In vitro osteogenic differentiation of adipose-derived mesenchymal stem cell spheroids impairs their in vivo vascularization capacity inside implanted porous polyurethane scaffolds

Abstract

Undifferentiated adipose-derived mesenchymal stem cell (adMSC) spheroids are attractive vascularization units for tissue engineering. Their osteogenic differentiation further offers the possibility of directed generation of bone constructs. The aim of this study was to analyze how this differentiation affects their in vivo vascularization capacity. Green fluorescent protein (GFP)-positive adMSCs were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of undifferentiated and differentiated spheroids using the liquid overlay technique. Subsequently, polyurethane scaffolds were seeded with these spheroids and successful osteogenic differentiation was proven by von Kossa staining and high-resolution microtomography. The scaffolds were then implanted into dorsal skinfold chambers of C57BL/6 wild-type mice to analyze their vascularization and incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with differentiated spheroids exhibited a markedly impaired vascularization. Immunohistochemical analyses revealed that this was caused by the lost ability of differentiated spheroids to form GFP-positive microvascular networks inside the scaffolds. This was associated with a reduced tissue incorporation of the implants. Moreover, they no longer exhibited a mineralized matrix after the 14day implantation period, indicating the dedifferentiation of the spheroids under the given in vivo conditions. These findings indicate that osteogenic differentiation of adMSC spheroids markedly impairs their vascularization capacity. Hence, it may be reasonable to combine adMSC spheroids of varying differentiation stages in scaffolds for bone tissue engineering to promote both vascularization and bone formation.