Cultivo de StemCells Sobre Composite

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Electrosprayed Hydroxyapatite on
Polymer Nanofibers to Differentiate
Mesenchymal Stem Cells to
Osteogenesis
a a a b
J. Venugopal , R. Rajeswari , M. Shayanti , Sharon Low ,
c d a b
Ariff Bongso , V. R. Giri Dev , G. Deepika , Aw Tar Choon &
a
S. Ramakrishna
a
Healthcare and Energy Materials Laboratory, Faculty of
Engineering, National University of Singapore, Singapore
b
StemLife Sdn BhD, 50450, Kuala Lumpur, Malaysia
c
Department of Obstetrics & Gynaecology, Yong Loo Lin School of
Medicine, National University of Singapore, Singapore
d
Department of Textile Technology, Anna University, Chennai,
India
Published online: 11 May 2012.
To cite this article: J. Venugopal , R. Rajeswari , M. Shayanti , Sharon Low , Ariff Bongso , V. R.
Giri Dev , G. Deepika , Aw Tar Choon & S. Ramakrishna (2013): Electrosprayed Hydroxyapatite
on Polymer Nanofibers to Differentiate Mesenchymal Stem Cells to Osteogenesis, Journal of
Biomaterials Science, Polymer Edition, 24:2, 170-184
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Journal of Biomaterials Science 24 (2013) 170–184
brill.nl/jbs
Electrosprayed Hydroxyapatite on Polymer Nanofibers to

Differentiate Mesenchymal Stem Cells to Osteogenesis
J. Venugopal a,∗ , R. Rajeswari a , M. Shayanti a , Sharon Low b , Ariff Bongso c ,

V. R. Giri Dev d , G. Deepika a , Aw Tar Choon b and S. Ramakrishna a
a
Healthcare and Energy Materials Laboratory, Faculty of Engineering,
National University of Singapore, Singapore

b
StemLife Sdn BhD, 50450 Kuala Lumpur, Malaysia
c
Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore
d
Department of Textile Technology, Anna University, Chennai, India
Received 10 November 2011; accepted 2 February 2012
Abstract
Electrospraying of hydroxyapatite (HA) nanoparticles onto the surface of polymer nanofibers provides a
potentially novel substrate for the adhesion, proliferation and differentiation of mesenchymal stem cells
(MSCs) into bone tissue regeneration. HA nanoparticles (4%) were electrosprayed on the surface of elec-
trospun polycaprolactone (PCL) nanofibers (420 ± 15 nm) for bone tissue engineering. PCL/HA nanofibers
were comparatively characterized with PCL/Collagen (275 ± 56 nm) nanofibers by FT-IR analysis to con-
firm the presence of HA. Fabricated PCL/HA and PCL/Collagen nanofibers and TCP (control) were used for
the differentiation of equine MSC into osteogenic lineages in the presence of DMEM/F12 medium supple-
mented with β-glycerophosphate, ascorbic acid and dexamethasone. Cell proliferation and differentiation
into an osteogenic lineage was evaluated by MTS assay, SEM observation, ALP activity, ARS staining,
quantification of mineral deposition and expression of osteocalcin. Proliferation of MSCs increased signif-
icantly (P 0.05) up to 12% in PCL/Collagen (day 15) compared to PCL/HA nanofibrous substrate. ALP
activity was increased 20% in PCL/HA by day 10 confirming the direction of osteogenic lineage from MSCs
differentiation. PCL/HA stimulated an increased mineral secretion up to 26% by day 15 on ARS staining
compared to PCL/Collagen nanofibers and showing cuboidal morphology by expressing osteocalcin. These
results confirmed that the specifically fabricated PCL/HA composite nanofibrous substrate enhanced the
differentiation of MSCs into osteogenesis.
© Koninklijke Brill NV, Leiden, 2012
Keywords
Electrospraying, electrospinning, hydroxyapatite, polycaprolactone, mesenchymal stem cells, ALP activ-
ity, mineralization
* To whom correspondence should be addressed. Tel.: (65) 6516 4272; Fax: (65) 6773-0339; e-mail:
nnijrv@nus.edu.sg
© Koninklijke Brill NV, Leiden, 2012 DOI:10.1163/156856212X629845

