Human IVD cell isolation and culture

Biopsies from patients undergoing spinal surgery due to disc herniation or DDD were obtained for in vitro experiments with human degenerated IVD cells. As described previously, NP, AF or mixed IVD tissue was excised intraoperatively, diced and digested enzymatically overnight with 0.2% collagenase NB4 (Nordmark) and 0.3% dispase II (Sigma-Aldrich) in 1 × Dulbecco's Phosphate Buffered Saline (DPBS, Cytiva) with 5% antibiotic–antimycotic (anti-anti, Gibco) at 37 °C, 5% CO2 [31]. After enzymatic tissue digestion, cells were kept in culture up to passages 1–2 in growth medium consisting of Dulbecco’s Modified Eagle’s Medium/Ham's F-12 medium (DMEM/F-12, Cytiva) supplemented with 10% fetal bovine serum (FBS, Cytiva) and 1% anti-anti. An overview of the patients characteristics can be found in Supplementary Table 1.

Transfection of miRNA mimics/inhibitors

In order to transfect human IVD cells with miRNAs, 3 uL HiPerFect Transfection Reagent (Qiagen, 301705) were mixed and incubated with 50 nM miRNA mimics/inhibitors or their corresponding non-targeting controls for 10 min at room temperature to allow for the formation of transfection complexes (hsa-miR-155-5p miRCURY LNA miRNA Mimic YM00472490-ADA, Qiagen, 339173; hsa-miR-155-5p miRCURY LNA miRNA Power Inhibitor YI04101510-DDA, Qiagen, 339131; negative control miRCURY LNA miRNA Mimic YM00479902-ADB, Qiagen, 339173; negative control B miRCURY LNA miRNA Power Inhibitor YI00199007-DDA, Qiagen, 339136). Human IVD cells were then reverse-transfected at a density of 20,000 cells/cm2 with miR-155-5p mimic/inhibitor or non-targeting complexes in no-serum media (DMEM/F12 with 0.1% anti-anti). After 24 h the medium was changed to growth medium in order to let the cells recover. The efficiency of the miRNA mimic/inhibitor transfection was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 72 h post transfection.

Induction of inflammation in IVD cells

Effects of miRNA gain- and loss-of-function on the inflammatory cell response were studied by first transfecting human degenerated IVD cells with miRNA mimics or inhibitors as described above. After 22 h of recovery in growth medium, cells were starved for 2 h in no-serum medium followed by treatment with 5 ng/mL recombinant human IL-1β (PeproTech, 200-01B). For the analysis of protein phosphorylation, cells were treated with 5 ng/mL IL-1β for 30 min before being lysed for protein extraction. Secretion of cytokines and catabolic factors as well as gene expression were analyzed after treating AF and NP cells for 24 h with 5 ng/mL IL-1β. To that end, cell culture supernatants were collected for cytokine/MMP arrays and IVD cells were lysed for miRNA and mRNA extraction 72 h post transfection.

Fabrication and characterization of stretching chambers

Polydimethylsiloxane (PDMS) stretching chambers were fabricated using a 1:1 mixture of Sylgard 184 (Dow, 2646340) and Sylgard 527 Silicone Dielectric Gel Clear 0.9 kg Kit (Dow, 1,696,742), cast using an aluminum mold with dimensions fitting the automated cell stretching system and cured at room temperature overnight, at 80 °C for 40 min or at 140 °C for 15 min. After demolding, the stretching chambers were cleaned in 70% ethanol for 5 min in an ultrasound bath, rinsed with deionized water, dried and plasma-treated with the plasma cleaner PDC-001 (Harrick Plasma) before being used for characterization or cyclic stretching.

The stiffness of the stretching chambers was determined by performing tensile testing with the UniVert tensile tester (CellScale Biomaterials Testing) at a crosshead speed of 10 mm/s until the point of failure using a 1–10 N and 10–100 N load cell. The Young’s Modulus was calculated using the stress/strain curve.

Digital image correlation (DIC) was used to determine the difference between engineering strain and true strain of the fabricated PDMS chambers during cyclic sinusoidal uniaxial loading with the automated cell stretching system (STB-140–10, STREX). To that end, fluorescent ink (Millennium Colorworks, Ink Glow UV) was incorporated into the PDMS mixture before casting and curing the chambers at 140 °C for 15 min. The stretching chambers were then imaged during cyclic sinusoidal uniaxial loading with the StrainMaster Portable System (LaVision) at an image sampling rate of 100 Hz using blue LED illumination. Calibration, data collection, image processing, and data analysis were performed using DaVis Software 10.2.1 (LaVision).

Cyclic stretching

Sterile PDMS stretching chambers were coated overnight with 50 μg/mL fibronectin (EMD Millipore, FC010) at 37 °C. The following day, human IVD cells, with or without simultaneous reverse-transfection of miRNA mimics/inhibitors, were seeded in the stretching chambers (20,000 cells/cm2, 10 cm2 cell culture surface). After the transfection and recovery phase (46 h post transfection), cells were starved in no-serum media for 2 h and then subjected to 8% cyclic sinusoidal uniaxial engineering strain for 24 h at a frequency of 1 Hz at 37 °C and 5% CO2. Control chambers were kept in identical conditions without stretching. Cell lysates and conditioned media were harvested for expression analysis, cytokine and MMP arrays 72 h post transfection (experimental timeline depicted in Supplementary Fig. 1).

