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A Comparison of Human Bone Marrow Derived Mesenchymal Stem Cells and Human Umbilical Cord-Derived Mesenchymal Stromal Cells for Cartilage Tissue Engineering

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Bone marrow-derived mesenchymal stem cells (BMSCs) have long been considered the criterion standard for stem cell sources in musculoskeletal tissue engineering. The true test of a stem cell source is a side-by-side comparison with BMSCs. Human umbilical cord-derived mesenchymal stromal cells (hUCMSCs), one such candidate with high potential, are a fetus-derived stem cell source collected from discarded tissue (Wharton's jelly) after birth. Compared with human BMSCs (hBMSCs), hUCMSCs have the advantages of abundant supply, painless collection, no donor site morbidity, and faster and longer self-renewal in vitro. In this 6-week study, a chondrogenic comparison was conducted of hBMSCs and hUCMSCs in a three-dimensional (3D) scaffold for the first time. Cells were seeded on polyglycolic acid (PGA) scaffolds at 25 M cells/mL and then cultured in identical conditions. Cell proliferation, biosynthesis, and chondrogenic differentiation were assessed at weeks 0, 3, and 6 after seeding. At weeks 3 and 6, hUCMSCs produced more glycosaminoglycans than hBMSCs. At week 6, the hUCMSC group had three times as much collagen as the hBMSC group. Immunohistochemistry revealed the presence of collagen types I and II and aggrecan in both groups, but type II collagen staining was more intense for hBMSCs than hUCMSCs. At week 6, the quantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed less type I collagen messenger RNA (mRNA) with both cell types, and more type II collagen mRNA with hBMSCs, than at week 3. Therefore, it was concluded that hUCMSCs may be a desirable option for use as a mesenchymal cell source for fibrocartilage tissue engineering, based on abundant type I collagen and aggrecan production of hUCMSCs in a 3D matrix, although further investigation of signals that best promote type II collagen production of hUCMSCs is warranted for hyaline cartilage engineering.
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Original Article
A Comparison of Human Bone Marrow–Derived
Mesenchymal Stem Cells and Human Umbilical
Cord–Derived Mesenchymal Stromal Cells
for Cartilage Tissue Engineering
Limin Wang, Ph.D.,
1,
*Ivy Tran,
2,
*Kiran Seshareddy, B.V.Sc., A.H.,
3
Mark L. Weiss, Ph.D.,
3
and Michael S. Detamore, Ph.D.
4
Bone marrow–derived mesenchymal stem cells (BMSCs) have long been considered the criterion standard for
stem cell sources in musculoskeletal tissue engineering. The true test of a stem cell source is a side-by-side
comparison with BMSCs. Human umbilical cord–derived mesenchymal stromal cells (hUCMSCs), one such
candidate with high potential, are a fetus-derived stem cell source collected from discarded tissue (Wharton’s
jelly) after birth. Compared with human BMSCs (hBMSCs), hUCMSCs have the advantages of abundant supply,
painless collection, no donor site morbidity, and faster and longer self-renewal in vitro. In this 6-week study, a
chondrogenic comparison was conducted of hBMSCs and hUCMSCs in a three-dimensional (3D) scaffold for the
first time. Cells were seeded on polyglycolic acid (PGA) scaffolds at 25 M cells=mL and then cultured in identical
conditions. Cell proliferation, biosynthesis, and chondrogenic differentiation were assessed at weeks 0, 3, and 6
after seeding. At weeks 3 and 6, hUCMSCs produced more glycosaminoglycans than hBMSCs. At week 6, the
hUCMSC group had three times as much collagen as the hBMSC group. Immunohistochemistry revealed the
presence of collagen types I and II and aggrecan in both groups, but type II collagen staining was more intense
for hBMSCs than hUCMSCs. At week 6, the quantitative reverse transcriptase polymerase chain reaction (RT-
PCR) revealed less type I collagen messenger RNA (mRNA) with both cell types, and more type II collagen
mRNA with hBMSCs, than at week 3. Therefore, it was concluded that hUCMSCs may be a desirable option for
use as a mesenchymal cell source for fibrocartilage tissue engineering, based on abundant type I collagen and
aggrecan production of hUCMSCs in a 3D matrix, although further investigation of signals that best promote
type II collagen production of hUCMSCs is warranted for hyaline cartilage engineering.
Introduction
Mesenchymal stem cells (MSCs) can differentiate
along a variety of cell lineages that can be used for
tissue engineering and regenerative medicine. MSCs reside
in many tissues, including bone marrow,
1
adipose tissue,
2
blood,
3
synovial fluid,
4
dermis,
5
muscle,
6
and dental pulp.
7
Human bone marrowderived MSCs (hBMSCs) are the most
commonly used MSCs for scientific and clinical purposes.
They are isolated via plastic adhesion or negative selection
from bone marrow aspirate that includes a highly hetero-
geneous cell population such as hematopoietic cells, endo-
thelial cells, and adipocytes.
8
However, there are some
limitations of hBMSCs. The relative number of hBMSCs
in the marrow and their differentiation potential decrease
significantly with age.
9
Moreover, the harvesting procedure
is painful and invasive and may lead to complications and
morbidity.
10
Because of the disadvantages associated with
hBMSCs, efforts have been made to find alternative sources
of MSCs that function as well as hBMSCs but overcome these
key limitations.
In recent years, umbilical cord–derived mesenchymal
stromal cells (UCMSCs) have been explored as a MSC source
with clear advantages over hBMSCs. UCMSCs are extracted
through enzyme digestion or the explant culture method
from Wharton’s jelly of umbilical cords,
11
a discarded tissue
1
Departments of Biomedical Engineering and
2
Chemistry, University of Michigan, Ann Arbor, Michigan.
3
Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas.
4
Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, Kansas.
*Limin Wang and Ivy Tran have contributed equally to this work and were considered first co-authors.
TISSUE ENGINEERING: Part A
Volume 15, Number 8, 2009
ªMary Ann Liebert, Inc.
DOI: 10.1089=ten.tea.2008.0393
2259
in abundant supply. The harvesting procedure is not invasive
or painful, and there is no donor site morbidity. The enzyme
extraction yields 1010
3
to 5010
3
cells per cm of cord,
12,13
corresponding to approximately 210
5
to 1010
5
primary
cells per cord, and these primary cells can be expanded 300
times as much as the original number for more than seven
passages without the loss of differentiation potential.
13
hUCMSCs strongly express surface markers that have been
identified with other MSCs, including CD10,
12
CD13,
12,14–16
CD29,
12,14,15,17
CD44,
12–18
CD49e,
12,19
CD51,
17
CD73=
SH3,
13,14,16–18
CD90 (Thy-1),
12,15,16,18,19
CD105=SH2,
12–14,16–18
CD106,
14
CD117,
16,18,20
CD146,
19
CD166,
14–16
and human
leukocyte antigen (HLA)-1=HLA-ABC,
12,14,15,18
and are neg-
ative for hematopoietic markers (e.g., CD14, CD31, CD34,
CD38, CD45, HLA-DR).
12–25
Moreover, the presence of Oct-4,
Nanog, and Sox-2 transcription factors indicates that a sub-
population of UCMSCs might share some properties of em-
bryonic and non-embryonic stem cells.
23
hUCMSCs have the
ability to differentiate into mesenchymal cell lineages, such as
osteogenic, chondrogenic, myogenic, and adipogenic.
13,17–19,25
Moreover, the number of fibroblast colony-forming units is
significantly higher in hUCMSCs than hBMSCs.
14
In the past 4 years, only five studies have demonstrated the
chondrogenic differentiation of hUCMSCs in cell pellet cul-
ture
13,17,19
or poly(glycolic acid) (PGA) scaffolds.
26,27
Wang
et al.