J. Venugopal et al. / Journal of Biomaterials Science 24 (2013) 170–184 171
1. Introduction
The interaction of cells and biocomposite nanofibrous substrates has been a novel
and exciting approach in tissue engineering for transplantation therapy of cer-
tain diseases. Mesenchymal stem cells (MSCs) are the popular cell source with
nanofibers for fabricating scaffolds to resolve the problems of preparing viable tis-
sues for the replacement of diseased organs or tissues. MSCs have the advantages
over hematopoietic stem cells capable of significant expansion and can be directed
towards adipogenic, chondrogenic, neurogenic, myogenic and osteogenic lineages
[1–4]. They are known to migrate in vivo to the sites of inflammation and home in a
variety of tissues playing an active role in tissue repair and regeneration through the
secretion of chemokines, as well as differentiation into desired cell types [5]. The
differentiation process has been reported to be likely initiated by MSCs sensing
changes in their micro-environment as they engraft the tissue [6]. An understand-
ing of cell–material interactions helps in the design of tissue-engineered medical
products with beneficial surface energy, modulus, crystallinity, surface roughness
and surface chemistry properties. These properties can influence a variety of cell re-
sponses to biomaterials. The cell responses include changes in surface adhesion that
activate biochemical pathways regulating cellular proliferation, differentiation and
survival. For example, surface hydrophilicity can affect cell adhesion and prolifer-
ation [7] and regulate expression of specific cell surface integrins [8] or modulate
the adsorption of extracellular matrix (ECM) proteins by the substrates [9, 10].
Nanofiber matrices with different fiber diameters exhibit a wide range of surface
properties (hydrophilicity, hydrophobicity), superior mechanical properties (stiff-
ness, tensile strength) and porosity compared with the other forms of materials.
Fiber properties such as surface functionality, electrical conductivity, weight and
tensile strength are greatly determined by the specific polymers used to fabri-
cate nanofibers [11]. Fabrication of nanostructured implants and scaffolds is one
of the most promising strategies to increase osseointegration and reduce implant-
associated infection in current orthopedic biomaterials. Nanostructured surfaces
have been shown to influence protein adsorption, cell adhesion, morphology and
differentiation, as well as the production and secretion of ECM molecules [12].
The process of differentiation has been subdivided into three stages: proliferation,
ECM synthesis, maturation and mineralization. Osteogenic activation of MSCs re-
quires the presence of β-glycerophosphate, ascorbic acid and dexamethasone [13].
Mineralized nodules are the end product of proliferation and differentiation of os-
teoprogenitor cells. Thus, the rationale for developing orthopedic applications via
nanofibrous substrate stems from the structure of bone itself, which is a composite
nanostructured tissue composed of hydroxyapatite (HA) crystals (20–80 nm long
and 2–5 nm thick) and collagen fibrils with diameters of less than 500 nm [14]. The
fact that bone cells are naturally adapted to nanophase materials has led to a motiva-
tion of studies investigating the response of bone cells to synthetic nanostructures.
Bone regeneration requires materials capable of providing mechanical and chem-
ical signals to promote biomineralization. Bioactive materials, such as HA, have
172 J. Venugopal et al. / Journal of Biomaterials Science 24 (2013) 170–184 3
become increasingly useful as a new generation of biomaterials [15, 16]. HA is

the mineral component of bone, and has been shown to be both osteoinductive
and osteoconductive, stimulating biomineralization in vitro and in vivo. Recent
studies on the mechanical properties of implant materials have shown that rough
surface nanocrystalline HA increases the adhesion, proliferation and differentiation
of osteoblasts in culture [17, 18]. Osteoblasts have been shown to undergo in vitro
biomineralization as evident by the formation of mineralized bone nodules. These
nodules are 35–100 µm in diameter and contain 3D aggregates of cells and collagen
ECM. Mineralization can occur via matrix vesicles which can be observed associ-
ated with ECM in bone nodules [19]. Our findings on osteo-progenitor cell response
to nanotopographies have presented compelling evidence that nanotopography is an
important factor in mesenchymal differentiation and in the design of electrospray

