[Effects of Endothelial Progenitor Cells on Vascularization and Osteogenesis of Tissue-engineered Bones in Beagle...

Objective The explore the effects of endothelial progenitor cells(EPCs)on vascularization and osteogenesis of tissue-engineered bones in Beagle dogs.Methods Bone marrow mesenchymal stem cells(BMSCs)and EPCs from bone marrow of Beagle dogs were isolated,cultured,and expanded and then seeded in β-tricalcium phosphate(TCP)scaffolds. The cell scaffold complexes(EPCs/ BMSCs/TCP group and BMSCs/TCP group)and the blank TCP scaffold were transplanted into the limb muscles of Beagle dogs,respectively. The relative CT values were calculated in the 3rd and 6th month through CT scan after operation. The vascularization and the osteogenesis were detected 6 months after operation by histological examination.Results Three months after the surgery,the relative CT value in the EPCs/BMSCs/TCP group was(366.67±19.51)HU,significantly higher than that[(163.00±30.81)HU] in the BMSCs/TCP group(t=2.10,P=0.0006);6 months after surgery,the relative CT value in the EPCs/BMSCs/TCP group was(553.34±26.86)HU,which was also significantly higher than that[(241.34±21.57)HU] in the BMSCs/TCP group(t=2.11,P=0.0006). The relative CT values at 6 months after operation were significantly higher than those at 3 months after operation in both BMSCs/TCP group(t=2.10,P=0.0255)and EPCs/BMSCs/TCP group(t=2.10,P=0.0006). Six months after the operation,HE staining showed no bone formation or calcium deposition in the blank TCP group,and osteoblasts,mature bone trabeculae,and calcium deposition were observed in EPCs/BMSCs/TCP group and BMSCs/TCP group. Immunohistochemical staining showed that the number of peripheral,central,and whole vessels in the EPCs/BMSCs/TCP group was 21.67±1.45,23.33±2.60,and 45.00±1.16,which were significantly higher than those in the BMSCs/TCP group[8.67±0.88(t=2.07,P=0.0016),9.33±0.67(t=2.07,P=0.0065),and 18.00±1.00(t=2.07,P=0.001),respectively]. The number of vessels was not significantly different between the BMSCs/TCP group and the blank TCP group(18.00±1.00 vs. 16.67±2.40;t=2.07,P=0.636). The osteogenic area of the EPCs/BMSCs/TCP group was(1 322 000±141 300)pixel,significantly higher than that of the BMSCs/TCP group[(874 900±49 430)pixel;t=2.10,P=0.04]. BMSCs/TCP group showed significantly higher bone area in the peripheral area than in the central region[(170 000±42 320)pixel vs.(613 900±90 290)pixel;t=2.10,P=0.02];in contrast,there was no statistically significant difference in bone area between the peripheral region and the central region in the EPCs/BMSCs/TCP group[(376 400±20 160)pixel vs.(310 400±6917)pixel;t=2.10,P=0.07].Conclusion EPCs as seed cells combined with BMSCs can promote vascularization and osteogenesis of tissue-engineered bone in Beagle dogs.