Acta Biomaterialia 7 (2011) 16091617

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Functional composite nanobers of poly(lactideco-caprolactone) containing

gelatinapatite bone mimetic precipitate for bone regeneration
Seung-Hwan Jegal a,c, Jeong-Hui Park a,c, Joong-Hyun Kim c, Tae-Hyun Kim c, Ueon Sang Shin c,
Tae-Il Kim d, Hae-Won Kim a,b,c,
a

Biomaterials and Tissue Engineering Laboratory, Department of Nanobiomedical Science and WCU Research Center, Dankook University, South Korea
Department of Biomaterials Science, School of Dentistry, Dankook Universitsy, South Korea
Institute of Tissue Regeneration Engineering, Dankook Universitsy, South Korea
d
Department of Periodontology, College of Dentistry, Seoul National University, South Korea
b
c

a r t i c l e

i n f o

Article history:
Received 6 September 2010
Received in revised form 28 October 2010
Accepted 1 December 2010
Available online 8 December 2010
Keywords:
Electrospun matrix
Apatitegelatin
Polymer nanober
Osteoblastic responses
Bone regeneration

a b s t r a c t
Functional nanobrous materials composed of gelatinapatitepoly(lactideco-caprolactone) (PLCL)
were produced using an electrospinning process. A gelatinapatite precipitate, which mimicked bone
extracellular matrix, was homogenized in an organic solvent using various concentrations of PLCL. A
brous structure with approximate diameters of a few hundred nanometers was successfully generated.
Apatite nanocrystallines were found to be effectively distributed within the polymeric matrix of the gelatinPLCL. The addition of a small amount of gelatinapatite into PLCL signicantly improved the tensile
strength of the nanober by a factor of 1.8. Moreover, tissue cell growth on the composite nanober was
enhanced. Osteogenic differentiation of the cells was signicantly stimulated by the composite nanober
compared with the pure PLCL nanober. When implanted in a rat calvarium for 6 weeks the composite
nanober supported defect closure and new bone formation better than the pure PLCL nanober, as
deduced from micro-computed tomography and histological analyses. Based on these results, the gelatinapatitePLCL composite nanober developed in this study is considered to be potentially useful as
a bone tissue regeneration matrix.
2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
The reconstruction of damaged hard tissues using biomedical
materials has shown some promise in the eld of regenerative
medicine [1,2]. Recently, nanobers have been developed as a
new type of scaffolding material via an electrospinning technique
[35]. A range of biopolymers, including poly(a-hydroxyl acids),
natural proteins and polysaccharides, have been produced as nanobrous structures with sizes of tens to hundreds of nanometers [6
13]. The ber morphology obtained has been considered far too
difcult to achieve by other conventional processing techniques.
Moreover, many biological tests have shown the merits of nanobrous structures in the adhesion and growth of cells and further
tissue development [14,15].
For the regeneration of hard tissues, including bone and tooth,
the recent research focus has been on composites combining polymeric matrices with inorganic components [1620]. Bone matrix is
a type of nanocomposite consisting of apatite nanocrystallites and
Corresponding author at: Biomaterials and Tissue Engineering Laboratory,
Department of Nanobiomedical Science and WCU Research Center, Dankook
University, South Korea. Tel.: +82 41 550 1926.
E-mail address: kimhw@dku.edu (H.-W. Kim).

collageneous brous protein, therefore, the composite approach is

considered to mimic the native bone structure [5]. Studies have
shown that nanocomposite biomaterials induced better bone cell
responses in vitro and bone formation in vivo compared with individual polymers [2126]. Specically, porous scaffolds made of
hydroxyapatite-precipitated gelatin showed signicantly enhanced bone cell responses [21]. Bioactive glass components in
composites containing degradable polymers have also been shown
to stimulate the gene expression and differentiation of osteogenic/
stem cells [2527]. Moreover, synthetic degradable polymeric
lms lled with an inorganic calcium phosphate phase have shown
better resistance to the rapid degradation associated with acidic
environments [24]. However, only limited studies have been made
on the production of nanobrous matrices composed of composites by the electrospinning process [1620]. This is because it is
far more difcult to construct a nanobrous network from composites than from the individual polymers [5].
Recently, a nanobrous membrane composed of apatite and
gelatin was produced by electrospinning [17]. The apatite nanocrystals were found to be evenly distributed within the gelatin matrix when the precipitated product was dispersed within an
organic solvent. In practice, this idea provides an important insight
into the fabrication of composite nanober systems. However, the

