J. Biomedical Science and Engineering, 2010, 3, 908-916
doi:10.4236/jbise.2010.39121 Published Online September 2010 (http://www.SciRP.org/journal/jbise/ JBiSE ).
Published Online September 2010 in SciRes. http:// www.scirp.org/journal/jbise
Accelerated chondrogenesis in nanofiber polymeric scaffolds
embedded with BMP-2 genetically engineered chondrocytes
Robert T. Gorsline1,2, Prasam Tangkawattana3, John J. Lannutti4, Mamoru Yamaguchi3, Christopher
C. Kaeding2, Alicia L. Bertone1,2
1Comparative Orthopaedic Research Laboratories, The Ohio State University, Columbus, USA;
2Department of Orthopaedics, College of Medicine, The Ohio State University, Columbus, USA;
3Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, USA;
4Department of Materials Science and Engineering, College of Engineering, The Ohio State University, Columbus, USA.
Received 28 June 2010; revised 19 July 2010; accepted 20 July 2010.
ABSTRACT
This study evaluated chondrogenesis within a nanofi-
ber polymeric scaffold seeded with isolated untreated
chondrocytes, isolated chondrocytes genetically engi-
neered with adenoviral (Ad) bone morphogenetic
protein (BMP)-2, or isolated chondrocytes genetically
engineered with green fluorescent protein (Ad-GFP).
Electrospun polycaprolactone scaffolds (150-200 m
thickness, 700 m fiber diameter, 30 m pore size)
were optimally seeded with 1 x 107 isolated chondro-
cytes by using a 20% serum gradient culture system.
Chondrocyte- scaffold constructs (untreated, Ad-B-
MP-2 and Ad-GFP) were generated from 5 adult ho-
rses, cultured in triplicate for 7 or 14 days, and quan-
titatively analyzed for cell proliferation (DNA content;
Hoechst assay), viability, morphology (confocal mi-
croscopy), matrix production (proteoglycan content;
DMMB assay), and mRNA expression of collagen I,
collagen II, and aggrecan. Chondrocytes transduced
with Ad-BMP-2 demonstrated greater cell numbers
and significantly greater expression of chondrogenic
markers including aggrecan, collagen II, and proteo-
glycan through 14 days of culture as compared to un-
transduced or Ad-GFP controls. This study demons-
trated that chondrocytes can be driven to seed a pol-
ycaprolactone nanofiber scaffold by serum gradient
and a polycaprolactone nanofiber scaffold containing
Ad-BMP2 transduced chondrocytes resulted in grea-
ter and accelerated chondrogenesis than controls. Th-
is cell engineered construct has potential use in one-
step cartilage repair in vivo.
Keywords: Nanofiber; Scaffold; Chondrogenesis;
BMP- 2; Adenovirus
1. INTRODUCTION
Articular cartilage defects heal poorly and combinations
of progenito r chondrocytes, bioactive factors, and matri-
ces are being applied as focal synthetic devices [1-3].
Synthetic biodegradable polymers have served as scaf-
folds for articular cartilage tissue engineering. Studies
have demonstrated that properties such as pore size, ma-
terial composition, and diameter of the fibers are impor-
tant for cell proliferation, adhesion, migration and main-
tenance of a chondrogenic phenotype of seeded cells
[4-12]. Successful biodegradable polymeric scaffolds
permissive to cell and tissue in-growth have been studied
both in vitro and in vivo, including for chondrocytes
[4,6-13]. Properties of successful scaffolds include in-
terconnected pores permissive to cell adhesion, prolif-
eration and migration, and fluid transport as well as bio-
compatibility. Scaffolds have been created from bioac-
tive materials such as plasma, starch, collagen, hydrogel,
hydroxyapatite, alginate, and periosteum [9,11,13-15] as
well as the synthetic materials such as porous poly
(l-lactic acid) [5,8,16], poly (glycerol sebacate) (PGS)[6],
poly (1,8 octanediol- co-citrate) (POC) [6], poly (ethyl-
ene glycol)-terephtha- late/poly (butylene terephthalate
(PEGT/PBT) [17] poly- caprolactone (PCL) [4 - 7 , 1 0 ,12 , 17- 2 3]
and poly (gamma- glutamic acid)-graft-chondroitin sul-
fate/PCL composites [24], polydioxone [25] and other PCL
composites[8,13].
Recent publications support the use of a nanoscale fib-
er size and PCL material for tissue engineering of chon-
drocytes [4-6,9-10,12,21,23]. Use of a fiber material in a
mesh pattern has been reported for chondrocytes an d the
optimum fiber material will consider fiber size and topo-
graphy. Nanofiber technology and its influence on cell
behavior is advancing the science of tissue engineering
by providing a fiber on the scale of biologic fibers [18-
20,23]. It is proposed that fibers which closely mimic

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
909
normal extra-cellular matrix or basement membrane str-
ucture will show improved cytocompatability. Typically,
naturally occurring extra-cellular matrices and basement
membranes are composed of proteins including fibrone-
ctin, collagen, hyaluronic acid, chondroitin sulfate, der-
matan sulfate and proteoglycans. These proteins are fib-
rous and are on a nanofiber scale in size. PCL fibers in
the 30-1500 nm diameter range have been demonstrated
to have optimal structural integrity and, particularly und-
er dynamic loading, supported a desirable cellular respo-
nse in culture, including chondrocyte proliferation and
matrix production [23]. Electrospinning is a well-estab-
lished process that can produce a random meshwork of
nanofibers, including PCL, with appropriate pore size to
support cellular infiltration of the scaffold, includ ing ch-
ondrocytes [4-5,9-10,12,23-24], stem cells [21,22,26],
glio- ma cells [27], and endothelial cells [28].
