Apatite-mineralized polycaprolactone nanofibrous web as a

bone tissue regeneration substrate

Hye-Sun Yu,1 Jun-Hyeog Jang,2 Tae-Il Kim,3 Hae-Hyoung Lee,1,4 Hae-Won Kim1,4
1
Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan, South Korea
2
Department of Biochemistry, College of Medicine, Inha University, Incheon, South Korea
3
Department of Periodontology, College of Dentistry, Seoul National University, Seoul, South Korea
4
Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan, South Korea

Received 14 May 2007; revised 9 August 2007; accepted 23 August 2007

Published online 20 March 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31709

Abstract: Degradable synthetic polymers with a nanofi-
brous structure have shown great promise in populating
and recruiting cells for the reconstruction of damaged tis-
sues. However, poor cell affinity and lack of bioactivity
have limited their potential usefulness in bone regeneration.
We produced polymeric nanofiber poly(e-caprolactone)
(PCL) with its surface mineralized with bone-like apatite for
use as bone regenerative and tissue engineering matrices.
PCL was first electrospun into a nanofibrous web, and the
surface was further mineralized with apatite following a se-
ries of solution treatments. The surface of the mineralized
PCL nanofiber was observed to be almost fully covered
with nanocrystalline apatites. Through mineralization, the
wettability of the nanofiber matrix was greatly improved.
Moreover, the murine-derived osteoblastic cells were
shown to attach and grow actively on the apatite-mineral-
ized nanofibrous substrate. In particular, the mineralized
PCL nanofibrous substrate significantly stimulated the
expression of bone-associated genes, including Runx2, col-
lagen type I, alkaline phosphatase, and osteocalcin, when
compared with the pure PCL nanofiber substrate without
mineralization. The currently developed polymer nanofi-
brous web with the bioactive mineralized surface is consid-
ered to be potentially useful as bone regenerative and tissue
engineering matrices. Ó 2008 Wiley Periodicals, Inc. J
Biomed Mater Res 88A: 747–754, 2009
Key words: nanofiber substrate; electrospinning; apatite
mineralization; bone regeneration matrix; osteoblast re-
sponses

