We propose an ultrasound approach which provides, with one single examination and one single device, access to three bone biomarkers: anatomy, tissue quality and blood flow. It unlocks ultrasound imaging inside bone by accounting for ultrasound wave speed heterogeneity and anisotropic wave refraction. This study reports the first in vivo evaluation with a comparison to peripheral Quantitative Computed Tomography (pQCT) and modulations of blood flow. Anatomical multi-layer bone-corrected reconstruction was validated at the tibia of healthy volunteers against pQCT and showed agreement on bone cortex interfaces. Estimation of axial and radial ultrasound wave speeds in cortical bone tissue (i.e. along the tissue symmetry axis and normal to it) demonstrated good reproducibility and positive correlation with bone mineral density measured by pQCT. Pulsatile blood flow was mapped and quantified in cortical and medullary regions. A directional ray selection method was developed to enhance blood signal extraction by reducing strong specular reflections originating from the outer and inner surfaces of the bone cortex. Physiological and non-physiological modulations of blood flow, namely head-up/head-down tilt table maneuvers and arterial occlusions, demonstrated the method sensitivity to blood flow variations. For the first time, reactive hyperemia was observed inside bone cortex. These results demonstrate the feasibility of a portable, non-ionizing, and quantitative ultrasound approach for structural, anatomical, and vascular characterization of bone tissue. This approach may offer new diagnostic capabilities for bone disorders, for instance osteoporosis, delayed fracture healing or osteonecrosis. ... Read more