1742-7061/$ - see front matter 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2010.12.003

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stiff nature of the product and premature dissolution must be overcome to obtain a scaffold suitable for cell cultivation and hard tissue formation. Within this context we utilized the mechanical
benets of a synthetic polymer, poly(lactideco-caprolactone)
(PLCL), within a bone-mimicking gelatinapatite system to produce a functional composite membrane composed of gelatinapatitePLCL. This composite nanobrous membrane is also
considered to provide better biological properties than PLCL biopolymer nanober. Herein, fabrication methods to produce composite membranes and their mechanical properties are described
and the biological performances in the presence of bone cells
in vitro and rat calvarium tissues in vivo are examined.
2. Materials and methods
2.1. Preparation of composite nanobers
The gelatinapatite solution was prepared through a precipitation reaction of Ca(NO3)2
4H2O and (NH3)2HPO4 within gelatin
(Type B, bovine skin), as described previously [17]. Briey, two separate solutions of calcium-containing gelatin (Ca-gelatin) and phosphate-containing gelatin (P-gelatin) were mixed at a ratio of [Ca]/
[P] of 1.67, with vigorous stirring at 40 C and a constant pH of
10, maintained using NH4OH. The apatite to gelatin ratio was maintained at an equivalent weight (apatite/gelatin = 1) in consideration
of the complete reaction of calcium and phosphate to form apatite.
The apatite-precipitated gelatin sol was frozen at 20 C and then
freeze-dried under vacuum. Next, the dried sample was washed
thoroughly with distilled water/ethanol to remove any salt byproducts, after which it was again freeze-dried. The freeze-dried sample
was then dispersed in triuoroethanol (TFE) (Aldrich) at 15% w/v
with ultrasonic vibration for a few minutes and then stirred vigorously for 24 h. Next, PLCL (Boelinger Ingelheim) dissolved in TFE at
15 wt.% was mixed with the gelatinapatite solution at two different mixing ratios (gelatinapatite:PLCL = 1:4 (high) or 1:6 (low)).
Each solution was then loaded into a syringe and injected onto a
mandrel rotating at a speed of 140 rpm under a high d.c. voltage
of 10 kV at a distance of 15 cm at an injection speed of 0.4 ml h1.
2.2. Characterizations
The morphology of the electrospun nanobers was evaluated by
scanning electron microscopy (SEM), and the ber diameter was
measured from the images. The phase of the apatite generated
within the gelatin matrix was conrmed by X-ray diffraction
(XRD). Transmission electron microscopy (TEM) was used to determine the presence and distribution of apatite nanocrystallines
within the nanober. The tensile mechanical properties of the
nanobrous membrane were measured using an Instron 3344.
Membranes were prepared with a thickness of 150200 lm
and then cut to a size of 30  4 mm (gauge length 10 mm), after
which a tensile load was applied. Stressstrain curves were recorded and the maximum tensile stress, elastic modulus and elongation at failure were determined. The thickness of each
membrane was determined from the average value observed in
the SEM images and a total of ve samples were tested for each
group. The water afnity of the nanober membrane was examined by measuring the water contact angle (contact angle analyzer
Phoenix300). Data were recorded for up to 1 h and ve samples
were tested for each group.
2.3. In vitro osteoblast responses
The in vitro biocompatibility of the composite nanobers was
observed using pre-osteoblast cells (MC3T3-E1, ATCC). The cells

