Hard Tissues and Materials

Journal of Biomaterials Applications

0(0) 1–12
Preparation and characterization of ! The Author(s) 2018
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DOI: 10.1177/0885328218772708
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Fernando Javier Aguilar-Perez1 , Rossana Vargas-Coronado1,

Jose Manuel Cervantes-Uc1, Juan Valerio Cauich-Rodriguez1,
nez2, Juan Jose Pavon-Palacio†,3,
Raul Rosales-Iba~
Yadir Torres-Hernandez4 and Jose Antonio Rodriguez-Ortiz4

Abstract
Segmented polyurethanes were prepared with polycaprolactone diol as soft segment and 4,4-methylene-bis cyclohexyl
diisocyanate and L-glutamine as the rigid segment. These polyurethanes were filled with 1 wt.% to 5 wt.% titanium
particles (Ti), physicochemically characterized and their biocompatibility assessed using human dental pulp stem cells and
mice osteoblasts. Physicochemical characterization showed that composites retained the properties of the semicrys-
talline polyurethane as they exhibited a glass transition temperature (Tg) between
35 C and
45 C, melting temper-
ature (Tm) at 52 C and crystallinity close to 40% as determined by differential scanning calorimetry. In agreement with
this, X-ray diffraction showed reflections at 21.3 and 23.6 for polycaprolactone diol and reflections at 35.1 , 38.4 , and
40.2 for Ti particles suggesting that these particles are not acting as nucleating sites. The addition of up to 5 wt.% of Ti
reduced both, tensile strength and maximum strain from 1.9 MPa to 1.2 MPa, and from 670% to 172% for pristine and
filled polyurethane, respectively. Although there were differences between composites at low strain rates, no significant
differences in mechanical behavior were observed at higher strain rate where a tensile stress of 8.5 MPa and strain of
223% were observed for 5 wt.% composites. The addition to titanium particles had a beneficial effect on both human
dental pulp stem cells and osteoblasts viability, as it increased with the amount of titanium in composites up to 10 days
of incubation.

Keywords
Segmented polyurethane, titanium particles, bone regeneration, dental pulp stem cells, osteoblasts

