Materials
Tri-PCG and tri-PCG-acryl were synthesized according to previously reported methods30. The materials for the synthesis and synthetic details are provided in the Supplementary Information (SI). A LabAssay ALP kit were purchased from FUJIFILM Wako Pure Chemical Co., Ltd. (Osaka, Japan). Water was purified using a Millipore Elix UV3 Direct-Q UV system (Merck, Darmstadt, Germany). Dulbecco’s phosphate-buffered saline (PBS) (−) was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F12), collagenase type Ⅷ, 1% BSA solution, and Coomassie protein assay reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ethylenediamine-N,N,N',N'-tetraacetic acid and disodium salt (EDTA×2Na) were purchased from Dojindo Laboratories (Kumamoto, Japan). The AdSC culture medium (KBM ADSC-1) and a lipid assay kit were purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Biowest (Nuaillé, France). FITC anti-mouse CD31, CD45, and CD90.2, FITC anti-mouse/human CD44, FITC anti-mouse/rat CD29, and PE anti-mouse CD34 antibodies were obtained from BioLegend (San Diego, CA, USA). NucleoSpin RNA and a PrimeScriptTM RT Reagent Kit (Perfect Real Time) were purchased from Takara Bio Inc. (Shiga, Japan). SsoAdvancedTM Universal SYBER Green Mix was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Hank’s balanced salt solution (HBSS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). StemXvivo® Osteogenic Supplement, Osteogenic/Adipogenic Base Media, and Adipogenic Supplement were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).
Animals
C57BL/6N mice (9 week-old, male) were purchased from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan). AdSCs were collected from the mice as reported previously43,49. The experiments were performed for mice under anesthesia with isoflurane using a small animal anesthesia station (DS Pharma Biomedical Co. Ltd., Osaka, Japan). The animal experiments were performed following the guidelines for animal experiments listed at Kansai University and were approved by the Ethical Committee for Animal Experiments of Kansai University (7 May 2019 Identification No.1903). This study was carried out in compliance with the ARRIVE guidelines.
Measurement
Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) was performed on a CFX96 deep-well real-time system (Bio-Rad Laboratories, Inc.). FCM was performed using a Gallios flow cytometer (Beckman Coulter, Inc., CA, USA) with an Ar-ion coherent beam laser (488 nm). The UV absorbance of the solutions in a microplate was measured using an iMark microplate reader (Bio-Rad Laboratories, Inc.).
Evaluation of surface expression markers for AdSC
AdSCs were collected from 9-week-old male C57BL/6N mice (SHIMIZU Laboratory Supplies Co., Ltd., Kyoto, Japan), as reported previously43,49. We then confirmed the expression of AdSC-specific surface markers in these cells. Mice were euthanized by blood removal from the epigastric cavity using a syringe with a 26–29G needle under isoflurane anesthesia. Subcutaneous inguinal adipose tissue was collected and placed on a culture dish (f = 3.5 cm). The adipose tissue was cut into small pieces with scissors and placed in a 15 mL centrifuge tube. HBSS (3 mL) containing BSA (1%) and collagenase VIII solution (2 mg/mL) were added to the tube at 37 °C. After incubation at 37 °C for 30 min with inversion and mixing, the tissue was added with 3 mL of DMEM/F12 medium containing 10% FBS to stop the enzymatic reaction. The top fat layer was removed by suction, and the supernatant was passed through a cell strainer (40 μm, BD Bioscience, Franklin Lake, NJ, USA) and collected in a 50 mL tube. Centrifugation (1,200 rpm × 5 min) was performed to remove the supernatant. The cell pellet was suspended in PBS (−) containing 1 mM EDTA (1 mL) up to a volume of 10 mL. Centrifugation was performed once more, and the supernatant was removed. The cell pellet was suspended in DMEM/F12 or KBM ADSC-1 containing 10% FBS, seeded in culture dishes, and cultured in a humidified atmosphere containing 5% CO2 for 3 d at 37 °C to collect AdSCs as adherent cells.
The cell suspension (2 × 105 cells/500 μL) was centrifuged (400× g) at 4 °C for 5 min, and the supernatant was removed. Antibody solution (100 μL) was added to the cells. Incubation in an ice bath was then performed for 30 min. FITC anti-mouse/rat CD29 (0.5 mg/mL), FITC anti-mouse/human CD44 (0.5 mg/mL), and FITC anti-mouse CD90.2 (0.5 mg/mL) antibodies were used as positive markers, whereas FITC anti-mouse CD31 (0.5 mg/mL), PE anti-mouse CD34 (0.2 mg/mL), FITC anti-mouse CD45 (0.5 mg/mL) antibodies were used as negative markers. After incubation, the cells were washed twice with PBS(−), added with PBS (500 µL), and then subjected to FCM measurements.
