FULL PAPER

Nanofiber Generation of GelatinHydroxyapatite Biomimetics

for Guided Tissue Regeneration**
By Hae-Won Kim,* Ju-Ha Song, and Hyoun-Ee Kim

The development of biomimetic bone matrices is one of the major goals in the bone-regeneration and tissue-engineering fields.
Nanocomposites consisting of a natural polymer and hydroxyapatite (HA) nanocrystals, which mimic the human bone matrix,
are thus regarded as promising bone regenerative materials. Herein, we developed a biomimetic nanocomposite with a
novel nanofibrous structure by employing an electrospinning (ES) method. The HA precipitate/gelatin matrix nanocomposites
are lyophilized and dissolved in an organic solvent, and then electrospun under controlled conditions. With this process, we can
successfully generate a continuous fiber with a diameter of the order of hundreds of nanometers. The internal structure of the
nanofiber features a typical nanocomposite, i.e., HA nanocrystals well distributed within a gelatin matrix. These nanocomposite
fibers improve the bone-derived cellular activity significantly when compared to the pure gelatin equivalent. This method of
generating a nanofiber of the biomimetic nanocomposite was effective in producing a biomedical membrane with a composi-
tion gradient, which is potentially applicable in the field of guided tissue regeneration (GTR).

1. Introduction biomineral phase. This problem is even more challenging when

The development of materials by mimicking the structure
and composition of human tissues, namely the biomimetic ap-
proach, has long been a major goal in the field of biomaterials
and tissue engineering. In bone-tissue regeneration, gaining an
insight into the hierarchical structure of the bone matrix, i.e.,
the nanocomposite consisting of mineralized hydroxyapatite
(HA) nanocrystals and collageneous fibers, has caused a con-
siderable amount of research to be directed toward the synthe-
sis of bio-inspired and biomimetic systems.[1,2] Particularly, with
increasing knowledge of the bone mineralization process oc-
curring in vivo, numerous in-vitro studies have been devoted to
the precipitation of HA nanocrystals within natural collage-
nous proteins or synthetic polymeric templates.[37] These bio-
mimetic nanocomposite matrices have been shown to improve
the bone-cell responses in vitro and the bone-forming ability in
vivo, in comparison with natural polymers alone, and are thus
regarded as a promising bone regenerative.[810]
Although the decisive role of the HA mineral phase in the
bone-forming process is well established, the utilization of HA
within the biopolymer matrix has remained limited. Unlike
biological polymers, which are relatively easy to process and
modify, it is difficult to produce well-defined structures, such as
spheres, films or three-dimensional (3D) scaffolds and fibers,
for specific applications in nanocomposites containing an HA
[*] Prof. H.-W. Kim,[+] J.-H. Song, Prof. H.-E. Kim
School of Materials Science and Engineering, Seoul National University
Seoul, 151742 (Korea)
E-mail: hwkim@snu.ac.kr
[+] Present Address: Department of Dental Biomaterials, College of
Dentistry, Dankook University, Cheonan, 330-714, Seoul, Korea.
[**] This work was supported by grants from the Korea Health 21 R&D Pro-
ject of the Ministry of Health and Welfare (02-PJ3-PG6-EV110002)
and from the Korea Research Foundation (R082004000101920).
dealing with the nanoscale formulation of the nanocomposite,
for example, nanoparticles, nanospheres, nanotubes, and nano-
fibers. Notwithstanding, the nanoscale exploitation of these
biomimetic nanocomposites should increase their regenerative
potential through the stimulation of protein mediation and cell
adhesion, and the subsequent matrix synthesis.[8]
Herein, we attempted to develop a biomimetic nanocomposite
with a one-dimensional nanofiber structure and, ultimately, to uti-
lize this nanofiber as a 3D biomedical membrane for guided tis-
sue regeneration (GTR). Gelatin was chosen as the natural poly-
mer matrix for the mineralization of the HA. Gelatin, which is
derived from collagen, has been widely used in clinics for wound
dressings, pharmaceuticals, and adhesives, because of its bioaffin-
ity, lack of antigenicity, and hydrogel characteristics, as well as its
formability and cost efficiency.[11] We first obtained the gelatin
HA nanocomposite by the biomimetic route and then generated
a nanofibrous structure by using the electrospinning (ES) process.
The ES technique has been utilized to produce nanoscale fibers
in numerous fields, including tissue engineering.[12,13] Numerous
polymers (natural or synthetic) have already been electrospun in
the form of nanoscale fibers for biomedical uses. However, little
is known about the ES of bioceramic-organized polymer nano-
composites and, to the best of our knowledge, the approach intro-
duced in this study is the first report on the generation of a bio-
mimetic nanocomposite fiber utilizing the ES process. Finally, we
highlighted the benefits of the process described herein for the
production of a functional biomedical membrane, which was de-
signed to have an HA/gelatin composition gradient, and, thus, to
guide bone-tissue regeneration more effectively.
2. Results and Discussion
The experimental scheme used in this study is described
in Figure 1. The materials produced at each step are shown

