- Culture and differentiation induction of AD-MSCs
1.1 Primary culture of AD-MSCs
Fresh adipose tissue was cut into pieces and digested with type I collagenase. After centrifugation, resuspension, and filtration, the obtained cells were added to DMEM/F12 complete culture medium, and the resulting P0 culture was placed in a 37°C, 5% CO2 incubator with saturated humidity. Cell growth and cell morphology were observed daily under an inverted phase-contrast microscope. The P0 AD-MSCs were digested with trypsin-EDTA, and the digestion was ended when the cells became oval and the intercellular space became larger. The adherent cells were suspended, added again to DMEM/F12 complete culture medium, and inoculated into a new culture bottle at a 1:3 ratio with complete culture medium with 10% fetal bovine serum. This became the P1 culture. Cells were later passaged using the same method.
1.2 Immunophenotype identification of AD-MSCs
AD-MSCs (P3) at 80% confluence were digested with 0.25% trypsin-EDTA. Cells were resuspended in phosphate-buffered saline (PBS) solution and distributed into six 0.5 ml Eppendorf tubes. Phycoerythrin (PE)-labeled rat anti-human cluster of differentiation (CD) 29, CD31, CD34, CD44, and human leukocyte antigen–DR isotype (HLA-DR) flow cytometry antibodies and immunoglobulin G1 (IgG1) isotype (negative control) were added to separate Eppendorf tubes. After mixing by pipetting, the tubes that had antibodies were incubated for 20 minutes in the dark and at room temperature, followed by a PBS wash, resuspension of the cell pellet in 0.5 ml PBS, and detection of the immunophenotypes of AD-MSCs in a flow cytometer.
1.3 Drawing the growth curve of AD-MSCs
AD-MSCs (P3) at 80% confluence was digested with 0.25% trypsin-EDTA, the density of AD-MSCs in the cell suspension was adjusted to 3 × 105 cells/ml, and the cell suspension was added to six wells of a 96-well plate at 100 μl/well that were included in the experimental group, while one well received 100 μl of complete medium as the blank control. A total of eight 96-well plates were inoculated. One 96-well plate was taken every 24 hours, 10 μl 0.5% 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, and the plate was incubated again for 4 hours and then removed again. DMSO was then added to the plate at 100 μl/well, and a microplate reader was used to measure the optical density (OD) value of each well at 490 nm. The wells that only had medium were the blank control group, which was used to adjust the baseline, and the data of six experimental wells were recorded and the average value was taken. The rest of the 96-well plates remained in the cell culture incubator, and the medium was changed every 2-3 days. One 96-well plate was taken every 24 hours to repeat the above steps. After recording the data for 8 days, the cell growth curve was drawn based on the average OD value obtained every day, and the shape of the cell growth curve was observed.
1.4 Determination of AD-MSC cell cycle
AD-MSCs (P3) at 80% confluence were digested with 0.25% trypsin-EDTA. After centrifugation, the supernatant was aspirated, and 1 ml of 70% ethanol stored at 4°C overnight was added to resuspend the cell pellet by pipetting. The cells were fixed in a refrigerator at 4°C overnight. On the second day, the cells were centrifuged for 5 minutes, the supernatant was aspirated, and the cells were resuspended by adding PBS at 4°C. After centrifugation again, the cell cycle detection kit was used to stain the fixed cells, and the cells were detected by flow cytometry to analyze the cell cycle of AD-MSCs.
1.5 Induction of osteogenic differentiation of AD-MSCs
AD-MSCs (P3) at 80% confluence were digested with 0.25% trypsin-EDTA and seeded at a density of 1.5 × 103 cells/ml into a six-well plate. After adding the cell suspension, the six-well plate was placed in a cell incubator. After 24 hours, the six-well plate was removed. After observing that most of the cells became adherent under an inverted phase-contrast microscope, the culture medium was aspirated and replaced with 2.5 ml of osteogenic induction medium. The six-well plate was placed in the cell incubator to continue the culture. The osteogenic induction medium was routinely replaced every 2-3 days. After 3 weeks of induction, AD-MSCs were stained with alizarin red S staining solution to observe the formation of calcium nodules.
1.6 Induction of adipogenic differentiation of AD-MSCs
AD-MSCs (P3) at 80% confluence were digested with 0.25% trypsin-EDTA and seeded at a density of 1 × 104 cells/ml into a six-well plate with 2.5 ml of cell suspension per well. The plate was placed in a cell incubator, and the medium was routinely replaced every 2-3 days. After AD-MSCs reached 100% confluence, 2.5 ml of adipogenic induction medium A was added. After 3 more days of culture, the adipogenic induction medium A was discarded and replaced with adipogenic induction medium B. After 24 hours of culture, the adipogenic induction medium B was replaced with the induction medium A, followed by incubation in a cell incubator for 3 days. After 12 days of repeating the above medium switches, media A and B each had been replaced three times. After observing obvious lipid droplets under an inverted phase-contrast microscope, oil red O staining was used to observe the results of adipogenic differentiation.
