Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone

Abstract

To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group ( n=6) and porous β-TCP scaffold as the control group ( n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel ( P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group ( P<0.05), except for area fraction at 4 weeks after implantation ( P>0.05). The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.