The lack of a functional vascular supply has, to a large extent, hampered the whole range of clinical applications of 'successful' laboratory-based bone tissue engineering strategies. To the present, grafts have been dependent on post-implant vascularization, which jeopardizes graft integration and often leads to its failure. For this reason, the development of strategies that could effectively induce the establishment of a microcirculation in the engineered constructs has become a major goal for the tissue engineering research community. This review addresses the role and importance of the development of a vascular network in bone tissue engineering and provides an overview of the most up to date research efforts to develop such a network.

The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on co-culture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell-cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on post-implantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structural proteins, homotypic interactions and angiogenic potential of human EC cultured on a scaffold made of a blend of starch and poly(caprolactone). The nano-network allowed cells to span between individual micro-fibers and influenced cell morphology. Furthermore, on nano-fibers as well as on micro-fibers ECs maintained the physiological expression pattern of the structural protein vimentin and PECAM-1 between adjacent cells. In addition, ECs growing on the nano/micro-fiber-combined scaffold were sensitive to pro-inflammatory stimulus. Under pro-angiogenic conditions in vitro, the ECM-like nano-network provided the structural and organizational stability for ECs' migration and organization into capillary-like structures. The architecture of nano/micro-fiber-combined scaffolds elicited and guided the 3D distribution of ECs without compromising the structural requirements for bone regeneration.

Blends of polysaccharides and proteins are a source for the development of novel materials with interesting and tailorable properties, with potential to be used in a range of biomedical applications. in this work a series of blended membranes composed by chitosan and soy protein isolate was prepared by solvent casting methodology. in addition, cross-linking was performed in situ with glutaraldehyde solutions in the range 5x10(-3)-0.1 M. Furthermore, the influence of the composition and cross-linking on the degradation behaviour, water uptake and cell adhesion was investigated. The obtained results showed that the incorporation of chitosan, associated to network formation by cross linking, promoted a slight decrease of water absorption and a slower degradability of the membranes. Moreover, direct contact biocompatibility studies, with L929 cells, indicate that the cross-linking enhances the capability of the material to support cell growth.