Skeletal tissue loss due to congenital defects, disease, and injury is normally treated by autologous tissue grafting, a method limited by the availability of the host tissue, harvesting difficulties, donor site morbidity, and clinician’s ability to manipulate delicate 3D shapes. The generation of autologous bone grafts in vitro avoiding the harvesting of autologous tissue at a second anatomic location is the ultimate goal in bone tissue engineering. We have developed a protocol for the isolation and culture of human adipose tissue- derived mesenchymal stem cells (AT-MSCs) which fulfils the strict European regulations concerning the Advanced Therapy Medicinal Products. AT-MSCs were grown inside a new 3D-scaffold based on equine bone grafts. This scaffold allows to maintain the adequate 3D-structure, promote cell adhesion and proliferation. The osteogenic differentiation of human AT-MSCs was obtained by an animal-free induction medium without the use of growth factors.