were sub-cultured in culture medium, consisting of a-minimal

essential medium supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 50 IU ml1 penicillin and 50 lg ml1
streptomycin. Electrospun nanobrous webs (pure PLCL as a control and two different composites) were prepared with dimensions
of 10  10 mm and placed in 24-well plates, after which the cell
suspension (at a density of 3  104 cells ml1) was plated on each
sample. The samples were then incubated at 37 C and the cell
growth level was assessed by MTS method at days 3 and 7. Next,
the cells were xed, dehydrated in a graded series of ethanol, treated with hexamethyldisilazane and coated with gold, after which
the cell morphology was observed by SEM. Osteoblastic differentiation of the cells was then observed by measuring the production
of alkaline phosphatase (ALP). Cells cultured on each nanobrous
sample for 7 and 14 days were assessed using an ALP activity kit
(Sigma), as described previously [17]. Triplicate samples in each
group were used for all cellular tests and groups were compared
by analysis of variance (ANOVA). Signicance was considered at
P < 0.05 and P < 0.01.
2.4. In vivo implantation in rat calvarium
Six 10-week-old male SpragueDawley rats were used in this
study. The animal surgery protocol was performed in accordance
with the Animal Care and Use Committee, Dankook University,
South Korea.
The animals were anesthetized by means of intramuscular
injection using ketamine (80 mg kg1) and xylazine (10 mg kg1).
The prepared membranes with dimensions of 10  10 mm were
sterilized prior to use. The skin hair on the cranium was shaved
and the surgical region was aseptically treated using povidone/
70% ethanol. A 15 mm skin incision was made and the periosteum
was elevated for trephining. Two critical sized full thickness bone
defects (5 mm diameter) were prepared in each rat at the center
of each parietal bone using a saline-cooled trephine drill. Care
was taken not to damage the underlying sagittal sinus and dura
matter. Each defect was randomly implanted with the two types
of membranes or kept empty as a negative control. The subcutaneous tissue was closed and the skin incisions sutured.
The animals were sacraced 6 weeks after implantation. The
skin was removed and the samples and the surrounding tissues
were withdrawn en bloc and xed in 10% neutral buffered formalin
solution for 24 h at room temperature and prepared for microcomputed tomography (micro-CT) analysis and histomorphometry.
Micro-CT (Skyscan 1072, Skyscan, Aartselaar, Belgium) was
used to observe the formation of new bone within the defect region. The harvested specimens were scanned, with each frame exposed for 20 ms. Scanning was performed in a direction parallel to
the coronal aspect of the calvarial bone surrounding the defect
area. A cylindrical region of interest (ROI) was precisely positioned
over the center of each defect, encompassing all new bone within
the defect site. Micro-CT images were reconstructed over the ROI
using a CTAn (Skyscan) and the data analyzed. The total volume
of newly formed bone within the ROI was measured using threedimensional (3-D) images by assigning a threshold for total bone
content (including trabecular and cortical bone) and subtracting
any contribution of the scaffold (determined previously). Four
samples for each group were measured and total volume of bone
is reported (mm3).
For histomorphometric analysis the xed samples were decalcied, dehydrated and embedded in parafn, then serially sectioned
with a microtome (LEICA) at 45 lm thickness and nally
mounted on microscope slides. Slides with tissue sections were
deparafnized and dehydrated with xylene and an ethanol series.
The slides were stained with hematoxylin and eosin (H&E) and
Massons trichrome (MT) and examined using a light microscope.

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3. Results and discussion

3.1. Generation of composite nanobers

Fig. 1. Schematic showing the experimental steps used to fabricate the gelatin
apatitePLCL composite nanober by electrospinning.

Fig. 1 shows a schematic illustration of the experimental steps

used to fabricate the functional composite membrane made of gelatinapatitePLCL. Although synthetic polymer nanobers such as
PLCL are good candidates for tissue regeneration, the addition of a
gelatinapatite composite improves the biocompatibility with
bone tissue. In addition, the apatite inorganic phase can stimulate
osteogenic differentiation and calcication when combined with
biopolymers [1618,28]. Moreover, calcium phosphate inorganics
are known to be highly effective in reducing the problems associated with the acidic products formed during polymer degradation,
such as a decrease in pH and inammation [29,30]. Additionally,
because the major weaknesses of synthetic polymers are hydrophobicity and poor cell afnity, adding a gelatin component should
improve the properties of synthetic polymers such as PLCL [31,32].
To introduce the gelatin and apatite compositions within the
PLCL phase we rst synthesized a gelatinapatite precipitate and

Fig. 2. (ac) SEM morphology of the electrospun nanobrous sheets: (a, b) PLCL containing gelatinapatite precipitate at (a) low (1/6) and (b) high (1/4) concentration, and (c)
pure PLCL. (d) TEM morphology of the composite nanober in (a) showing the precipitated apatite nanocrystallites dispersed in the polymeric matrix. (e) XRD analysis to
conrm the phase development of apatite formed in the presence of gelatin matrix (s, hydroxyapatite).

