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Novel ultrashort self-assembling peptide bioinks for 3D culture of muscle myoblast cells

donor tissues. network of short self-assembled peptides nanofibrous
Tissue engineering is an alternative approach for hydrogels has shown to support pluripotent stem cells
[6]
volumetric muscle loss treatment and organ fabrication , survival and differentiation [33] . Herein, we have tested
where the use of biological scaffolds are being explored. the biocompatibility of two different tetramer self-
These structures have been fabricated to deliver the assembling peptides for 3D cell culture of mouse
aesthetic and functional needs of skeletal muscle tissue myoblast (C2C12) to validate the effectiveness of
regeneration and have been tested in vitro and implanted these peptide bioinks for long-term cell viability in
in vivo to facilitate the native functions of the damaged 3D. We found that the 3D cultured cells within the
tissue [7–9] . However, there are a few negative outcomes hydrogels preserve cells viability and promote growth
associated with autologous cell-laden simple scaffolds and alignment of mouse myoblast cells. Moreover,
such as high complexity cell encapsulated scaffolds [10–14] . 3D cultured cells using our scaffold start replacing the
Three-dimensional bioprinting technology is a degrading hydrogel by secreting their matrix. We also
promising technique, constructing 3D structures to tested the printability of these peptide bioinks by using
fabricate platforms used for tissue engineering and a commercially available 3D bioprinter. We propose that
regenerative medicine [15,16] . The scaffold is a major these peptide bioinks could be promising biomaterials
component of a tissue-engineered construct that for 3D bioprinting and tissue engineering applications.
is made from biomaterials with 3D architecture. It 2. Materials and Methods
provides a microenvironment that accommodates
more cells, promotes cell adhesion and proliferation. Two tetramer self-assembling peptides CH-01 and
Despite its promise, the lack of suitable bioinks has CH-02 were procured from Bachem AG, (Budendorf,
constrained the development of bioprinting technologies. Switzerland). Mouse myoblast cells (C2C12) were
Biocompatibility, biodegradability, non-immunogenic, purchased from (ATCC, USA). Dulbecco’s Modified
non-mutagenic and non-hemolytic scaffolds are Eagle Medium (DMEM), fetal bovine serum (FBS),
essential properties for the long-term culture of cells Dulbecco’s phosphate-buffered saline (PBS) solution,
and engineered tissue for implantation to avoid adverse and penicillin-streptomycin antibiotics (P/S) were
physiological side effects. Several biomaterials have purchased from Gibco (Grand Island, USA). CellTiter-
®
been used in vitro as bioinks [17,18] . Scaffolds from Glo Luminescent 3D cell viability assay kit was
self-assembling peptides are of particular interest for procured from Promega (Madison, USA). MTT
bioprinting due to their synthetic but natural background. Cell Proliferation Assay kit, Cytotoxicity assay kit
They have been used as biomaterials for applications in (CytoTox96) and LIVE/DEAD Viability/Cytotoxicity
®
regenerative medicine and as matrices for the delivery Kit were purchased from ThermoFisher Scientific,
of encapsulated bioactive molecules for therapeutic USA, Promega, USA and Life TechnologiesTM,
applications [18-22] . The peptide scaffolds were used in USA, respectively. Immunostaining antibodies anti-
injectable forms enabling more accurate and localized vinculin and Rhodamine-Phalloidin were purchased
delivery of cancer drugs which in turn helped to reduce from (Invitrogen, USA), and anti-mouse IgG-FITC was
harsh side effects generally associated with cancer purchased from Sigma, USA. T175 or T75 cell culture
treatments [23] . Ultrashort amphiphilic peptides also flasks and 96-well plates were procured from Corning,
showed accelerated skin regeneration when used a burn USA.
[24]
wound dressings . 2.1 Hydrogel Preparation
Scaffolds aimed for skeletal muscle tissue engineering
must be capable of accommodating more cells and Two tetramer peptides CH-01 and CH-02 were used
promoting the formation of highly aligned myofibers in this study. Peptides were synthesized by Bachem
throughout the construct [25-28] . Porous scaffolds with AG, (Budendorf, Switzerland) using solid phase
aligned morphology in 3D, micro/nanofibrous struc- peptide synthesis and purified to above 95% via HPLC.
tures have been studied for skeletal muscle tissue Lyophilized peptide powder was dissolved in Milli-Q
[29]
engineering . It is imperative for a successful skeletal water and vortexed to get a homogenous solution.
muscle platform to present the right kind of structural Subsequently, 10× phosphate buffered saline at the final
cues to pre-align muscle cells to enhance myogenic concentration of 1× was added to the peptide solution
differentiation and myotube formation [30,31] . Many and vortexed briefly. Gelation occurred within a few
hydrogels have been used and evaluated for their seconds in CH-01 at 4 mg/mL and CH-02 at 3 mg/mL
cellular activity, mechanical properties, and myogenic peptide concentration. Alginate-gelatin was used as a
potential. There is still need to find the most appropriate positive control.
material which has the efficacy to maintain mechanical Alginate-gelatin was prepared by mixing an equal
stability and promote myotube formation [32] . The 3D amount (1:1) of each content; then serial dilution

2 International Journal of Bioprinting (2018)–Volume 4, Issue 2