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International Journal of Bioprinting 3D bioprinting of ultrashort peptides for chondrogenesis

prone to denaturation, which can lead to a decrease in its precipitated peptide was separated from the supernatant
biocompatibility and cell viability . by centrifugation and kept in a vacuum desiccator for
[25]
drying. The collected peptide was purified using Agilent
The field of 3D bioprinting has advanced rapidly
in recent years, with many promising applications in 1260 Infinity Prep-HPLC with Zorbax PrepHT SB-C18
column for 12 min at 20 mL/min flow rate. MilliQ water
biomedical engineering, regenerative medicine, and and acetonitrile containing 0.1% formic acid were used
tissue engineering . To further progress the field, as mobile phases. The purified peptide was collected with
[26]
researchers have explored various methods for optimizing more than 60% in yield.
3D bioprinting processes. One such method is the use
[26]
of ultrashort peptide bioinks . Our previous study 2.2. Characterization of ultrashort peptide hydrogel
demonstrated the potential of such bioinks for optimizing 2.2.1. Peptide gelation and hydrogel formation
a 3D bioprinting process, and using these bioinks results Peptide gelation and hydrogel formation potential for
in improved printability, enhanced mechanical properties, IIZK and IZZK peptides were evaluated as previously
[26]
and biocompatibility . Furthermore, using ultrashort described . Briefly, the peptide powder was dissolved
[29]
peptide bioinks in 3D bioprinting can result in a more cost- in 0.9 mL of MilliQ water and vortexed until a clear and
effective process. Overall, 3D bioprinting for chondrogenic homogeneous solution was observed. Then 0.1 mL of
applications is an exciting and rapidly advancing field of 10× phosphate-buffered saline (PBS; without Ca and
2+
[27]
research . It has the potential to revolutionize the way Mg ) was added to the peptide solution. The vial was kept
2+
we treat cartilage-related diseases and injuries and could undisturbed, and the soft solid hydrogel formation was
provide a more precise and efficient way to create tissue- observed using the vial inversion method. The time and
engineered cartilage . minimum concentration at which each peptide did form a
[28]
In this study, we aimed to investigate at the molecular hydrogel were identified.
level the potential of using two tetrameric ultrashort 2.2.2. Scanning electron microscopy
peptide bioink in cartilage tissue engineering. We Scanning electron microscopy (SEM) was used to identify
analyzed the printability of both ultrashort peptides at the nanofibrous topography of peptide hydrogel at
physiological conditions, studied their biocompatibility, different peptide concentrations. First, samples for SEM
and demonstrated their ability to induce chondrogenic imaging were prepared by dehydrating peptide hydrogels
differentiation of human bone marrow mesenchymal stem in a gradually increasing ethanol concentration. Then, the
cells (hBM-MSCs), which are ultimately to be used in dehydrated gel was transferred and dried in a Tousimis
cartilage tissue engineering. Automegasamdri-916B series C Critical Point Dryer. The
dried sample was sputter-coated with 5-nm Ir thickness
2. Materials and methods before imaging. SEM images were taken using an FEI
Magellan XHR Scanning Electron Microscope with an
2.1. Design and synthesis of self-assembling accelerating voltage of 3 kV.
ultrashort peptides
Two peptide sequences were used in this study: Ac-Ile- 2.2.3. Rheology measurements of ultrashort peptide
Ile-Cha-Lys-NH2 (IIZK) and Ac-Ile-Cha-Cha-Lys-NH2 hydrogels
(IZZK). Both ultrashort peptides (IIZK and IZZK) were Mechanical stiffness of IIZK and IZZK peptides was
synthesized by solid-phase peptide synthesis (SPPS) analyzed using a TA Ares-G2 Rheometer equipped with an
using CS136X CS Biopeptide synthesizer. The peptide advanced Peltier system (APS). The mechanical stiffness of
coupling was conducted on rink amide resin using a the peptide gels was measured at ambient temperature using
mixture of TBTU (3eq.), HOBt (3eq.), DIPEA (6 eq.), an 8-mm parallel plate with a gap of 1.8 mm, between the
and Fmoc-protected amino acid (3eq.). Piperidine/DMF upper and lower plates. The hydrogel samples were made
at 20% (v/v) was used to deprotect the Fmoc group on by mixing 13 mg/mL peptide solution with 7× PBS with a
the N-terminus of the ultrashort peptide sequence and ratio of two to one based on the flow rate ratio from Pump
proceed to the next coupling step. After coupling the 1 against Pump 2+3. These gels were prepared 1 day before
last amino acid to the peptide sequence, the sequence measurement using the ring-cast method. For each peptide,
was capped with an acetyl group. The peptide was six replicates were prepared to control the accuracy of the
then cleaved from the resin with an acidic solution of measurements. The stiffness was analyzed through two
trifluoroacetic acid (TFA), triisopropylsilane (TIS), and successive tests: frequency sweep and amplitude sweep.
water for 2 h. The peptide was subsequently collected, First, the frequency sweep was performed for a range of
and cold diethyl ether was added to induce peptide angular frequency of 0.1–100 rad/s with a strain of 0.1%.
precipitation that was kept standing overnight at 4°C. The Then, the amplitude sweep was performed by applying a

Volume 9 Issue 4 (2023) 64 https://doi.org/10.18063/ijb.719