Biomatter

ISSN: (Print) 2159-2535 (Online) Journal homepage: https://www.tandfonline.com/loi/kbim20

Preparation of laminated poly(ε-caprolactone)-

gelatin-hydroxyapatite nanocomposite scaffold
bioengineered via compound techniques for bone
substitution

Azhang Hamlekhan, Fathollah Moztarzadeh, Masoud Mozafari, Mahmoud

Azami & Nader Nezafati

To cite this article: Azhang Hamlekhan, Fathollah Moztarzadeh, Masoud Mozafari, Mahmoud
Azami & Nader Nezafati (2011) Preparation of laminated poly(ε-caprolactone)-gelatin-
hydroxyapatite nanocomposite scaffold bioengineered via compound techniques for bone
substitution, Biomatter, 1:1, 91-101, DOI: 10.4161/biom.1.1.17445

To link to this article: https://doi.org/10.4161/biom.1.1.17445

Copyright © 2011 Landes Bioscience

Published online: 01 Jul 2011.

Submit your article to this journal

Article views: 1098

View related articles

Citing articles: 11 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=kbim20
 Report Report
Biomatter 1:1, 91-101; July/August/September 2011; © 2011 Landes Bioscience

Preparation of laminated
poly(ε-caprolactone)-gelatin-hydroxyapatite
nanocomposite scaffold bioengineered via
compound techniques for bone substitution
Azhang Hamlekhan,1 Fathollah Moztarzadeh,1 Masoud Mozafari,1,* Mahmoud Azami2 and Nader Nezafati1
Biomaterials Group; Faculty of Biomedical Engineering (Center of Excellence); Amirkabir University of Technology; Tehran, Iran; 2Tissue Engineering and Cell Therapy
1

Department; School of Advanced Medical Technologies; Tehran University of Medical Sciences; Tehran, Iran

Key words: scaffold, poly(ε-caprolactone), gelatin, hydroxyapatite, mesenchymal stem cells, bone substitution

In this research, new bioactive nanocomposite scaffolds were successfully developed using poly(ε-caprolactone) (PCL),
cross-linked gelatin and nanoparticles of hydroxyapatite (HAp) after testing different solvents and methods. First, HAp
powder was synthesized via a chemical precipitation technique and characterized. Then, the nanocomposites were
prepared through layer solvent casting combined with freeze-drying and lamination techniques. According to the
results, the increasing of the PCL weight in the scaffolds led to the improvement of the mechanical properties. The
amount of ultimate stress, stiffness and also elastic modulus increased from 8 MPa for 0% wt PCL to 23.5 MPa for 50% wt
PCL. The biomineralization study revealed the formation of an apatite layer on the scaffolds after immersion in simulated
body fluid (SBF). The Ca-P ratios were in accordance to nonstoichiometric biological apatite, which was approximately

©2011L
andesBiosc
ienc
e.
1.67. The in vitro biocompatibility and cytocompatibility of the scaffolds were tested using mesenchymal stem cells
(MSCs), and the results indicated no sign of toxicity, and cells were found to be attached to the scaffold walls. The in vivo
biocompatibility and osteogenesis of these scaffolds in the animal experiments is also under investigation, and the result

Donotdi
str
ibut
e.
will be published at the end of the study.

Introduction Nowadays, the application potential of scaffolds made from

blends of synthetic and natural polymers for tissue regeneration
In the past decade, bone tissue engineering provided new medi- has been investigated by many researchers. For example, com-
cal therapy as an alternative to conventional transplantation posites of PCL and gelatin or collagen were used for tissue engi-
methods using polymeric biomaterials with or without bioactive neering applications such as the regeneration of cardiac muscle,
materials such as bioceramics, bioactive glasses or glass ceram- skin, nerves and skeletal muscle.3-7 Although these composite
ics. In particular, various nanocomposite scaffolds can be fab- materials exhibit improved cytocompatibility with adjustable
ricated with blends of synthetic and natural polymers by choice mechanical strength, the biopolymer components are often sub-
of suitable solvents and preparation methods. According to Kim ject to rapid degradation during cell culture or implantation,
et al.,1 most synthetic polymers can be modulated with respect to necessitating physical or chemical cross-linking steps after the
molecular weight, degradation rate and mechanical properties. fabrication process to covalently link reactive functional groups
However, they lack cell recognition sites, and therefore, matrix in biopolymers.8,9
scaffolds prepared with synthetic polymers often exhibit poor cell Gelatin is a natural biopolymer derived from collagens by
adhesion, migration and proliferation. In contrast, most natural controlled hydrolysis.10,11 Because of its many merits, such as its
polymers show excellent cellular affinity due to the presence of biological origin, biodegradability, biocompatibility and com-
proteins and polysaccharides of extracellular matrix (ECM) com- mercial availability at relatively low cost, gelatin has been widely
ponents but have poor mechanical properties. used in biomedical applications. In pharmaceutical and medi-
Hence, nanocomposite scaffolds that blend synthetic and cal fields, gelatin has long been used as a sealant for vascular
natural polymers can exploit the advantages of both polymer prostheses,12-14 carriers for drug delivery,15 dressings for wound
types.2 In addition, applying bioactive materials throughout the healing16 and so forth. Anyway, the approach of mixing gelatin
nanocomposite scaffold matrices, especially HAp, can drasti- with synthetic polymers has frequently been adopted by other
cally improve the bone regeneration ability of these materials. researchers. This is a feasible approach that may not only reduce

*Correspondence to: Masoud Mozafari; Email: [email protected]

Submitted: 04/07/11; Revised: 07/20/11; Accepted: 07/22/11
DOI: 10.4161/biom.1.1.17445

www.landesbioscience.com Biomatter 91
 Figure 1. The graphical sketch to describe the preparation procedure of the scaffolds.

the potential problem of cytotoxin as a result of using a chemical defect sites because of its brittleness and low plasticity.33-38 As
cross-linking reagent, but also provide a compromise solution for mentioned above, each of the materials that has been widely
overcoming the shortcomings of synthetic and natural polymers, used in the preparation of tissue engineering scaffolds has low
that is, producing a new biomaterial with good biocompatibility mechanical properties, so, for improving this weakness, many
and improved mechanical and physicochemical properties. works have been accomplished. For example Causa et al. used
Several nanocomposite scaffolds have been investigated for PCL-based composites with HA particles added at different
bone tissue engineering, including HAp, poly(α-hydroxyesters), volume/weight ratios. They showed both good mechanical per-
bioactive glasses and natural polymers, such as gelatin, collagen formance and appreciable biocompatibility. In another work by
and chitin. Also, several reviews have been published on the gen- Chang et al.,40 gelatin-HAp nanocomposites were fabricated as

