ARTICLE IN PRESS
Differentiation 79 (2010) 102–110
Contents lists available at ScienceDirect
Differentiation
journal homepage: www.elsevier.com/locate/diff
Bioactive nanofibers for fibroblastic differentiation of mesenchymal
precursor cells for ligament/tendon tissue engineering applications$
Sambit Sahoo a,1, Lay-Teng Ang a,2, James Cho-Hong Goh b,3, Siew-Lok Toh c,n
a
Division of Bioengineering, National University of Singapore, Singapore-117574, Singapore
Division of Bioengineering & Department of Orthopaedic Surgery, National University of Singapore, Singapore-119074, Singapore
c
Division of Bioengineering & Department of Mechanical Engineering, E3A-04-15, 7 Engineering Drive 1, National University of Singapore, Singapore-117574, Singapore
b
a r t i c l e in f o
a b s t r a c t
Article history:
Received 25 May 2009
Received in revised form
24 October 2009
Accepted 11 November 2009
Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue
engineering; however, for the stem cell-based approach to succeed, these cells would be required to
proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among
the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid
fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be
particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can
also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained
release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could
be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in
a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling
within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation,
upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and
deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/
ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.
& 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.
Keywords:
Functional tissue engineering
Biomimetic scaffolds
Electrospinning
Sustained release
Fibroblast growth factor
Bone marrow stromal cells
1. Introduction
Tendon and ligament injuries account for about half of all
musculoskeletal injuries and are associated with pain, suboptimal
healing and permanent loss of extremity function. Grafts and
prostheses, which are currently used for treatment, face problems
such as donor scarcity, donor-site morbidity, tissue rejection,
disease transmission and poor long-term performance. Tissue
engineered tendons and ligaments could overcome these shortcomings by regenerating a tissue that is biomechanically,
$
Work performed in: Tissue Repair Lab, Division of Bioengineering, National
University of Singapore, Singapore-117574, Singapore. Tel.: +65 6516 5985;
fax: + 65 6872 3069.
n
Corresponding author. Tel.: + 65 6874 2920; fax: + 65 6872 3069.
E-mail addresses: [email protected] (S. Sahoo), [email protected]
(L.T. Ang), [email protected] (J.C.H. Goh), [email protected]
(S.L. Toh).
1
Present address: Department of Orthopaedic Surgery, NUS Tissue Engineering Program, #04-01, DSO (Kent Ridge) Building, 27 Medical Drive, National
University of Singapore, Singapore-117510, Singapore. Tel.: + 65 6516 5447,
+ 65 6516 5985; fax: + 65 6776 5322, +65 6872 3069.
2
Tel.: + 65 6516 5985; fax: + 65 6872 3069.
3
Tel.: + 6772 4424; fax: + 6778 0720.
biochemically and morphologically similar to the normal tissue
(Butler et al., 2003, 2004; Woo et al., 2004). Though researchers
have developed and tried various scaffolds, there is still the need
for an ideal scaffold that could provide suitable mechanical
properties along with biological signals required for tendon/
ligament regeneration, especially in stem cell-based approaches.
Mesenchymal stem cells (MSCs) present an attractive way for
engineering tendon/ligament tissues as not only can they be
derived from replenishable sources like the bone marrow (unlike
differentiated tendon/ligament fibroblasts that would require
harvesting and enzymatic digestion of healthy donor tissues), but
their lack of immunogenicity also makes them suitable for
allogeneic implants (Gao and Caplan, 2003; Tuan et al., 2003;
Jorgensen et al., 2004; Ge et al., 2005). For the stem cell approach
to succeed, adequate biological signals would be required to be
delivered via the tissue engineering scaffold to encourage
proliferation and fibroblastic differentiation of the seeded precursor cells.
Though the exact environment or cocktail of signals necessary
to differentiate stem cells into tendon/ligament fibroblasts is still
unknown, stimuli like cyclic mechanical stretch applied through a
mechanical bioreactor, and specific growth and differentiation
factors have been shown to promote tendon and ligament cell
0301-4681/$ - see front matter & 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.
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doi:10.1016/j.diff.2009.11.001
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S. Sahoo et al. / Differentiation 79 (2010) 102–110
proliferation and matrix formation in several in vitro and in vivo
studies (Goh et al., 2003; Yamada et al., 2008; Jenner et al., 2007;
Hoffmann and Gross, 2007). Among the putative growth factors
such as basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-b, epidermal growth factor, platelet derived
growth factor, growth and differentiation factor-5 and insulin like
growth factor, bFGF is particularly relevant as it stimulates bone
marrow stem cell (BMSC) proliferation, self-renewal and differentiation into fibroblastic cells specific for tendon and ligament
lineages (Hankemeier et al., 2005; Petrigliano et al., 2006, 2007).