Introduction dental pulp stem cells expressed high levels of RunX2

In the quest of improving properties of segmented pol- osteogenic markers. In an effort to improve the
yurethanes (SPUs) for biomedical use, several mechanical and biological properties of glutamine-
approaches have been pursued. Some of them, made
use of biodegradable soft and rigid segment in the PU,
while others introduce an inorganic phase of proved 1
Centro de Investigacion Cientifica de Yucatan, Merida, Yucatan, Mexico
biocompatibility to obtain a composite based on 2
Universidad Nacional Autonoma de Mexico, Facultad de Estudios
PU.1,2 In previous studies, our group prepared SPU Superiores Iztacala, Ciudad de Mexico, Mexico
3
Universidad de Antioquia, Medellin, Colombia
with polycaprolactone diol (PCL) as soft segment and 4
Universidad de Sevilla, Escuela Politecnica Superior, Sevilla, Espa~
na
4,4-methylene-bis cyclohexyl diisocyanate (HMDI) and †
Deceased
L-glutamine (GLU) as the rigid segment and improved
their biocompatibility by the addition of either Corresponding author:
Juan Valerio Cauich-Rodriguez, Centro de Investigacion Cientifica de
hydroxyapatite or silica-based bioactive glasses.3,4 Yucatan A.C., Calle 43 No. 130, Colonia Chuburna de Hidalgo, C.P.
In the first case, alveolar odontoblast were able to 97205, Merida, Yucatan, Mexico.
adhere to these composites while in the second, Email: jvcr@cicy.mx
2 Journal of Biomaterials Applications 0(0)
based PUs, titanium particles (Ti) are used in
this study.
Titanium is a suitable metallic biomaterial for
bone tissue regeneration because of its balance of
mechanical, physical–chemical, and biofunctional
properties.5 It is considered inert in the human body
since it does not present either surface corrosion or
other reactions in physiological medium. When tita-
nium is implanted, good bone healing is observed
since it does not lead to the usual foreign body reac-
tions, typical of implanted materials such as encapsu-
lation or chronic inflammation.6 The titanium surface
has the ability to adsorb various proteins such as
glycosaminoglycans, fibronectin, fibrinogen, and
therefore, some authors suggest the possibility of a
titanium–protein complex formation, which enhances
its biocompatibility.7–9 Despite these good properties,
improvements in titanium biocompatibility have been
achieved through various types of surface treat-
ments.10–13 For example, pure titanium does not
adhere to the bone, since a biological layer between
5 nm and 10 nm thick separating the titanium
from the bone is formed.6 However, when an
apatitic layer is deposited on titanium, the bone
heals without encapsulation, and allows good bone
interlocking.
Composites of PU and titanium-based particles have
been reported with various applications including anti-
microbial coatings and substrates for bone regenera-
tion. Zhang et al.14 describe antimicrobial properties
of composites of PU and titanium, under ultraviolet
radiation. Hieda et al.15 reported to increase shear
bonding strength of Ti and SPU interface by modifying
terminal groups with a silane. A study by Pareta et al.16
report good adhesion of osteoblasts to titanium struc-
tures coated with PU. Similarly, the study by
Sakamoto et al.17 report good adhesion strength of
PU-coated titanium structures. On the other hand, da
Silva et al.18 reported composites of PU and titania and
concluded that the composites showed better mechan-
ical and thermal properties than pristine PU without
mentioning their biocompatibility.
Although good properties are expected for PU/Ti
composite biomaterials, no reports on their biocompat-
ibility with either HDPSC or osteoblasts have been
found in the literature. Therefore, the aim of this
study was to obtain composites made of segmented
PU and titanium particles by a low temperature
method, and to perform a physical–chemical and
mechanical characterization of them. Furthermore, as
there is little knowledge on the suitable scaffold prop-
erties to support HDPSC and osteoblast adhesion and
proliferation, we expect to determine their suitability
for dental tissue regeneration.
Materials and methods
Characterization of titanium particles (Ti)
The titanium (Ti) particles used in this study were grade
4 commercially pure (c.p.) Ti powder, synthesized by a
hydrogenation/dehydrogenation process as reported by
the supplier, SE-JONG Materials Co. Ltd. (Seoul,
Korea). In order to establish the chemical nature, struc-
ture, and morphology of the particles they were charac-
terized by several physical-chemical techniques. Particle
size was measured by suspending Ti particles in distilled
water using a Beckman Coulter LS100Q laser diffrac-
tion particle size analyzer. Particle morphology was
observed by scanning electron microscopy (SEM)
using a JEOL JSM 6360LV microscope. Elemental com-
position was obtained by energy-dispersive X-ray spec-
troscopy (EDX) by means of INCA X-sight Model
7582, Oxford Instruments coupled to the SEM. In addi-
tion, X-ray photoelectron spectroscopy (XPS) was con-
ducted with a Thermo Scientific K-Alpha X-ray
photoelectron spectrometer (with a monochromatic
source of Al Ka with an energy of 1486.6 eV) on both
etched (Ar ions for 30 s) and non-etched samples. X-ray
diffractograms (XRDs) were obtained with a Siemen
D5000 diffractometer with radiation CuKa (k ¼ 1.5416
Å), in the 2h range from 5 to 60 , with a step count of 3
s and a step size of 0.02 (2h).
PU synthesis
SPUs were synthesized by two-step polymerization
method with 1:2.05:1.05 (PCL:HMDI:GLU)
molar ratios. For this, PCL (molecular weight of
2000 g mol
1 from Aldrich) was dissolved in anhy-
drous dimethylformamide (DMF) (Sigma-Aldrich) and
then, an excess of 4,4-methylene-bis ciclohexyl diiso-
cyanate (Aldrich) was slowly added in the presence of
0.3% of stannous octoate (Sigma) during 4 h at 60 C.
During the second step, a nonessential amino acid,
GLU (Sigma), was added and left to react during 2 h
more. The PU was precipitated in water, and washed
several times before drying at 60 C during 24 h.
The molecular weight of the synthetized pristine
PU was Mw ¼ 77,649 g mol
1, as obtained by gel
permeation chromatography.
Composite preparation by solvent casting
Composites with 1, 3 and 5 wt.% of c.p. Ti powder were
prepared by dispersing the proper amount of titanium
particles in chloroform, and then adding this suspension
to a PU chloroform solution. The suspension was soni-
cated during 5 min and then poured in a Teflon mold.
Aguilar-Perez et al.
Films of the composites were obtained after slow solvent
evaporation at 25 C for a minimum of 24 h.
Physical-chemical and mechanical characterization
of PU/Ti composites
Fourier-transform infrared spectroscopy (FTIR). FTIR spec-
tra were obtained using attenuated total reflectance
(ATR) with ZnSe crystal in the 4000 and 650 cm
1
spectral range averaging 100 scans with a resolution
of 4 cm
1. For this, a Thermo Scientific Nicolet, 8700
spectrometer was used.
Raman spectroscopy. Spectra were acquired using an
inVia Renishaw Raman spectrometer. The system
was capable of collecting spectra over a Raman shift
spectral range of approximately 3200–100 cm
1. A 633
nm argon laser was used as the excitation radia-
tion source.
Differential scanning calorimetry (DSC). Melting point (Tm)
of the soft segment was determined with a Perkin
Elmer DSC-7, using 5–7 mg of the composite, after
heating from
5 C to 150 C with a heating rate of
10 C/min under nitrogen atmosphere. In addition, the
relative percent crystallinity (Xc) of the PCL in the PUs
was determined from the enthalpy of fusion using the
equation (1):
DHf
%Xc DSC ¼  100 (1)
Wss  DH f
where DHf is the enthalpy of melting of PUs obtained
experimentally by DSC during the first trace, Wss is the
theoretical mass fraction of the flexible segment, and

