Diffusion coefficients in scaffolds made with temperature controlled cryoprinting and an ink made of sodium alginate and agar

Temperature Controlled Cryoprinting (TCC), is a tissue engineering technique wherein each deposited voxel is frozen with precise control over cooling rates and the direction of freezing. This control allows for the generation of ice crystals with controlled shape and orientation. Recently we found that the macroscale fidelity of the TCC print is substantially improved by using a 3D printing ink composed of a mixture of two compounds: one that solidifies through chemical crosslinking (sodium alginate) and another that solidifies through physical (thermal) effects (agar). In this study we examine the hypothesis that the combination of sodium alginate and agar, affects also the fidelity of the microstructure and thereby the diffusivity of the scaffold. The ability of this technology to generate controlled diffusivity within the tissue scaffold was examined with a directional solidified TCC sample using fluorescence recovery after photobleaching (FRAP) and scanning electron microscope (SEM). We find that the diffusion coefficient in m²/s × 10⁻¹⁰ is: 1.62 $\pm$ 1.27 for the unfrozen sample, 2.40 $\pm 1$.54 for the rapidly frozen sample and $9.72\pm$ 4.50 for the slow frozen sample. This points to two conclusions. One is that the diffusivity is slow frozen samples is higher than that in unfrozen samples and in rapidly frozen sample. A second observation is that a relatively narrow range of diffusivity variance was obtained when using 2%w/v sodium alginate and 2%w/v of agar. However, when the concentration of agar was reduced to 0.5w/v a much wider spread of diffusivities was measure, $4.07\pm 1$.65. This suggests that the addition of agar has also an effect on the microscale fidelity, and consequently the diffusivity. The anisotropic diffusion properties of TCC-printed directional solidification samples were also validated through both FRAP and SEM.