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Owen, et al.
deposition and vascularization . PolyHIPE- internal phase. Therefore, it is essential to identify
[32]
based additive manufacturing techniques by a porogen that is insoluble in both emulsion
which this is achievable include emulsion phases whilst giving an open surface porosity. We
extrusion [33,34] and microstereolithography-based hypothesize that one such material is alginate, a
approaches [8,9,13,35,36] . The latter has the ability naturally derived polysaccharide commonly used
to rapidly polymerize the emulsions and, as in tissue engineering due to its biocompatibility,
we demonstrated recently, inclusion of light- biodegradability, and abundant availability . By
[45]
absorbers can afford tight control over the final incorporating alginate beads into the HIPE, then
architecture [8-10,13] . polymerizing and subsequently dissolving the
However, 3D printing emulsions using additive alginate, an additional, larger scale of porosity can
manufacturing technologies require expensive be quickly, easily and cheaply introduced to the
equipment and the trade-off for such architectural polyHIPE-based scaffolds.
fidelity is manufacture speed . In applications To test this hypothesis, we blended HIPEs
[9]
where high levels of control over architecture are with alginate beads at either 50 or 100 wt% of
essential, such as investigations into the effects the initial emulsion volume to create polyHIPE
of geometry or the production of patient-specific scaffolds with a multiscale porosity ranging from
scaffolds , clearly the slower speed of production 1 to 1000 μm. These were compared to standard
[37]
is worthwhile. Nevertheless, for more generic 3D polyHIPE materials with a hierarchy of porosity
cell culture applications using stereolithography ranging from 1 to 100 μm. To evaluate their
can have high initial setup costs and be time performance, MLO-A5 murine post-osteoblasts
intensive [38,39] . Therefore, identifying a simpler were cultured for 14 days, with cell proliferation
approach to introducing a multiscale porosity is and bone-like matrix deposition by histology and
warranted. One potential avenue is particulate lightsheet microscopy assessed.
leaching.
Solvent casting/particulate leaching is a 2 Materials and methods
conventional approach to creating scaffold
porosity. This process involves dissolving a Unless otherwise stated, all materials were sourced
polymer in a solvent such as dimethylformamide from Sigma-Aldrich, UK.
or chloroform then casting around a porogen such 2.1 Alginate bead synthesis
as crystals of sugar or salt [40-42] . These types of
porogen are readily available, cheap, and insoluble Sodium alginate was dissolved in deionized water
in hydrophobic solvents. However, this technique (diH O) at 3 wt% while being mixed (350 rpm)
2
can cause limited pore interconnectivity as there is on a magnetic stirrer hot-plate maintained at 30°C.
not sufficient contact between the porogens to have To create the beads, this solution was loaded into
a continuous porosity, resulting in “skin” forming a 3 mL fluid dispensing barrel (Nordson EFD),
around the pores during solvent evaporation. This sealed using a dispensing piston (InterTronic),
results in samples often having to be thin to ensure and injected through a 30 gauge tip (internal
even porogen dispersal and removal [43,44] . This diameter 0.15 mm, Nordson EFD) from a height
limitation is alleviated when casting a HIPE around of 100 mm into a cross-linking solution of calcium
a porogen as the water droplets are deformable chloride (20 wt% in diH O) using a mechanical
2
ensuring close contact with the porogen. syringe pump (Ultra 2800 Positive Displacement
Providing that the porogen material facilitates Dispenser, Nordson EFD) at 0.2195 mL/s. Beads
an open surface porosity, interconnectivity below 710 µm were selectively collected through
between the emulsion and the voids left by the sieving and stored in diH O until needed. To
2
porogen can be achieved . As sugar and salt are assess size distribution, 50 images were taken of
[19]
water-soluble, they are unsuitable porogens for bead populations (Motic Images Plus software)
polyHIPEs as they would dissolve in the aqueous and diameters measured using Fiji [46,47] .

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