AdSC culture in the IP hydrogels and quantification of gene expression
The preparation methods of the IP formulation (tri-PCG/DPMP + tri-PCG-acryl mixture containing 15–25 wt% of tri-PCG-acryl in the total polymer) and confirmation of the sol-gel transition of the IP formulation are described in the SI. The IP formulations (tri-PCG only or a mixture of tri-PCG-acryl and tri-PCG/DPMP aqueous solutions) were sterilized by filtration. The AdSC suspension (100 μL) and IP formulation (100 μL) were mixed and dispensed into sterilized Eppendorf tubes. After gelation by incubation at 37 °C for 30 min, KBM ADSC-1 (400 µL) was gently placed on top of the IP gel containing AdSCs. The cells were then further incubated in a humid atmosphere containing 5% CO2 at 37 °C. An aliquot of the supernatant (200 μL) was replaced with fresh KBM ADSC-1 every 2 d. Next, the IP gel was broken, and the homogeneous suspension was obtained by repeated pipetting with PBS(−) (3 mL). The cells were collected by centrifugation. This operation was repeated thrice to remove the IPs and DPMP.
mRNA and RT-qPCR extraction were performed using NucleoSpin RNA and PrimeScrip RT reagents, respectively, according to the manufacturer’s instructions. The mRNA levels of OCT452, Nanog53, and Sox254 as undifferentiated markers and AFP55, Brachyury56, and Pax657 as embryonic markers were quantified using the Bio-Rad CFX96 Deep Well Real-Time System, SsoAdvanced Universal SYBR Green Supermix, and primers obtained from Eurofin Genomics LLC (Louisville, KY, USA) (see SI). GAPDH was used as the housekeeping gene in all experiments. Relative RNA expression was determined using equation (1):
Rel ExP = 2−DCt, where DCt = Ct of the gene of interest – Ct (1)
Differentiation of AdSCs into osteoblasts in the IP hydrogel (Protocol (1))
Tri-PCG-acryl (20 wt%) and tri-PCG/DPMP (20 wt%) were prepared separately via the protocols described above using osteogenic differentiation medium (StemXVivo) instead of ADSC-1 and then mixed and stirred with a vortex mixer to induce osteogenic differentiation (+). The obtained mixture solution (IP formulation; 75 µL) was sterilized by filtration, mixed with the cell suspension in the same medium (25 µL), and dispensed into a sterilized Eppendorf tube (100 μL). After gelation by incubation at 37 °C for 30 min, the cells were further incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 3 d. The osteogenic differentiation medium (1,000 µL) was gently placed on top of the IP gel containing AdSCs, and the cells were further incubated for 14 or 21 d in a humidified atmosphere containing 5% CO2 at 37 °C. The supernatant was replaced with the medium every 2 d. Next, the IP gel was broken, and the homogeneous suspension was obtained by repeated pipetting with PBS(−) (3 mL). The cells were collected by centrifugation. This operation was repeated thrice to remove the IPs and DPMP. The collected cells were seeded in 24-well plates at a density of 2 × 104 cells/well and cultured in osteogenic differentiation medium. The medium was replaced every few days. AdSCs treated via the same protocol using KBM ADSC-1 instead of osteogenic differentiation medium were also prepared; these cells were considered osteogenic differentiation induction (−) cells. AdSCs cultured on TCPS dishes were prepared using osteogenic differentiation medium (osteogenic differentiation induction (+)) or KBM ADSC-1 (osteogenic differentiation induction (−)). The AdSC suspensions were used for ALP, total protein quantification, and RT-qPCR assays.
Differentiation of AdSCs into osteoblasts on TCPS after incubation in the IP hydrogel (Protocol (2))
AdSCs were incubated in the IP hydrogel via Protocol (1) using KBM ADSC-1 (osteogenic differentiation induction (−)) for 7 d. The obtained cells were seeded in 24-well TCPS plates at a density of 2 × 104 cells/well and cultured for 14 or 21 d in osteogenic differentiation medium (osteogenic differentiation induction (+)). The obtained cells were subjected to ALP assay, total protein quantification, and RT-qPCR assay. The ALP assay and total protein quantification procedures are described in the SI.
Differentiation of AdSCs into adipocyte on TCPS after incubation in the IP hydrogel
The AdSCs were cultured in the IP formulation containing KBM ADSC-1 (without differentiation) for 7 d. The obtained cells were then seeded in 24-well TCPS plates at a density of 2 × 104 cells/well and cultured for 14 or 21 d in StemXvivo® adipogenic differentiation medium. The culture medium was removed, and each well was washed with PBS(−) (500 µL). Neutral buffered formalin solution (10%, 500 µL) was added to the wells for fixation. After incubation at 25 °C for 20 min, the fixed cells were washed with water (500 μL). ORO solution (500 μL) from the lipid assay kit was dispensed into the wells, and the plate was incubated at 25 °C for 15 min. The ORO solution was then removed, and the wells were washed with water three times and dried. The extraction solution from the lipid assay kit (500 μL) was added to the wells, and the solution was incubated at 25 °C for 30 min to extract the dye. Finally, the absorbance of the extracted solution was measured at 570 nm using a spectrophotometer.