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then homogenized it with PLCL within a co-solvent, TFE. When

compared with directly adding the individual components (gelatin
and apatite) [33,34] the approach used herein, as developed in our
previous work [17], could produce a composite solution with improved mixing. The results of our previous study revealed that amino acid groups in gelatin facilitated the spatial and homogeneous
nucleation of apatite, which resulted in more uniform and ner
sized nanocrystallites [17]. As a result, the electrospun nanobers
produced from the precipitates had a better brous morphology
than those made by the direct mixing approach [17]. In practice,
during the electrospinning of inorganicorganic composites it is
important to utilize a solution with the appropriate mixing properties to ensure the production of uniform and bead-free nanobers
[5].
Herein, addition of the gelatinapatite precipitate to PLCL was
conducted at two different concentrations with respect to the
PLCL: low (1/6 ratio  14.3 wt.%) and high (1/4 ratio  20 wt.%).
The consequent levels of apatite were 7.2 and 10 wt.% with respect to the PLCLgelatin polymer phase.
3.2. Morphology and mechanical properties of membranes
The morphology of the generated gelatinapatitePLCL composite nanobers is shown in Fig. 2. At both concentrations of gelatinapatite spinning into a ber was possible under the adjusted
conditions. When a low concentration of gelatinapatite was used
(14.3 wt.%, Fig. 2a) a well-developed non-woven brous web with
bers a few hundreds of nanometers in size were produced. The
electrospun ber was continuous with no discrete beads, but some

regions appeared to be heterogeneous with a slightly thicker diameter. The hydrophilic gelatinapatite may become segregated to
some extent within the PLCL matrix during the electric eld-driven
process. When a high concentration (20 wt.%) of gelatinapatite
was used some discrete beads were noticed and the ber size became relatively non-uniform (Fig. 2b). Compared with the pure
PLCL nanobers (Fig. 2c) the composite nanobers had relatively
smaller diameters (average ber size 310 103 nm for the high
concentration and 291 51 nm for the low concentration of gelatinapatite vs. 517 232 nm for PLCL), moreover, some microbers
were noticed in the PLCL. The existence of an apatite inorganic
phase is evident in the TEM image (Fig. 2d). Additionally, highly
elongated apatite nanocrystallites were well distributed within
the PLCLgelatin matrix, and there appeared to be no sign of phase
separation between the gelatin and PLCL. The apatite phase produced in the presence of the gelatin matrix was conrmed by
XRD (Fig. 2e). The homogeneity of the gelatinapatite precipitate
was shown to be well preserved within the PLCL matrix. Thus,
the addition of gelatinapatite to PLCL is an effective method of
obtaining nanobers with well-homogenized inorganicorganic
components within the biopolymer matrix.
The mechanical properties of the composite nanobers are
compared with those of pure PLCL nanober in Fig. 3. The stress
strain curves of the nanober membranes were recorded on ve
individual samples, and a characteristic curve for each composition
is shown in Fig. 3a. All nanobrous membranes showed an initial
rapid increase in stress, which became less rapid as the maximum
stress value was approached, and then failure. The maximum
stress value (tensile strength), the initial slope (elastic modulus)

Fig. 3. Tensile mechanical tests of the nanobrous composite membranes and pure PLCL for comparison: (a) representative stressstrain curves of each membrane, (b)
maximum tensile stress, (c) elastic modulus, and (d) elongation at failure, as determined in 5 individual samples (mean SD for n = 5). The value obtained in the composite
nanobers was signicantly different from that in pure PLCL (P < 0.05, by ANOVA).

