4. Discussion
The identification and optimization of a suitable biomaterial that can have self-standing and vascular supportive properties are of great importance and are in demand for organ 3D printing. Though some of the single-phase biomaterials are being used in the preparation of bioinks, they often lack on the ability of accommodating multiple cell types suitable for organ engineering. Therefore, it is essential to develop multi-material-based bioinks in order to advance the field of 3D printing toward development of functional tissues and organs.
Keeping these points in view, in this study, a newly formulated multi-material hydrogel, based on the combination of A-SA-Gel, was developed and investigated their suitability in 3D printing of various simple and complex engineered shapes and structures applicable for tissue and organ engineering. As far as to our knowledge and through the literature surgery, this is the first study which explores the preparation and 3D printing of multi-material A-SA-Gel hydrogel using the combination of albumen, sodium alginate and gelatin.
Alginate is an anionic and hydrophilic polysaccharide derived from seaweeds. SA is one of the commonly used bioinks in 3D bioprinting due to its cell compatibility, and rapid and tunable gellation due to its excellent crosslinkability in the presence of ionic calcium solution or other divalent cations [13]. Although SA has excellent biocompatibility, it lacks bioactivity. Therefore, it is necessary to improve its bioactive functional properties suitable for engineering tissues and organs. Gelatin is a highly cell compatible and biologically active polymer derived from collagen by partial hydrolysis [14]. It is also widely used as a bioink either as such or in combination with other polymers, including alginate. The combination of alginate and gelatin often used as a bioink in 3D printing due to their enhanced functional properties as compared to their individual component. However, their ability of neovascularation is not satisfactory. In order to enhance their vascular supportive ability, the authors have introduced albumen, and its vascular supportive behavior has already been demonstrated in their earlier study [12]. In this study, a unique combination of albumin, SA, and gelatin has been developed in order to formulate a novel A-SA-Gel hydrogel to enhance their 3D printability, self-standability, and vascular supportivity of their individual component.
The A-SA-Gel hydrogel was prepared under optimized experimental conditions, and it was thoroughly characterized prior to printing. The current study employed an extrusion-based 3D printing. The hydrogel was subjected to 3D printing under various system and solution parameters in order to optimizing the conditions which could generate and extrude the continuous filaments, leading to various simple and complex engineered shapes and structures through layer-by-layer. As soon as the scaffolds were printed, they were cross-linked by means of chemical ions in order to further strengthen their structural and shape fidelity. The experimental conditions were optimized so that the scaffold’s integrity could be intact and maintained for the initial days during the course of study. The printed scaffolds exhibited high fidelity and robustness.
The viscoelastic properties of the A-SA-Gel hydrogel exhibited shear thinning behavior (Figure 2A), similar to the other bioinks,such as cellulose nanofibers (CNFs) and methacrylated gelatin (GelMA) composite bioink[15] and vascular-tissue-derived decellularized extracellular matrix (VdECM) and alginate-based bioink[16], which favor excellent printability and viability of the entrapped cells due to the alleviated shear stress when passing through printing nozzles at a certain flow rate [17]. The shear stress sweep results showed low loss modulus and high storage modulus of the crosslinked hydrogel (Figure 2B), which further confirms the ability of the A-SA-Gel hydrogel to retain its shape and structure.
After the pre-printing optimization, the A-SA-Gel hydrogel was subjected to print in order to evaluate its printing ability and integrity of the printed structure and shape fidelity. The printer head needle with 410 μm internal diameter was used and 2.8 ± 0.1 Psi stable air pressure as applied for this case. The results of the printed structures are shown in Figure 2C and 2D. It was quite interesting to observe the warping of the scaffold (Figure 2D), due to dehydration. The SEM micrographs shown the surface and cross-sections morphologies of the scaffolds (Figure 2E-2M), which revealed the integrity of mesh-like structure where the adjacent layers were perpendicularly stacked to construct a rectangular porous structure. It is known that the porous structure allows sufficient oxygen and nutrient mass transport into the scaffolding system that contribute to excellent cell growth by preventing or minimizing core necrosis [18]. In addition, the intersections of adjacent layers of the filaments were tightly connected with each other, which help increasing the strength of the scaffolding system overall. It should also be noted that the current formulated hydrogel not only could be used for 3D printing of simple scaffolding system as discussed here, but also could be used to engineer complex structures. For example, various shapes and structures, including a prototype of human ear, were printed out using the A-SA-Gel hydrogel as shown in Figure 3, which further demonstrate the feasibility and printability of the hydrogel suitable for various tissue or organ engineering applications.
The chemical functional groups of albumen, SA, gelatin, and A-SA-Gel hydrogel samples were analyzed using FTIR (see Figure 4A). As seen in the spectrum, all the major characteristic peaks were observed. The FTIR spectrum of the A-SA-Gel hydrogel showed a combined the features of those of albumen, SA, and gelatin. For instance, Amid I (related to C=O stretching vibrations; the peak range of 1700–1600 cm-1), amide II (related to 60% N-H bending and 40% C-H stretching vibrations; the peak range of 1575–1480 cm-1), and amide III (related to N-H bending and C-H stretching vibrations; the peak range of 1400–1200 cm-1) regions were clearly appeared. Moreover, the absorption band at 1021 cm-1 could be attributed to the C-O stretching vibration of SA [19]. From the peaks of the A-SA-Gel spectrum, it could be concluded that the mixture of albumen, SA, and gelatin did not cause any drastic alteration in the position of main peaks associated with the secondary structure of the protein present in the individual components. Moreover, it was obvious that there was no noticeable peak split was observed in the A-SA-Gel spectrum, indicating a homogeneous dispersion of albumen, SA, and gelatin.
