Langmuir 2008, 24, 14145-14150 14145

Coating Electrospun Poly(ε-caprolactone) Fibers with Gelatin and

Calcium Phosphate and Their Use as Biomimetic Scaffolds for Bone
Tissue Engineering
Xiaoran Li,†,‡ Jingwei Xie,† Xiaoyan Yuan,*,‡ and Younan Xia*,†
Department of Biomedical Engineering, Washington UniVersity, St. Louis, Missouri 63130, and School of
Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials,
Tianjin UniVersity, Tianjin 300072, P. R. China

ReceiVed September 10, 2008. ReVised Manuscript ReceiVed October 11, 2008

Electrospinning was employed to fabricate fibrous scaffolds of poly(ε-caprolactone) in the form of nonwoven mats.
The surfaces of the fibers were then coated with gelatin through layer-by-layer self-assembly, followed by functionalization
with a uniform coating of bonelike calcium phosphate by mineralization in the 10 times concentrated simulated body
fluid for 2 h. Transmission electron microscopy, water contact angle, and scanning electron microscopy measurements
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confirmed the presence of gelatin and calcium phosphate coating layers, and X-ray diffraction results suggested that
the deposited mineral phase was a mixture of dicalcium phosphate dehydrate (a precursor to apatite) and apatite. It
was also demonstrated that the incorporation of gelatin promoted nucleation and growth of calcium phosphate. The
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porous scaffolds could mimic the structure, composition, and biological function of bone extracellular matrix. It was
found that the preosteoblastic MC3T3-E1 cells attached, spread, and proliferated well with a flat morphology on the
mineralized scaffolds. The proliferation rate of the cells on the mineralized scaffolds was significantly higher (by
1.9-fold) than that on the pristine fibrous scaffolds after culture for 7 days. These results indicated that the hybrid
system containing poly(ε-caprolactone), gelatin, and calcium phosphate could serve as a new class of biomimetic
scaffolds for bone tissue engineering.

Introduction entrapment;7 cooperative electrostatic interactions;8 grafting via

In recent years, synthetic scaffolds capable of replacing
autologous or allogeneic bones have attracted broad interests.1
Besides the challenge of designing a scaffold that can mimic the
structure and biological function of bone extracellular matrix
(ECM), how to achieve a suitable bone-implant interface for the
host is also a key issue in bone tissue engineering.2
Electrospun fibers have been studied as a class of promising
scaffolds for tissue engineering, since they can mimic the
nanoscale features of the ECM. The nonwoven, fibrous mats
electrospun from biodegradable polyesters such as poly(lactic
acid) (PLA),3 poly(lactic-co-glycolic acid) (PLGA),4 and poly(ε-
caprolactone) (PCL)5 have all been intensively investigated for
bone tissue engineering due to their good mechanical properties
and controllable degradation. However, most polyesters are unable
to interact specifically with cells due to their relatively high
hydrophobicity as compared to natural ECM and lack of functional
groups for the attachment of biologically active molecules.6 The
biological polymers which can render innate biological informa-
tion guidance to cells are always introduced by making use of
surface modification to the synthetic polymers. Various ap-
proaches have been developed for surface modification, including
* To whom correspondence should be addressed. E-mail: xia@
biomed.wustl.edu (Y.X.); yuanxy@tju.edu.cn (X.Y.).
†
Washington University.
‡
Tianjin University.
(1) Stevens, M. M. Mater. Today 2008, 11, 18–25.
(2) Porter, A. E.; Patel, N.; Skepper, J. N.; Best, S. M.; Bonfield, W. Biomaterials
2004, 25, 3303–3314.
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A. S. Biomaterials 2006, 27, 596–606.
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plasma,9 γ-ray irradiation,10 etching,11 or chemical reaction;12
among others. Layer-by-layer (LBL) deposition provides a simple
means to generate polyelectrolyte multilayer coating on the surface
with a wide variety of different biofunctional properties, especially
for substrates with irregular shapes and inner structures where
traditional methods are generally ineffective.13 Moreover, the
multilayer surface coating formed by LBL deposition has been
shown with unique characteristics of improving cytocompatibility
to cells.14 To this end, Gao et al. studied electrospun poly(L-
lactic acid) (PLLA) scaffolds whose surfaces were coated with
chitosan using the LBL method. It was found that the attachment,
activity, and proliferation of human endothelial cells on the PLLA
scaffolds covered by three or five bilayers of poly(styrene
sulfonate) sodium salt (PSS)/chitosan (with chitosan as the
outermost layer) were better than those with one bilayer of PSS/
chitosan or the control, pristine PLLA.15 Vodouhê et al. also
reported that the viability of motoneurons on polyelectrolyte
(7) Liu, Z.; Jiao, Y.; Zhang, Z.; Zhou, C. J. Biomed. Mater. Res. 2007, 83A,
1110–1116.
