3D morphometric analysis of calcified cartilage properties using micro-computed tomography.

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (µCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a µ-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA.

3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography.

OBJECTIVE: Histopathological grading of osteochondral (OC) tissue is widely used in osteoarthritis (OA) research, and it is relatively common in post-surgery in vitro diagnostics. However, relying on thin tissue section, this approach includes a number of limitations, such as: (1) destructiveness, (2) sample processing artefacts, (3) 2D section does not represent spatial 3D structure and composition of the tissue, and (4) the final outcome is subjective. To overcome these limitations, we recently developed a contrast-enhanced µCT (CEµCT) imaging technique to visualize the collagenous extracellular matrix (ECM) of articular cartilage (AC). In the present study, we demonstrate that histopathological scoring of OC tissue from CEµCT is feasible. Moreover, we establish a new, semi-quantitative OA µCT grading system for OC tissue. RESULTS: Pathological features were clearly visualized in AC and subchondral bone (SB) with µCT and verified with histology, as demonstrated with image atlases. Comparison of histopathological grades (OARSI or severity (0-3)) across the characterization approaches, CEµCT and histology, excellent (0.92, 95% CI = [0.84, 0.96], n = 30) or fair (0.50, 95% CI = [0.16, 0.74], n = 27) intra-class correlations (ICC), respectively. A new µCT grading system was successfully established which achieved an excellent cross-method (µCT vs histology) reader-to-reader intra-class correlation (0.78, 95% CI = [0.58, 0.89], n = 27). CONCLUSIONS: We demonstrated that histopathological information relevant to OA can reliably be obtained from CEµCT images. This new grading system could be used as a reference for 3D imaging and analysis techniques intended for volumetric evaluation of OA pathology in research and clinical applications.

Pilot study on 18 F-FDG PET/CT for detection of inflammatory changes in blood-induced knee arthropathy in a rabbit model.

RATIONALE: 18 F-FDG-PET/CT has a potential role in the early detection of haemophilic arthritis, at a time when treatment may still avoid further joint degeneration. The purposes of this pilot study were to determine the ability of 18 F-FDG-PET/CT to detect inflammatory changes associated with blood-induced arthropathy in knees of a rabbit model. METHODS: Ten juvenile rabbits were imaged at baseline and weeks 5 and 17 post intraarticular autologous blood injections (ABI). Five rabbits in group 1 (G1) had ABI into the same knee joint every 2 weeks (total, eight injections). Five rabbits in group 2 (G2) had only two injections into the same knee, at weeks 5 and 17. Images were assessed visually and semi-quantitatively by measuring maximal standardized uptake values (SUVmax) and standardized uptake ratio (SUR = SUVmax in affected knee/SUVmax in non-affected knee). RESULTS: More rabbits in G1 than G2 presented with positive chronic inflammatory synovial scores at week 17. Mean iron staining scores in injected knees were greater for G1 than for G2 (P = 0.049). No increased uptake was identified in the injected knees in any of the rabbits at baseline or at week 5. At week 17, all G1 rabbits demonstrated increased uptake in their affected knees with higher mean SUVmax (1.5) than normal knees (1.0) (P < 0.02). None of the G2 rabbits showed asymmetric increased uptake. The SUR of G1 was higher at week 17 compared to baseline (P < 0.01) and week 5 (P < 0.01). The SUR at week 17 was higher for G1 than for G2 (1.13) rabbits (P < 0.01). CONCLUSION: 18 F-FDG-PET is able to detect the inflammatory changes associated with haemophilic arthropathy in this experimental model.

USPIO-related T1 and T2 mapping MRI of cartilage in a rabbit model of blood-induced arthritis: a pilot study.

