3D analysis and grading of calcifications from ex vivo human meniscus.

OBJECTIVE: Meniscal calcifications are associated with the pathogenesis of knee osteoarthritis (OA). We propose a micro-computed tomography (µCT) based 3D analysis of meniscal calcifications ex vivo, including a new grading system. METHOD: Human medial and lateral menisci were obtained from 10 patients having total knee replacement for medial compartment OA and 10 deceased donors without knee OA (healthy references). The samples were fixed; one subsection was imaged with µCT, and the adjacent tissue was processed for histological evaluation. Calcifications were examined from the reconstructed 3D µCT images, and a new grading system was developed. To validate the grading system, meniscal calcification volumes (CVM) were quantitatively analyzed and compared between the calcification grades. Furthermore, we estimated the relationship between histopathological degeneration and the calcification severity. RESULTS: 3D µCT images depict calcifications in every sample, including diminutive calcifications that are not visible in histology. In the new grading system, starting from grade 2, each grade results in a CVM that is 20.3 times higher (95% CI 13.3-30.5) than in the previous grade. However, there was no apparent difference in CVM between grades 1 and 2. The calcification grades appear to increase with the increasing histopathological degeneration, although histopathological degeneration is also observed with small calcification grades. CONCLUSIONS: 3D µCT grading of meniscal calcifications is feasible. Interestingly, it seems that there are two patterns of degeneration in the menisci of our sample set: 1) with diminutive calcifications (calcification grades 1-2), and 2) with large to widespread calcifications (calcification grades 3-5).

Quantitative three-dimensional collagen orientation analysis of human meniscus posterior horn in health and osteoarthritis using micro-computed tomography.

OBJECTIVE: Knee osteoarthritis (OA) is associated with meniscal degeneration that may involve disorganization of the meniscal collagen fiber network. Our aims were to quantitatively analyze the microstructural organization of human meniscus samples in 3D using micro-computed tomography (µCT), and to compare the local microstructural organization between OA and donor samples. METHOD: We collected posterior horns of both medial and lateral human menisci from 10 end-stage medial compartment knee OA patients undergoing total knee replacement (medial & lateral OA) and 10 deceased donors without knee OA (medial & lateral donor). Posterior horns were dissected and fixed in formalin, dehydrated in ascending ethanol concentrations, treated with hexamethyldisilazane (HMDS), and imaged with µCT. We performed local orientation analysis of collagenous microstructure in 3D by calculating structure tensors from greyscale gradients within selected integration window to determine the polar angle for each voxel. RESULTS: In donor samples, meniscus bundles were aligned circumferentially around the inner border of meniscus. In medial OA menisci, the organized structure of collagen network was lost, and main orientation was shifted away from the circumferential alignment. Quantitatively, medial OA menisci had the lowest mean orientation angle compared to all groups, -24° (95%CI -31 to -18) vs medial donor and -25° (95%CI -34 to -15) vs lateral OA. CONCLUSIONS: HMDS-based µCT imaging enabled quantitative analysis of meniscal collagen fiber bundles and their orientations in 3D. In human medial OA menisci, the collagen disorganization was profound with overall lower orientation angles, suggesting collagenous microstructure disorganization as an important part of meniscus degradation.

Proteomic comparison of osteoarthritic and reference human menisci using data-independent acquisition mass spectrometry.

