Enhanced repair of segmental bone defects of rats with hVEGF-165 gene-modified endothelial progenitor cells seeded in nanohydroxyapatite/collagen/poly(l-lactic acid) scaffolds

Abstract

A new type of tissue-engineered bone was constructed by seeding hVEGF165 gene-modified endothelial progenitor cells into the nanohydroxyapatite/collagen/poly(L-lactic acid) scaffolds. These were implanted into the segmental femoral defects of rats to explore the promotion of angiogenesis and osteogenesis. The bone marrow of Sprague Dawley rats was cultured and proliferated, and the endothelial progenitor cells were transfected with Ad5–hVEGF165–EGFP. The gene-modified endothelial progenitor cells were seeded into the nanohydroxyapatite/collagen/poly(L-lactic acid) scaffolds; the growth was observed by scanning electron microscope, and the proliferation was evaluated by methyl thiazolyl tetrazolium assay. In vivo, 80 Sprague Dawley rats were divided randomly into four groups; segmental femoral defects (5 mm) were made and allografted: group A with hVEGF165/endothelial progenitor cells–nanohydroxyapatite/collagen/poly(L-lactic acid), group B with mock endothelial progenitor cells–nanohydroxyapatite/collagen/poly(L-lactic acid), group C with endothelial progenitor cells–nanohydroxyapatite/collagen/poly(L-lactic acid), and group D with scaffolds only. Radiographic, histological, and microvessel density tests were performed to evaluate the angiogenic and osteogenic ability. Reverse transcription polymerase chain reaction and western blot results showed that the target gene was expressed by endothelial progenitor cells. The scanning electron microscope findings and methyl thiazolyl tetrazolium assay revealed that endothelial progenitor cells were attached and proliferated within the nanohydroxyapatite/collagen/poly(L-lactic acid) scaffolds. The average radiographic score and capillary density were the highest in group A, and those in groups B and C were higher than that of group D. The histology showed osteogenesis and scaffold degradation in group A, with less in groups B and C and little in group D. The hVEGF165 gene-modified endothelial progenitor cells, which promoted angiogenesis and osteogenesis in bone-defected areas and the hVEGF165/endothelial progenitor cells–nanohydroxyapatite/collagen/poly(L-lactic acid) composites, may have potential application in repair of segmental bone defects.

Keywords

segmental bone defects tissue engineering neovascularization endothelial progenitor cells gene transfer VEGF165

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