on the presence of layers. In 11 specimens, the lateral 3 cm of the PM tendon was carefully dissected from the footprint, whereas in 3 specimens, the tendon and humeral insertion were preserved and removed en bloc. Histology was performed in axial slabs along the medial-lateral length of the tendon and also evaluated for the presence of layers. RESULTS: The superior-inferior and medial-lateral lengths of the PM footprint were 75 ± 9 mm and 7 ± 1 mm respectively. In all specimens, the clavicular and sternal head muscles and tendons were identified, with the clavicular head tendon generally being shorter. The medial-lateral length of the clavicular head tendon measured 19 ± 8 mm superiorly and 9 ± 3 mm inferiorly. The medial-lateral length of the sternal head tendon measured 38 ± 8 superiorly and 41 ± 18 mm inferiorly. All specimens demonstrated a unilaminar, not bilaminar, enthesis with abundant fibrocartilage on histology. Three specimens demonstrated interspersed entheseal fat and loose connective tissue at the enthesis on MRI and histology. CONCLUSION: The PM tendon humeral insertion consists of a unilaminar fibrocartilaginous enthesis. US, MRI, and histology failed to identify true tendon layers at the enthesis. Delaminating injuries reported in the literature may originate from a location other than the enthesis.

Assessment of an in vitro model of rotator cuff degeneration using quantitative magnetic resonance and ultrasound imaging with biochemical and histological correlation.

PURPOSE: Quantitative imaging methods could improve diagnosis of rotator cuff degeneration, but the capability of quantitative MR and US imaging parameters to detect alterations in collagen is unknown. The goal of this study was to assess quantitative MR and US imaging measures for detecting abnormalities in collagen using an in vitro model of tendinosis with biochemical and histological correlation. METHOD: 36 pieces of supraspinatus tendons from 6 cadaveric donors were equally distributed into 3 groups (2 subjected to different concentrations of collagenase and a control group). Ultrashort echo time MR and US imaging measures were performed to assess changes at baseline and after 24 h of enzymatic digestion. Biochemical and histological measures, including brightfield, fluorescence, and polarized microscopy, were used to verify the validity of the model and were compared with quantitative imaging parameters. Correlations between the imaging parameters and biochemically measured digestion were analyzed. RESULTS: Among the imaging parameters, macromolecular fraction (MMF), adiabatic T1ρ, T2*, and backscatter coefficient (BSC) were useful in differentiating between the extent of degeneration among the 3 groups. MMF strongly correlated with collagen loss (r=-0.81; 95% confidence interval [CI]: -0.90,-0.66), while the adiabatic T1ρ (r = 0.66; CI: 0.42,0.81), T2* (r = 0.58; CI: 0.31,0.76), and BSC (r = 0.51; CI: 0.22,0.72) moderately correlated with collagen loss. CONCLUSIONS: MMF, adiabatic T1ρ, and T2* measured and US BSC can detect alterations in collagen. Of the quantitative MR and US imaging measures evaluated, MMF showed the highest correlation with collagen loss and can be used to assess rotator cuff degeneration.

Advanced magnetic resonance imaging of cartilage components in haemophilic joints reveals that cartilage hemosiderin correlates with joint deterioration.

INTRODUCTION: Evidence suggests that toxic iron is involved in haemophilic joint destruction. AIM: To determine whether joint iron deposition is linked to clinical and imaging outcomes in order to optimize management of haemophilic joint disease. METHODS: Adults with haemophilia A or haemophilia B (n = 23, ≥ age 21) of all severities were recruited prospectively to undergo assessment with Hemophilia Joint Health Scores (HJHS), pain scores (visual analogue scale [VAS]) and magnetic resonance imaging (MRI) at 3T using conventional MRI protocols and 4-echo 3D-UTE-Cones sequences for one affected arthropathic joint. MRI was scored blinded by two musculoskeletal radiologists using the International Prophylaxis Study Group (IPSG) MRI scale. Additionally, UTE-T2* values of cartilage were quantified. Correlations between parameters were performed using Spearman rank correlation. Two patients subsequently underwent knee arthroplasty, which permitted linking of histological findings (including Perl's reaction) with MRI results. RESULTS: MRI scores did not correlate with pain scores or HJHS. Sixteen joints had sufficient cartilage for UTE-T2* analysis. T2* values for cartilage correlated inversely with HJHS (rs  = -0.81, P < 0.001) and MRI scores (rs  = -0.52, P = 0.037). This was unexpected since UTE-T2* values decrease with better joint status in patients with osteoarthritis, suggesting that iron was present and responsible for the effects. Histological analysis of cartilage confirmed iron deposition within chondrocytes, associated with low UTE-T2* values. CONCLUSIONS: Iron accumulation can occur in cartilage (not only in synovium) and shows a clear association with joint health. Cartilage iron is a novel biomarker which, if quantifiable with innovative joint-specific MRI T2* sequences, may guide treatment optimization.

Quantitative Ultrasound and B-Mode Image Texture Features Correlate with Collagen and Myelin Content in Human Ulnar Nerve Fascicles.