HA nanoparticles on PCL nanofibers and ultimately implant structures for clinical
application. In this study, we investigated the influence of electrospray deposition of
HA nanoparticles on PCL nanofibers for directing MSCs to an osteogenic lineages
for bone tissue regeneration.
2. Materials and Methods

2.1. Materials
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (HAM), fetal bovine
serum (FBS), antibiotics, trypsin-EDTA, β-glycerophosphate, ascorbic acid, dex-
amethasone, alkaline phosphatase activity kit, polycaprolactone (PCL, 80 kDa),
Alizarin Red-S, Histopaque-1077, cetylpyridinium chloride, 1,1,1,3,3,3-hexafluoro-
2-propanol (HFP), chloroform and methanol were purchased from Sigma-Aldrich.
Atelocollagen purchased from Koken. CellTiter 96 AQueous One solution was
purchased from Promega. Crystalline hydroxyapatite was a gift provided by the
Department of Metallurgical and Materials Engineering, Indian Institute of Tech-
nology (Chennai, India) [20].
2.2. Fabrication of Nanofibrous (Electrospray) Scaffolds
PCL (8%, w/v) was dissolved in methanol/chloroform (1:3) and PCL/Collagen was
dissolved in HFP in a ratio of 1:1 to produce an 8% (w/w) solution. HA was soni-
cated (Vibra Cell™, Sonics & Materials) for 30 min (30% amplitude) to produce a
nanoparticular suspension in the solvent. For electrospinning, the polymer solutions
of PCL/Collagen were prepared separately and fed into a 3-ml standard syringe
attached to 27G blunt needle using a syringe pump (NE-1000, New Era Pump Sys-
tems) at a flow rate of 1.5 ml/h with an applied voltage of 12.5 kV (Gamma High
Voltage Research). On application of high voltage the polymer solution was drawn
into fibers, collected on 15-mm cover slips, spread on a collector plate wrapped with
aluminum foil, kept at a distance of 12.5 cm from the needle tip and subsequently
used for cell-culture studies.
For collecting PCL/HA nanofibers by the ‘spray’ method, a rotating mandrel

(20 rpm/min) set-up was used instead of collector plate (Fig. 1). The cylinder
was wrapped in aluminum foil and 15 mm cover slips were stuck on the rotat-
ing cylinder. PCL solution (8%) was fed in a syringe pump (1.5 ml/h) with the
same parameters used for electrospinning while in another, a nanoparticulate solu-
tion of 4% HA in methanol was fed at a rate of 0.5 ml/h and applied voltage of
9 kV was provided for spraying HA nanoparticles on the surface of PCL nanofibers
being formed on the rotating cylinder simultaneously. These nanofibers were dried
overnight under vacuum and used for characterization and cell-culture studies.
2.3. Characterization of Nanofibrous Scaffolds