1988  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/adfm.200500116 Adv. Funct. Mater. 2005, 15, 19881994
H.-W. Kim et al./GelatinHydroxyapatite Nanofibers for Tissue Regeneration

FULL PAPER
( HA precipitation ) ( Freeze-Drying ) ( Nanocomposite solution ) ( Electrospinning ) ( Cross-linking )

Ca+Gelatin P+Gelatin HFP EDC/NHS

HA-Gelatin HA-Gelatin

(a) (b) (c) (d) (e)

Figure 1. Schematic illustration of the processes used to produce the gelatinHA nanocomposite fiber through the biomimetic precipitation and electro-
spinning methods. The material obtained in each process is shown at the bottom of the illustrations. The HA precipitate/gelatin matrix nanocomposite
(a) was lyophilized (b) and dissolved in an organic solvent HFP (c) for the electrospinning process. The electrospun fibers (d) were subsequently cross-
linked with EDC/NHS solution (e). EDC: 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, NHS: N-hydroxysuccinimide.

below the illustrations (the 20 % HA formulation is

shown as a representative example). The gelatinHA (a)
nanocomposite sol (Fig. 1a) prepared using the
Intensity (arb.unit)

biomimetic precipitation process was freeze-dried

(Fig. 1b). The dried nanocomposite was dissolved in gelatin
an organic solvent, 1,1,1,3,3,3-hexafluoro-2-propanol
(HFP) (Fig. 1c). Both the 20 and 40 % HA solutions
maintained their initial emulsion states for several
hours without any segregation occurring (Fig. 1c).
This emulsion state of the nanocomposite solution is 20%HA
of particular importance in terms of the ability of the
electrospinning process to produce a continuous and
uniform nanofiber. Using the nanocomposite emul-
(002)

(210)

(211)

(300)
(112)
(102)

(310)

(222)
(113)

(213)
(312)
sion in HFP, ES was conducted under controlled pro-
cessing conditions to generate a nanofiber (Fig. 1d). 10 20 30 40 50
Finally, the electrospun nanofiber was crosslinked to 2 (degrees)
ensure its chemical and structural stability (Fig. 1e).
The characteristics of the gelatinHA nano-
(b)
composite obtained are shown in Figure 2. Data on
Transmittance (arb.unit)