OBJECTIVE: Recent research in knee osteoarthritis (OA) highlights the role of the meniscus in OA pathology. Our aim was to compare the proteomes of medial and lateral menisci from end-stage medial compartment knee OA patients, with reference menisci from knee-healthy deceased donors, using mass spectrometry. DESIGN: Tissue plugs of Ø3 mm were obtained from the posterior horns of the lateral and medial menisci from one knee of 10 knee-healthy deceased donors and 10 patients undergoing knee replacement. Proteins were extracted and prepared for mass spectrometric analysis. Statistical analysis was conducted on abundance data that was log2-transformed, using a linear mixed effects model and evaluated using pathway analysis. RESULTS: We identified a total of 835 proteins in all samples, of which 331 were included in the statistical analysis. The largest differences could be seen between the medial menisci from OA patients and references, with most proteins showing higher intensities in the medial menisci from OA patients. Several matrix proteins, e.g., matrix metalloproteinase 3 (MMP3) (4.3 times higher values [95%CI 1.8, 10.6]), TIMP1 (3.5 [1.4, 8.5]), asporin (4.1 [1.7, 10.0]) and versican (4.4 [1.8, 10.9]), all showed higher abundance in medial menisci from OA patients compared to medial reference menisci. OA medial menisci also showed increased activation of several pathways involved in inflammation. CONCLUSION: An increase in protein abundance for proteins such as MMP and TIMP1 in the medial menisci from OA patients suggests simultaneous activation of both catabolic and anabolic processes that warrants further attention.

Three-dimensional microstructure of human meniscus posterior horn in health and osteoarthritis.

OBJECTIVE: To develop and perform ex vivo 3D imaging of meniscus posterior horn microstructure using micro-computed tomography (µCT), and to compare specimens from healthy references against end-stage osteoarthritis (OA) using conventional section-based histology and qualitative µCT. DESIGN: We retrieved human medial and lateral menisci from 10 deceased donors without knee OA (healthy references) and medial and lateral menisci from 10 patients having total knee replacement for medial compartment OA. Meniscal posterior horns were dissected and fixed in formalin. One subsection underwent hexamethyldisilazane (HMDS) treatment and µCT imaging. Pauli's histopathological scoring was performed for 3 other subsections. The differences in histopathological scores were estimated using mixed linear regression, resulting in fixed effects estimates for within-knee comparisons and adjusted for age and body mass index for between-subjects comparisons. RESULTS: 3D visualization with µCT qualitatively revealed similar microstructural changes in the posterior horns as conventional histology. The mean histopathological score was higher for medial menisci from OA knees vs both medial reference menisci (mean difference [95% CI], 3.9 [2.6,5.3]), and lateral menisci from OA knees (3.9 [2.9,5.0]). The scores were similar between lateral menisci from OA knees and lateral reference menisci (0.8 [-0.6,2.2]), and between medial and lateral reference menisci (0.8 [-0.3,1.9]). CONCLUSIONS: HMDS-based µCT protocol allows unique 3D visualization of meniscus microstructures. Posterior horns of medial menisci from medial compartment OA knees had higher histopathological scores than both the lateral posterior horns from the same OA knees and medial reference menisci, suggesting a strong association between meniscus degradation and unicompartmental knee OA.

Osteoarthritis development related to cartilage quality-the prognostic value of dGEMRIC after anterior cruciate ligament injury.

OBJECTIVE: Rupture of the anterior cruciate ligament (ACL) increases the risk of developing osteoarthritis (OA). Delayed Gadolinium enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) investigates cartilage integrity through T1-analysis after intravenous contrast injection. A high dGEMRIC index represents good cartilage quality. The main purpose of this prospective cohort study was to investigate the prognostic value of the dGEMRIC index regarding future knee OA. METHOD: 31 patients with ACL injury (mean age 27 ± 6.7 (±SD) years, 19 males) were examined after 2 years with 1.5T dGEMRIC of femoral cartilage. Re-examination 14 years post-injury included weight-bearing knee radiographs, Lysholm and Knee Osteoarthritis Outcome Score (KOOS). RESULTS: At the 14-year follow up radiographic OA (ROA) was present in 68% and OA symptoms (SOA) in 42% of the injured knees. The dGEMRIC index of the medial compartment was lower in knees that developed medial ROA, 325 ± 68 (ms±SD) vs 376 ± 47 (51 (7-94)) (difference of means (95% confidence interval (CI))), in patients that developed symptomatic OA (SOA), 327 ± 61 vs 399 ± 42 (52 (11-93)), and poor knee function 337 ± 54 vs 381 ± 52 (48 (7-89)) compared to those that did not develop ROA, SOA or poor function. The dGEMRIC index correlated negatively with the OARSI osteophyte score in medial (r = -0.44, P = 0.01) and lateral (r = -0.38, P = 0.03) compartments. CONCLUSION: The associations between a low dGEMRIC index and future ROA, as well as SOA, are in agreement with previous studies and indicate that dGEMRIC has a prognostic value for future knee OA.

