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Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis

Significance It has been previously reported that hypertrophic cartilage tissues engineered from human mesenchymal stromal cells can efficiently remodel in vivo into bone organs, recapitulating developmental steps of endochondral ossification. We have here demonstrated that the extracellular matrix (ECM) of such engineered cartilage, even in the absence of a living cell component, retains frankly osteoinductive properties. The use of an apoptosis-driven devitalization technique revealed the importance of preserving the ECM integrity and, in particular, the embedded factors to trigger the regenerative process. Although exemplified in a skeletal context, our work outlines the general paradigm of cell-based but cell-free off-the-shelf materials capable of activating endogenous cells toward the formation of specific tissues. The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard “freeze & thaw” (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue.