mimicking nanofibrous hybrid bone substitute for mesenchymal stem cells differentiation into...
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MimickingNanofibrousHybridBoneSubstituteforMesenchymalStemCellsDifferentiationintoOsteogenesis
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JayaramaReddyVenugopal
NationalUniversityofSingapore
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NationalUniversityofSingapore
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SuganyaShanmugavel
UniversityofReading
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SeeramRamakrishna
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Full Paper
Biocompatible polycaprolactone/poly-
(a,b)-DL-aspartic acid/collagen nanofibrous
scaffolds are fabricated by electrospinning
and nanohydroxyapatite (n-HA) is depos-ited by a calcium phosphate dippingmethod. They are characterized for fiber
morphology, hydrophilicity, porosity, andtensile properties. Mesenchymal stem cell(MSCs) cultures on these nanofibrousscaffolds to facilitate cell adhesion, prolif-eration, mineralization, and osteogenicdifferentiation.
Mimicking Nanofibrous HybridBone Substitute for MesenchymalStem Cells Differentiation intoOsteogenesis
C. Gandhimathi, J. Reddy Venugopal,*R. Ravichandran, S. Sundarrajan,S. Suganya, S. Ramakrishna
Macromol. Biosci. 2013, 13, 000–000
Early View Publication; these are
NOT the final page numbers,
use DOI for citation !!
Contents
mabi.201200435C
Full Paper
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Mimicking Nanofibrous Hybrid BoneSubstitute for Mesenchymal Stem CellsDifferentiation into Osteogenesis
mabi.201200435
Chinnasamy Gandhimathi, Jayarama Reddy Venugopal,*Rajeswari Ravichandran, Subramanian Sundarrajan,Shanmugavel Suganya, Seeram Ramakrishna
Mimicking hybrid extracellular matrix is one of the main challenges for bone tissue engineering(BTE). Biocompatible polycaprolactone/poly(aa, bb)-DL-aspartic acid/collagen nanofibrous scaf-folds were fabricated by electrospinning and nanohydroxyapatite (n-HA) was deposited bycalcium phosphate dipping method forBTE. Human mesenchymal stem cells(hMSCs) were cultured on these hybridscaffolds to investigate the cell prolifer-ation, osteogenic differentiation byalkaline phosphatase activity, mineraliz-ation, double immunofluorescent stain-ing using CD90 and expression ofosteocalcin. The present study indicatedthat the PCL/PAA/collagen/n-HA scaf-folds promoted greater osteogenic differ-entiation of hMSCs, proving to be apotential hybrid scaffolds for BTE.
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1. Introduction
For an engineered osteogenic substitute several factors
have to be taken into consideration including, scaffolds
fabrication,[1] growth factor incorporation,[2] and chemical
composition.[3] Autografts and allografts have long been
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J. Raddy Venugopal, C. Gandhimathi, R. Ravichandran,S. Sundarrajan, S. Suganya, S. Ramakrishna&
Q1Please verify the corresponding author’s e-mail.&Center for Nanofibers and Nanotechnology, Nanoscience andNanotechnology Initiative, Faculty of Engineering, NationalUniversity of Singapore, SingaporeFax: 65 6773 0339; E-mail: [email protected]. Ravichandran, S. RamakrishnaDepartment of Mechanical Engineering, National University ofSingapore, Singapore
� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlin
Early View Publication; these are NOT
used for bone reconstructive surgery. However they have
disadvantages pertaining to inadequate tissue accessibil-
ity, risk of disease transmission, and immunological
response.[4] The ideal scaffolds for bone tissue engineering
(BTE) should be bioactive, biocompatible, biodegradable,
porous having a large surface area to volume ratio,
mechanically strong and capable of being fabricated into
preferred shapes.[5] Bone tissue scaffolds (BTSs) meet some
of the specific necessities such as porosity and adequate
pore size to facilitate cellular activities and diffusion of
both oxygen and nutrients throughout the scaffolds.[6]
Potential biomaterials must transmit the appropriate
signal to direct the process of osteogenesis such as cell
attachment, proliferation, differentiation, and mineraliza-
tionof extracellularmatrix (ECM).[7] BTE isbasedonthe idea
of seeding patients own cells during in vitro culture onto
a scaffold prior to transplantation into the defect site to
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C. Gandhimathi, J. Reddy Venugopal, R. Ravichandran, S. Sundarrajan, S. Suganya, S. Ramakrishna
form bone tissue.[8] Most of these material necessities are
fulfilled by polycaprolactone (PCL), which are biodegrad-
able andbiocompatiblewithgoodmechanical properties.[9]
Theyundergoenzymaticdegradation throughhydrolysis of
its esterbondsby lipases.[10] Themainapplicationsof PCL in
biomedical field includes tissue engineered skin,[11] drug
delivery,[12] axonal regeneration,[13] dermal substitute,[14]
and osteoblasts for BTE.[15] In the present study, poly(a,b)-
DL-aspartic acid (PAA) was employed as the cell binding
moiety along with PCL. PAA is a biodegradable and
completely synthetic water soluble ionic polymer with a
carboxylate contentmuch higher than poly(glutamic acid).
