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International Journal of Bioprinting Enhanced osteogenesis in gelatin releasing bioink

is suitable for processing by an automated biofabrication viscosity at T < T sol−gel (i.e., T sol−gel at 30°C) . Therefore,
[26]
technology [2,3] . Therefore, the bioink should include cells it also has suitable rheological properties that allow for
in different environments and forms (e.g., single cells, hydrogel extrusion during layer-by-layer deposition and
cell aggregates, etc.) Additionally, bioinks could, but not shape retention following extrusion to avoid structural
mandatory to, contain bioactive molecules (e.g., growth collapse. The printed structure must also be treated with
factors, DNA, miRNA, etc.) [1,3] . a crosslinker, which may introduce toxicity, for long-
term shape maintenance and stability at physiological
In general, hydrogels are being explored extensively as temperatures [18,27,28] .
bioinks in various tissue engineering applications due to
their favorable cell anchoring and metabolic activities [3,4] . In this study, alginate and gelatin were blended in a
An ideal bioink possesses biochemical and biophysical multi-component hydrogel; the two components were
qualities that are akin to those of natural tissues, as well as selected for their complementary characteristics. Recently,
high printability. It is hard for the current single-component the alginate–gelatin composite was developed and
hydrogels to satisfy all of these specific requirements investigated as bioink. However, these previous studies
and provide biofunctionality, which supports high cell were focused on rheological properties for high printability
viability and cell-instructive capacity as well as printability attributed to gelatin [29-31] . On the other hands, in this work,
supporting high print fidelity for shape retention . In multi-component hydrogel was designed to have high
[5]
brief, to maintain the desired shape and prevent the printed printability and promote cell adhesion and differentiation
construct from collapsing, high crosslinking density or through gelatin behavior. As shown in Figure 1, the bioink
high viscosity is required; however, such conditions need was designed to contain non-crosslinked gelatin that could
a relatively inflexible environment, which limits cellular be both retained by and released from the hydrogel. The
behavior. In contrast, the conditions required for soft release of gelatin from different hydrogel formulations with
matrices favor cell viability, although soft matrices tend different ratios of the components was studied. The effects
to be unstable and do not maintain the desired printed of gelatin on cell viability and activity were investigated in
structure [6,7] . In addition, even the natural biomaterials two ways. Briefly, the effects of gelatin on external cells and
that are widely used because of their high biocompatibility on cells encapsulated in the hydrogel were studied. The
cannot replicate the complexity of natural extracellular fabricable range of printing conditions for scaffold and
matrix (ECM) alone [8,9] . Due to these limitations, the osteogenic differentiation behavior of the fabricated
single-component hydrogel bioinks have a very narrow scaffolds were also investigated.
biofabrication window . To overcome the limitations of
[10]
single-component hydrogels, multi-component hydrogel 2. Materials and methods
bioink systems are being developed and gaining popularity. 2.1. Materials
They commonly incorporate biomimetic components (e.g., Sodium alginate powder (MW: 200,000–300,000
proteins, peptides, and growth factors) and a base polymer, mol/g, FMC BioPolymer, USA), gelatin (Sigma, USA),
and secondary polymers or nanoparticles are included to 1-[3-(dimethylamion-propyl)]-3-etylcarbodiimide
enhance biofunctionality and printability [2,10-14] . methiodide (EDC; Thermo Fisher Scientific, USA),

Among the various polymers available for 3D N-hydroxysulfosuccinimide (sulfo-NHS; Thermo Fisher
bioprinting, alginate was selected as the base polymer in Scientific, USA), 2-(diehylamino)ethyl methacrylate
this study for several reasons. This polysaccharide is well (AEMA, Tokyo Chemical Industry Co., Japan), and
known for its encapsulation and loading capacity (of 2-(N-morpholino)ethanesulfonic acid hydrate (MES
cells and bacteria) and for being easily tunable and cost- hydrate, Sigma, USA) were used.
effective [15-17] . However, due to the viscous characteristic that
causes the printed structures to spread, alginate solutions 2.2. Synthesis of alginate-2-aminoethyl
are incapable of forming stable 3D structures [15,18,19] . Also, methacrylate (MA-alginate)
alginate lacks the ability to adhere to cells due to the Sodium alginate was dissolved into MES buffer solution
absence of cell-binding ligands [20,21] . containing 0.3 M NaCl (pH 6.5) for producing a 1% (w/v)
solution. Then, 1.08 g of sulfo-NHS and 1.92 g of EDC were
Gelatin has low immunogenicity and outstanding added sequentially to the alginate solution and stirred for
biological features, such as cell adhesion and cell elongation, 5 min. Next, 0.82 g of AEMA were added to the mixture,
due to its natural cell-adhesive motifs (RGD peptide). It can and the solution was continuously stirred at room
also be completely resorbed in vivo without toxic products temperature overnight. The resultant solution was filtered
resulting from metalloproteinase-driven degradation [22-25] . through a 0.22 μm filter, dialyzed for 3 days, freeze-dried,
However, highly biocompatible gelatin has a water-like low and kept at 4°C until use.

Volume 9 Issue 2 (2023) 143 https://doi.org/10.18063/ijb.v9i2.660