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International Journal of Bioprinting 3D-Printed scaffolds for diabetic bone defects

3.2. Biocompatibility of PCL porous scaffolds and collagen, OPN, and ATP5A was significantly downregulated
BMSC adhesion and cell migration (P < 0.05) in BMSCs after the high-glucose-medium
The biocompatibility of porous scaffolds was examined intervention, as compared to the normal-medium group.
by live/dead staining and CCK-8 assay after co-culturing Then, BMSCs were co-cultured with four different porous
BMSCs with different groups of porous scaffold extracts for scaffolds in high-glucose medium; the PCL@SS31, PCL@
1–3 days. The CCK-8 results showed that PCL@SS31@E7 E7, and PCL@SS31@E7 porous scaffolds significantly
scaffold extracts significantly promoted the proliferation of promoted ALP activity and formation of calcium nodules,
BMSCs after 1–3 days of co-culture compared with the PCL as detected by the ALP activity assay and Alizarin Red
group, demonstrating good biocompatibility (Figure 3B). staining assay (Figure 4F, K, and L). Through quantitative
Live/dead staining results showed no significant cell death PCR and immunofluorescence experiments, we observed
after co-culture of BMSCs with PCL@SS31, PCL@E7, or higher mRNA and protein expression of OPN and type
PCL@SS31@E7 porous scaffolds for 48 h, indicating that I collagen in the PCL@SS31, PCL@E7, and PCL@SS31@
the porous scaffold materials had no inhibitory effect on E7 groups than in the PCL group. The mRNA expression
the growth of BMSCs (Figure 3A). of ATP5A was also significantly elevated, especially in the
PCL@SS31@E7 group, where the difference was the most
After BMSCs had been co-cultured with the four PCL
scaffolds for 48 h, cell adhesion on the scaffold surface was significant (Figure 4E and G–J).
observed by SEM. As the peptide modification improved In BMSCs, ATP synthesis is reduced upon the
the hydrophilicity of the PCL scaffold surface, the numbers occurrence of mitochondrial respiratory chain (MRC)
of adherent cells on the surface of the PCL@SS31, PCL@E7, dysfunction, which further inhibits cellular activity and
and PCL@SS31@E7 scaffolds were significantly higher than osteogenic differentiation capacity. Figure 5A shows
those on the PCL scaffolds alone (Figure 3D). In addition, the mitochondrial morphology of BMSCs observed by
the cell membranes of BMSCs were stained with F-actin transmission electron microscopy under normal-glucose
protein using a rhodamine phalloidin staining solution, and conditions, and the changes that occurred in the various
the nuclei were stained with DAPI. The attachment of cells on groups were noted: the PCL and PCL@E7 groups suffered
the surface of the four porous scaffold materials was observed from severe damage, with swollen outer mitochondrial
by confocal laser scanning microscopy, and the results were membranes and broken inner membrane cristae; the PCL@
consistent with those obtained using SEM (Figure 3C). SS31 group showed slightly less intracellular mitochondrial
damage, although there were some swollen mitochondria,
To verify whether surface modification of the porous
scaffolds with E7 peptide affected the recruitment of and the inner membrane cristae had disappeared; however,
the mitochondrial structure of the cells in the PCL@SS31@
BMSCs, we used scratch and Transwell assays to assess E7 group remained undamaged, with intact endomembrane
the migration properties of the cells. Both the PCL@E7 cristae and almost no swollen mitochondria. We examined
and PCL@SS31@E7 groups exhibited enhanced migration the rate of oxygen consumption in a cellular oxidative stress
of BMSCs compared with the PCL group, with the most model after 24 h of a high-glucose intervention using an
pronounced migration effect seen in the PCL@SS31@E7 OCR technique (Figure 5B); the results demonstrated that
group (Figure 3E–K). These data demonstrate that surface- the high-glucose intervention led to a decrease in the basal
modified E7 peptides afford porous scaffolds the ability to cellular respiration rate and an increase in proton leakage
recruit BMSCs, which is achieved through the promotion (Figure 5C–E), suggesting decreased function of the MRC
of cell migration.
of the cells. However, when BMSCs were co-cultured
3.3. Regulation of mitochondrial with PCL@SS31, PCL@E7, and PCL@SS31@E7, the
respiratory function and promotion of mitochondrial proton leakage caused by the high-glucose
osteogenic differentiation in BMSCs in intervention was reduced, and the ATP-generating capacity
high-glucose microenvironment was restored. Notably, stronger efficacy was achieved by
To further validate the role of peptide-modified PCL the synergistic effects of the SS31 peptide and E7 peptide
porous scaffolds in regulating the mitochondrial (Figure 5C–E).
respiratory function of BMSCs in a high-glucose To determine whether PCL porous scaffolds modified
microenvironment, we analyzed the expression of genes with peptides could activate the osteogenic signaling
and proteins related to osteogenic differentiation and pathway by improving mitochondrial respiratory function
mitochondrial function in BMSCs cultured in a high- and subsequently promote osteogenic differentiation of
glucose medium using Western blotting, quantitative BMSCs, we performed transcriptome gene sequencing
PCR, and immunofluorescence staining. As shown in analysis of BMSCs after co-culture with the four different
Figure 4A–D, the protein or gene expression of type I PCL porous scaffolds. The sequencing results showed

Volume 10 Issue 4 (2024) 211 doi: 10.36922/ijb.2379