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Limbs from the 9-month-old animals were harvested at 28 days postimplantation for blood vessel quantification and histological analysis. Mice were euthanized via CO2 inhalation. Immediately after death, the thoracic cavity was opened and a 22-gauge butterfly needle was inserted into the left ventricle. The inferior http://www.selleckchem.com/products/Y-27632.html vena cava was severed to allow the vasculature to be flushed. Heparinized saline (100?U/mL) was used to flush the vasculature followed by perfusion with 10% neutral buffered formalin to fix the tissue specimens. The vasculature was perfused using a radio-opaque silicone rubber compound (Microfil; Flow Tech, Inc., Carver, MA, USA) mixed in a 9:1 vol/vol ratio with Microfil curing agent. Following vascular perfusion, the injected Microfil compound was allowed to polymerize overnight at 4��C before histological processing. Fixed limbs were embedded in plastic. Sagittal tissue sections were cut and polished to a final thickness of 10 to 20??m and were stained with hematoxylin and eosin for histological analysis. Using commercially available image analysis software (Image-Pro http://www.selleck.cn/products/pexidartinib-plx3397.html Plus; Media Cybernetics, Bethesda, MD, USA), stained tissue sections were analyzed for bone-to-implant contact (BIC) using two different methods. First, the BIC where the implant surface was in direct contact with the femoral cortical bone surface was calculated for each animal for each implant type and the average total BIC was determined. We also measured the BIC where new bone had formed within the femoral intramedullary space for each animal for each implant surface type. For each in vitro experiment, data presented are mean?��?SEM for six independent cultures per variable. Experiments were repeated to ensure validity. Data are presented from one representative experiment of two experiments. For each in vivo study, six implants were harvested for each implant type. In vitro and in vivo data were analyzed by ANOVA and when statistical differences were http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html detected, Student's t test for multiple comparisons using Bonferroni's modification was used. Values of p?