DH f is the enthalpy of 100% crystalline PCL reported
as 136 J g
1.19
Thermogravimetric analysis (TGA). Mass loss was obtained
with 25 mg of the sample after heating from 50 C to
650 C at 10 C/min under nitrogen atmosphere by
means of a TGA-7 from Perkin-Elmer. From the
mass loss first derivative, the decomposition tempera-
ture (Td) was obtained.
Dynamic mechanical analysis (DMA). Storage modulus (E0 )
and Tan d were obtained with a Perkin Elmer DMA-7.
Rectangular samples of 20 mm long, 4 mm wide, and
0.1 mm thickness were deformed in the extension mode
with a static load of 80 mN and a dynamic load of 65
mN at frequency of 1 Hz while heated from
100 C to
75 C at 5 C/min.
3
X-ray diffraction (XRD). Crystallinity of the PCL and Ti
was observed by XRD using a Siemens D5000 diffrac-
tometer as mentioned above while Xc was calculated
using equation (2):
Ac
%Xc XRD ¼  100 (2)
Aa þ Ac
where Aa is the area under the amorphous hump, and
Ac is the area under the crystalline peak.
Scanning electron microscopy (SEM). Surface morphology
and elemental distribution Ti on the composites was
studied with a JEOL, JSM 6360LV with acceleration
voltage of 20 kV. Microanalysis mapping was con-
ducted with EDX (INCA X-sight Model 7582,
Oxford Instruments) during 300 s.
Tensile mechanical test. Elastic modulus (E), maximum
tensile strength (rmax), tensile strength at break (rbreak),
deformation at break (ebreak), and strength (roy) and
strain (eoy) to an offset yield at 2% of deformation
were obtained after tensile test according to ASTM
D882. For this, a Shimadzu AGSX with a cross head
speed of 250 mm/min was used. Films of 40 mm
long (effective gauge length of 25 mm), 5 mm wide, and
0.1 mm thickness, were used. Data were compared using
one-way analysis of variance (ANOVA) with post hoc
multiple comparison performed using Tukey’s test.
A minimum of n ¼ 5 were used and p < 0.05 was consid-
ered significant. Additionally, a low cross head speed test
(50 mm/min) was performed in order to compare
mechanical behavior of materials at different strain rates.
Biological studies
Isolation and characterization of human dental pulp
stem cells (HDPSCs)
Human dental pulp was obtained, with informed
and written consent, from the third molar of 16–20
years old individuals after routine tooth extraction.
HDPSC were isolated from dental pulp as follows.
Dental pulp was cut into small pieces, then washed
with phosphate buffered saline (PBS) (Biowest),
digested with 1% collagenase solution (Sigma-
Aldrich) for 30 min at 37 C and centrifuged at 1000
r/min to obtain a cell pellet. Then, cells were resus-
pended in low glucose Dulbecco’s modified Eagle
medium (DMEM) (Biowest), supplemented with 10%
fetal calf serum (Biowest) and 1% penicillin–strepto-
mycin (Sigma-Aldrich). Cells were cultured in 25 cm2
Nunc flasks (NunclonTM) at 37 C and 5% CO2 in a
humidified atmosphere. Medium was changed twice a
week during expansion. When cells reached 80–90%
4 Journal of Biomaterials Applications 0(0)
confluency, they were separated by magnetic cell sort-
ing (MACS), using CD44 micro beads, human
(Miltenyi, Biotec) as described elsewhere.20 The cellular
populations isolated were identified as mesenchymal
stem cells based on their ability to adhere to the plastic
culture plates, fusiform morphology, and expression of
surface markers like antihuman CD44. HDPSCs from
third passage, seeded at a density of 5  103 per well,
were used for cell viability and proliferation assays.
Isolation of mice osteoblasts. Osteoblasts were isolated
from the femurs of five female mice at postnatal fifth
day, after CO2 sacrifices in accordance to NOM-062-
ZOO-1999 standard under approval by the Committee
for the Use of Experimental Animals at FES-I. Tissue
was cut in small pieces of approximately of 2 mm2 in
preparation for enzymatic digestion in type I collage-
nase (Sigma-Aldrich) for 10 min, and this solution was
then filtered. After this, DMEM was added and cells
were incubated in a type “t” Falcon flask at 37 C with