Ultrasmall paramagnetic iron oxide (USPIO)-enhanced MRI is promising for evaluating inflammation. The aims of this study were to investigate the effect of USPIO on cartilage T1 and T2 mapping, and to evaluate a proposed rapid vs. conventional T2 map method for imaging cartilage in a blood-induced arthritis model. Knees of nine arthritic (induction by intra-articular autologous blood injection) and six control rabbits were imaged over time (baseline, weeks 1, 5, 10) by 1.5 T MRI. All rabbits had USPIO (35-75 µmol Fe/kg)-enhanced MRI at each time point. T1 and T2 (conventional and rapid) maps and signal-to-noise ratios (SNR) were obtained pre- and post-USPIO administration. Cartilage biochemistry and histology were compared with MRI. Excellent correlations were noted between T1 map values and histologic scores at week 10 pre-USPIO (medial, r = 0.93, P = 0.0007; lateral, r = 0.87, P = 0.005) in the arthritic group, but not between T2 map and histology. Marginally and significant differences were observed between pre- and post-USPIO T2 values at weeks 5 (P = 0.06) and 10 (P = 0.02), but only with the administration of high USPIO doses in the arthritic group using the conventional method. No significant differences were noted between pre- and post-USPIO T1 values at any imaging time points. Cartilage T2 maps with short-TR and conventional protocols provided similar T2 values [(decreased trend)] (P > 0.05). Concomitant use of USPIO to T1 and T2 mapping of cartilage would not impair the identification of interval changes of T1 and T2 maps. Rapid T2 map provides similar results compared to conventional method, but its validation warrants further investigation.

Magnetic resonance imaging characterization of osteochondral defect repair in a goat model at 8 T.

OBJECTIVE: This study was performed to non-invasively visualize and characterize osteochondral (OC) repair in ex vivo goat stifles using an 8 T magnetic resonance imaging (MRI) scanner and to compare the MR morphology with images obtained from 1.5 T, gross morphology and histology. METHODS: Mature, neutered male goats were assigned to an 8-week (n = 4) or 16-week (n = 4) study period. Two cylindrical OC defects (7 mm diameter, full cartilage thickness and 1mm into subchondral bone) were surgically created in the right stifle: one in the medial femoral condyle (MFC) and the other in the trochlear groove (TG). The implant matrices (non-woven or foam) were secured in the defect using a bottom anchored fixation device (FD). The contralateral left stifles served as time zero controls. At the day of necropsy, implants were placed at both defect sites (MFC and TG) on the normal left stifles. Following necropsy, the ex vivo goat stifles (intact and encapsulated) were disarticulated. Within 24 h postnecropsy, MR scans of the stifles along the mid-sagittal plane of the OC defect were acquired at 8 T and 1.5 T. MR relaxation times, T1 and T2, were measured at the region of repair tissue (RT) and adjacent native cartilage. Immediately after MR imaging, the stifles were dissected, grossly examined, and a sagittal OC block corresponding to the MR region of interest was prepared for formalin fixation. RESULTS: The high-resolution MR images enabled visualization of cartilage and bone integrity surrounding the implant as well as delineating the margins of RT/implant matrix and the FD. On spin echo sequence, the RT variably appeared as high, intermediate or low MR signal intensity; whereas, the FD always appeared as low signal intensity. In general, the MR signal intensity of 8-week RT was slightly higher compared to 16-week RT; however, there was no difference in RT morphology of stifles implanted with the non-woven matrix or foam matrix. Subchondral sclerosis appeared as low signal intensity. The 8 T MR images showed better delineation of the stifle tissues compared to the images acquired at 1.5 T. The T2 relaxation time of the RT appears to indicate (inconclusive due to small number of samples) a slight variation in the RT type between 8 weeks and 16 weeks. At both study times, the defects grossly appeared whitish to reddish but did not have the characteristic hyaline appearance typical of articular cartilage (AC). The gross appearance of the MFC and TG RT differed, which was predominantly mottled and recessed with fissuring of adjacent native AC in the MFC. Histologically, the RT of both 8-week and 16-week postsurgical defects predominantly comprised fibrovascular connective tissue with only few samples showing the presence of fibrocartilaginous and/or hypertrophic chondrocytes within the defect RT at 8 weeks. Also, compared to 8-week, the 16-week RT appeared to be more fibrotic. CONCLUSION: Using 8 T scanner, high-resolution MR images of ex vivo encapsulated goat stifles confirmed the capability of high-field MR imaging to distinguish the defect RT from the FD and adjacent joint tissues. The extent of OC repair and adjacent bone lesions (at 8 weeks and 16 weeks) observed in the MR images compared well with those observed on the corresponding histological sections.

MRI of the cartilaginous epiphysis of the femoral head in the piglet hip after ischemic damage.

