In Vitro and in Vivo Osteogenesis and Vasculogenesis in Synthetic Bone Grafts

Bone tissue is the second most common tissue transplant after blood transfusion and as people age, the need for bone transplants is expected to rise even more. Despite the great efforts in biomaterial research and development, autologous bone remains the benchmark among bone grafts whereas synthetic bone grafts are still less-used. Effective and reliable development of synthetic bone grafts require efficient cellular assessments in vitro including the evaluation of osteogenic as well as vasculogenic potential of the scaffolds. Adult stem cells, including human adipose stem cells (hASCs) and human bone marrow stem cells (hBMSCs), provide an excellent tool for this as they have been shown to differentiate to bone-forming cells and endothelial cells. However, efficient and cost-effective approaches to induce osteogenic and endothelial differentiation are still needed. This thesis focuses on the study and development of in vitro differentiation strategies for hASCs and hBMSCs in 3-dimensional (3D) culture in order to study and verify the feasibility of novel bone substitute materials.

Firstly, two clinically used synthetic bone graft scaffolds, BoneCeramic composed of biphasic calcium phosphate and BioRestore composed of bioactive glass (BaG), were compared in hASC culture and, the effectiveness of commonly used growth factors bone morphogenetic protein (BMP)-2, BMP-7 and vascular endothelial growth factor (VEGF) on hASC osteogenesis were compared to OM. As ceramics are hard and brittle making them difficult to implant and undesirable to load-bearing sites, an elastic β-tricalciumphosphate/poly(L-lactide-co-ε-caprolactone) (βTCP/PLCL) composite was developed using supercritical CO2 (scCO2) foaming. The capacity of the composites to support hASC osteogenesis and vasculogenesis were studied in vitro in hASC culture and in vivo in rabbit femur defect. Finally, 3D printed polycaprolactone (PCL)/copper-doped BaG composites were developed and their osteogenic and vasculogenic potential was studied in vitro in hBMSC culture and in a co-culture of hBMSCs and human umbilical vein endothelial cells (HUVECs).

The two biomaterial scaffolds BoneCeramic and BioRestore demonstrated differential effects on hASCs: BoneCeramic induced ALP activity and collagen production of hASCs, while BioRestore stimulated hASC proliferation. In comparison to the growth factors, OM was more effective in the osteogenic differentiation of hASCs in vitro in BoneCeramic and BioRestore. On the other hand, combining the growth factors with OM did not sensitize hASCs to the growth factors.

The β-TCP/PLCL composites supported the viability, proliferation and osteogenic differentiation of hASCs reliably in vitro. When studying the osteogenic and endothelial differentiation of hASCs in β-TCP/PLCL composites, hASCs differentiated towards both lineages when cultured in endothelial medium (EM) although the differentiation was left in a premature state. Culture in a cocktail medium of OM and EM supported osteogenic but not endothelial differentiation of hASCs. The in vivo study confirmed biocompatibility as well as osteoconductivity of the composite as no inflammation, fibrous tissue or cyst formation was detected and, native bone tissue was able to grow on and in the β-TCP/PLCL composite already at 4 weeks.

In the evaluation of 3D printed PCL/BaG-Cu composites, copper was observed to have a dose-dependent cytotoxic effect on hBMSCs in vitro. The PCL/BaG composite induced hBMSC osteogenesis while the addition of copper in BaG had an inhibiting effect on the osteogenic differentiation of hBMSCs. In co-culture of hBMSCs and HUVECs, both PCL/BaG and PCL/BaG-Cu scaffolds supported tubule formation, but the added copper did not boost the vasculogenic effect of the composite.

In conclusion, OM was shown to be a cost-effective osteogenic stimulant in 3D culture as compared to exogenously added growth factors. The feasibility of the scCO2 foamed β-TCP/PLCL composite as potential a bone graft for clinical use was evidenced in vitro and in vivo. The 3D printed PCL/BaG composite showed promise in supporting osteogenesis in vitro whereas the addition of copper hindered the osteogenic effect of the composite. Both PCL/BaG and PCL/BaG-Cu composites supported tubule formation in hBMSC+HUVEC co-culture but the copper did not induce the vasculogenic effect of the composite. To conclude, the elastic and bioactive synthetic polymer -based composites consisting osteoconductive ceramics are highly promising alternatives for synthetic bone grafts when compared to hard ceramics.