J. Biomedical Science and Engineering, 2010, 3, 1041-1049 JBiSE
doi:10.4236/jbise.2010.311135 Published Online November 2010 (http://www.SciRP.org/journal/jbise/).
Published Online November 2010 in SciRes. http://www.scirp.org/journal/jbise
Chondrogenic differentiation of stem cells in human umbilical
cord stroma with PGA and PLLA scaffolds
Liang Zhao1,2, Michael S. Detamore1,3
1Department of Spinal & Orthopaedic Surgery, Southern Medical University, Nanfang Hospital, Guangzhou, China;
2Department of Chemical & Petroleum Engineering, University of Kansas, Lawrence, KS, USA;
3Department of Mechanical Engineering, University of Kansas, Lawrence, KS, USA.
Received 9 September 2010; revised 25 September 2010; accepted 30 September 2010.
ABSTRACT
The stem cells in the umbilical cord stroma, or
Wharton’s jelly, are referred to as human umbilical
cord mesenchymal stromal cells (hUCMSCs) and
have been shown to differentiate along a chondro-
genic lineage. The aim of this study was to evaluate
the chondrogenic differentiation of hUCMSCs in ei-
ther polyglycolic acid (PGA) or poly-L-lactic acid
(PLLA) non-woven mesh scaffolds for cartilage tissue
engineering. PGA is widely known to degrade faster
than PLLA, and over longer time scales, and differ-
ences may be expected to emerge after extended cul-
ture periods. Therefore, the focus of this study was to
evaluate differences over a shorter duration. After 21
days of culture in PLLA or PGA scaffolds, hUCMSC
constructs were analyzed for biochemical content,
histology, and gene expression. Overall, there were
only minute differences between the two scaffold
groups, with similar gene expression and biosynthesis.
The most notable difference was a change in shape
from cylindrical to spherical by the PGA, but not
PLLA, scaffold group. The overall similar behavior
of the groups may suggest that in vivo application of
hUCMSC-seeded PLLA or PGA scaffolds, following
a 21-day pre-culture period, may yield similar con-
structs at the time of implantation. However, differ-
ences may begin to become more apparent with in
vivo performance following implantation, or with in
vitro performance over longer time periods.
Keywords: Umbilical Cord; Stem Cells; Cartilage Tis-
sue Engineering; Scaffold
1. INTRODUCTION
The treatment of articular cartilage injuries is a challenge
due to the very limited capacity for cartilage self-repair
and the limited surgical techniques that successfully treat
the damaged cartilage [1-3]. Stem cell-based tissue en-
gineering techniques have the potential to revolutionize
the ability to regenerate damaged cartilage [4-7].
Recently, a promising stem cell source residing in the
Wharton’s jelly of the umbilical cord, referred to as human
umbilical cord mesenchymal stromal cells (hUCMSCs),
appears to bear multi-potential mesenchymal stem cell
characteristics and can differentiate into cells resembling
adipocytes, osteoblasts, chondrocytes, neurons, and en-
dothelial cells [8-15]. Recently, our laboratory has suc-
cessfully induced hUCMSCs into osteogenic and chon-
drogenic lineages [14-16]. For engineering articular car-
tilage implants, a crucial consideration is the scaffolding
biomaterial. These biomaterials have included a variety
of natural gels and hydrogels based on collagen, glyco-
saminoglycans, hyaluronic acid, agarose, alginate and
gelatin [17-22], as well as a number of synthetic materi-
als used as scaffolds for chondrogenic differentiation of
stem cells, such as polyglycolic acid (PGA), among the
most common materials for cartilage tissue engineering
[23-25]. However, previous studies showed that PGA
scaffolds are limited by rapid degradation in vitro [26-
30].
Therefore, the aim of this study was to investigate the
incorporation of hUCMSCs with either PGA or PLLA
scaffolds under chondrogenic differentiation, and to
compare the relative performance of the two biomate-
rials under these conditions.
2. MATERIALS AND METHODS
2.1. Isolation and Culture of hUCMSCs
The hUCMSCs were harvested following our previous
method, with IRB approval (KU-Lawrence no.15402,
KU Medical Center no.10951) and informed consent
[14]. Four cords (2 males and 2 females, length: 20 ± 3
cm) were obtained from the University of Kansas Medi-

L. Zhao et al. / J. Biomedical Science and Engineering 3 (2010) 1041-1049
1042
cal Center (KUMC) and were processed within 24 hours.
