Pre-clinical validation of in vitro engineered but cell-free human tissue grafts for skeletal regeneration

Tissue engineering strategies predominantly consist in the autologous generation of living substitutes capable of restoring damaged body parts. Persisting challenges with patient-specific approaches include an inconsistent performance, high costs and delayed graft availability. Towards developing a one-for-all solution, a more attractive paradigm lies in the exploitation of dedicated cell lines for the fabrication of human tissue grafts. Following decellularization, this new class of biomaterials relies on the sole extracellular matrix scaffold and embedded growth factors instructing endogenous repair. This conceptual approach was previously validated by manufacturing human cartilage using a custom mesenchymal line, exhibiting remarkable osteoinductive capacity following lyophilization. However, key criteria to ensure clinical translation include a proper decellularization as well as stringent assessment of both immunogenicity and regenerative performance. Here, we report the engineering and decellularization of human cartilage tissue with minimal matrix impairment. Ectopic evaluation in immunocompetent and immunocompromised animals reveal preserved osteoinductivity which correlates with M0 to M2 polarization from Day 7 post-implantation. By establishing in vitro human allogeneic co-culture models, we evidenced the immuno-regulatory properties of cell-free human cartilages, suppressing T cell activation upon mounting of an immune response by activated macrophages, monocytes and dendritic cells. Last, the regenerative performance was stringently assessed in a competent rat orthotopic models whereby human cartilage grafts fully restored critical-sized femoral defects within six weeks, achieving native bone-like biomechanical properties by twelve weeks. Taken together, our study compiles safety and efficacy pre-requisites towards a first-in-human trial for engineered and decellularized human tissue grafts.