Femur

Bone marrow cells were collected from the femurs and tibiae of wild-type BALB/c mice (Taconic) by flushing the opened bones with Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen). Red blood cells were lysed in dH₂O followed by the addition of 10X PBS. After centrifugation, the cells were washed once in PBS containing 0.1% BSA. The bone marrow cells were cultured at 10⁶/mL in media containing RPMI 1640 (Invitrogen) with 20% FBS (Cambrex), 100 IU/mL penicillin and 10 μg/mL streptomycin (Cellgro), 2 mM glutamine (Invitrogen), 25 mM HEPES and 1x non-essential amino acids and 1 mM sodium pyruvate (Gibco) and 50 μM β-mercaptoethanol (Sigma) and supplemented with 100 ng/mL stem-cell factor (SCF; PeproTech) and 100 ng/mL FLT3-Ligand (FLT3-L; PeproTech) from day 0 to day 4. On day 4, the media containing SCF and FLT3-L was replaced with media containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5; R&D Systems) only. On day 8, the cells were moved to new flasks and maintained in fresh media supplemented with rmIL-5. Every other day, from this point forward, one-half of the media was replaced with fresh media containing rmIL-5, and the concentration of the cells was adjusted each time to10⁶ /mL. Cells were enumerated at day 0 and on days indicated thereafter in a hemocytometer, and 50,000 cells were subjected to cytospin (Thermo Shandon, Pittsburgh, PA). The cytospin preparations were fixed and stained using a modified Giemsa preparation (Diff Quik, Dade Behring AG, Dudingen, Switzerland).

Several methodological comparisons have been made regarding quantitative radio graphic data generated by the OAI (Supplementary Table 2 online). The findings emphasize, for example, the need to take radio-anatomic alignment of OAI fixed-flexion radiographs into account when analyzing change in JSW, and the need for central radiographic readings. Regarding semiquantitative scoring of articular tissue pathology using MRI images, two existing systems—WORMS (whole organ MRI score) and BLOKS (Boston Leeds osteoarthritis knee score)—were applied to a sample of images of 113 knees with radiographic OA and at risk of progression, from the OAI cohort. Both methods were shown to be reliable cross-sectionally (Supplementary Table 2 online). Longitudinally, BLOKS was found to be superior to WORMS for assessment of change in the meniscus, and WORMS was superior to BLOKS for scoring bone-marrow lesions (BMLs), in terms of predicting cartilage loss.^{4 (link)} A new hybrid method (MOAKS; MRI OA knee score) was hence proposed with the aim of combining the advantages of each scoring system.^{5 (link)} In assessing which sequence is better to detect such changes, more and larger focal cartilage defects and BMLs were detected with the intermediate-weighted fat-suppressed spin echo sequence than with DESS^{6 ,7 (link)} (Figure 2, Supplementary Table 2 online).
Semi-automated segmentation algorithms for quantitative measurement of cartilage, bone, meniscus, and thigh muscles (Figure 3) have been assessed. These studies have used different image analysis approaches and have reported, in part, the level of agreement with manual segmentation and/or the level of inter-observer reliability (Figure 1, Supplementary Table 2 online).
The sensitivity to change of cartilage thick ness over 1 year in the medial femorotibial compartment was found to be similar between sagittal DESS, coronal multiplanar reconstructed DESS, and coronal FLASH in 80 knees (Figure 2), with SRMs ranging from −0.34 to −0.38.^{8 (link)} The three protocols were also highly intercorrelated cross-sectionally (coefficient of correlation [r] ≥0.94); analysis of every second 0.7 mm DESS image provided similar sensitivity to change as analysis of every image.^{8 (link)} Change in the medial weight-bearing femur substantially exceeded that in the posterior aspect of the femoral condyle, suggesting that structural progression is faster in (commonly) weight-bearing regions of the joint.^{9 (link)}Measuring between-group differences using cartilage subregions (Figure 4) or atlases of cartilage thickness within anatomically defined cartilage plates has also been explored by several groups, alongside assessing whether such methods improve sensitivity to change (Supplementary Table 3 online). These studies generally identified the central subregion of the weight-bearing medial femoral cartilage plate as the region of interest with the greatest rate of cartilage loss and sensitivity to change (Figure 4).