Synthesis of photocrosslinkable polymers

Methacrylated HA (MAHA) was synthesized as previously reported25. Briefly, to prepare MAHA with a degree of substitution (DS) of 50%, methacrylic anhydride (8 mL, Sigma-Aldrich, St. Louis, MO) was added to a 1% w/v HA (Mw 500 kDa, Bioland, Korea) aqueous solution. The reaction mixture was allowed to react for 24 h with stirring at 4 °C after adjusting the solution pH to 8.0 using a 5 N NaOH solution. The reaction mixture was dialyzed against deionized water (DW) using a membrane with a cutoff molecular weight of 100 kDa for 3 days followed by lyophilization. 1H-NMR spectroscopy was used to determine the DS on HA. The DS was calculated from the ratio of the relative peak integrations of the methacrylate protons (peaks at ~6.1, 5.6, and 1.85 ppm) and methyl protons of HA (~1.9 ppm).

Methacrylated gelatin was prepared following the previously developed method26. Briefly, gelatin type B (4 g, Sigma-Aldrich, St. Louis, MO) was dissolved in phosphate-buffered saline (PBS, 10% w/v) at 60 °C and functionalized with methacrylic anhydride (3 mL) at pH 7.5 at 50 °C for 1 h. The reaction was then stopped by adding pre-warmed PBS. After dialysis against DW and lyophilization, the DS of methacrylated gelatin was measured as 90% using 1H-NMR spectroscopy.


Preparation of hydrogels

To prepare HA, gelatin, poly(ethylene glycol) (PEG) hydrogels, and polymer solutions of MAHA (0.5% w/v), methacrylated gelatin (7% w/v) and PEG diacrylate (Sigma-Aldrich, St. Louis, MO, 10% w/v) in PBS with Irgacure 2959 (final concentration 0.2% w/v) were prepared, and 40 μL of each polymer solution was placed in polydimethylsiloxane molds having a diameter of 5 mm and a depth of 2 mm and were exposed to UV light (365 nm, 60 mW/cm2, Sei Myung Vactron Co. LTD, Korea) for 10 s for MAHA and 50 s for methacrylated gelatin and PEG diacrylate.


Mechanical characterization of hydrogels

To characterize the mechanical properties of various types of hydrogel, we used a customized bulk-scale indenter consisting of a load cell (GS0-10, Transducer Techniques) and an automated stage (SM2-0803-3S and SZ-0604-3S, ST1) equipped with a microscope (AM4113, AnMo Electronics Corporation). A stainless steel spherical tip with a 2 mm diameter was attached to the load cell for indentation. The contact between the tip and the hydrogel was assumed to be Hertzian contact27. The maximum indentation depth was ~5% of the hydrogel thickness, and the indentation speed was 25 μm s−1. Both the applied force and the indentation depth were recorded at an acquisition rate of 10 Hz during the experiment. The effective modulus of the hydrogel was estimated by fitting a force-indentation curve to the Hertzian model given by equation (1)

$$F = \frac{4}{3} \cdot \frac{E}{{1 – \nu ^2}} \cdot \sqrt r \cdot \delta ^{\frac{3}{2}}$$

(1)

where F is the applied force, E is the Young’s modulus of the hydrogel, ν is the Poisson’s ratio of the hydrogel, which is assumed to be 0.5, r is the radius of the indentation tip, and δ is the indentation depth.


Isolation and expansion of hASCs

hASCs were isolated from adipose tissues in the articular fat pads around the patients’ knee28 with approval of the Ethics Committee at CHA University (IRB No. 2014-07-096). In brief, the adipose tissues were washed three times with phosphate-buffered saline (PBS) containing 2% v/v penicillin/streptomycin and digested using 0.5 mg/mL of collagenase type II (Sigma-Aldrich, St. Louis, MO) in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT) for 45 min at 37 °C with constant shaking. Digested tissues were collected in 50 mL conical tube, centrifuged (1000 × g, 10 min), and then supernatant fat was discarded, and the infranatant fluid was filtered through a cell strainer (pore size 40 μm). The filtered fluid was washed three times with DMEM, centrifuged (1000 × g, 10 min), and the cell pellet was resuspended with growth medium (DMEM supplemented with 20% v/v fetal bovine serum (FBS), and 1% v/v penicillin/streptomycin), and the cells were cultured on tissue culture plates at 37 °C in a 5% CO2 incubator. The culture medium was changed every 3 days.