the 20 % HA formulation are presented as a represen- gelatin

tative example. The X-ray diffraction (XRD) analysis
of the nanocomposite revealed that only apatite peaks
(including gelatin at ~20) were observed (Fig. 2a).
The apatite phase was poorly crystallized, as mani- 20%HA
fested by the weak splitting of the peaks (particularly
between (211) and (112)). This was attributed to the
low crystallinity and ultrafine size of the crystals, as is
usually observed in the organic-mediated crystalliza-
tion of an apatite.[3,4] The Fourier-transform infrared
(FTIR) spectrum of the nanocomposite clearly
showed the chemical bands associated with the apa-
4000 3200 1600 1200 800 400
tite, particularly PO4. Moreover, the amide bands re-
-1
lated to gelatin observed in the nanocomposite were Wave number (cm )
similar to those observed in pure gelatin (noted by
dotted lines in Fig. 2b).[14] The phase and structural Figure 2. Characteristics of the gelatinHA biomimetic nanocomposite fibers: a) XRD
pattern and b) FTIR spectrum. Data on the pure gelatin fiber are shown as a refer-
data of the gelatinHA nanocomposite were analo- ence. In (a), the apatite peaks are denoted by the cross symbol, and the lattice planes
gous to those of the collagenHA biomimetic materi- are provided. In (b), the bands related to phosphate, particularly PO4, are denoted by
al, as well as to those of natural bone matrix.[35,14] the cross symbol.