INTRODUCTION and ultimately to recover the function of damaged

Nanofibrous matrices produced by the electrospin-
ning technique have attracted great interest in the
fields of tissue regeneration.1,2 Many polymeric com-
positions, either in synthetic or natural base, have
been developed into fibers with diameters ranging
from tens of nanometers to a few micrometers
through the electrospinning method.3–8 Because of
the intriguing characteristics of the electrospun prod-
ucts, such as the large surface area and the direc-
tional fibrous network, the electrospun polymeric
fibers have recently been studied to find a suitable
matrix for tissue engineering. The nanofibrous sub-
strates can effectively direct and populate tissue cells
and trigger them to secrete appropriate tissue matrix
Correspondence to: H.-W. Kim; e-mail: kimhw@dankook.
ac.kr
Contract grant sponsor: 2006 Research Grant funded by
the Alumni Association of Dankook University, School of
Dentistry
Ó 2008 Wiley Periodicals, Inc.
tissues.
In the field of bone regeneration, the electrospun
polymeric fibers have also shown a number of
research outcomes in vitro.7–9 For example, poly(e-
caprolactone) (PCL) electrospun fiber was suggested
as a good matrix for the regeneration of tissues,
including bone, as the matrix was able to populate
bone marrow-derived mesenchymal cells (BMSCs)
and to mediate their differentiation into osteoblastic
cells.7,8 A recent study also developed composite
electrospun fibers made of poly(lactic acid) (PLA)
and bioactive inorganic hydroxyapatite (HA) to
improve cell affinity and osteoblastic responses.10
The HA incorporated within the PLA fiber was
shown to improve osteoblastic differentiation, such
as the secretion of alkaline phosphatase.
In practice, the effects of HA within the synthetic
polymers on the biological function of bone-associ-
ated cells, such as adhesive protein adsorption, cell
anchorage, signal transduction, osteoblastic differen-
tiation, and bone matrix synthesis, have been investi-
gated by many in vitro studies and are currently
regarded to be promising.10–14 As such, many bone
748
tissue engineering matrixes have been made as com-
posites, either by the introduction of HA nanopar-
ticles within polymeric matrices or by the minerali-
zation of HA on the surface of polymeric sub-
strates.15–17 The biologically beneficial characteristics
of HA, not just the fact that it is the major inorganic
component of bone matrix, such as its specific affin-
ity to many adhesive proteins and its direct involve-
ment in the bone cell differentiation and mineraliza-
tion processes, make HA especially suited for utiliza-
tion in the bone regeneration field.
In this study, the authors utilized the HA compo-
nent in the electrospun polymer PCL matrix. The
surface of PCL nanofiber was mineralized with
nanocrystalline HA to stimulate osteoblastic re-
sponses. A recent study has reported the HA miner-
alization approach on the PLA nanofibrous matrix.18
However, no systematic work on the biological
effects associated with the HA modification has yet
been carried out. Compared with PLA, the PCL is
known to be less absorbable and more hydrophobic
while more flexible, and it was more recently stud-
ied as a good matrix in the bone regeneration field.
As examined in our pilot study, it was hard to facili-
tate HA mineralization on the surface of PCL
through the simple approach used with PLA.
Herein, we activated the surface of PCL in the alka-
line solution and induced HA mineralization by
two-step processes. The osteoblastic growth and
expression of bone-associated genes on the HA-min-
eralized PCL were examined.
MATERIALS AND METHODS
Production of PCL nanofiber web
PCL (Mw, 80,000; Sigma-Aldrich) was dissolved at 10%
(w/v) in an organic solvent (dichloromethane:ethanol 5
4:1). From our pilot study, the concentration of PCL and
the selection of solvent were determined to produce the
PCL in a nanofibrous form through the electrospinning
technique. For the electrospinning process, the PCL solu-
tion was loaded into a syringe and injected under high DC
voltage through a laboratory-made multinozzle onto a
metal collector. To collect a nanofibrous web with a final
thickness of
200–300 lm, electrospinning was carried out
under the following conditions: voltage, 10 kV; distance,
10 cm; and injection rate, 0.5 mL/h.
Mineralization of nanofiber surface
The mineralization of the PCL nanofibrous web was car-
ried out in three sequential steps: activation, CaP induc-
tion, and mineralization. First, the surface of the nanofi-
brous web was activated by dipping the nanofiber into an
alkaline solution (2N NaOH). Specifically, the as-spun
nanofibrous web was dipped into the NaOH solution and
Journal of Biomedical Materials Research Part A
YU ET AL.
incubated at 248C for 12 h while gently stirring (80 rpm)
the solution. Next, for CaP induction, the nanofibrous web
was removed from the NaOH solution and washed with
distilled water, then dipped alternately into individual Ca
and P solutions, which were made from CaCl2 and
Na2HPO4, respectively. In detail, the nanofibrous web was
dipped into the 150 mM Ca solution for 30 s, washed with
distilled water for 5 s, and then directly dipped into the
150 mM P solution and washed again. This process was
repeated six times before drying the specimens. Finally,
for mineralization, the dried nanofibrous web was incu-
bated in simulated body fluid (SBF), which contained ions
in concentrations similar to those of human body plasma:
2.5 mM Ca2þ, 142 mM Naþ, 1.5 mM Mg2þ, 5.0 mM Kþ,
125.0 mM Cl2, 27.0 mM HCO32, 1.0 mM HPO422, and 0.5
mM SO422. The incubation time lasted up to 21 days to
investigate the mineralization behavior.
Characterization of nanofiber
The morphology of the electrospun nanofiber was exam-
ined by scanning electron microscopy (SEM). The average
diameter of the nanofiber was calculated based on the
arbitrarily selected SEM images. The mineralized product
formed on the nanofibrous web was analyzed by transmis-
