In vitro cell and culture models for osteoblasts and their progenitors

This thesis aimed to evaluate the relevancy of different in vitro cell and culture models for osteoblastic-linage cells. Cell lines provide a convenient and accessible alternative to primary human osteoblast cells. However, the direct comparison of these cells demonstrated limited similarity of cell lines to the primary human osteoblasts indicating that their use should be limited to appropriate and specific research questions. To investigate the paracrine regulation of osteogenic development, the immature human osteoblasts and human bone-derived mesenchymal stem cells (MSCs) were co-cultured in monolayer or high density culture. Results from this part of the study suggested the presence of an active signalling pathway between MSCs and osteoblasts. What is more, the effect of cell-cell crosstalk depended on the type of culture system. Co-culture in a 3D micromass system stimulated the osteogenic differentiation of progenitor cells, while in monolayer this was not seen. While the stimulation of MSCs with inflammatory and chemotactic factors successfully regulated the cell gene expression and secretion profile, no effect of the secrotome on the osteogenic differentiation of unstimulated cells in monolayer was demonstrated. Altogether, these results indicated the importance of cell-to-matrix interconnectivity. Therefore, the last part of this thesis focused on the assessment of osteogenic differentiation in 2D and 3D cell culture models, which are physiologically relevant. The progression in osteogenesis depends on the applied 3D culture model. While in both, micromass and type I collagen-hydroxyapatite gel, the differentiation is enhanced compared to monolayer, the regulation of this process is triggered in a different manner in these 3D culture models. Together these findings demonstrate how diverse outcomes can be obtained by the application of different models in in vitro research. Ultimately, the 3D in vitro models provide a better choice for a more in vivo-related osteogenic differentiation and its regulation

Combinatorial Delivery of Bioactive Molecules by a Nanoparticle-Decorated and Functionalized Biodegradable Scaffold

The combination of supportive biomaterials and bioactive factors to stimulate endogenous progenitor cells is of key interest for the treatment of conditions in which intrinsic bone healing capacities are compromised. To address this need a “scaffold-decoration platform” was developed in which a biocompatible, biotin-functionalised 3D structural polymer network was generated through a solvent blending process, and used to recruit avidin modified nanoparticles within its 3D structure through biotin-avidin conjugation. This was enabled via the generation of a suite of poly(lactic-co-glycolic acid) (PLGA) nanoparticles, encapsulating two bioactive factors, vascular endothelial growth factor (VEGF) and L-Ascorbic acid 2-phosphate (AA2P) and conjugated to streptavidin to allow attachment to the bone generating scaffold. The levels of encapsulated and released VEGF and AA2P were tailored to fall within the desired range to promote biological activity as confirmed by an increase in endothelial cell tubule formation and collagen production by osteoblast cells in response to nanoparticle release of VEGF and AA2P, respectively. The release of VEGF from the scaffolds produced a significant effect on vasculature development within the chick chorioallantoic membrane (CAM) angiogenic assay. Similarly, the scaffolds showed strong biological effects in ex vivo assays indicating the potential of this platform for localised delivery of bioactive molecules with applications in both hard and soft tissue engineering

A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing

Immortalized cell lines are used more frequently in basic and applied biology research than primary bone‐derived cells because of their ease of access and repeatability of results in experiments. It is clear that these cell models do not fully resemble the behavior of primary osteoblast cells. Although the differences will affect the results of biomaterials testing, they are not clearly defined. Here, we focused on comparing proliferation and maturation potential of three osteoblast cell lines, SaOs2, MG‐63, and MC3T3‐E1 with primary human osteoblast (HOb) cells to assess their suitability as in vitro models for biomaterials testing. We report similarities in cell proliferation and mineralization between primary cells and MC3T3‐E1. Both, SaOs2 and MG‐63 cells demonstrated a higher proliferation rate than HOb cells. In addition, SaOs2, but not MG‐63, cells demonstrated similar ALP activity, mineralization potential and gene regulation to HOb's. Our results demonstrate that despite SaOs‐2, MG63, and MC3T3 cells being popular choices for emulating osteoblast behavior, none can be considered appropriate replacements for HOb's. Nevertheless, these cell lines all demonstrated some distinct similarities with HOb's, thus when applied in the correct context are a valuable in vitro pilot model of osteoblast functionality, but should not be used to replace primary cell studies