Abstract
BackgroundIn bone tissue engineering (BTE), extensive research into vascular endothelial growth factor A (VEGFA)-mediated angiogenesis has yielded inconsistent results. The aim of this study was to investigate the influence on angio- and osteogenesis of adenoviral-mediated delivery of VEGFA alone or in combination with bone morphogenetic protein 2 (BMP2) in bone marrow stromal cells (BMSC) seeded onto a recently developed poly(LLA-co-CL) scaffold.MethodsHuman BMSC were engineered to express VEGFA alone or in combination with BMP2 and seeded onto poly(LLA-co-CL) scaffolds. Changes in angiogenic and osteogenic gene and protein levels were examined by quantitative reverse-transcription polymerase chain reaction (RT-PCR), PCR array, and alkaline phosphatase assay. An in vivo subcutaneous mouse model was used to investigate the effect on angio- and osteogenesis of VEGFA alone or in combination with BMP2, using microcomputed tomography (μCT), histology, immunohistochemistry, and immunofluorescence.ResultsCombined delivery of a lower ratio (1:3) of VEGFA and BMP2 (ad-BMP2 + VEGFA) led to upregulation of osteogenic and angiogenic genes in vitro at 3 and 14 days, compared with mono-delivery of VEGFA (ad-VEGFA) and other controls. In vivo, in a subcutaneous mouse model, both ad-VEGFA and ad-BMP2 + VEGFA scaffold explants exhibited increased angiogenesis at 2 weeks. Enhanced angiogenesis was largely related to the recruitment and differentiation of mouse progenitor cells to the endothelial lineage and, to a lesser extent, to endothelial differentiation of the implanted BMSC. μCT and histological analyses revealed enhanced de novo bone formation only in the ad-BMP2 + VEGFA group, corresponding at the molecular level to the upregulation of genes related to osteogenesis, such as ALPL, RUNX2, and SPP1.ConclusionsAlthough BMSC expressing VEGFA alone or in combination with BMP2 significantly induced angiogenesis, VEGFA alone failed to demonstrate osteogenic activity both in vitro and in vivo. These results not only call into question the use of VEGFA alone in bone regeneration, but also highlight the importance in BTE of appropriately formulated combined delivery of VEGFA and BMP2.
Highlights
In bone tissue engineering (BTE), extensive research into vascular endothelial growth factor A (VEGFA)-mediated angiogenesis has yielded inconsistent results
Significantly higher levels of bone morphogenetic protein 2 (BMP2) mRNA and secreted BMP2 protein were found in adVEGFA + BMP2 bone marrow stromal cells (BMSC) seeded onto scaffolds at day 3 and 14, compared with the control ad-GFP and adVEGFA BMSC (Fig. 1 g, i)
The analysis of variance (ANOVA) test with Bonferroni post hoc analysis showed significant upregulation of the osteogenic markers ALPL, BMP7, and BMP6 at day 3, and ALPL, RUNX2, SPP1, BGLAP, BMP6, and BMP7 at day 14 in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC (Fig. 2c, d)
Summary
In bone tissue engineering (BTE), extensive research into vascular endothelial growth factor A (VEGFA)-mediated angiogenesis has yielded inconsistent results. Bone tissue engineering (BTE) involves an interplay among mesenchymal stem cells, a supportive scaffold, and the controlled application of growth factors to produce vital bone grafts [1]. Early induction of vascularization is crucial, in large bone defects where the vitality of the implanted cells depends on the vascularity in the scaffolds. The blood vessels surrounding the scaffold-cell construct can provide nutrients by diffusion for distances of 100–200 μm [3]. In this context, various approaches to improve the vascularization of tissueengineered constructs have been explored [4]. An important research challenge is to harness these crucial events by therapeutic application of angiogenic molecules, such as vascular endothelial growth factor A (VEGFA) [6]
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