Bone is a highly vascularized tissue, and its development, maturation, remodeling, and regeneration are dependent on a tight regulation of blood vessel supply. This condition also has to be taken into consideration in the context of the development of artificial tissue substitutes. In classic tissue engineering, bone-forming cells such as primary osteoblasts or mesenchymal stem cells are introduced into suitable scaffolds and implanted in order to treat critical-size bone defects. However, such tissue substitutes are initially avascular. Because of the occurrence of hypoxic conditions, especially in larger tissue substitutes, this leads to the death of the implanted cells. Therefore, it is necessary to devise vascularization strategies aiming at fast and efficient vascularization of implanted artificial tissues. In this review article, we present and discuss the current vascularization strategies in bone tissue engineering. These are based on the use of angiogenic growth factors, the co-implantation of blood vessel forming cells, the ex vivo microfabrication of blood vessels by means of bioprinting, and surgical methods for creating surgically transferable composite tissues.

The neovascularization of three‐dimensional voluminous tissues, such as bone, represents an important challenge in tissue engineering applications. The formation of a preformed vascular plexus could maintain cell viability and promote vascularization after transplantation. We have developed a three‐dimensional spheroidal coculture system consisting of human primary endothelial cells and human primary osteoblasts (hOBs) to improve angiogenesis in bone tissue engineering applications. In this study, we investigated the survival and vascularization of the engineered implants in vivo. Endothelial cell spheroids were cocultured with hOBs in fibrin and seeded into scaffolds consisting of processed bovine cancellous bone (PBCB). The cell‐seeded scaffolds were evaluated for their angiogenic potential in two different in vivo assays: the chick embryo chorioallantoic membrane (CAM) model and the severe combined immunodeficiency disorder (SCID) mouse model. In both assays, the development of a complex three‐dimensional network of perfused human neovessels could be detected. After subcutaneous implantation into immunodeficient mice, the newly formed human vasculature was stabilized by the recruitment of murine smooth muscle α‐actin‐positive mural cells and anastomoses with the mouse vasculature. We conclude that this endothelial cell spheroid system can be used to create a network of functional perfused blood vessels in vivo. The finding that this process takes place with high efficacy in the presence of co‐implanted primary osteoblasts and in an osteoconductive environment provided by the PBCB scaffold, suggests that this system may be suitable for improving vascularization in bone tissue engineering.

Angiogenesis is a fundamental process in bone formation, remodeling, and regeneration. Moreover, for the regeneration of bone in tissue engineering applications, it is essential to support neovascularization. This can be achieved by cell‐based therapies using primary endothelial cells, which are able to form functional blood vessels upon implantation. In bone composite grafts, coimplanted endothelial cells do not only support neovascularization but also support osteogenic differentiation of osteoblasts and osteoprogenitor cells. In this study, we investigated the effect of endothelial cells on proliferation and cell survival of human primary osteoblasts (hOBs) and human mesenchymal stem cells (MSCs). Human umbilical vein endothelial cells (HUVECs) stimulated hOB and MSC proliferation, whereas proliferation of HUVECs was unaffected by cocultured hOBs or MSCs. The effect of HUVEC cocultivation on hOB and MSC proliferation was more pronounced in direct cocultures than in indirect cocultures, indicating that this effect is at least partially dependent on the formation of heterotypic cell contacts between HUVECs and hOBs or MSCs. Furthermore, HUVEC cocultivation reduced low‐serum induced apotosis of hOBs and MSCs by a mechanism involving increased phosphorylation and inactivation of the proapoptotic protein Bad. In summary, our experiments have shown that cocultured HUVECs increase the proliferation and reduce low‐serum induced apoptosis of hOBs and MSCs. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1682–1689, 2012

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co‐implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular‐derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE‐cadherin and vascular endothelial growth factor‐R2, and in their ability to endocytose acetylated low‐density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro, as assessed by in vitro Matrigel sprouting assay. However in vivo, a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.Conclusion:  Our results indicate that vascular‐derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.