The swelling profile curves of the A-SA-Gel and SA/Gel samples in PBS and culture medium are presented in Figure 4B and 4C, respectively. It can be seen from the graphs that the swelling patterns of all the samples were identical irrespective of the medium where the swelling experiments were carried out, and their swelling ratios increased over time during the course of the study. However,the swelling ratio of the SA/Gel sample was found to be higher than that of the A-SA-Gel sample in PBS as well as in culture medium. It was also noticed that the swelling ratio of SA/Gel and A-SA-Gel samples was rapidly increased in the first 15 minutes and then slowly stabilized. This trend indicates that the degree of deformation of the SA/Gel is higher than A-SA-Gel hydrogel, and the degree of water loss of SA/Gel is relatively higher than that of A-SA-Gel sample. Therefore, the time-dependent drying kinetics of the SA/Gel and A-SA-Gel samples were studied and the results are plotted in Figure 4D. Interestingly, as seen from the graph, the drying ratio of the SA/Gel was found to be faster than that of the A-SA-Gel. This can be attributed that the A-SA-Gel hydrogel sample has not only good hydrophilicity but also high water content which is due to the albumen’s high moisturizing functional property [20]. It is noteworthy to mention that the fabrication of scaffolds that provides a moist environment is always preferable for wound healing application where it can also mainly protect the wound against microorganisms as a kind of physical barrier [21].
The results of the degradation profile of the SA/Gel and A-SA-Gel samples are given in Figure 5 along with their morphological behavior. The results show that the dissolution rate of the A-SA-Gel sample is slightly higher than that of SA/Gel. Also, it was noticed that the degradation rate of A-SA-Gel in PBS was higher than in the culture medium (see Figure 5E). This is due to the fact that the albumen protein might be involved to regulate the degradation characteristics of the A-SA-Gel hydrogel. It was also clearly noticed from the series of SEM micrographs (see Figures 5A-5B) that the morphology of the A-SA-Gel hydrogel scaffolding system became fluffy, rougher and swelling over time when compared to SA/Gel scaffolds. These results are also in accordance with swelling measurement results (Figure 4B-4C).
The suitability of hydrogels as bioinks in tissue or organ engineering often depends on their mechanical properties, and structural integrity and shape fidelity. Therefore, the mechanical properties of the formulated hydrogels were thoroughly examined. The compressive strength and the strain of A-SA-Gel hydrogel sample exceeded 6 MPa and 55%, respectively, and was higher than SA/Gel sample (see Figure 6). The data are corroborated well in accordance with earlier studies [14, 22]. As for the tensile strength, the stress and strain of A-SA-Gel hydrogel samples were higher than that of SA/Gel sample. These results clearly indicate that the 3D printed A-SA-Gel hydrogel scaffolding systems have appropriate mechanical properties and they are tunable suitable for tissue or organ-specific application.
Though the structural and mechanical properties of a scaffolding system is important for organ engineering, the cellular compatibility is the most essential property because cells are the fundamental building blocks of tissues and thus organs and they must grow well on the scaffolding system. Moreover, the scaffolds should also support the vascularization process during the tissue organization [23]. Therefore, the cellular and vascular-supportive potential of 3D printed A-SA-Gel hydrogel samples were investigated using HUVECs as a model cell. The results of the in vitro cell culture study are given in Figures 7, 8 and 9. Figure 7 clearly shows that the cells were well attached to the A-SA-Gel hydrogel substrate and were uniformly distributed throughout the scaffolding system even after 4 days of culturing. In addition, there were endothelial sprouting and the formation of branched vessel networks observed on the A-SA-Gel hydrogel scaffolds (see Figure 8), which is a good sign that the scaffolds are vascular supportive in addition to mere cell compatible. The robust neovessels were also formed; the maximum diameter and length of endothelial sprouting are found to be 112μm and 476μm, respectively.
The fluorescent stained images showed dense endothelialized layer with sprouting on the surface of the A-SA-Gel hydrogel substrate (Figure 9A-A2 and 9B-B2). In addition, the SEM micrographs also supported the claim that the HUVECs were well attached on the hydrogel substrate and spread over it (Figure 9D-9F). The CCK 8 assay data (Figure 9G) show that cells were well proliferated onto the A-SA-Gel hydrogel sample as compared to SA/Gel and Petri dish.
All these results clearly demonstrated that the 3D printed A-SA-Gel hydrogel scaffolding systems are cell compatible and vascular supportive with adequate and tunable mechanical properties. Though the SA and Gel biomaterials were widely used as a tissue scaffolding system in our earlier study, and by other groups as reported in the literature, the present study advanced the utilization of those biomaterials as self-standing and vascular supportive in 3D printing of tissue/organ scaffolding systems. The further studies are necessary; however, particularly on how the newly formulated A-SA-Gel hydrogel perform under in vivo conditions to not only to validate its in vitro results and to further examine its efficacy in tissue regeneration which is under progress and the results will be published elsewhere.