However, delivering any bioactive growth factor locally at the
healing site in a sustained fashion has been a challenge, as most
growth factors have extremely small plasma half-lives and are
rapidly inactivated (Chan et al., 2000). Incorporation of growth
factors within the tissue engineering scaffold has been proposed
as a means for ensuring a sustained delivery of growth factors to
the repair site (Whitaker et al., 2001).
Our earlier studies have shown that a fibrous ‘‘nano-microscaffold’’ combining knitted microfibers and electrospun nanofibers, when seeded with BMSCs, could be used for tendon/
ligament tissue engineering (Sahoo et al., 2006, 2007). This hybrid
scaffold system combined the advantages of mechanical integrity
of microfibers and the large biomimetic surface offered by
nanofibers; due to their high surface area to volume ratio and
resemblance to the nanostructure of natural extracellular matrix
(ECM), the nanofibrous substrate facilitated cell attachment,
proliferation and ECM deposition. Nanofibrous scaffolds have
been demonstrated to also support MSC proliferation and
differentiation along multiple lineages (Bhattarai et al., 2004; Li
et al., 2005). A combination of nanofibrous substrate and
sustained growth factor release can be expected to make a tissue
engineering scaffold both structurally and functionally biomimetic. Protein growth factors have been incorporated into
electrospun nanofibers for continued release from scaffolds (Chew
et al., 2005; Liao et al., 2006; Li et al., 2006). We have recently
developed, using a technique of blend-electrospinning, PLGA
nanofibers that are capable of continued release of bioactive
bFGF over 1–2 weeks (Sahoo et al., 2009). Since injured tendons/
ligaments have increased tissue levels of bFGF and its receptors
during the 1st week of injury (Chang et al., 1998; Cool et al., 2004;
Kobayashi et al., 2006; Berglund et al., 2006; Wurgler-Hauri et al.,
2007), we hypothesise that an electrospun scaffold releasing bFGF
over 1 week would biomimic the ECM of injured tendon/ligament
in both structure and function, by providing a nanofibrous
topography as well as a week-long supply of bioactive bFGF to
the resident cells, and would be favourable for tendon/ligament
tissue engineering.
In this study, we demonstrate the feasibility of bFGF-releasing
blend-electrospun nanofibers for tendon/ligament tissue engineering applications. The scaffolds are characterised morphologically, the released bFGF is evaluated for its bioactivity on BMSCs
via demonstration of activation of intracellular signaling pathways, and the resulting proliferation and differentiation of seeded
BMSCs into tendon/ligament fibroblasts.
2. Materials and methods
2.1. Fabrication of bFGF-releasing nanofiber matrix
20 mg of lyophilized bFGF (Raybiotech, USA) in 333 ml of 5 mM
TRIS (pH 7.6) containing 0.1% Bovine Serum Albumin (BSA) was
blended with 1.5 ml of 6.1% PLGA (PLA85:PGA15; Purac Asia
Pacific, Singapore) solution in hexafluoro-2-propanol (HFIP; Fluka
Chemie GmBH, Germany). The resulting blend was electrospun
using a high voltage power supply unit (RR 30-2P/DDPM, Gamma
103
Fig. 1. Electrospinning setup used to fabricate bFGF-releasing nanofibers from a
blend of bFGF and PLGA solution.
High-Voltage Research, Ormond, USA) at 10–12 kV and a flowrate of 0.45 ml/h, onto glass cover-slips placed on a grounded
collector, about 15 cm from the positively charged spinneret
(Fig. 1). Control scaffolds not containing bFGF were also fabricated
by replacing the bFGF solution with 5 mM Tris containing 0.1%
BSA. The bFGF-containing scaffolds were termed bFGF(+ ) and
those without bFGF were termed as bFGF( ).
2.2. Scaffold characterization
The nanofiber matrices were characterized morphologically by
scanning electron microscopy (SEM) using both secondary
electron (SEM; JEOL JSM-5800 LV) and backscattered electron
imaging (Jeol JSM-6701 field-emission SEM). SEM images were
analyzed by image analysis software (Olympus MicroImage
v4.5.1, Olympus Optical Co., Germany) to determine the diameter
distribution of the fibers. bFGF release from the scaffolds was
studied over 2 weeks, using a release buffer comprising 1 PBS
with 0.1% BSA and 0.01% sodium azide at 37 1C. bFGF concentration in the buffer was estimated using a bFGF ELISA kit
(Calbiochem, Merck KGaA, Germany) on days 1, 3, 7, 10, 14 (n = 3).
