Clinical sample collection, cell culture, and osteogenic induction
A total of 20 OPLL patient samples and 18 cervical spine trauma patients' PLL samples were collected. All experiments in this study were approved by the ethics committee of Shanghai Changzheng Hospital. The patients signed an informed consent form before the operation. The OPLL samples came from patients who had received anterior cervical corpectomy and fusion (ACCF). After the vertebral body was removed during the operation, the ossified posterior longitudinal ligament of the posterior wall of the vertebral body was carefully separated, washed with normal saline, and stored aseptically in complete cell culture medium，which was prepared for primary OPLL cell culture. The normal posterior longitudinal ligament was derived from patients with cervical spine trauma who received ACCF. The posterior longitudinal ligament behind the posterior wall of the vertebral body was also separated for primary PLL cell culture. Specifically, OPLL or PLL was cut into pieces with a diameter of 1 mm approximately, and placed in a petri dish with the complete cell culture medium (90% high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA), 10% fetal bovine serum (FBS, Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA)). Put the petri dishes into a 37°C constant temperature incubator in a humidified atmosphere containing 5% CO2. The medium was changed in half every 3 days and in full every 7 days. After 10-14 days, mass of fibroblast-like cells migrated out of the tissue and grew adherently, which can be observed microscopically. When the cell convergence reached 80%, the cells were passaged according to 1:3.
The hMSCs used in this experiment were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The complete cell culture medium consisted of 90% low-glucose DMEM (Gibco, USA), 10% FBS (Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA). The culture environment was the same as that of OPLL cells.
Isolation and identification of exosomes
The exosomes were separated from the cell culture medium with gradient centrifugation. The cell supernatant was centrifuged at 300g for 15 min to remove floating cells, and then centrifuged again at 820g for 15min, 10,000g for 5 min, and cell debris was removed through a 0.8μm filter. Finally, centrifuge at 100,000g for 2 h (Beckman L-90K, USA) to obtain exosomes pellet. The exosomal pellet was resuspended in PBS and ultracentrifuged again for further experiments. All centrifugation was performed at 4°C.
The size distribution and concentration of exosomes were examined by nanoparticle tracking analysis with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software ZetaView 8.04.02. Exosome were diluted in PBS to measure the particle size and concentration. NTA measurement was recorded and analyzed at 11 positions. ZetaView system was calibrated using 110 nm polystyrene particles. All procedures were performed at room temperature. The exosome samples were prepared and observed with a transmission electron microscope (H-7650, Hitachi, Japan) as described before(39). The voltage was 80 kV.
Analysis of differential expression miRNAs and target prediction
Total exosomal RNA of OPLL or PLL cells was extracted using Total Exosome RNA Isolation Kit (Invitrogen, USA) according to the manufacturer’s instructions. MiRNA library construction and miRNA sequencing were performed as described before(40), intended for identifying the differentially expressed miRNAs derived from OPLL cell or PLL cell exosomes. Next, target genes of miR-140-5p were predicted by Targetscan (http://www.targetscan.org) and miRDB (http://mirdb.org). The intersection of these two databases was created by Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny).
Exosome labeling and confocal microscopy
Exosomes were collected and labeled with the green lipophilic fluorescent dye PKH67 (Sigma, USA)，then co-cultured with hMSCs for 1 h. After that, hMSCs were washed by PBS and fixed with 4% paraformaldehyde. The cell nuclear was stained by 4′6-diamidinophenylidole (DAPI, Beyotime, China). The samples were observed with confocal microscopy (FV10i, Olympus, Japan).
Luciferase report assay
With the help of restriction endonuclease site primers, we synthesized a human wild-type IGF1R 3'UTR fragment containing miR-140-5p conserved binding site by PCR, and cloned it into the pMIR reporter vector. This is called wild-type (tw) IGF1R 3'UTR. By mutating the miR-140-5p binding site of tw IGF1R 3'UTR, a mutant (mt) IGF1R 3'UTR was synthesized and cloned into the vector. Human 293T cells were transfected with the above two vectors and co-cultured with miR-140-5p-exo or control. A dual luciferase reporter gene detection system (Promega, USA) was used to detect luciferase activity.
