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Arab W, et al.

for 3D bioprinting was observed with both peptides. capsulated in peptide scaffolds illustrated prominently
Formation of ECM with a high level of viable cells was well-organized actin fibers and alignment of myoblast
observed with the 3D-culture cell. The formation of over the scaffold. Since the reorganization of F-actin
cell-laden, viable printed constructs was accomplished plays a crucial role in cell differentiation initiation,
the deposition process via cell embedded within we concluded that our peptide hydrogels promote
[42]
decellularize ECM hydrogel . It has been well known myoblast alignment and facilitate the synthesis of
that alignment of scaffold architecture plays a significant confluent myoblasts through stimulation of the ad-
role in the proper alignment of myofibers, which in turn hesion proteins and cytoskeletal pattern which lead to
induce the conduction of force and contractility towards prompt differentiation. These results do not eliminate
the regeneration of functional skeletal muscle [43] . Our the importance of studying the factors secretion by
results showed that the myoblast cells encapsulated in myoblasts and myotubes during culturing or embedded
[64]
the peptide hydrogel scaffolds demonstrated a high level in the ECM that may also alter the scaffold stiffness .
of cell viability, as well as structural stability without the Controlling the alignment of cells is critical for any
requirement for chemical cross-linking. The morphology tissue-engineered graft to enhance their functionality
[35]
and architecture of our hydrogels are similar to that of and to acquire a highly cellular organization . Different
the natural ECM as shown in the SEM micrographs. cells have been proofed for their high degree of
SEM results have revealed a dense fibrous mesh network alignment including, neural cells [65] , cardiac muscle [66] ,
with a 10–15 nm thickness of the fibers, mimicking skeletal muscle [67] , corneal tissue [68] and vascular
the architecture of the microenvironments found in tissue [69,70] . In particular, the alignment of skeletal muscle
the ECM [35] . Recently, the clinical importance of ECM cells is essential to maximize the contractile power of
based materials in tissue-engineering are highlighted the tissue [31] . Quantification of cellular alignment is
for different tissue regeneration applications [44,45] . The necessary to check the effectiveness of biomaterials and
ECM based material enables the remodeling of construct the engineered microenvironment on the organization
at the damaged site and encourages the formation of of cells. The alignment of myoblast cells was confirmed
a particular tissue rather than scar tissue formation [46] . using a two dimensional fast Fourier transform (FFT)
Hydrogels prepared from natural polymers, such as of the fluorescence images [34,35] . Our results showed that
alginate, gelatin, collagen, chitosan, etc., have been myoblast cells aligned at a particular angle in CH-01
used for bioprinting [47–52] . Alginate is a biomaterial from hydrogels after four days which could be attributed to
brown algae and widely used in various pharmaceutical the fact that CH-01 scaffolds provide a structural cue
and medical applications due to its biocompatibility and to the myoblast cells and help to align and proliferate.
[53]
low toxicity . However, a completely random alignment has seen in
Gelatin, a hydrolyzed form of collagen, has been the alginate-gelatin as illustrated in the alignment plot
widely used in wound dressing, as pro-angiogenic in Figure 7. Finally, as a step towards 3D bioprinting
matrices and absorbent pads for surgical appli- applications of these peptide bioinks, we showed the
cations [54–56] . Alginate-gelatin blends have been used printability of these peptides using extrusion based
as carriers in drug delivery [57,58] and wound dressing printing method. These results indicated that the peptide
fibers [59] . Also, alginate-gelatin blends have been used bioinks are printable and are a promising candidate
as bioinks for 3D bioprinting applications [60-62] . In this for 3D bioprinting of muscle myoblasts cells to create
study, we used alginate-gelatin blend bioink as a positive elastic designed and accurately defined structures with
control. 3D cell viability results confirmed that the cells a uniform distribution of cells within the construct
encapsulated in our hydrogels were healthy whereby that could lead to a better architectural organization of
the proliferation increased by day 8 and did not change muscle cells for the development of skeletal muscle
the bio-reconstruction. CH-01 preserved higher cell tissue engineering application.
numbers when compared to CH-02 and alginate-gelatin 5. Conclusion
which could be attributed to the fact that CH-01 may be
providing native cues and offers more surface area to Our results indicated that both peptide hydrogels offer
the cells to divide and grow. Also, this peptide hydrogel a substantial increase in cell viability and promote cell
has sufficient porosity to accommodate more cells and growth and expansion of myoblast cells. Furthermore,
help in viability. Actin is a major cytoskeletal protein we showed that high cell viability retained with 3D
present in eukaryotic cells which gave information about cultured constructs for at least eight days. We have also
cell shape and motility. This protein also has several shown that both peptides are printable which opens up
other functions such as direct regulation of different the possibility of 3D bioprinting of muscle myoblasts
[63]
transcription factors . and other cell types in the future. We believe that the
Immunofluorescence results of myoblast cells en- described results represent an advancement in the

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