Protein content of the nanofibers was estimated by basesurfactant mediated hydrolysis of the PLGA nanofibers (using
50 mM Tris extraction medium containing 0.1 N NaOH, 5 M Urea
and 0.08% SDS, at 37 1C for 3 h), neutralization with 0.1 N HCl and
centrifugation, followed by Bradford microassay of the supernatant (n= 3) (Gupta et al., 1997). Protein encapsulation efficiency
was calculated from the ratio of the estimated and the
theoretically obtained total protein content of the electrospun
scaffolds.
2.3. Cell culture and seeding on scaffolds
Bone marrow was obtained from iliac crests of New Zealand
White Rabbits under approval of the NUS Institutional Animal
Care and Use Committee, National University of Singapore. BMSCs
were isolated and cultured, using their property of short-term
selective adherence to tissue culture polystyrene, in Dulbecco’s
modified Eagle’s medium (DMEM) with low glucose, supplemented with 15% fetal bovine serum (FBS), at 37 1C with 5%
humidified CO2. Semi-confluent cells of second or third passage
were used for cell-seeding experiments. Nanofibrous scaffolds
were sterilized by exposure to formaldehyde gas and seeded with
rabbit BMSCs at a density of 104 cells/cm2 in DMEM-high glucose
(HG), supplemented with 5% FBS and 1% Penicillin–Streptomycin.
The constructs were cultured in a 5% CO2 incubator at 37 1C for 2
weeks, with the medium being replaced every 3 days.
Cell adhesion efficiency on the nanofibrous scaffolds was
estimated from the proportion of unattached cells in the culture
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medium, using a method previously described (Sahoo et al.,
2006). After incubating the cell-seeded scaffolds (n= 6, for each
group) for 18 h, the culture medium was collected separately and
centrifuged, cell pellets re-suspended in 100 ml of medium and
cell count performed. The cell adhesion efficiency was expressed
as the number of cells attached to the scaffold as percentage of the
number of cells seeded.
The morphology and proliferation of the cells on the scaffolds
was studied by live-cell staining using fluorescein diacetate (FDA,
Molecular Probes, Invitrogen Corporation) and by SEM. Cellseeded scaffolds were stained with 10 mg/ml FDA in 1 PBS for
30 min and visualized using an inverted fluorescence microscope
(IX71 Inverted Research Microscope, Olympus). For SEM, the
samples were fixed with 3.7% formaldehyde, dehydrated in
graded concentrations of ethanol, air-dried, sputter-coated with
gold and observed under the SEM at an accelerating voltage of
15 kV.
2.4. Demonstration of bioactivity of released bFGF: Western blot for
phosphorylated tyrosine kinases in BMSCs
bFGF acts through cell surface receptors that activate several
intracellular second messengers (ERK/ MAPK cascade) by phosphorylation of their tyrosine residues, before finally exerting their
effects on the genes. Increased activation of this signal transduction pathway in the BMSCs cultured on bFGF( +) nanofibrous
scaffolds, as compared to those cultured on bFGF( ) control
scaffolds, would confirm bioactivity of the released bFGF.
After 1 week of culture on the scaffolds (n =3) in DMEM-HG
with 5% FBS, the BMSCs were lysed by freeze-thawing and
treatment with NP-40 lysis buffer (20 mM Tris–HCl at pH 8,
100 mM NaCl, 10% Glycerol, 1% nonidet P-40, 5 mM Na2-EDTA)
supplemented with several protease inhibitors (10 mM sodium
fluoride, 1 mM sodium orthovanadate, 0.01 protease inhibitor
cocktail (Sigma Aldrich, USA)). Protein concentration in the
pooled lysates of each group was estimated using Bradford
method (Standard microplate assay, Bio-Rad Protein Kit). 100 mg
of total cell-lysates were separated on a 7.5% SDS–PAGE. After
semi-dry electrotransfer onto a nitrocellulose membrane, blocking was performed with Tris-buffered saline containing 1% BSA,
and the membrane incubated with phosphotyrosine antibody
(pY20, mouse IgG, BD Biosciences, USA). GAPDH was probed with
anti-GAPDH as a loading control on a duplicate membrane.