17
first revealed evidence of chondrogenesis by a posi-
tive immunohistochemical staining of type II collagen, which
was also observed with non-differentiated cells. Other than
a conference abstract,
28
our group was the first to apply
hUCMSCs in three-dimensional (3D) musculoskeletal tissue
engineering.
26,27
In these studies, we demonstrated that 3D
culture of hUCMSCs in PGA scaffolds led to a fibrocartilage
with intense type I collagen and moderate collagen type II and
aggrecan immunostaining.
26,27
Here, our objective is to com-
pare hUCMSCs and hBMSCs in vitro in a cartilage tissue en-
gineering application. Both types of cells were cultured in PGA
scaffolds, and their proliferation, biosynthesis, and chondro-
genic differentiation were evaluated over a period of 6 weeks.
Materials and Methods
Cell harvest
hUCMSCs were harvested following our previous meth-
ods, with institutional review board (IRB) approval and
informed consent.
12
The cells were plated and recorded as
passage 0 (P0) in 6-well plates containing a low-serum me-
dium at a density of 10,000 cells=cm
2
. The medium consisted
of low-glucose Dulbecco’s modified Eagle medium (DMEM-
LG; Invitrogen, Carlsbad, CA) and MCDB-201 medium
(Sigma, St. Louis, MO) supplemented with 1insulin-
transferrin-selenium (ITS; Invitrogen), 0.15% lipid-rich
bovine serum albumin (Albumax; Invitrogen), 0.1 nM dexa-
methasone (Sigma), 10 mM ascorbic acid-2-phosphate (Sig-
ma), 1penicillin=streptomycin (Fisher Scientific, Pittsburgh,
PA), 2% fetal bovine serum (FBS; Invitrogen), 10 ng=mL re-
combinant human epidermal growth factor (Invitrogen), and
10 ng=mL human platelet–derived growth factor BB (R&D
Systems, Minneapolis, MN). Cells in the well plates were fed
every 2 to 3 days and maintained in a cell culture incubator
(NuAire, Autoflow, 5% carbon dioxide, 378C, 90% humidity).
Cells were then detached at 80% to 90% confluence and
plated into 25-cm
2
flasks. At P1, cells were resuspended at
the density of 1 million cells per mL of freezing medium
composed of 90% FBS and 10% dimethyl sulfoxide (DMSO;
Fisher Scientific). The cell suspension was transferred into
cryotubes, which were stored in Mr. Frosty freezing con-
tainers (Nalgene, Rochester, NY) at 808C overnight and
then transferred to a liquid nitrogen cryogenic storage sys-
tem at 1968C for future use. P0 frozen hBMSCs were ob-
tained from StemCell Technologies (Vancouver, Canada),
with IRB approval. The bone marrow was extracted from the
posterior iliac crest from a maximum of four sites per donor
(25 mL=site). The phenotype of P0 hBMSC was evaluated
using flow cytometry, and cells included CD29 (>90%),
CD44 (>90%), CD105 (>90%), CD166 (>90%), CD14 (<1%),
CD34 (<1%), and CD45 (<1%).
Cell seeding
Both types of cells were thawed and expanded with a
plating density of 5000 to 6000 cells=cm
2
in 300-cm
2
flasks to
P4 in a medium containing DMEM-LG, 10% FBS (StemCell
Technologies), 1% penicillin=streptomycin (Invitrogen), and
1% non-essential amino acids (Invitrogen). Non-woven PGA
meshes (50 mg=cc; Synthecon, Houston, TX) were punched
to cylindrical scaffolds with a 5-mm diameter and 2-mm
thickness and then sterilized with ethylene oxide. After
sterilization, the scaffolds were aired under a fume hood for
1 day, then wetted with sterile filtered ethanol and two
washes of sterile PBS. The scaffolds were then soaked in the
medium for 1 day, and the medium was removed for cell
seeding. Both cell types, at P4, were seeded at 20 million cells
per mL of scaffold via orbital shakers onto PGA scaffolds
at 150 rpm for 24 h. Finally, the medium was replaced with
2 mL of chondrogenic differentiation medium consisting
of high-glucose DMEM (DMEM-HG; Invitrogen), 1% non-
essential amino acids, 1ITS premix (BD Biosciences, San
Jose, CA), 10 ng=mL transforming growth factor beta-1 (TGF-
b1; PeproTech, Rocky Hill, NJ), 100 nM dexamethasone (Sig-
ma), 50 mg=mL ascorbic acid 2-phosphate (Sigma), 100 mM
sodium pyruvate (Fishersci), and 40 mg=mL L-proline (Sigma).