Polypeptide moieties like PAA can bond to multiple ECM
proteins and growth factors in the medium, which may
further assist cell proliferation and migration into the
scaffolds. Collagen fibers are the main structural protein
comprising upto 30% dry weight of bone and 90–95%
organic non-mineral component in bone.[16,17] Studies
have proved that calcium phosphate-based materials like
hydroxyapatite (HA) provide rough surfaces that are
conductive to bone cell addition and proliferation termed
osteoconductivity.[18]HA is themajor inorganic component
of the bone matrix and has several advantages including
its specific affinity toward many adhesive proteins and
its direct involvement in bone cell differentiation and
mineralization.[18] Lee et al. reported PCL grafted HA
nanocomposites improved adhesion, proliferation, and
protein adsorption of fibroblasts compared to non-PCL
grafted HA and pure PCL scaffolds.[19] In the present study,
the in vitro response of hMSCs to the surface mineralized
PCL/PAA/Col/n-HA hybrid nanofibrous scaffolds were
investigated, in terms of cell adhesion, proliferation, and
further osteogenic differentiation and mineralization for
bone tissue regeneration.
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2. Experimental Section
2.1. Materials
Humanbonemarrowderivedmesenchymalstemcells (MSCs)were
obtained from Lonza (USA). Dulbecco’s modified eagle’s medium
(DMEM)/Nutrient Mixture F-12 (HAM), fetal bovine serum (FBS),
antibiotics, and trypsin-EDTA were purchased from GIBCO
Invitrogen (USA). CellTiter 96Aqueous one solutionwas purchased
from Promega, Madison (WI, USA). PCL (Mw 80 000),& Author:
Please add units for molecular weight throughout & 1,1,1,3,3,3-
hexafluoro-2-propanol (HFP), Alizarin Red-S (ARS), and Cetylpyr-
idinium chloride were purchased from Sigma. Collagen type I was
obtained from Koken Co, Tokyo (Japan).
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2.2. Fabrication of Nanofibrous Scaffolds
Polycaprolactone (PCL) pellets were dissolved in HFP at 10% w/v
and PCL/PAA solution was prepared 90:10w/w at the concentra-
tion of 10% in HFP. PCL/PAA/Col solution was also prepared at the
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ratio of 80:10:10 in HFP at the same concentration of 10%. All
solutionsweremagneticallystirredat roomtemperatureovernight
for better distribution and homogenization. The polymer solution
was then loaded into a 3ml standard syringe attached to 27Gblunt
needle using a syringe pump (KD 100 Scientific Inc., Holliston, MA,
USA) at a constant flow rate of 1.5 ml �h�1 with a high voltage
electric field of 15–16kV (DC high voltage power supply from
Gamma high-voltage research, Florida, USA). The electrospun
nanofibers were collected on aluminum foil wrapped on a flat
collector plate kept at a distance of 15.5 cm between the tip of the
spinneret and collector plate. Nanofibers are collected on 15mm
coverslips for cell culture experiments. Fabricated electrospun
nanofibrous scaffolds were consequently dried overnight under
vacuum oven to eliminate residual solvents and used for further
studies. Biomineralization procedure was then carried on the
PCL/PAA/Col scaffolds to precipitate n-HA by calcium phosphate
dipping method.[20]
2.3. Characterization of Nanofibrous Scaffolds
The fibermorphologywas analyzed under field emission scanning
electron microscope (FESEM) (FEI – QUANTA 200F, The Nether-
lands) at an accelerating voltage of 10 kV, after the specimenswere
coated with platinum using a sputter coater (JEOL, JFC-1200 Fine
Coater, Japan). EDXwas carried out by using Hitachi S4300 FESEM
analyzer. For measuring the fiber diameter of electrospun
nanofibers from the FESEM images, n¼10 fibers were selected at
random on each of the scaffolds. The average fiber diameter was
then calculated along with standard deviation (SD) by using
image analysis software Image J (Image Java, National Institutes
of Health, USA). Tensile properties of electrospun nanofibrous
scaffolds were determined with a table-top micro-tester (Instron
3345, USA) using load cell of 10N capacities. Test specimens of
dimension 10mm� 20mmwere tested at a cross head speed of 10
mm �min�1 at ambient conditions of 25 8Cand75%humidity.[21,22]
The pore size distribution and bubble point measurements of the
nanofibrous scaffolds were determined by using a capillary flow
porometer (PorousMaterials Inc, USA). Hydrophilic or hydrophobic
nature of the electrospun nanofibrous scaffolds was analyzed by
sessile drop water contact angle measurement using VCA optima
surface analysis system (AST Products, Billerica, MA, USA).
Functional groups present in the scaffolds were determined using
Fourier transform infrared (FTIR) spectroscopic analysis on Avatar
380 spectrometer (Thermo Nicolet, Waltham, MA, USA) over a
range of 400–4000 cm�1 at a resolution of 8 cm�1.