sion electron microscopy (TEM), including selected area
electron diffraction (SAED) pattern and energy dispersive
spectroscopy (EDS) measurement. The weight of the nano-
fibrous web was measured before and after mineralization
using an electronic balance.
Cell culturing
The murine-derived preosteoblast MC3T3-E1 cell line
(ATCC, CRL-2593) was cultured in a regular culture me-
dium consisting of a-modified minimum essential medium
supplemented with 10% fatal calf serum, 100 U/mL peni-
cillin G sodium, 100 lg/mL streptomycin sulfate, and
0.25 lg/mL amphotericin B in a humidified atmosphere
(5% CO2) at 378C.
Cell growth
The material samples for cell growth were prepared by
cutting them to a dimension of
10 3 10 3 0.2–0.3 mm3,
followed by sterilization with 70% ethanol and drying
overnight under a laminar flow. Prior to seeding the cells,
each sample was placed in an individual well of a 24-well
plate, and a sterilized glass ring-type holder (laboratory
design) was put on the specimen to prevent it from float-
ing.
Cells were seeded on each sample (PCL nanofiber, min-
eralized PCL nanofiber, and blank well plate) at a density
of 2 3 104 cells/cm2 and cultured in an osteogenic medium
(the regular culturing medium described earlier plus
10 mM b-glycerol phosphate and 50 lg/mL L-ascorbic
acid). After harvesting at each time point (days 1 and 3),
cell viability was measured as the mitochondrial NADH/
NADPH-dependent dehydrogenase activity, using an MTS
assay (CellTiter 96 Aqueous One Solution; Promega, USA).
APATITE-MINERALIZED PCL NANOFIBER AS A BONE REGENERATION MATRIX
In brief, the culture medium was removed and 100 lL of
MTS solution in 1 mL of culturing medium was added to
each well, and allowed to stand for 3 h. A colorimetric mea-
surement with 200 lL of sample of each solution was car-
ried out using a spectrophotometer at an absorbance of
490 nm.
The image of cell adhesion and spreading at a culturing
time of 8 h was observed with SEM after fixing the cells
with glutaraldehyde (2.5%) for 10 min, dehydrating them
with a graded series of ethanol (75, 90, 95, and 100%) each
for 7 min, critical point drying for 20 min, and Pt coating.
Gene expression by real-time RT-PCR
Total cellular RNA was isolated using easy-Blue
(iNtRON Biotechnology, Korea), following the manufac-
turer’s instructions. After culturing for 3 days on each
sample, the cells were harvested and mixed with 1 mL of
RNA lysis buffer (easy-Blue reagent). Chloroform (200 lL)
was added to the suspension and allowed to stand for
5 min at room temperature. After vortexing, the whole
suspension was transferred into 1.5-mL microtubes and
centrifuged for 10 min at 48C. The upper aqueous phase
containing cellular RNA was recovered and pooled back,
adjusted with 0.5 volume of isopropanol, and incubated
for 5 min at room temperature prior to centrifugation for
5 min at 48C. The pellet was washed with 1 mL of 75%
cold ethanol, air dried, and suspended in 50 lL of RNase-
DNase-free distillated water (Invitrogen). The total RNA
concentration was quantified spectrophotometrically at an
absorbance of 260 nm.
After isolating the RNA, first-strand cDNA was synthe-
sized from total RNA (2 lg) using the SuperScript first-
strand synthesis system for RT-PCR (Invitrogen), according
to the manufacturer’s instructions. Volume of the reaction
mixture was made up to 50 lL. Real-time quantitative
PCR was performed using SYBR GreenER qPCR SuperMix
reagents (Invitrogen) and a Bio-Rad iCycler. The fold
change in gene expression was calculated using the following
equation: fold change 5 22DDCT, where DDCT 5 (CT,Target 2
CT,GAPDH)mineralized PCL 2 (CT,Target 2 CT,GAPDH)PCL, which was
normalized to GAPDH. Primer sequences were as follows:
RUNX2 forward: 50 -GCATGGCCAAGAAGACATCC, reverse:
50 -CCTCGGGTTTCCACGTCTC; COL I forward: 50 -GC
ATGGCCAAGAAGACATCC, reverse: 50 -CCTCGGGTTTC
CACGTCTC; ALP forward: 50 -GCATGGCCAAGAAGA
CATCC, reverse: 50 -CCTCGGGTTTCCACGTCTC; OC for-
ward: 50 -CCGGGAGCAGTGTGAGCTTA, reverse: 50 -TA
GATGCGTTTGTAGGCGGTC; GAPDH forward: 50 -CCCT
GTTGCTGTAGCCGTA, reverse: 50 -CCGGTGCTGAGTAT
GTCG.
RESULTS
Fabrication of mineralized nanofiber
The change in morphology of the PCL nanofiber
following a series of pretreatment processes prior to
the mineralization step is shown in Figure 1. The
749
morphology of the as-spun nanofiber [Fig. 1(a,b)]
obtained with diameters of hundreds of nanometers
(average of 792 6 345 nm) was slightly changed
with the treatment of NaOH [Fig. 1(c,d)]. After the
CaP induction step, some discrete particles could be
observed on the nanofiber surface [Fig. 1(e,f)].
The pretreated nanofibrous web was mineralized
in SBF for periods of up to 21 days. The electron
morphologies of the nanofibers with incubation time
are shown in Figure 2(a–f). After incubation for
3 days, calcium phosphate precipitates were easily
observed on the surface, covering many parts of the
surface [Fig. 2(a,b)]. With incubation in SBF for a
prolonged period of 7 days, the nanofiber surface
was almost fully covered with the mineralized crys-
tals [Fig. 2(c,d)]. As a result, the nanofiber diameter
became much thicker. After incubation of the nano-
fiber for 21 days, the mineralization degree was
much pronounced, the fiber diameter was thickened
considerably, and some interspacings of the nanofib-
ers were merged [Fig. 2(e,f)].
As shown in Figure 3, through the mineralization
in SBF, the weight of the PCL nanofibrous web
exhibited an ongoing increase with incubation time
for up to 21 days. The weight increase by the miner-
alization was
170% of the initial weight after incu-
bation for 21 days.
The mineralized product on the PCL fibrous sur-
face with incubation for 7 days was analyzed with
TEM, as shown in Figure 4. The mineralized prod-
ucts [image in Fig. 4(a)] were shown to develop crys-
talline ring patterns from an SAED analysis, which
were similar to those of a crystalline apatite. The
atomic analysis of the crystalline apatite showed a
Ca/P ratio of
1.71, which is similar to but slightly
higher than that of the stoichiometric HA (Ca/P

