2011-Functional Composite Nanofibers of Poly(Lactide–Co-caprolactone) Containing Gelatin–Apatite...

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Functional composite nanofibers of poly(lactide–co-caprolactone) containing

gelatin–apatite 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 Koreab Department of Biomaterials Science, School of Dentistry, Dankook Universitsy, South Koreac Institute of Tissue Regeneration Engineering, Dankook Universitsy, South Koread Department of Periodontology, College of Dentistry, Seoul National University, South Korea

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

Apatite–gelatin

Polymer nanofiber

Osteoblastic responses

Bone regeneration

a b s t r a c t

Functional nanofibrous materials composed of gelatin–apatite–poly(lactide–co-caprolactone) (PLCL)

were produced using an electrospinning process. A gelatin–apatite precipitate, which mimicked bone

extracellular matrix, was homogenized in an organic solvent using various concentrations of PLCL. A

fibrous 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 gel-

atin–PLCL. The addition of a small amount of gelatin–apatite into PLCL significantly improved the tensile

strength of the nanofiber by a factor of 1.8. Moreover, tissue cell growth on the composite nanofiber was

enhanced. Osteogenic differentiation of the cells was significantly stimulated by the composite nanofiber

compared with the pure PLCL nanofiber. When implanted in a rat calvarium for 6 weeks the composite

nanofiber supported defect closure and new bone formation better than the pure PLCL nanofiber, as

deduced from micro-computed tomography and histological analyses. Based on these results, the gela-

tin–apatite–PLCL composite nanofiber 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 field of regenerative

medicine [1,2]. Recently, nanofibers have been developed as a

new type of scaffolding material via an electrospinning technique

[3–5]. A range of biopolymers, including poly(a-hydroxyl acids),natural proteins and polysaccharides, have been produced as nano-

fibrous structures with sizes of tens to hundreds of nanometers [6–

13]. The fiber morphology obtained has been considered far too

difficult to achieve by other conventional processing techniques.

Moreover, many biological tests have shown the merits of nanofi-

brous 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 poly-

meric matrices with inorganic components [16–20]. Bone matrix is

a type of nanocomposite consisting of apatite nanocrystallites and

collageneous fibrous 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 indi-

vidual polymers [21–26]. Specifically, porous scaffolds made of

hydroxyapatite-precipitated gelatin showed significantly en-

hanced 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 [25–27]. Moreover, synthetic degradable polymeric

films filled 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 nanofibrous matrices composed of compos-

ites by the electrospinning process [16–20]. This is because it is

far more difficult to construct a nanofibrous networkfrom compos-

ites than from the individual polymers [5].

Recently, a nanofibrous membrane composed of apatite and

gelatin was produced by electrospinning [17]. The apatite nano-

crystals were found to be evenly distributed within the gelatin ma-

trix when the precipitated product was dispersed within an

organic solvent. In practice, this idea provides an important insight

into the fabrication of composite nanofiber 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

⇑ 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: [email protected] (H.-W. Kim).

Acta Biomaterialia 7 (2011) 1609–1617

Contents lists available at ScienceDirect

Acta Biomaterialia

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a c t a b i o m a t

http://dx.doi.org/10.1016/j.actbio.2010.12.003mailto:[email protected]://dx.doi.org/10.1016/j.actbio.2010.12.003http://www.sciencedirect.com/science/journal/17427061http://www.elsevier.com/locate/actabiomathttp://www.elsevier.com/locate/actabiomathttp://www.sciencedirect.com/science/journal/17427061http://dx.doi.org/10.1016/j.actbio.2010.12.003mailto:[email protected]://dx.doi.org/10.1016/j.actbio.2010.12.003


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stiff nature of the product and premature dissolution must be over-

come to obtain a scaffold suitable for cell cultivation and hard tis-

sue formation. Within this context we utilized the mechanical

benefits of a synthetic polymer, poly(lactide–co-caprolactone)

