Introduction: The surge in clinical need for bone tissue restoration together with limitations of existing treatments calls for the development of alternative strategies. Tissue engineering has been proposed towards the formation of bone graft substitutes capable of driving repair. Previously, we demonstrated the possibility of generating engineered cartilage grafts using a human mesenchymal stromal cell (hMSC). Here, we aim to develop an efficient decellularization protocol and assess the osteoinductivity, immunogenicity and regenerative potential of decellularized cartilage in both immunodeficient (ID) and immunocompetent (IC) settings.
Method: Our graft consists of in vitro engineered cartilage tissue produced by human mesenchymal stromal cell (hMSCs) lines. After cartilage formation, the tissue was subsequently decellularized using a combination of hypertonic and hypotonic baths, Sodium dodecyl sulfate (SDS), and DNase to effectively remove cells, thus resulting in a cell-free graft aiming at instructing bone formation by endochondral ossification. The decellularized cartilage is implanted subcutaneously in the back of IC and ID animals for a maximum of 12 weeks. The early recruitment of immune cells (dendritic cells, monocytes, macrophages, natural killer, T and B cells) was assessed quantitively by flow cytometry at 3-, 7- and 10-days post-implantation (Immune prints).
Results: We demonstrated the reproducible engineering of decellularized human cartilage, as cell-free grafts capable of bone formation by exploiting a dedicated human Mesenchymal Stromal Cell line (MSOD-B). Following subcutaneous in vivo implantation, a complete remodeling into bone was achieved in immunodeficient mice (ID) through recapitulation of the endochondral ossification pathway. In contrast, only minor calcification was observed upon implantation into immunocompetent mice (IC). Initial immune responses during successful bone formation in ID seemed to correlate with an early M2 macrophages polarization and recruitment. Moreover, we showed that despite the absence of cellular material, human-derived grafts were able to induce a pro-inflammatory response in IC, detrimental to effective bone formation.
Conclusion and discussion: Using our decellularization method, we efficiently removed significant amount of cells without affecting the overall structure and composition of cartilage graft. Ectopic evaluation of decellularized tissues displayed excellent osteoinductive properties, correlating with early M2 polarization in ID mice but not in IC mice. This indicates ECM interspecies variations could still result in the immune rejection of cell-free tissues. Compiling immune prints may offer understanding the immunogenicity of engineered grafts, and help designing biomaterials with tailored immune profile for effective repair.