Adv. Funct. Mater. 2005, 15, 19881994 www.afm-journal.de  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1989
 H.-W. Kim et al./GelatinHydroxyapatite Nanofibers for Tissue Regeneration
FULL PAPER
Scanning electron microscopy (SEM) images of the morphol-
ogy of the nanofiber generated from the gelatinHA nanocom-
posite solution in HFP are shown in Figure 3. A nanofiber was
also produced from the pure gelatin for comparison purposes
(Fig. 3a). The nanocomposite with 20 % HA was successfully
electrospun to a nanofiber possessing a uniform and continu-
ous fiber morphology (Fig. 3b). The fiber diameter was ap-
proximately 200400 nm. When the amount of HA was in-
creased to 40 %, a continuous nanofiber was also generated
with a similar diameter to that produced with 20 % HA
(Fig. 3c). Although some beads started to appear with this
composition, mostly nanoscale fibers were produced. However,
in the case of the 60 % HAgelatin nanocomposite, the produc-
tion of beads was more obvious, and the fiber was not continu-
ous (Fig. 3d). The threshold amount of HA in the nanocompos-
ite with gelatin for the production of a continuous nanofiber is
thus regarded as being approximately 40 %. The formation of
beads was attributed to the precipitated HA crystals that were
not distributed efficiently in the gelatin matrix and therefore
agglomerated in clusters. Although the gelatin, particularly the
carboxyl anionic groups of the amino acids, could act as an effi-
cient nucleation site for the HA nanocrystals at a low concen-
tration of Ca and P, increasing their concentration would ham-
per this role.[7] However, through the biomimetic reaction, the
HA nanocrystals could be distributed uniformly in the gelatin
matrix at the molecular level and, as a result, it was possible to
accomplish the ES of the nanocomposite with amounts of HA
(a) (b)
1 m
(c) (d)
1 m
Figure 3. Field-emission SEM (FESEM) morphology of the gelatinHA nanocomposite
electrospun fibers: a) pure gelatin, b) 20 % HA, c) 40 % HA, and d) 60 % HAgelatin. The
material concentration in HFP was varied from 10 to 16 % w/v depending on the composi-
tion. The gelatin60 % HA fiber was not continuous and contained a number of beads.
1990  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
as high as ~40 %. In contrast, when we attempted to directly
disperse HA nanopowder in a gelatin sol, the powder sedimen-
ted rapidly without maintaining the emulsion state in HFP (in
contrast to the nanocomposite solution shown in Fig. 1c), mak-
ing it impossible to electrospin the composite solution even at
10 % HA. However, even in the case of the nanocomposite fi-
ber, the diameter could not be reduced below ~100 nm because
of the distinctive size of the precipitated apatite nanocrystal-
lites (a few nanometers in width by several tens of nanometers
in length, as shown in the transmission electron microscopy
(TEM) image in Fig. 4), whilst the pure polymeric fibers in-
cluding gelatin have often been reported as being several tens
of nanometers in diameter under carefully controlled condi-
tions.[12,13]
The internal structure of the nanocomposite fiber was ana-
lyzed with TEM, as shown in Figure 4. A uniform and con-
tinuous nanofiber (20 % HA) was generated, as seen at low
magnification (Fig. 4a). The high-magnification image of the
nanocomposite fiber (20 % HA) clearly reveals the existence
of the precipitated nanocrystals with an elongated shape
(Fig. 4b). The selected area electron diffraction (SAED) pat-
tern of the crystals (inset in Fig. 4b) revealed a diffuse ring pat-
tern, which is characteristic of the typical apatite structure as
regards the specific crystallographic planes, (112) and (002). In
the 40 % HAgelatin nanocomposite fiber, the apatite nano-
crystals were observed more readily, and their size and shape
were similar to those of the 20 % HAgelatin nanocomposite
fiber (Fig. 4c). Apatite crystals grown in a bio-
mimetic process normally have an elongated
shape because of the mediation of the amino acid
backbone in the collagen-based organics, which
leads to their preferential growth along the c-axis
direction.[14,15] The energy dispersive spectrosco-
py (EDS) profile of the crystals in Figure 4d
revealed a high concentration of Ca and P, and
a Ca/P ratio of ~1.71 0.05, which is very close
to the HA stoichiometry (Ca10(PO4)6(OH)2) of
1.67.[15]
The mechanical properties of the nanocompos-
ite fibers prepared in sheet form (5 mm 50 mm
1 m
~50 lm) were evaluated by applying a tensile
load. The stressstrain (SS) curve of the samples
was monitored, and representative examples are
shown in Figure 5. The different types of nano-
fibrous matrices all showed a similar SS pattern,
with an initial linear elastic region, followed by a
region of steady-state stress and subsequent fail-
ure. Compared to pure gelatin, the HAgelatin
nanocomposite fiber exhibited a higher initial
slope and lower strain at failure. The mechanical
properties of the nanofibrous matrices are sum-
marized below the graph. It is of note that the
1 m
20 % HAgelatin nanocomposite had the highest
tensile strength (5.2 MPa), suggesting that the
HA precipitates in gelatin contributed to the me-
chanical strength. This was attributed to the HA
minerals integrating well with the gelatin by
www.afm-journal.de Adv. Funct. Mater. 2005, 15, 19881994
H.-W. Kim et al./GelatinHydroxyapatite Nanofibers for Tissue Regeneration
(a) (b)
(002)
(112)
300 nm 10 nm
(c) (d)
10 nm Energy (keV)
Figure 4. TEM analysis of the gelatinHA nanocomposite electrospun nanofiber: TEM im-
age of a,b) 20 % HA at low and high resolution, respectively, and of c) 40 % HA at high
resolution. Numerous elongated apatite nanocrystals were well developed (dark areas in
(b,c)). Inset in (b) shows the SAED pattern of the nanocrystals, which is characteristic of
a typical apatite crystal pattern (diffused rings were observed for the (112) and (002)
planes). d) EDS profile of the nanocrystals in (b), showing a Ca/P ratio (1.71 0.05) simi- posite fiber obtained in this study is considered to
lar to apatite stoichiometry (1.67).
adopting the efficient nanocomposite structure of inorganicor-
ganic natural systems including bone. Practically, in convention-
ally mixed HAgelatin, the HA particulates have often been
reported to act as the source of failure, consequently reducing
the mechanical strength of the system. The higher elastic modu-
lus and lower strain at failure of the nanocomposites can be ex-
plained by the fact that the HA minerals render the nanofiber
matrix more stiff and less plastic in its deformation, in a manner
which is typical of hard inorganic phases. It is concluded that
the nanocomposite fibers (with a HA content of up to 40 %)
exhibited a stressstrain pattern quite similar to that of pure
gelatin and preserved or even improved the tensile strength of
gelatin and significantly increased its elastic modulus, but re-
duced the strain at failure.
The biocompatibility of the nanocomposite fibers was as-
sessed by measuring their bone-derived cell (MG63) responses,
as shown in Figure 6. In order to obtain precise measurements,
nanofibrous meshes electrospun on a glass cover slip were
used as shown in Figure 6a. The electron micrograph of the cells
grown on the 20 % HAgelatin nanocomposite fiber for 3 days
Adv. Funct. Mater. 2005, 15, 19881994 www.afm-journal.de
FULL PAPER
is shown in Figure 6b as a representative result.
The cells were observed to grow favorably on the
nanocomposite fibrous mesh. On closer examina-
tion, the cytoskeletal extension of the cells
was clearly observed and the cell membrane
was found to be in intimate contact with the
nanofiber. The cell-growth morphologies in the
case of the 40 % HAgelatin and pure gelatin
nanofibers were similar (data not shown). To
evaluate the bone forming ability of the cells
grown on the nanofibers, we measured the alka-
line phosphatase (ALP) activity expressed by the
cells. Since ALP is regarded as an important
marker of bone-forming cells at a relatively early
stage, it has been extensively used to assess the
performance of biomaterials in the bone forma-
tion process.[16] Of particular note is the observa-
tion that the cells on the nanocomposite fibers
(both 20 and 40 % HA) expressed significantly
higher ALP levels than those on the pure gelatin
fiber. When comparing the nanocomposites, the
ALP level appeared to be slightly lower in the
40 % HAgelatin, and this was considered to be
partially attributable to the different morphology
of these nanofibers, i.e., the less-uniform and less-
well-developed fiber morphology in the case of
the 40 % HAgelatin, which was associated with
the generation of some beads. The enhanced
bone-cell responses of HA-containing polymers
as compared to those of the polymer individual
equivalents have been observed in other HA-con-
taining biocomposite systems.[810,17] In the same
manner, the precipitated HAgelatin nanocom-
be more promising than the equivalent pure poly-
meric matrix in the bone regeneration field.
For the specific utilization of the gelatinHA
nanocomposite fiber and the methodology developed herein,
we produced a functional biomedical membrane, as shown in
Figure 7. A membrane with an internal nanofibrous structure
and a composition gradient is considered to guide tissue regen-
eration more effectively. Because the bone matrix is intimately
associated with different types of soft connective tissues, mus-
cles and nerves, a membrane structure with a composition gra-
dient is considered to be the optimum design for guiding tissue
regeneration so that it proceeds more effectively and specifical-
ly. One example of this is the periodontal membrane used in
dental surgery, which being placed in the defect region com-
prising the periodontium (soft tissue) and the alveolar bone
(hard tissue), acts initially as a defect coverage by preserving
the stability of autologous or synthetic bone grafts and ulti-
mately guides the specific cell migration and promotes the re-
generative potential of specific tissues.[18] Therefore, when the
membrane is designed to have a gelatin/HA composition gradi-
ent from one side to the other (as depicted in the image on the
left-hand side of Figure 7), its regenerative potential for both
the soft and bony tissues can be optimized by stimulating the
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1991
 H.-W. Kim et al./GelatinHydroxyapatite Nanofibers for Tissue Regeneration
FULL PAPER

