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.