1.67).
Osteoblastic responses
The electron micrographs of the cells that adhered
and spread for 8 h onto the nanofibrous PCL web
before and after mineralization for 7 days are shown,
respectively, in Figure 5(a,b). Cells were shown to
actively respond to the underlying mineralized
nanofibrous substrate, as the cells on the mineralized
PCL nanofiber had much better spreading behavior
than those on the pure PCL nanofiber.
The cell growth level, as measured by an MTS
assay, is represented in Figure 6. The pure PCL
showed a similar level of cell growth to the tissue
culture plastic used as a control. When compared
with the pure PCL nanofiber, the mineralized PCL
nanofiber showed significantly higher levels of cell
growth (p < 0.05) at both culturing periods.
Journal of Biomedical Materials Research Part A
750 YU ET AL.

Figure 1. SEM morphologies of the PCL nanofiber that underwent a series of treatments conducted prior to the minerali-
zation process: (a, b) as-spun sample, (c, d) after immersion in NaOH solution, and (e, f) after alternative dipping in Ca
and P solutions.

To observe the osteogenic potential of the mineral-
ized PCL nanofiber, the expression of representative
bone-associated genes, such as Runx2, collagen I, alka-
line phosphatase, and osteocalcin, was measured by
real-time RT-PCR. Figure 7 compares the gene expres-
sion of cells on the pure PCL nanofiber with those on
the mineralized PCL nanofiber at a culturing time of
3 days. All genes were expressed at a significantly
higher level (p < 0.05) on the mineralized PCL nano-
fiber with respect to those on the pure PCL nanofiber.

Journal of Biomedical Materials Research Part A

APATITE-MINERALIZED PCL NANOFIBER AS A BONE REGENERATION MATRIX 751

Figure 2. SEM morphologies of the mineralized PCL nanofiber in SBF after incubation for (a, b) 3, (c, d) 7, and (e, f)
21 days.

DISCUSSION from tens of nanometers to a few micrometers, have

Electrospun polymeric matrices have recently
gained great interest in the field of tissue regenera-
tion.1,2 The electrospun webs, typically being nonwo-
ven and fibrous structured with diameters ranging
been suggested as an effective substrate for tissue cells
to attach and populate because of their size-associated
high surface area and cell/tissue responsive sites, and
the capacity of the fibrous morphology to be tailored
for directional tissue regeneration.3–9

Journal of Biomedical Materials Research Part A

752 YU ET AL.

groups that are negatively charged, and thus acceler-

ates the surface to induce apatite minerals.20 On the
basis of the electron image (Fig. 2), mineralization
for about 7 days was considered to be appropriate to
cover the surface of the nanofiber almost completely.
However, prolonged mineralization resulted in an
excessive coverage of the apatite minerals, clogging
the interspacings. The mineralized products were
evidenced as apatite crystals at an immersion time
of 7 days (Fig. 4). In practice, the mineralized prod-
uct at a short time of immersion (<1–2 days) was
detected initially as amorphous, but later trans-
formed into a crystalline apatite phase.
The mineralization of the PCL nanofiber with apa-
tite was shown to significantly affect the MC3T3-E1
Figure 3. Weight change of the PCL nanofibrous matrix cell responses. The initial cell attachment and growth
after mineralization for up to 21 days.
were significantly improved on the mineralized
nanofibrous substrate, as confirmed by the enhanced
In the field of bone regeneration, some synthetic spreading behavior and higher cell growth level
polymeric nanofibers such as PCL and PLA, have
been proposed to support BMSCs and to trigger
them to differentiate into bone cells.7,8 However, lim-
itations in the use of those synthetic polymers for
bone regenerative matrices are still encountered,
mainly because of their lack of initial cell affinity
and poor osteogenic properties. In this context, more
recent studies, including that by our group, have
introduced a composite approach by incorporating
bioactive inorganics, such as HA nanocrystals,
within the polymeric nanofiber.10 Although some
level of improvement in the bone cell responses has
been achieved, one of the main problems encoun-
tered in the production of composite nanofiber has
been the dispersion of the inorganic particles within
the organic matrix and thus the limitation in the con-
tent of inorganic particles.
In this study, we modified the surface of the
degradable polymeric nanofiber with bioactive apa-
tite by the mineralization process. The nanofibrous
web tailored with a bone-mineral-deposited bioac-
tive surface was thought to improve the initial cell
responses and further bone cell differentiation. The
PCL, chosen as a nanofibrous substrate, is one of the
limited number of FDA-approved biopolymers, and
has a low degradation rate and a rubbery property
at application temperatures19; therefore, it is consid-
ered to perform well as a substrate for mineraliza-
tion and consequently as a cell supportive matrix.
To induce the spontaneous mineralization of an
apatite in SBF, the surface of the PCL was first acti-
vated with the alkaline solution and then calcium
phosphate was nucleated by alternating immersions
in the Ca- and P-concentrated solutions. By means of
these series of processes, apatite minerals could be
induced actively from the SBF solution onto the Figure 4. TEM analyses of the mineralized crystals on the
nanofibrous surface. It is known that the alkaline surface of PCL nanofiber after mineralization for 7 days:
treatment of polymers creates many carboxylated (a) crystal image, (b) SAED pattern, and (c) EDS profile.

