Chondroitin sulphate

Chondrocytes at P1 were seeded on coverslips onside 6‐well plates (2 × 10⁴/well) and treated by IL‐1β for 48 hours, then fixed by 4% (v/v) paraformaldehyde, stained with Safranin O (SO, Sigma) for histological evaluation of cell morphology and GAG production. Chondrocytes seeded in 6‐well plates were treated by IL‐1β for 96 hours, digested by proteinase K (Solarbio). The enzymatic hydrolysates were centrifuged. DNA content was measured by a spectrofluorometer using Hoechst 33258 dye at 460 nm (emission) and 360 nm (excitation). A series of diluted chondrocytes (1 × 10⁶ cells) were used as a control.24 The total intracellular sGAG secretion was qualitied spectrophotometrically at 525 nm using 1,9‐dimethylmethylene blue dye (Sigma) with chondroitin sulphate (Sigma) as a standard, and normalized to the total DNA content.

GAG content of the papain digested samples was determined by 1,9-dimethyl-methylene blue (Sigma-Aldrich) [43 (link)]. Absorbance was read at 600 nm on a plate reader, and GAG content was calculated with a standard curve from chondroitin sulphate (Sigma-Aldrich) and normalized to control IVDs.

Total sGAGs content was quantified with a 1,9-Dimethyl-Methylene Blue zinc chloride double salt (DMMB) assay based on reported protocols [26 (link),33 (link),34 (link)]. For this, 20–25 mg of ECM powder was incubated in 300 µL of 75 mM NaCl, 25 mM EDTA, 50 µL of 10% SDS, and 5 µL of proteinase K (19.9 mg/mL) (Thermo Scientific) at 60 °C overnight. Next, 20 µL of digested organ-derived ECM was mixed with 200 µL of DMMB solution (comprising 19 mg DMMB in 40 mM Glycine, 38 mM NaCl, 100 mM acetic acid pH 3) and the absorbance read at 525 nm immediately. Serial dilutions of chondroitin sulphate (Sigma-Aldrich) were used for the calibration curve, and the absorbance blank corrected with DMMB solution. The total sGAGs content (µg/mL) from each organ-derived ECM was calculated from four independent experiments each performed in triplicate (Figure 1d).

Synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK and TAMRA-AGVPGLGVG) were purchased from Proteogenix. Mouse anti-MMP-2 and anti-uPA antibodies were from Calbiochem. Y27632, blebbistatin, U0126, PD150606, lactose, chondroitin sulphate, nifedipine and EDTA were from Sigma-Aldrich. EGCG was purchased from Enzo Life Sciences. Rabbit anti-Hsp90, anti-cleaved caspase-3, anti-integrin αV, anti-p-ERM, mouse anti-αvβ3 and anti-αvβ5 integrin antibodies were from Ozyme. Rabbit anti-RPSA, anti-MMP-14, anti-calpain1, anti-ROCK2, anti-myosin light chain kinase, mouse anti-RPSA, anti-RhoA, anti-ROCK1 and mouse IgM isotype control antibodies were purchased from Abcam. Rabbit anti-p-LIMK-and goat anti-cofilin and anti-actin were from Santa Cruz. Anti-integrin αvβ3 antibody was purchased from Millipore. Annexin-5 alexa fluor^® 568, CellTrace Calcein Red-Orange, AM and DiOC18³ were from Invitrogen.

Total sulphated GAG content was determined in papain-digested samples by using 1,9-dimethylmethylene blue stain (DMB) (Polysciences, Eppelheim, Germany) [23 (link)]. Briefly, cell cultures were washed with 0.9% NaCl and incubated at 60°C for 16 hours in digestion buffer (100 mM sodium phosphate, pH 6.5, 5 mM L-cysteine HCl, 5 mM EDTA) containing 125 to 140 μg/ml papain (Sigma-Aldrich P3125). Samples were collected and centrifuged at 12,000 rpm for 5 minutes, and the supernatants were incubated with DMB solution (46 μM DMB, 40.5 mM glycine, 40.5 mM NaCl, pH 1.5). An absorption ratio of 540 nm/595 nm was determined within 5 to 10 minutes of adding DMB solution. A standard curve of chondroitin sulphate (Sigma-Aldrich) was included as a reference. OH-pro quantification was performed using a chloramine-T assay and compared to a trans-4-hydroxyproline standard essentially as described previously [24 (link)]. Both GAG and OH-pro content were normalized against cellular DNA content. DNA concentration in papain-digested samples was determined using SYBR Green stain (Eurogentec, Fremont, CA, USA) as described previously [14 (link)]. Quantification of DNA concentration was analysed against a genomic control DNA standard (calf thymus; Invitrogen).

At day 14, NP and AF samples were cut into small fragments, weighed and incubated overnight at 56 °C with 500 µL 0.5 mg/mL proteinase K solution for tissue digestion. The digests were stored at −20 °C for further analysis. DNA content in the AF and NP digests was determined using the Quant-iT PicoGreen dsDNA Assay kit. For sGAG quantification, the dimethylmethylene blue assay was applied as previously described [52 (link),56 (link)]. Chondroitin sulphate (Sigma-Aldrich) was used to generate a standard curve. The absorbance was determined at 525 nm. A predictive model, generated by linear regression-fitting to the values of the standard curve, was utilised to determine the sGAG concentration. For hydroxyproline precipitation, 100 µL tissue digest were incubated with an equal volume of 37% hydrochloric acid (Sigma-Aldrich) for 24 h at 110 °C [57 (link)]. Following centrifugation at 10,000× g for 3 min, 2 µL of the supernatant were transferred to a 96-well plate and the solvent evaporated at 60 °C. The amount of hydroxyproline was determined following the kit’s instructions (Sigma-Aldrich). Following 90 min of incubation at 60 °C, the absorbance was determined at 560 nm. Considering that the hydroxyproline corresponds to approximately 10% of the mass of collagen [58 (link),59 (link)], the collagen amount was extrapolated from the hydroxyproline measurements.