Detection was performed using horse radish peroxidase-conjugated secondary antibody (anti-mouse IgG) and TMB Substrate
(Sigma ProteoQwestTM Colorimetric Western Blotting Kit). The
blot was visualized using a gel documentation system (Syngene
G:BOX, Synoptics Ltd, Cambridge, UK) and differences in phosphotyrosine levels in the two cell populations were measured
using densitometric analysis (Quantity-One 4.4.0, BioRad), using
GAPDH as the normaliser.
2.5. Effect on BMSC number: PicoGreen assay
Cell number on the scaffolds was estimated by DNA quantitation using the PicoGreen assay (Molecular Probes, Invitrogen
Corporation) after 7 and 14 days of culture. Cells were lysed by a
cycle of freeze-thawing, freeze-drying and homogenization in a
lysis buffer. 20 ml of the cell-lysate was added to 80 ml of
PicoGreen dye in separate wells of a black 96-well plate, and
fluorescence intensity was measured at 520 nm wavelength using
a microplate reader (FLUOstar OPTIMA, BMG Labtech GmbH,
Germany) after excitation at 485 nm. BMSC numbers on bFGF( )
scaffolds and tissue culture polystyrene (TCP) were used as
controls (n = 3).
2.6. Effect on BMSC stemness: differentiation assays to demonstrate
loss of BMSC multipotentiality
It is desirable that bioactive bFGF from the nanofibrous
scaffolds, besides causing increased proliferation, should also
direct BMSC differentiation along a fibroblastic lineage. Since cellsurface markers for rabbit BMSC and tendon/ligament cells are
currently unknown (Doroski et al., 2007), lineage-specific differentiation of BMSCs can only be demonstrated indirectly through
differentiation assays, wherein the committed progeny should
exhibit a loss or reduction in multipotentiality, or ability to
differentiate into bone, cartilage and adipose tissues.
Primary rabbit BMSCs of P2 passage were cultured on bFGF(+ )
scaffolds in DMEM-HG with 5% FBS for 2 weeks, harvested by
trypsinization, replated and cultured on TCP substrates (6-well
plates), and then induced to differentiate along adipogenic,
osteogenic and chondrogenic lineages, using established protocols
(Pittenger et al., 1999; McBeath et al., 2004). 3 105 cells were
seeded per well in 6-well plates for adipogenic and osteogenic
differentiation, and 6 105 cells were grown in a pellet culture for
chondrogenic differentiation. Adipogenic differentiation was induced by 3 cycles of induction (with DMEM supplemented with
10% FBS, 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM dexamethasone, 10 mg/ml insulin, 0.2 mM indomethacin and antibiotics) and
maintenance treatment (with DMEM supplemented with 10% FBS,
10 mg/ml insulin and antibiotics). Differentiation was then
detected by Oil Red-O staining for lipid vacuoles. Chondrogenic
differentiation was induced by culturing the BMSC-pellet in
serum-free DMEM with TGF-b1, 50 mg/ml L-ascorbic acid 2phosphate, 1.25 mg/ml BSA, 0.1 mM dexamethasone, 1 ITS
(insulin–transferrin–selenium) and antibiotics supplements. After
3 weeks, the cell-pellet was sectioned and stained with Alcian
Blue for cartilage matrix-specific sulfated glycosaminoglycans and
acidic sulfated mucosubstances. Osteogenic differentiation of
BMSCs was induced under the influence of 10 mM b-glycerophosphate, 0.1 mM dexamethasone, 50 mg/ml L-ascorbic acid 2phosphate and 10 mg/ml insulin, and Alizarin Red staining was
carried out to detect calcium accumulation after 3 weeks. All the
induction reagents, except ITS (Gibco) and TGF-b1 (R&D Systems,
MN, USA), were from Sigma.
As a control, naı̈ve P3 BMSCs (obtained after sub-culturing P2
cells on TCP for 2 weeks) were induced to differentiate along the
three lineages, following the same protocols, and various staining
were performed to evaluate directed differentiation after the end
of 3 weeks.
2.7. Effect on collagen production: SirCol assay and immunostaining
Fibroblastic differentiation of BMSCs would be associated with
increased production and deposition of collagen, the major
component of tendon/ligament ECM. After 7 and 14 days of
culture, soluble collagen secreted into the culture medium was
determined by picrosirius red based colorimetric assay (SirCols
Assay, Biocolor Ltd, Northern Ireland), using previously described
methods (Sahoo et al., 2006). The total amount of soluble collagen
secreted per scaffold (n =4) was estimated from the collagen
concentration and the volume of culture media.
Insoluble collagen deposited in the ECM surrounding the cells
was detected by immunostaining for collagen type I and type III.
