Osteogenic induction medium

hDPSCs at P4 were differentiated into adipocytes and osteoblasts as follows in vitro. For adipogenic and osteogenic differentiation, hDPSCs were reseeded in the standard culture medium into 6-well culture plates at 2 × 10⁴ cells per well. The cells were incubated until they are 100% confluent or post-confluent. hDPSCs were exposed to adipogenic induction medium for 28 days. hDPSCs were exposed to osteogenic induction medium (Cyagen) for 28 days. The medium was replaced with fresh induction medium every 3 days. After 7 or 28 days, cells were fixed with 4% (w/v) paraformaldehyde solution. To assess adipogenic differentiation, cells were stained with oil red O working solution. To assess osteogenic differentiation, cells were stained with alizarin red working solution. Control cells were cultured in standard culture medium over the same period of time. The stained plates were visualized under light microscope (Leica DM IL LED Fluo, Leica Microsystems Inc, Germany) and captured images using Leica Application Suite Version 4.5.0 software.

Passage 3 BMSCs were seeded into 6-well plates (2 × 10⁵ cells per well), which were precoated with 0.1% gelatin and incubated for 14 days using a specific osteogenic induction medium (Cyagen Biosciences). For evaluating the effect of ASCs-exos on osteogenic differentiation, 200 μL of ASCs-exos with a concentration of 200 μg/mL and equal volumes of PBS and AEFS was supplemented with the osteogenic induction medium and refreshed every three days. In addition, 500 ng/mL of DKK-1 was applied for investigating the involvement of the Wnt/β-catenin pathway in ASCs-exos by promoting the differentiation of BMSCs. To evaluate the level of osteogenic differentiation, the cells were stained with alkaline phosphatase (ALP) staining and alizarin red staining, and were collected for Western blotting on day 14.

Six 4-week-old male SPF FVB/N mice were sacrificed by cervical dislocation and then placed into 75% ethanol solution for 5 min. The bilateral tibia and femur of the mice were dissected out using sterilized surgical instruments in the laminar flow hood. The metaphyses on both sides were cut off using ophthalmic scissors, and a 2-mL syringe containing PBS was used to flush the bone marrow from the long diaphysis into a 15-mL centrifuge tube. The centrifuge tube was then centrifuged at 500 × g for 15 min, the supernatant was discarded, and the cell pellets were resuspended in PBS. The cell concentration was adjusted to 1 × 10⁸ cells/mL to prepare the single cell suspension, and then separated using density gradient centrifugation.
The confluent passage P3 cells were collected and the expression of CD44 (eBioscience, CA, USA), CD105 (eBioscience, CA, USA), and CD34 (eBioscience, CA, USA) was detected using flow cytometry. The confluent passage P3 cells were cultured either in osteogenic induction medium or in adipogenic induction medium (Cyagen Biosciences Inc, CA, USA) for 21 or 14 days, respectively. Next, cultures were stained with either Alizarin Red or Oil Red O (Cyagen Biosciences Inc, CA, USA) to verify their osteogenic or adipogenic differentiation potential, respectively.

Res (99%) was obtained from Aladdin (Shanghai, China). Polyoxyethylene (40) stearate (Myrj 52) and dimethyl sulfoxide were purchased from Sigma-Aldrich (St Louis, MO, USA). Stearic acid and chloroform were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lecithin, ethylene diamine tetraacetic acid (EDTA), bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI) and 1% Alizarin Red S (ARS) solution at pH 4.1 were obtained from Solarbio Co. (Beijing, China). Cell counting kit-8 (CCK-8) and 4% paraformaldehyde were purchased from Biosharp (Shanghai, China). An osteogenic induction medium was obtained from Cyagen (Guangzhou, China). The alkaline phosphatase (ALP) staining kit was obtained from Yeasen (Shanghai, China). Osteocalcin (OCN) primary antibody, CD31 primary antibody and Cy3-labelled secondary antibody were obtained from Affinity Biosciences (Jiangsu, China). HiScript II QRT SuperMix for quantitative PCR (qPCR) and ChamQ SYBR Colour qPCR Master Mix were obtained from Vazyme (Nanjing, China). Dulbecco’s Modified Eagle’s Medium/Hams F12 (DMEM/F12, 1:1), foetal bovine serum and penicillin-streptomycin were obtained from Gibco (Grand Island, NY, USA). The GelMA hydrogel was obtained from EFL (Suzhou, China). All other chemicals were of analytical grade.