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Atra Malayeri, Colin Sherborne, Thomas Paterson, et al.

electrospinning is that the speed of making the scaf- compounds EHA, IBOA and triacrylate). The compo-
folds is much higher when using HIPEs. The scaffolds nents were mixed using a paddle stirrer (Pro40 Sci-
reported in this study are produced in minutes, while Quip) at 350 rpm while water was added drop by drop;
electrospinning would typically take hours to build the mixture was then left to mix for 5 minutes. The
similar thickness scaffolds. An additional advantage of HIPE was transferred to a glass vial for either the di-
using HIPEs for 3D structuring is the easy inclusion of rect laser write or bulk polymerisation. Bulk photopo-
nanoparticles in the formulations by using Pickering lymerisation of the PolyHIPE was carried out using a
HIPEs. Recently, we demonstrated that hydroxyapatite UV belt curer (GEW Mini Laboratory, GEW engi-
–2
particles can be incorporated in these resins and can neering UV) with a 100 W∙cm UV bulb. The sample
be used for 3D structuring [25] . was passed several times under the UV lamp at a
–1
The hierarchical porosity of these scaffolds plays a speed of 5 m∙min on both sides. The resulting mono-
crucial role as smaller pores limit the migration of liths were immersed in acetone (100 mL for 24 hours).
cells into the scaffold while improving mass transfer, Samples were dried in a vacuum oven and dried under
therefore constraining the cell growth to the outer sur- vacuum afterwards until reaching constant mass.
face [26] . Synthetic materials have been used for this Woodpile structures were manufactured from EH-
purpose as they can provide reproducibility in regards A80 using single photon direct laser write. A passively
to purity and tuneability of the material properties to Q-switched DPSS microchip laser (PULSELAS-P355-
control the tissues’ response [16] . Highly macro- and 300, ALPHALAS) emitting both 532 and 355 nm was
microporous polymers are appealing candidates for used as the light source. The 355 nm UV light was
tissue engineering applications due to their inherent separated using a Pellin-Broca prism (ADB-10, THO-
3D porous interconnected nature, structural strength RLABS), and expanded using a Galilean beam ex-
and tunable mechanical properties [27] . The aim of this pander to approximately 8 mm diameter. The on/off
research was to investigate the development of bio- stage of the light is controlled using the shutter (UN-
compatible non-degradable PolyHIPE materials that IBLITZ LS6, Vincent Associates) linked to a shutter
could present two levels of structural hierarchy, mi- driver (VCM-D1, Vincent Associates). An adjustable
croporosity to achieve optimal cell ingrowth and ma- pinhole was used to produce a uniform circular beam
croporosity to mimic larger tissue structural ordering. of UV light prior to entering the microscope objective
In this study we focus on building a structural mimic (EC-Plan NEOFLUAR 10x, ZEISS), which focused
of trabecular bone and we have studied the growth of the beam onto the sample holder affixed to a high pre-
osteosarcoma on these structures. cision xyz stage, (ANT130-XY, Aerotech for xy trans-
lation & PRO115, Aerotech for z translation), the mo-
2. Experimental Methods tion was controlled using the motion control software
2.1 Materials A3200 (Aerotech). This stage was used to translate the
HIPE-based resin relative to the objectives’ focal spot.
Monomers [isobornyl acrylate (IBOA) and 2-ethylh- HIPE (120 μL) was pipetted into a functionalised 13
exyl acrylate (EHA)], crosslinker (trimethylolpropane mm glass coverslip placed inside a temporary silicone
triacrylate) and the photoinitiator diphenyl(2,4,6-tri- well affixed on top of a glass slide. The laser was
methylbenzoyl) phosphine oxide/2-hydroxy-2-methy- passed over the top surface to polymerise the wood-
lpropiophenone were all purchased from Sigma-Ald- pile lines; 50 μL of HIPE was pipetted on top of the
rich (UK). Hypermer B246 (Croda, UK) was used as a previously cured layer PolyHIPE and the process was
surfactant. All materials were used without further repeated 3 times to produce the woodpile structures.
purification or modification. Cell culture media was The samples were washed in acetone for 24 hours, and
obtained from Invitrogen (Paisley, UK) and supple- then vacuum dried until reaching a constant weight.
ments from Sigma-Aldrich (UK). The samples were sterilised in 70% ethanol for 45
minutes and washed 3 times with phosphate buffered
2.2 PolyHIPE Sample Preparation
saline (PBS) prior to any cell culture.
Hypermer B246 surfactant (0.2 g) and the organic com- Plasma coating was performed in an in-house sys-
pounds EHA (3.7 g), IBOA (1.6 g) and triacrylate (1.4 g) tem formed from a cylindrical borosilicate chamber with
were mixed together until the surfactant had dissolved. stainless steel endplates. Chamber pressure was de-
The photoinitiator was added (5 wt % of the organic tected by an active Pirani gauge (APG-L-NW25 Ed-

International Journal of Bioprinting (2016)–Volume 2, Issue 2 69