(PLCL), within a bone-mimicking gelatin–apatite system to pro-

duce a functional composite membrane composed of gelatin–apa-

tite–PLCL. This composite nanofibrous membrane is also

considered to provide better biological properties than PLCL bio-

polymer nanofiber. Herein, fabrication methods to produce com-

posite 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 nanofibers

The gelatin–apatite solution was prepared through a precipita-

tion reaction of Ca(NO3)24H2O and (NH3)2HPO4 within gelatin

(Type B, bovine skin), as described previously [17]. Briefly, two sep-

arate solutions of calcium-containing gelatin (Ca-gelatin) and phos-

phate-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 main-

tained 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 byprod-

ucts, after which it was again freeze-dried. The freeze-dried sample

was then dispersed in trifluoroethanol (TFE) (Aldrich) at 15% w/v

with ultrasonic vibration for a few minutes and then stirred vigor-

ously for 24 h. Next, PLCL (Boelinger Ingelheim) dissolved in TFE at

15 wt.% was mixed with the gelatin–apatite solution at two differ-

ent mixing ratios (gelatin–apatite:PLCL = 1:4 (high) or 1:6 (low)).

Each solution was then loaded into a syringe and injected onto amandrel 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 h–1.

2.2. Characterizations

The morphology of the electrospun nanofibers was evaluated by

scanning electron microscopy (SEM), and the fiber diameter was

measured from the images. The phase of the apatite generated

within the gelatin matrix was confirmed by X-ray diffraction

(XRD). Transmission electron microscopy (TEM) was used to deter-

mine the presence and distribution of apatite nanocrystallines

within the nanofiber. The tensile mechanical properties of the

nanofibrous membrane were measured using an Instron 3344.

Membranes were prepared with a thickness of

150–200 lmand then cut to a size of 30 4 mm (gauge length 10 mm), afterwhich a tensile load was applied. Stress–strain curves were re-

corded and the maximum tensile stress, elastic modulus and elon-

gation at failure were determined. The thickness of each

membrane was determined from the average value observed in

the SEM images and a total of five samples were tested for each

group. The water affinity of the nanofiber membrane was exam-

ined by measuring the water contact angle (contact angle analyzer

Phoenix300). Data were recorded for up to 1 h and five samples

were tested for each group.

2.3. In vitro osteoblast responses

The in vitro biocompatibility of the composite nanofibers wasobserved using pre-osteoblast cells (MC3T3-E1, ATCC). The cells

were sub-cultured in culture medium, consisting of a-minimalessential medium supplemented with 10% fetal bovine serum

(FBS), 2 mM L-glutamine, 50 IU ml–1 penicillin and 50lg ml–1

streptomycin. Electrospun nanofibrous webs (pure PLCL as a con-

trol 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 ml–1) 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 fixed, dehydrated in a graded series of ethanol, trea-

ted with hexamethyldisilazane and coated with gold, after which

the cell morphology was observed by SEM. Osteoblastic differenti-

ation of the cells was then observed by measuring the production

of alkaline phosphatase (ALP). Cells cultured on each nanofibrous

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). Significance was considered at

P < 0.05 and P < 0.01.

2.4. In vivo implantation in rat calvarium

Six 10-week-old male Sprague–Dawley 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 kg–1) and xylazine (10 mg kg–1).

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 typesof membranes or kept empty as a negative control. The subcutane-

ous tissue was closed and the skin incisions sutured.

The animals were sacraficed 6 weeks after implantation. The

skin was removed and the samples and the surrounding tissues

were withdrawn en bloc and fixed in 10% neutral buffered formalin

solution for 24 h at room temperature and prepared for micro-

computed 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 re-

gion. The harvested specimens were scanned, with each frame ex-

posed 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 withinthe 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 three-

dimensional (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 fixed samples were decalci-

fied, dehydrated and embedded in paraffin, then serially sectioned

with a microtome (LEICA) at 4–5 lm thickness and finallymounted on microscope slides. Slides with tissue sections were

deparaffinized and dehydrated with xylene and an ethanol series.

The slides were stained with hematoxylin and eosin (H&E) andMasson’s trichrome (MT) and examined using a light microscope.

1610 S.-H. Jegal et al. / Acta Biomaterialia 7 (2011) 1609–1617


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3. Results and discussion

3.1. Generation of composite nanofibers

Fig. 1 shows a schematic illustration of the experimental steps

used to fabricate the functional composite membrane made of gel-

atin–apatite–PLCL. Although synthetic polymer nanofibers such as

PLCL are good candidates for tissue regeneration, the addition of agelatin–apatite composite improves the biocompatibility with

bone tissue. In addition, the apatite inorganic phase can stimulate

osteogenic differentiation and calcification when combined with

biopolymers [16–18,28]. Moreover, calcium phosphate inorganics

are known to be highly effective in reducing the problems associ-

ated with the acidic products formed during polymer degradation,

such as a decrease in pH and inflammation [29,30]. Additionally,

because the major weaknesses of synthetic polymers are hydro-

phobicity and poor cell affinity, 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 first synthesized a gelatin–apatite precipitate and

Fig. 1. Schematic showing the experimental steps used to fabricate the gelatin–

apatite–PLCL composite nanofiber by electrospinning.

