Increased chondrocyte sclerostin may protect against cartilage degradation in osteoarthritis
1Chan BY, 1Fuller ES, , 1Smith SS, 3Cake MA, 3Read RA, 2Bateman JF, 1Sambrook PN, 1Little CB

1Raymond Purves Bone and Joint Research Labs, Kolling Institute of Medical Research, Institute of Bone and Joint Research, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW 2065.
2Murdoch Childrens Research Institute, Parkville, Victoria 3052, Australia.
3School of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA 6150, Australia

Running Title: Sclerostin in osteoarthritic cartilage (no more than 40 characters).
Full length manuscript has to have extran blank line at the end of heading and paragraph, 2.0 used for reverse transcription (RT). Changes in mRNA expression were quantified using real time qRT-PCR as described (29) using specific primer sets (Table 1), corrected for total RNA and expressed as fold change from control. .

Statistical analysis
Throughout the text, data is reported as the mean with 95% confidence intervals (lower, upper). Ordinal data from the histological scoring is presented graphically in box plots showing median, upper and lower quartile (box), 10-90th percentile (whiskers) and maximum and minimum values. Changes in the number of positively stained cells, and the fold change in gene expression or GAG release compared with control or untreated samples are presented graphically as mean with 95% confidence intervals. Because some of the data (e.g. gene expression) was not normally distributed, all treatment effects were assessed using non-parametric analysis (Mann-Whitney U test) with P < 0.05 considered significant. Results
Sclerostin is increased in cartilage but decreased in subchondral bone in OA
Medial meniscectomy induced focal OA change in the sheep, including loss of proteoglycan, cartilage surface fibrillation and partial thickness erosion, and marginal osteophytosis (Fig 1A). Cartilage changes were restricted to the region previously protected by the meniscus that is subjected to high focal loading in meniscectomised joints (Area 2 – histopathological score 3.2 (2.4,3.9) for sham vs 10.4 (8.6,12.2) for MEN; p=0.0????), with no change in Area 1 (Fig 1B). In association with the overlying cartilage damage there was increased thickness and density of subchondral bone in Area 2 in meniscectomised joints (Fig 1C, 80.1% (74.1,86.1) for sham vs 92.8% (86.4,99.2) for MEN, p = 0.?????).

Sclerostin was immunolocalised to osteocytes and their canaliculi in the subchondral plate (Fig 1D) and deeper trabecular bone (not shown) in sham-operated joints. There was no difference in the number of SOST-positive osteocytes between Area 1 and 2 in sham-operated joints, although both were greater than the trabecular bone (Fig 1E; Area 1 = 71.0% (66.8,77.2), Area 2 = 67.9% (65.1,70.7) vs trabecular bone 29.5% (15.9,43), p = 0.0??? and 0.0??, respectively). There was little SOST staining of chondrocytes in the non-calcified cartilage in sham-operated joints (Fig 1D), while a few hypertrophic chondrocytes in the calcified cartilage were positive (not shown). In meniscectomised joints there was a loss of osteocyte SOST staining intensity in the subchondral bone of Area 2 but little change in Area 1 (Fig 1D) or the trabecular bone (not shown). The percentage of SOST-positive osteocytes did not change in trabecular bone but was decreased in meniscectomised versus sham-operated joints (Fig 1E) in Area 1 (MEN = 57.9% (49.5,66.3), p = 0.0???) and Area 2 (MEN = 46.2% (32.7,59.8), p=0.0???) such that Area 2 had fewer SOST-positive osteocytes than Area 1 (p = 0.????). In contrast to the bone, there was a marked focal increase in SOST staining intensity and the number of positive chondrocytes in the non-calcified cartilage in Area 2 in meniscectomised joints (Fig 1D and 1E; 15.0% (7.2,22.9) for sham vs 42.3% (23.1,61.4) for MEN, p=0.0???). There was no change in chondrocyte SOST