Cell-based therapy in conjunction with scaffolds for
tissue engineering of cartilage is supported in vitro [3,6,
8,10-12,23,24,26] and in vivo [7,14,17,21-22,25] and is
rapidly advancing toward clinical application [1-4]. [1-4]
Typically the cell source for cartilage engineering is
chondrocytes [3,6-12,14,21,23-25] or mesenchymal stem
cells. [17,21-22,26,29-30 ] Specifically for articular carti-
lage repair, animal studies support the use of biodegrad-
able scaffolds seed ed with morcelized cartilage [25], ch-
ondrocytes[7,14,21] or direct injection of mesenchymal
stem cells.[31] Supplementation w ith growth f actors, su-
ch as transforming growth factor-beta 3, a known regu-
lator of cell growth and differentiation, could enhance
chondrocyte density and integration [11]. Growth factor
members of the TGF beta superfamily, such as the BM-
Ps, are particularly beneficial in promoting chondrogen-
esis of mesenchymal stem cells [29-34] and can support
cartilage matrix production [35,36]. Specifically, bone
morphogenetic protein (BMP)-2 can regulate chondroc-
yte differentiation in progenitor cells [34], enhance bone
formation through the endochondral ossification pathw-
ay[31,37], and can increase chondrocyte extracellular ma-
trix production in vitro [35-36]. Mesenchymal stem cells
genetically engineered to produce BMP-2 can enhance
articular cartilage repair during articular fracture repair in
vivo [31]. Our study focuses on the ab ility of the BMP-2
gene to support phenotype, proliferation, and matrix pro-
duction of chondrocytes suspended in a biodegradable
nanofiber PCL scaffold. We investigated whether BMP-
2 could successfully promote chondrogenesis in this 3-
dimensional scaffold for potential use in articular carti-
lage tissue engineering.
2. METHODS
2.1. Study Design
Chondrocytes were isolated and expanded in primary mo-
nolayer culture. Chondrocytes were seeded onto the sur-
face of three dimensional polycaprolactone nanofiber sca-
ffolds and evaluated for scaffold penetration by histology
cryosection and morphology by confocal microscopy un-
der conditions of fetal bovine serum gradient (10 or 30%),
cell seeding density (5.0 × 105/ml, 1.0 × 106 ml, or 5.0 ×
106 ml) and duration of cell growth (day 2,7, or 14). The
best condition was selected and chondrocytes from 5 hor-
ses were seeded onto similar scaffolds, in duplicate, and
were evaluated at two time points (day 7 or 14) for three
cell preparations; isolated cells (untreated control), iso-
lated cells transduced with Ad-GFP (vector control), and
isolated cells transduced with Ad human (h) BMP-2 (ex-
perimental gene). Chondrocyte transduction and BMP-2
production were confirmed. Outcome assessments were
cell proliferation, cell morphology, cell viability, BMP-2
production, extracellular proteoglycan matrix production,
and chondrocyte gene expression of Type I and Type II
collagen as well as aggrecan.
2.2. Generation of Adenoviral Vector Constructs
Recombinant adenoviral vector containing these 1547
base-pairs of human BMP-2 under the cytomegalovirus
promoter were propagated [31]. Expression of transgene
was verified in cell culture.
2.3. Chondrocyte Preparation
Chondrocytes from 5 adult horses [5-9 years] were harv-
ested aseptically from articular cartilage of the femorop-
atellar joint and isolated by collagenase digestion. Cho-
ndrocytes were expanded in monolayer in Dulbecco’s
Modified Eagle Medium (DMEM; Gibco, Sigma-Aldri-
ch, St. Louis, MO) supp lemented with sodium penicillin
at a concentration of 50 units/ml, streptomycin at a con-
centration of 100 units/ml, and L-glutamine at a concen-
tration of 29.2 mg/ml (supplemented DMEM) with 10%
fetal bovine serum (FBS). At 75% confluence, cells were
lifted, counted and pooled in equal concentrations and
cultured in monolayer in flasks. Chondrocyte monolayer
flasks had Ad vector transduction with Ad-GFP or Ad-
BMP-2 performed at a multiplicity of infection (moi) of
17:1 (Adeno-XTM Rapid Titer Kit, BD Biosciences
Clontech, Palo Alto, CA) at 37˚C for a transduction time
of 2 h, washed and allowed to incubate overnight to ach-
ieve expression of transgene product. Transduction effi-
ciency was determined by calculating the percent of cells
fluorescing [525 nm wavelength] per microscopic field
(200X) in the monolayer chondrocytes treated with Ad-
GFP. Chondrocytes were harvested and allocated to PCL
culture systems.
