Translation of micro-CT imaging into a 3D bio printable bone model

Biomedical research requires representative models to drive innovation and knowledge. Traditionally, monolayer cell culture studies are equipped for early-stage research prior to animal studies, however, are rarely successfully translated. In replacement, dynamic and complex three-dimensional cell culture models are under development to bridge the gap. The research presented in this study investigated the methodology involved to translate ex-vivo murine bones imaged by high resolution micro-computed tomography into a three-dimensional bio printable in-vitro bone model to reflect ex-vivo morphometry. Including, the associated characterisation of the model compared to native in-vivo murine bone and traditional monolayer culture methods. Normality of native murine bone biology is very well characterised, including the tissue composition, morphometric parameters and cellular phenotypes. These factors were summarised to inform in-vitro model development. Model morphology was optimised by translating ex-vivo bones from mice and rats, 3D models from micro-computed tomography software: CTAn, into standard tessellation language files, with different meshing algorithm, code and unit explored. Following this, the resulting models were rendered in computer assisted design software’s Autodesk® Meshmixer and Fusion 360™ for the application of fused deposition modelling, stereolithography and extrusion based bioprinting. The ex-vivo morphology was successfully printed by both fused deposition modelling and stereolithography, inclusive of cortical and trabecular bone structures. For the application of extrusion based bioprinting, two commercially available biomaterial inks, Bone GelXA and TissueFAB, were characterised for flow behaviour, functionality and crosslinking, as well as an in-house generated laponite® crosslinked poly (N-isopropylacrylamide, N, N’-dimethylacetamide) co-polymer, containing hydroxy-apatite nanoparticles, known as ‘B-gel’. TissueFAB was removed from the study because of incompatible crosslinking, and Bone GelXA was removed due to batch-to-batch inconsistencies. In addition, to improve structural stability and the resulting fidelity of the 3D bioprinted model, a microparticle support slurry was generated and characterised. The microparticle slurry improved the bioprinted structural complexity of the bioinks. B-gel bioink was taken forward with incorporation of pre-osteoblast cell line, MC3T3-E1, with and without osteogenic differentiation media and compared against in-vitro monolayer cellular behaviour. The in-vitro model was unable to be bioprinted in the rendered ex-vivo morphology despite improvements in fidelity from the microparticle support slurry. In replacement, to assess cellular phenotype and material composition a scaffold structure was bioprinted. From micro-computed tomography imaging, the in-vitro B-gel bioprinted constructs increased in density in both cellular conditions, suggesting osteoblast differentiation eliciting remodelling therefore enhancing the bone-like environment of the 3D in-vitro model. Further research is required to improve the fabrication process of bioprinting B-gel to allow the replication of bone morphology, as well as characterisation of the in-vitro model over a longer period to assess the full potential of remodelling to generate a replica in-vivo bone for the aim of reducing animal models for early-stage biomedical investigations.