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Fig. 4. Osteoblastic cell responses to the composite nanobrous membranes: (a) cell growth morphology on the composite containing a low level of gelatinapatite at 3 and
7 days, (b) cell proliferation level for up to 7 days, as determined by MTS, and (c) ALP osteogenic differentiation on the nanobers at days 7 and 14. A signicant difference was
noticed on the composite nanober with respect to PLCL (P < 0.05 and P < 0.01, by ANOVA, n = 3). A signicant increase in ALP activity was observed on the composite with
a low content of gelatinapatite with respect to culturing time (++P < 0.01, 7 vs. 14 days).

and the percentage elongation (strain) at failure were obtained

from the stressstrain curves, as shown in Fig. 3bd. The composite
membrane with a low gelatinPLCL content showed the highest
strength (mean 10.1 MPa), which was almost double that of the
PLCL nanober (mean 5.7 MPa). However, the strength of the composite with a high gelatinapatite content was slightly lower
(mean 4.3 MPa) than that of pure PLCL. The elastic modulus of
the nanobers was also measured (Fig. 3c). The addition of gelatinapatite dramatically increased the elastic modulus of pure
PLCL, and the increase was more signicant with addition of the
low concentration of gelatinapatite (5.2 MPa for PLCL vs.
51 MPa for low and 25 MPa for high concentration composite).
While the percentage elongation at failure of the pure PLCL nanober was as high as 330%, the addition of gelatinapatite decreased this value in a concentration-dependent manner, as
indicated by elongation values of 230% and 90% for nanobers
that contained low and high concentrations of gelatinapatite,
respectively. In our previous study of a gelatinapatite nanober
system gelatin composite bers containing 20 and 40% apatite
had tensile strengths and elongations of approximately 45 MPa
and 47%, respectively, which are signicantly lower than the values obtained for the gelatinapatitePLCL composite ber [17]. The
addition of low concentration of gelatinapatite signicantly enhanced the strength (an approximately two times increase) and

stiffness (an approximately ten times increase) but simultaneously

slightly reduced the elongation rate (approximately 30% decrease).
However, the elongation obtained in the composite was high enough that the addition of gelatinapatite is not considered to
diminish the elongation property that is not appropriate for bone
regeneration.
In practice, the addition of inorganic particles to the polymeric
phase is known to enhance the mechanical strength when the inorganic particles are ne and evenly dispersed [35]. In biological systems such as bone the apatite nanocrystallites embedded in the
collagen bers strengthen and harden the bone tissue [36]. In the
present study the addition of a small amount of apatitegelatin
was found to be highly effective in improving the mechanical
strength of the PLCL nanobers. This was probably because the
ultrane apatite crystallites were evenly dispersed in the nanobers, which should have enabled the polymer to resist extension
in response to an applied load. However, the addition of a high concentration of apatitegelatin was found to decrease the strength of
the PLCL. This was believed to be due to agglomeration of the apatite nanocrystallites, which was revealed as the presence of some
large beads on the nanobers (as seen in Fig. 2b). The agglomerated
beads probably lead to premature failure rather than resistance to
an applied load, although some stiffening effect of the inorganic
phase was noticed, as determined from the initial slope of the

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Fig. 5. Micro-computed tomography (micro-CT) analyses of the harvest samples at 6 weeks post-operation. Sample groups are (a) blank control, (b) PLCL nanober, and (c)
composite nanober. To the right of the image of the surgical operation is a 2-D reconstructed micro-CT image including the 5 mm initial defect zone shown in yellow. Below
the image is a 3-D reconstructed micro-CT image, showing more clearly the formation of hard tissues within the defect region. (d) Bone volume determined from the 3-D
micro-CT data. A signicant difference was noticed on the PLCL and composite nanobers with respect to the blank control (P < 0.05 and P < 0.01, by ANOVA).

