Journal of Biomaterials and Nanobiotechnology, 2012, 3, 440-445 Published Online October 2012 (
In Vitro Evaluation of Polyurethane-Chitosan Scaffolds for
Tissue Engineering
Imelda Olivas-Armendariz1, Perla García-Casillas1, Adriana Martel Estrada1,
Alberto Martínez-Villafañe2, Laura A. A. de la Rosa3, Carlos A. Martínez-Pérez1*
1Institute of Engineering and Technology, Autonnomous University of the City of Juarez, Ciudad Juárez, México; 2Department of
Physics of Materials, Research Center of Advanced Materials, Chihuahua, México; 3Institute of Biomedical Science, Autonomous
University of the City of Juárez, Ciudad Juárez, México.
Email: *
Received July 17th, 2012; revised August 16th, 2012; accepted September 2nd, 2012
In this work the use of Polyurethane (PU)-Chitosan(CH) scaffolds prepared by thermal induced phase separation (TIPS)
for osteoblast proliferation and bone mineralization is described. Primary rat calvaria osteoblasts were seeded in the
scaffolds and it was shown that supported cell adhesion and growth. The behavior osteoblast cells growing in the scaf-
fold in function of the different ratio of PU and CH is presented. The results showed that TIPS is an appropriate tech-
nique for the production of PU-CH scaffolds with high potential for application as cell scaffolds in bone tissue engi-
Keywords: Biomaterials; Composite
1. Introduction
Tissue engineering combines cell biology, materials
chemistry, and processing to recreate viable tissue that
restore and maintain the function of the body. The scaf-
fold, a three dimensional porous material is created to
support the cell source and growth factors to facilitate the
differentiation of the cells and its proliferation. Much has
been done, but the state of the art is far from perfect
scaffolds production, and there is much more to do in
order to realize the potential of tissue engineering. The
requirements are complex and specific to the structure
and function of the tissue of interest [1]. Therefore, the
scaffold fabrication technique needs to be developed ap-
propriately to manufacture the scaffold with the desired
physical-chemical and mechanical properties. Also, fac-
tors such as porosity and pore size are very important for
vascularization and the tissue in growth but they are dif-
ficult to control precisely during the process [2]. In the
search to find the material that meet all this characteris-
tics it is common to make blends of two or more poly-
mers [3-6]. Chitosan, a natural polysaccharide, has gen-
erated an enormous interest due to its diverse advantages
like its low cost, easy access (by deacetylation of chitin,
for being the second most abundant natural polymer),
positive charge biocompatibility, and antimicrobial ac-
tiveity [7-9]. However, the poor flexibility in the regula-
tion of mechanical properties and the limits of degrada-
tion has restricted its use. On the other hand, the PU has
been utilized for the development of several kinds of
materials and devices for biomedical applications, the
preparation process and its velocity of degradation can be
controlled and the physical and mechanical properties
can be adjusted. In a previous work we present PU-CH
blends bonded by chemical interaction with tunable
properties [10]. Here in we present the evaluation for
application for bone tissue engineering by means of
seeding osteoblast rat calvaria cells into the scaffold; the
results in cell proliferation and growth as well its miner-
alization suggest that these scaffolds are potentially ap-
propriate for bone tissue engineering.
2. Materials and Methods
2.1. Scaffolds Preparation
The preparation of the Polyurethane prepolymer was
prepared following a methodology previously described
[2]. Briefly, Polycaprolactone diol (Mn 2000) and Poly-
caprolactone triol (Mn 900) were dissolved in 1.4 diox-
ane with c-hexane as a co-solvent. Once the reagents
were dissolved; 1.6 diisocyanatehexane was added and
0.5% in weight of dibutyltin dilaurate was used as cata-
lyst, the ratio PU/solvent was 20% w/v. Afterwards, chi-
tosan of high molecular weight dissolved in a 0.5 M ace-
*Corresponding a uthor.
Copyright © 2012 SciRes. JBNB
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
Copyright © 2012 SciRes. JBNB
In Vitro Evaluation of Polyurethane-Chitosan Scaffolds for Tissue Engineering
Figure 1. Cell adhesion on PU, chitosan, and PU/chitosan
Figure 2. Cell proliferation on PU, chitosan, and PU/chito-
san composites.
significantly from 10% to 30%. On the other hand, 90/10
and 50/50 composites duplicate them adherence, mean-
while, opposed to the fast increase in cell number ob-
served during the first hour, chitosan in creases only from
30% to 35% and the composite 70/30 from 17% to 22%,
respectively. These events are evidence that the rat cal-
varia cell adhesion was not only time dep endent, but also
a function of the material ratio in the composite. Chito-
san content in the composite plays an essential role in
osteoblast adhesion; since chitosan has a higher hydro-
philicity than polyurethane that deals with a more favor-
able topography and chemistry or surface energy for pure
osteoblast adhesion, chitosan has a higher adherence than
the PU and composites. Osteoblast proliferation in the
PU, chitosan and composites was evaluated by using the
indicator Alamar Blue. The number of cells in PU, chi-
tosan and composites increases over time in the evalu-
ated period. The representative graph for cell prolifera-
tion shows similar cell growth trend during cultivation. It
was seen that at early time point (d ay 3), the cell density
on composites is lower than PU; the presence of ch itosan
on the composite surface retards the proliferation of rat
calvaria osteoblast cells at early time point that it is in
good agreement with the results obtained in a previous
work by where they studied the responses of mesenchy-
mal stem cell on Chitosan- coraline composite [11]. After
14 days, the cell density on PU, chitosan, and composites
was higher than those at 3 days and the cell number in
the composites gradually became comparable to PU and
chitosan. Nevertheless, the osteoblast proliferation on all
polymeric composites showed a decrease after 14 days of
culture compare to the cell seeded initially. An explana-
tion for this behavior could be attributed to the low levels
of initial attachment of cells at the surface. On the other
hand, the cell density on th e PU/chitosan composites was
generally higher than PU and chitosan after 21 days;
however, there is not a significant difference suggesting
that the presence of chitosan in the composite does not
have influence in the cell proliferation at the long term.
