Single-layer graphene oxide nanosheets induce proliferation and Osteogenesis of single-cell hBMSCs encapsulated in Alginate Microgels

Microfluidics cell encapsulation offers a way to mimic a 3D microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Several studies have demonstrated the advantages of graphene oxide (GO) as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young's modulus of the alginate and alginate-slGO was obtained as 1414 kPa and 985.9 kPa, respectively. Live/dead assay and fluorescence microscopy images illustrate that slGO can maintain the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the encapsulated hBMSCs, so that microgels containing hBMSCs gradually became opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration. This finding offers a new method for creating injectable tissue transplants that are minimally invasive.