Angiogenic and inflammatory responses to biodegradable scaffolds were previously studied using the dorsal skinfold chamber for testing different scaffold materials. In this model, the angiogenic response originates from the soft tissue of the skin. Herein, we introduce a new model that allows the study of developing microcirculation of bone defects for testing tissue-engineered constructs. A bone defect was prepared in the femur of Balb/c mice by inserting a pin for intramedullary fixation, and a custom-made observation window fixed over the defect allowed constant observation. This study included three different groups: empty defect (control), defect filled with porous poly(l-lactide-co-glycolide), and beta-tricalcium-phosphate scaffolds. Starting from 6 days after surgery, angiogenesis, neovascularization, leukocyte-endothelial cell interaction, and microvascular permeability were analyzed over 22 days by using intravital fluorescence microscopy. The empty defects showed no signs of angiogenesis during the observation period, but a distinct increase of capillary density was detected in the scaffold-containing defects. Surprisingly, the histological sections of the scaffold-treated defects showed new bone formation 22 days after implantation. We present a new bone chamber model for intravital long-term study of scaffold materials suitable for bone reconstruction in mice by using fluorescence microscopy.