Osteogenic differentiation strategies of dental pulp pluripotent-like stem cells (dppsc) for their potential application in bone tissue engineering

Resumo

Bone defects due to trauma or disease affect millions of people worldwide, placing an even larger demand on the healthcare system to replace and restore bone loss. Recently, bone tissue engineering (BTE), combining biomaterials, cells and growth factors, has emerged as a promising therapeutic approach to treat large bone defects. For this purpose, pluripotent stem cells (PSC) are an attractive option. These cells can proliferate indefinitely in vitro, have self-renewal, high replicative capacity and are capable of differentiating into most cell types of the body. In addition, PSC have a higher value when testing the differentiation capacity of biomaterials; since these undifferentiated cells need to be guided merely by the biomaterial to their final fate. However, there is still a need to find a cell type with genetic stability, stemness characteristics and no ethical problems to be used in BTE approaches. In previous studies, our group described a pluripotent-like population of dental pulp stem cells derived from the third molars (DPPSC) that show genetic stability and share some pluripotent characteristics with embryonic stem cells. Until now, it has been studied the differentiation capacity of DPPSC into cells of different tissues from the three embryonic layers and it has been demonstrated they capacity to differentiate into bone-like tissue, even more than other dental pulp stem cells. This doctoral thesis introduces the use of DPPSC as a good alternative model for BTE approaches, either to direct bone regeneration therapy or to evaluate biomaterials before being applied. For this purpose, the thesis is divided in three main chapters. As a first step, the osteogenic differentiation process in DPPSC and their ability to grow, attach and differentiate were evaluated with different types of biomaterials commonly used in BTE studies, such as metals or natural scaffolds. Results reveal high osteogenic and adhesion potential of DPPSC on both biomaterials without acquiring genetic alterations. Thus, proposing the use of DPPSC as a good model to evaluate the biocompatibility and the osteogenic capacity of different biomaterials. Afterwards, different strategies were used to improve the osteogenesis of DPPSC for their potential application in BTE approaches: Firstly, a novel polymeric nanoparticle system was used as a non-viral gene delivery method to improve the osteogenic differentiation of DPPSC by silencing the expression of pluripotency genes (OCT3/4 and NANOG) and enhancing the expression of the osteogenic gene RUNX2. Thus, the combination of poly(β-amino ester)s (pBAEs) with natural oligopeptides was used in order to prove their biocompatibility with DPPSC and to simultaneously deliver anti-OCT3/4 siRNA, anti-NANOG siRNA, and RUNX2 plasmid in DPPSC. Results show that DPPSC can be transfected with these vectors whereas they maintain their viability and genetic stability. Furthermore, the delivery of siOCT3/4 in combination with pRUNX2 robustly accelerates the osteogenic differentiation of DPPSC. Secondly, different strategies were tested in order to induce the vascularization of the BTE constructs using DPPSC. This vascularization consequently, should improve the viability of the construct and the osteogenic differentiation process. Previous results showed that DPPSC also have a high endothelial potential. Thus, we suggested the combination of bone-like DPPSC and endothelial-like DPPSC to induce vascularized bone formation from the same stem cell population. Hence, different co-culture systems were analysed. Furthermore, in order to take this approach towards clinical assays, the osteogenic and endothelial medium compositions were tested using human serum (HS) to supplement the medium, instead of the commonly used animal-derived fetal bovine serum (FBS). In addition, the effect of bioactive glass (BaG) ions, characterized for their high osteogenic properties, was examined in DPPSC co-cultures and monocultures. Results demonstrate that endothelial medium with BaG extracts can provide an effective way to enhance both, osteogenic and endothelial processes, supporting the formation of vascular-like structures in DPPSC co-cultures. Therefore, the co-culture of pre-differentiated DPPSC in combination with BaG and xeno-free medium conditions provides a new promising system for the in vitro vascularization of the BTE constructs. Taken together, the findings described in this doctoral thesis propose DPPSC as a good alternative stem cell population for different BTE approaches.