Animals and ethical considerations
All animal procedures were followed in accordance with the guidelines of the Institutional Animal Care and Use Committee of China. The experiments were approved by the Ethics Committee of School of Stomatology, the Fourth Military Medical University. A total of thirty 8- to 9-week-old male Sprague-Dawley (SD) rats (Chengdu Dossy Experimental Animals Co., Ltd., Chengdu, China) were acquired for ADSCs isolation and animal experiments. SD rats were kept in the same pathogen-free conditions with the temperature of 25°C, 55% humidity, 12h of light alternating with 12h of darkness.
Isolation, culture, and identification of rat ADSCs
ADSCs were surgically isolated from the subcutaneous adipose tissue from the inguinal area and digested with an equal volume of 0.2% type I collagenase at 37°C for 60 minutes. Followed by filtering with a 200-mesh and centrifuged at 1000g for 5 min. After removing the supernatant, precipitate was resuspended with complete alpha-Mem (10% Fetal Bovine Serum from Gibco, USA, and 1% penicillin/streptomycin from Hyclone, USA) and cultured in T75 culture flasks in an incubator (37°C, 5% CO2). The culture medium was changed every 48 h and cells were passaged after reaching 80% confluence. The cells used in the following experiments were collected from passage 3. Cell morphology was photographed under an inverted phase-contrast microscope. To identify the characteristic of ADCSs, cell surface marker CD29, CD44, CD45, CD105 were identified by flow cytometry.
HIF-1α siRNA and cDNA transfection
Small interfering RNA (SiRNA) transfection was performed for HIF-1α gene silencing using SiRNA from GenePharma, with the sequence of GGGCCGUUCAAUUUAUGAATT (si-HIF-1α). ADSCSs were cultured on 6-well plates in complete alpha-MEM until reaching 60–70% confluence. Lipofectamine 3000 (Invitrogen, Waltham, MA) was used according to the manufacturer’s protocol to transfect HIF-1α siRNA Silencer (si-HIF-1α) or nontargeting siRNA as negative control (si-NC). After 6–8 hours incubation, the transfection media were changed to 100µmol/L CoCl2 media (2% O2) to explore the role of HIF-1α in ADSCs under hypoxia. Total RNA and protein were collected at 24 hours of cultivation. Plasmids containing HIF-1α cDNA (HIF-1α) or empty plasmid (pEX) was transfected into ADSCs using Lipofectamine 3000 (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. After 24h of transfection, the cells were washed with PBS, and the medium was replaced with complete-Alpha medium for the rest of experiments. Total RNA was extracted after 48h of transfection for qRT-PCR assays to detect the transfection efficiency of HIF-1α in ADSCs.
Cell proliferation assays
ADSCs were seeded into 96-well culture plates at a density of 3 x 103 cells per well and received different treatment according to the experimental purpose. Cell-counting kit-8 (CCK-8; 10µl per well; PCM, Xi’an, China) solution was used to assay the proliferation on hours 0, 24, 48, 72. Three repeated experiments were conducted, and the optical density of the medium was detected at 450nm using a multi-function enzyme labeling instrument (Thermo Fisher Scientific, Waltham, MA, USA).
Scratch wound healing assay and Transwell assay
The migration ability of ADSCs were measured using Scratch wound healing assay and Transwell assays (8.0µm pore size; Corning-Costar). For scratch assay, 1x106 cells per well of ADSCs were seeded into 6-well culture plates and received various treatment according to the experiment design. After reaching 100% confluence, a 200µL pipette tip was used to starch the monolayer and washed with PBS to rinse off floating cells. The cells were then cultured in serum-free alpha-MEM and photographed at 0, 12, and 24 h. For Transwell migration assay, a total of 2 x 105 ADSCs cells in 200µl serum-free alpha-MEM were added to the upper chamber, 800µl complete alpha-MEM was added in the bottom chamber. After 24h of cultivation, the migrated cells on the bottom chamber were taken out and were fixed using 4% PFA for 15 min and stain with 0.5% crystal violet for further evaluations.