The surface morphology of electrospun nanofibrous scaffolds were analyzed under

a field emission scanning electron microscope (FESEM, FEI-QUANTA 200F) at
an accelerating voltage of 10 kV, after sputter coating with gold (JEOL JFC-1200
fine coater). Diameter of electrospun nanofibers were analyzed from the SEM im-
ages using image analysis software (Image J, National Institutes of Health, USA).
Fourier Transform Infrared spectroscopic analysis (FT-IR) of electrospun nanofi-
brous scaffolds was performed on an Avatar 380 (Thermo Nicolet) over a range of
400–3800 cm−1 at a resolution of 2 cm−1 . Air plasma treatment was conducted
on PCL/HA nanofibrous scaffolds by electrode less radio frequency glow discharge
plasma cleaner (Model: PDC-001, Harrick Scientific). The samples, placed in a
glass Petri dish, were put in the chamber of the plasma cleaner. Plasma discharge
was applied to the samples for 2 min with the radio frequency power set at 30 W
under vacuum. Air plasma treatment modified the PCL/HA nanofiber surfaces to
increase surface charge, wettabilty and topography to encourage cell adhesion, pro-
liferation and differentiation.
2.4. Isolation and Expansion of MSCs
The equine bone marrow was collected from equine hospitals (Malaysia) accord-
ing to the ethical guidelines. The bone marrow was aspirated from the sternum
and ACD-A (Haemonetics) added to prevent coagulation. The samples were pro-
cessed within 24 h. Bone marrow was diluted in phosphate-buffered saline (PBS)
4 times and layered over 10 ml of histopaque solution (30/10 ml) and centrifuged
at 400 × g for 20 min to allow cell separation. The middle layer (buffy coat) con-
taining mononuclear cells were transferred to a 50-ml centrifuge tube and washed
twice with PBS. The mononuclear cells were seeded at 1.5 × 105 cells/cm2 in
DMEM/F12 nutrient medium (1:1), 10% FBS, 1× antibiotic mixture. Cells were
incubated at 37°C in a humidified atmosphere containing 5% CO2 in air for 15
days after seeding, and culture medium was changed once in every 5 days. The
attached MSCs were trypsinized and re-seeded into 75-cm2 cell-culture flasks and
medium was changed twice weekly. The MSCs were trypsinized at confluence and
passaged twice before being used for differentiation assays.
174 J. Venugopal et al. / Journal of Biomaterials Science 24 (2013) 170–184
2.5. Osteogenesis
Mesenchymal stem cells were seeded at a density of 10 000/cm2 in 24-well
plates containing PCL/Collagen, PCL/HA nanofibrous scaffolds and tissue-culture
plate (TCP) as a control. MSCs differentiation was induced with a DMEM/F12
medium supplemented with 10% FBS, β-glycerophosphate (10 mM), ascorbic acid
(5 µg/ml) and dexamethasone (10 nM). Osteogenesis was assessed after 15 days by
observation and testing for alkaline phosphatase activity and mineralization with
Alizarin Red-S assay and osteocalcin staining.
2.6. Cell Proliferation
Cell adhesion and proliferation on the nanofibrous substrates were determined us-
ing the colorimetric MTS assay (CellTiter 96 AQueous One solution). The reduction
of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2(4sulfophenyl)-2H tetrazolium) in MTS to form purple formazan crystals by
the dehydrogenase enzymes secreted by mitochondria of metabolically active cells
formed on the basis of this assay. The formazan dye shows the absorbance at 492 nm
and the amount of formazan crystals formed is directly proportional to the number
of cells. For MTS assay, samples were rinsed with PBS to remove unattached cells
and incubated with 20% MTS reagent in serum-free medium for a period of 3 h at
37°C. Absorbance of the obtained dye was measured at 490 nm using a spectropho-
tometric plate reader (FLUOstar OPTIMA, BMG Lab Technologies).
2.7. Cell Morphology
The morphology of cultured MSCs differentiates into osteogenic lineages on the
PCL/Collagen and PCL/HA nanofibrous scaffolds were studied after 15 days of
cell culture by FESEM. The scaffolds were rinsed twice with PBS and fixed in 3%
glutaraldehyde for 3 h. They were then rinsed in deionised water and dehydrated
with increasing concentrations of ethanol (50, 70, 90, 100%) twice for 10 min each.
The final washing with 100% ethanol was followed by treating the specimens with
hexamethyldisilazane (HMDS). Traces of HMDS were removed by air-drying the
samples in fume hood. Finally, the scaffolds were sputter coated with gold and
observed under SEM to observe the morphology of osteogenic cells.
2.8. Alkaline Phosphatase (ALP) Activity
ALP activity of MSCs differentiation into osteogenic lineages was measured using
the Alkaline Phosphate Yellow Liquid substrate system for ELISA (Sigma Life
Sciences). In this reaction, ALP catalyzes the hydrolysis of a colorless organic
phosphate ester substrate, p-nitrophenylphosphate (pNPP), to a yellow product,
p-nitrophenol, and phosphate. Nanofibrous scaffolds with osteogenic cells were
washed twice with PBS and 400 µl pNPP in liquid form was added to cells on
the scaffolds and incubated for 30 min until the color of solution became yellow.
The reaction was stopped by addition of 100 µl of 2 M NaOH solution following
which the yellow color product was aliquoted in 96-well plates and read in a spec-
trophotometric plate reader at 405 nm.
2.9. Mineralization of Osteogenic Cells
Alizarin Red-S (ARS) is a dye that binds selectively calcium salts and is widely
used for mineral staining. Nanofibrous scaffolds with osteogenic cells were washed
twice with PBS and fixed in ice-cold 70% ethanol for 1 h. These constructs were
then washed twice with dH2 O and stained with ARS (40 mM) for 20 min at room
temperature. After several washes with dH2 O, the scaffolds were observed under
an optical microscope and images were taken using image software (Leica DM
IRB). Later, the stain was extracted with 10% cetylpyridinium chloride for 1 h and
absorbance of the collected dye was read at 540 nm in a spectrophotometer (Thermo