5% of CO2 and 95% of humidity. Culture medium was
replaced twice a week until cells reached confluence at
day 5. Finally, cells of the cell passage number 2 were
harvested and seeded to a density of 5  103 per well for
each assay.
Cell viability. Pristine PU and its composites (PU/1% Ti,
PU/3% Ti, PU/5% Ti) were sterilized by ultraviolet
exposure for 20 min on each side. The viability
was assessed with a live/dead assay (Live/Dead,
InvitrogenTM) according to the manufacturer’s proto-
col. In this case, calcein AM (0.5 ml) and ethidium bro-
mide (2.0 ml) was dissolved in PBS (997.5 ml), then 100
ll of this solution were added to each sample and incu-
bated for 30 min while protected from light at 37  C in
a humidified atmosphere with 5% CO2. The cells were
counted using a fluorescence microscope (Zeiss
HXP 120C).
V
R not reveal the distribution and/or location of Ti par-
Cell proliferation. The MTT test (Roche Life Science)
was used to assess cell proliferation in the composites
seeded with 5  103 of HDPSCs after 24 h and 10 days
of culture. After the incubation time, 100 mL of dimeth-
yl sulfoxide was added to solubilize the formazan salts.
Afterward, the mixture was analyzed in a microplate
reader at 570 nm.
Osteoblasts proliferation was assessed by Alamar
blue assay (Sigma-Aldrich) as this assay does not lead
to cell death.21 For this, 20 mL of rezasurin salt were
diluted to 5.98 mL with DMEM without phenol red.
Cells were seeded at a density of 1  103 per well, in a
96-well plate and incubated 24 h at 37 C with 5% of
CO2 and 95% humidity. Then, 100 mL of rezasurin solu-
tion were added and incubated for 2 h. Absorbance was
measured using an ELISA BMG LAbtech POLARstar
OPTIMA (Ortenberg, Germany) at 550 nm. A compar-
ison between treatments at day 1 and 10 was made by
ANOVA, and followed by a Mann–Whitney test, where
p  0.05 was considered significant.
Results and discussion
Properties of titanium particles
Ti particle size showed an average particle size of
23.5 lm, median of 23.2 lm, and mode of 28.7 lm
within the range from 1.5 lm to 43.6 lm in agreement
with manufacturer data. By SEM (micrographs not
shown), it was observed that Ti particles were of irreg-
ular shape and with sizes in close approximation of
what is reported by manufacturer (average particle
size of 18 lm 3, max. of 45 lm).
XPS analysis (see Figure 1(a)) showed after 30 s
argon erosion that the amount of titanium (Ti2p at
459 eV) was 24.5 at%, 20.1 at% for carbon (C1s at
285 eV), and 55.3 at% for oxygen (O1s at 531 eV)
in agreement with Cai et al.22 who reported a similar
elemental composition of Ti particles by XPS.
Deconvolution of the peaks for Ti2p (Figure 1(b))
was associated to Ti4þ (90.8%) and Ti2þ (9.2%)
while deconvolution of O1s (Figure 1(c)) showed the
presence of O2
(66.7%) and other oxygen containing
species (33.3%). Overall, these results suggest that par-
ticles are from titanium with an oxidized surface layer,
TiO2. The presence of carbon can be explained by par-
ticle contamination or residuals from synthesis method.
Ti distribution in PU/Ti composites
Physical inspection of Ti composites showed that they
become opaque and darker as the concentration of Ti
increased in the translucent PU. SEM micrographs did
ticles as shown in Figure 2 (top). However, EDX map-
ping (see Figure 2 (bottom)), revealed that Ti particles
are well dispersed in composites, although rich areas of
Ti were detected, especially at Ti concentrations of
5 wt.%.
In addition to the distribution of Ti particles, EDX
microanalysis (see Table 1) showed that as the wt.% of
Ti is increased in the composite, this element increased
very close to the expected amount. The maximum con-
centration of Ti detected at the surface was 0.8 at.% for
1% composites, while it was 3.1 at.% and 4.4 at.% for
3% and 5% composites, respectively. This composi-
tional drift clearly reflects either the heterogeneous sur-
face distribution of the Ti particles or its possible
coating by the PU.
Aguilar-Perez et al. 5