Alkaline phosphatase (ALP) and alizarin red staining (ARS)
ALP activity was assayed using a BCIP/NBT alkaline phosphatase color development kit (Beyotime Biotech, Shanghai, China) and alkaline phosphatase (ALP) assay kit (Jiancheng, Nanjing, China) was purchased for quantification of ALP activity following the manufacturer’s protocol. Calcium deposits were detected by staining with 2% alizarin red S (Solarbio, Beijing, China). To quantify the stained nodules, solubilized stain was transferred to wells of a 96-well plate and measured at 620 nm of absorbance, all data are presented as means (n = 3).
ADSCs adhesion test and immunofluorescent staining
Prior to the cell adhesion test, prepared titanium sheets with a diameter of 12mm and 2mm in thickness (n = 3 in each group) were immersed in 5ml of 95% alcohol for 48 hours and place under ultraviolet irradiation for complete disinfection. Transfected ADSCs (3 x 103/well) were centrifuged and seeded into 24-well cultured plates with titanium sheets already placed in the wells. After 24 hours, the cultured medium was removed, and titanium sheet samples were washed with PBS buffer for 3 times. Samples were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized, and visualized using double fluorescent staining for cell cytoskeleton (AlexaFluro635phalloidin dye, Invitrogen) in green and nuclei (DAPI dye) in blue. The amount of adhesion cells, morphology, and spreading of ADSCs were observed under the confocal laser scanning microscopy (OLYMPUS, Tokyo, Japan).
Tube formation assay
HUVECs were grown and transfected with small interfering RNA of HIF-1α (si-HIF-1α), and plasmids containing HIF-1α cDNA (HIF-1α) for 24 h and then HUVECs (3 × 104 cells per well) were seeded on 96-well plate. Each well was coated with 200 µl matrigel (BD Biosciences, San Jose, CA, USA) and HUVECs were cultured for 18 h at 37°C in 5% CO2. Capillary-like structures were evident and counted using a phase-contrast microscope and the networks formed by HUVECs were quantified with VIDEOMET software (Videojet Technologies Inc., Chicago, IL, USA). Quantitation of the angiogenic activity on tube formation was performed by counting the number of junctions and total branch points. Three independent assays were performed. Data are summarized as means ± SD.
Quantitative real time qPCR
To determine the relative mRNA expression levels of HIF-1α, ALP, RUNX2, type I collagen (COL-I), BMP2 in ADSCs, 1x106 cells per well of ADSCs were seeded into 6-well culture plates and incubated with various treatment 24 h prior to total RNA extraction. cDNA was reverse transcribed from the extracted RNA (Applied Biosystems, Foster City, CA, USA) and SYBR Premix ExTaq II (TaKaRa, Tokyo, Japan) along with StepOne Plus Reat-Time PCR system (were used to performed qPCR analysis. The fold change in the mRNA expression levels of each target mRNA was calculated using the 2△△CT method, normalized to the internal reference glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of all primers (Sangon Biotech, Shanghai, China) used in the present study are shown in Table S1.
Protein extraction and Western blotting
Total proteins were lysed and extracted using RIPA buffer (ZHHC, China) after seeded (1x106 cells/well, 6-well culture plates) and incubated with various treatment for 24 h. BCA Protein Assay Kit (Beyotime Biotechnology, China) was used to quantify the protein concentration. All primary antibodies used for this study were obtained from Abcam Biotechnology (MA, USA), and listed as follows: anti-HIF-1α (1:2000, ab179483), anti-GAPDH (1:10000, ab181602), anti-ALP (1:2000,ab307726), anti-RUNX2 (1:2000, ab236639), anti-type I collagen (COL-I) (1:2000, ab260043), anti-BMP2 (1:2000, ab284387), anti-p-AKT (1:2000, ab81283), anti-AKT (1:2000, ab8805), anti-mTOR (1:2000, ab134903), anti-p-mTOR (1:2000, ab109268). The membrane was incubated at 4°C, and then was rinsed three times in TBS-T (5 min/wash) at RT. After 1 h incubated with a secondary antibody (Boster, Wuhan, China) at RT, followed by TBS-T rinsing, visualization was done by a chemical discharge imaging system (Bio-Rad GelDoc XR+, Hercules, CA, USA).
Fabricating ADSC Sheets.
