Engineering Cell Microenvironment Using Nanopattern-Derived Multicellular Spheroids and Photo-Crosslinked Gelatin/Hyaluronan Hydrogels

Cell cultures of dispersed cells within hydrogels depict the interaction of the cell–extracellular matrix (ECM) in 3D, while the coculture of different cells within spheroids combines both the effects of cell–cell and cell–ECM interactions. In this study, the cell co-spheroids of human bone mesenchymal stem cells/human umbilical vein endothelial cells (HBMSCs/HUVECs) are prepared with the assistance of a nanopattern, named colloidal self-assembled patterns (cSAPs), which is superior to low-adhesion surfaces. A phenol-modified gelatin/hyaluronan (Gel-Ph/HA-Ph) hydrogel is used to encapsulate the multicellular spheroids and the constructs are photo-crosslinked using blue light. The results show that Gel-Ph/HA-Ph hydrogels with a 5%-to-0.3% ratio have the best properties. Cells in HBMSC/HUVEC co-spheroids are more favorable for osteogenic differentiation (Runx2, ALP, Col1a1 and OPN) and vascular network formation (CD31+ cells) compared to HBMSC spheroids. In a subcutaneous nude mouse model, the HBMSC/HUVEC co-spheroids showed better performance than HBMSC spheroids in angiogenesis and the development of blood vessels. Overall, this study paves a new way for using nanopatterns, cell coculturing and hydrogel technology for the generation and application of multicellular spheroids.