Chondrogenic and vascular priming: an endochondral ossification approach to bone tissue regeneration

Abstract

Tissue engineering and regenerative medicine have significant potential to treat bone pathologies by exploiting the capacity for bone progenitors to grow and produce tissue constituents under specific biochemical and physical conditions. However, these approaches are limited and as such are not yet widely used for clinical treatment of large bone defects. The main limitations include degradation of the tissue engineered constructs, due to lack of vascularisation, and a lack of mechanical integrity to fulfil loading bearing functions. Recent studies have suggested that in vitro approaches, which mimic certain aspects of bone formation during embryogenesis (i.e. the endochondral ossification process), can promote mineralisation and vascularisation to a certain extent both in vitro and in vivo. However, in vivo endochondral ossification relies on the production of a cartilage template and the invasion of a vascular network into this template, and both events must occur before bone tissue can be produced. Although researchers have demonstrated the separate effectiveness of chondrogenic priming and prevascularisation, to date no tissue engineering strategy has sought to incorporate both of these crucial events. The global objectives of this Thesis are to investigate whether endochondral priming of stem cells in vitro can enhance (a) the osteogenic and vasculogenic potential in vitro and (b) ultimately enhance the angiogenic and mineralisation potential once implanted in vivo. The first study sought to determine the optimum period for chondrogenic priming of Mesenchymal Stem Cells (MSCs) in vitro that could enhance osteogenic differentiation. The results from this study found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have an optimum influence on their mineralization capacity and can produce an aggregate that is mineralised throughout the core. The findings of this study provided vital information in determining the optimum time (14-21 days) for chondrogenic priming to produce a fully mineralised bone tissue engineered construct in vitro. The second study of this Thesis sought to develop an in vitro bone regeneration strategy that mimics critical aspects of the endochondral ossification process, specifically (1) the formation of a cartilage template and (2) subsequent vascularization of this template. The results showed that chondrogenic priming provides a cartilage-like template that provides a suitable platform for Human Umbilical Vein Endothelial Cells (HUVECs) and MSC cells to attach, proliferate, and infiltrate for up to 3 weeks. More importantly this study showed that when both MSCs and HUVECs are added to the already formed cartilage template, rudimentary vessels were formed within this cartilage template and this strategy enhanced the mineralization potential of MSCs. The third study of this Thesis aimed to investigate whether mimicking both the chondrogenic and vascularisation aspects of the endochondral ossification process could induce osteogenesis, even without the use of any osteogenic supplements. The results from this study showed that the co-culture methodology enhanced both osteogenesis and vasculogenesis compared to osteogenic differentiation alone, whilst allowing for the formation of rudimentary vessels in vitro. Taken together, the results from Chapters 3-5 showed that the application of both chondrogenic and vascular priming of priming of human MSCs enhanced the mineralization potential of MSCs in vitro whilst also allowing the formation of immature vessels and can even obviate the need for osteogenic growth factors to induce osteogenesis by human MSCs in vitro. The final study of this Thesis investigated the in vivo potential of the endochondrally primed aggregates developed in Chapter 5. The results from this study found that in aggregates that were both chondrogenically primed and prevascularised viable human MSCs were identified 21 days after subcutaneous implantation. Most importantly these aggregates had mature endogenous vessels and mineralisation nodules, after 4 weeks subcutaneous implantation. In contrast aggregates that were not prevascularised had no vessels or mineralisation within the aggregate interior and human MSCs did not remain viable beyond 14 days. Taken together, the results from this Thesis provide a novel understanding of the optimum conditions needed to create a bone tissue engineered construct that when implanted in vivo may drive bone formation via an endochondral ossification-like process. Future bone tissue engineered constructs could be designed with these conditions in mind for the repair of non-union bone defects.