(a) 7E+04 Titanium Particles (b) 16000

O1s Ti2p3/2
Surface
30 s etching 14000
6E+04

12000
5E+04
Intensity (Counts/s)

Intensity (Counts/s)
4+
Ti2p 10000 Ti2p 1/2 Ti
4E+04
8000
Satellite 2+
Ti
3E+04 6000
C1s
2E+04 4000

2000
1E+04
0
0E+00
800 600 400 200 474 472 470 468 466 464 462 460 458 456 454 452 450
Binding energy (eV) Binding energy (eV)

(c) O1s
25000

O2-
20000
Intensity (Counts/s)

15000

10000

5000

544 542 540 538 536 534 532 530 528 526
Binding energy (eV)

Figure 1. XPS survey spectra (a) of non-etched Ti particles and 30 s argon etched Ti particles. Deconvoluted peaks of Ti2p (b), and
O1s (c).

Figure 2. SEM micrographs (top) of the cross section of PU/Ti composites, PU/1% Ti (a), PU/3% Ti (b), and PU/5% Ti (c). EDX
mapping for Ti on the corresponding composites (bottom).

Infrared/Raman spectroscopy. FTIR spectra of PU neat stretching vibration of CH2 groups from PCL,
films and composites are shown in Figure 3(a). HMDI and glutamine. The carbonyl stretching
The unfilled polymer showed absorption bands at, (C¼O) appeared in the interval between, 1680 and,
3373 cm
1 corresponding to the N–H bond in the ure- 1760 cm
1 which includes the ester group from the
thane/urea and at, 2932 cm
1 and, 2860 cm
1 those PCL, and the urethane group (NHCOO). Amide II
corresponding to the asymmetrical and symmetrical absorption (urethane N–H bending þ C–N stretching)
6 Journal of Biomaterials Applications 0(0)

was located at, 1530 cm
1 and, 1240 cm
1, while the
peak at, 1165 cm
1 was attributed to the C–O–C
stretching vibration in the soft segment.23,24 At
1636 cm
1, urea absorptions were detected in agree-
ment with previous works.4,10,25 In composites, infra-
red absorptions from the PU remain and bands from Ti
were not observed since metal–metal bonds cannot be
detected by conventional FTIR spectrometers using
ATR technique or because they were masked due to
their low concentration. For pristine titanium only
adsorbed water (3432 cm
1), carbon-containing spe-
cies (2921 and 2852 cm
1), and oxidized titanium
(705 cm
1) were observed.26
Raman spectroscopy can be used for the detection
of titanium (see Figure 3(b)). It is expected that rutile
phase of TiO2 shows a Raman shift at 141.1, 466.3,
614.5, and 819.1 cm
1, while anatase phase at 141.1,
154.4, 381.8, 503.7, 526.9, and 653.6 cm
1. In this
study, peaks at 417 and 610 cm
1 were detected and
assigned to TiO2, but not detected in composites prob-
ably because of their low concentration. Furthermore,
PU showed strong Raman absorptions at 1108 (C–O),
1305 (CH2 or C–N), 1440 (CH2 or C–N), 2867 (CH2
sym), and 2919 cm
1 (CH2 asym) and medium intensity
absorption at 1723 cm
1 (C¼O in ester or urethane).
Table 1. Elemental composition (at.%) of PU/Ti composites
obtained by energy-dispersive X-ray spectroscopy (EDX).
PU PU/1% Ti
C 66.1  1.3 65.3  0.8
N 7.6  2.9 8.9  0.3
O 26.3  0.9 25.0  1.4
Ti 00 0.8  0.7
PU: polyurethane.
Thermal properties
DSC thermograms showed that the PU and their com-
posites are semicrystalline as the exhibited a melting
temperature (Tm) of 52 C, while DMA thermograms
(data not shown) showed a significant drop in the stor-
age modulus of the composite between
40 C and

20 C, suggesting their alpha transition (Ta) at this
temperature range.3,4 As the Tg of PCL is located at

60 C, their shift to higher temperatures is suggesting
polymer movement restrictions when incorporated into
the PU. However, as this transition was not affected by
Ti particles, it is suggested that Ti particles are proba-
bly located either in the interface between soft and rigid
segment or in the rigid segment bulk.
As the melting temperature of the composites did
not change with Ti addition, this suggests that Ti par-
ticles are not acting as nucleating agents (see first DSC
trace on Figure 4(a)). In agreement with these observa-
tions, XRDs of the composites showed typical PCL
reflections at 23.6 and 21.3 (see Figure 4(b)).2,3,23 In
composites, PCL peaks become less intense while the
typical peaks of titanium at 35.1 , 38.4 , and 40.2
increased.12 The percentage of crystallinity of PUs, as
measured by DSC, was not to be affected by the inclu-
sion of Ti particles in the composites with values of
40%, 39%, 43%, and 37% for PU pure, PU/1% Ti,
PU/3% Ti, and PU/5% Ti, respectively. Crystallinity
percentage, measured by XRD showed values of 40%,
PU/3% Ti PU/5% Ti 39%, 42%, and 36%, respectively in agreement with
those obtained by DSC and literature.3
67.7  3.1 67.9  2.3 TGA thermograms of composites showed a main
3.9  2.3 3.9  2.9
decomposition temperature (Td) between 300 C and
25.3  1.4 23.8  2.6
350 C, which was related to degradation of urethane
3.1  0.5 4.4  0.3
bonds in the soft and rigid segments.27,28 A second
residual mass drop was observed near 450 C and it