Spectronics).
2.10. Immunofluorescent Staining for Osteocalcin
The osteogenic differentiation of MSCs was confirmed using immunofluorescent
staining by employing osteoblast specific marker protein osteocalcin. On day 15 of
the cell-culture study, cells were fixed in 100% ice-cold methanol for 15 min. The
samples were washed with PBS for 15 min and incubated in 0.5% Triton X-100 so-
lution for 5 min to permeabilize the cell membrane. Non-specific sites were blocked
by incubating the cells in 3% BSA (Sigma) for 1 h. The samples were incubated
with the osteoblast-specific marker protein osteocalcin (Sigma) in the dilution of
1:100 for 90 min at room temperature. Further the secondary antibody Alexa Fluor
594 (Invitrogen; Red) in the dilution of 1:250 was added for 60 min. The samples
were washed with PBS thrice to remove the excess staining and then incubated with
DAPI in the dilution of 1:5000 for 30 min at room temperature. The samples were
then removed and mounted over a glass slide using Vectashield mounting medium
and examined under a fluorescent microscope (Olympus FV1000).
2.11. Statistical Analysis
Data were expressed as mean ± SD. Levels of significance were calculated using
Student’s t-test (n = 6). Differences were considered statistically significant at P
0.05.
3. Results
3.1. Characterization of Nanofibers
Porosity and increased surface area of nanofibers are important parameters in tissue-
engineering applications. In the present study, these are achieved by electrospraying
of HA nanoparticles on electrospun PCL nanofibers which elicited a favorable
response towards MSC differentiation into oesteogenic lineages. Figure 1 shows
electrospraying of HA nanoparticles on nanofibers in a rotating drum; this system
Figure 1. Diagrammatic representation of the electrospraying and electrospinning system for fabricat-
ing PCL/HA nanofibers. This figure is published in colour in the online edition of this journal, which
can be accessed via http://www.brill.nl/jbs
Figure 2. FESEM images of electrospun composite nanofibers (a) PCL/Collagen (275 ± 56 nm, scale
bar = 20 µm) and PCL/HA nanofibers (420 ± 15 nm, scale bar = 50 µm).
was custom designed in our laboratory. PCL nanofibers are electrospun on the ro-
tating drum together with nanohydroxyapatite particles (52 nm) electrosprayed on
them gave diameters of 420 ± 15 nm (Fig. 2). The air plasma treatment to PCL/HA
nanofibers (2 min) increased the wettability suitable for cell adhesion and prolifer-
ation. The PCL/Collagen nanofibers fabricated separately for comparison of MSC
differentiation had fiber diameters of 275 ± 56 nm.
PCL, PCL/Collagen and PCL/HA nanofibers were further characterized by FT-
IR analysis (Fig. 3) to confirm HA nanoparticles present on the nanofibers. The
amide I peak was observed at 1632 cm−1 (C=O stretching), the amide II peak at
Figure 3. FT-IR analysis of PCL, PCL/Collagen and PCL/HA nanofibers. This figure is published in
colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs
1525 cm−1 (N–H deformation) and the amide III peak around 1210 cm−1 (N–H de-
formation) in PCL/Collagen. The characteristic vibrations of the phosphate group of
HA appeared at 1033, 603 and 560 cm−1 [21, 22]. PCL/HA nanofibrous substrate,
calcium (Ca2+ ) and phosphate ions (PO4 3− ) existed at the HA nanocrystal sur-
face. If self-organization had occurred from electrostatic interactions between such
ions and functional groups, e.g., Ca2+ vs. COO− or PO4 3− vs. NH3 + , their influ-
