The Impact of Perfusion Culture on hMSC Differentiation and Functional Maturation in a 3D-Bioprinted Bone Model

Volumetric 3D bioprinting is a light-based technique, which enables custom fabrication of complex objects within seconds. This emerging method has great potential to produce biomimetic bone models by printing cellular free-form constructs with high cell viability and bone tissue maturation. Bone formation and remodeling are heavily dependent on spatiotemporal biochemical and biophysical cues, which enable bone cells to locally interact and self-organize. Osteoblasts and osteocytes are derived from mesenchymal stem cells (MSC) and their differentiation is tightly regulated by mechanical stimuli to ensure bone formation that suits the structural and dynamic support needed. Shear stress is one of the most abundant and relevant cellular stress modalities. If applied to a bone, osteocytes are able to sense shear stress and transduce these mechanical signals into biochemical signals to adapt bone mass and structure. This shape-function relationship is fundamental for bone homeostasis, yet the complexity and dynamic characteristics of bone tissue provide a great challenge for in vitro modeling. A truly functional in vitro bone model that contains the major bone cell types in a 3D bone-like environment and to which biomechanical forces can be applied has not been achieved. Therefore, the aim of this thesis was to establish perfusable volumetric printed gelatin methacryloyl (GelMA) constructs in combination with adapted milifluid perfusion chambers to investigate the effect of fluid shear stress (FFS) on human MSC differentiation and functional maturation. To this end, mechanical properties and human MSC viability, differentiation, and maturation were studied using different batches and concentrations of synthesized GelMA. Results revealed that lower GelMA concentrations lead to higher cell viability and increased cell differentiation. A 7.5%GelMA-0.05%LAP resin was chosen as the best trade-off between sufficient mechanical properties and high cellular viability and differentiation. A milifluidic chip system was then set up and printing and perfusion parameters were optimized and validated. Perfusion with osteogenic medium was applied with a flow rate of 703.3 µL/min corresponding to a FFS of 0.2 Pa. Static cultures were then compared to dynamic cultures with perfusions done 3 x per week and for 1 hour each. Cultures were maintained over 3 weeks and analyzed for viability, ALP activity, osteogenic gene expression (RUNX2, ALPL, COL1a2, BGLAP, PDPN), and mineralization. Results did not reveal the expected increase in cell differentiation and maturation markers but the dynamic cultures revealed a higher content of mineralization. These observations are most consistent with the view that the applied FSS was too high and led to a hypoxic culture environment, which indirectly led to a suppression of cell viability and ALP activity but nevertheless promoted differentiation of osteoblasts and thus higher mineralization. Although perfusion conditions appeared suboptimal, the present achievement of establishing dynamic volumetric printed 3D bone cultures with enzyme and gene expression analysis to track bone formation is exciting and holds promise for future applications in regenerative bone medicine.