Journal of Biomedical Materials Research Part A

APATITE-MINERALIZED PCL NANOFIBER AS A BONE REGENERATION MATRIX 753

Figure 6. Effect of mineralization on the cell growth level

on the nanofibrous substrates. Data on mineralized PCL
nanofiber for 7 days were compared with those without
the mineralization for six replicate samples. One-way
ANOVA followed by Bonferroni was carried out to com-
pare data at each culturing time at a significance level of
*p < 0.05.

directly involved in the processes of osteoblastic dif-

ferentiation and mineralization. Because of these
findings, the apatite tailoring of the PCL surface is
considered as an appealing strategy to develop sub-
strates where the cells can be populated well and
differentiated into bone-developing cells. Current
results explained that the apatite-mineralized PCL
nanofibrous web provided favorable surroundings
Figure 5. Electron micrographs of the MC3T3-E1 cells
grown on the nanofibrous substrates for 8 h of culturing:
(a) mineralized PCL for 7 days and (b) pure PCL without
mineralization.

(Figs. 5 and 6). This improvement in cell adhesion

and growth on the mineralized PCL is deemed to
the specific biological roles of the apatite minerals. It
is well documented that apatite minerals are deeply
involved in the initial protein adsorption, particu-
larly in the selective attraction of many adhesion
proteins including fibronectin, vitronectin, and bone-
specific proteins.21,22 The adhesion proteins existing
within the serum, and consequently cells, should
favor the apatite-tailored surface rather than the
pure PCL, which is mainly known to lack the adhe-
sion domains for the specific proteins and cells.
More important is that the mineralized nanofi- Figure 7. Relative expression levels of genes by MC3T3-
brous surface stimulated the osteogenic properties of E1 cells on the substrates of pure PCL nanofiber and min-
the cells, as confirmed by the significantly improved eralized PCL nanofiber after 3 days of culturing. Real-time
expression of all the tested series of bone-associated RT-PCR data on two different samples were included to
genes. Twofold or greater increases were noticed in compare a series of bone-associated genes, such as Runx2,
collagen type I, alkaline phosphatase (ALP), and osteocal-
the collagen type I, osteocalcin, and ALP, and the cin. All genes were expressed significantly higher (*p <
Runx2 was also significantly enhanced (Fig. 7). The 0.05, ANOVA) on the mineralized PCL nanofiber than on
genes stimulated in this study are known to be the pure PCL nanofiber.

