Comparing intra-articular CTXII levels assessed via magnetic capture or lavage in a rat knee osteoarthritis model E.G. Yarmola, Y.Y. Shah, H.E. Kloefkorn,

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Comparing intra-articular CTXII levels assessed via magnetic capture or lavage in a rat knee osteoarthritis model E.G. Yarmola, Y.Y. Shah, H.E. Kloefkorn, J. Dobson, K.D. Allen Osteoarthritis and Cartilage Volume 25, Issue 7, Pages 1189-1194 (July 2017) DOI: 10.1016/j.joca.2017.01.009 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 CTXII in OA-affected and contralateral joints as measured by magnetic capture and lavage: in lavage, CTXII concentration was elevated in MMT-operated knees relative to contralateral knees at 1 week (P = 0.002), but lavage did not identify significant changes in CTXII at week 4 (P = 1.000). In magnetic capture, no significant difference was observed at 1 week (P = 1.000), but a significant increase was found in the OA-affected joint relative to the contralateral joint at 4 weeks (P = 0.016). Serum did not show changes in CTXII levels relative to historical values from rats of a similar age from our group9. While magnetic capture did not detect significant elevation of CTXII at week 1, five of the six rats assessed via magnetic capture had higher CTXII levels in the OA-affected joint relative to the contralateral limb. Moreover, one point in the contralateral knee in the magnetic capture group in Week 1 was identified as a potential outlier, well outside the 1.5× interquartile range fence. No differences were observed in CTXII levels between 1 week and 4 week in either the lavage or magnetic capture cohorts. Statistical differences between groups in CTXII were assessed via a 1-way nonparametric Kruskal Wallis ANOVA with a protected Mann–Whitney U post-hoc test (a statistical test of the differences between medians). Data are presented as a scatterplot, with a line indicating the median value. Osteoarthritis and Cartilage 2017 25, 1189-1194DOI: (10.1016/j.joca.2017.01.009) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Mixing of saline and synovial fluid is challenging: a demonstrative bench top experiment with a silicone tube imitating a joint illustrates the challenges of mixing saline and synovial fluid. Here, saline containing Evan's blue dye is injected into a tube containing bovine synovial fluid (clear). The tube is closed from both sides with hemostats and is then squeezed or flexed ten times. Panels A and B show that squeezing and flexing could not appropriately mix the saline with synovial fluid. Panel C demonstrates that aggressive mixing using 10 s of vortexing also fails to mix the two fluids. Osteoarthritis and Cartilage 2017 25, 1189-1194DOI: (10.1016/j.joca.2017.01.009) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 1 Study design: a total of thirty-two male Lewis rats underwent an MMT surgery. The first cohort (n = 16) was euthanized 1 week after surgery and the second cohort (n = 16) was euthanized 4 weeks post operation. At both time points, the CTXII levels were assessed in both the ipsilateral and contralateral limbs using either lavage or magnetic capture immediately after euthanasia. Due to an inability to aspirate lavage fluid (dry tap), only six MMT knees at 1 week, six contralateral knees at 1 week, and seven contralateral knees at 4 weeks could be analyzed for CTXII in the lavage group. For magnetic capture, one animal was humanely euthanized due to unintended complications from the MMT surgery, and one probe was mislabeled (human error), resulting in only six MMT and six contralateral samples analyzed in the 1 week cohort. Osteoarthritis and Cartilage 2017 25, 1189-1194DOI: (10.1016/j.joca.2017.01.009) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Supplemental Table I Histological scores according to the OARSI histopathology assessment scheme for the rat: differences between groups in histological scores were assessed via a 2-way ANOVA with a Tukey's HSD post-hoc test. Data are presented as mean ± 95% confidence intervals with associated P-values from the Tukey's HSD post-hoc test. Osteoarthritis and Cartilage 2017 25, 1189-1194DOI: (10.1016/j.joca.2017.01.009) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions