2.2.6 Determination of Lentivirus Multiplicity of Infection (MOI)
The lentiviruses evaluated included Fzd6(+), Fzd6(-), shRNA-1, shRNA-2, shRNA-3, and shRNA(-). The MOI value was set using the following gradient: 0, 20, 40, 60, 80, 100. The volume of virus solution needed in each group was calculated according to the following formula: virus volume = cell number × MOI/virus titer. After transfection for 72 h, the morphology and fluorescence expression of the cells was observed and recorded by fluorescence microscopy to determine the optimal MOI value for each lentivirus.
2.2.10 Osteogenic Induction
OP-ASCs were seeded onto 6-well plates at a density of 5×104 cells/mL. The medium was changed to osteogenic induction medium (Cyagen, Guangzhou, China) when OP-ASCs reached a fusion of 80%.
2.2.11 Alkaline Phosphatase Staining (ALP) and Alizarin Red Staining
After osteogenic induction for 7 days, OP-ASCs transfected with Fzd6 lentivirus were rinsed with PBS and fixed with 4% paraformaldehyde at 4°C for 30 minutes. ALP activity was detected using an Alkaline Phosphatase Assay Kit (Beyotime, Shanghai, China).
After 14 days of osteogenic induction, OP-ASCs were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Cells were then rinsed three times with PBS and incubated in 0.1% Alizarin red staining for 15 minutes at 37°C.
2.2.12 Extraction of RNA and qPCR
Total cellular RNA was isolated using the Total RNA Extraction Kit (Tiangen, Shanghai, China) and subsequently reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo, Waltham, USA). All primers in this study were synthesized by Shenggong Bioengineering Co., Ltd (Shanghai China). The specific primer sequences were as follows: Gapdh, (forward) 5′-GGGTGAAGGTCGGTGTGAACG-3′ and (reverse) 5′-CTCGCTCCTGGAAGATGGTG-3′; Fzd6, (forward) 5′-AGACAACATTAGCGGCGTTT-3′ and (reverse) 5′-AGAGGAGAGACAGCCCAACA-3′; Runx2, (forward) 5′-CCGAACTGGTCCGCACCGAC-3′ and (reverse) 5′-CTTGAAGGCCACGGGCAGGG-3′; Opn, (forward) 5′-GGATTCTGTGGACTCGGATG-3′ and (reverse) 5′-CGACTGTAGGGACGATTGGA-3′. The qPCR reaction was in accordance with the instructions of QuantiNova SYBR Green PCR Kit. The reaction system was added to qPCR 96-well plates. The reaction liquid was collected at the bottom of the plate by horizontal centrifugation and the plate was then placed in a real-time PCR instrument. After the reaction was completed, the amplification curve and dissolution curve of each gene was analyzed by 7900 System SDS software and the relative expression of the target genes was calculated and statistically analyzed.
2.2.13 Western Blotting Assay
Proteins were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h at 37°C, the membranes were incubated with primary rabbit monoclonal antibodies specific to the following targets: GAPDH (ab181602), 3-FLAG (ab125243), FZD6 (ab98933), OPN (ab8448), and RUNX2 (ab92336) (Abcam, Cambridge, UK). Then, each membrane was incubated with a secondary labeled anti-rabbit antibody (Beyotime, Shanghai, China). Membranes were developed using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, USA).
2.2.14 BCP Combined with OP-ASCs was Transplanted into a Mouse Model of Critical-sized Calvarial Defects
OP-ASCs transfected with Fzd6 overexpression lentivirus, silencing lentivirus, or untransfected lentivirus were prepared and cultured in osteogenic induction medium. 1 mL cell suspension (5×105 cells per 1 mL) was added to the surface of the BCP in 24-well plates. 24-well plates were cultured in a CO2 incubator for 48 h. The osteoporosis mice were treated with prone fixation, skin preparation, and disinfection at the top of the skull. The skin was cut at the midline of the skull, the skull was exposed, and the periosteum was bluntly separated. Then, full-thickness defects of 4 mm were created symmetrically. The BCP combined with OP-ASCs was transplanted into the skull defect area and the operation area was then sutured. After 4 and 8 weeks, mice were euthanized and the skull specimens were obtained.
2.2.15 Micro-CT, H&E staining, and Masson staining
Micro-CT scans were performed on mice with skull defects at 4 and 8 weeks. Then, tissues samples of mice skull defect were stained with H&E and Masson staining.