The
development of advanced techniques of fabrication of three-dimensional
(3D) microenvironments for the study of cell growth and proliferation
has become one of the major motivations of material scientists and
bioengineers in the past decade. Here, we present a novel residueless
3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS
emulsion casting and subsequent curing process in temperature-/pressure-controlled
environment. Scanning electron microscopy and X-ray microcomputed
tomography allowed us to investigate the impact of those parameters
on the microarchitecture of the porous structure. We demonstrated
that the optimized emulsion casting process gives rise to large-scale
and highly interconnected network with pore size ranging from 500 μm
to 1.5 mm that turned out to be nicely adapted to 3D cell culture.
Experimental cell culture validations were performed using SaOS-2
(osteosarcoma) cell lines. Epifluorescence and deep penetration imaging
techniques as two-photon confocal microscopy unveiled information
about cell morphology and confirmed a homogeneous cell proliferation
and spatial distribution in the 3D porous structure within an available
volume larger than 1 cm3. These results open alternative
scenarios for the fabrication and integration of porous scaffolds
for the development of 3D cell culture platforms.
In
nature, cells exist in three-dimensional (3D) microenvironments
with topography, stiffness, surface chemistry, and biological factors
that strongly dictate their phenotype and behavior. The cellular microenvironment
is an organized structure or scaffold that, together with the cells
that live within it, make up living tissue. To mimic these systems
and understand how the different properties of a scaffold, such as
adhesion, proliferation, or function, influence cell behavior, we
need to be able to fabricate cellular microenvironments with tunable
properties. In this work, the nanotopography and functionality of
scaffolds for cell culture were modified by coating 3D printed materials
(DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose
nanocrystals (CNCs). This general approach was demonstrated on a variety
of structures designed to incorporate macro- and microscale features
fabricated using photopolymerization and 3D printing techniques. Atomic
force microscopy was used to characterize the CNC coatings and showed
the ability to tune their density and in turn the surface nanoroughness
from isolated nanoparticles to dense surface coverage. The ability
to tune the density of CNCs on 3D printed structures could be leveraged
to control the attachment and morphology of prostate cancer cells.
It was also possible to introduce functionalization onto the surface
of these scaffolds, either by directly coating them with CNCs grafted
with the functionality of interest or with a more general approach
of functionalizing the CNCs after coating using biotin–streptavidin
coupling. The ability to carefully tune the nanostructure and functionalization
of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of
natural microenvironments, which are key to understanding their impact
on cells and developing artificial tissues.
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