In addition, immunostaining was also performed for tenascin-C, a
tendon/ligament-specific ECM molecule. After 10 days of culture,
paraformaldehyde-fixed cell-seeded scaffolds were labeled with
mouse monoclonal primary antibodies (anti-collagen type I, type
III and tenascin-C; ICN Biochemicals, Aurora, OH) at 1:200
dilution for 8 h at room temperature, alkaline phosphatase
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S. Sahoo et al. / Differentiation 79 (2010) 102–110
conjugated anti-mouse secondary antibodies at 1:100 dilution for
1 h, and detected using DAB substrate (IHC Select DAB Kit,
Chemicon, Millipore Corporation, MA, USA). The immunostained
nanofibrous scaffolds were then directly visualized under the
IX71 microscope.
2.8. Q-RT-PCR analysis for expression of ligament/tendon-related
ECM proteins from BMSCs
In addition to loss of multipotentiality, fibroblastic differentiation of BMSC would also be associated with upregulation of gene
expression for tendon/ligament-specific ECM proteins like collagen type I, collagen type III, fibronectin and biglycan. After 7 and
14 days of culture on bFGF( +) scaffolds as well as bFGF( ) and
TCP controls (n= 3), total RNA were extracted from the constructs
using Qiagen RNeasy Kits. Quantitative Reverse Transcriptasemediated-PCR (Q-RT-PCR) was performed using SYBR-Green
chemistry for collagen type I, collagen type III, fibronectin and
biglycan, using glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) and b-actin as reference genes. Primer sequences
(Table 1) were either obtained from published literature (Cooper
et al., 2006; Sobajima et al., 2005) or designed from rabbit gene
sequences obtained from the GenBank database, using Primer-3
software, and synthesized by Research Biolabs, Singapore. cDNA
synthesis and PCR expansion (using iScript and iQ SYBR Green
Supermix, Bio-Rad Laboratories, CA) were performed in a iCycler
iQ detection system (Bio-Rad Laboratories, CA). Data were
Table 1
Real time PCR primers used in the study. Primers for collagen type I, GAPDH and bactin were designed from NZWR gene sequences obtained from GenBank
(accession numbers D49399, NM_001082253 and AF309819 respectively) using
Primer3 software (http://frodo.wi.mit.edu). Primer sequences for collagen type III,
fibronectin and biglycan were obtained from published literature (Cooper et al.,
2006; Sobajima et al., 2005).
Primer
Sequence
Collagen I (a2)
F: GCA TGT CTG GTT AGG AGA AAC C
R: ATG TAT GCA ATG CTG TTC TTG C
F: AAG CCC CAG CAG AAA ATT G
R: TGG TGG AAC AGC AAA AAT CA
F: CTC ACC CGA GGC GCC ACC TA
R: TCG CTC CCA CTC CTC TCC AAC G
F: TGA ACA ACA AGA TCT CCA AGA T
R: ATT CAG GGT CTC TGG CAG A
F: GAC ATC AAG AAG GTG GTG AAG C
R: CTT CAC AAA GTG GTC ATT GAG G
F: CCC ATC TAC GAG GGC TAC G
R: CCA CGT AGC ACA GCT TCT CC
Collagen III (a1)
Fibronectin
Biglycan
GADPH
b-Actin
105
analyzed for relative expression using the DDCT method, and
normalized against the expression profile of BMSC grown on TCP
controls on day 7.
2.9. Data reduction and statistical analysis
Data were analyzed by single-factor ANOVA and post-hoc
Tukey tests for multiple comparisons. Results were presented as
mean7standard error and po0.05 was accepted as significant.
3. Results
3.1. Scaffold characterization
Scaffolds were composed of randomly oriented continuous
nanofibers of 200–700 nm diameter (from analysis of SEM
images, Fig. 2A). The growth factor was distributed as a random
dispersion within the fibers (Backscattered electron image on SEM
observation, Fig. 2B). Release kinetics results indicated that 55% of
the bFGF was incorporated into the scaffolds and released over a
period of 7 days (Fig. 2C); there was an initial rapid release
followed by a more gradual release (average bFGF concentration:
13.5 pg/ml on day 1, 6.5 pg/ml on day 3).
3.2. Demonstration of bioactivity of released bFGF
Several tyrosine-phosphorylated proteins could be observed in
the Western Blots of both groups, with a higher relative density in
the bFGF(+ ) group (Fig. 3). Sustained tyrosine phosphorylation
and activation of ERK1 and ERK2 (p44 and p42), as well as FRS2
(p89) were observed in the cells grown on the bFGF( + ) scaffolds,
indicating that the bFGF incorporated in the scaffolds was
bioactive over 7 days. p145 (FGF receptor) and p52 (shc)
phosphorylation levels were also slightly increased in the
bFGF(+ ) scaffold.
