Hypertrophic cartilage engineering for human bone and bone marrow regeneration

Abstract

Bone and bone marrow (BM) are major components of the human body, with a pivotal role in skeletal structure and hematopoietic cell function. Most bones in the human body form and heal through a process called endochondral ossification, whereby human mesenchymal stromal cells (hMSC) generate a hypertrophic cartilage (HyC) intermediate which progressively remodels into bone and BM. In my thesis, I propose to engineer HyC by recapitulating the key developmental features of endochondral ossification, and to exploit it for inducing bone repair and modelling human hematopoiesis. Part I of my thesis aims at generating devitalized HyC to be used as a graft for bone repair by engineering hMSC with an inducible apoptotic cassette. Apoptosis devitalization showed that by preserving the factors embedded in the extracellular matrix, endochondral bone formation could be achieved by osteoinduction in an ectopic environment. In a second phase, the process to generate devitalized HyC was upscaled and streamlined using a perfusion-based bioreactor system. The resulting devitalized HyC was assessed against a clinically used human processed allograft in a rabbit calvarial model. Efficient and homogenous production of devitalized HyC could be accomplished in a single step process. Finally, orthotopic implantation demonstrated the superiority of devitalized HyC compared to a clinical standard-of-care. Part II targets the exploitation of this developmental process to create a model for the study of interactions between human stroma and hematopoietic cells. Until now, most human hematopoiesis studies are carried out using humanized mice models, missing a human stromal compartment. By keeping alive the human stromal cellular fraction in the HyC, we generated an ectopic humanized bone organ (ossicle) in the mouse. The ossicles successfully engrafted long term functional human hematopoietic stem and progenitor cells (HSPC) and increased the fraction of quiescent human hematopoietic stem cells (HSC) as compared to the native mouse bone. Then, we asked whether the ossicles could be customized to visualize the human stroma and influence the hematopoietic environment. By engineering hMSC to overexpress a fluorescent reporter and the stromal cell-derived factor 1 alpha (SDF1α) cytokine, we visualized and quantified the human stromal cell fate in the ossicles and modified the hematopoietic cell homeostasis. Finally, this system allowed the visualization of putative human HSPC niches. Overall, by using engineered hMSC and HyC tissue, this work tackles both a clinical application by developing a new material for bone repair, and the generation of fundamental knowledge by establishing a model of human hematopoietic cells interacting with their stromal niche.