2.4. Polycaprolactone Scaffolds
Nanofibrous [~700 nm diameter] PCL [MW 40,000,

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
910
Sigma Aldrich, St. Louis, MO] matrices were created
through an electrospinning process [18-22] to form 150-
200 m thick 3 dimensional sheets containing ~88%
porosity and an average pore size of 30m.[18] Sheets
were cut into 25 mm diameter discs using a 25 mm cir-
cular leather punch and sterilized by ethanol treatment.
2.5. Chondrocyte Culture on PCL Matrices
The 25 mm diameter sterile 3D nano -fibrous PCL matri-
ces were placed into Corning Costar Snapwell 12 mm in-
sertsTM [Corning Inc., Corning, NY] designed to fit into
standard 6-well culture plates. Chondrocytes were seed-
ed onto the PCL scaffolds by placing cells in suppleme-
nted DMEM into the Snapwell® inserts. Inserts were pla-
ced into standard 6-well culture plates containing suppl-
emented DMEM with the assigned concentration of FBS
to create a gradient of FBS across the scaffold construct.
2.6. Preliminary Study
Isolated chondrocytes were cultured on PCL matrices in
triplicate for 18 different conditions representing 3 cell
seeding densities of 5.0 × 105/ml, 1.0 × 106/ml, or 5.0 ×
106/ml, 2 serum gradients of 10% or 30%, and duration
of culture of 2, 7 and 14 days. PCL/cell matrices were
embedded in OCT medium, snap frozen in liquid nitro-
gen, cryosectioned (10 microns), and stained with tolu-
idine blue for microscopy or fixed for surface scanning
electron microscopy (SEM) to assess fiber pattern and
cell distribution. The cell seeding density and serum
gradient that supported the greatest number and depth of
penetration of chondrocytes in the PCL matrix was se-
lected for use in subsequent experiments.
2.7. Chondrogenesis of Genetically Engineered
PCL/ Cell Matrices
Using the best seeding conditions, isolated chondrocytes
from 5 horses were cultured on PCL matrices in 12 repli-
cates each of untreated, transduced with Ad-GFP, or tra-
nsduced with Ad-hBMP-2 for 14 days. Media were
changed on days 2, 7, and 14 and stored at –80°C. Con-
st- ructs from at least 2 replicates from each horse from
each treatment (untreated, Ad-GFP-treated and Ad-BM-
P-2-treated) cultured for 7 or 14 days were quantitatively
evaluated for each parameter of cell proliferation (DNA
[g/ml]; Hoechst assay), % viability (live/dead stain)
and morphology (confocal microscopy), matrix proteo-
glycan expression (ng/ml; DMMB assay) and gene ex-
pression (mRNA quantitative RT-PCR) of aggrecan
(ddCT), collagen 1 (ddCT) and collagen II (ddCT) using
equine specific primers and probes. Aggrecan and col-
lagen gene expression intensity was expressed as a ration
to 18sRNA and between Ad-BMP-2 treated chondro-
cytes to untreated and Ad-GFP controls (ddCT). BMP-2
protein concentration (ng/ml; ELISA) in the media and
chondrocyte GFP expression intensity were compared
among untreated, Ad-GFP-treated and Ad-BMP-2-tre-
ated PCL/ cell matrices.
2.8. Transgene and Protein Expression
GFP fluorescence was quantified using an in vivo imag-
ing system (IVIS®, Xenogen Corporation, Alameda, CA)
at day 2 post-transduction. GFP production was quanti-
fied as flux (photons of light produced per second per
square centimeter per steradian, photons/s/cm2/sr) [31].
Aliquots of media from PCL/cell matrix culture sys-
tems were frozen at –80°C on days 2, 7, and 14. Produc-
tion of hBMP-2 was quantified using enzyme-linked
immunosobent assays (ELISAs) for recombinant human
(rh) BMP-2 (Quantikine®, R & D Systems, Minneapolis,
MN) and expressed as picograms/milliliter/day.
2.9. Cytomorphology of PCL/ Cell Matrices
Cell morphology and viability were quantified using
special stains [LIVE/DEAD® Viability Kit, Molecular
Probes Inc. OR] and confocal microscopy [Leica DM-
1RE2, Leica Microsystems Inc., Bannockburn, IL]. Cell
morphology was scored after staining the cytoskeleton
(actin) with phallotoxin (Alexa Fluoro 647 Phalloidin,
Molecular Cell Probes Inc. Oreg on) and the nu cleu s with
DAPI (Molecular Probes Inc. OR). Three representative
fields were scored 0-4 with 0 representing the most
healthy adherent cell and 4 representing the most pykno-
tic and crenated cell.
