Page 240 - IJB-10-1

International Journal of Bioprinting NIR-secretome release for nerve regeneration

microscope of Cytation 3. The number of viable cells was typical O-H stretching peak of GO in the broad band from
quantified using ImageJ software. For the detection of 3600 to 2400 cm . We also observed two peaks in the
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reactive oxygen species (ROS), the fluorinated derivative fingerprint region, at 1600 and 1422 cm , which are present
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of 2′,7′-di-chlorofluorescein (H DCFDA; Sigma-Aldrich) both in GO and, particularly, in alginate. The presence of
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was employed. This probe is non-fluorescent until the different absorption peaks in the spectroscopy of Figure 1F
acetate groups are removed by intracellular esterases confirmed the successful loading of AGO microbeads with
and oxidation occurs within cells. Thus, oxidation can secretome. We then investigated the thermal responsiveness
be detected by monitoring the increase in fluorescence of bioprinted AGO microbeads by setting our experiment
intensity. Cells were first treated with H O at 250 μM for to reach a mild increase in temperature, up to 39°C from
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30 min or with 1× PBS (control group). Microbeads were room temperature (Figure 1G). We used different power
then irradiated with an 808 nm laser for 1 min. After a densities of the infrared laser to achieve the same thermal
recovery time of 30 min, the medium was carefully washed increase after NIR irradiation. To evaluate the cumulative
and replaced with 1× PBS containing 10 μM H DCFDA. release of the MSCs secretome over time, we embedded a
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Cells were incubated for an additional hour at 37°C and fluorescent probe, FITC–Dextran, into the bioprinted AGO
5% CO . PBS containing H DCFDA was then removed, microbeads. We irradiated the microbeads and monitored
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and spheroids were resuspended in complete medium. the rate of increase in fluorescence over time (Figure 1H).
Fluorescence intensity of H DCFDA was recorded by We observed that the release of MSC secretome from AGO
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using a Cytation 3 by exciting at 495 nm and recording microbeads was dependent on the concentration of GO.
emission at 528 nm. Results were expressed as % of control Interestingly, the highest cumulative release was achieved
(untreated) cells. with AGO microbeads having a GO concentration of
0.5 mg/mL. Taken together, our results suggest that AGO
2.6. Statistical analysis microbeads have great biocompatibility and can be used
All measurements were performed in triplicate, and data for various biological applications. Additionally, the
are reported as mean ± standard deviation. Statistical release of molecules from AGO microbeads can be finely
analysis was performed using one-way analysis of variance controlled using NIR irradiation. This characteristic makes
(ANOVA), followed by Tukey’s post-hoc test. Differences AGO microbeads an excellent candidate for controlled
were considered significant when p < 0.05. drug delivery applications, where precise dosing is critical.

3. Results and discussion 3.2. Biological effect of near-infrared irradiation of
3.1. Characterization of alginate-graphene oxide microbeads on damaged neural cells
microbeads We conducted an evaluation on the impact of NIR
In this study, we aimed to stimulate regeneration of neural irradiation on damaged neural cells to assess the potential
cells through the controlled release of the secretome of of AGO microbeads in stimulating cell proliferation and
MSCs using bioprinted AGO microbeads. To achieve reducing the production of ROS. To accomplish this, we
this goal, we first bioprinted AGO microbeads with a initially subjected neural cells to H O incubation at a
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homogeneous size distribution, with a peak at 200 μm concentration of 250 μM. Subsequently, we introduced
(Figure 1A and B). To explore the surface structure of AGO microbeads to the cells and administered NIR
AGO microbeads, we acquired SEM images at different irradiation at previously characterized power densities.
magnifications: 3000× and 10,000× (Figure 1C, top and The outcomes of our investigation are presented in
bottom, respectively). We highlighted alginate polymers Figure 2. Upon NIR irradiation, the neural cells exhibited
having a fiber-like shape all over the surface of the sample sustained high cell viability compared to control cells
(green square), along with GO sheets having different lateral (Figure 2B). Nevertheless, following the induction of
size, as expected (red square). Different concentrations of toxicity through H O , no notable increase in cell viability
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GO, ranging from 0.1 to 0.5 mg/mL, were used to observe was observed across all tested concentrations of GO,
any potential differences in biocompatibility and the release demonstrating similar outcomes to the treatment with
of MSC secretome. Neural cells were then incubated with H O alone (Figure 2C). Notably, a slight enhancement
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AGO microbeads for 24 h, and viability was evaluated as in cell viability was observed at a GO concentration of
a percentage of control (untreated) cells (Figure 1D). We 0.5 mg/mL. The application of NIR irradiation at all
found that even the highest tested concentration of GO tested GO concentrations resulted in ROS levels within
did not cause any significant loss in viability, indicating the physiological ranges (Figure 2D). Conversely, after H O
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great biocompatibility of AGO microbeads. To evaluate treatment, ROS levels significantly increased for all
the surface chemical composition of AGO microbeads, samples (Figure 2E). Interestingly, AGO at a concentration
we performed FTIR analysis (Figure 1E). We depicted the of 0.5 mg/mL exhibited a strong reduction in ROS

Volume 10 Issue 1 (2024) 232 https://doi.org/10.36922/ijb.1045