Effect of interleukin-1β treatment on co-cultures of human meniscus cells and bone marrow mesenchymal stromal cells

Local ethical committee approval of the University of Alberta, Edmonton, Canada was obtained for this study. Bone marrow aspirates were acquired during routine orthopaedic procedures from the iliac crest of two male donors (age 45 and 57 years). The number of mononucleated cells (MNCs) in the aspirates was determined by crystal violet nuclei staining and cell counting on a haemocytometer. Thereafter, 15 million MNCs were seeded per 150 cm² tissue culture flask. Culture media was alpha MEM supplemented with 10% heat inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine (all from Invitrogen, Mississauga, Ontario, Canada) and 5 ng/ml of basic FGF or FGF-2 (Neuromics, Edina, MN, USA). Plastic adherent MNCs were allowed to attach and proliferate for 7 days before the first media change under normal oxygen tension (21% O₂; 95% air) at 37°C in a humidified incubator with 5% CO₂. Thereafter, media change was implemented twice per week until 70-80% cell confluence was attained. The plastic adherent MNCs populations now termed bone marrow mesenchymal stromal cells (BMSCs) were detached using trypsin-EDTA (0.05% w/v) and expanded until passage 2 prior to experimental use. We characterized the BMSCs as we have previously described using a panel of cell surface markers and flow cytometry analysis [35].

Local ethical committee approval of the University of Alberta, Edmonton, Canada was obtained to acquire menisci for this study. Both lateral and medial menisci were harvested from the knee joint of 4 male donors (age 56–76, mean age 66 ± 9 years) undergoing total knee arthroplasty because of osteoarthritis. Meniscus cells (MCs) were released via treatment with type II collagenase (0.15% w/v; Worthington, Lakewood, NJ, USA) after 16 h digestion of tissue at 37°C in a standard medium- high glucose Dulbecco’s modified Eagle’s medium containing 4.5 mg/ml D-Glucose (DMEM-HG), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine supplemented with 10% FBS - (all from Invitrogen, Mississauga, Ontario, Canada). The cell suspension obtained after digestion was passed through a 70 μm nylon-mesh filter. Isolated cells were plated at 10⁴ cells/cm² and cultured in standard medium for 48 h under normal oxygen tension (21% O₂; 95% air) at 37°C in a humidified incubator with 5% CO₂ before experimental use. Non-adherent cells were aspirated off while adherent cells were detached with trypsin-EDTA (0.05% w/v) (from Invitrogen).

Mono-cultures of MCs and BMSCs in the form of pellets were formed in 250 μl of serum-free chondrogenic culture medium consisting of standard medium supplemented with 0.1 mM ascorbic acid 2-phosphate, 10^-5 M dexamethasone, 1× ITS + 1 premix (Sigma-Aldrich, Oakville, Canada), 10 ng/ml TGF-β3 (Humanzyme-Medicorp Inc.) as described previously [36]. A total of 2.5 × 10⁵ cells were spun in 1.5 ml sterile conical polypropylene microfuge tubes (Enzymax LLC, Kentucky, USA) at 1500 rpm (433 g) for 3 minutes to form spherical cell pellets. Co-cultures in pellet form consisting of MCs: BMSCs were formulated by mixing the two cell types at a 1:3 ratio, respectively. Briefly, for each co-cultured pellet we mixed 62, 500 primary (unexpanded) meniscus cells with 187, 500 BMSCs at passage 2 (in vitro expanded cells). Primary meniscus cells from two donors were co-cultured with BMSCs at passage 2 from one donor and primary meniscus cells from the other two others were co-cultured with BMSCs at passage 2 from the other bone marrow aspirate donor. Control pellets were formed from either pure primary meniscus cells or pure BMSC (at passage 2) at cell density of 250,000 per pellet. Previous studies had shown that the co-cultured cell-cell ratio reproducibly resulted in enhanced matrix formation [21]. For each group, a minimum of six pellets were formed for subsequent histological, biochemical and molecular analysis. After 2 weeks of pellet culture with media changes 2 times per week, the pellets were cultured for 3 additional days in chondrogenic culture medium in the presence (i.e. MC+, BMSC + and MC:BMSC+) or absence (i.e. MC, BMSC and MC:BMSC) of 500 pg/ml of interleukin-1β (Humanzyme-Medicorp Inc.). After the culture period, pellets were processed biochemically for GAG and DNA contents, histologically, immuno-histochemically and by real-time quantitative RT-PCR for gene expression analyses. For each experimental repeat (4 in total), there was 1 experimental group (co-culture of MC:BMSC+) and 5 control groups (1 co-culture of MC:BMSC and 4 mono-cultures of BMSC; BMSC+; MC; MC+). In total there were 24 pellets per each group.