ence would be detectable with infrared spectroscopy. These composites expressed
PO4 3− stretch (1045 cm−1 ) and bending at 575 cm−1 , typical of HA present in the
nanocomposite of PCL/HA. The phosphate ions (PO4 3− ) are principal molecular
components of HA giving to IR absorbance in the 570–1210 cm−1 region (Fig. 3).
The major peaks of OH ions are identified by the observation of broad band from
about 3800 to 2700 cm−1 .
3.2. MSCs Differentiation into Osteogenesis
The proliferation rate of MSCs on PCL/Collagen nanofibers with supplements
increased significantly (up to 12%, P 0.05) on day 15 compared to PCL/HA
nanofibers (Fig. 4). The morphology of MSCs showed no significant difference in
TCP, PCL/Collagen compared to PCL/HA nanofibrous scaffolds (Fig. 5). Cell pro-
liferation varied in different scaffolds with higher rates in PCL/Collagen nanofibers
compared to TCP and PCL/HA nanofibers. ALP activity, determined using the ARS
assay, showed the secretion of minerals by MSCs for bone tissue regeneration.
ALP activity increased by 20% in PCL/HA substrates on day 10 and compara-
tively decreased by day 15 for the differentiation of MSCs into osteogenic lineages
(Fig. 6). Figure 7 shows ARS staining for the mineralization on MSCs cultured on
Figure 4. MTS assay for MSCs proliferation in composite nanofibrous scaffolds and TCP (control).
Asterisks indicate significant difference of proliferation (P 0.05).
PCL/HA substrates on day 15, and the amount of mineral deposition was greater
than PCL/Collagen nanofibers. ARS analysis showed that the HA in PCL/HA sub-
strate stimulated significantly (P 0.05) for the differentiation of MSCs with the
secretion of minerals on day 15 by an increase of 26% compared to PCL/Collagen
nanofibers (Fig. 8). The expression of osteocalcin was observed in MSCs differ-
entiated cells in Fig. 9, the cell morphology was changed to a cuboidal shape in
Fig. 9c, compared to all other samples. These results proved that the PCL/HA in-
ducing MSCs differentiating into osteogenic lineage for bone tissue engineering.
4. Discussion
There is a growing need for improved synthetic biomedical materials for bone
replacement in patients due to the increasing average life-span of the human popula-
tion and increased expectancy of the quality of life into old age [23]. One of the most
extensively investigated nanostructures for orthopedic tissue-engineering applica-
tion is composite polymeric nanofibrous scaffolds. Improving the porous structure
of the nanofibrous scaffold provides a larger surface area for cell attachment and
sufficient space for nutrient transportation. Electrospraying is a well-established
process expanding over a century and still being utilized increasingly in several ap-
plications. The process largely hinges on the formation of an electrically-induced
jet, which subsequently breaks up to form fine droplets in the nano- and micro-
meter range, depending on the processing conditions and properties of the medium
[24]. In the present study, electrospraying of HA nanoparticles on electrospun
PCL nanofibers elicited a favorable response towards MSC differentiation into os-
Figure 5. FESEM images showing the morphology of MSCs differentiated into osteogenic lineage on
(a) TCP, (b) PCL/Collagen and (c) PCL/HA (mineralization).
Figure 6. ALP activity of MSCs directing to osteogenic lineages on composite nanofibrous scaffolds
and TCP (∗ P 0.05).
teogenic lineages. PCL/HA (420 ± 15 nm) composite nanofibrous scaffolds (Fig. 2)

