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A new design of an electrospinning apparatus for tissue engineering applications

further analysis. The dsDNA quantification was per- flow rate of 3.17 mL/h, 12 cm distance between needle
formed using the Quant-iT PicoGreen dsDNA kit and collector and 10 kV of voltage; 15 wt% of gelatin
(Molecular Probes, Invitrogen, US), according to the dissolved in AA and 2% v/v of TEA, produced with a
manufacturer's protocol. Briefly, the samples were flow rate of 0.4 mL/h, a 12 cm distance between needle
thawed and lysed in 1% v/v Triton X-100 (in PBS) for and collector and 12 kV of voltage. Moreover as gelatin
1 hour at 250 rpm at 4 ºC. Then, they were transferred is a water-soluble protein, a crosslinking is needed to
to a black 96-well plate with clear bottom (Greiner) and improve its mechanical properties and to increase its
diluted in Tris-EDTA (TE) buffer (200 mM Tris–HCl, 20 stability in aqueous medium [18] . Gelatin fibers were in
mM EDTA, pH 7.5). Finaly, samples were incubated for situ crosslinked with BDDGE, according a protocol
5 min at room temperature in the dark, and fluorescence previously established [19] . The morphology of selected
was measured using a microplate reader (Ex at 480, Em meshes are shown in Figure 5A–C. The fiber diameter
at 520 nm). measurements, the reduced standard deviation observed

2.6 Statistical Analyses and the homogeneity of the obtained fibers demonstrates
the stability of the system in producing nanoscale
All data points were expressed as mean ± standard meshes. From Figure 5E, it is also possible to observe
deviation (SD). Statistical analysis (Levene’s and T-test) that the developed system improves fiber deposition in
was carried out using IBM SPSS Statistics 20.0 with the collector.
99% confidence level for cytotoxicity assays. The results FTIR-ATR spectra, used to evaluate possible structural
were considered statistically significant when p ≤ 0.05 changes in the electrospun meshes, are shown in
(*). Figure 5D. The spectrum of PCL meshes presents a
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3. Results and Discussion 1720.7 cm peak, corresponding to the C=O bond,
cha racteristic to esters, and additional peaks between
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750 and 1500 cm , corresponding to the CH groups
2
3.1 Microscopic and Macroscopic of PCL chain. Two other peaks at 2863.69 cm and
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Characterization of Electrospun Meshes 2941.57 cm can also be observed, corresponding to
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SEM images of the different produced meshes are the CH bonds. The FTIR spectrum of gelatin shows
presented in Figure 3 and Figure 4. For simplicity, only pro minent peaks in four different amide regions,
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the tested conditions that resulted in fibers without beads name ly at 1700‒1600 cm , corresponding to amide I;
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or drops are presented in these figures. 1565‒1520 cm , corresponding to amide II; 1240‒670
-1
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Results show that a stronger electric field increased the cm , corresponding to amide III; and 3500‒3000 cm ,
amount of produced fibers per time, which is correlated corres ponding to amide A. The absorption of amide I
to the higher amount of charges into the solution, con tains contributions from the C=O stretching vibration
there by increasing the jet velocity and, consequently, of amide group and a minor contribution from the C–
supplying more solution to the collector. The distance N stretching vibration. Amide II absorption is related
between the needle tip and the collector also determines to N–H bending and C–N stretching vibrations. Amide
the fiber diameter. By increasing this distance, the III presents vibrations from C–N stretching attached
flight time is longer, allowing the solvent to evaporate, to N–H in-bending with weak contributions from C–
resulting in higher polymer chain stretching, which leads C stretching and C=O in-plane bending. At 2930 and
−1
to a decrease in fiber diameter. These results show that 2890 cm , it is possible to observe two peaks associated
the designed electrospinning is able to process proper to the contribution of aliphatic moieties from BDDGE,
meshes, being particularly relevant the production of confirming the incorporation of BDDGE into the gelatin
gelatin meshes, which is strongly affected by ambient matrix (Figure 5D). For both samples, no solvent re-
parameters, namely the relative humidity. sidues were detected and no structural changes were
Concerning the PCL and gelatin meshes, according observed.
to the parameters tested significant differences were 3.4 Cytotoxicity of Nanofibers Produced
observed in terms of fiber diameter or morphology.
Thus, for further analyses, the following requirements Cytotoxicity of the produced electrospun meshes system
were selected, providing (i) longer distance to promote was assessed to demonstrate the process stability, as a
solvent evaporation and high chain stretching, (ii) high stable jet allows to produce electrospun meshes without
density of fibers per area, requiring less production solvent deposition. According to the obtained results
time and (iii) continuous and uniform fiber production (Figure 6), fibroblasts cultured on the electrospun
enhancing mechanical performance. Therefore, the meshes remained metabolically active for both PCL
follow ing fiber production parameters were selected: and gelatin meshes. After 24 hours, no cytotoxicity
17 wt% of PCL dissolved in DMK, produced with a was observed either in direct or indirect contact assays.

124 International Journal of Bioprinting (2017)–Volume 3, Issue 2