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