3.3. BMSC adhesion and proliferation on scaffolds (FDA staining,
SEM, PicoGreen assay)
Both scaffold groups showed similar cell adhesion, with more
than 90% of the seeded rabbit BMSCs adhering onto them in 18 h.
SEM (Fig. 4A, B) and fluorescent microscopy (Fig. 4C, D) after livecell staining with FDA revealed better cell proliferation and
spreading on bFGF( +) scaffolds. PicoGreen assay showed a
significant increase in cell population on the bFGF(+ ) scaffolds
between day 3 and 14 of culture (p o0.05), at the end of which,
Fig. 2. SEM images showing blend nanofibers of 200–700 nm diameter, with a random distribution of protein within. The bFGF was released over 1 week.
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cell lysis and DNA extraction from the 2D substrate of culture
flasks as compared to 3D nanofibrous scaffolds (Ng et al., 2005).
3.4. BMSC differentiation assay
Naı̈ve P3 BMSCs could be successfully differentiated into
adipocytic, osteocytic and chondrocytic lineages after 3 weeks of
specific induction. However, P2 BMSCs, after being passaged on
bFGF(+ ) nanoscaffolds for 2 weeks, failed to show any adipocytic
differentiation while osteocytic and chondrocytic differentiation
was markedly reduced (Fig. 5).
3.5. Collagen production: SirCol assay and immunostaining
Fig. 3. Western blot showing increased tyrosine phosphorlyation events (marked
by arrows in the blot) in BMSCs cultured for 7 days on bFGF( +) scaffolds,
indicating activation of bFGF signal transduction pathways. The relative increase
in band density, normalized against GAPDH intensities, is indicated next to each
band.
While Sircol assays showed that BMSCs on bFGF(+ ) and
bFGF( ) scaffolds produced similar amounts of soluble collagen
(36.673.56 mg/ bFGF( +) scaffold, 32.8 71.99 mg/ bFGF( ) scaffold, and 31.070.84 mg/TCP well, on day 14), immunostaining
revealed a denser deposition of collagen type I and III in the ECM
on bFGF(+ ) scaffolds as compared to control bFGF( ) scaffolds
after ten days of culture. In addition, a denser deposition of
tenascin-C, another tendon/ligament-specific ECM protein, was
also observed on bFGF(+ ) scaffolds (Fig. 6). Cellular outlines were
only faintly visible under the imaging conditions due of the
thickness and interference from the irregular surface of the
nanofibrous scaffolds.
3.6. Q-RT-PCR analysis for gene expression of ligament/tendonspecific ECM proteins
Gene expression analyses by single-factor ANOVA demonstrated upregulation of gene expression for various tendon/
ligament ECM proteins in the BMSCs cultured on nanofibrous
scaffolds compared to BMSCs on TCP, at the end of 1 week. By the
end of 2 weeks, bFGF(+ ) scaffolds showed significantly higher
gene upregulation compared to bFGF( ) scaffolds and TCP
controls (Fig. 7). Collagen type I expression was lower on the
bFGF(+ ) scaffolds than on the bFGF( ) control on day 7; but the
scenario was reversed by day 14. While the expression of collagen
type I and biglycan on the bFGF( +) scaffolds increased
significantly between day 7 and 14, that of fibronectin dropped
significantly; the decrease in collagen type III expression was not
statistically significant.
4. Discussion
A polymeric nanoscaffold was developed with the capability of
releasing an encapsulated bioactive growth factor over 1 week.
The scaffold facilitated BMSC attachment and subsequent proliferation, production and deposition of collagen as well as
tenascin-C, and upregulation of gene expression for tendon/
ligament-specific ECM proteins, suggesting BMSC differentiation
into a tendon/ligament phenotype.
Fig. 4. SEM and live cell imaging showing better cell proliferation and spreading
on bFGF( +) scaffolds (A, C). PicoGreen assay (E) confirmed significantly higher cell
population on bFGF(+ ) scaffolds compared to bFGF( ) scaffolds after two weeks.
