In vitro models for the evaluation of angiogenic potential in bone engineering

Abstract

Blood vessels have a fundamental role both in skeletal homeostasis and in bone repair. Angiogenesis is also important for a successful bone engineering. Therefore, scaffolds should be tested for their ability to favour endothelial cell adhesion, proliferation and functions. The type of endothelial cell to use for in vitro assays should be carefully considered, because the properties of these cells may depend on their source. Morphological and functional relationships between endothelial cells and osteoblasts are evaluated with co-cultures, but this model should still be standardized, particularly for distinguishing the two cell types. Platelet-rich plasma and recombinant growth factors may be useful for stimulating angiogenesis.

Figure 1

Neoangiogenesis on a porous scaffold.

Figure 2

Immunofluorescent staining of endothelial cells for proteins specific for cytoskeleton or for adhesion molecules. (A) F-actin stain of HUVEC. Actin was stained using rhodamine-phalloidin fluorescent dye. Nuclei were stained with Hoechst 33258. Magnification (×20). (B) Immunofluorescent staining for VE-cadherin. Nuclei were stained with Hoechst 33258. Magnification (×20).

Figure 3

Cell co-culture systems. In the direct contact model, cells are seeded together in 2D supports or 3D scaffolds or as 3-D multicellular spheroids. In the indirect contact model, a porous membrane can be used, with appropriate pore size which let the conditioned medium pass but not cells. Alternatively, the culture of one type of cells is supplemented with the conditioned medium of the other type. In the third method, one cell type is seeded on the ECM of the other type, which has been discharged after grown.