2.10. Cellular Content
Cellular content was determined by analyzing DNA con-
tent of PCL/cell matrices using a modification of the
previously described Hoechst 33258 Fluorometeric as-
say. Discs of PCL/matrices containing chondrocytes
cultured for 7 or 14 days were placed into a 2.0 ml Ep-
pendorf tube with 1 ml of papain (Acros Organics) dis-
solved at 125 g/ml in sterile 1X PBS pH 6.0, with 5mM
cysteine HCl and 5mM Na2EDTA and incubated for 24h
at 60˚C. Papain digested samples were centrifuged at
1500 rpm (0.2 rcf) for 30 seconds to pellet debris and th e
supernatant isolated. DNA standards of double stranded
calf thymus DNA (Sigma) dissolved in TN buffer (50 mM
Tris pH 7.5, 150 mM NaCl) and serially diluted ranged
from 10 g/ml to 500 g/ml. in a Costar® 96 well, black,
clear bottom assay plate (Corning, Inc.) 50l of papain
digested sample or standard was placed in 200l of
Hoechst 33258 dye (AnaSpec, Inc) diluted to 0.2 g/ml
in TN buffer. The plates were read on a UV/Vis spec-
trometer (Lambda 45, Perkin Elmer) at an excitation of
360 nm, and emission of 460 nm, with a 430 nm cutoff
filter. DNA concentrations of samples were determined

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
911
from the calf thymus DNA standard curve and reported
in g/ml.
2.11. Proteoglycan Production in PCL/ Cell
Matrices
Extracellular production of proteoglycan was determined
using a dimethylmethylene blue assay (DMMB assay)
on papain digested supernatant from samples. DMMB
reagent was prepared by adding 16 mg 1, 9 dimethyl-
methylene blue (Polysciences, Inc) to 5 ml ethanol fol-
lowed by the addition of 2 ml of formic acid and 2 g
sodium formate. The volume was then brought to 1000
ml with distilled water. Chondroitin Sulfate A from bo-
vine trachea (Calbiochem) was serially diluted to create
standards between 5 ng/ml and 50 ng/ml. the assay was
performed by combining 50 l of standard solution or
sample solution with 200 l of the DMMB staining re-
agent and immediately determining the absorbance at
550 nm with a BioMate 3 Spectrophotometer (Thermo
Electron Corporation). Proteoglycan concentrations were
determined from the Chondroitin Sulfate standard curve
and reported in ng/ml.
2.12. Chondrocyte Gene Expression
Discs of PCL/ Chondrocyte matrices were collected at
day 7 and 14 of culture and placed into a 2.0 ml Eppen-
dorf tube with 1 ml of a commercially available reagent
composed of a monophasic solution of phenol and gua-
nidine isothiocyanate (Trizol® reagent, GibcoBRL, Life
Technologies, Frederick, MD). The PCL completely dis-
solved in the Trizol® reagent and total mRNA extracted
with guanidine thiocyanate/phenol/chloroform and sto-
red at -80˚C for real time reverse transcription polym-
erase chain reaction (RT-PCR) analysis. Equine specific
primers and probes were designed and used (Primer Ex-
press software, Applied Biosystems Inc., Foster City,
CA) to amplify and detect equine aggrecan, equine col-
lagen type Ia, and equine collagen type II mRNA (Table
1). Relative gene expression was quantified by RT-PCR
using 18s ribosomal RNA for normalization (Taqman
7000 sequence Detection System, Applied Biosystems
Inc.). All Taqman prob es were labeled at the 5’ end with
6-carboxy-fluorescene (FAM) and at the 3’ end with a
minor groove binder non-fluorescent quencher. The
thermal cycle protocol was 50˚C for 2 in, 60˚C for 30
min, 95˚C for 5 min, followed by 40 cycles of 95˚C for
15 s and 60˚C for 1 m i n.
2.13. Statistical Analysis
Quantitative data (aggrecan [ddCT], collagen I [ddCT],
collagen II [ddCT], proteoglycan [ng/ml], % chondro-
cyte viability, BMP-2 concentration [pg/ml]) were ana-
lyzed for difference among the three groups across days
using a repeated two-factor analysis of variance with
statistical significance set at P < 0.05. Scored data (mor-
phology) was analyzed among groups with a Kruskal-
Wallis Rank test (P < 0.05).
3. RESULTS
3.1. Confirmation of Gene Transduction: GFP
Transduction efficiency (GFP positive cells) was > 80%
and only detected in Ad-GFP transduced chondrocytes.
GFP expression intensity was high and sustained for the
14 days of the study (Figure 1).
Table 1. Equus caballus primer and probe sequences for
RT-PCR analysis of gene sequences.
Forward Primer 5’-AAGAGCGGAGACT
GGATTGAC-3’
Reverse Primer 5’-TCCATGTTGCAGA
AAACCTTCA-3’
Type II collag en
Probe 5’-AACCAGGGCTGCA
CCTTAGACGCC-3’
Forward Primer 5’-CTGTCATTTCTCTA
CTGGCGAAAC-3’
Reverse Primer 5’-CCAGTTCTTGGCTG
GGATGT-3’
Type I collagen
Probe 5’-TGCATTCGGGCTCA
ACCTGAA-3’
Forward Primer 5’-CCGCTGGTCAGATG
GACACACT-3’
Reverse Primer 5’-GAAGAAGTTGTCG
GGCTGGTT-3’
Aggrecan
Probe 5’-CTTGCAATTTGAGA
ACTGGCGCCC-3’
Figure 1. IVIS analysis of positive GFP fluores-
cence in Ad-GFP transduced chondrocytes (cen-
tral 3 wells). Adjacent wells contained untransd-
uced cells and Ad-BMP-2 transduced cells which
had no GFP expression.