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Grzybowski, B. A. J. Am. Chem. Soc. 2007, 129, 15623–15630.
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1158.
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2008, 9, 1772–1781.
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33–47.
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127, 8865–8871. (b) Zhu, A.; Zhang, M.; Wu, J.; Shen, J. Biomaterials 2002, 23,
4657–4665. (c) Yan, M.; Ren, J. J. Mater. Chem. 2005, 15, 523–527. (d) Gu, H.;
Yang, Z.; Gao, J.; Chang, C. K.; Xu, B. J. Am. Chem. Soc. 2005, 127, 34–35.
(13) Hammond, P. T. AdV. Mater. 2004, 16, 1271–1293.
(14) Salloum, D. S.; Olenych, S. G.; Keller, T. C. S.; Schlenoff, J. B.
Biomacromolecules 2005, 6, 161–167.
(15) Zhu, Y.; Gao, C.; He, T.; Liu, X.; Shen, J. Biomacromolecules 2003, 4,
446–452.

10.1021/la802984a CCC: $40.75  2008 American Chemical Society

Published on Web 11/16/2008
14146 Langmuir, Vol. 24, No. 24, 2008 Li et al.

multilayers was higher compared to polyelectrolyte monolayers.16

In bone tissue engineering, collagen (a main structural protein
in bone ECM) is a well-known osteoconductive biomaterial.
Polyelectrolyte films based on LBL deposition of collagen have
been constructed. Human endothelial cells cultured on poly-
urethane covered by multilayers of collagen showed favorable
adhesion, spreading, and proliferation.17 Therefore, the mutilayer
coating formed by LBL deposition (with a biological polymer
as the outermost layer) seems to offer many attractive advantages
over the monolayer coating which can be prepared using many
other methods such as simple dip coating. In addition to its ability
to provide a more cell-friendly interface, many charged bioactive
molecules can be easily incorporated into a multiplayer system
without loss of activity during LBL self-assembly.18
With the desire to build an artificial analogue of native bone
ECM, which is mainly composed of hydroxyapatite (HAp)
dispersed within a fibrous collagen framework, many attempts
have been made to produce collagen/calcium phosphate com-
posites.19 Compared with pure polymers, the composite surface
can provide a bioactive environment and lead to improvement
of cell attachment, elevation of certain osteogenic biomarker
expression levels, and better integration with the host tissue.20
The composite fibrous scaffolds can be generated from their
blends using electrospinning21 or hybrid twin screw extrusion/
electrospinning techniques.22 For example, Venugopal et al.
prepared PCL/HAp/gelatin (1:1:2), PCL/HAp (1:1), PCL/gelatin
(1:2), and PCL fibrous scaffolds by electrospinning their blends
or solutions. It was found that PCL/HAp/gelatin composite fibrous
scaffolds not only showed highly flexible tensile property but
also allowed osteoblasts to penetrate into the scaffolds.21 However,
for the preparation of composite fibers via electrospinning, there
was difficulty in dispersing hydrophilic ceramic powders in
organic solvents, which could hamper the reduction of fiber size
and homogeneous distribution of the inorganic components within
the fibers.23 Biomimetic mineralization is an alternative route to Figure 1. SEM images of electrospun PCL fibers (A) before and (B)
the production of polymer/calcium phosphate composite in the after coating with 15 bilayers of alternating gelatin and PSS (with gelatin
simulated body fluid (SBF).24 By this means, bonelike apatite, as the outermost layer).