Pellets were rinsed in phosphate buffer saline (PBS; Invitrogen) prior to 16 h digestion in 250 μl of proteinase K (1 mg/ml in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide and 10 mg/ml pepstatin A – all from Sigma-Aldrich) at 56°C. The media collected in the last 3 days of pellet culture was analysed for GAG content. GAG contents in pellets and the collected media were measured spectrophotometrically after reaction with 1,9-dimethylmethylene blue binding (Sigma-Aldrich), with chondroitin sulfate (Sigma-Aldrich) as a standard [37]. The DNA content was determined spectrofluorometrically using the CyQuant cell proliferation assay kit (Invitrogen) with supplied bacteriophage λ DNA as standard. Based on experimental GAG per DNA values of mono-cultures of BMSC or MC pellets, the calculated GAG per DNA values were calculated as a linear function of the proportion (%) of BMSCs and MCs using the following equations [36]:

If the calculated GAG/DNA is significantly less than the experimentally determined GAG/DNA value of co-cultured pellets, then a synergistically enhanced chondrogenic GAG matrix formation is considered to have taken place [36].

Tissues generated from the pellet cultures were fixed in 4% (v/v) phosphate buffered formalin, processed into paraffin wax, sectioned at 5 μm and stained with 0.1% (w/v) Safranin O and counterstained with 1% (w/v) Fast Green stain, to reveal sulfated proteoglycan (GAG) matrix depositions. Other sections were probed with antibodies raised against collagen types I and II. Sections were digested with trypsin and then incubated with antibodies against collagen II (II-II6B3) from Developmental Studies Hybridoma Bank at University of Iowa, USA. Immuno-localised antigens were visualized with goat anti-mouse IgG biotinylated secondary antibody (Dako Canada Inc., Mississauga, Ontario, Canada) and a streptavidin-horseradish peroxidase labeling kit with 3,3′-diaminobenzidine (Dako). Images were captured on an Omano OM159T biological trinocular microscope (Microscope Store, Virginia, USA) fitted with an Optixcam summit series 5MP digital camera and Optixcam software and assembled in Adobe Photoshop (Adobe Systems Inc. San Jose, USA).

Total RNA was extracted from pellets using Trizol (Invitrogen) after grinding with Molecular Grinding Resin (Geno Technology Inc. St Louis, USA) in combination with the use of RNeasy mini kit (Qiagen, Mississauga, Ontario, Canada) and after removal of contaminating genomic DNA from the pellets by DNase treatment. To minimize changes in gene expression during cell pellet harvest, cell pellets were immediately (< 1 min) transferred into Trizol. Total RNA (100 ng) in a 40 μl reaction was reverse transcribed to cDNA using GoScript reverse transcriptase) primed with oligo(dT)₁₅ primer (Fisher Scientific, Whitby, Ontario, Canada). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in DNA Engine Opticon II Continuous Fluorescence Detection System (Bio-Rad) using hot start Taq and SYBR Green detection (Eurogentec North America Inc., San Diego, CA, USA). Primer sequences for aggrecan (AGG), collagen I (COL1A2), collagen II (COL2A1), collagen X (COL10A1), interleukin 1 receptor antagonist (IL1Ra), matrix metalloproteinase −1 (MMP-1), matrix metalloproteinase −2 (MMP-2), matrix metalloproteinase −13 (MMP-13), SOX9 and β-actin (Table 1) were taken from previously published work or were custom designed using the Primer Express software (Applied Biosystems, Foster City, USA) [38‐40]. All primers were obtained from Invitrogen. For each cDNA sample, the threshold cycle (Ct) value of each target gene was reduced by subtraction of Ct value of human β-actin to derive ΔCt. The level of gene expression was calculated as 2-^Δct[41]. Each sample was assessed at least in triplicates for each gene of interest.

