Chondrogenic induction medium

Briefly, 1 × 10⁶ GMSCs were suspended in a 15-ml centrifuge tube at 800 g for 5 min, and the cell pellets were then cultured in chondrogenic induction medium (Cyagen Biosciences) for 3 weeks. The chondrogenic induction medium was also refreshed every 3 days. The cell pellets were then fixed, cut into 4-μm sections and stained with Alcian blue (Yang et al., 2013 (link)).

To evaluate the in vitro osteogenic and chondrogenic differentiation of BMSCs on the gels, the 0.5 GO/PRP gel, which had low biotoxicity and significantly promoted cell proliferation, was selected for follow-up experiments, with the PRP gel as the control. The gels were placed in a 48-well plate and 1 × 10⁵ BMSCs were seeded onto each gel. The cells were cultured in BMSC medium for 3 days, followed by a period in either osteogenic or chondrogenic induction medium (Cyagen, China), as follows. After 3, 7 and 14 days of osteogenic induction, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyze the osteogenic-related gene expression of the samples, as previously described [24 (link)]. After 14 and 21 days of chondrogenic induction, chondrogenic-related gene expression was analyzed using the same method. Osteogenic-related genes included Runx2 and OCN, and chondrogenic-related genes included Sox9 and Col II. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. All primers were synthesized by Sangon Biotech (Shanghai, China; Table 1).

5 × 10⁵ hucMSCs were transferred to a 15 ml centrifuge tube and centrifuged at 800 g for 5 min at room temperature. The cellsform a pellet at the bottom of the centrifuge tube. Subsequently, the supernatant was gently removed and the pellet was transferred to chondrogenic induction medium (Cyagen Biosciences). The solution was changed every 3 days and the cells the cells were fixed on the 21st day. Paraffin-embedded tissue sections were Hematoxylin-Eosin (H&E) and immunohistochemical stained (Quan et al., 2017 (link)).

TDSCs were incubated with specific differentiation media to analyze the cell multipotency. For osteogenic differentiation, TDSCs were incubated with osteogenic induction medium (Cyagen Biosciences, Suzhou, China) for 14 days, and Alizarin Red staining was performed to confirm osteogenic differentiation. For chondrogenic differentiation, chondrogenesis was performed in pellet culture with chondrogenic induction medium (Cyagen Biosciences, Suzhou, China) for 21 days, and Safranin O staining was used to confirm chondrogenic differentiation. For adipogenic differentiation, TDSCs were incubated in adipogenic induction and maintenance medium (Cyagen Biosciences, Suzhou, China) for 14 days, and Oil Red staining was performed to confirm the adipogenic differentiation.

The ADSCs were identified by flow cytometry. The cells were harvested with 0.25% trypsin and centrifuged at 1000 rpm for 5 min. Then, 1 × 10⁶ cells were resuspended in PBS and incubated using the following antibodies at 4 °C: CD29, CD90, CD31, and CD45 (Biolegend, USA).
To confirm the multipotency of ADSCs, they were subjected to induced differentiation by culturing them in osteogenic induction medium (OIM, Cyagen Biosciences, USA), adipogenic induction medium (AIM, Cyagen Biosciences, USA), and chondrogenic induction medium (Cyagen Biosciences, USA) and then verified with Alizarin Red, Oil Red O, and toluidine blue staining, respectively.

For chondrogenic differentiation, 4×10⁵ DPSCs were placed into a 15-mL polypropylene tube and centrifuged for 4 min at 250×g. The cell pellets were resuspended in the chondrogenic induction medium (Cyagen Biosciences, Guangzhou, China). The induction medium was replaced every 3 days. After 28 days of induction, pellets were formalin fixed and paraffin embedded. Thin sections were stained with Alcian blue (Cyagen Biosciences) to visualize proteoglycans in the extracellular matrix.

To verify the multi–lineage differentiation potential of GMSCs and metformin–treated GMSCs, we added metformin to the osteogenic, adipogenic, and chondrogenic induction medium (all from Cyagen, CA, USA), respectively, and the control group was a conditioned induction medium without metformin.
For osteogenic differentiation, the uncharacterized cells were seeded in 12–well plates (Corning, NY, USA) at a density of 5 × 10⁴ cells/well and cultured in the proliferating medium until reaching 70% confluence. For osteogenic differentiation, GMSCs were replaced with DMEM supplemented with 10% FBS, 0.1 mM dexamethasone, 10 mM β–glycerophosphate, and 50 mM ascorbic acid for 4 weeks. The medium was changed every 3 days. The calcification of an extracellular matrix was assayed by Alizarin red (Cyagen, CA, USA) staining and captured by the Compact Cell Culture Microscope, CKX3 (Olympus).
For chondrogenic differentiation, GMSCs were induced by the chondrocyte differentiation basal medium for 3 weeks and then assayed by 1% Alcian blue (Cyagen, CA, USA) staining to detect the synthesis of proteoglycans by chondrocytes.
For adipogenic differentiation, GMSCs were induced by the adipogenic differentiation basal medium for 3 weeks and then assayed by Oil Red O (Cyagen, CA, USA) staining to detect the formation of lipid droplets by adipocyte.