Transfection of lentivirus and oligonucleotide
Agomirs, Antagomirs, and siRNAs were synthesized by Sangon Biotech (Shanghai, China). The IGF1R vector for cDNA delivery was synthesized by Sangon Biotech (Shanghai, China). Lentivirus production, purification, and titration were performed as described before(41). Transfection or miR-140-5p mimic or inhibitor into cells was performed by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocols. Transfection or miR-140-5p mimic or inhibitor into exosomes was performed by using Exo-Fect Exosome Transfection Kit (SBI, CA) according to the manufacturer’s protocols.
In vivo Bio-Oss Collagen scaffold implantation and bone formation
After MiR-140-5p-exo, miR-NC-exo, sponge-exo, and sponge-NC-exo were collected, they were co-cultured with hMSCs for 48 hours, and then replaced by osteogenic induction culture for 14 days. The cells were resuspended, thoroughly mixed with Bio-Oss Collagen (Geistlich, GEWO GmbH, Germany) and co-cultured for 48 hours. Finally, the mixture of scaffold and cells was implanted into the back of nude mice (4-week-old BALB/c homozygous nude) subcutaneously. After 8 weeks, the stent specimen was removed and fixed in 4% paraformaldehyde for further immunohistochemical analysis.
The scaffold samples were decalcified in 10% ethylene diamine tetraacetic acid (EDTA) for 1 month. Then, they were dehydrated and embedded in paraffin. The paraffin tissue block was cut into 5 μm slices for hematoxylin and eosin (H&E) staining. The sections were blocked with 3% BSA for 30 min, and then incubated with primary antibodies (Abcam, USA), including anti-OCN, anti-COLIA1, anti-RUNX2 anti-ALP, and anti-TGF1R. 95℃ citrate buffer was used to retrieve antigen for 10 min. Subsequent sections were incubated again with primary antibodies at 4℃ overnight. Sections were observed with Olympus microscope equipped with Olympus DP70 camera (Olympus, Japan).
The scaffold samples were harvested after intervention and fixed in 4% paraformaldehyde. Subsequently, the samples were scanned by micro-CT (Quantum FX microCT, PerkinElmer, USA), RigakuTM software (Rigaku, Japan) and RadiAnt DICOM ViewerTM (Medixant, Poland) were used for 3D reconstruction and image processing.
ALP and alizarin red staining
Osteogenesis Induction Medium (Cyagen, China) contains 10% FBS, 1% Penicillin-Streptomycin, 10mM L-glutamine, 50μM L-ascorbic acid, 10mM β-glycerophosphate, and 100nM dexamethasone. When hMSCs fusion degree reached 60%-70%, the culture medium was replaced with the osteogenic medium for osteoinduction. The medium was changed every 3 days. After 14 days, the induction medium was removed. Then the cells were washed with PBS and fixed with 4% paraformaldehyde. Cells were stained by using ALP staining kit (Beyotime, China) as the manufacture’s instruction. Similarly, cells were stained by Alizarin red S (Cyagen, China) 14 days after induction.
RNA extraction and qPCR
Briefly, total RNA from cultured cells was isolated by TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. Then extracted RNA was reverse transcribed into cDNA by a ReverTra Ace® qPCR RT Kit (Toyobo, Japan). QPCR was performed with real-time PCR (ABI 7500, Applied Biosystems, USA) and expressions of several osteogenesis-related genes, including OCN, COLIA1, RUNX2 and ALP, were calculated by the 2−ΔΔCt method.
Firstly, cells were lysed by high-efficiency RIPA Lysate (Solarbio, China) and the protein content was determined by a BCA protein assay kit (Thermo Fisher Scientific, USA). Secondly, protein samples were separated by Bis-Tris gel (Invitrogen, USA), transferred to a nitrocellulose filter membrane (Bio-Rad, USA), blocked with 5% w/v skimmed milk in TBST buffer (1X Tris-buffered Saline and 0.1% Tween 20) for 1 hour at room temperature, and incubated with primary antibodies overnight at 4°C. Thirdly, membranes were washed and incubated with secondary antibodies for 2 hours at room temperature. Finally, the Odyssey imaging system (Li-Cor, Lincoln, USA) was used to determine the fluorescent signals. Anti-GAPDH or anti-β-actin was used as endogenous control.
All data analysis used SPSS 21.0 software (Chicago, IL, USA). All data were expressed as Mean ± SD. Student’s t test was selected for the comparison of the two independent variables. One-way ANOVA was selected for the comparison of multiple independent variables. P value<0.05 was considered to be statistically significant.