This time point was recorded as week 0. Medium was chan-
ged every other day over a period of 6 weeks.
Biochemical analysis
At weeks 0, 3, and 6, constructs (n ¼4) were digested by
adding 1.1 mL papain solution (120 mg=mL) at 608C overnight
and centrifuged at 10,000gfor 5 min to remove the PGA
debris. The supernatant was stored at 208C for future assays.
DNA content was measured using a PicoGreen kit (Invitro-
gen). A conversion factor of 8.5 pg DNA=cell determined in
preliminary studies can be used to convert DNA content to
cell number. Biosynthesis was evaluated by measuring total
glycosaminoglycan (GAG) and hydroxyproline (HYP) con-
tents. GAG content was measured using a dimethylmethylene
blue (DMMB) dye binding assay kit (Biocolor, Belfast, UK).
From each sample, 100 mL was added to 1 mL of DMMB and
allowed to bind for 30 min. Solutions were then centrifuged,
supernatant was discarded, and the pellet was resuspended
and read at 656 nm. HYP content was determined using a
modified HYP assay.
29
Briefly, 400 mL of each sample was
hydrolyzed with an equal volume of 4 N sodium hydroxide at
1218C for 30 min, neutralized with an equal volume of 4 N
hydrochloric acid, and then titrated to an approximate pH
2260 WANG ET AL.
range between 6.5 and 7.0. One mL of this solution was
combined with 0.5 mL chloramine-T (14.1 g=L) in the buffer
(50 g=L citric acid, 120 g=L sodium acetate trihydrate, 34 g=L
sodium hydroxide, and 12.5 g=L acetic acid), and the result-
ing solution was then combined with 0.5 mL of 1.17 mM
p-dimethylaminobenzaldehyde in perchloric acid and read at
550 nm. A conversion factor of 11.5 can be used to convert
HYP mass to collagen mass based on our preliminary studies.
Hematoxylin staining and immunohistochemistry
for types I and II collagen and aggrecan
Scaffolds were frozen and sliced into thin sections (10 mm)
that were fixed in chilled acetone (48C) for 10 min before
staining. Hematoxylin staining was performed using VEC-
TOR hematoxylin QS (Vector Laboratories, Burlingame, CA),
according to the manufacturer instructions. In brief, slides
were rinsed in tap water and immersed in the hematoxy-
lin reagent for 2 min. After the staining, slides were rinsed in
tap water, dehydrated, and mounted. Immunohistochemical
analysis (n ¼2) was performed in a BioGenex i6000 auto-
stainer (BioGenex, San Ramon, CA). Slides were rehydrated
with PBS for 5 min. Endogenous peroxidase activity was in-
hibited using 1% hydrogen peroxide in methanol for 30 min.
Each section was blocked in 3% horse serum for 20 min and
incubated with a primary antibody for 1 hour. Primary anti-
bodies used in this study included the mouse monoclonal im-
munoglobulin (Ig)G anti-collagen I (1:1500 dilution; Accurate
Chemical and Scientific, Westbury, NY), mouse monoclonal
IgG anti-collagen II (1:1000 dilution; Chondrex, Redmond,
WA), and mouse monoclonal IgG anti-aggrecan (1:50 dilution;
Abcam, Cambridge, MA). After primary antibody incuba-
tion, the sections were incubated with a streptavidin-linked
horse anti-mouse IgG secondary antibody (Vector Labora-
tories) for 30 min. After secondary antibody incubation, the
sections were incubated with an avidin-biotinylated enzyme
complex (ABC complex; Vector Laboratories) for 30 min, and
then VIP substrate (purple color) (Vector Laboratories) was
applied on sections for 6 min. Protocols run with the primary
antibody omitted served as negative controls.