In brief, isolated hUCMSCs were cultured in a complete
culture medium consisting of low-glucose Dulbecco’s
modified Eagle’s medium (DMEM-LG), 10% MSC-
qualified fetal bovine serum (FBS), and 1% penicil-
lin/streptomycin (PS) (Invitrogen, Carlsbad, CA). Cells
were plated in cell culture flasks at 3000 cells/cm2 (P0
cells). Non-adherent cells were rinsed off 3 days after
plating. When attached hUCMSCs reached 80% conflu-
ence, the cells were detached using 0.05% trypsin-EDTA
(Invitrogen) and expanded (P1). Culture medium was
changed every 2 days, and the cells were split approxi-
mately 1:4 at each passage thereafter. Passage 4 (P4)
hUCMSCs were used for all experiments.
2.2. Identification of hUCMSCs
The P4 hUCMSCs were characterized by flow cytome-
try to analyze the specific surface antigens of cells in-
cluding CD13, CD29, CD34, CD45, CD49e, CD73,
CD90 and CD105. All supplies were purchased from BD
Biosciences (San Jose, CA), except CD73 and CD105
from eBioscience (San Diego, CA). In brief, approxi-
mately 0.5 × 106 cells per vial were used to stain. The
nonspecific binding was blocked with a staining buffer
including PBS and 2% FBS for 15 minutes on ice, while
the cells were incubated with single label antigen for 20
minutes on ice. Mouse isotype antigens served as the
control. The analysis was measured using a FACscan
(Becton Dickinson, San Jose, CA).
2.3. Seeding hUCMSCs Into PGA and PLLA
Scaffolds
Non-woven PGA and non-woven PLLA meshes (50
mg/cc; > 95% porosity; Biomedical Structures LLC, RI)
were punched to create cylindrical scaffolds (5 mm di-
ameter, 2 mm thick). Scaffolds were sterilized following
our previous methods with ethylene oxide [14]. The P4
hUCMSCs were seeded at a cell density of 25 × 106 cells
per ml, 0.98 × 106 of hUCMSCs in 35 μl of the complete
medium were seeded in a dropwise manner into each
scaffold. Cell-seeded scaffolds were incubated in a cell
culture incubator for 3 hours to allow for cell attachment.
To keep constructs hydrated, 10 μl of complete medium
was added every 1 hour. After the 3-hour period, 2 ml of
either chondrogenic medium was added into each culture
well. Chondrogenic medium consisted of high-glucose
DMEM (DMEM-HG; Invitrogen), 1% non-essential
amino acids (NEAA; Invitrogen), 1X sodium pyruvate
(Invitrogen), 1X insulin transferring selenium premix
(ITS premix; BD Biosciences), 50 μg/ml ascorbic acid
2-phosphate (AA2P; Sigma, St Louis, MO), 40 μg/ml
L-proline (Sigma), 100 nM dexamethasone (Sigma) and
10 ng/ml transforming growth factor beta-1 (TGF-β1;
R&D system, Minneapolis, MN). For the control, a
group of cell-seeded scaffolds were cultured in the com-
plete culture medium. All of the medium was changed
every two days for 21 days [15].
2.4. Adhesion Assay
Approximately 200,000 P4 hUCMSCs were suspended
in 30 μl of the complete culture medium and then seeded
in the PGA and PLLA scaffolds as previously described
[15,31]. Seeded scaffolds were incubated in a humidified
atmosphere containing 5% CO2 at 37°C for 6 hours to
allow for cell attachment. At three and six hours, four
scaffolds were rinsed with 2 ml phosphate-buffered sa-
line (PBS) and the cells in PBS were counted. By sub-
tracting the number of washed out hUCMSCs from
200,000 cells per scaffold, the number of cells adhering
to the scaffold was calculated [27].
2.5. Biochemical Assays
At 0, 14 and 21 days, PGA and PLLA scaffolds were
processed for biochemical assays including DNA, gly-
cosaminoglycan (GAG) and hydroxyproline (HYP) con-
tent as in our previous work [15]. To measure DNA,
GAG, and HYP contents, four scaffolds per group and
time point were digested in 1.5 ml papain solution (120
μg/ml) at 60°C overnight. DNA contents were fluoro-
metrically determined using a PicoGreen kit according
to the manufacturer’s instruction (Invitrogen). GAG
contents were measured using a dimethylmethylene blue
(DMMB) dye binding assay kit according to the manu-
facturer’s instructions (Biocolor, Belfast, UK). In brief,
100 μl solution of each sample was measured after
binding with 1 ml of DMMB by using chondroitin sul-
fate as the standard. Solutions were analyzed at 656 nm
using a Fluoroskan Ascent plate reader (Thermo Elec-
tron Corporation, Waltham, MA). HYP content was de-
termined using a modified HYP assay protocol [15]. In
brief, 400 μl of specimen solution was hydrolyzed and
neutralized, then analyzed at 550 nm using a Fluoroskan
Ascent plate reader (Thermo Electron Corporation).