the gelatin and 20 % HAgelatin solutions in a simple manner

6 20%HA (Fig. 7, right-hand side). Practically, the successful exploitation
of a biological membrane requires the precise design and con-
5 40%HA Gelatin trol of its structural and compositional parameters. In particu-
lar, consideration based on the gradient layer concept should
Tensile stress (MPa)

4 be envisaged with regard to the pore structure, in order to de-

termine whether the system requires a more dense structure on
3 one side to suppress the ingrowth of selective tissues or else re-
quires more open spaced pores to induce improved vasculari-
2 zation and cellular ingrowth. This aspect, associated with the
designing of the layered structure appropriate to specific appli-
1 cations, remains a future area of research.
Based on the findings described above, the nanocomposite
0 ES approach has the advantage of allowing the layers to be re-
0 1 2 3 4 5 6 7 8 9 alized in such a way as to produce a composition gradient in
Strain (%) one membrane structure simply by changing the composition
of the solution. Moreover, a given spatial area can be locally
Materials Gelatin 20%HA 40%HA electrospun with the appropriate composition. Currently, al-
Ultimate strength (MPa) 4.6 (1.4) 5.2 (1.7) 4.4 (2.4) though the uniform generation of a nanofiber at a higher HA
Elastic modulus (MPa) 204 (52) 326 (88) 412 (120) concentration (over 40 %) still remains as a challenge, this
Strain at failure (%) 7.6 (2.1) 6.5 (3.3) 4.3 (1.9) nanocomposite ES strategy is believed to overcome the limita-
tions associated with the other techniques, such as that in which
Figure 5. Representative stressstrain curves of the nanofibrous matrices the gelatin fiber is normally generated first followed by its
in response to an applied tensile load. Below the graph are summarized
the mechanical properties (tensile strength, elastic modulus, and strain at post-treatment with HA crystals. Furthermore, other biomi-
failure) obtained from the curves (mean 1 std). metic nanocomposite systems are expected to be produced
based on the methodology developed in this study.