Ultra-high field magnetic resonance imaging parameter mapping in the posterior horn of ex vivo human menisci.

OBJECTIVE: To investigate the relationship between meniscus magnetic resonance (MR) relaxation parameters and meniscus degradation through quantitative imaging of ex vivo posterior horns of menisci from subjects with and without knee osteoarthritis (OA). DESIGN: We sampled medial and lateral menisci from ten medial compartment knee OA patients (mean age 63 years) undergoing total knee replacement and from ten deceased donors (references, mean age 51 years). MR relaxation parameters T2*, T2 and T1 of the posterior horn were measured at a 9.4 T scanner. Comparisons were made between OA patients and references (with adjustment for age) as well as between medial and lateral menisci from the same knees. RESULTS: Mean values (standard deviation) of mean T2* were 13 (3.8), 6.9 (2.3), 7.2 (1.9) and 7.2 (1.7) ms for the medial and lateral patient menisci and the medial and lateral reference menisci, respectively. Corresponding values were 17 (3.7), 9.0 (2.2), 12 (4) and 9.0 (1.3) ms for T2 and 1810 (150), 1630 (30), 1580 (90) and 1560 (50) ms for T1. All three relaxation times were significantly longer in medial OA menisci compared to the other groups. Among medial reference menisci, relaxation times (mainly T1) tended to increase with age. CONCLUSIONS: MR relaxation times T2*, T2 and T1 in the posterior horn are longer in the medial menisci of patients with end-stage medial compartment knee OA compared to the corresponding lateral menisci and to reference menisci. The meniscus seems to undergo intrasubstance alterations related to both OA and ageing.

Association between delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and joint space narrowing and osteophytes: a cohort study in patients with partial meniscectomy with 11 years of follow-up.

OBJECTIVE: To examine the association between the relaxation time (T1Gd) of delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) and grade of tibiofemoral joint space narrowing (JSN) and osteophytosis 11 years later, in a cohort of meniscectomized patients. DESIGN: Patients (n = 45) aged 35-50 who had undergone an arthroscopic partial medial meniscectomy 1-6 years earlier, due to degenerative meniscal tear, were examined using dGEMRIC. These patients had no cartilage changes defined as deep clefts or visible bone at the time of arthroscopy. Eleven years later (12-16 years after surgery) 34 of these subjects (76%) were evaluated by weight-bearing knee radiography, and tibiofemoral joint changes were graded according to the Osteoarthritis Research Society International Atlas. RESULTS: Lower T1Gd in the medial compartment was associated with higher grade of medial JSN (grade 0, 351 ms; grade 1, 386 ms; grade 2, 342 ms; grade 3, 259 ms [P for trend < 0.001]) and more osteophytosis (score 0, 371 ms; score 1, 389 ms; score 2, 354 ms; score 3, 289 ms; score 4, 265 ms; score 5, 275 ms [P for trend = 0.001]). Lower T1Gd in the lateral compartment was associated with higher grade of lateral JSN (grade 0, 436 ms; grade 1, 346 ms [P for trend = 0.026]). CONCLUSION: The current study suggests that lower T1Gd measured with dGEMRIC of medial and lateral femoral cartilage is associated with higher grade of JSN 11 years later, and medially, also with more osteophytosis.

Longitudinal assessment of femoral knee cartilage quality using contrast enhanced MRI (dGEMRIC) in patients with anterior cruciate ligament injury--comparison with asymptomatic volunteers.