RNA isolation and gene expression analysis
Real-time reverse transcriptase polymerase chain reaction
(RT-PCR) was used to quantify the relative gene expression
level of types I and II collagen and aggrecan. The constructs
(n ¼4) were collected at weeks 3 and 6 and homogenized
with an electrical homogenizer in 1 mL of Trizol reagent
(Invitrogen) to extract messenger RNA (mRNA) according
to the manufacturer’s protocol. Total mRNA was reverse
transcribed to complementary DNA (cDNA) using a high-
capacity cDNA Archive kit (Applied Biosystems, Foster City,
CA). Real-time RT-PCR reactions were run in an Applied
Biosystems 7500 Fast Sequence Detection System. TaqMan
gene expression assay kits, including two pre-designed
primers and one probe, were used for transcript levels of the
proposed genes. Two mL of cDNA from each sample was
mixed with 7 mL of RNase=DNase free water, 1 mLof20
TaqMan gene expression assay reagent, and 10 mLof2
TaqMan universal PCR master mix. Assay IDs of TaqMan
gene expression assays were Hs00164004_m1 for type I col-
lagen, Hs00156568_m1 for type II collagen, Hs00153936_m1
for aggrecan, and Hs99999905_m1 for glyceraldehyde 3-
phosphate dehydrogenase (GAPDH). Relative mRNA ex-
pression level for each target gene was evaluated using a
2
DDCt
method.
30
The Ct values of target genes were first
normalized by subtracting the Ct values of the TaqMan hu-
man housekeeping gene GAPDH to obtain DCt values. They
were then normalized by subtracting the Ct value of the
calibrator sample, the hBMSC group at week 3, to obtain
DDCt values, which were finally calculated to acquire the
fold changes. Each sample was analyzed in triplicate.
Statistical analysis
All data were expressed as means one standard devia-
tion and analyzed using analysis of variance (ANOVA) fol-
lowed by Tukey’s honestly significant difference (HSD) post
hoc tests. Two-way ANOVAs with interaction were per-
formed with time-points and cell sources as statistical factors.
A statistical threshold of p<0.05 was used to indicate whe-
ther there were statistically significant differences between
different groups.
Results
Seeding efficiency and DNA content
At week 0, a seeding efficiency (defined as the percent-
age ratio of the DNA content of attached cells to the DNA
FIG. 1. Seeding efficiency
at week 0 (A) and DNA
content at weeks 0, 3, and 6
(B)(n¼4). Human umbilical
cord–derived mesenchymal
stromal cells (hUCMSCs)
outperformed human
bone marrowderived mes-
enchymal stem cells
(hBMSCs) in seeding effi-
ciency and in DNA content
at all time points. *Statistically
significant difference
between hUCMSC and hBMSC groups. #Statistically significant difference between weeks 0 and 3. DNA content can be
converted to cell number using a conversion factor of 8.5 pg DNA=cell. Error bars represent standard deviations.
UMBILICAL CORD MESENCHYMAL STROMAL CELLS FOR CARTILAGE TISSUE ENGINEERING 2261
content of seeded cells) of 75% was achieved with hUCMSCs,
whereas a seeding efficiency of only 62% was observed with
hBMSCs ( p<0.05) (Fig. 1A). After the initial seeding period,
the hUCMSC and hBMSC groups both maintained their
DNA content over 6 weeks, except for a drop ( p<0.05) at
week 3 in the hBMSC group. However, after the drop, a
recovery increase in DNA content occurred without signifi-
cant difference. In comparison, the DNA content in the
hUCMSC group was significantly higher than in the hBMSC
group throughout the culture period ( p<0.05) (Fig. 1B).
