Page 358 - IJB-9-6

International Journal of Bioprinting Surface modification of PCL scaffolds

3.3. Effect of surface modification on cell 3.6. Surface modification mediated osteogenesis via
morphology and adhesion integrinα2/β1-PI3K-AKT signaling pathway
Cell morphology and adhesion were analyzed through Transcriptomics analysis identified 2116 differentially
double staining of vinculin (red) and F-actin (green). expressed genes (DEGs), with 1413 upregulated and 713
The cytoskeletons of BMSCs were observed to form a downregulated DEGs (Figure 6A and B). The underlying
ring-shaped structure and interweave within the pores mechanisms were then analyzed using KEGG analyses,
of all scaffolds (Figure 3A). In addition, the study found which revealed the top 20 KEGG pathways (Figure 6C).
that BMSCs on M-PCL scaffolds produced a greater Previous studies have shown that the use of hierarchical
amount of vinculin compared to those on PCL scaffolds. microgroove/nanopore topography on titanium implants
This was confirmed through quantitative analysis of can improve osseointegration through the activation of the
the average fluorescence intensity (Figure 3A and B). PI3K-AKT signaling pathway . Therefore, it was chosen
[45]
SEM results showed that BMSCs on M-PCL scaffolds for subsequent study. Further analysis demonstrated a
displayed more and longer filopodial protrusions significant upregulation of gene expression in the PI3K-Akt
compared to relatively short and less filopodia on PCL signaling pathway in the M-PCL group compared to the
scaffolds (Figure 3C). PCL group (Figure 6D). Western blotting confirmed the
activation of the PI3K-Akt signaling pathway (Figure 6E).
3.4. Effect of surface modification on osteogenic These findings suggest that the NaOH-modified surface can
differentiation enhance osteogenic differentiation through the integrinα2/
The study utilized qRT-PCR to detect the expression of β1-PI3K-Akt signaling pathway (Figure 6F).
osteogenesis-related genes such as collagen type 1 (Col1),
osteocalcin (Ocn), osteopontin (Opn), and runt-related 3.7. Effect of surface modification on bone
transcription factor 2 (Runx2). Results showed that, formation in vivo
except for the Col1 gene, the expressions of the other In order to evaluate the effect of surface modification
three genes were significantly higher in BMSCs cultured on bone formation of critical-sized bone defects, the rat
on M-PCL scaffolds than in BMSCs on PCL scaffolds cranial defect model was utilized. After 1 and 3 months of
after 7 days (Figure 4A), whereas the expression levels implantation, micro-CT 3D reconstruction was performed
of all four genes were significantly higher at 14 days in to assess new bone regeneration on scaffolds. The resulting
M-PCL scaffolds compared to PCL scaffolds (Figure 4B). micro-CT 3D images showed that there was a higher
Immunofluorescence staining was used to measure the amount of newly formed bone on the M-PCL scaffolds than
protein expression of OCN, OPN, and RUNX2. The results on the PCL scaffold at both 1 and 3 months (Figure 7A;
of the staining were consistent with those obtained from Figure S1 in Supplementary File). The results were further
qRT-PCR. Specifically, the expressions of OCN, OPN, confirmed through quantitative analysis. It was observed
and RUNX2 proteins were observed to increase on PCL that M-PCL scaffolds had a higher BV/TV value in
scaffolds following alkaline treatment (Figure 4C). comparison to PCL scaffolds (Figure 7B). Additionally, the
new bone formed on M-PCL scaffolds exhibited improved
3.5 Effect of surface modification on mineralization
and ALP activity trabecular structural features, which was confirmed by the
The study measured mineralization and ALP activity using quantitative analysis of Tb.Th and Tb.Sp (Figure 7C and D).
ARD and ALP staining. BMSCs were cultured in basic To measure new bone formation, fluorescent double
medium without osteoinductive factors to determine the labeling of calcein (green) and Alizarin Red (red) was
osteogenic capacity of the scaffolds. The depth of ARD performed. The M-PCL scaffold showed a bigger distance
staining was higher in M-PCL scaffolds compared to PCL between the two fluorescent signals (Figure 7E). Further
scaffolds on days 14 and 21. Furthermore, there was more quantitative analysis revealed that the mineral apposition
and larger calcium ion deposition and mineralization in the rate (MAR) of M-PCL scaffolds was higher than that of
M-PCL scaffolds compared to PCL scaffolds (Figure 5A). PCL scaffolds (Figure 7F).
These findings were also confirmed by quantitative analysis After a duration of 1 month, the control group showed
(Figure 5C). Similarly, the study found that M-PCL thin fibrous tissue at the defect site. However, there was a
scaffolds had a higher level of ALP activity compared to stable integration between M-PCL scaffolds and host bone
PCL scaffolds after 7 and 14 days of culture (Figure 5B). at the edge of the bone defect. Only a few scattered bone
Meanwhile, additionally, quantitative analysis of ALP formations were noted in PCL scaffolds. After a period of 3
staining confirmed that alkaline treatment of PCL scaffolds months, a higher amount of bone formation was observed
resulted in increased ALP activity (Figure 5D). in M-PCL scaffolds, resulting in a bony bridge at the defect

Volume 9 Issue 6 (2023) 350 https://doi.org/10.36922/ijb.1071