1.7 Induction of endothelial differentiation of AD-MSCs
The digested P3 suspension was added to a 15-ml sterile centrifuge tube and centrifuged at 1000 r/min for 5 minutes. The upper layer in the centrifuge tube was discarded, the cells were washed with 1 × PBS and centrifuged again, and then the endothelial cell support solution 100% EGM2-MV was added to suspend the cells. After resuspension, the cells were added to a culture flask. The morphological changes of the cells were continuously observed every day using an inverted phase-contrast microscope. The culture medium was replaced every 3 days, and the cell growth was closely observed. After 4 weeks, CD31-PE was used to label cells that were induced and cells that were not induced to undergo differentiation. Immunophenotypes were identified by flow cytometry.
- Preparation of functionalized self-assembling nanopeptide hydrogel and three-dimensional culture of combined AD-MSCs
2.1 Preparation of peptide solution and detection by atomic-force microscopy
The three peptide powders were dissolved in a 10% glucose solution to obtain three kinds of peptide solutions, each having a mass concentration of 10 g/L (1%). The peptide was completely dissolved by ultrasound treatment. The aqueous solutions of RADA16-I polypeptide, KLT functionalized polypeptide, and arginylglycylaspartic acid (RGD) functional polypeptide were mixed at a volume ratio of 2:1:1 and mixed well by ultrasound to obtain a functionalized self-assembling polypeptide solution (RADA/KLT/RGD). After diluting 100-fold and mixing by ultrasonic treatment, 10 μl of the sample was dropped onto a freshly peeled mica sheet, dried, and observed under an atomic-force microscope.
2.2 Preparation of functionalized self-assembling nanopeptide hydrogels and observation of the microstructure of the gels by scanning electron microscopy
A Transwell chamber was used to complete the self-assembly of the peptide solution. The chamber was placed in a 24-well culture plate with PBS and incubated at 37°C overnight, in order to permeate the basement membrane. Then, 100 μL of the functionalized self-assembling peptide solution (RADA/KLT/RGD) was added into the chamber, the chamber was removed after gelation, and the basement membrane was cut off to remove the hydrogel, which was fixed in 2.5% glutaraldehyde and dehydrated to prepare samples for scanning electron microscopy.
2.3 Three-dimensional culture of functionalized self-assembling nanopeptide hydrogel combined with AD-MSCs
After digesting the P3 AD-MSCs, the cell suspension was adjusted to 1 × 106/ml, and 100 μL of the suspension was inoculated into the hydrogel soaked with the culture medium. After overnight incubation, the hydrogel was rinsed. After 5 days of culture, the morphology and migration status of AD-MSCs were observed under an inverted phase-contrast microscope. The hydrogel in the chamber was then digested with digestive enzymes and quickly stained with acridine orange/ethidium bromide (AO/EB) for 30 seconds, followed by observation under a fluorescence microscope to exam the conditions of cell growth and apoptosis and to count cells.
- Establishment of a rabbit hindlimb ischemia model and stem cell transplantation
3.1 Rabbit hindlimb ischemia model and angiography
Each rabbit was anesthetized with 3% sodium pentobarbital solution. After disinfection, the operation area was covered with sterile drapes. The skin from 1 cm above the midpoint of the inguinal ligament to the knee joint line was longitudinally cut, the femoral artery was dissected, the arterial collaterals were ligated separately from the inguinal ligament to the knee, and the femoral artery in this area was resected. A longitudinal incision was made in the midline of the neck, slightly leading towards the right side, to cut open the skin and subcutaneous tissue. A section of the common carotid artery approximately 2 cm long was dissected and placed with an arterial sheath entering the level of the aortic arch. Then 10 ml of contrast agent was quickly injected. Digital angiography of bilateral hindlimbs was conducted for at least 10 seconds to observe the arteries of the hindlimbs of rabbits after operation.
3.2 Transplantation of functionalized self-assembled nanopeptide hydrogel-loaded AD-MSCs into the rabbit model of hindlimb ischemia
At 48 hours after operation on rabbit hindlimbs, rabbits in the experimental group (group A) were transplanted with the self-assembled polypeptide hydrogel combined with AD-MSCs at multiple sites in the right gastrocnemius muscle. Rabbits in the positive control group (group B) received multi-point injections of a suspension of PBS-washed AD-MSCs at the same location and with the same number of cells. Rabbits in the negative control group (group C) were injected with the same amount of PBS at the same sites. Limb movements, skin color, and the presence or absence of skin ulcers, loss of toenails, and limb gangrene in the hindlimbs on both the operation side and the healthy side were observed daily, and blood pressure, skin temperature, and arterial pulsation were measured and recorded.
3.3 Histological examination after stem cell transplantation
At 30 days after AD-MSC transplantation, muscle samples were taken from the same sites of both hindlimbs (gastrocnemius muscles) and sectioned for hematoxylin-eosin (HE) staining. We observed whether the muscle tissues had necrosis, whether the muscle fibers were arranged neatly, and whether the muscle space was widened under the microscope. Immunofluorescence staining of factor VIII was conducted on the abovementioned frozen sections. The sections were fixed in 4% paraformaldehyde solution, dried, permeabilized, and blocked in 5% goat serum blocking solution. The sections were then added to factor VIII primary antibody diluted 1:100 and FITC-labeled secondary antibody in drops and incubated in a humidified box at room temperature. The slides were sealed after drying, and the number of cells positive for immunofluorescence staining in a single field were counted under a fluorescence microscope.
The recorded experimental data were processed using SPSS18.0. The data are expressed as the mean ± standard deviation and were analyzed using pairwise comparisons in analysis of variance (ANOVA) within SPSS. The test standard for statistical significance was P < 0.05.