In Situ Release Of VEGF Enhances Osteogenesis In 3D Porous Scaffolds Engineered With Osterix-modified Adipose-derived Stem Cells

Adipose-derived stem cells(ADSCs)can differentiate into various cell types and thus have great potential for regenerative medicine.Herein,rat ADSCs were isolated and identified through CCK-8 assay,cell cycle detection,colony-forming assay,cell surface markers detection and multi-lineage induction;then transduced with lentiviruses expressing Osterix(Osx),a transcriptional factor essential for osteogenesis.Osx overexpression up-regulated key osteogenesis-related genes,such as special AT-rich binding protein 2(Satb2),alkaline phosphatase(ALP),osteocalcin(OCN)and osteopontin(OPN),at both the m RNA and protein levels.In addition,mineral nodule formation and alkaline phosphatase activity were enhanced in Osx-overexpressing ADSCs.The expression of dickkopf-related protein 1(Dkk1),a potent Wnt signaling pathway inhibitor,was also increased,whereas that of ?-catenin,an intracellular signal transducer in the Wnt pathway,was decreased.?-catenin expression was partially recovered by treatment with lithium chloride,a canonical Wnt pathway activator.The Osx-expressing ADSCs were then combined with 3D gelatin-coated porous poly(?-caprolactone)scaffolds with a unique release prolife of entrapped recombinant human vascular endothelial growth factor(VEGF).The controlled release of VEGF promoted osteogenic differentiation capacity in vitro.When the scaffold-ADSC complexes were transplanted into rat calvarial critical-sized defects,more bone formed on the gelatin/VEGF-coated scaffolds than on other scaffold types.Taken together,the results indicate that,Osx-overexpression promotes ADSCs' osteogenesis both in vitro and in vivo,which could be enhanced by release of VEGF,which may advance our understanding of ADSC fate and facilitate the clinical translation of ADSC-mediated bone therapies.