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.