PU PU/1% Ti PU/3% Ti PU/5% Ti Ti

(a) 240 (b) 160000
PU
220
140000
PU/1% Ti
200 PU/3% Ti
180 120000 PU/5% Ti
Transmittance (a.u.)

2919
Ti
160
Intensity (a.u.)

100000
140 417 610

2867
120 80000
100
60000 1108 1440
80 1305 1723

60 40000

40
20000
20

0 0
4000 3500 3000 2500 2000 1500 1000 500 500 1000 1500 2000 2500 3000

Wavenumber (cm–1) Raman shift (cm–1)

Figure 3. Vibrational spectra of PU/Ti composites. IR (a), and Raman (b).

Aguilar-Perez et al. 7

(a) 52°C (b) 3000 21.3°

32 PU
PU
PU/1% Ti
PU/1% Ti
28 PU/3% Ti 2500
PU/3% Ti
PU/5% Ti PU/5% Ti
24 2000
Ti

Intensity (a.u.)
Heat flow (W/g)

20
1500

23.6°
16
40.2°
1000

12
500 38.4°
35.1°
8

0
–20 0 20 40 60 80 100 120 140 160 0 10 20 30 40 50 60
Temperature (°C) 2q

Figure 4. Effect of titanium on crystallinity of composites. DSC thermograms (a) and XRD patterns (b).

(a) (b)
100
6 0

First derivative (residual mass % / °C)

–5
4
80
–10
Residual mass (%)

2
60
–15

0
40 –20
PU
PU/1% Ti 600 650 PU
–25
PU/3% Ti PU/1% Ti
20
PU/5% Ti PU/3% Ti
–30
PU/5% Ti
0
–35

100 200 300 400 500 600 100 200 300 400 500 600
Temperature (°C) Temperature (°C)

Figure 5. TGA thermograms of PU/Ti composites. Residual mass (a) and first derivative of residual mass (b).

Table 2. Properties of polyurethane (PU)/Ti composites obtained by thermal analysis.

DMA DSC TGA

Ta Tm DHf Crystallinity Td1 Td2 Residual


C 
C J g
1 % 
C 
C mass %

PU
40 52 40.9 40 351 461 0.9

PU/1% Ti
39 52 39.8 39 346 442 2.1
PU/3% Ti
43 52 43.5 44 344 438 2.5
PU/5% Ti
37 52 37.7 39 346 436 5.9
DSC: differential scanning calorimetry; DMA: dynamic mechanical analysis; TGA: thermogravimetric analysis.

was related to decomposition of carbon chains of rigid
segment. Both decomposition temperatures seem not to
be affected by the increase of Ti concentration in con-
trast to some reported catalytic activity that accelerates
decomposition.26 Figure 5 summarizes these findings,
where the residual mass (Figure 5(a)) and first deriva-
tive (Figure 5(b)) are plotted against temperature. The
maximum temperature used during TGA was 650 C,
which is far from the Tm of titanium reported at
1668 C. As observed in the inset of Figure 5(a), the
final mass is, as expected, in close agreement with the
original weight composition of Ti particles in compo-
sites i.e. 2.1% of residual mass for PU/1% Ti, 2.5% for
PU/3% Ti, and 5.9% of residual mass for PU/5% Ti.
8 Journal of Biomaterials Applications 0(0)