which is very close to natural bone with low crystallinity and
Experimental Section
nanoscale sizes, can be formed on the surface.25 Further study
showed that the growth behavior as well as the dimensions of Materials. Poly(ε-caprolactone) (PCL, Mn ) 42 500 g/mol),
crystals were also similar to biological apatite present in human gelatin (type A, form porcine skin), poly(styrene sulfonate) sodium
salt (PSS, Mw ) 7000 g/mol), dichloromethane (DCM), dimeth-
bones.26 ylformaldehyde (DMF), acetic acid, and all the chemicals for
In the present study, we aim to demonstrate a hybrid fibrous preparation of the 10 times concentrated simulated body fluid (10SBF)
scaffold that can closely mimic bone ECM and provide a friendly were obtained from Sigma-Aldrich (St. Louis, MO). All chemicals
interface with the host. We achieve this goal by fabricating fibrous were used as received. The water used in all experiments was purified
scaffolds from PCL by electrospinning, followed by surface by passing through a Millipore system.
modification with gelatin via a LBL method and deposition of Fabrication of PCL Fibrous Scaffolds by Electrospinning.
calcium phosphate using a mild mineralization procedure. The electrospinning setup used in the present study was described
in our previous publication.27 PCL solution at a concentration of
20% was obtained by dissolving it in a solvent mixture of DCM and
(16) Vodouhê, C.; Schmittbuhl, M.; Boulmedais, F.; Bagnard, D.; Vautier, D.; DMF with a volume ratio of 80:20. The solution was loaded into
Schaaf, P.; Egles, C.; Voegel, J.; Ogier, J. Biomaterials 2005, 26, 545–554.
(17) Zhu, Y.; Sun, Y. Colloids Surf., B 2004, 36, 49–55. a 5 mL plastic syringe with a 24-gauge needle attached and injected
(18) (a) Müller, K.; Quinn, J. F.; Johnston, A. P. R.; Becker, M.; Greiner, A.; using a syringe pump at a flow rate of 0.5 mL/h. The collector was
Caruso, F. Chem. Mater. 2006, 18, 2397–2403. (b) Ma, L.; Zhou, J.; Gao, C.; a piece of aluminum foil. The distance between the tip of needle and
Shen, J. J. Biomed. Mater. Res. 2007, 83B, 285–292. the collector was about 15 cm, and a voltage of 15 kV was applied.
(19) Zou, C.; Weng, W.; Deng, X.; Cheng, K.; Liu, X.; Du, P.; Shen, G.; Han,
G. Biomaterials 2005, 26, 5276–5284. Deposition of Gelatin. The deposition of gelatin onto electrospun
(20) Yao, J.; Radin, S.; Leboy, P. S.; Ducheyne, P. Biomaterials 2005, 26, PCL fibers via LBL self-assembly was conducted as follows:
1935–1943. electrospun PCL fibers were immersed in a 2 mg/mL gelatin solution
(21) Venugopal, J. R.; Low, S.; Choon, A. T.; Kumar, A. B.; Ramakrishna, in 20 mM acetic buffer (pH ) 4.0) and kept for 20 min. Thereafter,
S. Artif. Organs 2008, 32, 388–397.
(22) Erisken, C.; Kalyon, D. M.; Wang, H. Nanotechnology 2008, 19, 165302. the fibers were thoroughly rinsed with water for 10 min and then
(23) Kim, H.; Lee, H.; Knowles, J. C. J. Biomed. Mater. Res. 2006, 79A, immersed in a 3 mg/mL PSS solution in 20 mM acetic buffer (pH
643–649. ) 4.0) for another 20 min. The sample was thoroughly rinsed with
(24) Yuan, X.; Mak, A. F. T.; Li, J. J. Biomed. Mater. Res. 2001, 57, 140–150. water for 10 min. The deposition cycle was repeated until the desired
(25) Abe, Y.; Kokubo, T.; Yamamuro, T. J. Mater. Sci.: Mater. Med. 1990,
1, 233–238. number of layers (or film thickness) was reached. The scaffolds
(26) Müller, F. A.; Müller, L.; Caillard, D.; Conforto, E. J. Cryst. Growth
2007, 304, 464–471. (27) Li, D.; McCann, J. T.; Xia, Y. J. Am. Ceram. Soc. 2006, 89, 1861–1869.
