Abstract
Three dimensional printable
formulation of self-standing and vascular-supportive structures using
multi-materials suitable for organ engineering is of great importance
and highly challengeable, but, it could advance the 3D printing scenario
from printable shape to functional unit of human body. In this study,
the authors report a 3D printable formulation of such self-standing and
vascular-supportive structures using an in-house formulated
multi-material combination of albumen/alginate/gelatin (A-SA-Gel)-based
hydrogel. The rheological properties and relaxation behavior of
hydrogels were analyzed prior to the printing process. The suitability
of the hydrogel in 3D printing of various customizable and self-standing
structures, including a human ear model, was examined by extrusion-based
3D printing. The structural, mechanical, and physicochemical properties
of the printed scaffolds were studied systematically. Results supported
the 3D printability of the formulated hydrogel with self-standing
structures, which are customizable to a specific need. In vitrocell experiment showed that the formulated hydrogel has excellent
biocompatibility and vascular supportive behavior with the extent of
endothelial sprout formation when tested with human umbilical vein
endothelial cells. In conclusion, the present study demonstrated the
suitability of the extrusion-based 3D printing technique for
manufacturing complex shapes and structures using multi-materials with
high fidelity, which
have
great potential in organ engineering.
Keywords: Multi-material
hydrogel; extrusion-based 3D printing;
self-standing
structures; endothelial sprouting; organ engineering.1. Introduction
The recent advances in 3D printing alias additive manufacturing
technology opens tremendous possibilities of engineering tissues and
organs, which mimic native structure and function to some extent. The
emerging 3D printing technology enables even to fabricate
anatomy-specific 3D shapes and structures suitable for organ
engineering[1] [2]. Although rapid progress has been witnessed
in the past decade, there are limitations to the existing printing
technology. For instance, conventional 3D printing often employs bioinks
made up of single-phase biomaterial, which may not be sufficient to
construct precise biomimetic architecture. Therefore, there is a need to
develop a 3D printing technology that utilize printing of multiple
biomaterials to construct a complex tissue architecture that could
accommodate various cell types and biomolecules, required for emulating
physiologically relevant tissues or organs.
Biomimetic approach in 3D bioprinting often requires self-standing and
vascular supportive multi-material bioinks to realize the translational
potential of 3D bioprinting towards clinical applications, from bench to
bedside. Atabak et al., proposed a
method to develop alginate-based self-supportive scaffolds for creating
tubular structures and employed a crosslinking strategy to strengthen
the scaffolds [3]. However, the application of this strategy was
limited by the crosslinking agents and the precise regulation of degree
of crosslinking in each step. Alternatively, thermosensitive
and
photoactivated materials were applied to improve the printability of
large-size tissue-engineering structures. The self-supporting properties
of the thermo- /photo- activated materials, such as gelatin,
poly(ethylene glycol), and gelatin methacryloyl, were improved by
controlling the temperature or light at the extrusion nozzle exit to
modulate the bioink’s pre-crosslinking, but, this strategy is limited by
its versatility, such as the accurate control over the degree of
crosslinking and availability of thermo-/photo- activated materials
[4]. Bin et al. utilized the alginate/gelatin-based multi-material
hydrogel formulation to improve the printing accuracy and biomechanics
of printed structures; heterogeneous aortic valve conduits, for example
[5]. Alexandra et al., demonstrated a multi-material bioink-based
printing method using polyethylene glycol crosslinking for expanding the
biomaterial palette required for bioprinting of customizable tissue and
organ scaffolds [6]. The printed scaffolds facilitated high cell
viability during the course of the study. To obtain a mechanically and
biologically enhanced cell-laden structure, Yeo et al., employed a
coaxial extrusion printing method using collagen-based bioink (core) and
pure alginate-based bioink (shell) to prepare cell-laden mesh structure
[7]. The fabricated cell-laden 3D core-shell structure exhibited
excellent cell viability and efficient hepatogenic differentiation were
observed. All these experimental studies, and others, were clearly
demonstrated the efficacy of multi-material formulation strategy and
it’s potential in creating complex shapes and structures suitable for
tissue or organ engineering.
Among others, vascularization is one of the essential factors for organ
engineering and therefore, the biomaterials that are used for 3D
printing should have the ability to support and promote sufficient
neovascularization within the engineered tissue and organ. Several
studies reported different strategies to facilitate vascularization
within the bioengineered constructs, including growth factor and
co-culture strategy [8, 9]. The growth factor strategy often causes
atherosclerosis and abnormal blood vessels because the amount of growth
factors cannot be precisely controlled [10]. The co-culturing of
different cells, such as endothelial cells, adipose stem cells,
mesenchymal stem cells, etc. were commonly used to study the
self-promoted vascularization [11]. However, high operational
difficulty, low cell viability, and high-cost are some of the common
factors that greatly influence the effectiveness of this method.
In a previous study, the authors were inspired by the processes of
chicken eggs hatching; lots of angiogenesis were formed in egg white,
transfer nutrition in egg white to embryo. Then authors reported the 3D
printing ability of a novel multi-material formulation, based on albumen
(egg white) and alginate, to promote angiogenic sprouting and vascular
network formation [12]. Though the albumen/alginate-based hydrogel
formulation has excellent 3D printability, it has its own limitations
and challenges in printing large-scale structures. The present research
is therefore intended to improve the efficiency of the albumin-rich
hydrogel formulation with unique combination of A-SA-Gel, having
self-standing and vascular-supportive properties suitable of engineering
complex shapes and structures with high fidelity.