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
912
3.2. Transgene Protein Expression
Gene transduction and protein expression were confir-
med for the Ad-BMP-2-transduced chondrocytes in the
PCL matrices on days 7 and 14 of culture. BMP concen-
tration was > 150,000 pg/ml in Ad-BMP-2-transduced
PCL/ cell matrices and <100 pg/ml in untreated and Ad-
GFP PCL/ cell matrices.
3.3. Scanning Electron Microscopy
Scanning electron microscopy of PCL scaffolds demon-
strated a random woven pa ttern of fiber s in the 300-1000
nm range with an average pore size of ~30 nm (Figure
2). Toluidine blue stained frozen cross sections of the
PCL/ cell scaffolds demonstrated penetration to ¾ depth
by 5 × 105and 1 × 106 cells/ scaffold in a 30% serum gr-
adient by day 7 (Figures 3A-B). Cell seeding density of
1 × 105 cells, 10% serum gradient and 2 days were insu-
fficient to produce chondrocyte penetration of PCL ma-
trices. Confocal microscopy of stained untreated chon-
drocytes and GFP expressing chondrocytes seeded on
PCL matrices demonstrated an even distribution of cells
with normal morphology, sustained gene expression, and
cell viability in situ for 14 days (Figures 4A-B).
3.4. Cytomorphology of PCL/ Cell Matrices
Cell morphology score (median 4; range 3-4) and nucl-
ear morphology score (median 3; rang e 3-4) w as not d if-
ferent (P > 0.05) among untreated, Ad-GFP, and Ad-
BMP-2 PCL/ cell matrices at both days 7 and 14.
Figure 2. Scanning electron microscopy of the scaffold surface
(1000X).
(a) (b)
Figure 3. Toluidine blue positive chondrocytes (a) on the sur-
face of the PCL at day 2. Toluidine blue positive chondrocytes
(b) penetrating most of the scaffold with a 20% FBS gradient
and 1 × 106 cell seeding density.
(a) (b)
Figure 4. Cells transduced with Ad-GFP and suspended in
scaffold demonstrated fluorescence under confocal microscopy
(a) and had similar cell numbers and morphology to other
transduced and untransduced cells at day 7 (b).
3.5. Cell Content
DNA content (g/ml) was not different among untreated,
Ad-GFP, and Ad-BMP-2 PCL/cell matrices at day 7, but
was significantly decreased in Ad-GFP and increa- sed
in Ad-BMP-2 by day 14 (P < 0.01) (Figure 5A).
3.6. Proteoglycan Production in PCL/ Cell
Matrices
Proteoglycan content (ng/ml) within the PCL/cell matri-
ces was significantly greater in the Ad-BMP-2 treated
matrices by day 7 and continued to significantly in crease
only in the Ad-BMP-2 matrices (P < 0.05) (Fi gure 5B ).
3.7. Chondrocyte Gene Expression
Quantitative gene expression was expressed as the inve-
rse of Delta CT values so that positive values correlated
to increased gene expression. Ad-BMP-2 matrices has
significantly greater Type II collagen (P < 0.002) and ag-
grecan (P < 0.001) gene expression than untreated and
Ad-GFP matrices by day 7 and sustained until at least
day 14. There was no difference in type I collagen gene
expression among groups (Figures 6A-C).

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
913
(a)
(b)
Figure 5. DNA content (a) decreased on day 14 as com-
pared to day 7 except in the Ad-BMP2 treated group (p <
0.01) which sustained their cell numbers. Cells transduced
with Ad-BMP2 (b) had significantly greater proteoglycan
content by day 7 (p < 0.05), which had further increased by
day 14 (p < 0.03).
(a)
(b)
(c)
Figure 6. Cells transduced with Ad-BMP2 had significantly gr-
eater gene expression of chondrogenic markers including Type
II collagen (a) and aggrecan (b) and significantly less gene
expression of Type I collagen (c).
4. DISCUSSION
Our data demonstrated that a serum gradient can be used
to attract chondrocytes to seed into an electrospun PCL
nanofiber scaffold and sustain chondrogenic phenotype
in standard media. Nanofiber scaffolds offer the adva nta-
ge of smaller biologic fiber diameters, but this woven me-
sh can be a challenge for cells to permeate the scaffold.
Our work demonstrated this as the chon drocytes remain-

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
914
ed on the surface in the control specimens. This is partic-
ularly challenging for relatively nonmobile ch ondrocytes
that must dedifferentiate to a fibroblastic phenotype to
migrate along fibrils and into pores. In our study, BMP2
sustained chondrogenic phenotype and extracellular ma-
trix production while permitting these cells to enter the
scaffold. For cartilage engineering, migration is particu-
larly relevant to accommodate the thickness of a scaffold
desired for cartilage regeneration (100-300 uM). Althou-
gh it is possible to incorpor ate cells into a scaffold using
an in situ cell seeding protocol [38], or by modifying the
electrospinning protocol to produce larger fibers (in the
micrometer range with larger pore sizes of >100 uM
[23,29]), these methods are potentially handicapped by
injury or contamination of cells or loss of the bioactivity
advantage of the nanofibrous scale. In vivo generation of
a serum gradient in th e base of a cartilage defect is pote-
ntially achievable w ith the use of autologous serum pro-
ducts placed in the bed of debrided cartilage defects.[40]
Commercially available platelet rich plasma or plasma
concentrate products have been shown to have increased
growth factor concentration of TGFbeta1 and 2 as well
as other growth factors that may serve as a chemoattrac-
tant to chondrocytes layered on the surface of a scaffold
in vivo. [41] Studies to further investigate this are war-
ranted. These biologic plasma/fibrin products offer the
additional advantage of serving as a biologic glue that
may be able to help secure the scaffold into the defect.