©2011L
andesBiosc
ienc
e.
eral properties and design features of biodegradable and biore- foams. The mechanical properties of the foams and, in the same
sorbable polymers and scaffolds.17 research by Lia et al.,41 the biological and mechanical properties
Among synthetic polymers, PCL has been considered an of gelatin-apatite nanofibers were examined, and the result was

Donotdi
str
ibut
e.
appropriate choice for mixing with gelatin due to the fact that
PCL is a semicrystalline linear resorbable aliphatic polyester and
has been subjected to hydrolytic degradation because of the sus-
better than using the gelatin alone, and the scaffolds also showed
excellent biocompatibility and mechanical stability.
One of the most important aspects of bone tissue engineering
ceptibility of its aliphatic ester linkage to hydrolysis.18-20 On the is the introduction of bioactive cells into the scaffolds.42-46 For
other hand, PCL is regarded as a soft and hard biocompatible the application of MSCs in bone regeneration, understanding the
material for tissue engineering applications, such as resorbable interactions between cells and scaffold materials is particularly
suture, drug delivery system and also bone-graft substitutes.21 important, because the cell-material interaction plays an impor-
Although applications of PCL might be limited because its degra- tant role in bonding implant materials to native bone tissue and
dation and resorption kinetics are considerably slower than other in preventing the formation of a fibrous capsule. It is obvious that
aliphatic polyesters, due to its hydrophobic character and high MSCs present more advantages than other cells and have already
crystallinity, PCL is currently under study for use as a potential been widely used in bone tissue engineering.47-49
material for bone regeneration. Marra et al., in their research on In this study, HAp powder was synthesized by chemical
in vitro analysis of biodegradable polymer blend/HAp composites precipitation through aqueous solutions of the reactants. Then,
for bone tissue engineering, reported that PCL is a comparable to mimic the mineral and organic component of natural bone,
substrate for supporting cell growth resulting from two-dimen- the PCL-Gelatin-HAp nanocomposites were prepared via layer
sional bone marrow stromal cell culture. Moreover, PCL/PLA- solvent casting combined with freeze-drying and lamination
blend discs incorporated with HAp are feasible as scaffolding for techniques. Finally, glutardialdehyde (GA) was used as a cross-
bone tissue engineering. linking agent. The increasing of the PCL weight through the
From another point of view, applying bioactive ceramics is scaffold samples caused the improvement of mechanical prop-
one of the most famous methods for improving scaffolds for erties. Eventually, the cellular responses of the scaffolds were
bone repair, and bioactive ceramics based on calcium phosphates examined. The proliferation of the MSCs in direct contact with
(CaP), particularly in the composition of tricalcium phosphate the scaffolds was qualitatively determined by Scanning Electron
[TCP: Ca3 (PO4)2] and HAp [Ca10 (PO4) 6 (OH)2], have been Microscope (SEM) analysis and quantitatively with MTT assay.
studied extensively and used clinically.23-25 The biomaterials
research field has focused on the synthesis of these ceramics for Results and Discussion
three decades for applications in orthopedics and dentistry.26-32
Previous applications of porous HAp include graft applications HAp powder. The XRD data of the nanocrystalline HAp pow-
in maxillofacial surgery as alveolar ridge augments and as bone der is presented in Figure 2A. The XRD analysis was performed
defect filler. However, HAp is difficult to handle and keep in using an X-ray diffractometer. The straight base line and the

92 Biomatter Volume 1 Issue 1

 Table 1. The best preparation method of the nanocomposite scaffolds
PCL solvent Gelatin solvent Method Result
Acetone Distilled water Lamination of PCL layers between gelatin layers PCL-Gelatin blend was obtained

sharp peaks of the diffractogram confirmed that the product was chemical bonding between HAp nanoparticles and gelatin in the
well crystallized. The XRD patterns indicated that HAp was nanocomposites has three major steps:
formed in this sample, and traces of other calcium phosphate (1) A critical complex reaction between Ca 2+ ions of HAp
impurities were not detected by this technique. The XRD pat- nanoparticles and gelatin molecules.55,56
tern of sintered samples can be completely indexed with HAp in (2) The Ca 2+ ions the of complex with gelatin molecules
the standard card (JCPDS No. 09-0432), the only phase found assembled with PO43- ions from HAp nanoparticles.
present. No processing residue or secondary phases were found (3) The -COOH and -NH2 groups in the gelatin mole-
in the materials. cule form chemical bonds with P-O and O-H groups of HAp
Figure 2B shows the FTIR spectrum in the 500–4,000 cm-1 nanoparticles, resulting in gelatin chains firmly attaching to the
spectral range of the HAp powder. The HAp powder exhibited surface of HAp nanoparticles.
five important infrared bands located at 560, 605, 622, 1,040 There are two main sources of stabilization that prevent the
and 3,555 cm-1. Among these bands, two bonds were observed occurrence of the HAp particle agglomeration. The first one is
at 3,555 and 622 cm-1 due to the stretching mode of hydrogen- electrostatic stabilization and another is spatial stabilization.
bonded OH- ions and the liberational mode of hydrogen-bonded Here, electrostatic stabilization is mainly due to adsorption of
OH- ions, respectively. In addition, the band at 1,040 cm-1 arises Ca 2+ ions on the surface of HAp particles. The adsorption would
from υ3 PO4, and the bonds at 605 and 560 cm-1 arise from υ4 produce an electrical double layer. In other words, the ionization
PO4.50 The FTIR analysis showed all typical absorption char- of carboxyl is enforced while that of amino is restrained, and
acteristics of the HAp powder, and, according to these explana- the carboxyl ions of gelatin act, consequently, as counter ions in
tions, it is obvious that the synthesized powder is certainly HAp. the electrical double layer. The spatial stabilization in this reac-

©2011L
andesBiosc
ienc
e.
The result of measurement of elemental composition (Ca and tion system is mainly due to chemisorption of gelatin molecules
P content) and Ca/P molar ratio were chemically analyzed by on the HAp particles.57 Also, the bond at 2,363 cm-1 appeared
quantitative chemical analysis via EDTA titration technique after cross-linking of gelatin with GA. Therefore, this bond arises