For each experimental repeat and donor, at least triplicate specimens were assessed and the data were presented as mean ± standard error of mean (SEM) of measurements. All statistical analyses were performed using SPSS version 20 (IBM SPSS Statistics 20; Chicago, IL, USA) unless stated otherwise. Differences between experimental groups were assessed by one-way ANOVA with Tukey’s multiple comparison post-tests and considered significant with p < 0.05 (i.e. *p < 0.05, ** p < 0.01 and ***p < 0.001).

Firstly, we established that chondrogenic co-culture of MCs and BMSCs resulted in a synergistically enhanced cartilaginous matrix formation relative to mono-cultures of MCs and BMSCs as previously reported [21]. After a total of 17 days of cell culture in 3D cell pellet format in the presence of TGFβ3 supplemented chondrogenic media, primary human MCs, passaged 2 expanded BMSCs or in co-culture with each other at 1 to 3 ratio, respectively, formed hyaline-like cartilaginous tissue with varied staining intensity for GAG (as detected by Safranin O stain) and collagen II (Figure 1A). Qualitatively, the GAG and collagen II staining intensity was least in mono-cultured meniscus cell pellets and highest in co-cultured cell pellets of MCs and BMSCs. Biochemical analysis for the accumulated GAG amount after normalization β to the cellular DNA content, revealed statistically significant differences between the GAG content of co-cultured and mono-cultured pellets of MCs (p value of 0.005; Figure 2A). The GAG/DNA content was 3.40 ± 0.19 μg/μg (for MCs pellets), 6.43 ± 0.32 μg/μg (for co-cultured MCs:BMSC pellets) and 6.42 ± 0.30 μg/μg (for mono-cultured BMSC pellets) (Figure 2A; Table 2).

Addition of IL-1β (500 pg/ml) on day 14 of the 17 days of chondrogenic pellet culture resulted in tissues with reduced Safranin O staining appearance relative to control pellet cultures without IL-1β treatment (Figure 1A and B). In contrast, the collagen II staining intensity appeared similar between control and experimental pairs although there was some evidence of altered distribution of the matrix protein within pellets. Biochemical analysis for GAG/DNA content confirmed the reduced GAG staining intensity in pellets formed from mono-cultured MCs relative to mono-cultured pellets of BMSCs or co-cultured pellets of MCs and BMSCs (Table 2). Although the GAG/DNA value for pellets from mono-cultured MCs (2.87 ± 0.46 μg/μg) and co-cultured cells (6.11 ± 0.31 μg/μg) were lower relative to control pellets without IL-1β treatment, the values were not significantly different (p values of 1.00 and 0.98, respectively) from that of control pellets (Figure 2A). There was no significant difference between the GAG/DNA contents of BMSCs pellets treated with or without IL-1β (p value 1.00; Figure 2A).

Based on the experimental GAG/DNA values of mono-cultured MCs and BMSCs, the calculated GAG/DNA content of co-cultured pellets of MCs and BMSCs was determined as a linear function of the proportion of MCs and BMSCs in the co-cultured pellets. In the absence of IL-1β, the calculated GAG/DNA content was 5.66 ± 0.05 μg/ug, while in the presence of IL-1β the calculated GAG/DNA content was 5.44 ± 0.04 μg/ug. The experimentally determined GAG/DNA contents of the co-cultured pellets were 6.43 ± 0.32 μg/ug, in the absence of IL-1β, and 6.11 ± 0.31 μg/ug after treatment with IL-1β. The experimental GAG/DNA value for co-cultured pellets in the absence of IL-1β was significantly higher than calculated (1.14-fold; p value of 0.001). Similarly, there was a significant difference (1.12-fold; p value of 0.002) between the calculated and experimental GAG/DNA values of the co-cultured pellets after IL-1β treatment i.e. 5.44 ± 0.04 μg/ug (calculated) vs. 6.11 ± 0.31 μg/ug (experimental) (Figure 2B).