MRI of the cartilaginous epiphysis (CE) of piglet femoral head was performed after ischemic damage to study the changes in the CE found on MRI and to correlate these changes with histologic findings. Avascular necrosis of the femoral head was induced with a suture ligature in nine piglets; one piglet was killed postoperatively on day 3 and on weeks 1, 2, 3, 4, 6, 7, and 8 (two piglets were killed on week 3). MRI of the ischemic and contralateral nonischemic hip joints were obtained with a 60-mm field of view (low resolution MRI). Biopsy cores of the femoral heads were imaged with a 15-mm field of view (high resolution MRI) and correlated with histologic sections. The CE for all operated hips, except for the 3-day postoperative specimen, showed evidence of ischemic changes on histologic assessment; the severity of damage increased with time. The MRI appearance of ischemic and nonischemic CE was clearly different by 2 weeks after the operation. No trilaminar signal pattern was evident in the high resolution T2-weighted (T2W) imaging of the ischemic CE from 2 weeks after the operation. In the 3- to 8-week postoperative specimens, focal areas of low signal intensity on high resolution T1-weighted (T1W) and T2W imaging corresponded to the areas of chondronecrosis found on histologic assessment. The regions of high signal intensity on T2W imaging corresponded to the areas of chondrocyte clusters with increased safranin-O staining. High resolution MRI can demonstrate changes in the CE associated with ischemic injury and may have a role in the assessment of the CE and its development after ischemic injury.

High-resolution magnetic resonance imaging of normal porcine cartilaginous epiphyseal maturation.

The aim of this study was to determine whether high-resolution magnetic resonance (MR) imaging could differentiate epiphyseal and articular cartilage in the cartilaginous epiphysis and demonstrate its developmental changes. T1- and T2-weighted (T1W and T2W) spin-echo sequences at 50-mm field of view (FOV) of hip joints were obtained from 14 piglets (newborn to 6 months). Subsequently, high-resolution MR images (15-mm FOV) of a biopsy core of the proximal femoral cartilaginous epiphysis were correlated with histology. Newborn cartilaginous epiphysis demonstrated homogeneous signal intensity on T1W and T2W imaging with abundant cartilage canals. From 2 weeks of age, the cartilaginous epiphysis showed a diminution of cartilage canals, with three zones evident on T2W imaging consisting of a low-signal middle zone separating two higher signal zones. Histologic evaluation demonstrated four distinct morphologic laminas with a decrease in overall cartilage thickness with age. The laminas were not as well defined in the newborn compared with the older piglets. No simple correlation was found between the MR zonal pattern and the morphological laminas on histology. No distinct demarcation between the articular cartilage and epiphyseal cartilage was present. MR can visualize cartilage canals and demonstrate changes in the cartilaginous epiphysis that occur with maturation. What component of the cartilaginous epiphysis that accounts for the MR differences seen between newborn and older piglets remains unclear.

Imaging of immature articular cartilage using ultrasound backscatter microscopy at 50 MHz.

A high frequency sonographic technique-ultrasound backscatter microscopy-was used to visualize the subsurface structure of immature porcine articular cartilage from the knee joint. In 20-week-old pigs, all parts that were scanned, except the weight-bearing regions of the femoral condyles, demonstrated heterogeneous ultrasound backscatter characteristics within the articular cartilage. A trilaminar pattern consisting of hypoechoic, hyperechoic, and anechoic layers ranging from superficial to deep generally was observed, except in the weight-bearing regions of the femoral condyles, where a homogeneous anechoic pattern was seen. In the younger pigs (6 and 10 weeks old), the trilaminar backscatter pattern was not observed. Small, highly echogenic structures that correlated with vascular channels in histologic assessment were observed frequently in the cartilage of younger pigs, but they were seldom present in the cartilage of 20-week-old pigs. Structural details, such as disruption of the subchondral bone and presence of a thickened fibrous layer on the articular surface at the chondrosynovial junction, also were detected with the ultrasound backscatter microscope. We concluded that high frequency ultrasound can be used to visualize the subsurface structure of immature articular cartilage and some of its developmental changes. Further research is required to explain the mechanism underlying the observed backscatter characteristics of immature articular cartilage and to study its potential for the imaging of pathologic changes.

Osteoarthritis staging: comparison between magnetic resonance imaging, gross pathology and histopathology in the rhesus macaque.

