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Scaffolds produced by combining porogen leaching and emulsion templating
in highly porous samples where cells reside deep encourages deeper cell penetration into the
within the scaffold encased in extracellular matrix polyHIPE network as cells have greater nutrient
than in plain polyHIPE scaffolds where cells are availability and waste transport in comparison to
all the outer edges. Therefore, it is likely that cell plain scaffolds at these deeper locations.
number could be increasingly underestimated as In summary, we have shown that alginate-bead
scaffold porosity increases. porogen-leaching of polyHIPEs can be performed
At day 7, mineralized matrix deposition in a single-step process. This quickly and easily
was lower in porogen leached polyHIPEs than produces multiscale porosity scaffolds with
plain ones (Figure 4B and C). As osteoblasts pore sizes spanning three orders of magnitude
do not begin to deposit mineralized matrix until (1 – 1000 μm). This approach enhances initial cell
confluent , the increased potential for ingrowth seeding efficiency, promotes ingrowth and uniform
[54]
into porogen leached samples likely meant that matrix deposition, and allows cells to remain
MLO-A5 were still in a proliferative state, at this viable deep within the scaffold. Overall, these
time point, hence the lower calcium deposition. findings have implications in tissue engineering of
These differences were no longer present by day both bone and other tissues due to the ability to
14, which indicate a faster rate of matrix deposition recreate the hierarchical porosities observed in a
in the porogen leached scaffolds in the 2 week wide range of natural biological tissues.
nd
than the plain. Furthermore, a similar quantity of
mineralized matrix was deposited in the alginate- Authors’ contributions
leached scaffolds to the plain scaffolds by day
14 despite having a significantly reduced culture Conceptualization (RO, GR, FC), data curation
area due to the presence of alginate beads during (RO), formal analysis (RO, CS, RE), funding
polymerization (up to 50% less total material in acquisition (RO, GR, FC), investigation (RO,
the case of the 100 wt% scaffolds). Considering all CS, RE), methodology (RO, CS, RE), project
scaffolds had the same exterior dimensions (5 × 5 × administration (RO, CS, RE, GR, FC), resources
5 mm), this indicates a better distributed neo-tissue (GR, FC), supervision (RO, CS, GR, FC),
formation throughout the alginate leached scaffolds. validation (RO, CS, RE, GR, FC), visualization
Photographs of scaffolds before destaining show (RO), writing – original draft (RO), and writing –
how mineral distribution was more uniform in review and editing (RO, GR, FC).
the porogen leached scaffolds at both time points, Conflicts of interest
whereas it was confined to the exterior surfaces
in plain scaffolds. These observations on matrix The are no conflicts of interest to declare
distribution from low magnification photographs
agree with the histology and lightsheet microscopy Acknowledgments
(Figures 5 and 6). In both techniques, cells and We acknowledge funding from the Engineering
matrix in plain polyHIPEs are only apparent on and Physical Sciences Research Council (Grant
the outer perimeter of the scaffold. In contrast, no. EP/L505055/1 and EP/N509735/1) and
infiltration readily occurred on porogen leached Biotechnology and Biological Sciences Research
scaffolds, with the greatest ingrowth occurring on Council (Grant no. BB/F016840/1). We also
the most porous scaffolds and viable cells being acknowledge the EPSRC Henry Royce Institute
present in the scaffold millimeter(s) from the outer
surface. This deeper infiltration likely occurs for funding (Grant No. EP/P02470X/1) for the light
two reasons. First, the connected macroporosity sheet microscope.
left by the alginate beads provides a facile route References
for cells to enter the bulk of the scaffold during
seeding. Second, improved diffusion throughout 1. Wu S, Liu X, Yeung KW, et al., 2014, Biomimetic Porous
the scaffold due to the additional macroporosity Scaffolds for Bone Tissue Engineering. Materials Sci Eng R

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