The study protocol has been reviewed and approved by the Ethics Committee of Northwestern Switzerland (Number 147/12). Written consent was obtained from all patients. Facet joint specimens were obtained from patients suffering from symptomatic stenosis of the lumbar spine for >6 months. These patients underwent single-level decompression and fusion surgery (L1-S1 levels). In all cases the routine transforaminal lumbar intercorporal fusion (TLIF) technique was applied. In all nineteen patients (70 ± 14 years, 11 female) the processus articularis superior of the dissected facet joint (T10 n=1, L2 n=2, L3 n=3, L4 n=8, L5 n=5) was collected and transported in saline solution for immediate processing for histology (n=10, 5 female) or explant cultures (n=9, 6 female). All patients reported pain radiating down at least one leg and self-assessed walking distance varied between 20-1000 meters. Pain medication included paracetamol (n=1), metamizole (n=1), conventional NSAIDs (n=4), opioids (n=4), or none (n=9).

MRI of the lumbar spine was performed on a 1.5-T scanner using a standard protocol comprising, T1-, T2- and fat-saturated T2-weighted sequences. MRI images were evaluated twice by a senior spine consultant with 15 years of experience in reading MRI of the spine. FJOA was graded T1- and T2-weighted sequences in sagittal and axial planes using the Weishaupt grading system (12). Grade 1 = joint space narrowing (JSN) and/or small osteophytes and/or mild hypertrophy of articular processes. Grade 2 = JSN and/or moderate osteophytes and/or moderate hypertrophy of the articular process and/or mild subarticular bone erosions. Grade 3 = JSN and/or large osteophytes and/or severe hypertrophy of the articular process and/or severe subarticular bone erosions and/or subchondral cysts. Synovitis was defined as T2 hyperintensity in and adjacent to facet joints in axial fat-saturated T2-weighted images. Severity of lumbar spinal stenosis was graded on T2-weigthed axial images according to Schizas (13), grade A = no or minor stenosis, B = moderate stenosis, C = severe stenosis and D = extreme stenosis.

The medial portion of the processus articularis superior comprising the facet joint and capsular tissue (n=10, 68 ± 16 years, 5 female) was collected by partial facetectomy and formalin-fixed at 4°C for 2 days. Specimens were decalcified in 5% formic acid for 5-7 days, rinsed in PBS twice for 1 hour and embedded in paraffin. Cartilage degeneration and proteoglycan loss was assessed on 6 μm sagittal sections stained with Safranin-O/Fast Green. Quantitative assessment of Safranin-O staining was performed using ImageJ (version 1.53c, National Institutes of Health, USA) by measuring area fraction on tresholded images after color deconvolution (Masson/Trichrome filter). Regions of interest (ROIs) were placed on cartilage tissue only.

After deparaffinization, tissue sections were subsequently incubated in 3% H₂O₂ for 10 minutes and antibody diluent for IHC for 45 minutes (Catalogue no. 559148, BD Pharmingen, Eysins, Switzerland) to block endogenous peroxidase activity and non-specific antibody binding, respectively. Consecutive 6 μm tissue sections were incubated overnight at 4°C in antibody diluent with the following antibodies: monoclonal mouse anti-TrkA (Clone OTI5B6, dilution 1:150, Novus Biologicals, Centennial, CO, USA), monoclonal rabbit anti-NGF (Clone EP1320Y, dilution 1:250, Abcam, Cambridge, UK), mouse monoclonal anti-SP (Clone 266815, dilution 1:50, Novus Biologicals). Macrophage staining was performed after heat-mediated antigen retrieval (10 mM citrate buffer pH 6.0 at 70°C for 25 min) using monoclonal mouse anti-CD68 (Clone KP1, dilution 1:100, Zytomed Systems, Berlin, Germany) for 1 hour at RT. Immunoreactivities were visualized using mouse- and rabbit-specific peroxidase kits according to the manufacturer’s instructions (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Negative controls were performed with isotype control antibodies ( Supplementary Table and Supplementary Figure 1 ). Stained tissue sections were counterstained with Papanicolaou (Merck AG, Dietikon, Switzerland). NGF area fraction in cartilage tissue was quantified on tresholded images after color deconvolution (Hematoxylin/DAB filter) using ImageJ.

