ABSTRACT
Three-dimensional (3D) bioprinting is a potential therapeutic method for
tissue engineering owing to its ability to prepare cell-laden tissue
constructs. The properties of bioink are crucial to accurately control
the printing structure. Meanwhile, the effect of process parameters on
the precise structure is significant. We investigated the correlation
between process parameters of 3D bioprinting and the structural response
of κ-carrageenan-based hydrogels to explore the controllable structure,
printing resolution, and cell survival rate. Small-diameter
(<6 mm) gel filaments with different structures were printed
by varying the shear stress of the extrusion bioprinter to simulate the
natural blood vessel structure. The cell viability of the scaffold was
evaluated. The in vitro culture of human umbilical vein
endothelium cells (HUVECs) on the κ-carrageenan (kc) and composite gels
(carrageenan/carbon nanotube and carrageenan/sodium alginate)
demonstrated that the cell attachment and proliferation on composite
gels were better than those on pure kc. Our results revealed that the
carrageenan-based composite bioinks offer better printability,
sufficient mechanical stiffness, interconnectivity, and
biocompatibility. This process can facilitate precise adjustment of the
pore size, porosity, and pore distribution of the hydrogel structure by
optimising the printing parameters as well as realise the precise
preparation of the internal structure of the 3D hydrogel-based tissue
engineering scaffold. Moreover, we obtained perfused tubular filament by
3D printing at optimal process parameters.
Keywords: 3D bioprinting; cell viability; process parameter;
tissue engineering scaffold
Introduction
Recent research on the replacement of large arteries with
larger-diameter artificial blood vessels has been comparatively
mature[1-2]. However, research on small-diameter artificial blood
vessels (of <6-mm diameter) remain formidable and crucial
owing to problems such as low compliance in the body and unsatisfactory
long-term patency (often resulting in thrombosis and neointimal
thickening)[1,3-6]. Cardiovascular tissue engineering is a promising
method to improve the traditional vascular graft replacement scaffolds,
especially for small-diameter blood vessels[7-9]. Various
microfabrication techniques for tissue engineering, such as
electrospinning, cell sheet engineering, and mould-casting, have been
widely studied to develop complex multi-layered architecture for
artificial functional tubular tissues. However, these approaches not
only are ineffective in creating the tubular structure with a
target-specific mechanical property but also restrict shape-freedom
owing to the related technical limitations[10]. The
three-dimensional (3D) bioprinting technique is an emerging alternative
to overcome the limitations of fabrication in terms of building tubular
tissues to mimic the native blood vessel[11-13]. 3D bioprinting has
also been utilised for higher-complexity structures with printable bio
composite inks containing living cells and natural and synthetic
polymers that can be controlled in a spatial position[14]. However,
suitable bioinks for producing translationally relevant tissue with
complex geometries have not been identified yet.
An ideal bioink simultaneously offers the properties required for 3D
bioprinting of complex tissues as well as specific biological cues to
support both in vitro and in vivo tissue maturation.
Natural polymer hydrogels with shear-thinning properties are the most
widely used materials in bioinks[15-16]. Carrageenan is a class of
natural polymers that are extracted from red algae and consist of
repeated (1-3)-linked β-D-galactose and (1-4)-linked α-D-galactose
units[20-24]. The composition of carrageenan demonstrates similarity
with mammalian glycosaminoglycans in a component of the extracellular
matrix[25]. In addition, carrageenan inhibits the inflammatory
responses because of the presence of negatively charged carboxyl and
sulphate groups[26].
However, it is well understood that a single-component hydrogel cannot
meet the rigorous requirements of a bioink. Composite hydrogel with
multi-component is thus an alternative idea. Alginates are an attractive
hydrogel for bioprinting applications as their printability can be
changed by altering the polymer density and adding calcium chloride
(CaCl2) for cross-linking[17-19]. Furthermore, we
can introduce an inorganic component to obtain an ideal bioink. We
propose multi-walled carbon nanotubes (MCNTs)[27], with unique
electrical, mechanical, and surface properties, for this purpose. MCNTs
appear well suited as biomaterials to enhance the properties and
functions of medical devices, for example, improving the tracking of
cells, sensing of microenvironments, delivering transfection agents, and
providing nanostructured surfaces for optimal integration with the host
body[28-34], which is not only because of their ability to simulate
dimensions of proteins that comprise native tissues using their unique
properties but also because of their higher reactivity for cell
interactions to improve the cellular functions. Terada et al.[35]
proved that rat osteoblast-like MC3T3-E1 cells attached better on MCNTs
than on collagens.
In this study, we directly printed small-diameter gel filament with
hollow tubular structure through extrusion-based 3D bioprinting instead
of the conventional 3D bioprinting method, such as those involving
deposition or the use of a roller to simulate the tubular structure of
the blood vessel. The feasibility of precise fabrication of
carrageenan/sodium alginate(kc-s) and carrageenan/ MCNTs (kc-c)
composite scaffolds was assessed by analysing the printability, shear
stress, porosity, and pore size distribution. Printing resolutions of
carrageenan-based hydrogels in different printing parameters (such as
printing speed and pressure) were presented in the printability maps.
Small-diameter gel filaments with different structures are printed by
changing the printing parameters of the extrusion bioprinter. We
analysed the porosity and pore size distribution of gel filaments by
using a scanning electron microscope.
On the other hand, the cell viability of the composite scaffold was
evaluated and compared. Then, the biocompatibility of the 3D
carrageenan-based composite scaffolds printed with the cells was
evaluated with optimised printing parameters by fluorescence staining
and imaging. Finally, we analysed the histocompatibility of bioinks with
different components under in vivo conditions.
Methods and materials