Aggrecan

Cells were harvested and lysed on ice using 100 μL RIPA buffer (Thermo Fisher Scientific, CA, USA) containing Phenylmethylsulfonyl fluoride. After separation on 10% or 5% polyacrylamide gels, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with different primary antibodies (collagen II, aggrecan, and collagen X were from Novus, CO, USA; p38 MAPK was from Boster Biological Technology, CA, USA; p-p38 MAPK was from Santa Cruz, CA, USA; MMP-3, IL-1β, TNF-α, and NF-κB were from Abcam, CA, USA) overnight at 4 °C. The next day membranes were incubated with HRP-linked secondary antibodies (goat anti-rabbit and goat anti-mouse were from Beijing Zhongshan Jinqiao Biotechnology) for 1 h at room temperature. The protein signal was detected using an ECL chemiluminescence kit (Thermo Scientific CA, USA) according to the manufacturer’s instructions and observed using a gel image analyzer. The relative protein expression was compared to the control group. The phosphorylation level of p38 MAPK is given as the ratio of phosphorylated p38 MAPK to total p38 MAPK protein.

Normal SIS, D-SIS, recellularized SIS at 7, 14 and 28 days, and DD-SIS were washed with PBS three times, fixed in PFA (4%, 24 h), followed by standard paraffin embedding and sectioning. For in vivo treatment, the IVD were harvested from the rabbits three months after injection of DD-SIS, fixed in PFA (4%, 48 h), decalcified (30 days) and embedded in paraffin. Hematoxylin and eosin staining was performed to reveal the whole structure and cell distribution. Alcian blue staining was performed to evaluate retained GAG content. Specimens for immunohistochemistry were processed as previously described by our group. [47 (link)] Epitopes of interest included collagen type I (Abcam, Cambridge, MA, USA), collagen type II (Novus Biologicals, Littleton, CO, USA) and aggrecan (Novus Biologicals). 4,6-Diamidino-2-phenylindole (DAPI, Sigma) fluorescent nuclear staining was performed to evaluate the efficiency of decellularization and NPC distribution at different time points.

The prepared tissue sections were processed through dewaxing, antigen retrieval, and inactivation of endogenous peroxidase. Then, they were incubated overnight at 4°C with primary antibodies (aggrecan: Novus, NB120-11570, diluted 1:200; collagen II: Abcam, ab34712, diluted 1:200) and corresponding HRP-conjugated secondary antibodies (goat antimouse IgG and goat antirabbit IgG, ZSGB-BIO, China, diluted 1:200) for 2 h at 37°C. After color development with diaminobenzidine (DAB), the sections were counterstained with Hematoxylin. The positive staining was observed under a light microscope (Olympus BX51) and quantitated using the ImagePro Plus software (version 5.1, Media Cybernetics, Inc.) to calculate the staining intensity.

After the indicated treatments, NP cells were washed twice with PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) supplemented with phosphatase and protease inhibitors. The lysates were electrophoresed by 10–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Then, the membranes were blocked with NcmBlot blocking buffer (NCM Biotech, Jiangsu, China) for 20 min at room temperature and incubated overnight at 4 °C with primary antibodies against HSP70 (1:1000, ABclonal), Mfn1 (1:1500, Proteintech), Mfn2 (1:1500, Proteintech), OPA1 (1:1000, Proteintech), DRP1 (1:1000, Affinity Biosciences), MFF (1:1000, Proteintech), Fis1 (1:1000, Proteintech), cleaved caspase 3 (1:1000, Affinity Biosciences, OH, USA), caspase 9 (1:1000, Proteintech), Bcl-2 (1:1000, Abcam, Cambridge, MA, USA), Bax (1:1000, Proteintech), Collagen II (1:500, Affinity Biosciences), Aggrecan (1:500, NOVUS, USA), SIRT3 (1:1000, ABclonal), and β-actin (1:2000, Cell Signaling Technology, Danvers, MA, USA). Then, the PVDF membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using an ECL kit (Affinity Biosciences).