2.4. Human Mesenchymal Stem Cells (hMSCs)
Human mesenchymal stem cells (hMSCs) (Lonza, USA) were
cultured in DMEM containing 10% FBS with 1% antibiotics in a
75 cm2 cell-culture flask. The hMSCs were incubated at 37 8Chumidified atmosphere containing 5% CO2 for 7 d and the culture
mediumwas changed once in every 3 d. The cultured cells (passage
4) were trypsinized by trypsin-EDTA and replated after counting
with trypan blue using hemocytometer. The electrospun nanofi-
bers were collected on coverslips of 15mm diameter were placed
in a 24-well plate with a stainless steel ring to prevent lifting of
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Mimicking Nanofibrous Hybrid Bone Substitute for Mesenchymal Stem Cells Differentiation
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nanofibers. The fibers were sterilized under UV light for 3 h and
the scaffolds were again sterilized with 70% ethanol for 30min
and washed thrice with phosphate buffered saline (PBS) for
15min each in order to remove any residual solvent and
subsequently immersed in complete medium overnight before
cell seeding. The hMSCswere seeded on the electrospunnanofibers
at a cell density of 10 000 cells/well and tissue culture polystyrene
(TCP) was used as a control.
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2.5. Cell Proliferation
The cell proliferation was determined using the colorimetric MTS
assay (cell titer 96 Aqueous one solution Promega, Madison, WI,
USA). The principle of this assay is that the reduction of
yellow tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2 (4 sulfophenyl)-2H tetrazolium]& Author:
Please check the presentation of compound.& in MTS to form
purple formazan crystals by dehydrogenase enzymes secreted
by mitochondria of metabolically active cells. The formazan dye
shows the absorbance at 490nm and the quantity of formazan
crystalsproduced isdirectlyproportional to thenumberof livecells.
After culturing thecells foraperiodof 7, 14, and21d, themediawas
removed from the 24 well plates and the scaffolds were washed
with PBS to remove unattached cells. The scaffolds were then
incubated in a 1:5 ratio mixture of MTS reagent in serum free
DMEMmedium for 3h at 37 8C in 5%CO2 incubator. Thereafter, the
samples were pipetted into 96well plates and the absorbancewas
measured at 490nm using a microplate reader (Fluostar optima,
BMG Lab Technologies, Germany).
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2.6. Cell Morphology
The cell morphology was analyzed using FESEM (Hitachi S4300,
FESEM). After 21 d of seeding cells on the scaffolds, the media was
removed fromculturewells and the cell scaffoldswere rinsed twice
with PBS for 15min to remove non-adherent cell and then the
samples were fixed with 3% glutaraldehyde in PBS for 3 h at room
temperature. The scaffolds were again rinsed in distilled water for
15min and then dehydrated with increasing concentrations of
ethanol (30, 50, 70, 90, and 100% v/v) for 10min. Finally the cell
scaffolds were treated with hexamethyldisilazane (HMDS, Sigma)
solution and air dried in fume hood overnight. Dried cellular
constructs were coated with platinum in an automatic sputter
coater and the cell morphology was observed under FESEM at an
accelerating voltage of 10 kV. Additionally, the electron beamwas
used to scan small areas to obtain compositional information from
well-defined regions of the nanofibrous substitute by energy
dispersive X-ray analysis (EDX).
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2.7. Immunofluorescent Staining for Osteocalcin
(OCN)
Osteogenic differentiation of hMSCs was confirmed using
immunofluorescence staining by employing both hMSCs specific
marker protein CD 90 and osteoblast specific marker protein OCN.
The cells were primarily fixed with 100% ice-cold methanol for
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15min. After fixing, the scaffolds were washed with PBS once for
15minand incubated in0.5%TritonX-100 solution in PBS for 5min
to permeabilize the cellmembrane.Non-specific siteswere blocked
by incubating the cells in 3% bovine serum albumin (BSA) for 1 h.
Primary antibody hMSCs specific marker protein CD 90 (Abcam,
USA) was added in the dilution (1:100) for 90min at room
temperature. This was followed by adding secondary antibody
Alexa Fluor 488 (Invitrogen-Green) in the dilution 1:250 for 60min.
ThescaffoldswerewashedthricewithPBSandthenincubatedwith
osteoblast specific protein OCN (Sigma) in the dilution 1:100 for
90min. Further secondary antibody Alexa Fluor 594 (Invitrogen-
Red)was added in the dilution 1:250 for 60min. The scaffoldswere
washed thrice with PBS for 15min to remove the excess staining.
Finally, the cells were incubated with DAPI (4,6,diamino-2 phenyl
indole) (Invitrogen Corp, Carlsbad, CA, USA) in the dilution 1:5000
for 30min. The samples were removed from 24-well plates and
mounted over glass slide using vectashieldmountingmediumand
examined under the fluorescence microscope (Olympus FV 1000).
2.8. Alkaline Phosphatase (ALP) Activity
The osteogenic differentiation of hMSCs was evaluated by
determining the ALP activity. ALP activity was measured by using
alkaline phosphate yellow liquid substitute system for ELISA
(Sigma Life Science, USA). In this reaction, ALP catalyzes hydrolyses
of colorless organic phosphate ester substitute, p-nitro phenyl
phosphate (PNPP) to a yellow product p-nitrophenol and phos-
phate.After seedingthecells foraperiodof7, 14, and21d themedia
was removed from 24-well plates and the scaffolds were washed
thrice with PBS. Then 400ml of PNPP liquid solution was added to
each scaffolds and incubated for 30min till the color of solution
becomesyellow.The reactionwas thencompletedbyadding200ml
of 2M NaOH solution. Then the yellow color product was pipetted
out into 96 wells plate and the absorbance was read at 405nm in
microplate reader.
