(A-11) Simple and Tunable Macroporous Hydrogels for Regenerative Medicine

Introduction:: Hydrogel-based biomaterials are widely used as scaffolds in regenerative medicine, as they can provide a suitable environment for cell attachment, proliferation, and differentiation. However, traditional hydrogel scaffolds typically have small pore sizes that limit cellular activity and mass transport. This can lead to immune response when implanted, hindering tissue regeneration. To address this challenge, macroporous hydrogels have been developed as a solution. Various methods have been used to create these hydrogels, including cryogelation or annealing of microgel building blocks. However, these strategies are often complex and time-consuming, making them unsuitable for translation into clinical applications.


Materials and Methods:: A macroporous hydrogel is formed through enzymatic crosslinking of foam-based bioinks. This involves mixing gelatin (5-10%w/v; Sigma) and transglutaminase (0.5-2%w/v; Modernist Pantry) with a controlled air volume (1:1-1:5 ratio) through repeated passing through meshes (10-400µm size), using a double syringe approach. The resulting foam biomaterial can be used to create a scaffold by either injecting or printing it in situ. The method's pore content, pore size, crosslinking kinetics, mechanical properties, printability, and cellular behavior were all characterized and optimized. Images were captured using a fluorescent microscope (Zeiss Axio Observer) and analyzed with ImageJ software. Biocompatibility was evaluated using Live/Dead staining and PrestoBlue analysis. Figure 1 illustrates the method's schematic.





Results, Conclusions, and Discussions:: The air volumetric ratio had the most significant effect on the pore size and structure. Controlling the pore size enables adjusting mass transport through the scaffold structure as well as infiltration of different cells. The mechanical properties could be controlled in the range of 3-25 KPa based on the gelatin and transglutaminase concentrations. These values demonstrate the macroscopic mechanical properties for recapitulation of the soft tissue properties. Biomimetic mechanical properties improve tissue integration, specifically for moving body parts. On the other hand, our results demonstrate a higher mechanical young modulus (30-80 KPa) for the bulk scaffolds without foaming. This is Young's modulus of the scaffold regions between the bubbles that resemble the microscale Young's modulus of tissues such as muscle (individual muscle fibers), which is previously shown to enhance cellular behavior.
Furthermore, the viability and metabolic activity of the cells significantly increased when encapsulated in macroporous foam compared to the bulk hydrogel. This can be due to the enhanced transport of nutrients, oxygen, and waste, as well as enhanced cell proliferation without the need for scaffold degradation. Interestingly, the colloidal nature of the foam significantly improved the printability. Finally, subcutaneous injection in mice demonstrated improved cell infiltration and remodeling for foam scaffolds.

The developed macroporous scaffolds are highly translational, cell permissive, and convenient to use anywhere, such as in an operating room.


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