For critical size bone defects and bone non-unions, bone tissue engineering using osteoblastic differentiated adipose mesenchymal stem cells (AMSCs) is limited by the need for a biomaterial to support cell transplantation. An osteoblastic three-dimensional autologous graft made of AMSCs (3D AMSC) was developed to solve this issue. This autograft was obtained by supplementing the osteoblastic differentiation medium with demineralized bone matrix. Two surgical models were developed to assess the potential of this 3D osteogenic AMSC autograft. A four-level spinal fusion using polyetheretherketone cageswas designed in six pigs to assess the early phase of ossification (8e12 weeks postimplantation). In each pig, four groups were compared: cancellous bone autograft, freeze-dried irradiated cancellous pig bone, 3D AMSC, and an empty cage. A critical size femoral defect (n ¼ 4, bone non-union confirmed 6 months postoperatively)was used to assess the 3DAMSCs’ ability to achieve bone fusion. Pigs were followed by CTscan and explanted specimens were analyzed for bone tissue remodeling by micro-CT scan, micro-radiography, and histology/histomorphometry. In the spine fusion model, bone formation with the 3D AMSC was demonstrated by a significant increase in bone content. In the critical-size femoral defect model, the 3DAMSC achieved newbone formation and fusion in a poorly vascularized fibrotic environment. This custom-made 3D osteogenic AMSC autograft is a therapeutic solution for bone non-unions and for critical-size defects. 2013 Elsevier Ltd. All rights reserved.