Journal of Biomedical Materials Research Part A

754
for cell attachment and growth and osteogenic regu-
lation.
Although further assessments, such as gene
expression variation with culturing time, are re-
quired to confirm the osteogenic stimulation of the
apatite-mineralized surface, this study suggests the
potential role of the mineralized apatite in the osteo-
genic modulation of the MC3T3-E1 cells when sup-
ported on the nanofibrous substrate.
CONCLUSIONS
The surface of the electrospun PCL nanofibrous
web was covered by apatite minerals through a series
of in vitro mineralization processes. Preosteoblastic
MC3T3-E1 cells were shown to adhere, spread, and
grow better on the mineralized PCL nanofiber com-
pared with those on the pure PCL nanofiber. Real-
time RT-PCR showed significantly enhanced expres-
sion of bone-associated genes, including Runx2, col-
lagen type I, alkaline phosphatase, and osteocalcin.
The apatite-tailored PCL nanofibrous web is consid-
ered to find potential applications in the bone regen-
eration and tissue engineering area.
References
1. Pham QP, Sharma U, Mikos AG. Electrospinning of poly-
meric nanofibers for tissue engineering applications: A
review. Tissue Eng 2006;12:1197–1211.
2. Li MY, Mondrinos MJ, Gandhi MR, Ko FK, Weiss AS, Lelkes
PI. Electrospun protein fibers as matrices for tissue engineer-
ing. Biomaterials 2005;26:5999–6008.
3. Song JH, Kim HE, Kim HW. Production of electrospun gela-
tin nanofiber by water-based co-solvent approach. J Mater Sci
Mater Med 2008;19:95–102.
4. Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electro-
spinning of collagen nanofibers. Biomacromolecules 2002;3:
232–238.
5. Zhang YZ, Ouyang HW, Lim CT, Ramakrishna S, Huang
ZM. Electrospinning of gelatin fibers and gelatin/PCL com-
posite fibrous scaffolds. J Biomed Mater Res B 2005;72:156–
165.
6. Chen M, Patra PK, Warner SB, Bhowmich S. Role of fiber di-
ameter in adhesion and proliferation of NIH 3T3 fibroblast
on electrospun polycaprolactone scaffolds. Tissue Eng 2007;
13:79–587.
Journal of Biomedical Materials Research Part A
YU ET AL.
7. Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS. Multilineage
differentiation of human mesenchymal stem cells in a three-
dimensional nanofibrous scaffold. Biomaterials 2005;26:5158–
5166.
8. Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradable
nanofiber scaffold by electrospinning and its potential for
bone tissue engineering. Biomaterials 2003;24:2077–2082.
9. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-
BMP-2 scaffolds for bone tissue engineering. Biomaterials
2006;27:3115–3124.
10. Kim HW, Lee HH, Knowles JC. Electrospinning biomedical
nanocomposite fibers of hydroxyapatite/poly(lactic acid) for
bone regeneration. J Biomed Mater Res A 2006;79:643–649.
11. Rizzi SC, Heath DJ, Coombes AGA, Bock N, Textor M,
Downes S. Biodegradable polymer/hydroxyapatite compo-
sites: Surface analysis and initial attachment of human osteo-
blasts. J Biomed Mater Res 2001;55:475–486.
12. Wei G, Ma PX. Structure and properties of nano-hydroxyapa-
tite/polymer composite scaffolds for bone tissue engineering.
Biomaterials 2004;25:4749–4757.
13. Kim HW, Kim HE, Salih V. Stimulation of osteoblast
responses to biomimetic nanocomposites of gelatin–hydroxy-
apatite for tissue engineering scaffolds. Biomaterials 2005;26:
5221–5230.
14. Zhao F, Grayson WL, Ma T, Bunnell B, Lu WW. Effects of
hydroxyapatite in 3-D chitosan–gelatin polymer network
on human mesenchymal stem cell construct development.
Biomaterials 2006;27:1859–1867.
15. Taboas JM, Maddox RD, Krebsbach PH, Hollister SJ. Indirect
solid free form fabrication of local and global porous, biomi-
metic and composite 3D polymer-ceramic scaffolds. Biomate-
rials 2003;24:181–194.
16. Schek RM, Taboas JM, Segvich SJ, Hollister SJ, Krebsbach
PH. Engineered osteochondral grafts using biphasic compos-
ite solid free-form fabricated scaffolds. Tissue Eng 2004;10:
1376–1385.
17. Zhang Y, Ni M, Zhang M, Ratner B. Calcium phosphate–
chitosan composite scaffolds for bone tissue engineering.
Tissue Eng 2003;9:337–345.
18. Chen J, Chu B, Hsiao BS. Mineralization of hydroxyapatite
in electrospun nanofibrous poly(L-lactic acid) scaffolds.
J Biomed Mater Res A 2006;79:307–317.
19. Pitt CG. Poly e-caprolactone and its copolymers. In: Langer
R, Chasin M, editors. Biodegradable Polymers as Drug Deliv-
ery Systems. New York: Marcel Dekker; 1990. p 71–120.
20. Kawashita M, Nakao M, Minoda M, Kim HM, Beppu T,
Miyamoto T, Kokubo T, Nakamura T. Apatite-forming ability
of carboxyl group-containing polymer gels in a simulated
body fluid. Biomaterials 2003;24:2477–2484.
21. Kilpadi KL, Chang PL, Bellis SL. Hydroxylapatite binds more
serum proteins, purified integrins, and osteoblast precursor
cells than titanium or steel. J Biomed Mater Res 2001;57:258–
267.
22. Hoang QQ, Sicheri F, Howard AJ, Yang DSC. Bone recogni-
tion mechanism of porcine osteocalcin from crystal structure.
Nature 2003;425:977–980.