Electrospun Poly(ε-caprolactone) Fibers
Figure 2. TEM images of (A) PCL/(gelatin/PSS)15/gelatin fibers and
(B) nanotubes obtained by dissolving the inner PCL fibers.
used in both mineralization and cell culture were nonwoven mats
of PCL fibers whose surfaces had been coated with five bilayers of
gelatin and PSS, with gelatin on the outermost surface (thereafter,
they are referred to as gelatin-covered scaffolds). As a control,
nonwoven mats of pristine PCL fibers were also used for miner-
alization and cell culture studies.
Coating of Calcium Phosphate. We followed Tas and Bhaduri’s
method to prepare the 10 times concentrated simulated body fluid
(10SBF).28 A stock solution containing NaCl, KCl, CaCl2, MgCl2,
and NaH2PO4 · H2O with a pH value of about 4.1 was prepared in
advance. The stock solution could be kept at 4 °C for several weeks
without precipitation. At the beginning of a coating process, NaHCO3
was added at room temperature under a stirring speed of 500 rpm,
and the pH value rose to about 6.5. The electrospun PCL and gelatin-
covered PCL scaffolds were immersed in 10SBF hosted in a tightly
capped plastic tube and kept at room temperature for 1-8 h. The
10SBF solution was changed every 2 h. After being removed from
buffer solution, the samples were gently washed with water and then
dried in air at room temperature.
Characterization. The morphologies of the electrospun PCL
fibers, the gelatin-covered PCL fibers, and the scaffolds after
mineralization in 10SBF for different periods of time were examined
by scanning electron microscopy (SEM, FEI Nova 200 NanoLab).
(28) Tas, A. C.; Bhaduri, S. B. J. Mater. Res. 2004, 19, 2742–2749.
Langmuir, Vol. 24, No. 24, 2008 14147
The microstructures of gelatin-covered PCL scaffolds were studied
by transmission electron microscopy (TEM, Hitachi H-7500). Water
contact angles of PCL and gelatin-covered PCL scaffolds were
measured with a contact angle meter (Ramé-Hart Inc.) at ambient
temperature. In order to determine the crystallographic structure, we
examined the gelatin-covered PCL scaffolds after mineralization in
10SBF for 8 h (to make sure there was a sufficient amount of mineral
phase coating) by using a Rigaku Geigerflex D-MAX/A diffrac-
tometer with Cu KR radiation (50 kV, 50 mA). The scanning range
was from 10° to 40° with a step size of 0.02°.
Cell Culture. Mouse calvaria-derived, preosteoblastic cells
(MC3T3-E1; ATCC CRL-2593) were cultured in alpha minimum
essential medium (R-MEM, Invitrogen, Grand Island, NY), supple-
mented with 10% fetal bovine serum (FBS, Invitrogen) and 1%
antibiotics (containing penicillin and streptomycin, Invitrogen). The
medium was changed every other day, and the cultures were incubated
at 37 °C in a humidified atmosphere containing 5% CO2.
Both pristine and mineralized PCL fibrous scaffolds were cut into
circular discs of 15 mm in diameter and placed in the wells of a
24-well plate (Corning). The samples were sterilized in 70% ethanol
overnight and then washed with phosphate buffer saline (PBS) three
times. Approximately 105 cells were seeded in each well and cultured
in 300 µL of osteogenic medium containing FBS for different periods
of time up to 7 days.
The cell viability was measured using the 3-(4,5-dimethylthiazol)-
2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on
the mitochondrial conversion of tetrazolium salt. After 1, 3, and 7
days of incubation, the medium was removed, and 270 µL of fresh
medium and 30 µL of MTT (5 mg/mL in PBS) (Invitrogen) were
added to each well and incubated at 37 °C and 5% CO2 for 3 h. After
removal of the medium, the converted dye was dissolved with
isopropyl alcohol. The absorbance at a wavelength of 560 nm was
measured using a microplate reader (Tecan).
The cell morphology at day 7 was characterized by fluorescent
microscopy. The cells on the samples were washed twice with PBS,
fixed in 3.7% formaldehyde solution (Sigma-Aldrich) in PBS for 30
min at room temperature, and dyed with Alexa Fluor1 488 phalloidin
(Invitrogen) and 4′-6-diamidino-2-phenylindole (DAPI, Invitrogen)
for 1 h. The fluorescent images were taken using a QICAM Fast
Cooled Mono 12-bit camera (Q Imaging, Burnaby, BC, Canada)
attached to an Olympus microscope with Capture 2.90.1 (Olympus).
