In Vitro hMSC Differentiation

Human mesenchymal stem cells (Rooster Bio) were encapsulated in slow and fast relaxing hydrogels at a final concentration of 15 million cells/mL gel, gels were punched into disks, and placed into 24-well plates. The encapsulated cells were cultured in osteogenic differentiation medium (Stempro, Life Technologies) and cell culture medium was changed every 3–4 days for two weeks.

At two weeks, samples were fixed in 4% paraformaldehyde for 45 min on an orbital shaker, exposed to increasing concentrations of OCT in a sucrose solution, and flash frozen for cryosectioning. Gels were sectioned at a thickness of 50 μm before von Kossa Staining. Briefly, sections were incubated in a 1% silver nitrate solution under ultraviolet light for 20 seconds, rinsed with DI water, and incubated in 5% sodium thiosulfate for 5 min.