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International Journal of Bioprinting G40T60@WNT5A promotes osteoblast differentiation

Figure 4. Selection of critical factors in CTO&BD. (A) Protein–protein interaction (PPI) network of proteins encoded by differentially expressed genes.
Nodes represent proteins, and different colors represent degree values, with colors changing from blue to red indicating the increasing degree values. Edges
represent the interactions between proteins. (B) Bar graph showing the top 30 genes ranked by the degree values. The red arrow points to SCGB1A1 and
WNT5A. (C) Box plot showing the differential expression of SCGB1A1 and WNT5A in high-throughput sequencing analysis. Control group: n = 6, disease
group: n = 6. *P < 0.05, **P < 0.01 compared to the control group.

3.4. Bioinformatics screening identified WNT5A as stiffness of the hydrogel also increased (Figure 5B). We
the key factor of CTO&BD conducted an analysis of the hydrogel’s gelation point in
Next, we imported the proteins encoded by the DEGs relation to its temperature to determine the stability of the
obtained from high-throughput sequencing into the material for bioprinting applications. The four groups were
String database for PPI network analysis. The PPI network all in a gel state below 31°C, and the addition of β-TCP
relationships were visualized using Cytoscape software had no significant effect on the printability of the hydrogel
(Figure 4A). The degree values of each protein in the PPI at room temperature (Figure 5C). Gels, G80T20, G60T40,
network were calculated and sorted. It was found that both and G40T60 displayed uniform compression at pressures
SCGB1A1 and WNT5A were among the top 30 proteins of 230, 360, 380, and 430 kPa, respectively, as observed
in the degree ranking. The degree value of WNT5A (18) optically (Figure 5D). As the content of β-TCP increases,
was much higher than that of SCGB1A1 (6) (Figure 4B). higher pressure is required to obtain uniform chains.
Differential analysis results of high-throughput sequencing The pressure required for bioprinting material extrusion
showed that WNT5A and SCGB1A1 were downregulated depends on the material’s stiffness. Therefore, the G40T60
in CTO&BD, and the differential expression of WNT5A hydrogel, which had the highest hardness, required a
was more significant (Figure 4C). According to published higher pressure. Under the microscope, the observation of
literature, deferoxamine induces Wnt5a expression to the composite scaffold prepared under optimized pressure
promote osteogenic differentiation, while overexpression confirmed a strut thickness of 500 µm (Figure 5E).
of miR-148a inhibits osteogenic differentiation by
suppressing the Wnt5a/Ror2 pathway. Transcriptional 3.6. The chemical composition, crystallinity,
activation of Wnt5a in fibroblasts could promote VEGF- surface morphology, compressive strength, and
independent angiogenesis. 33,65,66 Therefore, WNT5A may degradation characteristics of the scaffolds were
be essential in regulating osteogenesis and angiogenesis characterized
processes in CTO&BD. We further characterized the scaffolds’ chemical
composition, crystallinity, surface morphology,
3.5. Printability assessment of 3D-printed compressive strength, and degradation properties.
biodegradable brackets Figure 6A shows the composite scaffold’s Fourier-
To evaluate the printability and mechanical properties transform infrared (FTIR) spectra cross-linked with 2.5%
of gelatin/β-TCP composite hydrogel, we first analyzed glutaraldehyde, which confirmed the presence of collagen
its rheological data. For 3D bioprinting, hydrogels must and β-TCP in the scaffold and stable interactions between
have shear-thinning and dilution properties. According these components. The XRD pattern of the scaffold is
to the shear rate measurements of viscosity, all groups shown in Figure 6B. At 2θ = 20–25°, gelatin exhibits a
showed shear-thinning behavior, confirming that the large and broad peak, indicating the presence of the peak
hydrogel is a deformable material (Figure 5A). To β-TCP in the amorphous region of the gelatin phase in
evaluate the mechanical properties of hydrogels based all composite scaffolds. With an increase in the gelatin
on the proportion of β-TCP, G’ was measured during the amount, the intensity of these peaks decreases due to an
frequency sweep process. With the increase of β-TCP increase in amorphous gelatin. The surface morphology of
content, the G’ value also increased, indicating that the the freeze-dried scaffold is shown in Figure 6C. We found

Volume 10 Issue 2 (2024) 238 doi: 10.36922/ijb.1461