progenitor cells to be differentiated into specific types of cells

and thereby inducing further matrix production and wound 3. Conclusions
healing. Herein, this concept was partially validated by sand-
wiching the pure gelatin and 20 % HAgelatin nanofibers Biomimetic nanocomposites of gelatinHA were successfully
together, which was achieved by sequentially electrospinning converted into a nanofibrous structure by means of the electro-

(a) (b)

gelatin 20%HA 40%HA

(c) 0.6
P < 0.01

10 m
ALP activity (mol/mg/h)

P < 0.001

0.4

0.2

0.0
500 nm
Gelatin 20%HA 40%HA

Figure 6. Osteoblastic cellular responses to the gelatinHA nanocomposite electrospun nanofibers: a) samples electrospun on a glass coverslip; b) elec-
tron micrograph of the MG63 cells grown on 20 % HA nanofiber after 3 days of culturing (at low (upper) and high resolution (lower)); c) ALP activity of
the cells after 7 days of culturing. Data on pure gelatin fiber are included for comparison. The cell-seeding density was 1 104 mL1. The data are repre-
sented as mean 1 std for n= 5, and the data comparison was carried out using ANOVA one-way analysis.

1992  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.afm-journal.de Adv. Funct. Mater. 2005, 15, 19881994
H.-W. Kim et al./GelatinHydroxyapatite Nanofibers for Tissue Regeneration
Hard tissue
Compositional
gradient
(HA / Gelatin)
Soft tissue
Figure 7. Schematic illustration of the design of the functional biomedical membrane with a composition gradient between the gelatin and HA, for use in
guided hard/soft tissue regeneration. On the right is the layered membrane (gelatin/20 % HAgelatin) successfully produced by the electrospinning of
both solutions.
spinning technique. The precipitated HA nanocrystalgelatin
sol was lyophilized and dissolved in an organic solvent, in order
to generate a continuous nanofiber with a diameter of the or-
der of hundreds of nanometers. The HA ultrafine nanocrystals
were observed to be homogeneously distributed within the
gelatin matrix. The nanocomposite fibrous mesh significantly
improved the osteoblastic cellular activity in comparison with
the pure gelatin equivalent. The electrospun nanofibrous mem-
brane based on the gelatinHA nanocomposite solution is be-
lieved to be potentially applicable in the field of guided tissue
regeneration.
4. Experimental
GelatinHA nanocomposite sols were prepared from gelatin
(Type B, bovine skin, Aldrich), calcium nitrate [Ca(NO3)24H2O, Al-
drich], and ammonium hydrogen phosphate [(NH3)2HPO4, Aldrich].
The Ca and P precursors were dissolved in distilled water separately by
stirring for 1 h, at a ratio of Ca/P= 1.67. The gelatin powders were
added to each solution and stirred for an additional 12 h while kept at
40 C. The amounts of gelatin with respect to Ca and P were adjusted
so as to obtain the nanocomposite compositions: HA/(gelatin
HA) = 20, 40 and 60 wt.-%. The pH of both the Cagelatin and Pgela-
tin sols was adjusted to a value of approximately 10 by adding ammoni-
um hydroxide (NH4OH, BDH chemicals). The Pgelatin sol was added
to the Cagelatin sol dropwise while keeping the pH constant and
stirred at 40 C for 24 h to allow it to mature. For the purpose of com-
parison, conventional gelatinHA composites were also prepared by
directly mixing the gelatin and HA powders (Alfa-Aesar Co.) in dis-
tilled water at 40 C with stirring for 24 h. The nanocomposite sols were
frozen at 70 C for 24 h, followed by freeze-drying for 48 h under vac-
Adv. Funct. Mater. 2005, 15, 19881994 www.afm-journal.de
FULL PAPER
5 m
Gelatin
20%HA
20 m
5 m
uum. The dried samples were washed profusely with distilled water
to remove any salt by-products, and then freeze-dried again. The
dried samples were subjected to thermogravimetirc analysis (TGA,
Seteram), in order to observe the amount of minerals precipitated
in the gelatin matrix, where the mineral phase was considered to
maintain the initial weight during the thermal treatment at 900 C. The
amounts of mineral phase remaining were ~16, 35, and 53 wt.-% for
the solution stoichiometries of 20, 40, and 60 wt.-% of HA with
respect to gelatinHA, respectively. Throughout this study, we denoted
the nanocomposites as 20, 40, and 60 % HA for the sake of conveni-
ence.
The freeze-dried samples were dissolved in HFP (Aldrich) at 10
16 % w/v, depending on the composition (the higher the material load-
ing, the higher the HA amount), by stirring for 24 h, to prepare precur-
sors for electrospinning. The state of the HFP-dissolved samples was
examined, in order to determine whether any sedimentation or fluctua-
tion occurred. The solution was loaded into a syringe (with a capacity
of 5 mL and a needle diameter of 500 lm) and injected into a rotating
mandrel under a high direct current field strength (12 kV/8 cm) at an
injection rate of 0.05 mL h1. In particular, a layered membrane struc-
ture consisting of pure gelatin and gelatin20 %HA was fabricated by
electrospinning the gelatin solution for 5 h and subsequently the 20 %
HA solution for an additional 5 h. The electrospun nanofibers were
crosslinked using an EDC/NHS agent based on the experimentally de-
termined conditions: 100 mM EDC and 100 mM NHS in 95 % ethanol
