In Vitro Evaluation of Polyurethane-Chitosan Scaffolds for Tissue Engineering 441
tic acid solution with a ration CH/solvent of 5% w/v was
added and stirred to obtain different homogeneous mix-
tures of 90/10, 80/20, 70/30, 60/40, 50/50 (PU/Chitosan
wt%). Once homogenized the solution was templated at
–78˚C, then the solvent was extracted by a freeze drying
system for 48 h afterwards the composite was cured at
60˚C under reduced pressure for 48 h.
2.2. Osteoblast Cell Culture
The ability of scaffolds to support osteoblast cell adhe-
sion and growth was evaluated for the different PU/CH
composites, the scaffolds were sterilized by immersion in
70% ethanol for 5 minutes, followed by washing with
sterile DI water and finally exposure under UV light in a
laminar flow hood for 12 hours each side of the scaffold .
After that, the scaffolds were incubated at 37˚C in
α-MEM and 10% v/v FBS overnight in order to degas
them. Primary rat calvaria osteoblasts were obtained by
collagenase digestion of calvaria bone from 14 days old
Wistar rats. In all the experiments, cells at first passage
were used. The cells were seeded onto the PU, chitosan,
and PU/chitosan scaffo lds at a density of 50,000 cells per
sample. Cells were cultured in α-MEM supplemented
with 10% FBS, 3% penicillin-streptomycin, 3 mM β-
glycerophosphate and 10 µg/ml ascorbic acid, and then
placed in an incubator at 37˚C with 5% CO2 and humidi-
fied air for a periods of 3, 7, 14, 21 an d 28 days.
At the end of this period the scaffolds were gently
rinsed with PBS and then the adherent cells were tryp-
sin-detached and counted using a hemocytometer (blood
counting chamb er neubauer improved doub le ruled). The
number of adherent cells was expressed as the percentage
of those originally seeded on each respective sample
surface. Cell proliferation was assessed using colorimet-
ric indicator Alamar Blue assay (Alamar BioSciences,
Monterrey, México). After the incubation time the sam-
ples were washed with PBS and placed into fresh, sterile
12-well culture plates. 2 ml of α-MEM containing 1%
v/v FBS and 10% v/v Alamar Blue indicator were added
to each plate containing samples, and the plates were
incubated for 4 h at 37˚C. Absorbance of the extracted
dye, which is proportional to the number of cells attached
to the scaffold, was measured spectrophotometrically
with a microplate reader (microplate spectrophotometer
Bench mark plus, BIO-RAD) at wavelengths of 570 and
600 nm. In order to quantify the number of cells attached
to the scaffolds, a calibration curve from a known num-
ber of osteoblast cells reacting with the Alamar Blue in-
dicator was generated.
Also, alkaline phosphatase activity of the cells was
measured as an early marker of the osteoblastic phenol-
type using an alkaline pho sphatase substrate kit (PIERCE
biotechnology, Monterrey, México). Cells were lysed
with 1% Triton X-100 in DEPC-treated water and three
freeze-thaw cycles. The lysed cells were collected and
stored at –70˚C. A volume of the sample was added to
100 µl of the p-nitrophenyl phosphate solution and incu-
bated at room temperature for 30 minute and then the
reaction was stopped by the addition of 50 µl of 2 N
NaOH. The production of p-nitrofenol was determined
by the absorbance at 405 nm. The results of alkaline
phosphatase activity were normalized by the number of
cells on the scaffolds. At designated time points, cell
attachment and proliferation were visualized qualitatively
using Scanning Electron Microscopy. The cells on sam-
ples were fixed at room temperature with 1% and 3% of
gluteraldehyde for 1 and 24 h, respectively. The samples
were then dehydrated sequentially using ethanol series
(50%, 70%, 90%, 95%, and 100%) for 10 minutes each.
Samples were dried overnight, and analyzed by SEM. In
addition, energy-dispersive spectroscopy was performed
to assess the elemental composition of the inorganic
elements deposited on the scaffolds.
3. Results and Discussion
Polyurethane Chitosan composites had a high and inter-
connected porosity, a necessary requirement for tissue
formation in vitro and in vivo. It is well know n the proc-
essing parameters like template temperature and polymer
concentration have an enormous influence in the mor-
phological and properties of the materials; in this work
the temperature was hold at –78˚C. Therein the chitosan
scaffolds can have a bigger porosity than PU under the
same processing conditions so it can be observed a dif-
ferent morphology and structure since the Chitosan and
blends show greater porosity than pure polyurethane,
therefore the union of the chitosan and polyurethane has
an influence in the morphology of the composite. This
can be tuned in a way that the scaffold maintains the
form desired and the indispensable space for the forma-
tion of new tissue via proliferation and differentiation of
cells or secretion of its extracellular matrix. There are
compatibility and miscibility between both polymers
according with our previous results and can be prepared
according to the require conditions [10].
Rat calvaria osteoblast cells were cultured onto the
scaffolds to evaluate the cell-surface interactions such as
adhesion, proliferation, cell viability and spreading. The
results of adhesion and proliferation are shown in Fig-
ures 1 and 2, respectively. The results of cell adhesion
revealed that during the first hour the osteoblast attach-
ment on chitosan is higher than that on PU and the com-
posites; the osteoblast adherence was similar for PU and
the composites (10% - 17% approximately), meanwhile
for chitosan was around 30%. However, important dif-
ferences were evident after 4 h. PU increases its adherence
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