Donotdi
str
ibut
e.
and AAS. The Ca and P content and bulk Ca/P molar ratio was
determined as 38.63 (wt%), 17.48 (wt%) and 1.71, respectively.
The measured Ca/P ratio for this synthesized powder was higher
from the C-H bond in C3H6 alkene, which remains in the final
cross-linked gelatin chains after GA reaction, as shown:

than the stoichiometric ratio (1.667) expected for a pure HAp Gelatin-NH2 + C5H8O2 + NH2-Gelatin → Gelatin-N = CH -
phase that can arise from local presence of carbonate apatite in C3H6 - CH = N = Gelatin
which the Ca/P molar ratio can be as high as 3.33.51
TEM analysis was used to examine and estimate HAp crys- In addition, according to Ghasemi-Mobarakeh et al., PCL
tallites. TEM micrographs of the HAp powder in low and high and gelatin chains can be chemically bonded. Herein, it is worth
magnifications are shown in Figure 3. The crystalline structure mentioning that the appearance of the amide groups in the FTIR
of HAp particles has an elliptical shape with a grain size in the spectra of the nanocomposite scaffolds, which is shown with
range of 8–12 nm. arrows in Figure 4, indicates that the PCL chains were chemi-
Nanocomposite scaffolds. FTIR analyses and chemical bonds. cally bonded to gelatin sidewalls, leading to the introduction of
In order to study the chemical bonds in the nanocomposite scaf- functional groups such as NH2 and COOH on the surface of
folds, FTIR results obtained from two samples were investigated. prepared scaffolds, which was also reported by former research-
Figure 4 shows the FTIR spectra of the samples with 20% wt ers. In conclusion, all these chemical bonds caused a strong and
PCL and 50% wt PCL; the peaks that appeared give information effective structure for the prepared scaffolds.
about detected chemical bonds. The FTIR spectra of 20% wt SEM observations. Figure 5 shows the morphologies of the
PCL were nearly same as that of 50% wt PCL. PCL, gelatin and nanocomposite layers in different aspects. The
Three series of obtained peaks, like 1,236, 1,450, 1,555, 1,670, morphological analysis of the gelatin layers showed a uniform
2,930, 3,048 cm-1 related to gelatin; peaks at 560, 868, 1,040, distribution of pores, with an average size around 150 μm, which
3,558 related to HAp chemical structure, and infrared spectra for is shown in Figure 5A. Also, it can be seen that pores are either
PCL-related stretching modes were also observed for both scaf- interconnected or separated via thin walls. During the prepara-
folds, including 2,930, 2,870, 1,670, 1,293 and 1,240 cm-1.52 But tion process, it was expected that the HAp nanoparticles would be
there are two peaks related to chemical bonds that were formed distributed homogeneously throughout the gelatin network. The
after the mixing of gelatin-HAp and then cross-linking of com- distribution of the HAp nanoparticles is not affected by gravity,
ponents, which stood at about 1,345 and 2,363 cm-1, respectively. because the viscous gelatin was kept in an ultrasonic place and
The first one indicates the formation of a chemical bond between then was rapidly frozen to form a homogeneous nanocomposite
the carboxyl group from gelatin and the Ca+2 ion from HAp, layer. According to Figure 5B and C, a porous structure is even
which has been mentioned in former studies too.53,54 Herein, the seen in PCL layers, especially in areas away from the gelatin-PCL

www.landesbioscience.com Biomatter 93
 ©2011L
andesBiosc
ienc
e.
Donotdi
str
ibut
e.

Figure 2. (A) The XRD pattern of the synthesized nanocrystalline HAp powder. (B) The FTIR spectrum of the synthesized HAp powder.

boundary. The pore configurations in the PCL layers are more technique lets macro-pores of the PCL layers make the gelatin
complex, and the pore size is nearly smaller. It is notable that the layers continuity possible throughout the nanocomposite scaf-
PCL and gelatin layers are mechanically bound in most areas to folds (see Fig. 5F).
form a gelatin-PCL boundary (see Fig. 5D and E). As was men- In vitro biomineralization studies. The in vitro biomineral-
tioned previously, evaporation of acetone during the lamination ization of the prepared scaffolds immersed in SBF is shown in

94 Biomatter Volume 1 Issue 1

Figure 6. The SEM micrographs showed Table 2. Ion concentrations of SBF and human blood plasma29
the deposition of newly formed apatite Ion Plasma (mmol/l) SBF (mmol/l)
particles on the surface of the scaffolds. Na +
142.0 142.0
Figure 6B–D shows the SEM micrographs K+ 5.0 5.0
of the scaffolds after immersion for 3, 7 Mg +2
1.5 1.5
and 14 d, respectively. According to these
Ca+2 2.5 2.5
observations, scattered and small par-
Cl- 103.0 147.8
ticles covered the surface of the scaffolds
after 3 d of immersion, which is shown HCO3 -
27 4.2
in Figure 6B. After 7 d, a substantial HPO 4
-2
1.0 1.0
amount of apatite microparticles formed SO4 -2
0.5 0.5
on the surfaces (Fig. 6B). Also, after 7 d
of immersion, the whole inner pore-wall
surfaces of the scaffolds were covered by a layer of apatite, and the fusiform shape attached to the surface of the scaffolds with their
underlying surfaces were not clearly observable (Fig. 6D). pseudopodia. Wide distribution of these traces on the surface of
We also confirmed the formation of apatite layers on the sur- scaffolds is an indication of good cellular migration and osteo-
face of scaffolds by EDX analysis, which is shown in Figure 6. conductivity of the scaffolds, and the continuous increase in cell
The results from EDX analysis revealed the gradual development aggregation on the scaffolds during the 3 d incubation also indi-
of the apatite layer on the surfaces and pores of scaffolds after cated the ability of the scaffolds to support cell growth.
immersion in SBF solution. Furthermore, EDX analysis showed Both the MSCs cultured on the standard plastic plate
that, after 14 d immersion in SBF solution, the Ca-P ratios were (control) and MSCs cultured on the nanocomposite scaffolds
in accordance to nonstoichiometric biological apatite, which was were evaluated in the osteogenic medium. The cell proliferation
approximately 1.67. in each group was evaluated using MTT test, which is shown in
Mechanical properties of the nanocomposite scaffolds. The Figure 8. In 6-d cell culture, the cell number increased with the

©2011L
andesBiosc
ienc
e.
mechanical properties of the prepared porous scaffolds have been culture time in both tested groups. Statistical analysis indicated
of particular concern for many tissue engineering applications no significant difference in the cell number between the cells
due to the necessity of the structure to withstand stress during seeded on the scaffold samples and cells cultured on the standard

