Bone marrow MSCs from five healthy donors were obtained from the Institut für Klinische Transfusionsmedizin und Immungenetik of Ulm, Germany. The isolation and validation procedure of these cells following Good Manufacturing Practices (GMP) was previously described by Rojewski et al.27. Specific information on each donor cells used in the present study can be found as supplemental data in the previously cited article. Bone marrow was collected after written informed consent was obtained according to the Declaration of Helsinki and approved by the Ethics Committee of Ulm University (ethical approval numbers 21/10 and 24/11). Trilineage differentiation capacity (adipogenic, chondrogenic and osteogenic) and surface marker analysis (negative for CD3, CD34, CD45 and MHC cI, and positive for CD73, CD90, CD105 and MHC cII) ensured that isolated cells followed the minimal criteria for mesenchymal stem cells28. Karyotyping as well as microbial, mycoplasma and endotoxin testing were also performed as part of the GMP quality control. Donors are encoded as follow, their age and sex specified in brackets: APS 7554 (22, male), APS 7553 (22, male), APS 7537 (28, female), ALA 7543 (23, male) and APA 7535 (26, female). Cells were grown in αMEM (Gibco, 22571020) containing 1% Penicillin/Streptomycin mixture (P/S, Eurobio, CABPES010U) and 5% pooled human platelet lysate with heparin (PLP, 1 IU/ml of final media).
For 3D experiments, 400,000 cells were seeded in serum free media (αMEM, 1%P/S), either on 50 mg of MBCP+ or in 15 mL Falcon tubes and centrifuged (500 g, 5 min) to form spheroids. For experiments using staurosporine (STS, Santa Cruz, sc-3510), cells were seeded in T75 flasks and incubated until 80 to 90% confluence was reached. The treatment consisted of 4 h in 20 mL serum-free media containing 0.1 µM STS or without STS as a control (untreated, UNT). The flasks were then washed 3 times with phosphate buffer saline (PBS) to remove excess STS and 20 mL of fresh serum-free media was added. The supernatants were collected after 48 h, filtered at 0.22 µm, aliquoted and stored at − 20 °C until use.
Osteoclasts and MNGCs differentiation
Circulating monocytes were isolated from concentrated peripheral blood of healthy individuals, provided by the Etablissement Français du Sang as leftovers of platelet donation. The blood was flushed out of the sorting system and diluted with PBS. This diluted solution was carefully deposited on an equivalent volume of Ficoll-Paque Premium (GE Healthcare) and centrifuged (800 g, 20 min). Peripheral blood mononuclear cells at the interface were recovered using a Pasteur pipette, wash 3 times with PBS and counted. CD14 positive cells were isolated using hCD14 MicroBeads (Miltenyi Biotec, 130-050-201) and LS Columns (Miltenyi Biotec, 130-042-401) according to the manufacturer’s instruction. CD14+ enriched cells were stored for up to six months in liquid nitrogen in vials of 10 million cells until use.
CD14+ monocytes were plated at 150,000 cells/cm2, either in 48-well plates for TRAP staining or in 24-well plates for RNA extraction. The media consisted of αMEM, 1% P/S, 5% fetal bovine serum supplemented with recombinant human Macrophage Colony-Stimulating Factor (rhMCSF, Miltenyi Biotec) at 25 ng/mL. This media was changed at day 2 and 5, and supplemented with 20% conditioned media from MSC culture and either of 100 ng/mL recombinant human Receptor Activator of Nuclear factor Kappa-B Ligand (rhRANK-L, Miltenyi Biotec) for osteoclast differentiation or, recombinant human Granulocyte–Macrophage Stimulating Factor and Interleukin-4 (rhGM-CSF & rhIL-4, R&D Systems, both 50 ng/mL) for multinucleated giant cell formation. Neutralizing antibodies were also added at the media change, at 5 µg/mL (human CXCR-1, human CXCR-2 and human gp130 antibodies, R&D Systems). The non-interference of a control IgG2A with basal osteoclast differentiation was confirmed on one donor. After 8 days, cells were either fixed in formalin for 20 min for TRAP staining or lysed for RNA extraction.
Viability and apoptosis assays
The LIVE/DEAD viability kit for mammalian cells (Invitrogen, L3224) was used to visualize the status of cells on the biomaterial and in spheroids. The samples were washed 3 times with PBS and incubated in culture conditions for 30 min in a PBS solution of the two reagents (calcein-AM for living cells and ethidium homodimer-1 for dead cells) diluted 2000 times. The staining solution was replaced by PBS and images were taken within 30 min.
Metabolic activity was measured with a resazurin assay (Sigma-Aldrich, R7017). A 2 mM solution was prepared and diluted at 0.2 mM in culture media. The preparation was incubated 3 h with the cells in culture conditions and the end-point fluorescence was measured on a microplate reader (Berthold). For Crystal Violet staining, cells were fixed for 5 min by addition of glutaraldehyde to the culture media (1% final concentration). Fixed cells were washed twice in distilled water and stained for 5 min with a solution of Crystal Violet (Sigma-Aldrich, HT901) at 0.1% in 20% ethanol. After several washes in water, plates were let to dry before image acquisition.