miRNA extraction and RT-qPCR

MiRNA extraction for the expression analysis was performed with the miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, 217604). Following IL-1β treatment or mechanical loading, cells were rinsed twice with ice-cold 1 × DPBS, lysed with 260 uL lysis buffer, and the samples were further processed according to the manufacturer’s instructions. The concentration and quality of miRNA were analyzed with the UV–Vis spectrophotometer NanoPhotometer N50 (Implen). Reverse transcription of miRNAs was achieved with the miRCURY LNA RT Kit (Qiagen, 339340) according to the manufacturer’s protocol using 20 ng total RNA per 20 μL reaction followed by quantitative PCR with the miRCURY LNA SYBR Green PCR Kit (Qiagen, 339347). The respective miRCURY LNA miRNA PCR Assays were used for the analysis of the expression of specific miRNAs (YP00204308—hsa-miR-155-5p, YP00204063—hsa-miR-103a-3p, Qiagen, 339306). Furthermore, spike-ins were used to monitor the consistency of the extraction process and reverse transcription (RNA Spike-In Kit, Qiagen, 339390). The results are shown as 2−∆∆Ct values relative to the housekeeping miRNA miR-103a-3p and control conditions (cells transfected with non-targeting miRNA mimics or inhibitors).

Cytokine and MMP array

In order to analyze the secretion of cytokines and MMPs, conditioned media was collected immediately after treatment and centrifuged at 500 g for 5 min for the removal of cell debris. The resulting supernatants were used undiluted for the Human Cytokine Array GS1 (RayBiotech Life, GSH-CYT-1–4) and Human MMP Array GS1 (RayBiotech Life, GSH-MMP-1–4), which were performed according to the manufacturer’s instructions. The concentration range of the cytokine and MMP array proteins was detected at a range of 1 pg/mL – 1 ng/mL. The median fluorescent signal was normalized to the plate background and array’s positive control. Fold changes of fluorescence were calculated relative to the non-targeting control conditions.

Phosphorylation array

The phosphorylation of proteins associated with the MAPK pathway was determined with the Human/Mouse MAPK Phosphorylation Array (RayBiotech Life, AAH-MAPK-1–8). To that end, cells were rinsed with ice-cold 1 × DPBS and lysed on ice with 60 µL radioimmunoprecipitation assay (RIPA) buffer supplemented with 100 × phosphatase and protease inhibitor cocktail, which was diluted to a final concentration of 1 × in RIPA buffer (Thermo Fisher Scientific, 89,900 and 78,440). The lysate was incubated for 30 min at 4 °C with gently shaking followed by centrifugation at 14,000 g for 10 min at 4 °C. Protein concentrations were quantified with the BCA protein assay (Thermo Fisher Scientific, 23,225). The samples were then diluted to a final concentration of 75 µg/mL in RIPA buffer containing phosphatase and protease inhibitor cocktail and 1 mL of the diluted samples was used for the MAPK Phosphorylation Array, according to the manufacturer’s instructions. The membranes were scanned with the chemiluminescence imaging system Odyssey XF imaging system (LI-COR Biosciences) and analyzed with the Empiria Studio Software (LI-COR Biosciences). Signal intensities were normalized to the positive control and membrane background. The results are shown as fold changes relative to the non-targeting controls.

RNA extraction and gene expression analysis (RT-qPCR)

Analysis of gene expression was performed by co-extracting mRNA with the miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, 217604) according to the manufacturer’s recommendations. After determining the quantity and quality of RNA with the NanoPhotometer N50 (Implen), 500–1000 ng RNA were used for reverse transcription into cDNA with the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific, 4374967). Finally, gene expression was quantified by qPCR with the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, 4444963) and the respective Taqman Gene Expression Assay (Hs00153936_m1 ACAN,

Hs00171458_m1 NGF, Hs02718934_s1 BDNF, Hs00174103_m1 CXCL8/IL-8, Hs00153133_m1 PTGS2/COX2, Hs00427620_m1 TBP, Thermo Fisher Scientific, 4331182) using the QuantStudio™ 3 (Thermo Fisher Scientific). The results were calculated as 2−∆∆Ct values relative to the housekeeping gene TBP and control conditions.

Cell viability assay

The effect of PDMS on cell viability was determined with the alamarBlue assay. Cells were seeded on PDMS stretching chambers and kept in culture for 72 h. Thereafter, cells were incubated with a 1:10 dilution of alamarBlue Cell Viability Reagent (Thermo Fisher Scientific, DAL1025) for 4 h at 37 °C. Cells seeded in standard 6-well tissue culture plates were used as a positive control and cells lysed in a 6-well tissue culture plate were used as a negative control. Furthermore, the cytotoxicity of cyclic stretching was tested using the CyQUANT™ LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, C20301) following the manufacturer’s instructions. Briefly, conditioned media of cells undergoing cyclic stretching for 24 h was collected and 50 µL of each sample was used for the LDH cytotoxicity assay. Non-stretched cells were used as a positive control and lysed cells were used as a negative control.

Statistical analysis

To check normality, Saphiro-Wilks test was used and Levene’s test to test variance homogeneity. Bootstrap-t tests with and without the assumption of variance homogeneity were used to test means. 95% bias-corrected and accelerated (BCa) confidence intervals were computed to estimate the difference of means. All reported tests were two-sided, and p-values < 0.05 were considered statistically significant. All statistical analyses in this report were performed by use of NCSS (NCSS 10, NCSS, LLC. Kaysville, UT), and Wolfram Research, Inc., Mathematica, Version 13.1, Champaign, IL (2022).