Retroviral transduction

The day before transduction, Gp2 cells (Clontech) were seeded at a concentration of 3 × 106 cells per 100 mm dish. pMX-GFP (Addgene, Cambridge, MA) was transfected with retrovirus packaging vector VSV-G (Invitrogen) and Convoy transfection reagent (ACTGene Inc., Piscataway, NJ). A day after transfection, the medium was replaced with 10 mL of fresh media. After 48 h of transfection, the supernatants were collected, centrifuged at 1300 rpm for 3 min, and filtered with a 0.45 μm filter (Millipore). Then, retro-X concentrator was added at a volume equal to 1/3 of the media. The mixture was incubated at 4 °C overnight with constant inversion. After centrifugation at 1500 × g for 45 min, the concentrated pellet was resuspended with fresh hASC culture media. A day before infection, cell cycle regulated hASCs from the 2D and 3D systems were seeded into 6-well plates at a density of 3 × 104 cells per well. For infection, 2 mL of concentrated retrovirus and 1 μL mL−1 polybrene (final concentration 8 μg mL−1; Sigma-Aldrich) were added to the cells. After incubation for 24 h, the medium was replaced with 2 mL of fresh growth medium. Three days after, green fluorescent protein (GFP) fluorescence images of the infected cells were observed with a Nikon TE2000-U fluorescence microscope (Nikon, Japan).


Cell-cycle regulation on 2D tissue culture plate

To determine the cell-cycle regulation on 2D tissue culture polystyrene (TCPS) plates, we followed the conventional cell cycle regulating method of serum starvation followed by the addition of serum16,29,30. For cell cycle arrest at the G1 phase, hASCs were seeded at a density of 3 × 105 cells per well in a 6-well plate and cultured with growth medium for 24 h. Cells were washed with PBS, and serum-free medium (0.5% v/v FBS) was added. After serum starvation for 1, 3, 6, 12, 24, or 48 h, cells were passaged and released into cell cycle by the addition of growth medium.


3D primed cell-cycle regulation

For 3D primed cell cycle arrest, harvested hASCs were suspended in MAHA solution (final concentration 0.5% w/v) at a density of 2 × 106 cells mL−1, UV irradiated in the presence of Irgacure 2959 (final concentration 0.2% w/v), and cultured in growth medium at 37 °C in a 5% CO2 incubator. After 0.5–24 h of incubation, the cells were released from the hydrogels by treatment with hyaluronidase (2000 U mL−1 in growth media, Sigma-Aldrich) for 40 min at 37 °C. Released hASCs from the 3D physical cue were seeded on tissue culture plates and cultured with growth medium.


Cell-cycle analysis

At each time point, cells were collected and stained with propidium iodide (PI, Sigma-Aldrich). Briefly, cells were harvested by treatment with trypsin or hyaluronidase in the case of the 2D or 3D system, respectively. Cells were collected and washed with PBS by centrifugation at 1000 × g. Collected cells were fixed with cold 70% ethanol for at least 10 min and washed with PBS three times. Fixed cells were resuspended in 200 μL of PBS containing 100 μg mL−1 of RNase A (Thermo ScientificTM, Lithuania, EU) and 10 μL of PI solution (1 mg mL−1), and the cell suspensions were then held for 15 min in the dark at room temperature. The DNA content of individual cells was examined by flow cytometry (BD AccuriTM C6, BD Biosciences, San Jose, CA).


Apoptosis assay

FITC Annexin V and PI double labeling was used to determine the apoptosis-inducing effect of the cell cycle arrest at the G1 phase. Briefly, cells were harvested by treatment with trypsin 24 and 48 h after 2D arrest or with hyaluronidase 1 h after 3D primed arrest. Collected cells were resuspended in 100 μL of 1 × Annexin V binding buffer (BioLegend, San Diego, CA). Then, 5 μL of FITC Annexin V (100 μg mL−1, BioLegend, San Diego, CA) and 5 μL of PI solution (1 mg mL−1, Sigma-Aldrich) were added to the cells. Fifteen minutes after incubation in the dark at room temperature, the stained cells were washed with PBS three times. The percentage of apoptotic cells was measured by flow cytometry (BD AccuriTM C6, BD Biosciences).


Gene expression analysis

To confirm cyclin D1 gene expression, the total cellular RNA was extracted using TRizol (Ambion) reagent. PrimeScript RT Reagent Kit (Perfect Real Time, TAKARA) was used to synthesize complementary DNA from 1 μg of the total RNA. For quantification of gene expression level, qRT-PCR was performed using Power SYBER Green PCR Master Mix (Applied Biosystems) with a StepOnePlus Real-Time PCR System (Applies Biosystems). The PCR condition was 40 cycles at 95 °C for 15 min and 60 °C for 1 min, and the melting curve stage was 95 °C for 15 min and 60 °C for 1 min. The PCR primer sequences are shown in Supplementary Table S1. Target gene expression was normalized to the 18 s (housekeeping gene) gene for quantification.


Statistical analysis

All statistical analyses were implemented with Graphpad prism ver. 5.0 (Graphpad software, San Diego, CA). One-way ANOVAs with Tukey’s multiple comparison posttest were implemented to compare the samples. Statistical significance was set at p < 0.05.

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