Cell culture and co-culture system
All experiments were approved by the ethics and research committee of Nanjing Medical University (Permit Number: 2018-190). Informed consent was obtained from all the participants. HAMSCs were obtained from the discarded amniotic membrane, and HBMSCs were collected from patients undergoing sagittal split ramus osteotomy (SSRO). The cells were isolated and maintained as reported (16, 17). The cells in three to five passages were used in this study. An HAMSCs/HBMSCs Transwell co-culture system was established as previously described (5). HBMSCs were seeded at an initial cell density of 5 × 104 cells/cm2 in six-well culture plates. Transwells were placed in other six-well culture plates, and HAMSCs were seeded at the same ratios (5 × 104 cells/Transwell). Following the attachment of the cells (approximately 24 h), HBMSCs were subjected to a 24-h treatment with serum-free medium or Escherichia coli LPS (1 μg/mL) to induce inflammatory responses. After washing with phosphate-buffered saline, Transwells containing HAMSCs were transferred to the corresponding wells of the six-well culture plate containing HBMSCs.
Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR)
RNA isolation and cDNA transcription were performed using TRIzol reagent (Invitrogen, NY, USA) and reverse transcription kit (Applied Biosystems, CA, USA). RT-PCR was conducted as previously reported (18). The primer sequences used are listed in Table 1. Human glyceraldehyde-3-phosphate dehydrogenase was used as a reference for estimating the levels of lncRNAs and mRNAs, whereas human U6 was used to normalize miRNAs. The fold changes in gene expression were determined by the 2−ΔΔCt method.
Reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity
Flow cytometry was used to determine LPS-induced ROS by measuring the intensity of 2’,7’-dichlorofluorescin fluorescence after 48 h as previously reported (5). The SOD activity was detected using a xanthine oxidase assay kit (Jiancheng Corp., Nanjing, China) following the manufacturer’s protocols (19).
Cell transfection
Recombinant lentiviruses containing full-length ANRIL, scramble control (NC), targeting ANRIL, and scramble control (shNC) were obtained from GenePharma Company (Shanghai, China). Those lentiviruses were named Lenti- ANRIL, Lenti-NC, Lenti-sh ANRIL, and Lenti-shNC, respectively. HBMSCs transfected with miRNA plasmids (RiboBio, Guangzhou, China) were prepared using transfection reagent riboFECTTM CP (RiboBio). The mutated binding sites of miR-125a in luciferase reporter vectors containing APC were constructed by site-directed mutagenesis.
Cell proliferation assay
HBMSCs were collected after 1, 3, and 5 days. Flow cytometry (BD Biosciences, NJ, USA) was performed to determine the cell viability as previously reported (20). G0, G1, S, and G2 M phases were determined using MODFIT LT 3.2 (Verity Software House, ME, USA).
Alkaline phosphatase (ALP) and Alizarin red assay
After 7 days of osteogenic induction, ALP staining was used to detect the activity with an NBT/BCIP staining kit (CoWin Biotech, Beijing, China) and an ALP assay kit (Jiancheng Corp, Nanjing, China) as previously reported (21, 22). Mineralized matrix formation was determined after 14 days of osteogenic induction as previously reported (23).
Western blot analysis
Western blot analysis was performed as previously reported (24). The primary antibodies were as follows: anti-ALP (ab83259) (1:1000), anti-osteocalcin (OCN) (ab133612) (1:1000), and anti-Osterix (OSX) (ab209484) (1:1000) (all from Abcam, MA, USA; and RUNX2 (D1L7F) rabbit mAb #12556 (1:1000), APC antibody #2504 (1:1000), 𝛽-catenin (D10A8) XP Rabbit mAb #8480 (1:1000), and 𝛽-actin (8H10D10) mouse mAb #3700 (1:1000) (all from Cell Signaling Technology MA, USA). 𝛽-Actin served as an internal control. Relative densitometry analysis of the Western blot was carried out using ImageJ software. Relative protein levels were quantified as the ratio of the level of target protein to the level of 𝛽-actin, in each group.
In vivo bone formation assay
Approximately 105 cells (5 × 104 HAMSCs and 5 × 104 HBMSCsNC/HBMSCsANRIL/ HBMSCsshNC/HBMSCsshANRIL pretreated with LPS) were attached to each HA/TCP biomaterial (Φ 5 × H 2 mm, Sichuan University, Chengdu, Sichuan, China). After 12 hours, the complexes were subcutaneously implanted into the rat mandibular defect area designed as previously reported (four female nude rats per group, with an average weight of 280 g) (23). All animal experiments were conducted in compliance with the regulations and guidelines of Nanjing Medical University institutional animal care.
Micro-computed tomography (micro-CT) analysis
Mandibles were harvested for micro-CT analysis after 8 weeks of implantation as previously reported (25). Bone volume ratio (BV/TV, %) was calculated.
Histological observation
Mandible samples were harvested and analyzed using hematoxylin and eosin (H&E), Masson trichrome, and immunohistochemical staining. Primary antibodies against RUNX2 (1:300 dilution) were used for immunohistochemical analysis as previously reported (23). Positive areas were observed under the microscope.
Dual-luciferase reporter assay
Luciferase assays were performed using the Lipofectamine 2000 and Dual-Luciferase Reporter Assay System as previously reported (26).
Immunofluorescence staining
Primary antibody [𝛽-Catenin (D10A8) XP Rabbit mAb #8480 (1:100), Cell Signaling Technology] and DAPI were used to perform immunofluorescence staining as previously reported (23). Images were captured under an inverted fluorescence microscope (Olympus, Japan).
Statistical analysis
The data were expressed as the mean and standard deviation of at least three independent samples. Comparisons between two groups were analyzed using the two-tailed, unpaired Student t test. Comparisons among ≥3 groups were performed using one-way analysis of variance followed by Tukey's multiple comparisons. P < 0.05 indicated a statistically significant difference.