Table 2 summarizes the thermal properties of the
PU/Ti composites.
Tensile mechanical properties
Figure 6 shows typical stress–strain curves obtained
during the mechanical test of composites at high defor-
mation rate, while the corresponding mechanical prop-
erties are summarized in Table 3. In general, at low
strain rate, tensile strength decreased from 1.9 MPa
to 1.2 MPa with Ti addition suggesting a poor inter-
face, while deformation followed the same trend, i.e., a
reduction from 670% for pristine PU to 172% for PU/
5% Ti. However, as shown in Table 3, Young’s mod-
ulus did not show a significant difference between the
pristine PU and most Ti composites being the highest
(19 MPa) when 1% of titanium was used. In agreement
with this, the storage modulus (E0 ) obtained by DMA
showed that both PU/1% Ti and PU/3% Ti composites
showed higher modulus at 25 C that unfilled PU (see
Table 3). Results of tensile tests at high deformation
rates showed a higher Young’s modulus (77–83 MPa),
higher tensile strength (7.1–8.5 MPa) but lower strain to
break (185–223%). Nevertheless, no statistical differen-
ces were observed between materials in any mechanical
9
8
7 HDPSC proliferation on composites increased from
6 day 1 to day 10 as measured by the MTT assay (see
Stress (MPa)
property studied. In general, the mechanical properties
of composites of PU/Ti were lower than PU/bioactive
glass composites in spite of their higher rigidity, except
for the Young’s modulus that was significantly higher at
low strain rates.4 However, the mechanical properties of
these composites are far from those exhibited by trabec-
ular bone from human femur (17.5  1.12 GPa) or cor-
tical bone (22.1 GPa) measured by ultrasonic
technique.29
Biocompatibility of composites
Primary cultures can provide information more rele-
vant to clinical outcomes when compared to immortal-
ized cell lines such as Saos-2 or MG63, and are also
increasingly used in the biomaterials field with tissue
engineering applications, and for these reasons were
chosen for this study.30 Although several reports exist
on stem cell/osteoblast biocompatibility of either tita-
nium31–35 or PU,36–39 no studies were found on PU/Ti
composites. Therefore, the potential cytotoxicity of
those used in this study was assessed with both
HDPSC and osteoblasts.
HDPSC adhesion to PU and composites was good
at day 1, as shown by the live/dead assay in Figure 7(a).
This figure also shows that there are a higher number of
live cells when the content of Ti was increased.
However, at day 10, there was a significant reduction
in cell viability at the surface for any composition.
Figure 8(a)). However, at day 1, there was no difference

5
between pristine PU and Ti composites. However,
4 PU
PU/ 1% Ti
there was a statistical difference between composites
3 PU/ 3% Ti at day 10, increasing with Ti content in composites,
2
PU/ 5% Ti except on PU/3% Ti, which is higher than PU, but
lower than PU/1% Ti.
1
In contrast to HDPSC behavior, osteoblast viability
0 at both 1 and 10 days improved with Ti concentration
0 20 40 60 80 100 120 140 160 180 200 220
(see Figure 7(b)), cells count at 24 h of incubation were
Strain (%)
297, 452, 656, and 480 cells per field, and at 10 days
Figure 6. Tensile stress-strain curves of PU/Ti composites were 28, 255, 370, and 440 cells per field for PU, PU/
obtained at 250 mm/min. 1% Ti, PU/3% Ti, and PU/5% Ti, respectively.

Table 3. Tensile mechanical properties of polyurethane (PU)/Ti composites.

DMA Cross head speed (250 mm/min) Cross head speed (50 mm/min)

E0 25 C E rmax roy rbreak eoy ebreak E rmax roy rbreak eoy ebreak
Material MPa MPa MPa MPa MPa % % MPa MPa MPa MPa % %

PU 112.6 78.5  6.0 8.2  0.9 7.4  0.1 7.2  0.8 11.5  0.3 223  34 17.3  1.2 1.9  0.3 1.3  0.1 1.9  0.3 8.7  0.5 671  77
PU/1% Ti 170.8 77.2  4.1 7.8  0.6 7.4  0.6 7.1  0.6 11.0  0.3 185  56 19.9  3.9 1.8  0.1 1.3  0.2 1.8  0.1 8  1.9 537  40
PU/3% Ti 172.4 83.6  4.4 8.1  0.5 7.9  0.5 7.4  0.4 10.5  0.6 221  53 16.1  2.5 1.7  0.1 1.3  0.1 1.7  0.1 9.3  0.5 499  29
PU/5% Ti 116.1 80.7  3.5 8.5  0.8 6.3  1.1 8.5  0.8 10.2  2.7 223  42 15.0  0.6 1.2  0.1 0.9  0.2 1.2  0.8 6.9  1.2 172  38

DMA: dynamic mechanical analysis.

Aguilar-Perez et al. 9

(a) (b)
24 hours 10 days 24 hours 10 days

PU

PU
1%
Ti

PU
3%
Ti

PU
5%
Ti

Figure 7. Fluorescent microscopy images of (a) HDPSCs at 20 and (b) osteoblasts at 4, after 24 h and 10 days on
PU/Ti composites.

(a) 0.6 (b) PU ∗

∗ PU/1% Ti
PU 0.10 PU/3% Ti
0.5 PU/1% Ti PU/5% Ti
PU/3% Ti
PU/5% Ti 0.08
∗
Absorbance (a.u.)

∗
Absorbance (a.u.)

