Regeneration of complex bone defects presents challenging scenarios for the surgeons. Implants based on autologous Mesenchymal Stem/Stromal Cells (MSC) provide promising approaches for the repair of such defects. However, several issues are still associated with the clinical application of this approach, namely (i) the limited vascularization of the implants, (ii) the costly manufacturing, (iii) the limited reproducibility in the implant performance. MSC have been used to generate bone tissue by a process resembling intra-membranous ossification, i.e. by direct osteoblastic differentiation. However, most bones develop by endochondral ossification, i.e. via remodeling of hypertrophic cartilaginous templates. Endochondral bone formation could solve the issue of vascularisation, due to the intrinsic capability of hypertrophic chondrocytes to survive in hypoxic conditions and to produce potent angiogenic factors. The goal of the project is to standardize the engineering of clinically relevant sized hypertrophic cartilage tissues, which support the efficient formation of vascularized bone by endochondral ossification. Devitalization of engineered hypertrophic cartilage tissues will allow to assess whether signals accumulated in the extracellular matrix per se may be sufficient for vascularized bone formation. The specific aims are: 1.Identify conditions for reproducible hypertrophic differentiation by MSC and test osteoinductivity of devitalized grafts. 2.Scale-up tissues to a clinically relevant size using porous scaffolds and a perfusion-based bioreactor system 3.Assess the efficiency of vascularisation and osteoinductivity of scaled-up engineered grafts in a rabbit model MSC are expanded and differentiated towards a hypertrophic chondrocyte phenotype in micromass culture systems using standard supplements, as well as agonists of the indian hedgehog pathway or reduced oxygen percentages. The osteoinductive nature of the resulting tissues, devitalized or not, is assessed by ectopic implantation in nude mice. The possibility to devitalize the constructs by inducing apoptosis of MSC carrying an inducible promoter associated with a death cassette is evaluated, and if found effective, an established immortalized MSC cell line will be transduced. A perfusion-based bioreactor system is used to generate scaled-up hypertrophic cartilage tissues, which will be tested in a previously established rabbit model of bone formation and vascularization. The possibility to use devitalized grafts, engineered within automated bioreactor systems, will open the perspective of cost-effective and standardized manufacturing of off-the-shelf biological bone substitutes.