Donotdi
str
ibut
e.
culturing in vitro and as in vivo implants. Mechanical proper- plastic plate in the same osteogenic condition. During the cell
ties also influence specific cell functions within the engineered culturing in both groups, the number of cells grew dramatically.
tissues. This is why, in the present study, compressive properties This similar ascendant tendency of the cell population demon-
of scaffolds were examined. As can be seen in Table 3, increasing strated that the nanocomposite scaffolds have little influence on
the PCL weight caused improvement of the scaffolds’ mechanical cell growth.
properties. In addition, the elastic modulus increased from 8 MPa According to the results, we came to the conclusion that the
to 23.5 MPa for 50% wt PCL. Also, the amount of ultimate stress prepared nanocomposite scaffolds have no negative effect on the
and stiffness increased from 1.83 MPa and 38 N/mm to 3.73 attachment and proliferation of MSCs, and they are in vitro bio-
MPa and 131 N/mm, respectively. At the same time, a decrease in compatible and noncytotoxic to cells.
ultimate strain was observed according to the stress-strain curves.
Biocompatibilty evaluation using MSCs. MSCs represent an Materials and Methods
attractive and promising field in tissue regeneration and engi-
neering for treatment applications in a wide range of trauma and Materials. Calcium nitrate tetra-hydrate [Ca(NO3)2 0.4H2O,
orthopedic conditions. In the bone tissue engineering field, there 98%], di-ammonium hydrogen phosphate [(NH4)2HPO4, 99%]
has been a special interest in MSCs loaded on scaffolds, which and sodium hydroxide [NaOH, 99%] were purchased from
provide a prospective approach for the reconstruction of even Merck Inc. to synthesize the HAp powder by precipitation tech-
large bone defects.58-60 nique. PCL used in this study were obtained from Solvay, with
In the present study, MSCs derived from the bone marrow of an average molecular weight of 50,000 (CAPA-6500). Also, gela-
neonatal rabbits were cultured, expanded and seeded on the pre- tin for microbiology (Merck No. 104070) was used. The gelatin
pared nanocomposite scaffolds. The proliferation of the MSCs foams were cross-linked by immersing into Glutardialdehyde
in direct contact with the scaffolds was qualitatively determined (Merck No. 820603). Distilled water, acetone extra pure,
with SEM and quantitatively with MTT assay. The biocompat- Dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), N,N-
ibility of the nanocomposite scaffolds was evaluated in vitro by Dimethylformamide (DMF) and acetic acid 100% (glacial) were
observing the behavior of the cells cultured in close contact with used as polymer solvents.
the scaffolds. According to Figure 7, the SEM micrographs of the Synthesis of nanocrystalline HAp powder. The HAp powder
cells cultured on the surface of nanocomposite scaffolds showed was prepared by chemical precipitation through aqueous solu-
well-spread cells on the scaffolds, an indication of good attach- tions of the reactants. A total of 0.09 M diammonium hydrogen
ment and penetration to the surface of the scaffolds. It also shows phosphate solution and 0.15 M calcium nitrate tetrahydrate solu-
that, after 3 d of cell culturing, the cells with a predominantly tion were prepared, and the pH of the both solutions was brought

www.landesbioscience.com Biomatter 95
 to 10 by adding a small amount of sodium hydrox-
ide solution. The phosphate solution was added
drop-wise into the calcium nitrate solution, result-
ing in the precipitation of HAp. The precipitation
of HAp can be described by the following reactions
(1) and/or (2): 61

10 Ca+2 + 6 H2PO4- + 2 OH- → Ca10 (PO4) 6 (OH)2 +

6 H+ (1)

10 Ca+2 + 6 H2PO4- + 2 OH- → Ca10 (PO4) 6 (OH)2 +

12 H+ (2)

After ripening for a specified period of time

(24 h) at room temperature, the precipitates were
recovered by centrifuge and then washed with
deionized water. Five cycles of washing and centri-
fuging were repeated to ensure complete removal
of the by-product. The calcination of the synthe-
sized powders was performed at 800°C for 1 h in
Figure 3. The TEM micrographs of the HAp powder, (A) low and (B) high magnifica- air using a heating rate at 3.0°C/min in an electri-
tions. cal tube furnace from room temperature to 800°C
after drying the sample in freeze-drier for 10 h.
Preparation of the nanocomposite scaffolds. The

©2011L
andesBiosc
ienc
e.
gelatin of 10% (w/v) was added into distilled water
and stirred at 40°C for 1 h. HAp nanoparticles were
added to the solution at 40 wt% of the gelatin. The

Donotdi
str
ibut
e. mixture was homogenized in an ultrasonic place by
a stirrer at 40°C for 1 h. The gelatin-HAp solution
was poured into plastic dishes and left in ambient
conditions for 10 min to form the first gelatin-HAp
layer. Then, as can be seen in Table 1, a solution of
10% (w/v) PCL in acetone was poured on the gel-
atin-HAp layer, and the complex was left in ambi-
ent conditions for 20 min to form the PCL layer.
After evaporation of the acetone, the gelatin-HAp
solution was poured again to fabricate the next gel-
atin-HAp layer. Lamination was continued to form
the fourth gelatin-HAp layer. After evaporation of
the acetone, lots of macro-porosities were observed
in the surface of PCL layers, which made gelatin
able to influence all the nanocomposite layers. Five
samples were prepared in which PCL amounts were
0, 20, 30, 40 and 50 wt% of gelatin. The samples
were frozen in the freezer at -20°C for 24 h and then
moved to the freeze-drier for 72 h. To cross-link
gelatin polymeric chains and to reduce biodegrada-
tion and enhance the biomechanical properties of
the scaffolds for tissue repair, the prepared samples
Figure 4. The FTIR spectra of the nanocomposite samples with 20% wt PCL (top) and were immersed into 1% glutardialdehyde aqueous
50% wt PCL (bottom). Triangles, Hap; stars, 1,345 (cm-1) related to the gelatin-HAp solution for 24 h to cross-link the gelatin-HAp lay-
bond; sunbursts, 2,363 (cm-1) related to the cross-linking process. ers. Finally, the cross-linked nanocomposite scaf-
folds were carefully washed several times to remove
the rest of GA and then dried at room temperature.
The graphical sketch of the preparation procedure
of scaffolds is presented in Figure 1.

96 Biomatter Volume 1 Issue 1

 Table 3. The mechanical properties of the nanocomposite scaffolds
Samples 0% wt PCL 20% wt PCL 30% wt PCL 40% wt PCL 50% wt PCL
E0.02 (MPa) 8 ± 1.2 16 ± 3.3 19 ± 1.6 20.5 ± 1.3 23.5 ± 4.8
Gult (Mpa) 1.83 ± 0.5 3.13 ± 0.4 3.40 ± 0.1 3.71 ± 1.1 3.73 ± 1
K (N/mm) 38 ± 2 79 ± 4.1 93 ± 3.9 114 ± 2.7 131 ± 7.1

