3D culture of HGFs
Gingival tissue specimens were obtained from healthy premolar teeth of patients (age 12-25 years) who had planned to undergo orthodontic treatment at the affiliated stomatology hospital of Guangxi Medical University. The patients had no systemic diseases, gum inflammation, or gum hyperplasia. This research was reviewed and approved by the Medical Ethics Committee of Guangxi Medical University (approval number: 20150304-22) and patients and their families were fully informed and offered written consent. The research was performed according to the principles of the revisory Declaration of Helsinki (World Medical Association General Assembly, 2008).
The gingival tissue was scraped from the root surface and cut into small tissue blocks (1 mm3). These tissue pieces were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen; Thermo Fisher Scientific,Inc., MA, USA ), supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., MA, USA) and 1% (v/v) penicillin and streptomycin (Gibco; Thermo Fisher Scientific,Inc., MA, USA) and maintained in a humidified atmosphere at 37°C with 5% CO2 . The PLGA scaffold (size, 1cm × 1cm × 300 µm; molar ratio of lactic acid to glycolic acid, 75:25; pore size, 100–120 µm; porosity, 85%) was synthesised by solvent casting/particle leaching technology. The 4th generation (1 × 105) HGFs in good growth conditions in the logarithmic growth phase with a single cell suspension were inoculated on a single sheet of PLGA scaffold to establish a co-culture model of HGFs and PLGA scaffolds.
Application of mechanical force
The above HGFs-PLGA co-culture model was placed into a sterile well plate and incubated for 24 h and then moved to a new well plate. The cells in the PLGA co-culture model were subjected to a compressive force of 25 g/cm2 by placing a sterilized cylindrical bottle containing lead granules for 6, 24, 48, and 72 h. The stress value was determined based on previous studies, under a force of 25g/cm2, the proliferation index of HGFs was at the maximum4. In addition, the stress value was also in line with the level reported by Schwarz46, who suggested that the proper force for moving teeth should be within the scope of capillary pressure, which is 7–26 g/cm246. The cells in the control group were cultured under the same conditions without applying a mechanical force. In another experiment, the 3D-cultured cells were infected with CRT-siRNA lentivirus and NC lentivirus (GeneChem, Shanghai, China). After 3 d of infection, green fluorescent protein (GFP) expression was observed by fluorescent microscopy, and a mechanical force of 25 g/cm2 was applied to the cells for 24 h. Quantitative real-time PCR and western blot analysis were performed to determine the knock-down efficiency, and a dose-response assay using the Cell Counting Kit-8 (CCK8; Dojindo, Japan) was performed to assess cytotoxicity.
Laser scanning usingconfocal microscope
The 3D-cultured cells that were treated with compressive force were washed twice with phosphate-buffered saline (PBS). Then, 4% paraformaldehyde was applied for cell fixation for 30 min and washed with PBS three times for 5 minutes each time. The complex was treated with DAPI (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 10 min at 37°C. Immunofluorescence micrographs (Olympus Corporation, Japan) were obtained using a laser confocal microscope (light wavelength of 405nm).
Quantitative real-time PCR
Total RNA was extracted from the HGFs-PLGA Complex using the Trizol kit (Aidlab Biotechnology Co., Ltd., Beijing, China) in each group. The obtained RNA was reverse-transcribed to cDNA using the ExScript RT kit (Takara Bio, Inc., Otsu, Japan), RT-qPCR was performed based on the (SYBR) manufacturer’s instruction. The 2−ΔΔCt was considered to be the relevant expression of the target gene in each group. Each experiment was repeated three times, and the average value was recorded.
The primers used had the following sequences:
GAPDH-forward: 5’-TGTGTCCGTCGTGGATCTGA-3’
GAPDH-reverse: 5’-TTGCTGTTGAAGTCGCAGGAG-3’
CRT-forward: 5’-GCACTTGGATCCACCCAGAA-3’
CRT-reverse: 5’-GAAGTTGTCAAAGATGGTGCCAGA-3’
RCaN1-forward: 5’-CCAGACAAGCAGTTTCTGATCTCC-3’
RCaN1-reverse: 5’-AGTCGCTGCGTGCAATTCATA- 3’
COL-I-forwar: 5’-CTACTCTCTCAACACTCACTCTTTCACTTA-3
COL-I-reverse: 5’-TCACTCACCACCCTATCTTCCA-3
Western blotting analysis for protein expression
To separate the HGFs from the HGFs-PLGA 3D culture model, 0.25% trypsin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used. The cells were harvested in ice-cold RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktails. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were immersed in 5% skimmed milk at 25 ℃ for 2 h. The primary antibodies were then incubated at 4 °C overnight, including the anti-CRT (ab92516, Abcam), anti-CaN (ab140131, Abcam), anti-NFAT3 (CST, USA), anti-p-NFAT3 (Santa, USA), and anti-COL-I (ab138492, Abcam). The membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Cell Signalling Technology, Boston, USA) at 25 ℃ for 2h. Enhanced chemiluminescence (ECL) was used for development. ChemicDoc MP all-round imaging system (Bio-Rad, Cal., USA) was used for photography, while the Image Lab v3.0 software (Bio-Rad, Cal., USA) was used for the gray-value analysis of the protein bands.
Statistical analysis
Statistical analyses were performed using SPSS 20.0 (IBM, Chicago, IL, USA) statistical software. The results were expressed as mean ± standard deviation (SD), while t-test methods were used for comparison between two groups, with one‐way analysis of variance used for comparison among multiple groups. The inspection level of α=0.05, P<0.05 were considered to be statistically significant.