Preparation of notochordal cell conditioned medium (NCCM)
We have reported on the use of NCCM extensively in our past publications [17, 21, 25]. In the current study, notochordal cell-rich IVD NPs were obtained from 10 different NCD dogs within an age range of 12–18 months in collaboration with a licensed animal facility and all practices were in accordance with the animal care policies and ethics approval board of The University Health Network, Toronto, Ontario, Canada. Briefly, after humane euthanasia, the T6-L6 spinal segments were removed aseptically en bloc and a wide laminectomy performed separating the posterior elements from the vertebral bodies. Next, we removed the NPs in accordance with our established methods and developed NCCM by culturing the NPs in advanced Dulbeccos’s modified Eagle’s medium F/12 (ADMEM/F12) supplemented with 2 % fetal bovine serum (FBS) (v/v) and penicillin/streptomycin (PS) (100 U/mL) [18]. We collected the NCCM at 24-hour intervals for 5 days and at each interval the NCCM was centrifuged for 20 minutes at 3000 g and filtered using 0.2-μm filters (Corning, USA). Next the media were transferred to 15-mL aliquots and frozen at –80̊ C. Consistent with our established methods once the media were collected, filtered and centrifuged they were pooled in order to remove any batch-to-batch variability and all culture conditions were performed with identical conditioned medium. We generated 2 % FBS (v/v)-supplemented ADMEM/F12 as the control medium (ctr).
Healthy chondrocyte culture
Cartilage sample collection, and cell isolation and expansion
In the first experiments cartilage tissue was harvested from nine donors (18–68 years of age), from macroscopically healthy-looking areas of their knee joints. Two samples were taken from an 18-year-old man and a 68-year-old man during diagnostic arthroscopy and seven samples were taken from cadavers within 24 hours after donors had deceased (age range 41–64 years). Either patients or relatives gave informed consent for tissue harvest. All tissue samples were minced with a scalpel into small pieces that were then digested overnight in 0.2 % collagenase II (300 U/mg, Worthington Biochemical Corp, Lakewood, NJ, USA) in an orbital shaker at 37 °C. The isolated chondrocytes were then expanded for two passages in basal medium (BM, DMEM, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/mL streptomycin, and 0.29 mg/mL glutamate (all from Invitrogen)) supplemented with 10 % FBS, 5 ng/mL fibroblast growth factor-2 (FGF-2) and 1 ng/mL transforming growth factor (TGF)ß1 in a humidified incubator (37 °C, 5 % CO2, 19 % oxygen) as previously described [26].
Chondrogenic differentiation in pellets
Chondrogenic differentiation was induced by culturing the expanded chondrocytes in pellets using defined serum-free medium as previously described [27]. Briefly, cells were re-suspended in chondrogenic medium (BM, 1.25 mg/mL human serum albumin, ITS-A (Invitrogen), 10 ng/mL TGF-ß1 (R&D Systems), and 10-7 M dexamethasone and 0.1 mM ascorbic acid 2-phosphate (Sigma-Aldrich). Aliquots of 2.5 × 105 cells/250 μL were centrifuged at 250 g for 5 minutes in 1.5 mL screw-cap Eppendorf tubes. Pellets were cultured for 2 weeks in a humidified incubator (37 °C, 5 % CO2, 19 % oxygen) with a change of medium twice a week.
Inflammation and recovery cultures
After 2 weeks of culture, the pellets were harvested for baseline histological and biochemical assessments, while the remaining were exposed to IL-1β (Sigma, I9401) and TNF-α (Peprotech), each at 1 ng/mL, for 72 hours. During this inflammatory phase, pellets were cultured in chondrogenic medium deprived of TGF-β1 and dexamethasone. Pellets were then harvested for analysis or cultured for 2, 7, and 14 days in NCCM or 2 % FBS-supplemented ADMEM/F12 (ctr) (Fig. 1).
To assess possible age-dependent differences in the response of chondrocytes to the inflammatory factors and post-inflammatory NCCM treatment, donors were assigned to two arbitrary age groups: group 1, ≤55 years of age (n = 4) and group 2, >55 years of age (n = 5).
Osteoarthritic chondrocyte culture
Based on the findings generated with the healthy chondrocytes, in a second experiment we investigated the effects of NCCM on chondrocytes obtained from six patients (ages 60–82 years, all female), who suffered from end-stage OA and were undergoing total knee arthroplasty. Within 2 hours of tissue harvest, pieces of grossly visible degraded osteoarthritic cartilage were collected and directly processed as described above. Isolated OA chondrocytes were also expanded and chondrogenically cultured as described for the healthy chondrocytes. After 2 weeks of chondrogenic differentiation, baseline pellets were collected for histological, immunohistochemical and biochemical analyses. Hereafter, the remaining pellets were directly cultured in NCCM or control medium. Medium was changed twice a week and pellets were harvested at 2, 7, and 14 days (see Fig. 1 for the experimental plan). Culture medium was also collected from baseline pellets and pellets cultured in control and NCCM medium, to quantify the amounts of released cytokines.