Results and Discussion
Preparation of PCL Fibers. In the present study, PCL was
chosen as a model polymer due to its relatively slow degradation
in ViVo, which is useful to bone tissue engineering.29 Figure 1A
shows a typical SEM image of PCL fibers fabricated by
electrospinning. The PCL fibers were smooth and free of beads,
with an average diameter of 1.2 µm. The fibers were randomly
oriented to form a porous scaffold. It has been reported that the
fibrous scaffold could serve as a much better support for cell
attachment and proliferation, in comparison with a solid film
cast from the same polymer solution.30
Gelatin Deposition. In addition to the topographical structure,
the biological activity is also critical to the performance of a
scaffold. Decoration of synthetic polymers with natural polymers
via surface modification has been developed to improve the
biomaterial/cell interaction. In the present study, positively
charged gelatin was deposited onto the electrospun PCL fiber
surface in a LBL assembly manner using PSS as the negatively
charged polyelectrolyte. This approach, which is free from gelatin
cross-linking, can avoid the potential problem of cytotoxicity
caused by the chemicals employed during the process of cross-
linking gelatin. Gelatin derived from collagen has been exploited
for a variety of biomedical applications due to its biocompatibility
(29) Bölgen, N.; Menceloğlu, Y. Z.; Acatay, K.; Vargel, |$$İ.; Pişkin, E.
J. Biomater. Sci., Polymer Ed. 2005, 16, 1537–1555.
(30) Sombatmankhong, K.; Sanchavanakit, N.; Pavasant, P.; Supaphol, P.
Polymer 2007, 48, 1419–1427.
14148 Langmuir, Vol. 24, No. 24, 2008
Figure 3. SEM images of electrospun PCL fibers after their surfaces had been coated with gelatin and then incubated in 10SBF for (A) 1, (B) 2,
(C) 3, (D) 4, and (E) 5 h. (F) SEM image of electrospun PCL fibers which were directly incubated in 10SBF for 2 h without gelatin treatment. The
scale bars in the insets are 2 µm.
and low cost.31 In addition, PSS, a component widely used in
LBL assembly, exhibited good cytocompatibility and stability
in culture medium.32 After coating, the scaffold still maintained
the fibrous and porous structure (Figure 1B).
Figure 2A shows a TEM image of a single PCL fiber after
coating with 15 bilayers of gelatin and PSS, with gelatin on the
outermost surface. In order to clearly resolve the coating thickness,
we deposited 15 bilayers rather than 5 bilayers on the PCL fibers.
A core/shell structure could be observed under TEM due to the
density difference between PCL and the polyelectrolyte layer,
with a corresponding average shell thickness of 76 nm. To further
confirm the deposition of gelatin and PSS, the fibers were
immersed in DCM, resulting in dissolution of PCL cores and
(31) Sun, J.; Wu, S. Y.; Lin, F. Biomaterials 2005, 26, 3953–3960.
(32) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir
2002, 18, 5047–5050.
Li et al.
formation of hollow fibers made of gelatin and PSS (Figure 2B).
Water contact angle measurements provided additional evidence
to support the LBL coating. The hydrophobic PCL scaffolds
exhibited a high water contact angle of 117°, whereas the surface
of gelatin-covered PCL fibers was completely hydrophilic with
a water contact angle of almost zero. From these results, it is
clear that gelatin had been deposited on the fiber surface via LBL
assembly, and the amount of gelatin could be tailored easily by
controlling the number of LBL deposition cycles. If needed,
various bioactive agents capable of stimulating cell adhesion,
proliferation, and differentiation, such as DNA and growth
factors,18 could also be directly incorporated into the polyelec-
trolyte layers during LBL deposition.
Biomimetic Coating. SBF has been widely used for biomi-
metic calcium phosphate coating on bioinert materials. This
Electrospun Poly(ε-caprolactone) Fibers
Figure 4. X-ray diffraction pattern taken from a scaffold of electrospun
PCL fibers whose surfaces were coated with gelatin and then mineralized
by incubation in 10SBF for 8 h.