2.6. Histology and Immunohistochemistry
At 14 and 21 days, PGA and PLLA scaffolds were fro-
zen and sectioned at a thickness of 10 μm using a cry-
ostat (Microm, Thermo Scientific, Waltham, MA). For
immunohistochemical analysis, two scaffolds were used
to detect types I and II collagen and aggrecan using a
BioGenex i6000 autostainer (BioGenex, San Ramon, CA)
as in our previous study [15]. Primary antibodies in-
cluded the mouse monoclonal IgG anti-collagen type I
(1:1500 dilution; Accurate Chemical and Scientific,
Westbury, NY), mouse monoclonal IgG anti-collagen

L. Zhao et al. / J. Biomedical Science and Engineering 3 (2010) 1041-1049
1043
type II (1:1000 dilution; Chondrex, Redmond, WA), and
mouse monoclonal IgG anti-aggrecan (1:50 dilution;
Abcam, Cambridge, MA). In brief, endogenous peroxi-
dase activity was inhibited using 1% hydrogen peroxide,
blocked in horse serum and incubated with a primary
antibody. Then the sections were incubated with a strep-
tavidin-linked horse anti-mouse IgG secondary antibody
(Vector Laboratories, Burlingame, CA). After secondary
antibody incubation, the sections were incubated with an
avidin-biotin ylated enzyme complex (ABC complex;
Vector Laboratories) and VIP substrate (Vector Labora-
tories). Histological analyses were performed using Sa-
franin O/fast green staining with Harris hematoxylin
(Sigma) to visualize GAG distribution [32].
2.7. Quantitative Real-Time Reverse
Transcription Polymerase Chain Reaction
Total RNA of PGA and PLLA scaffolds were extracted
with TRIzol reagent according to the manufacturer’s
protocol (Invitrogen) and reverse-transcribed into cDNA
using a high-capacity cDNA Archive kit (Applied Bio-
systems, Foster city, CA). Real-time RT-PCR reactions
were performed using an Applied Biosystems 7500 Fast
System. TaqMan gene expression assay kits (Applied
Biosystems), including two pre-designed specific prim-
ers and probes, were used to measure the transcript lev-
els of the proposed genes including human type I colla-
gen (Hs00164004), type II collagen (Hs00156568), ag-
grecan (Hs00153936) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; Hs99999905). Relative ex-
pression level for each target gene was evaluated using
the 2−ΔΔCt method [33]. The control samples at the first
day served as the calibrator. The fold of change was ob-
tained with n = 4.
3. RESULTS
3.1. Phenotype of hUCMSCs
The P4 hUCMSCs were characterized with respect to the
expression of surface antigens by flow cytometry. The
hUCMSCs that were characterized with respect to the
expressions of CD34 and CD45 were less than 1%,
whereas hUCMSCs had positive expressions for CD73
(96 ± 2%), CD90 (98 ± 4%) and CD105 (99 ± 3%). The
hUCMSCs also expressed high levels of CD13 (96 ±
4%), CD29 (95 ± 4%) and CD49e (98± 4%) (Figure 1).
3.2. Morphology and Adhesion Assay of PGA
and PLLA Scaffolds
After 21 days of chondrogenic differentiation culture,
the shapes of PGA and PLLA scaffolds were observed to
be different. The shape of PGA scaffolds significantly
changed from cylindrical to spherical over the 21 day
period (Figures 2(a), (c)). The contraction of PLLA
scaffolds was relatively negligible during the same
process and no significant change of the PLLA scaffolds’
morphology was observed (Figures 2(b), (d)).
For the adhesion assay, three hours after seeding
hUCMSCs, the adherent cell percentage was 60 ± 9% in
PGA scaffolds and was 58 ± 6% in PLLA scaffolds. Af-
ter 6 hours, the adherent cell percentage increased to 73
± 8% in PGA scaffolds and to 75 ± 4% in PLLA scaf-
folds. The results from the adhesion assay showed no
significant difference between PGA and PLLA scaffolds
(p > 0.1) (Figure 2(e)).
3.3. Biochemical Assays
Throughout the entire period of the chondrogenic dif-
ferentiation culture, the DNA content decreased in both
scaffolds as shown in Figure 3(a). The DNA content
decreased approximately 30% in the PGA scaffolds and
21% in the PLLA scaffolds over the 21 day period. At 21
days, the DNA content in PLLA scaffolds was 18.8%
higher than that of PGA scaffolds (p < 0.05).