We evaluated the expression of a panel of genes (i.e. aggrecan, collagens I and II, Sox9 that we had previously reported to be synergistically up-regulated in chondrogenic co-cultures of meniscus cells and bone marrow mesenchymal stromal cells, and the expression collagen X that had been shown to be down-regulated in co-cultures of meniscus cells and BMSCs [21, 33, 34]. Additionally, we investigated the expression of a panel of catabolic genes, MMP-1, MMP-3 and MMP-13, which have been reported to be modulated at both the mRNA and protein levels by IL-1β in chondrocytes to [42]. Finally, we investigated the expression of IL1Ra as it had been shown to mediate the anti-inflammatory and anti-fibrotic response of BMSCs to IL-1α in the lungs of mice, and inhibition of IL-1β mediated matrix degradation in human intervertebral discs [43, 44]. The following observations were made in pellets cultured in the absence or after short term treatment of pellets with IL-1β: (1) the transcript expression of aggrecan and Sox9 were significantly upregulated in co-cultured pellets than in mono-cultured pellets of MC in the absence of IL-1β. The expression of aggrecan and Sox9 in the co-cultured pellets was not significantly different from their expression in pure BMSC pellets. However, after short-term treatment of all pellets with IL-1β, the level of expression of both genes declined (relative to pellets without IL-1β treatment) to levels that were not statistically different between the different pellet groups (Figure 3A and B); (2) in the absence of IL-1β, the expression of collagen I was significantly higher in co-cultured pellets relative to its expression in mono-cultured pellets. After IL-1β treatment, the expression of collagen I declined in all pellets groups, however, its expression (although decreased) remained significantly higher in the co-cultured pellets than in pure MC pellets. In contrast, collagen I’s expression was similar in co-cultured pellets and pure BMSCs pellets after IL-1β treatment (Figure 3C). Similarly, the expression of collagen II was significantly higher in the co-cultured pellets compared to its expression in either pure MC or pure BMSC pellets in the absence of IL-1β. After treatment of all pellets with IL-1β, the expression of collagen II declined relative to control pellets without IL-1β treatment but its expression remained significantly higher in the co-cultured pellets relative to its expression in pure MC and pure BMSCs pellets (Figure 3D); (3) in the absence of IL-1β, the level of collagen X expression in pure BMSC and co-cultured pellets was significantly higher (26-fold; p value of 0.001) than its expression in pure MC pellets. In contrast, the expression of collagen X decreased to the same level in both co-cultured and mono-cultured pellets of BMSC after IL-1β treatment. Collagen X expression remained at the same level of expression in MC pellets before and after treatment with IL-1β (Figure 3E); (4) in the absence of IL-1β, the level of MMP-1 expression was similar in all pellets. However, after IL-1β treatment, we observed a statistically significant (p value of 0.02) increase in MMP-1 expression in mono-cultured MC pellets relative to other pellets (Figure 3F). Additionally, MMP-1 expression was significantly up-regulated (21-fold; p value of 0.006) in co-cultured cell pellets after IL-1β treatment relative to co-cultured controls without IL-1β treatment (Figure 3F). In contrast, the level of MMP-3 expression remained the same in all pellets before and after treatment with IL-1β (Figure 3G). The expression of MMP-13 in pure MC pellets was not significantly modulated by IL-1β. However, IL-1β increased the expression of MMP-13 in pure BMSC and co-cultured pellets (Figure 3H). The expression of IL1Ra was highest in co-cultured pellets without IL-1β treatment (Figure 3I). After treatment with IL-1β, the expression of IL1Ra declined significantly by 24-fold in co-cultured pellets relative to its expression in co-cultured pellets without IL-1β treatment. IL-1β did not significantly modulate the expression of IL1Ra in pure MC or pure BMSC (Figure 3I).