The ADSCs of third generation were seeded at 1 × 106 cells/well in a plate containing 6 wells. After reaching about 90% confluence, the sheet-forming inducing medium was used instead of the basic medium. The composition of the culture medium is α-MEM (Gibco, USA), 10% bovine fetal serum (Sijiqing, China), 1% penicillin/streptomycin (HyClone, USA), and 50 mg/mL vitamin C (NCM, China). ADSCs were cultured in sheet-inducing medium for 10 days, and the nutrient solution was replaced every 3 days. When the curly edge appeared at the plate rim, the whole cell sheets were peeled off with a scraper(50).
Surgical procedure and treatment
The experiments were approved by the Ethics Committee of School of Stomatology, the Fourth Military Medical University (K9-2022-004). The guidelines of the Institutional Animal Care and Use Committee of China was followed. The experiment consists of four groups (n = 5 in each group): the (1) Blank controlled group: rats receiving only implant placement, the (2) si-HIF-1α ADSCs-sheets-wrapped group, SD rats receiving ADSCs sheets transfected with silencing HIF-1α wrapped around implants and subsequent implant placement, the (3) pEX ADSCs-sheets-wrapped group, SD rats receiving ADSCs sheets transfected with non-targeting HIF-1α wrapped around implants and subsequent implant placement, and the (4) HIF-1α ADSCs-sheets-wrapped group, SD rats receiving ADSCs sheets transfected with overexpressed HIF-1α wrapped around implants and subsequent implant placement. The implants used in this study adopted from Kontour Medica (Xi’an, China) with a length of 5 mm and a diameter of 2 mm. The rats received general anesthesia with 1% pentobarbital solution (45mg/kg rat weight). The animals were placed on a heating pad for maintenance of body temperature. After shaving the hind limb, the femoral condyles were exposed by longitudinal incision on the lateral side of the knee joint. The hole was created in the femoral condyles parallel to the long axis of the femora, and implants were first wrapped with transfected ADSCs sheets then placed into the holes. The incisions were sutured in layers carefully. At the tenth and third day before sacrifice, a sequence of alizarin red S (30mg/kg) and calcein (20mg/kg) (Sigma-Aldrich, St, Louis, MO) was administrated intraperitoneally. Animals were sacrificed after 4 weeks after implantation and the femurs were harvested for radiographic and histological analysis.
Micro-CT analysis
The specimens were fixed overnight in 70% ethanol (n = 3 in each group) and scanned by microcomputed tomography (Siemens Inveon, Erlangen, Germany) to determine alterations in the peri-implant tissue. The region of interest (ROI) of 40µm away from the implant surface were reconstructed into three-dimensional structures to analyze the related morphometric parameters of bone around implants, which including the bone volume percentage (BV/TV, %), trabecular thickness (Tb. Th, mm), and trabecular number (Tb. N, 1/mm), and trabecular separation (Tb. Sp, mm).
Sequential fluorescence labeling and histological
The specimens were undergone through a series of ethanol gradient dehydration (75–100%) and were infiltrated in methyl methacrylate, then embedded in poly-methyl-methacrylate resin (n = 3 in each group). A hard tissue slicer (Leica SP1600, Nussloch, Germany) was used to cut each specimen through the center of the implant parallel to their long axis, each section was roughly 300µm in thickness. The slices were then grinded and polished to a sheet of 80 µm and can be directly observed under the confocal laser scanning microscopy (OLYMPUS, Tokyo, Japan) with different wavelengths of spectral excitation fluorescence. To quantify the speed of osteoid mineralization during the observation period in between four groups, the mineral apposition rate (MAR) was measured.
Histological evaluation
Methylene Blue acid fuchsin staining (VG staining), toluidine blue staining, Masson-trichrome staining, and Safranin O staining were performed on the undecalcified sections to revealed new bone formation (n = 3 in each group).
Statistical analysis
Quantitative data was described as means ± SD of at least three independent experiments and statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Images were analyzed using Image J, Image Pro. Quantitative data across all groups were analyzed using one-way analysis of variance (ANOVA) and t-test. P value less than 0.05 indicated statistical significance. The comparison results were labeled with * for P-value < 0.05; ** for P-value < 0.01 and *** for P-value < 0.001.