The GAG content in both scaffolds decreased with
culture time (Figure 3(b)). In the PGA scaffolds, the
highest GAG content of 11.1 ± 2.4 μg/scaffold was
measured at 0 days. The GAG content in the PGA scaf-
folds decreased by 51% at 14 days and decreased by
80% at 21 days when compared to the 0 day values. In
the PLLA scaffolds, the highest GAG content was 10.9 ±
2.3 μg/scaffold at 0 days. The GAG content in PLLA
scaffolds decreased by 43% at 14 days and decreased by
70% at 21 days when compared to the 0 day values. At
21 days, the GAG content in PLLA scaffolds was higher
than that in PGA scaffolds (p < 0.05).
Cumulative HYP production in the PGA and PLLA
scaffolds was measured at 0, 14, and 21 days. The higher
HYP content was observed at 14 days in both scaffolds.
In the PGA scaffold group, the HYP content was 1.5 ±
0.1 μg/scaffold at 14 days and 1.2 ± 0.1 μg/scaffold at 21
days. In the PLLA scaffold group, the HYP content was
1.4 ± 0.2 μg/scaffold at 14 days and 1.1 ± 0.1 μg/scaffold
at 21 days. The HYP content measurements showed no
significant difference between the HYP contents of the
PGA and PLLA scaffolds (p > 0.1) (Figure 3(c)).
3.4. Histology and Immunohistochemistry
At 14 days, both PGA and PLLA sections showed a
moderate amount of collagen type I staining with a trace
amount of collagen type II and aggrecan staining. There
was no significant difference in staining intensity present
between the PGA and PLLA groups (Figure 4). Differ-
ences in immunostaining were generally minimal be-
tween 14 days and 21 days. Only Saf-O staining appeared

L. Zhao et al. / J. Biomedical Science and Engineering 3 (2010) 1041-1049
1044
(a)
(b)
Figure 1. Flow cytometric analysis of surface-marker expression of P4 hUCMSCs. (a) Surface phenotypic characterization of
hUCMSCs from a representative sample. The hUCMSCs were positive for CD13, CD29, CD49e, CD73, CD90 and CD105, but
negative for CD34 and CD45; (b) The histogram plot of expression of cell surface markers (mean ± standard deviation; n = 4).
to increase in intensity from 14 days to 21 days (Figure
4).
3.5. Gene Expression
As shown in Figure 5, at 14 days and 21 days, the cells
in both PGA and PLLA scaffolds were observed to sig-
nificantly activate expression of genes encoding for col-
lagen type I, collagen type II and aggrecan (p < 0.05). In
PGA scaffolds, collagen I showed a 3-fold increase at 14
days and a 15-fold increase at 21 days when compared to
0 day (p < 0.05). The collagen II expression in PGA
scaffolds increased 8-fold at 14 days and 9-fold at 21
days when compared to 0 days (p < 0.05). Aggrecan in
the PGA scaffolds increased 3-fold at 14 days and 6-fold
at 21 days when compared to 0 day (p < 0.05). In PLLA
scaffolds, collagen I showed a 4-fold increase at 14 days
and a 16-fold increase at 21 days when compared to 0
day (p < 0.05). The collagen II in PLLA scaffolds in-
creased 7-fold at 14 days and 10-fold at 21 days when
compared to day 0, while aggrecan expression increased

L. Zhao et al. / J. Biomedical Science and Engineering 3 (2010) 1041-1049
1045
(a) (b)
(c) (d)
Figure 2. Morphology and adhesion assay of PGA and PLLA
scaffolds. (a) The morphology of a representative PGA scaffold
at 1 day after seeding; (b) The morphology of a representative
PLLA scaffold at 1 day when seeded with hUCMSCs; (c) The
morphology of a representative PGA scaffold after 21 days of
chondrogenic differentiation culture; (d) The morphology of a
representative PLLA scaffold at 21 days. The scale bars are 5
mm; (e) Adhesion assay of PGA and PLLA scaffolds (mean ±
standard deviation; n = 4).
3-fold at 14 days and 5-fold at 21 days when compared
to day 0 (p < 0.05). However, there was no significant
difference in gene expression between the PGA and
PLLA scaffold groups (p > 0.1) (Figure 5).