Fig. 2. (a–c) SEMmorphologyof theelectrospunnanofibrous sheets: (a, b) PLCL containing gelatin–apatiteprecipitateat (a)low (1/6) and(b) high (1/4) concentration, and(c)

pure PLCL. (d) TEM morphology of the composite nanofiber in (a) showing the precipitated apatite nanocrystallites dispersed in the polymeric matrix. (e) XRD analysis toconfirm the phase development of apatite formed in the presence of gelatin matrix (s, hydroxyapatite).

S.-H. Jegal et al. / Acta Biomaterialia 7 (2011) 1609–1617 1611


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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 im-

proved mixing. The results of our previous study revealed that ami-

no acid groups in gelatin facilitated the spatial and homogeneous

nucleation of apatite, which resulted in more uniform and finer

sized nanocrystallites [17]. As a result, the electrospun nanofibers

produced from the precipitates had a better fibrous morphology

than those made by the direct mixing approach [17]. In practice,

during the electrospinning of inorganic–organic composites it is

important to utilize a solution with the appropriate mixing proper-

ties to ensure the production of uniform and bead-free nanofibers

[5].

Herein, addition of the gelatin–apatite 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 re-

spect to the PLCL–gelatin polymer phase.

3.2. Morphology and mechanical properties of membranes

The morphology of the generated gelatin–apatite–PLCL com-

posite nanofibers is shown in Fig. 2. At both concentrations of gel-

atin–apatite spinning into a fiber was possible under the adjusted

conditions. When a low concentration of gelatin–apatite was used

(14.3 wt.%, Fig. 2a) a well-developed non-woven fibrous web with

fibers a few hundreds of nanometers in size were produced. The

electrospun fiber was continuous with no discrete beads, but some

regions appeared to be heterogeneous with a slightly thicker diam-

eter. The hydrophilic gelatin–apatite may become segregated to

some extent within the PLCL matrix during the electric field-driven

process. When a high concentration (20 wt.%) of gelatin–apatite

was used some discrete beads were noticed and the fiber size be-

came relatively non-uniform (Fig. 2b). Compared with the pure

PLCL nanofibers (Fig. 2c) the composite nanofibers had relatively

smaller diameters (average fiber size 310 ± 103 nm for the high

concentration and 291 ± 51 nm for the low concentration of gela-

tin–apatite vs. 517 ± 232 nm for PLCL), moreover, some microfibers

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 PLCL–gelatin matrix, and there appeared to be no sign of phase

separation between the gelatin and PLCL. The apatite phase pro-

duced in the presence of the gelatin matrix was confirmed by

XRD (Fig. 2e). The homogeneity of the gelatin–apatite precipitate

was shown to be well preserved within the PLCL matrix. Thus,

the addition of gelatin–apatite to PLCL is an effective method of

obtaining nanofibers with well-homogenized inorganic–organic

components within the biopolymer matrix.

The mechanical properties of the composite nanofibers are

compared with those of pure PLCL nanofiber in Fig. 3. The stress–

strain curves of the nanofiber membranes were recorded on five

individual samples, and a characteristic curve for each composition

is shown in Fig. 3a. All nanofibrous 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 nanofibrous composite membranes and pure PLCL for comparison: (a) representative stress–strain 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 compositenanofibers was significantly different from that in pure PLCL (⁄P < 0.05, by ANOVA).