Although osteoarthritis (OA) is the most common cause of articular skeletal disability in humans, assessing progression (staging) with noninvasive methods remains a major clinical problem. Using the rhesus macaque animal model, the objective of this study was to compare OA staging by noninvasive magnetic resonance imaging (MRI) against gross pathology and histopathology. Right knee joints from 18 rhesus macaques were used in this study. Using a four-point ordinal scale for each of the above-mentioned modalities, the lateral and medial femoral condyle and tibial plateau of each knee joint was independently scored for OA severity, i.e. normal, mild OA, moderate OA and severe OA. Correlation between each staging system was performed using Stuart's Tau-c correlation coefficient. By our criteria, MRI staging correlated as well with gross pathology (tau = 0.75) and histopathology (tau = 0.80) as did gross pathology with histopathology (tau = 0.78). Our study shows that MRI is a promising noninvasive modality to evaluate the severity of OA. MRI appears to be sensitive for demarcating the presence and extent of focal OA cartilage lesions. However, at this time, while MRI is sensitive for detecting OA change it cannot distinguish between certain lesions such as superficial cartilage matrix fibrillation and hypertrophy both of which show elevated signal intensity.

Osteoarthritis in rhesus macaque knee joint: quantitative magnetic resonance imaging tissue characterization of articular cartilage.

OBJECTIVE: To assess cartilage matrix quality variation by anatomical location and extent of osteoarthritis (OA) using quantitative magnetic resonance imaging (MRI) and to compare the anatomic MR morphologic features with corresponding histological findings. METHODS: We studied 18 fully encapsulated right knee joints from a population of rhesus monkeys with a high incidence of degenerative arthritis resembling human OA. Relaxation times (T1 and T2) spin density, and cartilage thickness were determined along 8 contiguous anteroposterior segments of articular cartilage. Histological slides, prepared in the same plane as the MR image, were assessed for OA severity. Using a modification of Mankin's OA classification, each quadrant was grouped into normal (0), mild (1), moderate (2), or severe OA (3). Histopathological scores served as the standard and corresponding MR quadrants were classified accordingly. RESULTS: Cumulative results revealed a significant decrease in T1 relaxation time (p = 0.04) and an increase in T2 relaxation time (p = 0.03) in the mild and severe OA groups, respectively. Statistically significant changes in spin density and cartilage thickness measurements were not observed. MR signal intensity abnormalities in selected regions of interest were demarcated and studied histologically. Regions with histological proliferating chondrocytes or fibrillated cartilage showed bright signal intensity on MR images (TR = 3000 ms; TE = 10 ms) and corresponded with elevated T1 and T2 values. Histological regions of collagen condensation showed low signal intensity on MR images (TR = 3000 ms; TE = 10 ms) and corresponded with decreased T1 and T2 relaxation times. CONCLUSION: Topological quantitative MRI relaxation time assessment demonstrates increasing cartilage matrix quality variation with OA progression.

Analysis of collagens solubilized from cartilage of normal and spontaneously osteoarthritic rhesus monkeys.

Osteoarthritis (OA) is a disorder which results in the destruction of the articular cartilage and the remodeling of the subchondral bone in synovial joints. We have analyzed the cartilage collagen from normal and osteoarthritic free-ranging rhesus monkeys from the Cayo Santiago colony. The cartilage samples were assigned a severity score based on histological staging system and were divided into four groups (normals, mild OA, moderate OA and severe OA). After a 4.0 M guanidinium chloride (GuCl) extraction, the remainder of the cartilage was digested with pepsin and the collagen was salt precipitated at 2.5 M and 4.3 M NaCl. The GuCl solubility of the osteoarthritic cartilage increased compared to normals. Collagen extractability by GuCl also increased with the severity of disease. Pepsin digestion followed by salt precipitation shows that collagen from rhesus osteoarthritis cartilage is more easily extracted than from normal cartilage. With an anti-type I collagen antibody we have detected the presence of type I collagen in the severe OA cartilage samples but not in the milder OA groups or in normal cartilage. Total collagen content decreases with severity of OA, which is not due to changes in propyl hydroxylation because examination of collagen hydroxylation, based on hydroxyproline analysis, shows no difference between OA and normal cartilage.

Osteoarthritis in rhesus macaques: assessment of cartilage matrix quality by quantitative magnetic resonance imaging.

To assess cartilage quality with anatomical location and extent of osteoarthritis (OA), magnetic resonance imaging (MRI) of the knee joint from rhesus monkeys was performed. MRI measurements--T1 and T2 relaxation times, proton density and cartilage thickness were taken from 8 equal segments of articular cartilage volumes extending from anterior to posterior edge of 1 mm sagittal slices. To grade the severity of OA in each quadrant of the knee joint a modified Mankin score was applied on the histological sections. Our results suggest that MR relaxation times and proton density values vary with the severity of osteoarthritis.