4. DISCUSSION
hUCMSCs may be considered to be a promising, inex-
haustible and low-cost source of mesenchymal stem
cells. In previous studies, hUCMSCs were successfully
induced for chondrogenic differentiation [15,16,30]. The
present study investigated the influence of seeding
hUCMSCs into PGA and PLLA scaffolds on the poten-
tial ability of chondrogenesis in vitro. The results showed
(a)
(b)
(c)
Figure 3. Biochemical analyses are shown for the chondro-
genic differentiation of hUCMSCs in PGA and PLLA scaffolds
(mean ± standard deviation; n = 4). (a) DNA content; (b) Gly-
cosaminoglycan (GAG) content; (c) Hydroxyproline (HYP)
content. *= statistically significant difference between the PGA
and the PLLA scaffolds (p < 0.05).

L. Zhao et al. / J. Biomedical Science and Engineering 3 (2010) 1041-1049
1046
Figure 4. Immunohistochemical staining for type I and II collagen and aggrecan, and Safranin-O staining (n = 2) at 14 days and 21
days. With chondrogenic differentiation, both the PGA and PLLA scaffolds stained faintly for cartilage-specific proteins collagen II
and aggrecan. Safranin-O staining was observed in both PGA and PLLA scaffolds. The scale bar is 250 μm. CI = collagen type I, CII =
collagen type II, Saf O = Safranin-O.
some degree of chondrogenic differentiation of hUCMSCs,
as evidenced by the expression of collagen type II and
aggrecan genes.
The present study showed that hUCMSCs could dif-
ferentiate along a chondrogenic lineage in both PGA and
PLLA scaffolds. The similar chondrogenic differentia-
tion patterns were quantitatively characterized by the
extracellular matrix, including GAG production (Figure
3(b)) and collagen production (Figure 3(c)). Histology
showed staining for GAGs (Figure 4), although these
GAGs did not aggregate to form a large amount of ag-
grecan as indicated by only a trace aggrecan staining.
Quantitative RT-PCR showed the up-regulation of carti-
lage marker gene expressions, specifically collagen type
II and aggrecan (Figure 5(b), (c)). This upregulation of
aggrecan and collagen II gene expression may show po-
tential in longer-term studies for greater cumulative lev-
els of aggrecan and collagen II production.
The physicochemical characteristics and mechanical
performances of PGA are well established in clinical
practice. However, previous studies showed that PGA
fibers degraded relatively quickly, losing their integrity
and becoming fiber fragments in the cell culture medium
as quickly as within 2-4 weeks [14,34-36]. PLLA pro-
vides more space for cellular, biochemical, and even
biomechanical development [37,38]. In the present study,
during the 21 days chondrogenic differentiation culture,
PGA scaffolds were observed to morphologically change
in shape more than the PLLA scaffolds (Figure 2(c),
(d)). Moreover, the DNA of the PLLA group was rela-
tively higher than that of the PGA group after 14 days of
culture (Figure 3(a)). The reason may be that the PGA
scaffolds, which are expected to degrade faster, led to a
greater structural shift. Our results showed that the
chondrogenic potential of hUCMSCs in PLLA scaffolds
was similar to that in PGA scaffolds. PLLA scaffolds
showed a relatively stable morphology during the same
chondrogenic differentiation conditions.

L. Zhao et al. / J. Biomedical Science and Engineering 3 (2010) 1041-1049
1047
(a)
(b)
(c)
Figure 5. Quantitative relative gene expression profile of
hUCMSCs in both PGA and PLLA scaffolds at 0, 14 and 21
days (mean ± standard deviation; n = 4). (a) Collagen type I
gene expression. (b) Collagen type II gene expression. (c) Ag-
grecan gene expression. CI = collagen type I, CII = collagen
type II. *= statistically significant difference among the PGA
and the PLLA groups compared to day 0 (p < 0.05).
5. CONCLUSIONS
In the present study, the chondrogenic differentiation of
hUCMSCs in both PGA and PLLA scaffolds has been
evaluated. Compared with the PGA scaffolds, PLLA
scaffolds showed a relatively stable morphology during
the same period (3 wks) and conditions. The results in-
dicated that hUCMSCs had some degree of chondro-
genic potential in both PGA and PLLA scaffolds. Dif-
ferences between the PLLA and PGA scaffold groups
were minimal over a 21-day period, indicating that an in
vivo application requiring an in vitro pre-culture time on
the order of 21 days would find these two cell-seeded
biomaterials in a similar state. However, differences may
begin to become more apparent in the longer term (e.g.,
6 weeks).
6. ACKNOWLEDGEMENTS
We gratefully acknowledge Dr. Xinkun Wang for his guidance in per-
forming the RT-PCR. We also thank Dr. Limin Wang for his RT-PCR
assistance, and Lauren Byers for her assistance in proofreading the
manuscript. This study was supported by NIH R21 grant DE017673-01,
Arthritis Foundation (National and Kansas Chapters), and the State of
Kansas.
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