2.9. Mineralization of Differentiated Osteogenic Cells
Alizarin red-S (ARS) staining was used to determine and quantify
themineralization of differentiated osteogenic cells.[23] On day 14,
the scaffolds seeded with hMSCs was washed three times in PBS
and fixed in 70% ice cold ethanol for 1 h. These constructs were
washed three times with distilled water and stained with ARS
(40mM) for 20min. After several washes with distilled water, the
scaffolds were observed under inverted optical microscope and
image were taken using Leica FW 4000 (versionV 1.0.2). The stain
was eluted by incubating the scaffolds with 10% cetylpyridinium
chloride for 1h. The dye was collected and absorbance read
at 540nm in spectrophotometer (Thermo Spectronics, Waltham,
MA, USA).
2.10. Statistical Analysis
Experiments were run in triplicates and the data presented were
expressed as mean� SD. Statistical differences were determined
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C. Gandhimathi, J. Reddy Venugopal, R. Ravichandran, S. Sundarrajan, S. Suganya, S. Ramakrishna
using ANOVA variance. Difference was considered statistically
significant at p� 0.05.
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3. Results and Discussion
3.1. Characterization of Nanofibrous Scaffolds
Nanofibrous substitute provides the optimum surface
architecture for promoting cell adhesion, proliferation,
and differentiation for tissue engineering applications.
FESEM micrographs of electrospun nanofibrous scaffolds
revealed beadless, porous, uniform interconnected fibrous
Figure 1. FESEM micrographs of the electrospun nanofibers. (a) PCL, (scaffolds.
Table 1. Characterization of the electrospun nanofibers for fiber diameof the nanofiber membranes.
Nanofiber
construct
Pore
size
[mm]
Porosity
[%]
Te
str
[M
PCL 0.58–1.11 85
PCL/PAA 1.32–1.54 88
PCL/PAA/Col 1.33–1.58 92
PCL/PAA/Col/n-HA 1.17–1.35 96
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structures formed under controlled conditions (Figure 1).
The fiber diameter, water contact angle, porosity, and
tensile properties of the nanofibrous scaffolds were
measured and tabulated as shown in Table 1. The fiber
diameter of the nanofibrous scaffolds were obtained
around 150–250nm and porosity estimated around 85–
96%. PCL scaffolds were found to be highly hydrophobic
with a contact angle of 129.28� 2.118; upon incorporation
of protein biomolecules of PAA, collagen, n-HA scaffolds
become more hydrophilic and the contact angle of
25.548� 5.098 for PCL/PAA/Col/n-HA scaffolds (Table 1).
The pore size and porosity as high as >90% is desirable for
the transport of nutrient and oxygen in the scaffolds
b) PCL/PAA, (c) PCL/PAA/Col, and (d) PCL/PAA/Col/n-HA nanofibrous
ter and water contact angle, pore size, porosity, and tensile strength
nsile
ength
Pa]
Tensile
break
[%]
Fiber
diameter
[nm]
Water
contact
angle [-]
3.12 38 233� 48.22 129.2� 2.11
2.46 43 222� 27.13 116.15� 1.89
1.28 39 179� 14.1 42.23� 3.36
1.07 40 197� 21.58 25.54� 5.09
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Figure 2. Tensile stress–strain curves of PCL, PCL/PAA, PCL/PAA/Col, and PCL/PAA/Col/n-HA nanofibrous scaffolds.
Figure 3. FTIR spectroscopic analysis of PCL, PCL/PAA, PCL/PAA/Col, and PCL/PAA/Col/n-HA nanofibrous scaffolds.
Mimicking Nanofibrous Hybrid Bone Substitute for Mesenchymal Stem Cells Differentiation
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for restoring ECM.[24] Normally, scaffolds for tissue
engineering have definite temporary mechanical proper-
ties to withstand the stresses until new tissue formed.
Figure 2 shows the elongation at break of PCL, PCL/PAA
and PCL/PAA/Col, and PCL/PAA/Col/n-HA nanofibrous
scaffoldswere found to be 38, 43, 39, and 40%, respectively.
The tensile strength of PCL/PAA, PCL/PAA/Col, and
PCL/PAA/Col/HA nanofibrous scaffolds were higher than
the tensile strength of PCL scaffolds. Developed bio-
composite nanofibrous scaffold with improved tensile
propertiesmainly suitable forBTE. Previous studies showed
the tensile strength of electrospun PLLA/Coll/HA scaffolds
were higher than the collagen fibrous matrix (1.68MPa)
prepared by Thomas et al. (2007)[25] and even PCL/HA
scaffolds fabricated by Venugopal et al. (2008).[26] The
nanofibers are produced in an electrostatic field and
randomly deposited layer by layer on the target site to
form various diameters of pore size.[27] Martinez et al.
revealed the surface roughness of fibrous scaffolds is
desirable for cell adhesion and growth, it can be improved
Figure 4. Cell proliferation study on TCP, PCL, PCL/PAA, PCL/PAA/Col, and PCL/PAA/Col/n-HA nanofibers using hMSC on day 7, 14, and 21. � Indicates significant difference ofp�0.05; �� indicates significant difference of p�0.01.
