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Electrohydrodynamic Printed Sub-microscale Fibrous Architectures Improved Cell Attachment and Collagen Type I Deposition
constructs were further incubated with Alexa Fluor manufacturer’s protocol. Briefly, the cell-scaffold
488-conjugated goat anti-rabbit IgG H&L (ab150077, constructs were washed with PBS for 3 times and fixed
Abcam, USA) secondary antibodies for 60 min. Finally, with 4% formaldehyde for 30 min. Then the constructs
F-actin was stained with Alexa Fluor 594 phalloidin for were incubated in BCIP/NBT solutions for 60 min. For
30 min and cell nuclear was stained with DAPI for 5 min. measuring the ALP activity, the cell-scaffold constructs
Cells were visualized by laser confocal fluorescence were washed with cold PBS (4°C) for 3 times and lysed in
microscope. For each sample, the fluorescence images RIPA lysis buffer (strong, without inhibitors, Beyotime,
were captured with a total thickness of 60 μm and a z-step China). After centrifugation at 11,000 relative centrifugal
of 5 μm. The fluorescence intensity was then measured force (rcf) for 20 min, the supernatant was collected and
on the maximum projection stacks of the vinculin channel the amount of converted p-nitrophenol was measured
by Image J software . using the p-nitrophenyl phosphate (PNPP) assay. The
[7]
total amount of protein was evaluated by a bicinchoninic
2.5. MC3T3-E1 morphology, migration on acid protein assay kit (BCA, Beyotime, China) with
scaffolds with micro/sub-microfibers BSA serving as standard protein samples. The ALP
activities of MC3T3-E1 cells were finally quantified by
Cell morphology and migration were visualized by standardization of the amount of converted p-nitrophenol
staining cellular F-actin and nucleus with phalloidin and with the corresponding reaction time and the total amount
DAPI after 4 h, 24 h, 4 days, and 7 days of culture using of proteins.
previously described methods. The fluorescence images
of the cells on the scaffolds were captured with a total 2.7. Statistical analysis
thickness of 25 μm and a z-step of 5 μm. Cell projection
area and cell aspect ratio were defined as critical The quantified and semi-quantified data were presented
parameters for the evaluation of cellular morphology. as mean ± standard deviation. Statistical analysis was
Cell projection area was measured by manually drawing performed using one-way statistical analysis of variance
the outlines of F-actin on the maximum projection stacks (ANOVA) followed by Tukey post hoc test with GraphPad
of the F-actin channel and cell aspect ratio was obtained software. P < 0.05 (*) was considered as statistical
by calculating the ratio of the major to minor axis of difference.
the cells . The cell-scaffold constructs were gradually 3. Results and discussion
[18]
dehydrated by ethyl alcohol and the cellular morphology
was further observed with SEM . 3.1. Characterization of micro/sub-microscale
[28]
fibrous scaffolds
2.6. Osteogenic differentiation of MC3T3-E1 on
scaffolds with micro/sub-microfibers Figure 2 shows the SEM images of EHD-printed M, MS, and
MSN scaffolds with orthogonal microfibers (Figure 2A),
The osteogenic differentiation of MC3T3-E1 cells on microscale and sub-microscale fibers (Figure 2B) as well
scaffolds with micro/sub-microscale fibers was analyzed as microscale and sub-microscale fibers containing nHA
by COL-I immunofluorescence staining, ALP staining, (Figure 2C), respectively. It can be observed that the
and ALP activity. Cells were seeded at a density of microfibers can be regularly deposited according to the
2 × 10 cells per scaffold and cultured for 14 days. designed trajectory with a smooth surface morphology
5
Osteoblastic phenotypic maturation of MC3T3-E1 cells in each type of scaffold. In contrast, the deposited sub-
was analyzed by immunofluorescence staining of COL-I. microscale fibers exhibited a less regular organization, as
After being fixed with 4% formaldehyde, followed by the ultrafine fibers are very tiny and extremely susceptible
permeabilized by 0.3% Triton X-100 and blocked by 5% to electrostatic repulsion and attraction force derived from
BSA, the cell-scaffold constructs were incubated with previously deposited fibers. Besides, the sub-microscale
primary antibodies (COL1A1 (E8F4L) XP Rabbit mAb, fibers with nHA possessed a larger diameter compared
72026, and Cell Signaling Technology, USA) overnight to PCL-PEO sub-microscale fibers (Figure 2C). The
at 4°C. Next, the samples were incubated with Alexa diameters of EHD-printed microfibers, sub-microscale
Fluor 555-conjugated anti-rabbit IgG H&L (4413, Cell fibers, and sub-microscale fibers with nHA were measured
Signaling Technology, USA) secondary antibodies for as 20.51 ± 1.70 μm, 0.58 ± 0.13 μm, and 0.62 ± 0.11 μm,
60 min. Cell nucleus was stained with DAPI for 5 min. respectively (Figure 2D). The size of the sub-microscale
The COL-I was viewed using the confocal laser scanning fibers with or without nHA showed no statistical differences
microscope, and the amount of the deposited COL-I was and was close to the scale of collagen fibrils (<500 nm) .
[30]
quantified using Image J software . The presence of nHA in sub-microscale fibers was further
[29]
ALP was stained using BCIP/NBT ALP color verified using EDS profiles. Calcium and phosphorus
development kit (Beyotime, China) according to the elements were detected in sub-microscale fibers with nHA
4 International Journal of Bioprinting (2022)–Volume 8, Issue 2