curve. Conversely, the composite nanobers with low gelatinapatite content showed a greater elongation, almost comparable with
that of pure PLCL. Apart from a strengthening effect, the maintenance a high degree of exibility should favor the use of this composite as a tissue regeneration membrane or a scaffold for cell
growth.
The afnity to water of the composite membranes was shown
to be signicantly higher than that of PLCL. The initial water contact angle to the nanober surface was 80 for PLCL and 70
for both composites. Additionally, the water droplet was shown
to spread completely over the composites, penetrating into the
nanober matrix within 1 (high gelatinapatite content) and
5 min (low gelatinapatite content). However, no such water permeation was observed when the pure PLCL nanober was evaluated, even after 1 h. This high water afnity of the PLCL
nanobers containing gelatinapatite should allow effective and
rapid uid contact with the material surface, thereby enhancing
the reaction with biological molecules and cells.
3.3. In vitro cellular responses
The biological role of the gelatinapatite within the PLCL nanofibers was addressed in terms of cell growth and osteogenic development in vitro. MC3T3-E1 murine-derived pre-osteoblast cells
were cultured on the nanobrous membranes and cell proliferation of and ALP activity in the cells were then examined. Fig. 4a
shows the morphology of cells grown on the composite (low content gelatinapatite) and PLCL nanobrous substrates. On the composite nanobers the cells were very viable initially (at day 3) with

good cytoplasmic extensions, and the cells almost reached conuence, forming a thick cell layer by day 7. On the pure PLCL the cells
on day 3 exhibited less spreading than those on the composite, but
showed similar behavior on day 7. The level of cell growth on the
nanobers was also measured by MTS assay (Fig. 4b). At day 3 cell
proliferation was signicantly higher on the composite membranes
than on the pure PLCL (P < 0.01). No signicant difference was observed between the two composite nanobers. It is believed that
the increase in initial cell spreading and growth on the composites
was primarily due to the enhanced hydrophilicity, which allowed
the rapid adsorption of proteins and facilitated the cell adhesion
process [10]. Moreover, ion (such as calcium and phosphate) release from the apatite component within the nanober can alter
cell behavior, such as cell proliferation and osteoblastic differentiation [37,38].
It is important to note that the ALP level was signicantly greater on the composite nanobers (Fig. 4c). ALP enzymatic activity
produced by cells on the nanober membranes was measured during culture for 7 and 14 days. The ALP activity of cells grown on the
composite nanobers was signicantly higher than that of cells
grown on pure PLCL at day 7, while no signicant difference was
observed between the two composite nanobers. At this point
the cells had reached conuence and formed a thick layer, therefore, they may have undergone osteoblastic differentiation, which
is generally associated with the saturated proliferative potential
of MC3T3-E1 cells. After prolonged culture for 14 days ALP stimulation on the composite with a low content of gelatinapatite was
signicant (almost double), while only a slight increase was noticed on the other membranes. As a result, the ALP level in cells

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Fig. 6. Hematoxylin and eosin (H&E) and Massons trichrome (MT) staining of the tissues formed within the defect with the help of the nanobrous membranes: (a) H&E
stain, PLCL nanober, (b) H&E stain, composite nanober, (c) MT stain, PLCL nanober and (d) MT stain, composite nanober. Defect margins are indicated by arrows. (e)
Enlarged image of inset in (d) showing the formation of bony tissue. Defect closure was signicantly different between the groups based on the histomorphometric analysis
(64.7% for composite >57.7% for PLCL >40.4% for control, P < 0.01, by ANOVA, n = 4). Scale bars: (ad) 500 lm; (e) 30 lm.

on the composite with a low content of gelatinapatite was significantly higher (by a factor of 2) than that in other membranes.
The apatite dispersed within the PLCLgelatin matrix in the nanober should enhance osteogenic differentiation, particularly during
culture for 714 days. It is also believed that apatite synthesized in
the presence of a gelatin network largely mimics native bone

[33,39]. Previous studies have shown that apatite created within

gelatin greatly enhances bone cell differentiation and ALP production [17]. Furthermore, the added gelatin may also improve the
biological potential of the PLCL polymer during osteoblast growth
and matrix synthesis [8]. An increase in the initial event can lead
to increases in the overall processes, including cell division and