Figure 5. Proliferation of MC3T3-E1 cells seeded on membranes of
electrospun PCL fibers without and with surface functionalization (n )
4). Bar represented means ( SD. A statistically significant difference
was observed after 7 days of culture (Student’s t-test, *p < 0.05).
process is rather slow, and it normally takes up to several weeks.33
The in Vitro mineralization in 10SBF was developed as an
effective and robust approach to calcium phosphate coating.28,34
Figure 3 (A-E) shows morphological changes of the gelatin-
covered PCL fibers after different periods of mineralization in
10SBF. After incubation for 1 h, a few tiny particles appeared
on the surfaces of the fibers, showing a fast precipitation of
calcium phosphate (Figure 3A). A prolonged incubation sig-
nificantly changed the surface morphology. The calcium phos-
phate growth occurred preferentially along the longitudinal
direction of the fibers. After 2 h of incubation, the scaffold was
fully covered with nanotextured precipitates but maintained its
porous and fibrous structure, indicating a homogeneous nucleation
(Figure 3B). The deposition gradually grew to become globules
after 3 and 4 h (Figure 3C and D). The fibers were completely
wrapped by thick calcium phosphate layers after 5 h; meanwhile,
the fibrous and porous structures disappeared (Figure 3E). Figure
3F shows an SEM image of a pristine PCL scaffold after
incubation in 10SBF for 2 h, and it can be seen that the mineral
(33) Zhang, R.; Ma, P. X. Macromol. Biosci. 2004, 4, 100–111.
(34) Yang, F.; Wolke, J. G. C.; Jansen, J. A. Chem. Eng. J. 2008, 137, 154–
161.
Langmuir, Vol. 24, No. 24, 2008 14149
deposition over individual fibers was uneven and bare fibers
inside the scaffold could still be easily found. This ineffectiveness
in coating could be ascribed to the relatively inert surface of
PCL, which lacks the ability to bind to calcium phosphate.34
Previous studies have reported that anionic groups such as
-COO- on the surface of organic polymers could lead to
enrichment of Ca2+, resulting in local supersaturation and
nucleation of crystallites.35 Several methods have been employed
to modify the surfaces of substrates toward activating the
polymers, and typical examples include NaOH36 and plasma37
treatment. However, either toxic reagents (e.g., NaOH) or
dedicated equipment (e.g., plasma cleaner) was required. In
addition, for polymer fibers, which were not as strong as the bulk
materials, the harsh reaction conditions such as NaOH and plasma
treatment could possibly impound intrinsic mechanical and
chemical properties, and even result in degradation and/or
damage.38 In this work, the deposition of gelatin via the LBL
technique followed by incubation in 10SBF successfully induced
fast and uniform calcium phosphate coating on the surface of
PCL fibers. The increased hydrophilicity and introduction of
functional groups such as -COOH and -NH2 could be the
possible reasons for the effective calcium phosphate deposition.
Yao et al. have studied the in situ formation of nanohydroxyapatite
on a chitosan-gelatin network.39 It was found that the carboxyl
groups of gelatin, and carbonyl and amino groups of gelatin and
chitosan played a crucial role in HAp formation.
It has been reported that calcium phosphates in different forms
such as HAp40 and octacalcium phosphate41 could grow on a
gelatin matrix. The crystal structure of the mineral phase
developed from the gelatin-covered PCL fibers after 8 h of
incubation in 10SBF was determined by X-ray diffraction (XRD).
As indicated in Figure 4, the characteristic peaks of dicalcium
phosphate dehydrate (DCPD) appeared clearly in the diffraction
pattern (labeled with /), and the peak labeled with a dot could
be assigned to apatite. It can be concluded that the deposition
consisted of a mixture of DCPC and apatite. It has been established
that DCPD is a potential starting material for bone substitute.42
Cell Response. It is well-known that the porous structure of
a scaffold is very important to bone tissue engineering. The
pores favor the inward growth of bone, and the interconnective
porous structure facilitates in Vitro nutrient/waste transportation
and in ViVo vascularization.43 In this study, the gelatin-covered
PCL fibers after mineralization for 2 h in 10SBF were selected
as the scaffolds for cell culture because the calcium phosphate
could effectively coat on the surfaces of individual fibers while
leaving the inherent porous structures of the scaffolds unchanged.