by the presence of functional groups and
surface hydrophilicity.[28] The FTIR ana-
lysis showed the peak of carbonyl group
at 1720 cm�1 in PCL and PCL/PAA
nanofibers (Figure 3). The characteristic
peak of PAAwas noticed at 1365 cm�1 on
PCL/PAA and PCL/PAA/Col and PCL/PAA/
Col/n-HA nanofibrous scaffolds. The
amide bonds (N�H) characteristic of
collagen showing the peaks at 1091
and 1127 cm�1 on PLLA/PAA/Col and
PCL/PAA/Col/n-HA scaffolds, respec-
tively. The wave numbers for phosphate
groups were characterized at 800 cm�1
and carbonate groups obtained at 866
cm�1. Stretching vibration of PO3�4 group
for n-HA was detected at 1184 cm�1 and
bands corresponding to CO2�3 and PO3�
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groups in HA were obtained at 1450 cm�1 similar to
bone.[29]
3.2. Interaction of Cells and Scaffolds
hMSCs are particularly critical for long-term stability and
differentiation; thus the capability for nanofiber scaffolds
to support hMSCs adhesion and proliferation in tissue
engineering. The proliferation of hMSCs on TCP, PCL,
PCL/PAA, PCL/PAA/Col, and PCL/PAA/Col/n-HA nano-
fibrous scaffolds on day 7, 14, and 21 was determined by
cell proliferation assay as shown in Figure 4. The results
observed that the rate of proliferation on PCL/PAA/Col
scaffolds was significantly higher (p� 0.01) than PCL
nanofibrous scaffolds on day 7 and 21. This phenomenon
demonstrates that the chemicalmodification of nanofibers
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C. Gandhimathi, J. Reddy Venugopal, R. Ravichandran, S. Sundarrajan, S. Suganya, S. Ramakrishna
by adding PAA can be as critical as the nanofiber
architecture for increasing cell penetration and prolifera-
tion. PCL/PAA and PCL/PAA/Col scaffolds were found
amino groups on the surface of the nanofibrous scaffolds,
provided ligands for supporting the cell proliferation and
further differentiation of hMSCs to osteogenesis. However,
the rate of proliferation was significantly (p� 0.05)
increased in PCL/PAA/Col/n-HA compared to all other
scaffolds on day 14 and 21. This is because greater surface
roughness owing to the inclusion of n-HA, provides greater
surface area with more complex geometry of the nano-
fibrous matrix.[30] HA is desirable for regulating cell
function and stimulating osteogenesis and mineralization
Figure 5. FESEM images showing the cell–biomaterial interactions onn-HA nanofibers at 1 000� magnification. Arrows indicate the ECM
Macromol. Biosci. 2013, DOI: 1
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of bone. Studies have shown that the HA combination in
poly(l-lactic acid)/poly-benzyl-L-glutamate/collagen scaf-
folds increased surface roughness and subsequent protein
absorption from themedium on the surface; thus resulting
in more cells attaching to the nanofibrous scaffolds.[20] It
can be noticed that from day 14 to 21, the proliferation
starts to decrease on PCL/PAA/Col/n-HA samples, the
hMSCs start to differentiate into osteogenic lineages, and
they switch from the state of proliferation to differentiated
phenotype that is defined by a decrease in proliferation
owing to matrix maturation and followingmineralization.
The FESEM images of hMSCs (Figure 5a–e) showed normal
cell morphology on these scaffolds with the formation
(a) TCP, (b) PCL, (c) PCL/PAA, (d) PCL/PAA/Col, and (e) PCL/PAA/Col/secreted by hMSCs.
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Figure 6. Confocal microscopy images to confirm the osteogenic differentiation of hMSCs using MSC specific marker protein CD 90(a,d,g,j,m) and osteoblasts specific marker protein OCN (b,e,h,k,n). Merged image showing the dual expression of both CD 90 and OCN,characteristic of hMSCs cells which have undergone osteogenic differentiation (c,f,i,l,o) on TCP (a–c), PCL (d–f), PCL/PAA (g–i), PCL/PAA/Col(j–l), and PCL/PAA/Col/n-HA (m–o) at 20� magnification.
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Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200435
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C. Gandhimathi, J. Reddy Venugopal, R. Ravichandran, S. Sundarrajan, S. Suganya, S. Ramakrishna
of minerals on the surface of cells after day 21. Cell
activities such as adhesion, spreading, and proliferation
represent the initial phase of cell–scaffold communication
that consequently influences differentiation and miner-
alization.[31] Moreover, the cells were observed to migrate
slowly into the fibers and indulge in cell to cell interaction
via extension of filopodia. As indicated in Figure 5d,e, the
nanofibrous scaffold surfaces were covered with multi-
layers of cells as well as cell secreted ECM, representing
predominant cell–biomaterial interactions. HA possesses
the effective affinity for regulating cell function and
promoting osteogenesis and mineralization of bone.
The collagen influences cell proliferation and HA acts as
a chelating agent for the mineralization of osteoblasts for
bone regeneration. Studies reported that biomaterials
reveal the best primary cell attachment characteristic after
7 h of culture and encourage higher cell activity or
proliferation at the longer time interval.[32] The observed
results proved that the cell morphology was more or less
relatively similar in all nanofibrous scaffolds, except on
PCL/PAA/Col/n-HA nanofibrous scaffolds which showed
osteoblastmorphologyandalso significant level of increase
in proliferation and mineralization compared to all other
nanofibrous scaffolds.
