Abstract
IntroductionA major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation.MethodsMSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining.ResultsThe results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks.ConclusionsThis study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.
Highlights
A major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization
In Endothelial cell (EC)/Mesenchymal stem cell (MSC) constructs, endothelial microvascular networks were observed after 1 week of dynamic culture in vitro (Figure 1A, left)
Functional grouping showed that genes related to skeletal development and the extracellular matrix (ECM), were expressed predominantly in monocultured MSCs, whereas higher expression of genes was found related to cell growth and differentiation in cocultures (Table 1)
Summary
A major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic geneexpression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation. The reconstruction of bone defects by using stem cells seeded onto biodegradable carrier materials requires timely formation of functional blood vessels. The establishment of a biological vasculature within a tissue-engineered construct influences differentiation of cells present and, subsequently, development of the tissue [7]. The addition of ECs to osteogenic cells has been shown to cause increased bone formation in calvarial defects as well as after subcutaneous implantation [8,10]. Prevascularization, in which three-dimensional EC microvascular networks are developed in vitro through coculture of ECs and MSCs before in vivo implantation, is an approach whereby the phenotype of MSCs delivered to tissue defects might be altered
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