Angiogenesis is the formation of new blood vessels occurring in an adult through migration and proliferation of endothelial cells, and tubular structures formation. Angiogenesis is modulated by growth factors, cytokines, adhesion molecules, integrins, and enzymes. Angiogenesis plays a role in many physiological processes (i.e. remodeling of ischemic muscle, woumd healing, fracture repair) as well as in pathological process such as rheumathoid arthritis and metastases. In bone, vasculature is essential for cartilage resorption and angiogenesis temporally precedes osteogenesis: the origin of bone is the artery carrying calcium and phosphate ions. Osteogenesis takes place near newly formed vessels, that mediate delivery of osteoprogenitor cells, secrete mitogens for osteoblasts, and transport nutrients and oxygen. Inadequate bone vascularity is associated with decreased bone formation and bone mass. In animals, inhibition of angiogenesis during fracture repair results in the formation of fibrous tissue. A poor blood supply is therefore considered as a risk factor for an impaired bone healing. Angiogenesis is vital in tissue engineering, especially when matrices are colonized by cells with an aerobic metabolism. The scaffold must not only support the growth of the cells making up the organ which should be replaced in vivo (i.e. osteoblasts); it must also support the growth of endothelial cells and develop an effectively functioning vasculature to supply the cells with oxygen. Osteogenesis of tissue engineered materials could be limited by a lack of vascularization, and the bioengineered graft may be potentially resorbed in the same way as a conventional bone graft. In rats, angiogenesis in coralline materials implanted in ectopic muscular sites, was higher when the biomaterial was combined with a vascular pedicle or was coated with bone marrow stromal cells. A combination of both enhanced vascularization and osteogenesis to a greater extent. Endothelial cells release growth factors and cytokines promoting bone deposition: PDGF-AB, TGF-beta 1 and 2, FGF-2, EGF, BMP. However, under inflammatory stimula, endothelial cells release bone resorbing cytokines: IL-6, M-CSF, GM-CSF. Bone marrow stromal cells release angiogenetic proteins such as VEGF, FGF-2, PDGF, TGF, and, after induction with BMP, PlGF. A conversation between bone marrow stromal cells and endothelial cells may therefore be hypothesized. Cultures of bone marrow stromal cells with endothelial cell conditioned medium showed significantly higher phosphatase alkaline activity and osteocalcin production. It was also be hypothesized that stromal cells may acquire immunophenotypic characteristics consistent with endothelial cells. Therefore scaffold requirements are also the ability to favour angiogenesis; endothelial cells growing on the artificial scaffold should mantain a normal phenotype and should not exhibit a pro-inflammatory and bone.resorbing phenotype. Endothelial cell cultures are useful supplementary in vitro tests for the evaluation of scaffolds for bone tissue engineering. Endothelial cell cultures are derived both from animals (usually ox, calf or pig vessels) and from human tissues, mainly the human umbilical vein and the vessels of microcirculation (derma or subcutaneous fat). Endothelial cells in non-human species show different reactions: they have usually a faster replication rate and grow better on the artificial substrata. Endothelial cells from different organs are intrinsically different and exhibit different responses to stimula. if the use of endothelial cells from bone microcirculation should be desirable, they require transfection with viral vectors to be immortalized. To study the response of endothelial cells cultured in vitro on artificial scaffolds, their adhesion, growth, viability and production of metabolites should be evaluated. Adhesion and growth on the materials may be evaluated indirectly by the uptake of Alamar Blue, which measures the amount of oxido-reduction reactions in the cell. A direct evaluation may be obtained by fluorescence microscopy using specific staining for the different cell structures. By studying the expression of adhesins and integrins, the interference of the scaffold with the cell/cell and cell/substrate adhesion should be verified. The release of substances in conditioned medium, as well as the evaluation of specific mRNAs in cells, should be assayed. Among the metabolites released by endothelial cells, the substances promoting bone deposition or favouring resorption, should be investigated. In particular, the release of growth factors may be explored, as they favour cell proliferation and the incorporation of the engineered scaffold within tissues. For the enhancement of bone formation, growth factors may be delivered in different ways: through incorporation on the scaffold, through transfection of bone marrow stromal cells, through platelet gel. Angiogenic growth factors are stored in platelet alpha granules and released during activation. A significant increase in the proliferation of bovine bone endothelial cells was demonstrated after 72 hour incubation with platelet gel in comparison with serum free conditions; the proliferation was similar to the growth induced by the fetal calf serum supplementation (platelet gel: 82.2B18.1x103 cells; serum free: 19.5B11.1x103 cells; fetal calf serum: 72.4B12.4x103 cells). However, the platelet gel inhibited the formation of tubular structures on Matrigel

In conclusion, the development of newly formed vessels on the bone cell engineered scaffold improves the incorporation in the host tissues and the success of the device. The use of exogenous growth factors or of platelet gel favours angiogenesis, besides osteoblast differentiation. The in vitro evaluation of the scaffold should be supplemented by tests on the adhesion, growth and functionality of endothelial cells.