For preparing a creamy white sauce, we usually can use butter, milk, flour, cheese, garlic, and salt. During the preparation, if we see that the sauce is getting too thick, one option we have is to add more milk. On the contrary, if the sauce is too thin (or too liquid), we can add more flour to make it thicker. We can visually observe what the sauce may be missing by mixing and checking how it is moving or how it is flowing from a spoon. Doing a parallel with the formulation of samples in biomedical research, the concentration of specific components (ingredients) can be increased or decreased to tune the mechanical properties of the sample, making it less flowable (thicker) or more flowable fluid (thinner). The visual evaluation, in this case, is not so straightforward, and changing the formulation is more complex. Analytical tools are needed to quantify the mechanical properties of the sample in question in order to guide the adjustment of its formulation.
A group of researchers from Trinity College Dublin led by Dr. Daniel J. Kelly investigated how the formulation of an alginate bioink can modulate the mechanical properties of a 3D printed scaffold. Alginate is a natural biomaterial obtained from algae and is widely used in the medical and pharmaceutical industries due to its viscosifying, gelling, and biocompatible properties. In biomedical research, alginate is commonly used as bioink in 3D bioprinting to produce 3D structures (scaffolds) often containing cells for repairing or replacing damaged tissues (field of tissue engineering), or for delivering drugs or bioactive molecules to the body. Alginate solutions can quickly crosslink (have their molecules bonded together) in the presence of ions (e.g., Ca2+) resulting in a cohesive hydrogel that can be prepared in different shapes and with tunable mechanical properties.
In their published study entitled “Tuning Alginate Bioink Stiffness and Composition for Controlled Growth Factor Delivery and to Spatially Direct MSC Fate within Bioprinted Tissues” , the authors evaluated the influence of the alginate molecular weight (28000 and 75000 g/moL), type of crosslinker (calcium chloride CaCl2, calcium sulphate CaSO4 and calcium carbonate CaCO3), alginate:crosslinker ratios (6:1,19:5, 25:9, 49:10), and gelling conditions (PBS versus DMEM) on the mechanical properties of alginate-based scaffolds using a compression tester. After that, the authors also studied how this difference in stiffness of a given hydrogel (alginate in this case) can influence stem cells differentiation (transformation of stem cells into specific types of cells). This differentiation is important for tissue engineering purposes.
The authors started by checking how the many components involved in the preparation of alginate constructs can affect their final mechanical properties. Firstly, they found similar results compared to literature concerning the MW: the highest MW resulted in a higher Young’s modulus. The type of crosslinker was shown to affect the mechanical properties: CaSO4 led to stiffer scaffolds when compared to those prepared with CaCO3 and CaCl2. This difference was suggested to be related to the speed of gelation that was slower for the CaSO4 and can allow a more uniform crosslinking positively contributing to the mechanical properties. Varying the alginate:crosslinker ratio also changes the mechanical properties of the scaffold: the stiffness increases with the relative increase in available crosslinker (6:1 < 49:10 < 19:5 < 25:9). Finally, preparing alginate solution in PBS resulted in less stiff scaffolds compared to DMEM just for the high MW sample prepared with CaSO4. The phosphate groups can transiently bind to Ca2+ ions reducing their availability for crosslinking.
It is known that the mechanical properties of hydrogels affect stem cell differentiation. The group then printed a cylindrical construct loaded with stem cells with spatially varying mechanical stiffness using high MW alginate, CaSO4, and two alginate:crosslinker ratios (6:1 and 25:9). In the softer region of the structure (Young Modulus ~ 0.8 kPa), they observed that half of the stem cells appeared to undergo osteogenesis (bone cell type) and the other half adipogenesis (fat cells). On the other hand, in the stiffer part (Young Modulus ~ 4 kPa), the majority of the cells underwent osteogenesis which was in accordance with the literature.
In this study, it was possible to see how important it is to have analytical tools to quantify the mechanical properties of alginate hydrogels allowing their tuning by simply changing their formulation. As seen in the results, the softer and stiffer regions of the cell-laden alginate construct varied by only changing the alginate:crosslinker ratio was enough for changing the fate of stem cells. Understanding the correlation of mechanical properties and cell differentiation as well as cell viability, cell function, stability of the construct over time at 37 °C in physiological fluids, among others are critical for the development of the tissue engineering field.
 Freeman, F. E., & Kelly, D. J. (2017). Tuning alginate bioink stiffness and composition for controlled growth factor delivery and to spatially direct MSC fate within bioprinted tissues. Scientific reports, 7(1), 1-12.
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