Dissected facet joint specimens (n=9, 72 ± 12 years, 6 female) were processed aseptically and cut in two equal-sized coronal 2 mm samples using a scalpel and cultured as previously described (14). Briefly, specimens were cultured for 7 days at 37˚C in 8 mL αMEM medium (10% fetal bovine serum, 10 mM HEPES, 4 mM L-glutamine, 50 μM L-ascorbic acid-2-phosphate and 10 mM sodium β-glycerophosphate pentahydrate, 10^-7 M dexamethasone) in the presence and absence of 1 μg/mL lipopolysaccharide from Escherichia coli O111:B4 (Sigma-Aldrich, Buochs, Switzerland). LPS treatment was added on day 1 and 4. Supernatants were collected, centrifuged, aliquoted and stored at -80˚C until ELISA measurement. Concentrations of human interleukin-6 (IL-6), NGF and SP were determined in the supernatants using ELISA kits (Abcam ab178013, ab99986 and ab133029, respectively). Secreted protein levels were normalized for wet tissue weight and expressed as pg protein/mg tissue.

Statistical analyses were performed using GraphPad Prism (v6.2, Graphpad Software Inc., San Diego, CA, USA). Data following normal distribution were reported as means ± SD and non-parametric data as median with interquartile range. Significant differences between treatment groups were calculated using ratio paired t-test. Association between quantitative histological assessments and MRI-based severity grades were determined using Spearman’s rank correlation. P-values less than 0.05 were considered significant.

Lumbar facet joint specimens were obtained from patients with moderate to severe radiologic OA ( Figure 1A ), indicated by a median Weishaupt grade of 2 [IQR: 1.75–3] ( Table 1 ). Safranin-O-stained tissue sections displayed hallmarks of severe cartilage degeneration including surface damage, fissures and loss of proteoglycan content ( Figure 1B ). NGF expression was detected in nine out of ten FJOA specimens, predominantly in cartilage (n=8) and occasionally in marrow tissue (n=2) ( Table 1 ). Microscopic analysis of NGF expression and proteoglycan distribution in consecutive tissue sections revealed that NGF localized almost exclusively to damaged regions of cartilaginous tissue ( Figure 1C ). High magnification views showed NGF localization in the extracellular matrix as well as pericellular chondrocyte staining ( Figure 1D ). Correlation analyses demonstrated that NGF area fraction did not significantly correlate with the extent of proteoglycan loss (r=0.25, p=0.50, Figure 2A ) or Weishaupt grade (r= –0.46, p=0.15, Figure 2B ). Radiologic OA severity was negatively correlated with the cartilage proteoglycan content in Safranin-O-stained cartilaginous tissue (r= –0.69, p=0.02, Figure 2C ). Presence of synovitis was positively correlated with NGF area fraction (r= 0.78, p= 0.03), but not with radiological severity (r= –0.09, p= 0.38) or Safranin-O area fraction (r= –0.26, p= 0.28).

Next, we analysed distribution of NGF, its high affinity receptor TrkA and downstream neuropeptide SP in consecutive tissue sections. TrkA expression was detected in neither chondrocytes, nor extracellular matrix of cartilaginous tissues. SP was expressed in seven out of eight specimens displaying cartilaginous NGF expression. Extracellular staining patterns for SP and NGF were largely overlapping in consecutive tissue sections ( Figure 3A ). In contrast to cartilaginous tissue, TrkA and SP displayed respectively abundant and focal expression in subchondral bone marrow cells in eight out of ten specimens. CD68+ macrophages showed a focal expression pattern in all specimens. Specimens displaying NGF expression in bone marrow (n=2) revealed abundant cellular expression ( Figure 3B ).

Finally, we sought to determine whether NGF and SP are secreted by FJOA specimens under basal and inflammatory conditions. For this, fresh osteochondral explants were left either untreated or stimulated with 1 μg/mL LPS (n=9 patients, n=18 explants). This treatment activates tissue macrophages and mimicks inflammatory signalling induced by damage-associated molecular patterns in joint tissues (15). As a positive control, LPS challenge led to four-fold elevated tissue secretion of IL-6 compared with untreated specimens ( Figure 4A ). Low level NGF secretion was detected in explants from four patients (n=2 untreated, n=2 LPS-treated) ( Figure 4B ). In contrast, SP was not detected in FJOA explant-conditioned medium (data not shown).