The faith of tissue engineered bone replacing constructs depends on their early supply with oxygen and nutrients, and thus on a rapid vascularization. Although some models for direct observation of angiogenesis are described, none of them allows the observation of new vessel formation in desmal bone. Therefore, we developed a new chamber model suitable for quantitative in vivo assessment of the vascularization of bone substitutes by intravital fluorescence microscopy. In the parietal calvaria of 32 balb/c mice a critical size defect was set. Porous 3D-poly(L-lactide-co-glycolide) (PLGA)-blocks were inserted into 16 osseous defects (groups 3 and 4) while other 16 osseous defects remained unequipped (groups 1 and 2). By placing a polyethylene membrane onto the dura mater, the angiogenesis was mainly restricted to the osseous margins (groups 2 and 4). Microvascular density, angiogenesis, and microcirculatory parameters were evaluated repetitively during 22 days. In all animals, only a mild inflammatory reaction was observed with a climax after 2 weeks. The implantation of PLGA scaffolds resulted in a vascular growth directed towards the center of the defect as demonstrated by the significantly (p < 0.05) enhanced central microvascular densitiy from day 3 to day 22 when compared with unequipped chambers. The additional application of polyethylene membrane was found to reduce significantly the microvessel density mainly in the center of both scaffolds and defects. The present calvaria bone chamber allows for the first time to assess quantitatively the angiogenesis arising from desmal bone directly in vivo. Therefore, this chronic model may support the future research in the biological adequacy of bone substitutes.