Journal: Biomedicines

Article Title: Mechanistic Illustration: How Newly-Formed Blood Vessels Stopped by the Mineral Blocks of Bone Substitutes Can Be Avoided by Using Innovative Combined Therapeutics

doi: 10.3390/biomedicines9080952

Figure Lengend Snippet: Vasculoneogenesis in bone substitutes subcutaneously implanted in nude mice. ( A , B ) H&E staining of bone substitutes (green lines) with either empty (NF-) ( A ) or HEP/VEGF- ( B ) SNCs, at 12 dpi. Yellow arrowheads indicate blood vessels. Scale bars: 200 µm. The images at the top right show enlargements of blood vessels. Scale bars: 20 µm. ( C ) Quantitative analysis of the vessels found in the implanted bone substitutes. The average number and diameter of the vessels found, together with the average surface covered are given at 4 and 12 dpi. Values are expressed as mean ± SD of at least five images/section and four sections/sample. **: p ≤ 0.05. ( D , E ) Ultrastructural view of a blood vessel found in the HEP/VEGF-SNCs bone substitute, shown as a transverse section. Mural cells enveloping the blood vessels (e.g., the pericyte indicated with a red asterisk) were found in the nano-active bone substitute. ( E ) This is the enlargement of the area delimited by the yellow frame in ( D ). No blood cells are in the lumen of the vessel, as a contrast agent was perfused (residues indicated with a blue asterisk). E: endothelial cell; eN: nucleus of the endothelial cell; P: pericyte; pN: nucleus of the pericyte. Scale bars: 5 µm in d, 2 µm in e. ( F ) Endothelial cells of human origin in the bone substitute, as revealed with anti-human (top panels) or anti-human/mouse PECAM1 antibody (mid panel). Mouse control bone was negative to the anti-human PECAM1 antibody (lower panel). Scale bars: 200 µm.

Article Snippet: Rabbit anti-human PECAM1/CD31 (Microm, Brignais, France) and non-species-specific anti-PECAM1/CD31 (Abcam, Paris, France), then secondary antibody raised against rabbit antibodies and coupled with Alexa 594 fluorochrome (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Illkirch-Graffenstaden, France), diluted 1:200 in PBS 1% BSA, were used.

Techniques: Staining