Patients and tissue acquisition
A total of 17 facet joints from 14 patients with AS (12 male, 2 female; mean age ± standard deviation (SD) 51 ± 8.12 years) undergoing surgical correction of rigid hyperkyphosis were included in the analysis. These joints belonged to stages I–III of AS joint remodeling as previously described by us. Joints without cartilage, due to complete replacement of the joint by trabecular bone, were excluded from this study. Of 14 AS patients, 11 were treated with nonsteroidal anti-inflammatory drugs (NSAIDs), none of the AS patients received disease-modifying antirheumatic drugs or TNF-alpha blocking agents at the time of tissue acquisition. In addition, 22 facet joints from 12 patients with OA (1 male, 11 female; mean age ± SD 69.83 ± 6.01 years) who underwent surgery of the lumbar spine because of neurological deficits in the lower limbs caused by compression of nerve roots were acquired and 11 facet joints of 10 non-AS control patients (4 male, 6 female; mean age ± SD 68.90 ± 14.91 years) who had no history of rheumatic diseases and died of cardiovascular diseases, were removed in toto at autopsy.
All patients gave informed consent to the study. Permission for this study was given by the local ethics committee of the Charité University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany.
Tissue preparation
After acquisition, all joints were fixed, decalcified with ethylenediaminetetraacetic acid and embedded in paraffin as described [4]. Sections 4–6 μm thick were used for stainings. For overview the sections were stained using safranin O/light green.
Immunohistochemistry
For immunohistochemistry, fixed, decalcified tissue sections were deparaffinized in xylene and rehydrated before either heat-induced epitope retrieval with citrate buffer at pH 6.0 for beta-catenin, BMP-2, BMP-7 and wif-1 detection or enzymatic retrieval for sclerostin, DKK-1, Runx2, osteocalcin, sex determining region Y (SRY)-box 9 (Sox9), type II collagen (COL2) and COL10 detection. After blocking of nonspecific binding by serum-free protein block (Dako, Glostrup, Denmark) sections were incubated with the respective primary antibodies overnight at 4 °C. After blocking with an endogenous avidin/biotin-blocking kit (Invitrogen, Paisley, UK), the slides were incubated with species-specific biotinylated immunoglobulin (Dianova, Hamburg, Germany) and alkaline phosphatase streptavidin (Vector Laboratories, Burlingame, CA, USA). Alkaline phosphatase was visualized using Chromogen Red (Dako REAL Detection System Kit, Dako, Glostrup, Denmark) before counterstaining with Meyer’s hematoxylin.
Primary antibodies
Monoclonal mouse antibodies against osteocalcin (clone 190125, dilution 1:100; R&D Systems, Minneapolis, MN, USA), wif-1 (clone 133015, dilution 1:30; R&D Systems, Minneapolis, MN, USA), COL2 (clone II-4C11, dilution 1:75; Acris Antibodies GmbH, Herford, Germany), COL10 (clone X53, dilution 1:25; Quartett, Berlin, Germany), polyclonal rabbit antibodies against beta-catenin (dilution 1:50; Thermo Fisher Scientific, Waltham, MA, USA), DKK-1 (dilution 1:50; Abcam, Cambridge, UK), sclerostin (dilution 1:100; Abcam, Cambridge, UK), Runx2 (dilution 1:40; Quartett, Berlin, Germany), BMP-2 and BMP-7 (dilution 1:20; PeproTech, Rocky Hill, NJ, USA) and polyclonal goat antibodies against MMP13 (dilution 1:20) and Sox9 (dilution 1:20) both obtained from R&D Systems (Minneapolis, MN, USA) were used.
As negative controls, experiments were performed (i) with isotype controls for immunoglobulin G (IgG) (mouse IgG1 DAK-GO1, mouse IgG2a DAK-GO5, mouse IgG2b DAK-GO9 and rabbit IgG all Dako, Glostrup, Denmark) and (ii) by omitting the primary antibodies.
The expression of the respective markers was evaluated by calculating the percentage of positively stained chondrocytes, i.e., the number of positively stained chondrocytes per total number of chondrocytes. The joint sections of AS patients contained 2635 ± 397 chondrocytes (mean ± SD) and as a minimum 337 chondrocytes, joint sections of OA patients 1461 ± 296 chondrocytes (minimum 332 chondrocytes) and sections of autopsy controls (CO) 2294 ± 517 (minimum 589) chondrocytes. The cartilage area was defined beforehand by safranin O staining.
Microscope, camera and software
Immunohistological analysis was performed using an Olympus BX60 microscope (Hamburg, Germany). Pictures were taken with a digital camera (Color View II; Soft Imaging System, Hamburg, Germany) and analyzed using image analysis software (Soft Imaging Software Cell D, Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).
Statistical analysis
Statistical analyses were performed using GraphPad Prism software (GraphPad Prism 5 for Windows, Version 5.01, GraphPad Software Inc., San Diego, CA, USA). Individual measurements and the median are shown in the graphs. For multiple group comparisons the Kruskal-Wallis test and the Dunn’s multiple comparison test as post test were used; a p value less than 0.05 was considered significant. For correlation analysis the Spearman correlation coefficient was calculated.