Preparation of SBF solution. The SBF solution was prepared directly connected to SEM was used to investigate semi-quanti-
by dissolving reagent-grade NaCl, KCl, NaHCO3, MgCl2.6H2O, tatively chemical compositions.
CaCl2 and KH2PO4 into distilled water and buffered at pH = 7.25 Compression strength tests were performed on the porous
with TRIS (trishydroxymethyl aminomethane) and HCl 1 N at foams. Each sample was loaded using Zwick-Roell (MCT-25-
37°C. Its composition is given in Table 2 and is compared with 400, Germany) at a crosshead speed of 2 mm/min with a load
the human blood plasma as previously described by Mozafari of 500 N cell in ambient conditions. The stress-strain curve was
et al.29 obtained to determine mechanical properties, including elastic
Characterization. HAp powder characterization. The result- modulus, compression strength, maximum stress and strain at
ing powder was analyzed by X-ray diffraction (XRD) with a maximum stress. The elastic modulus was obtained from the
Siemens-Brucker D5000 diffractometer. This instrument works slope at the initial stages of loading (2% strain). Five specimens
with voltage and current settings of 40 kV and 40 mA, respec- were tested for each composite.
tively and uses Cu-Kα radiation (1.540600 Å). For qualitative Porosity measurement. Pore volume (Vp) was calculated
analysis, XRD diagrams were recorded in the interval 20 ≤ 2θ ≤ according to the following equation:
60° at a scan speed of 2°/min, 0.02° being the step size and 1 sec
being the step time. Vp = Vsk - VBG - VGel = Vsk - (WBG/ρBG) - (WGel/ρGel)
The HAp sample was examined by Fourier transform infrared
spectroscopy with a Bomem MB 100 spectrometer. For IR analy- where Vsk is the skeletal volume of the nanocomposite (cm3),

©2011L
andesBiosc
ienc
e.
sis, first 1 mg of the powder sample was carefully mixed with VBG the actual volume of BaG (cm3) and VGel the actual vol-
300 mg of KBr (infrared grade) and pelletized under vacuum. ume of gelatin (cm3); in addition, VBG and VGel were measured
Then, the pellet was analyzed in the range of 500–4,000 cm-1 at with attention to the mass and the density of BaG and gelatin,

Donotdi
str
ibut
e.
a scan speed of 23 scan/min with 4 cm-1 resolution.
In order to calculate the Ca/P molar ratio of the precipitated
powder, the content of Ca and P were chemically analyzed by
which were used in each sample. The density of BaG and gelatin
are ρ = 2.7 and ρ = 1.35, respectively. So, the calculation formula
of porosity (%) is defined as follows:
quantitative chemical analysis via EDTA titration technique
and atomic absorption spectroscopy (AAS) with a Shimadzu porosity (%) = (Vp/Vsk) x 100
UV-31005 instrument, respectively.
Transmission electron microscopy (TEM; CM200-FEG- In vitro biomineralization studies. Three scaffolds of equal
Philips) was used for characterizing the particles. For this pur- weight and shape were immersed in SBF solution and incubated
pose, particles were deposited onto Cu grids, which supported a at 37°C in a closed Falcon tube for 3, 7 and 14 d. After the speci-
carbon film. The particles were deposited onto the support grids fied times, the scaffolds were removed and then carefully washed
by deposition from a dilute suspension in acetone or ethanol. The four times with deionized water to remove adsorbed minerals.
particles’ shape and size were characterized by diffraction (ampli- Finally, the scaffolds were lyophilized, viewed and analyzed using
tude) contrast and, for crystalline materials, by high-resolution SEM and EDX for mineralization.
(phase contrast) imaging. In vitro cell attachment and proliferation. MSCs seeding.
Nanocomposite scaffolds characterization. The functional First, MSCs were aspirated from the bone marrow of neonatal
groups of nanocomposite samples were ascertained by Fourier rabbits, gradient centrifuged and plated into flasks containing
transform infrared spectroscopy (FTIR). For IR analysis, first Dulbecco’s modified Eagle’s medium low glucose containing
samples were powdered and then dispersed into pellets of KBr 10% fetal bovine serum and 2% antibiotics (200 μg/ml peni-
(infrared grade) and the spectra recorded by Bomem MB 100 cillium and 200 μg/ml streptomycin). MSCs at passage three
spectrometer in the range 400–4,000 cm-1 at a scan speed of were removed into the culture medium containing osteogenic
23 scan/min with 4 cm-1 resolution. factors (50 μg/l α-ascorbic acid, 10-8 mol/l dexamethasone and
Microstructure and morphology of porous nanocomposite 10 mmol/l β-glycerophosphate, 10 mmol/l VitD3, 100 μg/ml
(pore morphology) and measurement of pore size were evalu- penicillium and 100 μg/ml streptomycin, 0.3 μg/m amphoteri-
ated using SEM analysis. For this purpose, the nanocomposite cin, 2.2 g/l sodium bicarbonate and 15% fetal bovine serum).
samples were coated with a thin layer of Gold (Au) by sputtering Medium was changed every 3 d.
(EMITECH K450X, England), and then the morphology of the Then, the prepared nanocomposite scaffolds were steril-
samples were observed on a scanning electron microscope (SEM- ized by ethylene oxide gas and presented in the osteogenic cul-
Philips XL30) that operated at the acceleration voltage of 15 kV. ture medium for 24 h. MSCs that were cultured in osteogenic
An energy dispersive X-ray analyzer (EDX, Rontec, Germany) medium for 1 week were seeded onto the tops of the pre-wetted

www.landesbioscience.com Biomatter 97
 ©2011L
andesBiosc
ienc
e.
Donotdi
str
ibut
e.

Figure 5. The SEM micrographs of (A) the gelatin layer in the nanocomposite containing 20% wt PCL, (B) the PCL layer in the nanocomposite contain-
ing 20% wt PCL, (C) the PCL layer in the nanocomposite containing 50% wt PCL, (D) the gelatin-PCL boundary in the nanocomposite containing
20% wt PCL, (E) the gelatin-PCL boundary in nanocomposite containing 50% wt PCL and (F) the continuity of the gelatin layers in the nanocomposite
containing 20% wt PCL.

scaffolds (2.0 x 106 cells/scaffold), and then the scaffold/cell con- MTT assay. The proliferation of MSCs on the nanocompos-
structs were placed in the wells of tissue culture plates. The scaf- ite scaffolds was determined using the MTT assay. Thus, MSCs
folds were left undisturbed in an incubator for 3 h to allow the were cultured on the nanocomposite scaffolds for 1, 3 and 6 d,
cells to attach to them, and then an additional 1 ml of culture and their proliferation rates were compared with the cells cul-
medium was added into each well. The cell/scaffold constructs tured on standard plastic culture surfaces. For this purpose, six
were cultured in a humidified incubator at 37°C with 95% air samples were prepared in each group. The test was performed
and 5% CO2 for 7 d. The medium was changed every 3 d. according to the manufacturer’s (Sigma) instructions, and the

98 Biomatter Volume 1 Issue 1

 ©2011L
andesBiosc
ienc
e.
Donotdi
str
ibut
e.