0.4
∗
0.3
0.06 ∗

0.2 0.04

0.1 0.02

0.0 0.00
24 hours 10 days 24 hours 10 days

Figure 8. HDPSC proliferation on composites by MTT (a) and osteoblast proliferation by Alamar Blue on PU/Ti composites (b).
(*p  0.05).

However, when comparing the same composition after
10 days, osteoblast number decreased with exception of
PU/5% Ti, which showed similar amount of live cells
present at both times.
Osteoblasts proliferation increased from day 1 to day
10 as determined by Alamar Blue (see Figure 8(b)).
At 24 h, osteoblast proliferation on pristine PU was
significantly lower than PU/1% Ti, and in this compos-
ite, osteoblast proliferation was significantly higher than
PU/3% Ti, while PU/5% Ti was significantly higher
than pristine PU and PU/3% Ti. At 10 days, composites
showed an increase in cell proliferation when higher
10
amounts of Ti were used in the composites, where PU/
3% Ti and PU/5% Ti were significantly higher than PU
and PU/1% Ti.
It is not clear why HDPSC detached after 10 days
from the PU/Ti composites while osteoblast remained
as shown in Figure 7. A possible explanation is that
they almost reached confluency after 1 day, leading to
contact inhibition and detaching at longer times, as
seen in Figure 7(a). This is in contrast with previous
reports where either HDPSC or osteoblast were cul-
tured, in the absence of a polymer, on laser sintered
or acid etched titanium alloys and where it was
observed no difference in cell adhesion.31
Surface roughness,32 surface chemistry,33,34 zeta
potential, and surface energy22,30 have been proposed
as the main factors contributing to osteoblast attach-
ment to titanium implants, but to our knowledge, there
is no similar study on HDPSC.35
Although it has been reported that committed cells
(human osteoblasts) display better proliferation, distri-
bution, and adhesion in 2D cultures on roughened tita-
nium surface compared to HDPSC,40 it is possible that
HDPSC exhibited higher proliferation at shorter times
either due to the higher proliferative index as many
multipotent cells or due to the stimulating effect
of titanium.
In has been reported that collagen sponges, porous
hydroxyapatite/tricalcium phosphate ceramics, and
titanium meshes support the attachment, growth, and
differentiation of human DPSC equally well in vitro
but with little amounts of hard tissue formed in
vivo.41 A similar result was obtained during the
course of this study where HDPSC adhere well at
short times to a PU/Ti composite but also it was
found that osteoblast proliferate better in a 2D cell
culture model. Overall, these composites were not cyto-
toxic but depending on the final application the use of
differentiated or undifferentiated cells should be used.
For tissue engineering purposes (3D cultures), HDPSC
should be used due to their higher proliferation rate
while for biomaterials development or screening, 2D
osteoblasts cultures are recommended.
Conclusions
Semicrystalline polymeric composites were obtained by
mixing metallic titanium powder and a segmented PU
based on glutamine. In these composites, Ti particles
did not act as nucleating agents as their crystallinity,
demonstrated by DSC and XRD, remained the same.
Titanium particles, however, had an effect on mechan-
ical properties. At low strain rates, both tensile strength
and maximum strain of composites tend to decrease
when higher Ti concentration while a low Young’s
modulus was observed; at higher strain rates, there is
Journal of Biomaterials Applications 0(0)
no significant difference in tensile mechanical behavior
between materials but a higher modulus was achieved.
This indicates that Ti particles could be acting as stress
concentrators due to their large particle size and that
they have a slight reinforcing effect on PU matrix, espe-
cially when used at 1%. Both, viability and prolifera-
tion were good in composites containing 1%, 3%, and
5% of Ti up to 10 days, for mice osteoblasts, and
HDPSCs, and therefore, biocompatibility tends to
improve at higher content of Ti particles. Based on
these results, composites of PU and Ti in the form of
films may be suitable for membrane-guided bone
regeneration.
Acknowledgements
The authors want to thank Patricia Quintana and Daniel
Aguilar Trevi~
no for XRD and XPS experiments at
Laboratorio Nacional de Nano y Biomateriales (LANBIO),
Cinvestav-IPN, Unidad Mérida (Projects FOMIX-Yucatan
2008–108160 and CONACYT LAB-2009–01 no. 123913).
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article.
Funding
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: This research was supported by Consejo Nacional de
Ciencia y Tecnologia through grants 248378, 1360 and
268595. The authors Y. Torres and J.A. Rodrı́guez-Ortiz
would like to thanks the support of the Ministry of
Economy and Competitiveness of the State General
Administration of Spain under the grant MAT2015-71284-P.
ORCID iD
Fernando Javier Aguilar-Perez http://orcid.org/0000-
0003-4266-0464
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