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differentiation. The in vitro biological stimulation by the apatite

and gelatin within the PLCL composition appeared to be similar
for both composite nanobers. Although the nanobers containing
the high concentration of gelatinapatite can stimulate biological
activity, the large beads formed at this concentration may have
an adverse effect on cellular events [17].
3.4. In vivo bone regeneration ability
After calvarial implantation rats were killed at 6 weeks and defect sites were harvested to evaluate tissue recovery and bone
regeneration. Fig. 5 shows micro-CT analyses of the samples.
Two-dimensional (2-D) and 3-D images were constructed for a
blank control without the membrane (Fig. 5a) and for the membrane groups: PLCL nanober (Fig. 5b) and composite nanober
(Fig. 5c). Based on the 2-D images, hard tissue formation occurred
from the outer margin to the central region. In the blank control
the defect region remained primarily empty throughout the study,
demonstrating the negative control as a critical size defect. However, when the PLCL or composite nanobrous membrane was implanted there was considerable in-growth of hard tissue. The 3-D
images show more clearly the bone in-growth and the effectiveness of the membranes. As summarized in Fig. 5d, hard tissue formation was better in the order composite membrane > PLCL
membrane >> blank control.
The newly formed tissues within the calvarium defect were further analyzed by histological staining, as shown in Fig. 6. H&E
staining was rst carried out to reveal cell or/and tissue in-growth
within the defect region (Fig. 6a and b). In both the PLCL nanober
(Fig. 6a) and composite nanober membranes (Fig. 6b) the defect
site was observed to be almost completely lled with dense connective tissue. The defect closure measured from the histological
images was in the order composite nanober (64.7 3.6) > PLCL
nanober (57.7 1.6) > blank control (40.4 5.1), with signicant
differences between the groups (P < 0.01, one-way ANOVA, n = 4).
After Massons trichrome staining the formation of bony tissue
was more clearly revealed (Fig. 6c and d). Compared with the PLCL
nanober (Fig. 6c), the composite nanober (Fig. 6d) showed a
much thicker layer of new bone formation. The newly formed bone
was well integrated with the edge of the host bone. In contrast to
these membrane groups, the control group showed the formation
of very thin and loose connective tissue with little new bone formation originating from the defect margins (not shown here), supporting the micro-CT data. A higher magnication of the newly
formed bone revealed osteoid regions, appearing pale blue, and
mineralized bone regions, showing much darker blue in color
(Fig. 6e).
The results on the in vitro and in vivo behaviors combined with
the mechanical properties suggest that a small content of gelatin
apatite should provide the optimal substrate conditions for
osteogenic differentiation and bone tissue regeneration, and the
composite nanobers could nd practical application in orthopedics and dentistry, such as guided bone regeneration membranes
in periodontal pockets.
4. Conclusions
Composite nanobers made of gelatinapatitePLCL were produced by electrospinning. A precipitate of gelatinapatite was
added to the PLCL in TFE solvent to prepare a homogeneous precursor solution. At a low concentration of gelatinapatite (14.3 wt.%) a
bead-free, non-woven nanobrous web was produced, while a
considerable number of beads were noticed with a high concentration of gelatinapatite (20 wt.%). Apatite nanocrystallites were observed to be well distributed within the gelatinPLCL organic

matrix. Moreover, the addition of a small amount of gelatinapatite led to bers with a signicantly improved tensile strength
(nearly double) when compared with those composed of pure
PLCL, without considerable loss in exibility. Additionally, cell proliferation and osteogenic development were signicantly higher on
the composite nanobers than on the pure PLCL nanober. When
the composite nanober membrane was implanted in a rat calvarium defect new bone formation and defect closure were signicantly enhanced with respect to pure PLCL or a negative control.
Overall, the results demonstrate that the gelatinapatitePLCL
nanobrous matrix developed here could potentially be useful in
the regeneration of hard tissues, such as a guided bone regeneration membrane in periodontology.
Acknowledgements
This work was supported by the Priority Research Centers Program (grant no. 2009-0093829) and the World Class University
Program (grant no. R31-10069) through the National Research
Foundation funded by the Ministry of Education, Science and
Technology.
Appendix A. Figures with essential colour discrimination
Certain gures in this article, particularly Figures 3, 5, and 6, are
difcult to interpret in black and white. The full colour images can
be found in the on-line version, at doi: 10.1016/j.actbio.2010.
12.003.
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