We investigate the usefulness of quantitative ultrasound and B-mode texture features for characterization of ulnar nerve fascicles. Ultrasound data were acquired from cadaveric specimens using a nominal 30-MHz probe. Next, the nerves were extracted to prepare histology sections. Eighty-five fascicles were matched between the B-mode images and the histology sections. For each fascicle image, we selected an intra-fascicular region of interest. We used histology sections to determine features related to the concentration of collagen and myelin and ultrasound data to calculate the backscatter coefficient (-24.89 ± 8.31 dB), attenuation coefficient (0.92 ± 0.04 db/cm-MHz), Nakagami parameter (1.01 ± 0.18) and entropy (6.92 ± 0.83), as well as B-mode texture features obtained via the gray-level co-occurrence matrix algorithm. Significant Spearman rank correlations between the combined collagen and myelin concentrations were obtained for the backscatter coefficient (R = -0.68), entropy (R = -0.51) and several texture features. Our study indicates that quantitative ultrasound may potentially provide information on structural components of nerve fascicles.

Ultrashort echo time magnetic resonance imaging (UTE-MRI) of cortical bone correlates well with histomorphometric assessment of bone microstructure.

Ultrashort echo time magnetic resonance imaging (UTE-MRI) techniques have been increasingly used to assess cortical bone microstructure. High resolution micro computed tomography (µCT) is routinely employed for validating the MRI-based assessments. However, water protons in cortical bone may reside in micropores smaller than the detectable size ranges by µCT. The goal of this study was to evaluate the upper limit of UTE-MRI and compare its efficacy to µCT at determining bone porosity ex vivo. This study investigated the correlations between UTE-MRI based quantifications and histomorphometric measures of bone porosity that cover all pores larger than 1 µm. Anterior tibial midshaft specimens from eleven donors (51 ±â€¯16 years old, 6 males, 5 females) were scanned on a clinical 3 T-MRI using UTE magnetization transfer (UTE-MT, three power levels and five frequency offsets) and UTE-T2* sequences. Two-pool MT modeling and bi-component exponential T2* fitting were performed on the MRI datasets. Specimens were then scanned by µCT at 9 µm voxel size. Histomorphometry was performed on hematoxylin and eosin (H&E) stained slides imaged at submicron resolution. Macromolecular fraction from MT modeling, bi-component T2* fractions, and short component T2* showed strong correlations (R > 0.7, p < 0.01) with histomorphometric total and large-pores (>40 µm) porosities as well as with µCT-based porosity. UTE-MRI could also assess small pores variations with moderate correlations (R > 0.5, p < 0.01). The UTE-MRI techniques can detect variations of bone porosity comprised of pores below the range detectable by µCT. Such fine pore variations can contribute differently to the development of bone diseases or to the bone remodeling process, however, this needs to be investigated. In scanned specimens, major porosity changes were from large pores, therefore the µCT employment was likely adequate to validate UTE-MRI biomarkers.

Rotator cuff tendon assessment using magic-angle insensitive 3D ultrashort echo time cones magnetization transfer (UTE-Cones-MT) imaging and modeling with histological correlation.

BACKGROUND: Rotator cuff tendons (RCTs) are challenging to image due to the "magic angle effect" and their short T2 . PURPOSE: To assess the degree of magic angle sensitivity of human RCTs and to utilize a 3D ultrashort echo time Cones sequence with magnetization transfer preparation (UTE-Cones-MT) and two-pool quantitative MT modeling with histological correlation. We hypothesized that MT parameters would be less sensitive to the magic angle compared with conventional T2 measurements. STUDY TYPE: Prospective imaging pathologic correlation. SPECIMEN: Twenty cadaveric rotator cuff tendons were imaged at five sample orientations ranging from 0-90° relative to the B0 field. FIELD STRENGTH/SEQUENCE: 3T/3D UTE-Cones-MT and Carr-Purcell-Meiboom-Gill (CPMG). ASSESSMENT: Two-pool quantitative MT modeling parameters and T2 values were calculated in regions of interest drawn by a medical physicist. Histopathological analysis was performed and mild and severe tendinopathy groups were assigned by a histopathologist and histotechnician. STATISTICAL TESTS: Coefficients of variations (CVs) were calculated for measures between the different orientations and group means were compared for each measure. RESULTS: CVs of T2 and macromolecular fractions between orientations were 26.14 ± 16.82% and 6.18 ± 2.77% (mean ± SD), respectively. T2 measurements at 0°, 27°, 70°, and 90° showed significant differences between the two histological groups (P = 0.004, 0.008, 0.003, and 0.015, respectively), but not at 55° (P = 0.611). Mean T2 value ranges between orientations for the mild and severe tendinopathy groups were 15.27-30.32 msec and 20.81-35.85 msec, respectively, showing overlap despite statistically significant differences (P = 0.003). Macromolecular fractions at all angles showed significant differences between the two groups (P < 0.0001). Mean fraction ranges between orientations for the mild and severe tendinopathy groups were 14.32-17.17% and 10.00-13.75% respectively (P < 0.0001) with no overlap. DATA CONCLUSION: Compared with T2 , macromolecular fraction obtained with the 3D UTE-Cones-MT technique is resistant to the magic angle effect and is more sensitive to RCT degeneration. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017.