Figure 6. The SEM micrographs of the nanocomposite scaffolds before and after immersion in SBF (A) before immersion, (B) 3 d, (C) 7 d and (D) 14 d.

www.landesbioscience.com Biomatter 99
 Figure 8. The MTT cell proliferation on the standard plastic plate
(control) and scaffolds showing biocompatibility of the nanocomposite
Figure 7. The SEM micrographs of the MSCs cultured on the surface of scaffolds.
the nanocomposite scaffolds.

colorimetry was performed at 570 nm. A blank OD value was effective. The nanocomposite scaffolds were macroporous in
reduced from each sample’s reading. nature, and pore size ranged from 150 to 500 μm. The results
Statistical analysis. All experiments were performed in fifth from biomineralization studies revealed the gradual develop-

©2011L
andesBiosc
ienc
e.
replicate. The results were given as means ± standard error (SE). ment of the apatite layer on the surfaces and pores of scaffolds
Statistical analysis was performed by using one-way ANOVA and after immersion in SBF. Furthermore, EDX analysis showed
Tukey test, with significance reported when p < 0.05. Also for that, after 14 d immersion in SBF solution, the Ca-P ratios were

was used. Donotdi

str
ibut
e.
investigation of group normalizing, Kolmogorov-Smirnov test in accordance to nonstoichiometric biological apatite, which
was approximately 1.67. The in vitro biocompatibility results
showed that the scaffolds were biocompatible, and MSCs seeded
Conclusion on the scaffolds attached to the pore walls. Therefore, we con-
cluded that the PCL-gelatin-HAp nanocomposite scaffold could
In this study, after testing different solvents and preparation be used as an appropriate alternative for bone tissue engineering
methods, the novel PCL-gelatin-HAp nanocomposite scaf- applications.
folds were successfully prepared using a new effective lami-
nation technique. The chemical bonds between gelatin and Disclosure of Potential Conflicts of Interest
HAp and also between gelatin and PCL made it mechanically No potential conflicts of interest were disclosed.
5. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed 10. Rose PI. In: Mark HF, Bikales NM, Overberger CG,
References Menges G, Kroschwitz JI, Editors. Encyclopedia of
M, Nasr-Esfahani MH, Ramakrishna S. Electrospun
1. Kim MS, Jun I, Shin YM, Jang W, Kim SI, Shin H. poly(3-caprolactone)/gelatin nanofibrous scaf- polymer science and engineering, New York: Wiley
Heungsoo Shin, The development of genipin-cross- folds for nerve tissue engineering. Biomaterials 1987; 488.
linked poly(caprolactone) (PCL)/gelatin nanofibers 2008; 29:4532‑9; PMID:18757094; DOI:10.1016/ 11. Johns P, Courts A. In: Ward AG, Courts A, Editors.
for tissue engineering applications. Macromol Biosci j.biomaterials.2008.08.007. The science and technology of gelatin, London:
2010; 10:91-100; PMID:19685497; DOI:10.1002/ 6. Heydarkhan-Hagvall S, Schenke-Layland K, Academic 1977; 138.
mabi.200900168. Dhanasopon AP, Rofail F, Smith H, Wu BM, et 12. Guidoin R, Marceau D, Rao TJ, King M, Merhi
2. Barnes CP, Sell SA, Boland ED, Simpson DG, Bowlin al. Three-dimensional electrospun ECM-based Y, Roy PE, et al. In vitro and in vivo character-
GL. Nanofiber technology: Designing the next genera- hybrid scaffolds for cardiovascular tissue engineering. ization of an impervious polyester arterial prosthe-
tion of tissue engineering scaffolds. Adv Drug Deliv Rev Biomaterials 2008; 29:2907-14; PMID:18403012; sis: The Gelseal Triaxial_graft. Biomaterials 1987;
2007; 59:1413-33; PMID:17916396; DOI:10.1016/ DOI:10.1016/j.biomaterials.2008.03.034. 8:433-41; PMID:3427141; DOI:10.1016/0142-
j.addr.2007.04.022. 7. Zong X, Bien H, Chung CY, Yin L, Fang D, Hsiao 9612(87)90079 2.
3. Blackwood KA, McKean R, Canton I, Freeman CO, BS, et al. Electrospun fine-textured scaffolds for 13. Jonas RA, Ziemer G, Schoen FJ, Britton L, Castaneda
Franklin KL, Cole D, et al. Development of bio-degrad- heart tissue constructs. Biomaterials 2005; 26:5330- AR. A new sealant for knitted Dacron prostheses mini-
able electrospun scaffolds for dermal replacement. 8; PMID:15814131; DOI:10.1016/j.biomateri- mally cross-linked gelatin. J Vasc Surg 1988; 7:414-9;
Biomaterials 2008; 29:3091-104; PMID:18448164; als.2005.01.052. PMID:2450210.
DOI:10.1016/j.biomaterials.2008.03.037. 8. Schiffman JD, Schauer CL. Cross-Linking Chitosan 14. Marois Y, Chakfe¥ NL, Deng X, Marois M, How T,
4. Choi JS, Lee SJ, Christ GJ, Atala A, Yoo JJ. The Nanofibers Biomacromolecules. Biomacromolecules King MW, et al. Carbodiimide cross-linked gelatin:
influence of electrospun aligned poly(varepsilon- 2007; 8:594-601; PMID:17291083; DOI:10.1021/ a new coating for porous polyester arterial prosthe-
caprolactone)/collagen nanofiber meshes on the bm060804s. ses. Biomaterials 1995; 16:1131-9; PMID:8562788;
formation of self-aligned skeletal muscle myotubes. 9. Ye P, Xu ZK, Wu J, Innocent C, Seta P. Nanofibrous DOI:10.1016/0142-9612(95)93576-Y.
Biomaterials 2008; 29:2899-906; PMID:18400295; Membranes Containing Reactive Groups: 15. Tabata Y, Hijikata S, Ikada Y. Enhanced vasculariza-
DOI:10.1016/j.biomaterials.2008.03.031. Electrospinning from Poly(acrylonitrile-co-maleic acid) tion and tissue granulation by basic fibroblast growth
for Lipase Immobilization. Macromolecules 2006; factor impregnated in gelatin hydrogels. J Control
39:1041-5; DOI:10.1021/ma0517998. Release 1994; 31:189-99; DOI:10.1016/0168-
3659(94)00035-2.

