In vivo vascularization of MSC-loaded porous hydroxyapatite constructs coated with VEGF-functionalized collagen/heparin multilayers

Rapid and adequate vascularization is vital to the long-term success of porous orbital enucleation implants. In this study, porous hydroxyapatite (HA) scaffolds coated with vascular endothelial growth factor (VEGF)-functionalized collagen (COL)/heparin (HEP) multilayers (porosity 75%, pore size 316.8 ± 77.1 μm, VEGF dose 3.39 ng/mm(3)) were fabricated to enhance vascularization by inducing the differentiation of mesenchymal stem cells (MSCs) to endothelial cells. The in vitro immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting results demonstrated that the expression of the endothelial differentiation markers CD31, Flk-1, and von Willebrand factor (vWF) was significantly increased in the HA/(COL/HEP)(5)/VEGF/MSCs group compared with the HA/VEGF/MSCs group. Moreover, the HA/(COL/HEP)(5) scaffolds showed a better entrapment of the MSCs and accelerated cell proliferation. The in vivo assays showed that the number of newly formed vessels within the constructs after 28 d was significantly higher in the HA/(COL/HEP)(5)/VEGF/MSCs group (51.9 ± 6.3/mm(2)) than in the HA (26.7 ± 2.3/mm(2)) and HA/VEGF/MSCs (38.2 ± 2.4/mm(2)) groups. The qRT-PCR and western blotting results demonstrated that the HA/(COL/HEP)(5)/VEGF/MSCs group also had the highest expression of CD31, Flk-1, and vWF at both the mRNA and protein levels.