GAG and HYP content
There was a continuing increase in GAG content per con-
struct in the hUCMSC group over 6 weeks ( p<0.05), whereas
the GAG content decreased in the hBMSC group from weeks 0
to 3 ( p<0.05) and then increased from weeks 3 to 6 ( p<0.05)
(Fig. 2A). However, the increases did not lead to a signifi-
cant difference between weeks 0 and 6 for either group. The
hUCMSC group had a 51% and 28% higher GAG content per
construct than the hBMSC group at weeks 3 and 6, respec-
tively ( p<0.05). With regard to GAG content per unit DNA,
the hUCMSC and hBMSC groups both had a significant in-
crease between weeks 0 and 6 (Fig. 2B) ( p<0.05). There was
also a significant increase in GAG content for the hBMSC
group from weeks 3 to 6 ( p<0.05). The hUCMSC group at
week 3 had a higher GAG content per unit DNA than the
hBMSC group ( p<0.05), although there was no statistically
significant difference between them at week 6.
HYP content per construct and per unit DNA increased
markedly from weeks 0 to 3 in the hUCMSC and hBMSC
groups (Fig. 2C, D), whereas there was no increase in HYP
FIG. 2. Glycosaminoglycan
(GAG) and hydroxyproline
(HYP) content per construct
and per unit DNA at weeks
0, 3, and 6 (n ¼4). Human
umbilical cord–derived mes-
enchymal stromal cells
(hUCMSCs) outperformed
human bone marrow–derived
mesenchymal stem cells
(hBMSCs) in GAG and HYP
biosynthesis, most notably
with nearly three times as
much collagen per construct
at week 6 as evidenced
by HYP content. *Statistically
significant difference between
hUCMSC and hBMSC groups
at a specific time point. #Sta-
tistically significant differ-
ence from week 0 hUCMSC or
hBMSC group. @Statistically
significant difference between
weeks 3 and 6 in hUCMSC or
hBMSC group. Error bars rep-
resent standard deviations.
FIG. 3. Hematoxylin staining (n ¼2). The scale bar is
100 mm. The human bone marrow–derived mesenchymal
stem cell (hBMSC) group had a higher cell density than the
human umbilical cord–derived mesenchymal stromal cell
(hUCMSC) group. Color images available online at www
.liebertonline.com=ten.
2262 WANG ET AL.
content between week 3 and 6 for either group. The
hUCMSC group had 2.5 and 2.9 times as much HYP per
construct as the hBMSC group at weeks 3 and 6, respectively
(p<0.05). Similar to HYP content per construct, the HYP
content per unit DNA increased from weeks 0 to 3 ( p<0.05)
and then had no significant changes until week 6 for both
groups. The hUCMSC group possessed 1.7 and 2.1 times as
much HYP per unit DNA as the hBMSC group ( p<0.05).
Histology and immunohistochemistry
At week 6, hematoxylin staining revealed that the hBMSC
group had a higher cell density (cell number per volume)
than the hUCMSC group (Fig. 3). The PGA debris density in
the hBMSC group was higher than that of the hUCMSC
group as well. Immunohistochemistry revealed positive
staining for types I and II collagen and aggrecan in the
hUCMSC and hBMSC groups through the 6-week period
(Fig. 4). At week 3, the hBMSC and hUCMSC groups both
showed an abundant amount of type I collagen, a small
amount of type II collagen, and a moderate amount of ag-
grecan. Stronger staining of type I collagen and aggrecan
was present in the hUCMSC group than in the hBMSC
group. At week 6, the hUCMSC and hBMSC groups pre-
sented a concentrated staining for type I collagen and a
more-intense staining for aggrecan than at week 3. There was
a vast increase in type II collagen staining in the hBMSC
group from weeks 3 to 6, whereas only a trace of type II
collagen was detected in the hUCMSC group at week 6.