Fibrin glue is reported to secure cells within full-thick-
ness cartilage defects in animal models [42].
In our study, the chondrocytes engineered to express
BMP-2 had accelerated and amplified chondrogenesis
within the scaffold as compared to control cells. In a pr e-
vious in vitro study, chondrocytes incorporated in a hyd-
rogel had enhanced matrix generation when the media
was supplemented with TGFbeta3, a known chondrog-
enic growth factor. [11] Supplementing cells within a
scaffold with a growth factor solution is technically dif-
ficult in vivo, other than by using a serum/plasma prod-
uct as described above. Saturation of the scaffold with a
TGFbeta solution is likely to have the TGFbeta rapidly
leave the site or be diluted by join t fluid. Engineering of
chondrocytes with chon drogenic genes offers an alterna-
tive for sustained trophic influences on the cells within
the scaffold as demonstrated with BMP2 in our study.
Additionally the release of soluable BMP2 will have a
paracrine effect on other cells migrating into the site to
heal the defect such as bone marrow-derived cells or sy-
novial fibroblasts. Other genes such as insulin-like gro-
wth factor or TGFbeta may function similarly and could
be used in chondrocyte engineering. The process of cho-
ndrocyte transduction with adenovirus did not nullify
this trophic effect in our study. Methods of cell transdu-
ction other than use of adenovirus may offer advantages,
most notably a more sustained gene expression [43]. Our
data provided evidence that engineering cells can pro-
vide enhanced chondrogenesis in Electrospun PCL nan-
ofiber scaffolds for use in cartilage tissue eng ineering.
Current techniques under investigation for autologous
chondrocyte transplantation include direct intraoperative
processing of autologous articular cartilage to expose or
isolate chondrocytes for immediate reimplantation [25]
or chondrocyte expansion in culture prior to reimplanta-
tion at a second surgery. G ene transduction with BMP-2
can occur in under 2 hours and could be a practical ge-
netic engineering technique to augment autologous cells
with genes promoting chondrogenesis in the operating
room. Other techniques that focus on autologous chondr-
ocyte expansion prior to reimplantation at a second surg-
ery would also be readily amenable to genetic engineer-
ing as described in this report. This study provides evi-
dence that this process holds merit for potential clinical
application. Our study was limited to in vitro processing
and further studies to confirm this potential in an in vivo
animal model are warranted.
REFERENCES
[1] Simon, T.M. and Jackson, D.W. (2006) Articular carti-
lage: Injury pathways and treatment options. Sports
Medicine and Arthroscopy Review, 14(3), 146-154.
[2] Chung, C. and Burdick, J.A. (2008) Engineering carti-
lage tissue. Advanced Drug Delivery Reviews, 60(2),
243-262.
[3] Kino-Oka, M., Maeda, Y., Yamamoto, T., Sugawara, K.
and Taya, M. (2005) A kinetic modeling of chondrocyte
culture for manufacture of tissue-engineered cartilage.
Journal of Bioscience and Bioengineering, 99(3), 197-207.
[4] Thorvaldsson, A., Stenhamre, H., Gatenholm, P. and
Walkenstrom, P. (2008) Electrospinning of highly porous
scaffolds for cartilage regeneration. Biomacromolecules,
9(3), 1044-1049.
[5] Li, W.J. and Tuan, R.S. (2009) Fabrication and applica-
tion of nanofibrous scaffolds in tissue engineering. Cur-
rent Protocols in Cell Biology, Chapter 25(25), Unit
25.2.
[6] Jeong, C.G. and Hollister, S.J. (2010) A comparison of
the influence of material on in vitro cartilage tissue en-
gineering with PCL, PGS and POC 3D scaffold archi-
tecture seeded with chondrocytes. Biomaterials, 31(15),
4304-4312.
[7] Martinez-Diaz, S., Garcia-Giralt, N., Lebourg, M., et al.
(2010) In vivo evaluation of 3-dimensional polycaprolac-
tone scaffolds for cartilage repair in rabbits. American
Journal of Sports Medicine, 38(3), 509-519.
[8] He, L., Liu, B., Xipeng, G., et al. (2009) Microstructure
and properties of nano-fibrous PCL-b-PLLA scaffolds
for cartilage tissue engineering. European Cells and Ma-
terials, 18, 63-74.
[9] Martins, A., Pinho, E.D., Faria, S., et al. (2009) Surface

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
915
modification of electrospun polycaprolactone nanofiber
meshes by plasma treatment to enhance biological per-
formance. Small, 5(10), 1195-1206.