100 Biomatter Volume 1 Issue 1

16. Ulubayram K, Cakar AN, Korkusuz P, Ertan C, Hasirci 31. Sepahvandi A, Moztarzadeh F, Mozafari M, Ghaffari 46. Kasten P, Luginbuhl R, van Griensven M, Barkhausen
N. EGF containing gelatin-based wound dressings. M, Raee N. Photoluminescence in the characterization T, Krettek C, Bohner M. Comparison of human
Biomaterials 2001; 22:1345-56; PMID:11336307; and early detection of biomimetic bone-like apatite bone marrow stromal cells seeded on calcium-deficient
DOI:10.1016/S0142-9612(00)00287-8. formation on the surface of alkaline-treated titanium hydroxyapatite, beta-tricalcium phosphate and demin-
17. Schnettler R, Alt V, Dingeldein E, Pfefferle HJ, Kilian implant: State of the art. Colloids Surf B Biointerfaces eralized bone matrix. Biomaterials 2003; 24:2593-
O, Meyer C, et al. Bone ingrowth in bFGF-coated 2011; 86:390-6; PMID:21592746; DOI:10.1016/j. 603; PMID:12726713; DOI:10.1016/S0142-
hydroxyapatite ceramic implants. Biomaterials 2003; colsurfb.2011.04.027. 9612(03)00062-0.
24:4603-8; PMID:12951003; DOI:10.1016/S0142- 32. Nezafati N, Moztarzadeh F, Hesaraki S, Mozafari 47. Wang H, Li Y, Zuo Y, Li J, Ma S, Cheng L.
9612(03)00354-5. M. Synergistically reinforcement of a self-setting cal- Biocompatibility and osteogenesis of biomimetic nano-
18. Bezwada RS, Jamiolkowski DD, Lee I, Vishvaroop A, cium phosphate cement with bioactive glass fibers. hydroxyapatite/polyamide composite scaffolds for
Persivale J, Treka-Benthin S, et al. a new ultra-pliable Ceram Int 2010; 37:927-34; DOI:10.1016/j.cera- bone tissue engineering. Biomaterials 2007; 28:3338-
absorbable monofilament suture. Biomaterials 1995; mint.2010.11.002. 48; PMID:17481726; DOI:10.1016/j.biomateri-
16:1141-8; PMID:8562789; DOI:10.1016/0142- 33. Peng J, Li X, Guo G, Yi T, Fu S, Liang H, et als.2007.04.014.
9612(95)93577-Z. al. Preparation and Characterization of Poly(vinyl 48. Salgada AJ, Coutinho OP, Reis RL. Bone tissue
19. Darney PD, Monroe SE, Klaisle CM, Alvarado A. alcohol)/Poly(caprolactone)-Poly(ethylene glycol)- engineering: state of the art and future trends.
Clinical evaluation of the Capronor contraceptive Poly(caprolactone)/Nano-Hydroxyapatite Composite Macromol Biosci 2004; 4:743-65; PMID:15468269;
implant—preliminary report. Am J Obstet Gynecol Membranes for Tissue Engineering. J Nanosci DOI:10.1002/mabi.200400026.
1989; 160:1292-5; PMID:2497647. Nanotechnol 2011; 11:2354-60; PMID:21449393; 49. Martin BJ, Pittenger MF. Bone marrow-derived stem
20. Rohner D, Hutmacher DW, Cheng TK, Oberholzer DOI:10.1166/jnn.2011.3139. cell for myocardial regeneration: preclinical experience,
M, Hammer B. In vivo efficacy of bone-marrow- 34. Tyagi P, Catledge SA, Stanishevsky A, Thomas V, In: Nabil D, Taylor DA, Diethrich EB, Editors. Stem
coated polycaprolactone scaffolds for the reconstruc- Vohra YK. Nanomechanical Properties of Electrospun cell therapy and tissue engineering for cardiovascular
tion of orbital defects in the pig. J Biomed Mater Res Composite Scaffolds Based on Polycaprolactone and repair from basic research to clinical applications, USA:
B 2003; 66:574-80; PMID:12861610; DOI:10.1002/ Hydroxyapatite. J Nanosci Nanotechnol 2009; 9:4839- Springer US 2006; 137-57.
jbm.b.10037. 45; PMID:19928159; DOI:10.1166/jnn.2009.1588. 50. Rameshbabu N, Kumar TSS, Prasad Rao K. Synthesis
21. Kweon H, Yoo MK, Park IK, Kim TH, Lee HC, 35. Sadjadi MAS, Meskinfam M, Sadeghi B, Jazdarreh of nanocrystalline fluorinated hydroxyapatite by micro-
Lee HS, et al. A novel degradable polycaprolactone H, Zare K. In Situ Biomimetic Synthesis and wave processing and its in vitro dissolution study. Bull
network for tissue engineering. Biomaterials 2003; Characterization of Nano Hydroxyapatite in Gelatin Mater Sci 2006; 29:611-5; DOI:10.1007/s12034-006-
24:801-8; PMID:12485798; DOI:10.1016/S0142- Matrix. J Biomed. Nanotechnol 2011; 7:450-4. 0012-3.
9612(02)00370-8. 36. Sadjadi MAS, Jazdarreh H, Zare K. Biocompatibility 51. Wei M, Evans JH, Bostrom T, Grondahl L. Synthesis
Evaluation of Nano Hydroxyapatite-Starch and characterization of hydroxyapatite, fluoride-sub-
22. Marra KG, Szem JW, Kumta PN, DiMilla PA,
Biocomposites M. Meskinfam. J Biomed Nanotechnol stituted hydroxyapa-tite and fluorapatite. J Mater
Weiss LE. In vitro analysis of biodegradable poly-
2011; 7:455-9. Sci Mater Med 2003; 14:311-20; PMID:15348455;
mer blend/hydroxyapatite composites for bone tissue
37. Sahithi K, Swetha M, Prabaharan M, Moorthi DOI:10.1023/A:1022975730730.
engineering. J Biomed Mater Res 1999; 47:324-
35; PMID:10487883; DOI:10.1002/(SICI)1097- A, Saranya N, Ramasamy K, et al. Synthesis and 52. Catledge SA, Clem WC, Shrikishen N, Chowdhury

©2011L
andesBiosc
ienc
e.
4636(19991205)47:3<324::AID-JBM6>3.0.CO;2-Y. Characterization of Nanoscale-Hydroxyapatite-Copper S, Stanishevsky AV, Koopman M. An electrospun
for Antimicrobial Activity Towards Bone Tissue triphasic nanofibrous scaffold for bone tissue engineer-
23. Jones F. Teeth and bones: Applications of surface
Engineering Applications. J Biomed. Nanotechnol ing. Biomed Mater 2007; 2:142-50; PMID:18458448;
science to dental materials and related biomaterials.
2010; 6:333-9; PMID:21323106; DOI:10.1166/ DOI:10.1088/1748-6041/2/2/013.
Surf Sci Rep 2001; 42:75-205; DOI:10.1016/S0167-