RT-PCR analysis
hUCMSCs had a lower mRNA level of type I collagen
than hBMSCs at weeks 3 and 6 ( p<0.05), a lower mRNA
level of type II collagen at week 6, and the same level of
aggrecan at weeks 3 and 6 (Fig. 5). From weeks 3 and 6, in
the hUCMSC and hBMSC groups, type I collagen mRNA
level decreased ( p<0.05), and aggrecan mRNA level re-
mained at the same level. Type II collagen mRNA level in-
creased 3.8 times in the hBMSC group from weeks 3 to 6
(p<0.05), whereas no change was observed in the hUCMSC
group.
Discussion
It has been previously shown that hUCMSCs can differ-
entiate toward a chondrogenic lineage.
13,17,19,26
However, the
application of these cells in musculoskeletal tissue engi-
neering has scarcely been investigated.
26–28
This study, for
the first time, specifically focused on the comparison of
hUCMSCs and hBMSCs in a biomaterial-based 3D environ-
ment. This comparison is vital to the validation of hUCMSCs
as a formidable cell source for cartilage—and more broadly,
musculoskeletal—tissue engineering.
It has been reported that hUCMSCs have a faster prolifer-
ation rate in monolayer culture than adult stem cells.
31
In-
deed, in the current study, hUCMSCs reached 80% to 90%
confluence 2 to 3 days earlier than hBMSCs, and both cell
types had similar cell numbers per flask at this confluence.
FIG. 4. Immunohistoche-
mical staining for types I and
II collagen, and aggrecan
(n ¼2). Although collagen
I staining was comparable
between human umbilical
cord–derived mesenchymal
stromal cells (hUCMSCs) and
human bone marrow–
derived mesenchymal stem
cells (hBMSCs), collagen II
staining was more intense
with hBMSCs. The scale bar
is 500 mm. CI, type I collagen;
CII, type II collagen; @3WK,
at week 3; @6WK, at week
6. Negative control groups
(not shown) confirmed the
absence of non-specific stain-
ing. Color images available
online at www.liebertonline
.com=ten.
UMBILICAL CORD MESENCHYMAL STROMAL CELLS FOR CARTILAGE TISSUE ENGINEERING 2263
The faster proliferation provides for a larger number of cells in
a shorter time period to better meet the needs of tissue engi-
neering and clinical practice. The higher seeding efficiency
with the hUCMSCs at week 0 indicated that hUCMSCs were
more adherent to PGA scaffolds than hBMSCs. This higher
seeding efficiency resulted in a higher DNA content (cell
number) for the hUCMSC group, which was maintained over
6 weeks and probably contributed in part to the higher matrix
production in the hUCMSC group. Although the cell density
in the hBMSC group was higher than in the hUCMSC group,
as revealed by hematoxylin staining (Fig. 3), smaller scaffold
volume with the hBMSC constructs than with the hUCMSC
constructs, as revealed by the immunohistochemical staining,
explains this seemingly conflicting result (Fig. 4). The decrease
in DNA content with hBMSCs from weeks 0 to 3 may be due
to the degradation of the PGA scaffold, as we have noted in the
past.
27,32
The hUCMSC group also experienced a slight loss of
DNA (not statistically significant). The ability of hUCMSCs to
proliferate at a higher rate and the higher matrix content that
helps to retain cells in scaffolds can explain the maintenance
of DNA content with hUCMSCs. To overcome cell loss
caused by PGA degradation, slowly degrading scaffolds such
poly-L-lactic acid and poly-lactic-co-glycolic acid will be in-
vestigated in the future.
In this study, the hUCMSC group produced more GAGs
than the hBMSC group. The higher GAG content per con-
struct with hUCMSCs was due to the larger number of cells,
because the GAG content per unit DNA at week 6 was
similar for the two groups. With regard to collagen pro-
duction, the hBMSC and hUCMSC groups both showed an
increase in collagen content throughout the 6-week culture,
with the largest increase occurring between weeks 0 and 3.
