Endochondral ossification - towards a clinical translation

Bone is a rigid mineralized organ and forms part of the human skeleton. It is the only tissue of the human body which can completely heal without any scar formation. However, this is only possible if conditions are just right. Large missing bone volumes, non-unions and treatment of osteoporotic fractures are examples of challenging clinical scenarios, for which a suitable bone substitute is necessary.
The aim of my thesis was to validate the use of in vitro engineered hypertrophic cartilage as a bone substitute. I explored two available options for a rapid clinical translation. First, I engineered hypertrophic cartilage from bone marrow derived mesenchymal stromal cells for autologous use. I identified a suitable production method for the generation of large grafts and a way of monitoring and assuring the quality of the resulting grafts. Second, I produced allogeneic hypertrophic grafts, devitalized them and seeded them with different cell types to enhance their osteoinductive properties.
Hypertrophic cartilage was generated in a perfusion bioreactor system with rabbit bone marrow derived mesenchymal stem cells and in vivo bone formation was tested in order to generate protocols and guidelines for a future pre-clinical trial in a rabbit orthotopic model. Glycosaminoglycan and alkaline phosphatase content in culture supernatants were monitored to collect information on tissue integrity and maturity. The monitored parameters correlated with tissue quality and in vivo bone formation. This correlation might be used to maximize the efficiency, reproducibility and most importantly quality of the manufacturing process.
A major difficulty of autologous use was the variability of cartilage formation with different rabbit donors, which is in line with reports in human MSCs. To address this issue, pre-production of allogeneic devitalized tissue was considered. Engineered hypertrophic matrix subjected to different modes of devitalization was characterized for preservation of growth factors and components of the extracellular matrix and in vivo bone formation. It was demonstrated that suitably devitalized matrix, could deliver the set of factors necessary to induce formation of bone and bone marrow. The findings outlined a paradigm relying on the engineering of cell-based but cell-free niches, which could recruit and instruct endogenous cells to form predetermined tissues.
Devitalized constructs remained inferior to vital ones, which could be due to a deficient remodelling by host cells. To actively stimulate tissue degradation, thus releasing chemoattractant factors and ultimately improving bone formation, osteoclastogenic monocytes were seeded on both living and devitalized matrix. In vitro, the formation of osteoclasts, the secretion of factors in supernatants, the attraction of monocytes, endothelial cells or mesenchymal stem cells and the differentiation of mesenchymal stem cells in the presence of secreted factors were analyzed. In vivo, the presence of osteoclasts, macrophages, endothelial cells and mesenchymal stem cells was described at an early time point, as well as the bone formation at a late time point. In vitro and in vivo the observations were consistent. No improvement of bone formation was observed in living or devitalized grafts. MSC were not attracted and vascularization was only correlated with the presence of recruited M2 macrophages. While monocytes could be attracted by devitalized matrix, their survival and differentiation into osteoclasts strongly depended on interactions with hypertrophic chondrocytes.
As a way to provide multiple progenitor lineages and enhance bone formation, stromal vascular fraction (SVF) cells from human adipose tissue were used. Multiple pellets of devitalized matrix were combined together with different amounts of SVF and implanted subcutaneously. The contribution of SVF to the bone formation, vascularization and bone resorption was analyzed. SVF activation of the hypertrophic cartilage strongly enhanced the bone formation efficiency, both in subcutaneous ectopic implantation and calvarial defect models. In particular, the density of SVF cells was correlated with that of osteoclasts in the grafts, and the percentage of SVF-derived endothelial lineage cells was correlated with the amount of deposited mineralized matrix. These findings support a novel strategy for bone repair or augmentation, whereby allogeneic engineered and devitalized hypertrophic cartilage is clinically used as an off-the-shelf material in combination with autologous SVF cells.
Taken together, the results of my thesis provide a stepping stone for the use of hypertrophic cartilage as a tool for bone regeneration.