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

Table 2. Shape fidelity assessment score after 3D bioprinting and IIZK peptides, and then the cell-laden constructs
were stained for cytoskeleton, and z-stack 3D images were
Score Description taken using confocal microscopy. The distribution and
1 • Extremely poor resolution arrangement of cells were evaluated within cuboids with
• Slow gelation 1.0 cm edges and 0.26 cm height. After 21 days of culture,
• Forms clumps
• Does not form consistent thread of hydrogel cells in both ultrashort peptides’ cell-laden constructs
• Cannot identify details of structure retained their fibroblast-like morphology, with actin
2 • Poor resolution fibers well-defined. This cell elongation demonstrates
• Slow gelation the connection between cells and the ultrashort peptide
• Clumpy and inconsistent hydrogel, pointing to the high cytocompatibility of
• Structure details are visible but blurry the ultrashort peptide biomaterial and a high level of
• Begins to fall apart as structure gets taller interaction with the ultrashort peptide hydrogel.
3 • Good resolution
• Gelation time is reasonable Z-stack images and (x, y, z) projected area images
• Forms a consistent thread of hydrogel with occasional demonstrated the 3D distribution of the cells throughout
clumping the printed structures. Using both ultrashort peptide
• Details of structure are clearly visible but weak bioink, the cells were found to be dispersed throughout
• Difficult to hold shape for taller structures
4 • Very good resolution the entire printed constructs with cytoplasmic extensions
and cell–cell interaction. A summary comparing the
• Gel forms immediately
• Consistent thread of hydrogel two ultrashort peptide bioink in terms of mechanical
• Details of structure are cleary visible properties, printability, shape fidelity, and biocompatibility
• Holds shape for tall structures but may have gap in is listed in Table 3.
layers due to excess water
5 • Excellent resolution 3.4. Ultrashort peptide bioink supports
• Gel forms immediately chondrogenic differentiation of hMSCs
• Consistent thread of hydrogel is very fine
• Sharp details are clearly visible The differentiation of MSCs is governed by their
• Holds shape for tall structures without sagging due to surrounding microenvironment, including growth
excess water stimulation, activation of intracellular signaling, and
interaction of cells with the ECM. Besides stimuli of the
that the brief delay in gelation resulted in slightly weaker growth factors, it has been shown that matrix stiffness can
walls as compared to IZZK which resulted in a score of 4 regulate and guide the differentiation of these cells toward
for IIZK. This was expected as it reflected the difference a specific lineage. For instance, soft substrates were found
in stiffness and elasticity in both ultrashort peptides, as to promote the differentiation of MSCs toward adipogenic
seen in rheology readings. Overall, IZZK and IIZK both lineage, whereas stiff substrates were found to promote
achieved printability and maintained strong shape fidelity. the differentiation toward osteogenic lineage [41,42] . MSCs
sense the mechanical signals and biophysical cues from
3.3. Assessment of 3D-bioprinted cell-laden the surrounding ECM, eliciting intracellular signaling
constructs pathways that influence and guide cell fate decisions [43,44] .
Long-term cell viability postprinting is one of the most Accordingly, mechanical stiffness is an essential factor
crucial parameters in evaluating the potential use of bioink for consideration when designing material for tissue
for tissue engineering. We had previously demonstrated the engineering applications.
high cell viability rate upon 3D bioprinting using peptide- For the first time, we investigated at the molecular
based bioink and the in-house developed robotic arm level which ultrashort peptide hydrogel (IIZK or IZZK) is
bioprinting , which was further confirmed in this study. better suited for the chondrogenic differentiation of MSCs
[29]
The cell viability of 3D-bioprinted cell-laden constructs and can be preferably used in cartilage tissue regenerative
was assessed using the LIVE/DEAD cell imaging assay. medicine applications. Accordingly, to study chondrogenic
A high percentage of viable cells was observed for both differentiation, the MSCs were 3D-bioprinted using IIZK
ultrashort peptide bioink (Figure 3A). Using the ultrashort or IZZK, and cells were supplemented with chondrogenic
peptide bioinks, instant gelation was achieved without induction media. Then, gene expression analysis of
needing harmful crosslinking reagents.
cartilage-specific markers using RT-PCR was studied at
We also investigated the 3D distribution of cells within different time points (days 7 and 14) upon differentiation
both ultrashort peptides after bioprinting (Figure 3B). In (Figure 4). Chondrogenic biomarkers such as collagen
this regard, hBM-MSCs were 3D-bioprinted using IZZK type II (Col-II), aggrecan, and SRY-related high mobility

Volume 9 Issue 4 (2023) 68 https://doi.org/10.18063/ijb.719