Apoptosis was evaluated by measuring caspases activity with the kit Apo-ONE Homogeneous Caspase-3/7 Assay (Promega, G7791). Proteins were extracted in RIPA buffer from attached cells and potential cells in the supernatant, retrieved by centrifugation. Samples were incubated at room temperature overnight with the kit’s reagent diluted at 1/100 in the buffer before fluorescence measurement. Proteins were dosed with a bicinchoninic acid assay and bovine serum albumin was used as a standard.
After fixation, cells were stored up to 2 weeks in PBS at 4 °C or immediately stained. The wells were incubated for 30 min at 37 °C in a buffer containing 40 mM Sodium Acetate, 10 mM Sodium Tartrate at pH = 5. The buffer was removed and a 1:1 mixture of acetone and 100% ethanol was applied to the cell layer for 30 s. The samples were left to dry for 2 min before being incubated in a staining solution in buffer of 0.6 mg/mL Fast Red Violet LB salt (Sigma-Aldrich, 3381) and 100 µL/mL of a solution at 10 mg/mL Naphthol-AS-MX phosphate (Sigma-Aldrich, N4875) in N,N-dimethylformamide (Sigma-Aldrich, 227056) for 30 min. Wells were then washed with distilled water and dried for image acquisition.
All steps followed the manufacturer’s recommendations. RNA was extracted using the NucleoSpin RNA Plus kit (Macherey-Nagel, 740984.250). Reverse transcription was performed with the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, K1652). Real time PCR was carried out on a CFX96 (Bio-Rad) with SYBR Select Master Mix (Applied Biosystems, 4472920) with HPRT as a reference gene and using the primers in Table 1.
For immunoassay detection of cytokine levels in the CM (UNT and STS) from five MSC donors, the Human XL Cytokine Discovery Base Kit (R&D Systems LUXLM000) was used on a Bio-Plex® 200 system (Bio-Rad), following the manufacturer’s recommendations.
MS-based quantitative proteomics
UNT-CMs and STS-CMs from three MSC donors were produced as previously described and concentrated 20-fold (from 20 to 1 mL) in Amicon Ultra-15 3kD filters (Millipore). The final protein concentration was estimated by the bicinchoninic acid assay and samples were stored at -20 °C, diluted in 1X Laemli buffer (from 4X stock solution). The proteins from each sample were stacked in a single band in the top of a SDS-PAGE gel (4–12% NuPAGE, Life Technologies) and stained with Coomassie blue R-250 (Bio-Rad) before in-gel digestion using modified trypsin (Promega, sequencing grade) as previously described29. The resulting peptides were analyzed by online nanoliquid chromatography coupled to tandem MS (Ultimate 3000 RSLCnano and Q-Exactive HF, Thermo Scientific). Peptides were sampled on a 300 μm × 5 mm PepMap C18 precolumn (Thermo Scientific) and separated on a 75 μm × 250 mm C18 column (Reprosil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch) using a 240-min gradient. MS and MS/MS data were acquired using the Xcalibur software (Thermo Scientific).
Peptides and proteins were identified using Mascot (version 2.6.0, Matrix Science) through concomitant searches against Uniprot database (Homo sapiens taxonomy, September 2019 version), homemade classical contaminant database and the corresponding reversed databases. Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at respectively at 10 and 25 mmu. Peptide modifications allowed during the search were: Carbamidomethyl (C, fixed), Acetyl (Protein N-term, variable) and Oxidation (M, variable). The Proline software30 was used to filter the results: conservation of rank 1 peptides, peptide-spectrum-match score ≥ 25, peptide length ≥ 7 amino acids, false discovery rate of peptide-spectrum-match identifications < 1% as calculated on peptide-spectrum-match scores by employing the reverse database strategy, and minimum of 1 specific peptide per identified protein group. Proline was then used to perform a compilation, grouping and MS1 quantification of the protein groups based on specific peptides.
Statistical analysis was performed using ProStaR31. Proteins identified in the reverse and contaminant databases, and proteins exhibiting less than three abundance values in one condition were discarded from the list. After log2 transformation, abundance values were normalized by median centering before missing value imputation (slsa algorithm for partially observed values in the condition and DetQuantile algorithm for totally absent values in the condition). Statistical testing was conducted using limma test. Differentially expressed proteins were sorted out using a log2 (fold change) cut-off of 1 and a p-value cut-off of 0.03, allowing to reach a FDR inferior to 5% according to the Benjamini–Hochberg procedure.
Proteins found differentially abundant between UNT-CM and STS-CM were submitted to functional classification using PANTHER v.15.032. Their enrichment in Reactome pathways was tested using Fisher’s exact test. Enrichment was considered for a Bonferroni adjusted p value < 0.05.
Data representation and statistical analysis were performed on GraphPad Prism software 6.0. Paired t-test was used to compare two groups of matching data. For comparison of three groups and more, repeated measure one- or two-way ANOVA was carried out, followed by Tukey’s multiple comparison test. Differences were considered significant for p value < 0.05 (*), very significant for p value < 0.01 (**) and extremely significant for p value < 0.001 (***) and p value < 0.0001 (****).