High-Resolution 3D Printing of Bone Tissue Engineering Scaffolds

Background and Aim: Extrusion-based 3D printing has been usedfor fabrication of bone tissue engineering (TE) scaffolds. As for hardbiomaterials like bioceramics, it has been quite challenging to 3D printuniform and accurate scaffolds as the inks experience nozzle jamming/clogging and need exceedingly high pressure to impel ink to flow througha fine nozzle. Minimum nozzle size has been used for 3D printing ofbioceramics is currently limited to 200 μm. The initial goal of this project focused on 3D printing of consistent and highly uniform bioceramicscaffolds for in vitro testing. The main objective was to optimize theprocess in order to increase the resolution. Hydroxyapatite (HA) scaffoldscould be 3D printed using a nozzle diameter of 50 μm for the first timeby carefully adjusting the process parameters.Methods: An extrusion-based 3D printer was designed and set up andseries of experiments were conducted using the design of experiment(DOE) approach to analyze the effects of paste solvent content, extrudatevelocity and die design on extrusion pressure using the Benbow’s model.The printed samples were subjected to further analysis using a scanningelectron microscope (SEM) and computed tomography (CT). Cell viabilityand adherence were assessed by incubation and fluorescent imagingof Cell Tracker Green and Ethidium Homodimer (Molecular Probes,Oregon, OR, USA), respectively. Chorioallantoic Membrane Model(CAM) was also used to investigate blood vessel generation.Results: Different scaffolds with a high level of accuracy (+/-20 μm) werefabricated from HA, PLA/HA, β-TCP, alumina, and zirconia using thebespoke developed 3D printer. Fig. 1 depicts the bespoke 3D printer,the bioceramics paste characterization, and the very high-resolution 3Dprinted HA scaffold using 50 μm nozzle by proper adjustment of printingparameters and nozzle design. Excellent cell adhesion and viabilitywere observed by Cell Tracker Green/Ethidium Homodimer staining. Thereally interesting bit of results was that cells tend to row/bridge from oneportion of the filament to the adjacent filament, even after just one day(Fig. 2a). Microscopic images at day 7 proved widespread cell growth,proliferation and pores filling (Fig. 2b). The scaffolds with 250 μm poresize were implanted for 7 days into CAM (Fig. 2c). Location intimate withyolk sac indicates good CAM integration and the membrane is visiblesurrounding HA scaffold. Fig. 2d depicts a typical scaffold with generatedblood vessels after CAM implantation. As seen, there is a good bloodvessel formation and adhesion.Conclusion: Optimized 3D printing allowed production of the highestresolution bioceramic scaffolds has been reported so far while retaininga reasonable level of detail and accuracy. In vitro tests of the 3D printedHA scaffolds proved cell attachment, proliferation, and blood vesselformation.

کلیدواژه ها:

3D printing ، Bioceramics ، Tissue engineering scaffolds