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Wei Long Ng, Wai Yee Yeong and May Win Naing

determine the highest possible printing resolution and manner till the pH of the mixture reaches ~ 6.5 to in-
printing accuracy at room temperature. Lastly, bio- itiate the pH-dependent crosslinking using a pH meter
compatibility tests were conducted to evaluate the (HM Digital. Inc.).
potential use of PGC hydrogels for bioprinting of skin
constructs. These outcomes will provide valuable in- 2.3 FTIR Characterization
sights into development of printable hydrogels for The interactions between chitosan and gelatin within
bioprinting of 3D tissue constructs. the polymer blend were investigated with dried gelatin-

2. Materials and Methods chitosan hydrogels using a Fourier Transform Infrared
(FTIR) Spectrometer (Bruker Vertex 80v, Germany).
2.1 Materials and Cells Each dried gelatin-chitosan hydrogel was placed
within the enclosed vacuum chamber one at a time
Chitosan (low molecular weight, 75–85% deacetyla- and FTIR spectra were collected within the range of
tion) and gelatin (porcine skin, Type A) powders were 800–2000 cm via attenuated total reflectance (ATR)
−1
obtained from (Sigma Aldrich, Singapore). Other rea-
gents like acetic acid, sodium hydroxide (NaOH) and technique. The measurements were conducted in trip-
phosphate buffered saline (PBS) solution (pH 7.4 at licate and presented in the transmittance mode.
0.01 M) were sterile-filtered before use. Neonatal hu- 2.4 Rheological Characterization
®
man foreskin fibroblasts (HFF-1 from ATCC SCRC-
1041 TM ) were used in this study. The cell line was The rheological properties of PGC hydrogels were
cultured in a HERAcell 150i cell incubator (Thermo evaluated using the Discovery hybrid rheometer (TA
Scientific) at 37°C in 5% CO 2 using ATCC-formulated instruments, USA). The values of the strain amplitude
Dulbecco’s Modified Eagle’s Medium (DMEM) sup- were first verified to ensure that all measurements
plemented with 15% fetal bovine serum (HyClone TM were performed within the linear viscoelastic region.
from GE Healthcare). Culture media was changed Next, the viscosities of PGC hydrogels were evaluated
-1
every 3 days and the cells were routinely passaged in for shear rates ranging from 0.1 to 100 s at a constant
tissue culture flasks (cells were not used after Passage temperature of 27°C (room temperature). To evaluate
6). The adherent HFF-1 cells were harvested using the sol-gel transition state of the hydrogels, (i) storage
0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) modulus (G’) and (ii) loss modulus (G”) of the 2.5%,
(Invitrogen) at 90% confluency. 5% and 7.5% PGC were then measured at varying
temperatures from 20 to 40°C at a fixed shear strain of
2.2 Synthesis of Polyelectrolyte Gelatin-chitosan 2%. The sol-gel transition state can be determined by
Hydrogels the G’/G” ratio, whereby G’/G” = 1 is the gelling
Modification of chitosan was carried out via the addi- point. All measurements were conducted in triplicate.
tion of gelatin to create a polyelectrolyte gelatin-chi- 2.5 Bioprinting of Biomaterials
tosan hydrogel [30] . 2.5% w/v chitosan was dissolved in
®
acetic acid and mechanically agitated for three hours A 3-D bioprinter, Biofactory (regenHU Ltd., Swit-
to obtain a homogeneous gel. Varying concentration of zerland), was used for printing of PGC hydrogels. The
gelatin solutions (2.5%, 5% and 7.5% w/v) were dis- PGC bio-ink was loaded into a sterile printing car-
solved in sterile PBS solution and stirred at 40°C for tridge and the printing process was conducted using
complete dissolution of gelatin powder. The gelatin an extrusion-based print-head. The hydrogel was de-
solution of varying concentration was then added sep- posited via extrusion-based printing approach and the
arately to the chitosan gel at a pH greater than 4.7 to material flow for each print-head was controlled by
initiate the formation of polyelectrolyte complex be- individual pressure regulators. Pre-defined structures
tween the positively-charged chitosan and negatively were input into BioCAD (regenHU Ltd., Switzerland).
charged gelatin and they were designated hereafter as The printability of different PGC hydrogels was eva-
2.5%, 5% and 7.5% PGC respectively. Equal volume luated using a combination of different printing pres-
of gelatin solution was added to the chitosan gel in a sures (1–3.5 bars) and feed rates (600–1000 mm/min)
drop-wise manner under constant mechanical agitation, using a constant nozzle diameter of 210 µm. Adjacent
followed by subsequent addition of NaOH solution to filaments of 2 cm length at inter-spacing of 1 mm (n =
the gelatin-chitosan polymer blend in a drop-wise 6) were printed and measured in terms of filament

International Journal of Bioprinting (2016)–Volume 2, Issue 1 55