OBJECTIVE: In this observational longitudinal study we estimate knee joint cartilage glycosaminoglycan (GAG) content, in patients with an acute anterior cruciate ligament (ACL) injury, with or without a concomitant meniscus injury. METHODS: 29 knees (19 men/10 women) were prospectively examined by repeat delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), approximately 3 weeks and 2.3±1.3 (range 4.5) years after the injury. We estimated the GAG content (T1Gd) in the central weight-bearing parts of the medial and lateral femoral cartilage and compared results with a reference cohort (n=24) with normal knees and no history of injury examined by dGEMRIC at one occasion previously. RESULTS: The healthy reference group had longer T1Gd values compared with the ACL-injured patients at follow-up both medially: 428±38 vs 363±61ms (P<0.0001) and laterally: 445±41 vs 396±48ms (P=0.0002). At follow-up T1Gd was lower in meniscectomized patients compared to those without a meniscectomy, both medially (-84ms, P=0.002) and laterally (-38ms, P=0.05). In the injured group, the medial femoral cartilage showed similar T1Gd at the two dGEMRIC investigations: 357±50 vs 363±61ms (P=0.57), whereas the lateral femoral cartilage T1Gd increased: 374±48 vs 396±48ms (P=0.04). CONCLUSIONS: The general decrease in cartilage T1Gd in ACL-injured patients compared with references provide evidence for structural matrix GAG changes that seem more pronounced if a concomitant meniscal injury is present. The fact that post-traumatic OA commonly develops in ACL-injured patients, in particularly those with meniscectomy, suggests that shorter T1Gd may be an early biomarker for OA.

Relationship between cartilage glycosaminoglycan content (assessed with dGEMRIC) and OA risk factors in meniscectomized patients.

OBJECTIVE: To study the relationship between cartilage integrity, assessed with [delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC)] and epidemiologic risk factors for knee osteoarthritis (OA) in meniscectomized patients. METHODS: Body mass index (BMI) was calculated in 45 patients (16 women), mean age 46, who underwent an arthroscopic medial meniscectomy 1-6 years earlier. The cartilage glycosaminoglycan (GAG) content was estimated by dGEMRIC Index and tests of isokinetic muscle strength and functional performance (one-leg hop test) were conducted. RESULTS: BMI ranged from 20.0 to 34.3 (mean: 26.5). The dGEMRIC Index was 14.4% lower in the medial index compartment (374+/-61 ms, mean+/-SD) than in the lateral reference compartment (437+/-59 ms, mean+/-SD) (P<0.001). The dGEMRIC Index of the medial diseased compartment correlated positively with both knee flexor (r=0.50, P=0.001) and knee extensor strength (r=0.47, P=0.001) relative to body weight and with the one-leg hop test (r=0.42, P=0.004). Furthermore, a negative correlation was found between the dGEMRIC Index of the medial compartment and BMI (r=-0.35, P=0.019). No significant correlations were found in the lateral reference compartment. CONCLUSION: The lower dGEMRIC Index of the medial compartment suggests decreased cartilage GAG content after medial meniscectomy, indicating an early stage OA. Furthermore, results suggest that overweight is a factor that deteriorates cartilage, whereas strong and co-ordinated thigh muscles may have a protective effect on the cartilage integrity.

Delayed gadolinium-enhanced MRI of cartilage (dGEMRIC): intra- and interobserver variability in standardized drawing of regions of interest.

PURPOSE: To establish the reproducibility of a standardized region of interest (ROI) drawing procedure in delayed gadolinium-enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC). MATERIAL AND METHODS: A large ROI in lateral and medial femoral weight-bearing cartilage was drawn in images of 12 healthy male volunteers by 6 investigators with different skills in MRI. The procedure was done twice, with a 1-week interval. Calculated T1-values were evaluated for intra- and interobserver variability. RESULTS: The mean interobserver variability for both compartments ranged between 1.3% and 2.3% for the 6 different investigators without correlation to their experience in MRI. Post-contrast intra-observer variability was low in both the lateral and the medial femoral cartilage, 2.6% and 1.5%, respectively. The larger variability in lateral than in medial cartilage was related to slightly longer and thinner ROIs. CONCLUSION: Intra-observer variability and interobserver variability are both low when a large standardized ROI is used in dGEMRIC. The experience of the investigator does not affect the variability, which further supports a clinical applicability of the method.