for 4 h at 4 C .
The phase and chemical bonding structure of the nanocomposite
fiber were analyzed with X-ray diffraction (XRD; M18XHF-SRA, Mac
Science Co.) and Fourier-transform infrared (FTIR; System 2000, Per-
kinElmer) spectroscopy, respectively. The morphology of the nanofi-
ber was characterized with field-emission scanning electron spectrosco-
py (FESEM; JSM6330F, JEOL) and transmission electron microscopy
(TEM; CM20, Philips). The internal structure of the nanocomposite
fiber was examined with high-resolution TEM (JEM 3000F, JEOL).
The composition and crystal pattern of the precipitated nanocrystal
were analyzed from the TEM measurements.
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1993
 H.-W. Kim et al./GelatinHydroxyapatite Nanofibers for Tissue Regeneration
FULL PAPER
For the mechanical tests, nanofibrous matrices prepared in a thin
rectangular form (5 mm 50 mm ~50 lm) were mounted on mechan-
ical gripping units leaving a gauge length of 30 mm. The tensile proper-
ties of the specimens were evaluated using an Instron (model 4505) at a
cross-head speed of 10 mm min1. The load-deformation data recorded
were converted to stressstrain curves. Based on these curves, the ten-
sile strength (maximum stress until failure), elastic modulus (slope
within the initial elastic region), and strain at failure were obtained.
Eight to ten specimens were tested for each condition.
For the in-vitro biocompatibility test of the nanocomposite fiber, hu-
man osteoblastic cell responses were observed, as described previously
[8]. MG63 cells were sub-cultured in a culture medium consisting of
Dulbecco's modified Eagle medium (DMEM) supplemented with
10 % fetal calf serum (FCS), 2 mM L-glutamine, 50 IU mL1 of penicil-
lin, and 50 lg mL1 of streptomycin. The nanofibrous meshes for the
cell tests were prepared on a glass coverslip. The samples were steril-
ized with ethanol (70 % in distilled water) for 30 min. The cells were
plated at a density of 1 104 mL1 on all specimens in individual wells
of a 12-well plate and cultured in an incubator kept at 37 C. The cells
were observed to reach confluence after approximately 67 days of cul-
turing in all cases. The morphology of the cells grown on each sample
for 3 days was observed by SEM at an accelerating voltage of 5 kV,
after fixing, dehydrating, and gold coating the cells.
The ALP level produced by the cells was measured, in order to ob-
serve the functional activity of the cells cultured on the samples [8].
After culturing for 7 days, the cells were washed with PBS and de-
tached with trypsin/EDTA (EDTA: ethylenediaminetetraacetate).
After centrifugation, the cell pellets were resuspended by vortexing
them with 0.1 % Triton X-100. The cell pellets were disrupted via a cy-
clic freezing/thawing process to prepare cell lysates, which were kept
for the determination of the ALP activity. Prior to the ALP assay, each
sample was normalized to the total protein content, which was mea-
sured using a commercial DC protein assay kit (BioRad, Hercules).
The ALP activity was measured colorimetrically using p-nitrophenyl
phosphate as the substrate. The enzyme ALP, expressed by the cells,
hydrolyzes the substrate to p-nitrophenol and an inorganic phosphate.
Under alkaline conditions, the p-nitrophenol was converted to a yellow
______________________
1994  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.afm-journal.de
product and its absorbance was subsequently measured at 410 nm
using a spectrophotometer. The absorbance was directly converted to
ALP activity level based on a protein standard curve, which was ob-
tained with bovine serum albumin in the range of 0.21.2 mg mL1.
Received: May 30, 2005
Final version: July 28, 2005
Published online: October 31, 2005
[1] S. Weiner, H. D. Wagner, Annu. Rev. Mater. Sci. 1998, 28, 271.
[2] W. F. Neuman, M. W. Neuman, The Chemical Dynamics of Bone
Mineral, University of Chicago Press, Chicago 1958.
[3] S. Weiner, L. Addadi, J. Mater. Chem. 1997, 7, 689.
[4] J.-H. Bradt, M. Mertig, A. Teresiak, W. Pompe, Chem. Mater. 1999,
11, 2694.
[5] S. I. Stupp, P. V. Braun, Science 1997, 277, 1242.
[6] J. D. Hartgerink, E. Beniash, S. I. Stupp, Science 2001, 294, 1684.
[7] A. L. Boskey, J. Phys. Chem. 1989, 93, 1628.
[8] H.-W. Kim, H.-E. Kim, V. Salih, Biomaterials 2005, 26, 5221.
[9] G. Wei, P. X. Ma, Biomaterials 2004, 25, 4749.
[10] K. Yamauchi, T. Goda, N. Takeuchi, H. Einaga, T. Tanabe, Biomateri-
als 2004, 25, 5481.
[11] G. G. Word, A. Courts, The Science and Technology of Gelatin, Aca-
demic Press, London 1977.
[12] U. Dzenis, Science 2004, 304, 1917.
[13] L. A. Smith, P. X. Ma, Colloids Surf., B 2004, 39, 125.
[14] M. Kikuchi, T. Ikoma, S. Itoh, H. N. Matsumoto, Y. Koyama, K. Taka-
kuda, K. Shinomiya, J. Tanaka, Comp. Sci. Tech. 2004, 64, 819.
[15] R. Z. LeGeros, Prog. Cryst. Growth 1981, 4, 1.
[16] G. A. Rodan, S. B. Rodan, in Bone and Mineral Research (Ed: W. A.
Peck), Vol. 2, Elsevier, Amsterdam 1984, pp. 244285.
[17] H.-W. Kim, J. C. Knowles, H.-E. Kim, J. Biomed. Mater. Res. A. 2005,
72A, 136.
[18] T. V. Scantlebury, J Periodontol. 1993, 64, 1129.
Adv. Funct. Mater. 2005, 15, 19881994