Donotdi
str
ibut
e.
jbn.2010.1138. 53. Itoh S, Kikuchi M, Koyama Y, Matumoto HN,
5729(00)00011-X.
38. Zeng S, Fu S, Guo G, Liang H, Qian Z, Tang X, Takakuda K, Shinomiya K. Development of a novel
24. Kikuchi M, Itoh S, Ichinose S, Shinomiya K, Tanaka
et al. Preparation and Characterization of Nano- biomaterial, hydroxyapatite/collagen (HAp/Col) com-
J. Self-organization mechanism in a bonelike hydroxy-
Hydroxyapatite/Poly(vinyl alcohol) Composite posite for medical use. J Biomed Mater Eng 2005;
apatite/collagen nanocomposite synthesized in vitro
Membranes for Guided Bone Regeneration. J Biomed 15:29-41; PMID:15623928.
and its biological reaction in vivo. Biomaterials 2001;
Nanotechnol 2011; 7:549-57. 54. Masanori KB, Hiroko N, Matsumotob C, Takeki Y,
22:1705-11; PMID:11396873; DOI:10.1016/S0142-
39. Causa F, Netti PA, Ambrosio L, Ciapetti G, Baldini Yoshihisa K, Kazuo T, et al. Glutaraldehyde cross-linked
9612(00)00305-7.
N, Pagani S, et al. Poly-α-caprolactone/hydroxyapatite hydroxyapatite/collagen self-organized nanocompos-
25. Liou SC, Chen SY, Liu DM. Synthesis and char- ites. Biomaterials 2004; 25:63-9; PMID:14580909;
composites for bone regeneration: in vitro charac-
acterization of needlelike apatitic nanocomposite terization and human osteoblast response. J Biomed DOI:10.1016/S0142-9612(03)00472-1.
with controlled aspect ratios. Biomaterials 2003; Mater Res A 2006; 76:151-9; PMID:16258959; 55. Teng S, Shi J, Peng BL, Chen F. The effect of algi-
24:3981-8; PMID:12834593; DOI:10.1016/S0142- DOI:10.1002/jbm.a.30528. nate addition on the structure and morphology of
9612(03)00303-X.
40. Chang MC, Ko CC, Douglas WH. Preparation of hydroxyapatite/gelatin Nanocomposites. Compos Sci
26. Poursamar SA, Azami M, Mozafari M. Controllable hydroxyapatite-gelatin nanocomposite. Biomaterials Technol 2006; 66:1532-8; DOI:10.1016/j.compsci-
synthesis and characterization of porous polyvinyl alco- 2003; 24:2853-62; PMID:12742723; DOI:10.1016/ tech.2005.11.021.
hol/hydroxyapatite nanocomposite scaffolds via an in S0142-9612(03)00115-7. 56. Chang MC, Ko CC, Douglas WH. Conformational
situ colloidal technique. Colloids Surf B Biointerfaces change of hydroxyapatite-gelatin nanocom-
41. Liu X, Smith LA, Hu J, Maa PX. Biomimetic nano-
2011; 84:310-6; PMID:21310596; DOI:10.1016/j. posite by glutaraldehyde. Biomaterials 2003;
fibrous gelatin/apatite composite scaffolds for bone
colsurfb.2011.01.015. 24:3087‑94; PMID:12895581; DOI:10.1016/S0142-
tissue engineering. Biomaterials 2009; 30:2252-
27. Hamlekhan A, Mozafari M, Nezafati N, Azami M, 8; PMID:19152974; DOI:10.1016/j.biomateri- 9612(03)00150-9.
Hadipour H. A proposed fabrication method of novel als.2008.12.068. 57. Minfang C, Junjun T, Yuying L, Debao L. Preparation
PCL-GEL-HAp nanocomposite scaffolds for bone tis- 42. Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang of Gelatin coated hydroxyapatite nanorods and the
sue engineering applications. Adv Compos Lett 2010; ZM. Electrospinning of gelatin fibers and gelatin/PCL stability of its aqueous colloidal. Appl Surf Sci 2008;
19:123-30. composite fibrous scaffolds. J Biomed Mater Res B 254:2730-5; DOI:10.1016/j.apsusc.2007.10.011.
28. Azami M, Jalilifiroozinezhad S, Mozafari M. Synthesis Appl Biomater 2004; 72:156-63; PMID:15389493; 58. Kon E, Muraglia A, Corsi A. Electrospun poly(α-
and solubility of calcium fluoride/hydroxy-fluorapatite DOI:10.1002/jbm.b.30128. caprolactone)/gelatin nanofibrous scaffolds for nerve tis-
nanocrystals for dental applications. Ceram Int 2011; 43. Majumdar MK, Keane-Moore M, Buyaner D, Hardy sue engineering. J Biomed Mater Res 2000; 49:4532-9.
37:2007-14; DOI:10.1016/j.ceramint.2011.02.025. WB, Moorman MA, Mclntosh KR. Characterization 59. Barry FP, Murphy JM. Autologous bone marrow stro-
29. Mozafari M, Rabiee M, Azami M, Maleknia S. and functionality of cell surface molecules on human mal cells loaded onto porous hydroxyapatite ceramic
Biomimetic formation of apatite on the surface of mesenchymal stem cells. J Biomed Sci 2003; 10:228- accelerate bone repair in critical-size defects of sheep
porous gelatin/bioactive glass nanocomposite scaffolds. 41; PMID:12595759; DOI:10.1007/BF02256058. long bones. Int J Biochem Cell Biol 2004; 36:328-37.
Appl Surf Sci 2010; 257:1740-9; DOI:10.1016/j. 44. Sun JS, Tsuang YH, Liao CJ, Liu HC, Hang YS, 60. Wang YW, Wu Q, Chen GQ. Attachment, prolif-
apsusc.2010.09.008. Lin FH. The effects of calcium phosphate particles eration and differentiation of osteoblasts on ran-
30. Razavi A, Moztarzadeh F, Mozafari M, Azami M, on the growth of osteoblasts. J Biomed Mater Res dom biopolyester poly(3-hydroxybutyrate-co-
Baghbani F. Synthesis and characterization of high-pure 1997; 37:324-34; PMID:9368137; DOI:10.1002/ 3-hydroxyhexanoate) scaffolds. Biomaterials 2004;
nanocrystalline forsterite and its potential for soft tissue (SICI)1097-4636(19971205)37:3<324::AID- 25:669-75; PMID:14607505; DOI:10.1016/S0142-
applications. Adv Compos Lett 2011; 20:41-7. JBM3>3.0.CO;2-N. 9612(03)00561-1.
45. Murphy WL, Hsiong S, Richardson TP, Simmons CA, 61. Morales JG, Burgues JT, Boix T, Fraile J, Clemente
Mooney DJ. Effects of a bone-like mineral film on RR. Precipitation of Stoichiometric Hydroxyapatite by
phenotype of adult human mesenchymal stem cells in a Continuous Method. Cryst Res Technol 2001; 36:15-
vitro. Biomaterials 2005; 26:303-10; PMID:15262472; 22; DOI:10.1002/1521-4079(200101)36:1<15::AID-
DOI:10.1016/j.biomaterials.2004.02.034. CRAT15>3.0.CO;2-E.

www.landesbioscience.com Biomatter 101