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3.3. Expression of Osteocalcin (OCN)
Osteocalcin (OCN) is a bone specific protein related to bone
formation and unlike other bone related proteins, such as
osteonectin and osteopontin.[31,32] OCN plays a significant
role in modulating mineralization as it has glutamic
acid rich regions with strong binding affinities to both
Figure 7. Alkaline phosphatase (ALP) activity showing the osteogenic differentiation ofhMSCs on TCP, PCL, PCL/PAA, PCL/PAA/Col, and PCL/PAA/Col/n-HA nanofibers usinghMSCs on day 7, 14, and 21. � Indicates significant difference of p�0.05; �� indicatessignificant difference of p�0.01.
Ca2þ and HA.[35] HA incorporation sti-
mulating initial cell adhesion and osteo-
genic gene expression during osteo-
blastic differentiation.[36] Figure 6a,d,g,j,m
showed that the cells express hMSCs
specific marker protein CD90 (green
color). These cells differentiated into
osteogenic lineage to express OCN (red
color) marker protein (Figure 6b,e,h,k,n)
in addition to CD90. The obtained results
confirmed that the osteogenic differen-
tiation of hMSCs by dual expression of
both CD90 and OCN in Figure 6c,f,i,l,o.
The functionalized nanofibrous scaffolds
are more attractive for increased cell
adhesion, proliferation, differentiation,
and ECM production.[37,38] The results
observed that the hMSCs cultured
on PCL/PAA/Col/n-HA (Figure 6m–o)
scaffolds exhibiting the characteristic
cuboidal morphology of osteoblasts and
OCN expression representing complete
Macromol. Biosci. 2013, DOI: 1
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osteogenic differentiation compared to all other nano-
fibrous scaffolds. These results proved that the n-HA
incorporation stimulates not only early cell adhesion but
also osteogenic protein expression during osteoblastic
differentiation for BTE.[36]
3.4. Mineralization of Differentiated Osteogenic Cells
An effective bone scaffolds must support improved bone
development containing organic and inorganic constituent
of natural tissues. ALP is a main component of bone
matrix vesicles because of its part in the formation of
apatite calcium phosphate and also primary indicator of
immature osteoblast activity.[39,40] Figure 7 shows the ALP
activity was significantly (p� 0.01) increased on PCL/PAA,
PCL/PAA/Col scaffolds compared to PCL scaffolds on days 7,
14, and 21. However, the ALP activity was significantly
(p� 0.05) increased in PCL/PAA/Col/n-HA nanofibrous
scaffolds compared to all other scaffolds on day 7, 14,
and 21. This is because HA particularly known to induce
in vitro osteogenic differentiation of precursor cells as
well as improve in vitro bone formation.[41–44] Calcium
mineralization was determined qualitatively and quanti-
tatively as shown in Figure 8a and 8b. The capacity
to deposit minerals is a marker for mature osteoblasts,
which can be used to confirm that the hMSCs seeded onto
nanofibrous scaffolds differentiated and entered into
the mineralization phase to deposit mineralized ECM.
Compared to PCL nanofibrous scaffolds, the other scaffolds
had considerable calcium phosphate deposition due to
enhanced differentiation detected in these scaffolds
in the presence of PAA/n-HA biomolecules. Compared to
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Figure 8. (a) Opticalmicroscope images showing the secretion of ECMby hMSCs using Alizarin red staining on day 14 (a–e) on TCP (a), PCL (b),PCL/PAA (c), PCL/PAA/Col (d), and PCL/PAA/Col/n-HA (e) nanofibers at 10�magnification. (b) Quantitative analysis of the mineralization byhMSCs on TCP, PCL, PCL/PAA, PCL/PAA/Col, and PCL/PAA/Col/n-HA nanofibers using MSCs on day 7, 14, and 21. � Indicates significantdifference of p�0.05; �� indicates significant difference of p�0.01.
Mimicking Nanofibrous Hybrid Bone Substitute for Mesenchymal Stem Cells Differentiation
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PCL/PAA/Col scaffolds, cells cultured on the PCL/PAA/
Col/n-HA scaffolds were in intimate contact with the
deposited minerals. ARS staining was quantitatively
observed showing significantly (p� 0.05) increased
mineral deposition in PCL/PAA/Col/n-HA scaffolds com-
pared to PCL/PAA, PCL/PAA/Col, and PCL scaffolds on
days 7, 14, and 21 [Figure 8(b)]. ALP precedes OCN in
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the differentiation process; as ALP supports to prepare the
ECM for the deposition before the onset of mineralization
that coincide with OCN expression.[45,46] EDX results
showed that the presence of higher amount of calcium
and phosphorous deposition in PCL/PAA/Col/n-HA nano-
fibrous scaffolds than PCL scaffolds as shown in Figure 9.
Elements of mineral particles were examined for PCL
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Figure 9. EDX analysis for the detection of mineralization in hMSCs on 21 d culture in PCL and PAA/PAA/Col/n-HA nanofibrousscaffolds.
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C. Gandhimathi, J. Reddy Venugopal, R. Ravichandran, S. Sundarrajan, S. Suganya, S. Ramakrishna
and PCL/PAA/Col/n-HAwith cells in EDX shown in Table 2.
