Page 35 - IJB-3-2

Juliana R. Dias, et. al.

horizontal configurations; were analyzed and fifty measurements per image were
- Keeping the jet stable, not presenting secondary jets; carried out.
due to the selection of non-conductive materials the jet
is kept stable, and not presenting secondary jets; 2.4.2 Structure
- Providing accurate regulation of voltage due to the Fourier transform infrared (FTIR) spectroscopy with
addition of a controller to the high voltage source. attenuated total reflectance (ATR) was used to evaluate

2.2 Materials the chemical composition of the materials and to detect
possible structural changes. FTIR analyses were carried
Poly (ɛ-caprolactone) (PCL) (Mw 50,000 (g/mol), bulk out using an Alpha-P Brucker FTIR-ATR spectrometer,
-3
density: 1.1 g.cm ) was kindly supplied by Perstorp in the range of 4000–500 cm , at a 4 cm resolution
−1
−1
(UK) and dissolved in dimethyl Ketone (DMK) (Sigma- with 64 scans.
Aldrich), and acetic acid (AA) (PanReac AppliChem).
Gelatin powder from pig skin (type A, 300 bloom, 60 2.5 In Vitro Studies
mesh) were kindly supplied by Italgelatine (Italy). For Human dermal neonatal fibroblasts (hDNF) isolated
polymers dissolution, different solvents were explored as from the foreskin of healthy male newborns (ZenBio,
indicated in Table 1. In order to increase the conductivity US) were cultured, expanded, and maintained in Dul-
of the solutions prepared with acetic acid, 2% v/v of becco’s modified eagle medium (DMEM) (Gibco, US),
triethylamine (TEA, Sigma Aldrich) was added. After at 37 ºC in a humidified atmosphere of 5% CO . The
2
optimization, 1,4-butanediol diglycidyl ether (BDDGE, culture medium was changed twice a week and cells
Alpha Aesar, Germany) was used as the gelatin cross- were trypsinized (0.25% trypsin/0.05% ethylenediamine
linker. tetraacetic acid (EDTA)/0.1% glucose in PBS (pH 7.5))

2.3 Electrospinning of Nanofiber Meshes when they reached 70%–80% of confluence. Cells from
passages between 8 and 11 were used in this study.
Polymeric meshes were processed using a single jet To assess cytotoxicity, electrospun meshes were tested
approach. Table 1 presents the processing parameters in direct (samples) and indirect (leachables) contact
used to produce the meshes. Non-woven electrospun with different pre-conditions (washed and non-washed
meshes were obtained at room temperature and relative in ultrapure water). Samples were sterilized with UV
humidity of 40%–50%. Crosslinking of electrospun light followed by washing during 24 hours. hDNF cells
gelatin fibers were produced through the incorporation were seeded in culture wells for 24 hours at a density of
of BDDGE on the gelatin solution immediately before 2×10 cells/well. 24 hours later, samples (direct contact)
4
fiber electrospinning to avoid the loss of configuration and culture medium in contact with samples (indirect
that is induced through a crosslinking bath after fiber contact) were incubated with cells for another 24 hours.
production. The culture medium was then removed from the wells
2.4 Physico-chemical Characterization and fresh basal medium with 20% v/v resazurin (Sigma-
Aldrich) was added. Cells were incubated (37 ºC, 5% v/v
CO ) for an additional period of 2 hours, after which 300
2.4.1 Morphology and Fiber Diameter 2
µL per well were transferred to a black 96-well plate and
The morphology of the produced meshes was examined measured (Ex at 530 nm, Em at 590 nm) using a micro-
by scanning electron microscopy (SEM) using a Quanta plate reader (Synergy MX, BioTek, US). The control
400 FEG ESEM/EDAX Genesis X4M (FEI Company, consisted in cells alone.
USA). Prior to examination, samples were coated with For the quantification of the total double-stranded
a gold/palladium (Au/Pd) thin film, by sputtering, using DNA (dsDNA) content, the cell pellets were recovered
the SPI module sputter coater equipment. SEM images from the wells and washed with phosphate-buffered
were also used to measure the fiber diameter using Image saline (PBS). The suspension was then centrifuged
J software. For each condition, three individual samples (10,000 rpm, 5 min) and then stored at ‒20 ºC until

Table 1. Parameters tested to optimized the electrospun mesh production
Solution parameters Processing parameters
Polymer
Solvent system Polymer concentration (wt%) Distance between collector and needle (cm) Voltage (kV) Flow rate (mL/h)
AA/TEA (2% v/v)
PCL 6, 11, 17 0.72, 3.17
DMK 7, 10, 12 7, 10, 12
Gelatin AA/TEA(2% v/v) 5, 10, 15 0.4, 0.72

International Journal of Bioprinting (2017)–Volume 3, Issue 2 123