4.1. Electrospun nanofibers as carriers for sustained release of
bioactive molecules
bFGF(+ ) scaffolds had significantly higher cell population than
bFGF( ) scaffolds (Fig. 4E). Cells cultured on TCP gave
prominently higher PicoGreen readings compared to the
nanofibrous scaffolds (significant difference based on Tukey test
on day 3 and day 14); this may be the result of higher efficiency of
Several recent studies have also reported growth factorreleasing electrospun nanofibers for tissue engineering scaffolds
(Chew et al., 2005; Liao et al., 2006; Li et al., 2006). It has been
hypothesised that a combination of passive diffusion across
nanopores on the nanofiber surface and material degradation of
the nanofibers causes protein release (Jiang et al., 2005, 2006;
Ramakrishna et al., 2006). Chain scission of PLGA molecules
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107
Fig. 5. Oil red O (for fat deposits, indicated by black arrows), alizarin red (for calcium deposits, indicated by white arrows) and alcian blue staining (for sulfated
mucosubstances), showing adipogenic, osteogenic and chondrogenic differentiation of BMSCs. Data demonstrate a reduction of multilineage differentiation potential of
BMSCs after culture on bFGF(+ ) nanoscaffolds (bottom) as compared with untreated naı̈ve BMSCs (top). (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
Fig. 6. Immunostaining for collagen type I, collagen type III and tenascin-C showing a denser deposition (indicated by arrows) of the ECM proteins in the BMSC-seeded
bFGF(+ )scaffolds, compared to bFGF( )scaffolds, on day 10. Cell outlines were not clearly visible in the stained samples compared to unstained controls under the imaging
conditions.
during electrospinning increases the rate of hydrolytic degradation of PLGA nanofibers, particularly in the amorphous regions,
allowing small proteins to diffuse through (Zong et al., 2003; Kim
et al., 2009). Protein release from the nanofibers developed in this
study is also expected to have the underlying mechanisms of
diffusion and degradation. Growth factor bioactivity, in previous
studies, has only been indirectly demonstrated by increased
proliferation of cells when cultured in media supplemented with
supernatant containing the released growth factor (Chew et al.,
2005; Liao et al., 2006), or by enhanced differentiation of MSCs
when cultured on the scaffolds (Li et al., 2006). In this study,
bioactivity of the released bFGF was directly demonstrated
though specific signal activation in BMSCs. In vitro studies have
shown that optimal differentiation of BMSCs into tendon/
ligament fibroblasts requires the simultaneous and sequential
administration of multiple growth factors (Moreau et al., 2005a,
2005b). It would therefore be desirable to fabricate nanofibrous
scaffolds incorporated with several relevant growth factors; the
initiation, duration and concentration of release of the different
growth factors could be controlled by modifying the amount of
growth factor loaded into the nanofibers, by choosing biomaterials of different degradation rates and by addition of porogens
such as PEG in the biomaterials used for the different nanofibers,
and by using techniques like coaxial electrospinning (Liao et al.,
2006; Jiang et al., 2006).
4.2. Biomimiking the ECM in structure and function
The ECM in a tissue is structurally and functionally integrated
with resident cells, providing them support and anchorage, as
well as regulating their survival, differentiation and function. In
addition, the ECM sequesters several cellular growth factors,
thereby stabilizing and protecting them and acting as their local
depot. Tissue engineering scaffolds that present a combination of
nanofibrous substrate and sustained growth factor release would
thus be both structurally and functionally biomimetic. Nanofiber
coating on scaffolds can provide a large biomimetic surface aiding
in BMSC differentiation into a tendon/ligament lineage, even
without any supplementation of growth or differentiation factors
(Sahoo et al., 2006, 2007). Aligned nanofibers have been
demonstrated to induce fibroblast alignment and collagen
production (Lee et al., 2005), suggesting that nanotopographic
cues from electrospun scaffolds affect cell behavior and fate. The
current study also demonstrated gene upregulation of tendon/
ligament matrix proteins in BMSCs after culture on control
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Fig. 7. Q-RT-PCR analysis showing a significant gene upregulation of tendon/ligament ECM proteins on bFGF(+ ) scaffolds after 2 weeks.
nanofibrous scaffolds without any bFGF, at the end of 1 week. A
significantly higher gene upregulation was observed on bFGF(+ )
nanoscaffolds suggesting a synergistic effect of nanotopography
and growth factor release on the cells.
4.3. BMSC proliferation and differentiation on nanofibrous scaffolds
Compared to the control scaffolds, enhanced cell proliferation
and fibroblastic differentiation of BMSCs (indicated by loss of
multipotency, gene upregulation of tendon/ligament ECM proteins, and increased collagen production and deposition) were
observed on the bFGF(+ ) nanoscaffolds. The temporal pattern of
collagen type I gene expression on the bFGF(+ ) scaffolds (lower
expression on day 7, but higher expression by day 14) suggests
that while high levels of bFGF initially maintained BMSCs in an
actively proliferating undifferentiated phenotype, sustained bFGF
levels resulted in BMSC differentiation by day 14. Collagen type III
and fibronectin showed earlier gene upregulation (day 7)
compared to collagen type I and biglycan (day 14).