[10] da Silva, M.A., Crawford, A., Mundy, J., et al. (2009)
Evaluation of extracellular matrix formation in poly-
caprolactone and starch-compounded polycaprolactone
nanofiber meshes when seeded with bovine articular cho-
ndrocytes. Tissue Engineering Part A, 15(2), 377-385.
[11] Maher, S.A., Mauck, R.L., Rackwitz, L. and Tuan, R.S.
(2010) A nanofibrous cell-seeded hydrogel promotes in-
tegration in a cartilage gap model. Journal of Tissue En-
gineering and Regenerative Medicine, 4(1), 25-29.
[12] Li, W.J., Danielson, K.G., Alexander, P.G. and Tuan,
R.S. (2003) Biological response of chondrocytes cultured
in three-di- mensional nano fibrous p oly (epsilon-cap rolactone)
scaffolds. Journal of Medical Materials Research, 67(4),
1105-1114.
[13] Lee, J.J., Yu, H.S., Hong, S.J., Jeong, I., Jang, J.H. and
K im, H.W. ( 2009 ) Nanofibrou s membrane of collagen- poly-
caprola- ctone for cell growth and tissue regeneration.
Journal of M aterials Sc ience: Materials in Medicine, 20(9),
1927-1935.
[14] Mierisch, C. M., Wilson, H.A., Turner, M. A., et al. (2003)
Chondrocyte transplantation into articular cartilage de-
fects with use of calcium alginate: the fate of the cells.
Journal of Bone and Joint Surgery, 85A(9), 1757-1767.
[15] I to, Y., Fitzsimmons, J.S., Sanyal, A., Mello, M.A., Mukher-
jee, N. and O'Driscoll, S.W. (2001) Localization of chon-
drocyte precursors in periosteum. Osteoarthritis and
Cartilage, 9(3), 215-223.
[16] Li, J., Chen, Y., Mak, A.F., Tuan, R.S., Li, L. and Li, Y.
(2009) A one-step method to fabricate PLLA scaffolds
with deposition of bioactive hydroxyapatite and collagen
using ice- based microporogens. Acta Biomaterialia, 6(6),
2013-2019.
[17] Mendes, S.C., Bezemer, J., Claase, M.B., et al. (2003) Ev
aluation of two biodegradable polymeric syste ms as sub-
strates for bone tissue engineering. Tissue Engineering, 9
(Suppl 1), 91-101.
[18] Duling, R.R., Dupaix, R.B., Katsube, N. and Lannutti, J.
(2008) Mechanical characterization of electrospun poly-
caprolac- tone (PCL): A potential scaffold for tissue en-
gineering. Journal of Biomechanical Engineering, 130(1),
011006.
[19] Gaumer, J ., Pr asa d, A ., Le e, D. and Lannu tti, J. (2009) Struc-
ture-function relationships and source-to-ground distance
in electrospun polycaprolactone. Acta Biomaterialia, 5(5),
1552-1561.
[20] Johnson, J., Niehaus, A., Nichols, S., et al. (2009) Electr-
ospun PCL in vitro: a microstructural basis for mechani-
cal property changes. Journal of Biomaterials Sicence,
Polymer Edition, 20(4), 467-481.
[21] Li, W.J., Chiang, H., Kuo, T.F., Lee, H.S., Jiang, C.C.
and Tuan, R.S. (2009) Evaluation of articular cartilage
repair using biodegradable nanofibrous scaffolds in a
swine model: A pilot study. Journal of Tissue Engineer-
ing and Regenerative Medicine, 3(1), 1-10.
[22] Fecek, C., Yao, D., Kacorri, A., et al. (2008) Chondro-
genic derivatives of embryonic stem cells seeded into 3D
poly- caprolactone scaffolds generated cartilage tissue in
vivo. Tissue Engineering Part A, 14(8), 1403-1413.
[23] Nam, J., Rath, B., Knobloch, T.J., Lannutti, J.J. and
Agarwal, S. (2009) Novel electrospun scaffolds for the
molecular analysis of chondrocytes under dynamic com-
pression. Tissue Engineering Part A, 15(3), 513-523.
[24] Chang, K.Y., Cheng, L.W., Ho, G.H., Huang, Y.P. and
Lee, Y.D. (2009) Fabrication and characterization of
poly (gamma- glutamic acid)-graft-chondroitin sulfate/
polycaprolacto- ne porous scaffolds for cartilage tissue
engineering. Acta Biomaterialia, 5(6), 1937-1947.
[25] Frisbie, D.D., Lu, Y., Kawcak, C.E., DiCarlo, E.F., Bi-
nette, F. and McIlwraith, C.W. (2009) In vivo evaluation
of autologous cartilage fragment-loaded scaffolds im-
planted into equine articular defects and compared with
autologous chondrocyte implantation. American Journal
of Sports Medicine, 37 (Suppl 1), 71-80.
[26] Kang, X., Xie, Y., Powell, H.M., et al. (2007) Adipogenesis
of murine embryonic stem cells in a th r ee - d i m e n s i on a l c u l -
ture system using electrospun polymer scaffolds. Bio-
materials, 28(3), 450-458.