In order to assess whether the soluble glycosaminoglycans (GAGs) contained in NCCM could have served as active factors, chondrogenic pellets obtained from one of the NCCM-responder donors were also cultured with control medium supplemented with 1 mg/mL chondroitin sulphate (Sigma, CS9819) for 2 weeks; the amount of chondroitin sulphate used corresponded to the concentration of GAG measured in the basal NCCM medium used in this study).
Analytical methods
Biochemical analysis
To measure the amounts of sulfated GAG and DNA, pellets (n = 3) from each group and condition were digested with Proteinase K (0.5 ml of 1 mg/mL protease K in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide, and 10 mg/mL pepstatin-A) for 15 hours at 56 °C. The GAG content was measured spectrophotometrically after reaction with dimethylmethylene blue and chondroitin sulfate as a standard (Sigma, C8529), using the Blyscan color assay kit (Biocolor Ltd, Carrickfergus, UK). For further analysis the mean amount of GAG for each condition was normalized to the corresponding mean amount of DNA, which was measured spectrofluorometrically using the CyQUANT cell proliferation assay kit (Molecular Probes, Eugene, OR, USA), with bovine DNA as a standard. GAG and DNA content is reported as μg GAG/μg DNA for samples and supernatants or normalized to baseline values, respectively. Additionally, GAG was also quantified in the basal NCCM medium.
Histologic and immunohistochemical analysis
Pellets (n = 2) for each condition were fixed in 4 % formalin for 24 hours, washed with PBS, and then embedded in paraffin blocks. For (immuno-) histological staining the samples were sectioned at a thickness of 5 μm. Sections were then stained with Safranin O for sulfated GAGs or processed for more detailed immunohistochemical analyses with antibodies against collagen type I collagen (mouse-anti-human, Clone I-8H5, MP Biomedicals) and type II collagen (mouse-anti-human, clone II-4 CII, MP Biomedicals, Santa Ana, CA, USA).
Western blotting
The expression of anabolic (SOX-9, HAPLN1, and fibromodulin), and catabolic proteins (MMP-3, MMP-13, and COX-2) in either NCCM-treated or Ctr-treated pellets was detected by western blot with β-actin used as a loading control. Total protein was extracted from OA pellets of every donor for each condition. We used the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockwood, IL, USA) together with radioimmunoprecipitation assay (RIPA) lysis buffer for protein extraction and measurement of the protein concentration according to the manufacturer’s instructions. Results were then read with a Perkin Elmer Victor3 Multilabel Counter (model 1420). Proteins were resolved on 10 % sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) and then electro-transferred onto polyvinyledendifluroide (PVDF) membranes (BioRad, Hercules, CA, USA). SDS-PAGE gels were then transferred to nitrocellulose membrane in transfer buffer (BioRad) containing 20 % methanol. Membranes were washed with TBS-T (TBS, 0.1 M, pH = 7.4; 0.1 % Tween 20, Bioshop) and then blocked with 5 % non-fat milk extract in a TBS blocking buffer for 1 hour in an orbital shaker at room temperature. We then incubated the membranes with the primary antibody of each protein of interest (see Additional file 1). The next morning, membranes were washed three times with Tween 20 (0.1 %)-TBS-T for 5 minutes each in an orbital shaker and thereafter, incubated with goat anti-rabbit IgG (H + L)-HRP conjugate (Bio-Rad cat. 170-6515), goat anti-mouse IgG(H + L)-HRP conjugate (Bio-Rad cat. 170-6516) and rabbit anti-goat F(AB)2 HRP XADs (NOVEX cat. A24452) secondary antibodies, respectively, diluted at an appropriate dilution in 1 % non-fat powdered milk for 1 hour at room temperature. Conjugates were matched with the appropriate antibody, i.e., rabbit polyclonal anti-Sox9 (Abcam cat. ab71762) 1:200 was matched with goat anti-rabbit IgG conjugate, etc. Blots were then washed three times with Tween 20 (0.1 %)-TBS. Signals were visualized by exposing the membranes to enhanced chemiluminescence (ECL) reagent (Biorad) according to the manufacturer’s instructions. Densitometry values were calculated for each band using ImageJ (National Institute of Health). Results of western blot analysis are presented as fold-change for pellets after 14 days treatment (NCCM or Ctr) compared to baseline.
Enzyme-linked immunosorbent assay
We determined differential OA chondrocyte pellet cytokine secretion using supernatants obtained from two representative responder donors using the multi-analyte cytokine profiling ELISA kit according to the manufacturer’s instruction (Qiagen, ML, USA) evaluating the expression of IL-6 and IL-8 in particular. We performed each experiment in triplicate.
Statistical analysis
Statistical evaluation was performed using SPSS 21 (IBM, Armonk, NY, USA). Values are presented as mean with corresponding standard deviation (SD) or as normalized to baseline values. Differences between experimental groups were assessed using the Mann-Whitney U test for non-parametric samples. P values <0.05 were considered significant.