Characterisation and quantification of angiogenesis in beta-tricalcium phosphate implants by immunohistochemistry and transmission electron microscopy.

The aim of this study is the histological characterisation of angiogenesis in a macroporous biomaterial with quantification techniques used in oncology. Porous tricalcium phosphate implants were seated in the tibias of 12 rabbits. This work allows (1) morphological study with photonic microscopy, transmission electron microscopic and immunohistochemistry labelling for (2) quantification of vascularisation using anti-CD31 monoclonal antibody (3) quantification of proliferation using anti-PCNA polyclonal antibody (4) study of two angiogenic growth factors: VEGF and FGF-2. Quantification of angiogenesis revealed an outbreak kinetic with early vascular growth in first several days and a second growth phase after 4 weeks. This study reveals in macropores many isolated cells without adjacent vascular lumen, with endothelial phenotype. Expression of angiogenic growth factors reveals that all endothelial cells were VEGF-negative throughout the test period. FGF-2 expression by endothelial cells began 2 weeks post-implantation. Osteoblasts strongly expressed two markers throughout the test period. Furthermore, the procedure described here can be used to compare angiogenesis in different biomaterials or in the same biomaterial with the influence of macroporosities.