[27] Johnson, J., Nowicki, M.O., Lee, C.H., et al. (2009)
Quantitative analysis of complex glioma cell migration
on electrospun polycaprolactone using time-lapse mi-
croscopy. Tissue Engineering Part C, 15(4), 531-540.
[28] Drilling, S., Gaumer, J. and Lannutti, J. (2009) Fabrica-
tion of burst pressure competent vascular grafts via elec-
trospinning: Effects of microstructure. Journal of Bio-
medical Materials Research Part A, 88(4), 923-934.
[29] Xian, C.J. and Foster, B.K. (2006) Repair of injured ar-
ticular and growth plate cartilage using mesenchymal
stem cells and chondrogenic gene therapy. Current Stem
Cell Research & Therapy, 1(2), 213-229.
[30] Puetzer, J.L., Petitte, J. and Loboa, E. (2010) Compara-
tive review of growth f actors for in duction of 3-Dimensional
in vitro chondrogenesis in human mesenchymal stem
cells isolated from bone marrow and adipose tissue. Tis-
sue Engineering Part B, 16(4), 435-444
[31] Zachos, T., Diggs, A., Weisbrode, S., Bartlett, J. and
Bertone, A. (2007) Mesenchymal stem cell-mediated
gene delivery of bone morphogenetic protein-2 in an ar-
ticular fracture m odel. Molecular Th erapy, 15(8), 1543-1550.
[32] van der Kraan, P.M., Davidson, E.N. and van den Berg,
W.B. (2010) Bone morphogenetic proteins and articular
cartilage to serve and protect or a wolf in sheep clothing's?
Osteoarthritis and Cartilage, 18(6), 735-741.
[33] Kaps, C., Bramlage, C., Smolian, H., et al. (2002) Bone
morphogenetic proteins promote cartilage differentiation
and protect engineered artificial cartilage from fibroblast
invasion and destruction. Arthritis & Rheumatism, 46(1),
149-162.
[34] Majumdar, M.K., Wang, E. and Morris, E.A. (2001)
BMP-2 and BMP-9 promotes chondrogenic differentia-
tion of human multipotential mesenchymal cells and
overcomes the inhibitory effect of IL-1. Journal of Cell
Physiology, 189(3), 275-284.
[35] Glansbeek, H.L., van Beuningen, H.M., Vitters, E.L.,
Morris, E.A., van der Kraan, P.M. and van den Berg,
W.B. (1997) Bone morphogenetic protein 2 stimulates
articular cartilage proteoglycan synthesis in vivo but does
not counteract interleukin-1alpha effects on proteoglycan
synthesis and content. Arthritis & Rheumatism, 40(6),
1020-1028.
[36] Smith, P., Shuler, F.D., Georgescu, H.I., et al. (2000)
Genetic enhancement of matrix synthesis by articular

R. T. Gorsline et al. / J. Biomedical Science and Engineering 3 (2010) 908-916
916
JBiSE
chondrocytes: Comparison of different growth factor
genes in the presence and absence of interleukin-1. Ar-
thritis & Rheumatism, 43(5), 1156-1164.
[37] Zachos, T.A., Shields, K.M. and Bertone, A.L. (2006)
Gene-mediated osteogenic differentiation of stem cells
by bone morphogenetic proteins-2 or -6. Journal of Or-
thopaedic Research, 24(6), 1279-1291.
[38] Stankus, J.J., Guan, J., Fujimoto, K. and Wagner, W.R.
(2006) Microintegrating smooth muscle cells into a bio-
degradable, elastomeric fiber matrix. Biomaterials, 27(5),
735-744.
[39] Nam, J., Huang, Y., Agarwal, S. and Lannutti, J. (2008)
Materials selection and residual solvent retention in bio-
degradable electrospun fibers. Journal of Applied Poly-
mer Science, 107(3), 1547-1554.
[40] Lopez-Vidriero, E., Goulding, K.A., Simon, D.A., San-
chez, M. and Johnson, D.H. (2010) The use of plate-
let-rich plasma in arthroscopy and sports medicine: Op-
timizing the healing environment. Arthroscopy, 26(2),
269-278.
[41] Sutter, W.W., Kaneps, A.J. and Bertone, A.L. (2004)
Comparison of hematologic values and transforming gro-
wth factorbeta and insulin-like growth factor concentra-
tions in platelet concentrates obtained by use of buffy
coat and apheresis methods from equine blood. American
Journal of Veterinary Research, 65(7), 924-930.
[42] Wilke, M.M., Nydam, D.V. and Nixon, A.J. (2007) En-
hanced early chondrogenesis in articular defects follow-
ing arthroscopic mesenchymal stem cell implantation in
an equine model. Journal of Orthopaedic Research,
25(7), 913-925.
[43] Santangelo, K.S., Baker, S.A., Nuovo, G., Dyce, J., Bart-
lett, J.S. and Bertone, A.L. (2007) Detectable reporter ge-
ne expression following transduction of adenovirus and
adeno-associated virus serotype 2 vectors within
full-thickness osteoarthritic and unaffected canine carti-
lage in vitro and unaffected guinea pig cartilage in vivo.
Journal of Orthopaedic Research, 28(2), 149-155.