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and the percentage elongation (strain) at failure were obtained

from the stress–strain curves, as shown in Fig. 3b–d. The composite

membrane with a low gelatin–PLCL content showed the highest

strength (mean 10.1 MPa), which was almost double that of the

PLCL nanofiber (mean 5.7 MPa). However, the strength of the com-

posite with a high gelatin–apatite content was slightly lower

(mean 4.3 MPa) than that of pure PLCL. The elastic modulus of

the nanofibers was also measured (Fig. 3c). The addition of gela-

tin–apatite dramatically increased the elastic modulus of purePLCL, and the increase was more significant with addition of the

low concentration of gelatin–apatite (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 nano-

fiber was as high as 330%, the addition of gelatin–apatite de-

creased this value in a concentration-dependent manner, as

indicated by elongation values of 230% and 90% for nanofibers

that contained low and high concentrations of gelatin–apatite,

respectively. In our previous study of a gelatin–apatite nanofiber

system gelatin composite fibers containing 20 and 40% apatite

had tensile strengths and elongations of approximately 4–5 MPa

and 4–7%, respectively, which are significantly lower than the val-

ues obtained for the gelatin–apatite–PLCL composite fiber [17]. The

addition of low concentration of gelatin–apatite significantly en-hanced 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 en-

ough that the addition of gelatin–apatite 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 inor-

ganic particles are fine and evenly dispersed [35]. In biological sys-tems such as bone the apatite nanocrystallites embedded in the

collagen fibers strengthen and harden the bone tissue [36]. In the

present study the addition of a small amount of apatite–gelatin

was found to be highly effective in improving the mechanical

strength of the PLCL nanofibers. This was probably because the

ultrafine apatite crystallites were evenly dispersed in the nanofi-

bers, which should have enabled the polymer to resist extension

in response to an applied load. However, the addition of a high con-

centration of apatite–gelatin was found to decrease the strength of

the PLCL. This was believed to be due to agglomeration of the apa-

tite nanocrystallites, which was revealed as the presence of some

large beads on the nanofibers (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 inorganicphase was noticed, as determined from the initial slope of the

Fig. 4. Osteoblastic cell responses to the composite nanofibrous membranes: (a) cell growth morphology on the composite containing a low level of gelatin–apatite at 3 and7 days, (b)cell proliferation level for up to 7 days, as determined by MTS, and(c) ALPosteogenic differentiationon thenanofibers at days 7 and14. A significant difference was

noticed on thecomposite nanofiber with respect to PLCL (⁄P < 0.05 and ⁄⁄P < 0.01, by ANOVA, n =3). A significant increase in ALPactivity wasobserved on the composite with

a low content of gelatin–apatite with respect to culturing time ( ++P < 0.01, 7 vs. 14 days).



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curve. Conversely, the composite nanofibers with low gelatin–apa-

tite content showed a greater elongation, almost comparable with

that of pure PLCL. Apart from a strengthening effect, the mainte-

nance a high degree of flexibility should favor the use of this com-

posite as a tissue regeneration membrane or a scaffold for cell

growth.

The affinity to water of the composite membranes was shown

to be significantly higher than that of PLCL. The initial water con-

tact angle to the nanofiber 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

nanofiber matrix within 1 (high gelatin–apatite content) and

5 min (low gelatin–apatite content). However, no such water per-

meation was observed when the pure PLCL nanofiber was evalu-ated, even after 1 h. This high water affinity of the PLCL

nanofibers containing gelatin–apatite should allow effective and

rapid fluid 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 gelatin–apatite within the PLCL nanof-

ibers was addressed in terms of cell growth and osteogenic devel-

opment in vitro. MC3T3-E1 murine-derived pre-osteoblast cells

were cultured on the nanofibrous membranes and cell prolifera-

tion of and ALP activity in the cells were then examined. Fig. 4a

shows the morphology of cells grown on the composite (low con-

tent gelatin–apatite) and PLCL nanofibrous substrates. On the com-posite nanofibers the cells were very viable initially (at day 3) with

good cytoplasmic extensions, and the cells almost reached conflu-

ence, 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

nanofibers was also measured by MTS assay (Fig. 4b). At day 3 cell

proliferation was significantly higher on the composite membranes

than on the pure PLCL (P < 0.01). No significant difference was ob-

served between the two composite nanofibers. 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) re-

lease from the apatite component within the nanofiber can alter

cell behavior, such as cell proliferation and osteoblastic differenti-ation [37,38].

It is important to note that the ALP level was significantly great-

er on the composite nanofibers (Fig. 4c). ALP enzymatic activity

produced by cells on the nanofiber membranes was measured dur-

ing culture for 7 and 14 days. The ALP activity of cells grown on the

composite nanofibers was significantly higher than that of cells

grown on pure PLCL at day 7, while no significant difference was

observed between the two composite nanofibers. At this point

the cells had reached confluence and formed a thick layer, there-

fore, 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 stimu-

lation on the composite with a low content of gelatin–apatite was

significant (almost double), while only a slight increase was no-ticed on the other membranes. As a result, the ALP level in cells

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 nanofiber, and (c)

composite nanofiber. To the right of the image of the surgical operation is a 2-D reconstructed micro-CT image including the 5 mm initial defectzone 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 significant difference was noticed on the PLCL and composite nanofibers with respect to the blank control ( ⁄P < 0.05 and ⁄⁄P < 0.01, by ANOVA).