In addition, the osteoblast proliferation was faster during
day 3 and 14 of culture but slow down considerably
thereafter. The proliferation behavior could be explained
for the changes that occurred in the composition and or-
ganization of the extracellular matrix in order to be
competent for the mineralization process [12]. Another
possible explanatio n for this fact is that cells could reach
confluence on the places where they were seeded, the
rate of cellular death and proliferation is equal and h ence
the proliferation is stopped [13].
Figures 3 and 4 show SEM images of PU, CH, and
Composites seeded with osteoblast cells for 7, 14, and 21
days of culture. The appearance and size of the os-
teoblasts on the polymers is consistent with the mor-
phology of osteoblast cells. After 7 days of culture (Fig-
ures 3(a)-(c)) cells were attached to the pores and the
osteoblasts could be observed in clusters and randomly
attached on the material surface. At day 14, more cells
can be seen into the samples where it can be appreciated
that the cells have proliferated at the adjacent areas
through the pores. It can be seen several layers of cells,
as well as extracellular matrix (ECM) secreted by the
cells, like microvilli forming fibers between them. In
Figure 3(f), the morphology of individual osteoblast cell
on the 70/30 composite after 14 days of culture can be
appreciated where it presents the typical morphology that
exhibit osteoblast cells and they are anchored to the sur-
face by filopodia. In Figure 4 it can be appreciated that
after three weeks of culture the osteoblast cells covered
the whole composite and filled the pores with uniform
The SEM images reveal the existence of calcium-
phosphate mineralization with morphology in form of
spherulites or nodules that it is a biomineralization phase
and it was characterized by energy-dispersive spectros-
copy (EDS). The EDS spectra from the composite-cell
surface showed both calcium and pho sphate signals from
cells on the PU, chitosan and composites where it was
shown higher mineralization on 90/10, 70/30, and 50/50
composites. These results suggest that the proliferation of
Copyright © 2012 SciRes. JBNB
In Vitro Evaluation of Polyurethane-Chitosan Scaffolds for Tissue Engineering
Copyright © 2012 SciRes. JBNB
Figure 3. SEM micrographs of osteoblasts cultured on PU, chitosan, and composites. Osteoblasts cells on (a) 90/10 composite,
(b) 70/30 composite and (c) Chitosan after 7 days of culture; osteoblasts cells on (d) 70/30 composite and (e) PU after 14 days
of culture; (f) Individual osteoblast cell on 70/30 composite after 14 days of culture.
0 1 2 3 4
0 1 2 3 4
0 1 2 3 4
0 1 2 3 4
0 1 2 3 4
Figure 4. SEM micrographs of osteoblasts cultured on PU, chitosan and composites after 21 days of culture. EDS spectrum of
U, chitosan and composites after 21 days of culture. P
In Vitro Evaluation of Polyurethane-Chitosan Scaffolds for Tissue Engineering
rat calvaria osteoblast cells is directly related to the po-
rosity of the material. It is generally accepted than the
open and interconnectivity porosity improve cell seeding
and proliferation in the scaffolds; moreover, it is crucial
for nutrient and waste exchange [14]. On the other hand,
the calcium content in the composites shows a similar
trend of the results obtained in the cell proliferation. So,
the obtained results in the mineralization and prolifera-
tion suggest that the composites are good candidates for
applications in bone tissu e regeneration.
Alkaline phosphatase (ALP) activity was measured as
an indicator of osteoblast cell differentiation. Figure 5
exhibits the general behavior of ALP activity for PU,
chitosan, and composites during a 21-day culture period.
All samples expressed alkaline phosphatase activity th-
roughout the study period where the highest activity of
ALP was presented at seven days of culture in all the
samples. The decrease in ALP activity after the second
week is typically attributed to its down-regulation in
more mature osteoblast phenotypes that is in agreement
with others works [15]. The alka line phosphatase activity
of cells increases as the chitosan content in the composite
increase. These results suggest that the presence of chi-
tosan improved ALP activity of individual cells, which is
commonly used as a signal of normal osteoblast devel-
opment and phenotypic expression that it is very impor-
tant for bone mineralization. Through the alkaline phos-
phates, the osteoblast cells generate phosphates ions,
which are secreted into the extracellular medium. These
ions bond to calciu m and g enerate nucleatio n sites for the
production of calcium phosphates similar to the mineral
phase of bone.
4. Conclusion
The synthesis and processing of polyurethane and chito-
Figure 5. Alkaline phosphatase activity for osteoblasts cul-
tured on PU, chitosan and PU/chitosan composites.
san composite as scaffolds shown to have physico-
chemical properties that are attractive for future applica-
tion in bone tissue engineering. Future investigations
with these materials will be made involving evaluation in
5. Acknowledgements
The authors are very gratefully to National Science
Council of México, CONACyT for their financial sup-
port trough the project sep-conacyt 2007-84339.
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