Towards 3D-Printed Bioactive Calcium Phosphate Scaffolds for Bone Tissue Engineering

Bone defects can arise as a consequence of trauma, cancer, or overloading. The reconstruction of critical size bone defects remains a challenge. Currently, the “golden standard” for this type of defects is the use of autologous bone grafts. Autologous bone grafts provide both an osteoconductive and osteogenic environment, as a result of the presence of living cells and the cocktail of growth factors in the matrix. This method, however, has drawbacks: bone grafts are only available in limited volumes and this procedure can cause donor site morbidity and pain. These drawbacks could be overcome by alternative approaches, such as bone tissue engineering Biological components such as growth factors can be used as chemical stimuli aiding bone tissue engineering. The kinetics of delivery of biological components will influence the effect of the component, or the occurrence of side effects. In chapter 2, we shed light on the continuing debate whether short, burst or sustained release is the best mechanism for the application of biological components for bone tissue engineering. We mimicked the in vivo situation by seeding human adipose stem cells (hASCs) on clinically applicable biphasic calcium phosphate (BCP) particles, allowing precise manipulation of doses and timing of application. We investigated which application of vitD3 i.e. mimicking short exposure of cells ([200 nM] for 30 minutes), burst release from scaffolds ([100 nM] for 2 days), or sustained release from scaffolds ([10 nM] for 20 days), leads to optimal osteogenic differentiation of hASCs. Our results suggest that mimicking sustained release seems to have more effect on hASCs than the other applications. Bone tissue engineering constructs send signals to cells in the bone to form more bone and to recruit stem cells, which can differentiate bone forming cells. Cells from different skeletal sites may react differently to such signals. Therefore, in chapter 3 we analyzed the degree of differentiation of bone cells derived from human long bone and alveolar bone. The results show that under basic culturing conditions long bone cells seemed more differentiated compared to alveolar bone cells. Furthermore, long bone-derived cells have stronger osteoclastogenic potential than alveolar bone cells. Taken together, these differences reveal that skeletal sites generate site-specific differences between the cells. 3D-printing can be used to make scaffolds for bone tissue engineering. By using this technique patient-specific scaffolds can be created that fits the defect perfectly. Calcium phosphates are often used in bone tissue engineering. 3D printing of calcium phosphates with a chemical stimulus such as growth factors is a challenge since methods using high temperatures or aggressive chemicals, impede the bioactivity of the biological components. In chapter 4, we developed a method to 3D print calcium phosphate with a biological binder allowing for the incorporation of biologically active components as chemical stimuli. Finally, in chapter 5, as the next step towards a clinical application, we assessed if constructs printed with the method described in chapter 4, and enhanced with 1,25(OH)2vitD3 as a biological component, can be clinically relevant. In order to meet clinical relevance, the material should have the following features: The material should have a certain rigidity, so the surgeon can handle the construct and it should be tolerated by cells. We investigated these features by measuring the printability, compressive stiffness, and cytotoxicity of the material and the effect of the material on human osteosarcoma-derived cells when cultured in close proximity to the print material. The results show that the material is printable, is rigid enough to handle, and did not have an adverse effect on the proliferation of cells when co-cultured. We also showed that the vitD3 is released from the material, and is still bioactive.