Hascs

Cells (hBMMSCs and hASCs) were purchased from ScienCell (San Diego, CA, USA). All materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 100× penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). Cells from three donors at passages 4–6 were used for in vitro experiments. For the study on hASCs, all experiments were repeated at least three times using cells from two donors, and the mean value was calculated from three independent experiments. For the study on hBMMSCs, all experiments were also repeated at least three times using cells from another donor, and the mean value was again calculated from three independent experiments. Proliferation medium (PM) comprised fresh DMEM containing 10% (v/v) FBS, 100 U/mL penicillin G and 100 mg/mL streptomycin. PM supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate and 50 mg/ml L-ascorbic acid constituted osteogenic medium (OM). Culture medium was changed every 3 days.

hASCs and hBMMSCs were purchased from ScienCell Research Laboratories (San Diego, CA, USA), whereas hGFs were obtained from the attached gingiva of human premolars. The cells were cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin for proliferation. DMEM, FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin were purchased form Gibco (Grand Island, NY, USA). For the osteogenic differentiation, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL l-ascorbic acid were added to the medium. Cells were cultured in a controlled environment at 37 °C in an incubator (95% air, 5% CO₂, 100% relative humidity). All subsequent in vitro and in vivo experiments were performed using cells at the third and fourth passage. Moreover, all experiments were carried out in triplicate with cells extracted from three different patients.

P2–P4 of adipose tissue-derived stem cells (hASCs) from ScienCell (Carlsbad, CA, USA) were used for cell cultures. Cells were plated in T75 culture-treated flasks with approximately 1 million cells per flask, and culture media was changed every 3–4 days for the duration of the culture. For the polymer solution, 125 mg gelatin and 125 mg pullulan were dissolved in 50 ml serum-free media. Media was warmed to 37 °C for gelatin dissolution. At the time of electrospinning, each mL of the Pullulan/Gelatin stock solution was mixed with another mL of serum-free media and was added to the cell pellet. Cell electrospraying content was aseptically transferred to a sterile 10 mL syringe, and a sterile 18-gauge syringe needle tip was secured. The collector plate, which was a Petri dish (Fisherbrand, polystyrene), was positioned 7 cm from the end of the needle tip. The syringe pump settings were adjusted to produce readings for a plastic 10 mL syringe pump. The pump volumetric flow rate was set to 200 μL/min. Electrospraying was performed at 10 and 15 kV. Control experimentation was performed without applying any voltages. Some electrosprayed samples were made using PKA-specific inhibitor H89 treated cells. The electrospraying setup is pictured in Supplementary Fig. S5.

Primary human BMMSCs and human adipose-derived mesenchymal stem cells (hASCs) were purchased from ScienCell Company (San Diego, CA, USA). All cell-based in vitro studies were repeated three times using MSCs from three donors. All materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. This study was approved by the Institutional Animal Care and Use Committee of the Peking University Health Science Center (LA2014233), and all experiments were performed in accordance with the approved guidelines.
FBS, MEM, DMEM, and 100× penicillin and streptomycin mixture were purchased from Gibco (Grand Island, NY, USA). Human BMMSCs and ASCs were cultured in proliferation medium (PM), consisting of 10% (v/v) FBS, penicillin/streptomycin, and fresh MEM (for hBMMSCs) or DMEM (for hASCs), with 5% CO₂ atmosphere at 37 °C. The OM comprised fresh DMEM or MEM containing 10 nM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM l-ascorbic acid, 10% (v/v) FBS, and penicillin/streptomycin. TNF-α was purchased from R&D Systems (Minneapolis, MN, USA), and BAY117082 was purchased from Selleck (Houston, TX, USA). Cells at the fourth to sixth passage were used for the in vitro experiments.

Primary human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs) were obtained from ScienCell (San Diego, CA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) or α-minimum essential medium (α-MEM). The proliferation medium (PM) contained 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotics. The osteogenic medium (OM) contained 10% (v/v) FBS, 1% (v/v) antibiotics, 10 nM dexamethasone, 200 μM ascorbic acid, and 10 mM β-glycerophosphate. The adipogenic medium (AM) contained 10% (v/v) FBS, 1% (v/v) antibiotics, 50 nM insulin, 100 nM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine, and 200 μM indomecin.

Primary human adipose derived stem cells (hASCs), human bone marrow mesenchymal stem cells (hBMSCs), and mouse MC3T3-E1 cells were purchased from ScienCell (Carlsbad, CA, USA). Human osteosarcoma U2OS cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China). Cells were cultured using a proliferation medium (PM) consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (Gibco), and 1% penicillin/streptomycin (Gibco). For osteogenic differentiation, hASCs and hBMSCs were cultured in osteogenic medium (OM) containing standard PM supplemented with 0.2 mM ascorbic acid (Sigma, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma), and 100 nM dexamethasone (Sigma). All experiments were repeated at least three times.