(M1530-02-09) Tomographic Characterization of Alginate Foam Scaffolds for 3D Cell Growth

Purpose: Alginate is a versatile biomaterial to engineer cell culture platforms for disease modeling and tissue engineering. Alginate is bioinert and biocompatible, and with the addition of divalent cations, can form hydrogel structures for use as 3D cell culture scaffolds, drug delivery platforms, and injectables. To ensure consistent cell encapsulation in a defined alginate network, an alginate-based porous foam scaffold was developed where, when hydrated with a suspension of cells in alginate solution, it forms a hydrogel culture. The dry foam design provides a versatile base scaffold, where the molecular weight, viscosity, and functionalization of the alginate in the hydrating solution can be modulated to support optimization for the culture of various cell types. This macroporous foam scaffold is designed to allow cell suspension ingress from its dry state, which then allows hydration and encapsulation within a defined foam network. In order to identify cell construct targets for seeding and growth within the foam pores, we desired to characterize the dry foam dimensional properties such as pore size distribution, wall thickness, and overall permeability. X-ray tomography with virtual reconstruction was chosen as a non-destructive technique to obtain a quantitative 3D characterization of the porous scaffold characteristics.

Methods: Alginate-based foam scaffolds were produced through mechanical aeration and slow internal gelling followed by casting and drying.   Foam structural properties were assessed by stacking five discs in a custom sample holder and imaging them using X-ray tomography (Carl Zeiss Microscopy, USA) [Clark et al., 2023]. The scans were acquired at a voxel resolution of 5 µm within a sample volume of 5.0 × 5.0 × 5.0 mm³. DigiM’s I2STM was used to perform AI-driven segmentation and analysis to delineate and quantitatively characterize pore fraction, wall thickness, and the pore network.

Results: X-ray tomography method was able to distinguish foam walls separately from pores, and segmentation confirmed a highly porous alginate sample. Once a segmented 3D model was constructed, foam dimensions could be quantified.  Size distribution analysis showed the foam discs contained pores with mean diameter of 660 µm, with sizes ranging from ~50-1100 µm.  Wall dimension analysis showed a mean wall thickness of ~6 µm, and analysis of pore shape showed a mean roundness of ~0.62, indicating oblong pore shapes.  Spatial distribution and volume fraction in individual alginate discs showed average porosity values from 92-95%, with a uniform distribution along an individual disc’s diameter.  Liquid flow simulation utilizing the Navier-Stokes equation showed a highly permeable structure, with an overall permeability of 220 Darcy.

Conclusion: Alginate-based foam scaffolds were successfully characterized by X-ray tomography, with large pore structures observed in the alginate foam disc.  The pores in the higher resolution data are uniformly distributed.  The pore volume is comparable in the five alginate samples analyzed, indicating a uniform structure.  High permeability to liquid ingress is verified through simulation.  Overall, the pore sizes and interconnectivity is indicative of a versatile cell growth scaffold capable of hosting a wide range of cell constructs such as spheroids or organoids.

References: Clark, Andrew G., et al. "Investigating structural attributes of drug encapsulated microspheres with quantitative X-ray imaging." Journal of Controlled Release 358 (2023): 626-635.

Reconstructed simulation of foam disc wall structure from tomographic analysis (bottom left). Structure quantitation showed a pore size distribution with most pores in the 200-1000 µm range (top left) and a porosity value from 94-96% over most of the disc radius.