Human dental pulp stem cells: characterisation and in vitro 3D bone ontogeny

Tissue engineering has emerged as a practical approach to tackle with the prosthetic industry limitations. Its methods merge aspects from developmental biology, engineering, material sciences and medicine, with the aim to produce fully-functional bone tissue ex vivo, to further replacement or regeneration of real bone injuries and/ or defects. Traditional bone tissue engineering cell culture technique, includes the seeding of SCs in three-dimensional matrices, cultured in fed-batch rotating bioreactors, working jointly with biological cues to produce biomimics. Nonetheless, fed-batch bioprocessing has found some difficulties. Namely, mass transport limitations in the seeded scaffolds and accumulation of cellular waste, yielding poor nutrition and oxygenation of the cells. Thus producing heterogeneous distributed cell/ bone constructs. Perfusion of media has been proven to improve mass transport in the culture system, along with removing cellular waste. To overcome heterogeneity, finding the adequate cells and proper cues to drive osteogenic differentiation, are as important as the bioprocess to host the culture. hDPSCs are a promising source of stem cells for the production of bioactive biomaterial for skeletal tissue reconstruction. They lodge immunosuppressive and regenerative functions, high proliferation rates and ease in access. Their source and the subtle nature of the extraction procedure, harbour less moral concerns and variability than ESCs and most of the MSCs. In this study, the characterisation of hDPSCs as MSCs under the minimal criteria set by The International Society for Cellular Therapy was performed. Further, the osteogenic differentiation of hDPSCs encapsulated in alginate/ gelatin hydrogel subjected to suspended culture in a novel perfusion-RWV bioreactor was studied, and compared with traditional fed-batch and static culture methodologies. Finally, the effect of osteogenic cues as physiological BMP2 and simvastatin were studied to enhance the designed bioprocess. The characterisation results demonstrated that passage 4 donor hDPSCs were ideal to perform the 3D osteogenic differentiation. These cells, allowing enough production of cells while maintaining the multipotent phenotype of the parental cells under several conditions, including highly dense long-term culture. These cells were able to undergo osteogenesis in 2D and 3D. The novel high throughput perfusion-RWV bioreactor bioprocessing, demonstrated to be successful in the mitigation of nutrient and oxygen transport limitations, external to three-dimensional cell/alginate constructs, performing above fed-batch RWV bioreactor and static culture, and able to produce more homogeneous, denser and functional bone constructs, rich in mature osteoblasts and mineralising osteocytes. Both BMP2 and simvastatin, demonstrated to enhance the quality of bone constructs produced by the perfusion-RWV unit, yielding more homogeneous constructs, with higher alkaline phosphatase activity, mineralisation and showing a more mature gene pattern. Interestingly. BMP2 produced constructs rich in mature osteoblast, while simvastatin, constructs rich in osteocytes. In particular, this experiment proved effective in producing osteogenic differentiation with minimal use of BMP2, offering a potential mean to avoid dosage dependant safety risks of BMP2. In conclusion, this thesis reports the development of a novel bioprocess to produce homogeneous bone tissue constructs able to support the transfer of the “on the bench” research, to the clinical facility.Open Acces