The in Vitro biocompatibility of the electrospun hybrid fibers
was assessed in terms of the proliferation of MC3T3-E1 cells
with pristine electrospun PCL fibers as a control. Figure 5 shows
proliferation data of the cells seeded on pristine and mineralized
PCL fibrous scaffolds. The mineralized scaffolds were composed
of electropsun PCL fibers covered with five bilayers of gelatin/
(35) Huang, S.; Zhou, K.; Zhu, W.; Huang, B.; Li, Z. J. Appl. Polym. Sci. 2006,
101, 1842–1847.
(36) Oyane, A.; Uchida, M.; Choong, C.; Triffitt, J.; Jones, J.; Ito, A. Biomaterials
2005, 26, 2407–2413.
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J. Biomed. Mater. Res. 2005, 75A, 138–145.
(38) Ma, Z.; Kotaki, M.; Yong, T.; He, W.; Ramakrishna, S. Biomaterials
2005, 26, 2527–2536.
(39) Li, J.; Chen, Y.; Yin, Y.; Yao, F.; Yao, K. Biomaterials 2007, 28, 781–
790.
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Mater. Res. 2002, 59, 709–714.
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(42) Tas, A. C.; Bhaduri, S. B. J. Am. Ceram. Soc. 2004, 87, 2195–2200.
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14150 Langmuir, Vol. 24, No. 24, 2008
Figure 6. Fluorescence micrographs of MC3T3-E1 cells that were cultured for 7 days on (A, B) membranes of electrospun PCL fibers and (C, D)
membranes of electrospun PCL fibers whose surfaces had been derivatized with gelatin and then calcium phosphate. The F-actin was stained with
fluorescein isothiocyanate-phallodin (green color), while the cell nucleus was stained with 4′-6-diamidino-2-phenylindole (DAPI) (purple color).
PSS (with gelatin as the outermost layer), followed by a bonelike
calcium phosphate coating. They showed more favorable adhesion
and higher proliferation rate of MC3T3-E1cells. The ratios of
cell growth on the mineralized fibers to pristine PCL fibers at
day 3 and day 7 were 1.4-fold and 1.9-fold, respectively. The
cell proliferation on the mineralized fibers was significantly (p
< 0.05) higher compared with that on pristine PCL fibers after
7 days of cell culture.
After culturing for 7 days, we also observed the cell morphology
by fluorescent microscopy, as shown in Figure 6. The cells were
observed to attach and spread well on both types of PCL scaffolds.
However, the cells seemed to prefer expanding on the mineralized
fibers. After 7 days of culture, the surface of the mineralized
scaffold was covered with multilayers of cells, which was in
good agreement with our proliferation result. It could thus be
concluded that the PCL scaffolds functionalized with gelatin
and calcium phosphate might offer a more favorable microen-
vironment for MC3T3 cell growth. In this study, the PCL/gelatin/
calcium phosphate fibrous scaffolds combine all the attractive
features and unique properties of synthetic polymers, natural
polymers, and mineral deposition: that is, PCL for the mechanical
properties, and coatings of gelatin and calcium phosphate for the
bioactivity and osteoconductivity, respectively. The fibrous matrix
also exhibits a structure and components similar to those of bone
ECM. All these attributes should make the nonwoven mats of
Li et al.
mineralized PCL fibers as a new class of promising scaffolds for
bone tissue engineering.
Conclusion
A class of hybrid scaffolds based on PCL, gelatin, and calcium
phosphate was developed through surface modification on the
electrospun PCL fibers. Gelatin was immobilized by LBL
assembly, and calcium phosphate was deposited on the surface
of gelatin-covered fibers by mineralization in 10SBF. We found
that the presence of gelatin facilitated a homogeneous calcium
phosphate coating. After 2 h of incubation, fibers were uniformly
covered by a thin layer of mineral deposition. XRD results
indicated that the composition of the deposited mineral was a
mixture of dicalcium phosphate dehydrate (a precursor to apatite)
and apatite. The hybrid scaffolds were then evaluated for the
culture of MC3T3-E1 cells. Cell proliferation was significantly
higher than that on pristine PCL scaffolds, which were used as
a control. A multilayered film of cells was observed on the
mineralized scaffolds after 7 days of culture. It can be concluded
that the introduction of gelatin and calcium phosphate coatings
was effective in enhancing the cytocompatiblity. The scaffold
developed in this study can also accommodate the incorporation
of drugs such as bone morphogenetic proteins (BMP) and other
bioactive species in the fibers or the polyelectrolyte films to
empower them with more functions.
LA802984A