expressed PO4 3− stretch (1045 cm−1 ) and bending 575 cm−1 , typical of HA present
in the nanocomposite of PCL/HA. The phosphate ions (PO4 3− ) are the principal
molecular components of HA yielding an IR absorbance at 570–1210 cm−1 region
Figure 7. ARS staining of mineral deposition observed in MSCs differentiated into osteogenic
lineage (a) TCP, (b) PCL/Collagen and (c) PCL/HA (magnification 100×, scale bar = 100 µm).
This figure is published in colour in the online edition of this journal, which can be accessed via
http://www.brill.nl/jbs
Figure 8. Quantification of mineral deposition in MSCs differentiated into osteogenic lineage mea-
sured by the method of Alizarin Red-S staining (∗ P 0.05).
Figure 9. Osteoblast marker protein osteocalcin expression on nanofibrous scaffolds. (a) TCP,
(b) PCL/Collagen were showing the normal elongated morphology of MSCs, (c) PCL/HA nanofi-
brous scaffold induced differentiation of MSCs into osteogenic lineage showing cuboidal morphology
and expression of osteocalcin. This figure is published in colour in the online edition of this journal,
which can be accessed via http://www.brill.nl/jbs
to initiate mineralization (Fig. 3). PCL/HA nanofibrous scaffolds were treated with
air plasma to increase the surface roughness and wettability of the substrate. The
results of the present study are consistent with other studies that showed increased
hydrophilicity or surface energy of biomaterials to improve cell adhesion and pro-
liferation [25, 26]. These electrosprayed HA nanoparticles directly come in contact
with MSCs to initiate differentiation into osteogenic lineages for the mineralization
in bone tissue engineering.
Particle size, shape and surface roughness affect cellular adhesion, prolifera-
tion and phenotype of MSCs. Specifically, cells are sensitive and respond to the
chemistry, topography and surface energy of the material substrate with which they
interact. In this respect, the type, amount and conformation of specific proteins
which adsorb onto materials surfaces subsequently modulated cell function [27, 28].
Nanohydroxyapatite, which is a ceramic mineral, has its mainstream applications in
healthcare specifically for osteological applications via the mechanism of cellular
interaction. Such nanoparticles sprayed on the surface of PCL nanofibers to fab-
ricate composites directly initiates MSCs differentiation into osteogenic lineages.
HA has been considered as a structural template for the bone mineral phase and also
a major inorganic mineral component of bone and commonly used as bioceramic
filler in polymer-based bone substitutes because of its high bioactivity and bio-
compatibility [29]. Our studies showed that the proliferation of MSCs significantly
increased up to 12% in PCL/Collagen on day 15 compared to PCL/HA nanofibers.
The results observed that the HA (4%) present in PCL/HA substrate was suffi-
cient to initiate MSC differentiation into osteogenesis in bone tissue engineering
for transplantation therapies. Collagen, a major ECM component, possesses a fi-
brous structure with fiber bundles of varying diameter (50–500 nm). When HA was
not used, PCL/Collagen and TCP required supplementation in the culture medium
to initiate osteogenic differentiation. HA possesses the effective affinity for regu-
lating cell function and promoting osteogenesis and mineralization of bone. The
nanometer size feature influences cell behavior by allowing cells to attach to diam-
eters smaller than the cell size.
In Fig. 5c one can see the different morphology and mineral deposition on the
surface of osteogenic cells, confirming that HA stimulates MSCs for differentiation
and mineralization. When MSCs were seeded on the nanopatterned surfaces show-
ing filapodial interactions they were able to specifically sense the nanotopographical
features, and their cytoskeletal arrangement was important in cell adhesion function
and differentiation into an osteogenic lineages (Fig. 5). ALP activity was observed
in PCL/Collagen and PCL/HA nanofibrous scaffolds showing higher level of ac-
tivity on day 10 and slowly decreasing after 15 days (Fig. 6). These results were
further confirmed with mineral formation on day 15 compared to day 10. Ko et
al. demonstrated maximum ALP activity at 14 days and then decreases thereafter
in hMSCs-Osteogenic medium (hMSCs-OS) cultured on poly(L-lactide) (PLA) and
PLA/DBP scaffolds [30]. ALP is one of the early osteogenic differentiation markers
of MSCs and previous studies have reported the decline in ALP activity after pro-
longed culture [31]. ALP precedes osteocalcin (OCN) in the differentiation process,
as ALP helps to prepare the ECM for deposition before the onset of mineralization
that coincides with OCN expression [32, 33]. The decrease in ALP activity observed
in our study could be attributed to the terminal osteogenic differentiation of MSCs
over 15 days of culture. Figures 7 and 8 show ARS staining for the mineraliza-
tion of MSCs cultured on PCL/HA on day 15, and the amount of mineral secretion
increased significantly (P 0.05) to 26% compared to PCL/Collagen substrates.
Mineralization of MSCs is one of the key processes for bone tissue regeneration
and our results suggest that the incorporation of HA up-regulated the mineraliza-
tion process, yielding mineral deposition as seen in Figs 5c and 7c. OCN plays a
significant role in modulating mineralization, as it has glutamic-acid-rich regions
with strong binding affinities to both Ca2+ and HA [34]. OCN is an ECM protein
related to bone formation [35] and unlike other bone-related proteins, such as os-
teonectin and osteopontin, which are also present in other tissues, osteocalcin is
bone specific protein [36–38]. The present study shows that the osteogenic differ-
entiation was confirmed by the expression of OCN in PCL/Collagen and PCL/HA
nanofibrous scaffolds. Figure 9c shows the morphology of MSCs on PCL/HA scaf-
folds, exhibiting the characteristic cuboidal morphology of osteoblasts, indicating
osteogenic differentiation of MSC on these scaffold, compared to PCL/Collagen
nanofibers (Fig. 9b). Venugopal et al. demonstrated the efficacy of biocomposite
nanofibrous scaffolds in enhancing osteoblast adhesion, proliferation and mineral-
ization [26, 39]. The enhanced performance has been attributed to the ability of the
matrix to closely mimic the structure of natural ECM [16, 26]. Studies attributed
the enhanced performance of osteoblasts on fibers with diameters of >100 nm,
to their dimensional similarity to HA crystals found in bone [40]. The observed re-
sults prove that the MSCs undergo osteogenic differentiation through a well-defined
pathway secreting ECM and expressing osteoblast-associated genes and mineral-
ization in vitro, by the induction of HA. The observed results of morphological
evidence, ARS staining, OCN expression showed the HA nanoparticles directing