The obtained results proved by EDX analysis, the presence
of calcium phosphate on hMSCs which have undergone
osteogenic differentiation on PCL/PAA//Col/n-HA nano-
fibrous for BTE.
Creation of the native nanostructure of bone, which
is composed of n-HA and collagen fibers, has long been
a goal of many tissue engineers. Biomaterial scaffolds
that are employed for BTE should provide temporary
structural and functional support within a bone defect.
Incorporation of protein biomolecules of PAA, collagen,
n-HA, the scaffolds becomemore hydrophilic, the pore size
and porosity as high it is desirable for the transport of
nutrient and oxygen in the scaffolds for restoring ECM. For
being fully efficient system for BTE, the targeted system
shouldassociate simultaneouslymultiple functionalitiesof
cell binding, calcium binding, differentiation capabilities,
and osteoconductivity. The PAA/n-HA introduced PCL
nanofibers to improve specific biological functions like
adhesion, proliferation, and differentiation in bone tissue
regeneration.
Table 2. Normal weight and atomic percentage of C, O, P, Cl, Ca,elements in PCL/osteoblasts and PCL/PAA/Col/n-HA/osteoblasts.&Please check the presentation of table.&
Elements PCL/Osteoblasts PCL/PAA/Col/
n-HA/Osteoblasts
Atomic
[%]
Weight
[%]
Atomic
[%]
Weight
[%]
C 80.11 67.09 74.67 63.70
O 10.96 12.23 21.44 24.36
P – – 1.08 2.38
Cl – – 0.39 0.97
Ca 1.48 3.66 2.27 6.46
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4. Conclusion
Electrospun biocomposite porous PCL/PAA//Col/n-HA
nanofibrous scaffolds have good mechanical properties,
biocompatibility, and composition comparable to that of
bone matrix. The interconnecting porous structures of the
biocomposite nanofibrous scaffold provided more struc-
tural space for the adhesion, proliferation, and mineraliza-
tion of hMSCs and allowed efficient exchange of nutrients
and metabolic wastes. The importance of this study is the
application of bioactive macromolecules PAA/n-HA that
have been introduced into the polymeric nanofiber to
improve specific biological functions like adhesion, pro-
liferation, and differentiation in bone tissue regeneration.
Therapeutic potentials of hMSCs cultured on PCL/PAA/Col/
n-HA composite nanofibrous scaffold hold great potential
for cellular activities ranging fromcell adhesion,migration,
proliferation, differentiation, and mineralization for the
treatment of bone defects in BTE.
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Acknowledgements: This study was supported by the NRF-Technion (R-398-001-065-592), and NUSNNI, National Universityof Singapore, Singapore. There are no conflicts of interest todeclare.& Author: Is it necessary to include this last comment reconflicts of interest &
2425
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Received: November 29, 2012; Revised: January 30, 2013;Published online: DOI: 10.1002/mabi.201200435Keywords: bone tissue engineering; mineralization; nanofibrousscaffolds; nanohydroxyapatite; osteogenic differentiation; poly-aspartic acid& Author: Please check the keywords; as perjournal style more than five keywords are not allowed and twoshould be matched from the keyword list.&
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[1] J. Hu, X. Liu, P. X. Ma, Biomaterials 2008, 29, 3815.[2] F. Basmanav, G. T. Kose, V. Hasirci, Biomaterials 2008, 29,
4195.
0.1002/mabi.201200435
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e numbers, use DOI for citation !!
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Mimicking Nanofibrous Hybrid Bone Substitute for Mesenchymal Stem Cells Differentiation
www.mbs-journal.de
[3] P. Muller, U. Bulnheim, A. Diener, F. Luthen, M. Teller, E. D.Klinkenberg, J. Cell. Mol. Med. 2008, 12, 281.
[4] S. S. Liao, F. Z. Cui, Y. Zhu, J. Biol. Comp. Polym. 2004, 19, 117.[5] Y. Zhang, M. Q. Zhang, J. Biomed. Mater. Res. 2002, 61, 1.[6] S. H. Hsu, H. J. Yen, C. S. Tseng, C. L. Tsai, J. Biomed. Mater.
Res., B 2007, 80, 519.[7] J. R. Jones, O. Tsigkou, E. E. Coates, L. L. Hench, Biomaterials
2007, 28, 1653.[8] S. C. Mendes, J. M. Tibbe, M. Veenhof, S. Both, F. C. Oner,
J. Mater. Sci. Mater. Med. 2004, 10, 1123.[9] H. Kweon, M. K. Yoo, I. K. Park, T. H. Kim, C. S. Cho, Bio-
materials 2003, 24, 801.[10] Z. Gan, Q. Luang, J. Zhang, X. Jing, Polym. Degrad. Stab. 1997,
56, 209.[11] J. Venugopal, Y. Z. Zhang, S. Ramakrishna, Artif. Organs 2006,
30, 438.[12] E. Luong-Van, K. W. Leong, V. Nurcombe, Biomaterials 2006,
27, 204.[13] T. J. Galla, S. V. Vedecnik, J. Halbgewachs, S. Steinmann,
C. Friedrich, G. B. Stark, Int. J. Artif. Organs 2004, 27, 127.[14] J. Venugopal, L. L. Ma, S. Ramakrishna, Tissue Eng. 2005, 11,
847.[15] F. Causa, P. A. Netti, L. Ambrosio, G. Ciapetti, D. Martini,
A. Giunti, J. Biomed. Mater. Res., A 2006, 76, 151.[16] M. J. Vallet-Regi, Chem. Soc. Dalton Trans. 2001, 100, 97.[17] W. J. Landis, M. J. Song, A. Leith, B. F. McEwen, J. Struct. Biol.