Collagen type III is an embryonic form of collagen, characterized by short disorganized fibrils, that is typically over-expressed
during the early inflammatory and proliferative stages of tendon
healing. It is gradually replaced by longitudinally aligned long
collagen type I fibrils with remodeling and maturation of the
healing tissue (Lin et al., 2004). A similar relationship between the
expression of collagen type I and III was observed in this study.
Fibronectin is known to be a marker for active reparative
connective tissue processes and regulates initial cell attachment
and survival (Venugopal et al., 2006). Down-regulation of
fibronectin transcript levels in the second week, in this study, is
consistent with other studies where cells were observed to
synthesize fibronectin during proliferation and early differentiation; once the cells reached maturation and accumulated
collagenous ECMs, they sharply reduced the production of
fibronectin (Venugopal et al., 2006; Chen et al., 2006). Biglycan
and tenascin-C are known to modulate growth factor activity
during tendon development and repair; biglycan also controls the
diameter and assembly of ECM collagen fibrils (Berglund et al.,
2006) and tenascin-C modulates cell–ECM interactions like cell
adhesion and migration (Doroski et al., 2007). They are generally
over-expressed during the reorganization phases of tissue development and repair, and coincide with collagen type I overexpression in this study.
In contrast to 2-dimensional cultures on tissue culture
polystyrene, where bFGF helps in BMSC self-renewal and maintains their multipotency (Tsutsumi et al., 2001), 2 weeks’ culture
on bFGF-releasing nanoscaffolds resulted in their differentiation,
further suggesting that the nanofibrous substrate plays a crucial
role in determining cell behavior and fate. While previous in vitro
studies have demonstrated significant effects of bFGF on BMSC
proliferation, self-renewal and differentiation using twice-weekly
replenished culture media supplemented with 0.1–10 ng/ml of
bFGF (Hankemeier et al., 2005; Tsutsumi et al., 2001), in this
study, sustained picogram concentrations of bFGF over 1 week
was shown to be sufficient to induce the same effects. These
results are supportive of our hypothesis that a biomimetic
nanofibrous scaffold allowing a biomimetic sustained release of
bFGF over 1 week would be suitable for tendon/ligament tissue
engineering. The bFGF( +) scaffold developed in this study
biomimics the ECM of injured tendons in both structure and
function by providing a nanofibrous topography as well as a
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S. Sahoo et al. / Differentiation 79 (2010) 102–110
week-long supply of bioactive bFGF to the resident cells. Some of
the cellular response to bFGF stimulation were observed on the
bFGF(+ ) scaffolds in the second week, when the incorporated
bFGF had already been released from the underlying scaffolds.
This suggests that the released bFGF may have resulted in
persistent, long-term signaling events in the BMSCs resulting in
their proliferation and differentiation in the second week. A
similar long-term response has been reported with TGF-b elsewhere (Wormstone et al., 2006).
There were several limitations in this study that could be
addressed in future studies. The current study used primary
rabbit BMSCs, for which surface antigenic and gene markers are
currently unknown. Therefore, demonstration of multipotency
(and its loss) could only be demonstrated through differentiation
assays. Also the relative contributions of the nanotopographic
substrate and the released growth factor on cell fate has not been
studied. Since multiple growth factors coordinate the healing of
tendon/ligament injuries (Moreau et al., 2005a, 2005b), nanofibrous scaffolds incorporated with several relevant growth factors
could be a more effective model than the single growth factor
model used in this study. As nanofibers alone cannot provide
sufficient mechanical support required for healing tendons/
ligaments, hybrid scaffolds have been fabricated by coating
nanofibers on microfibrous scaffolds (Sahoo et al., 2006, 2007)
growth factor releasing nanofibers developed in this study could
be suitable candidates for such coating.
5. Conclusion
bFGF could be successfully incorporated, randomly dispersed
within the electrospun nanofibers and released in a bioactive
form over 1 week. The released bioactive bFGF could activate
tyrosine phosphorylation signaling within seeded BMSCs. The
bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, production and deposition of collagen and tenascin-C, and
induced tendon/ligament-like fibroblastic differentiation, suggesting their potential in tendon/ligament tissue engineering
applications.
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