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Owen, et al.
into the calcium chloride solution, thus forming and therefore will have a thin layer of water
larger beads. surrounding them. This means that the emulsion
In initial experiments (data not shown), we interface around these large alginate beads should
produced very large millimeter-sized beads when be the same as if it were a water droplet – it will be
a slower speed or larger gauge needle was used. surrounded by a thin film of surfactant stabilized
Furthermore, very large beads were often created monomer that on polymerization would contract
when setting up and removing the syringe as the to create an open surface porosity. As the alginate
alginate solution could occasionally drop into beads have been made in a separate process to the
the calcium chloride solution; hence, all alginate emulsion, both the porosity in the polyHIPE from
beads above 780 μm were sieved out after the water droplets and the macropores from the
bead manufacture was complete. The alginate alginate beads can be controlled independently
beads were not sieved further, as we wanted to to each other, unlike when heating or solvent
preserve the size distribution to create a range of destabilization is used .
[23]
macropores within the polyHIPE; a polydisperse As expected, when porosity increased
bead distribution should have a better packing compressive modulus decreased (Figure 3).
efficiency than a monodisperse one yielding an Interestingly, as porosity increased the standard
interconnected macropore network throughout the plasma treatment applied to polyHIPEs to increase
scaffold. However, alternative direct fabrication hydrophilicity in our group resulted in sample
methods to have tighter control over alginate bead charring (data not shown) . We hypothesize that
[17]
size and shape may be desirable. These could this is due to the high energy ions in the plasma
include electrospraying and microfluidics to impacting and accumulating within the polyHIPE
[54]
[53]
produce a more monodisperse size distribution. macropores introduced by alginate bead leaching,
The ratios of alginate beads (0, 50, and resulting in localized heating and charring. This
100 wt%) relative to the initial HIPE weight is supported by greater charring occurring in
were chosen as this broad range affords us a the 100 wt% than the 50 wt% alginate porogen
baseline understanding of the feasibility of leached polyHIPE scaffolds, and the apparent
combining alginate bead porogen leaching with localization of the charring to the macropores. In
emulsion templating and their effect on the the plain polyHIPEs which do not possess these
polyHIPE scaffold morphology. Here, alginate macropores, these ions are carried over the surface
beads were incorporated into the HIPE at up to of the polyHIPE meaning that no charring occurs.
100 wt% of the emulsion with no visual signs of To alleviate this, the combination of lower power,
emulsion destabilization. The polymer struts of reduced starting pressure to increase air flow rate,
the polyHIPE surface that has cured against the and shorter treatment times were tested, finding that
alginate beads have a smooth surface composed a 70% reduction in power eliminated the charring
of numerous pores of different sizes. Rather than entirely whilst still reducing the hydrophobicity of
the classical polyHIPE morphology observed in the scaffold.
the bulk of the plain polyHIPE, the pore shape Seeding efficiency was significantly greater in
at this interface appears different as the water the 100 wt% scaffolds than the plain polyHIPEs as
droplets in the initial emulsion have been pressed the larger pores allowed cells and media to penetrate
and deformed against the smooth alginate surface the porous network more easily (Figure 4A).
before polymerization. Regardless of the shape of Although there was no significant difference at
the alginate bead the polyHIPE retained an open day 7, metabolic activity was significantly higher
pore surface and overall mirrored the curved shape in 100 wt% samples than plain polyHIPEs by day
of the alginate it cured against. The polymerized 14. PrestoBlue measures the reduction of resazurin
boundary layer between the alginate surface and to fluorescent resorufin. This must be completely
polyHIPE had open connected pores. We assume eluted from the samples for the fluorescence to
this is because the alginate beads are 97% water correlate to the cell number. This is more difficult

International Journal of Bioprinting (2020)–Volume 6, Issue 2 109