1993, 110, 39.[18] Y. Liu, G. Wang, Y. Cai, H. Ji, X. Zhao, J. Biomed. Mater. Res.
2009, 90A, 1083.[19] H. J. Lee, S. E. Kim, H. W. Choi, S. C. Lee, Eur. Polym. J. 2007, 43,
1602.[20] R. Ravichandran, J. Venugopal, S. Sundarrajan, M. Shayanti,
S. Ramakrishna, Biomaterials 2012, 33, 846.[21] M. P. Prabhakaran, J. Venugopal, C. K. Chan, S. Ramakrishna,
Nanotechnology 2008, 19, 455102.[22] J. R. Woodard, A. J. Hilldore, S. K. Lan, J. A. Eurell, S. G. Clark,
M. B. Wheeler, R. D. Jamison, A. J. Wagoner Johnson, Bio-materials 2007, 28, 45.
[23] L. D. Shea, D. Wang, R. T. Franceshi, D. J. Mooney, Tissue Eng.2000, 6, 605.
[24] R. Murugan, S. Ramakrishna, Tissue Eng. 2007, 13, 1845.
www.MaterialsViews.com
Macromol. Biosci. 2013, DOI: 1
� 2013 WILEY-VCH Verlag Gmb
Early View Publication; these are NOT
[25] V. Thomas, D. R. Dean, M. V. Jose, B. Mathew, S. Chowdhury,Y. K. Vohra, Biomacromolecules 2007, 8, 631.
[26] J. Venugopal, S. Low, A. T. Choon, A. B. Kumar, S. Ramakrishna,J. Biomed. Mater. Res. 2008, 85A, 408.
[27] W. J. Li, C. T. Laurencin, E. J. Caterson, R. S. Tuan, F. K. Ko,J. Biomed. Mater. Res. 2002, 60, 613.
[28] E. C. Martinez, J. L. E. Ivirico, M. I. Criado, M. M. Pradas, M. S.Sanchez, J. Mater. Sci. Mater. Med. 2007, 18, 1627.
[29] A. Boskey, N. P. Camacho, Biomaterials 2007, 28, 2464.[30] S. J. Heo, S. E. Kim, J. Wei, Y. T. Hyun, H. S. Yun, Tissue Eng.
2009, 15, 977.[31] J. R. Venugopal, S. Low, A. T. Choon, A. B. Kumar, S. Ramakrishna,
Artif. Organs 2008, 32, 388.[32] R. Ravichandran, S. Liao, C. C. H. Ng, C. K. Chan, M. Raghunath,
S. Ramakrishna, World J. Stem Cells 2009, 1, 55.[33] M. Riminucci, P. Bianco, J. Braz. J. Med. Biol. Res. 2003, 36, 1027
& Author: Please provide citations for ref. [33] and [34] inthe text.&
[34] P. Ducy, T. Schinke, G. Karsenty, Science 2000, 289, 1501.[35] M. F. Young, J. M. Kerr, K. Ibaraki, A. M. Heegaard, P. G. Robey,
Clin. Orthop. Relat. Res. 1992, 281, 275.[36] K. Kim, D. Dean, A. Lu, A. Mikos, Acta Biomater. 2011, 7,
1249.[37] R. Ravichandran, J. R. Venugopal, S. Sundarrajan, S. Mukherjee,
S. Ramakrishna, Tissue Eng., Part A 2010, 17, 1363.[38] Y. Z. Zhang, J. Venugopal, Z.M.Huang, C. T. Lim, S. Ramakrishna,
Biomacromolecules 2005, 6, 2583.[39] H. C. Anderson, J. B. Sipe, L. Hessle, R. Dhanyamraju, E. Atti,
N. P. Camacho, Am. J. Pathol. 2004, 164, 841.[40] F. Liu, L. Malaval, J. E. Aubin, J. Cell Sci. 2003, 116, 1787.[41] B. Chuenjitkuntaworn,W. Inrung, D. Damrongsri, K.Mekaapiruk,
J. Biomed. Mater. Res. 2010, 94, 241.[42] H. Wang, Y. Li, Y. Zuo, J. Li, S. Ma, L. Cheng, Biomaterials 2007,
28, 3338.[43] K. C. Popat, K. I. Chatvanichkul, G. L. Barnes, T. J. Latempa, Jr.,
C. A. Grimes, T. A. Desai, Biomed. Mater. Res., A 2007, 80, 955.[44] K. C. Popat, R. H. Daniels, V. Hardev, T. A. Desai, J. Orthop. Res.
2006, 24, 619.[45] M. Mizuno, Y. Kuboki, J. Biochem. 2001, 129, 133.[46] G. S. Stein, J. B. Lian, A. J. vanWijnen, J. L. Stein, M. Montecino,
A. Javed, Oncogene 2004, 23, 4315.
4243
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