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on the composite with a low content of gelatin–apatite was signif-

icantly higher (by a factor of 2) than that in other membranes.

The apatite dispersed within the PLCL–gelatin matrix in the nano-

fiber should enhance osteogenic differentiation, particularly during

culture for 7–14 days. It is also believed that apatite synthesized inthe 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 produc-

tion [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 leadto increases in the overall processes, including cell division and

Fig. 6. Hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining of the tissues formed within the defect with the help of the nanofibrous membranes: (a) H&E

stain, PLCL nanofiber, (b) H&E stain, composite nanofiber, (c) MT stain, PLCL nanofiber and (d) MT stain, composite nanofiber. Defect margins are indicated by arrows. (e)

Enlarged image of inset in (d) showing the formation of bony tissue. Defect closure was significantly 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: (a–d) 500lm; (e) 30 lm.



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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 nanofibers. Although the nanofibers containing

the high concentration of gelatin–apatite 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 de-

fect 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 mem-

brane groups: PLCL nanofiber (Fig. 5b) and composite nanofiber

(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. How-

ever, when the PLCL or composite nanofibrous membrane was im-

planted there was considerable in-growth of hard tissue. The 3-D

images show more clearly the bone in-growth and the effective-ness of the membranes. As summarized in Fig. 5d, hard tissue for-

mation was better in the order composite membrane > PLCL

membrane >> blank control.

The newly formed tissues within the calvarium defect were fur-

ther analyzed by histological staining, as shown in Fig. 6. H&E

staining was first carried out to reveal cell or/and tissue in-growth

within the defect region (Fig. 6a and b). In both the PLCL nanofiber

(Fig. 6a) and composite nanofiber membranes (Fig. 6b) the defect

site was observed to be almost completely filled with dense con-

nective tissue. The defect closure measured from the histological

images was in the order composite nanofiber (64.7 ± 3.6) > PLCL

nanofiber (57.7 ± 1.6) > blank control (40.4 ± 5.1), with significant

differences between the groups (P < 0.01, one-way ANOVA, n = 4).

After Masson’s trichrome staining the formation of bony tissue

was more clearly revealed (Fig. 6c and d). Compared with the PLCL

nanofiber (Fig. 6c), the composite nanofiber (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 for-

mation originating from the defect margins (not shown here), sup-

porting the micro-CT data. A higher magnification 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 nanofibers could find practical application in orthope-

dics and dentistry, such as guided bone regeneration membranes

in periodontal pockets.

4. Conclusions

Composite nanofibers made of gelatin–apatite–PLCL were pro-

duced by electrospinning. A precipitate of gelatin–apatite was

added to the PLCL in TFE solvent to prepare a homogeneous precur-

sor solution. At a low concentration of gelatin–apatite (14.3 wt.%) a

bead-free, non-woven nanofibrous web was produced, while a

considerable number of beads were noticed with a high concentra-

tion of gelatin–apatite (20 wt.%). Apatite nanocrystallites were ob-served to be well distributed within the gelatin–PLCL organic

matrix. Moreover, the addition of a small amount of gelatin–apa-

tite led to fibers with a significantly improved tensile strength

(nearly double) when compared with those composed of pure

PLCL, without considerable loss in flexibility. Additionally, cell pro-

liferation and osteogenic development were significantly higher on

the composite nanofibers than on the pure PLCL nanofiber. When

the composite nanofiber membrane was implanted in a rat calvar-

ium defect new bone formation and defect closure were signifi-

cantly enhanced with respect to pure PLCL or a negative control.

Overall, the results demonstrate that the gelatin–apatite–PLCL

nanofibrous matrix developed here could potentially be useful in

the regeneration of hard tissues, such as a guided bone regenera-

tion membrane in periodontology.

Acknowledgements

This work was supported by the Priority Research Centers Pro-

gram (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 figures in this article, particularly Figures 3, 5, and 6, are

difficult 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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