MSCs differentiation into osteogenic lineages when used with PCL/HA nanofibrous
scaffolds for bone tissue regeneration.
5. Conclusions
Electrospraying is a simple, economical, room temperature process, capable of pro-
ducing surface coating of nanoparticles that are helpful for direct contact of cells for
adhesion, proliferation and differentiation. Increased surface area and porosity pro-
vided by nanofibrous substrate in the presence of HA nanoparticles enhances cell
adhesion and differentiation. The combination of bioactive and selective hydroxya-
patite surface coating of nanofibers could provide both physical and chemical cues
to enhance and direct MSCs to osteogenic lineages. Biocomposites of PCL/HA
nanofibrous substrate have different pore structures and the loose peripheral regions
of scaffolds are favorable for cell infiltration and provided enough space for MSC
ingrowth and differentiation into osteogenic lineage for in vitro bone tissue forma-
tion. These results confirmed that the electrosprayed hydroxyapatite nanocrystals
for coating PCL nanofibers can be used as an alternate composite biomaterial for
bone tissue regeneration.
Acknowledgements
This study was supported by NRF-Technion (R-398-001-065-592), NUSNNI, Fac-
ulty of Engineering, National University of Singapore and StemLife Sdn Bhd,
50450 Kuala Lumpur, Malaysia.
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Electrosprayed Hydroxyapatite on Polymer Nanofibers to

J. Venugopal a,∗ , R. Rajeswari a , M. Shayanti a , Sharon Low b , Ariff Bongso c ,

National University of Singapore, Singapore

© Koninklijke Brill NV, Leiden, 2012 DOI:10.1163/156856212X629845

become increasingly useful as a new generation of biomaterials [15, 16]. HA is

important factor in mesenchymal differentiation and in the design of electrospray

2. Materials and Methods

For collecting PCL/HA nanofibers by the ‘spray’ method, a rotating mandrel

2.3. Characterization of Nanofibrous Scaffolds

The surface morphology of electrospun nanofibrous scaffolds were analyzed under

2.4. Isolation and Expansion of MSCs

absorbance of the collected dye was read at 540 nm in a spectrophotometer (Thermo

teogenic lineages. PCL/HA (420 ± 15 nm) composite nanofibrous scaffolds (Fig. 2)

evidence, ARS staining, OCN expression showed the HA nanoparticles directing

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