The higher porosity of cell–scaffold constructs in the first
3 weeks might contribute to higher collagen production,
because it provides a large space for matrix synthesis and
nutrient diffusion. When compared directly with each other,
the hUCMSC group produced 2.5 and 3.5 times as much
collagen per construct as the hBMSC group at weeks 3 and 6,
respectively. More importantly, the hUCMSC group had
more collagen per unit DNA than the hBMSC group, indi-
cating that hUCMSCs are better able to produce collagen
than hBMSCs under the prescribed conditions. Thus, the
high collagen content produced by hUCMSCs is attractive
for cartilage tissue engineering.
Exposed to chondrogenic media, both types of cells pro-
duced a cartilage-like tissue with types I and II collagen and
aggrecan. A down-regulation of type I collagen gene expres-
sion during this period accompanied the chondrogenic dif-
ferentiation of both cell types. Higher type II collagen
expression in the hBMSC group at the mRNA and protein
levels suggested that chondrogenic differentiation was more
extensive with hBMSCs under the prescribed conditions. Al-
though the hBMSC group had a higher gene expression of
types I and II collagen at week 6, the total collagen content in
the hUCMSC group was higher than in the hBMSC group.
Although at first glance these results may appear contradic-
tory, the gene expression level represents a snapshot of the
cellular disposition at that exact point in time, whereas the
hydroxyproline assay is cumulative, representing the total
collagen protein synthesized. The hydroxyproline data ap-
pear to be relatively consistent with the intensity and con-
struct size with collagen immunostaining, which is also a
cumulative assay. With regard to aggrecan, mRNA and pro-
tein expression were comparable between the two groups,
which suggests that hUCMSCs were at least in one capacity
matching the apparent chondrogenesis of hBMSCs.
In previous hUCMSC pellet culture,
13,17
plentiful type II
collagen
13,17
was observed with a small amount of type I
collagen
13
around the periphery. Limited oxygen (hypoxia)
exists in the center of cell pellets because of a low diffusion
coefficient,
33
which may have contributed to the production
of type II collagen.
34
It was unexpected that only a trace
amount of type II collagen
13
was observed in hBMSC pellets
because hBMSCs generally produced dominant type II col-
lagen in pellets.
35
The constructs based on highly porous
PGA scaffolds (>95% porosity) in the current study had
better diffusion than cell pellets, especially at the early stages.
Moreover, the differences in chondrogenic media (TGF-b1vs
b3) and harvesting methods might explain these conflicting
results between the pellet culture in the literature and the
biomaterial-based culture in this study.
In conclusion, hUCMSCs were seeded more efficiently and
demonstrated superior biosynthesis, with nearly three times
as much collagen production as hBMSCs. The differentiation
profile of hUCMSCs, consisting of a large amount of type I
collagen, a small amount of type II collagen, and moderate
aggrecan content, clearly indicated that hUCMSCs may be
highly desirable for fibrocartilage tissue engineering such as
for the temporomandibular joint disc or the knee meniscus.
In contrast, hBMSCs appeared to have progressed further
down a chondrogenic lineage based on superior collagen II
gene expression and protein production. However, one may
expect that the hUCMSCs will require a modified set of
signals to provide for optimal chondrogenesis. With the
identification of this set of signals, it would be expected
that hUCMSCs would be capable of producing a similar
FIG. 5. Gene expression for types I and II collagen, and
aggrecan (n ¼4). Human bone marrow–derived mesenchy-
mal stem cells (hBMSCs) significantly outperformed hu-
man umbilical cord–derived mesenchymal stromal cells
(hUCMSCs) in collagen II gene expression at the week 6
snapshot. CI, type I collagen; CII, type II collagen. *Statisti-
cally significant difference between hUCMSC and hBMSC
groups at a specific time point. #Statistically significant dif-
ference between weeks 3 and 6 in hUCMSC or hBMSC
group.
2264 WANG ET AL.
tissue in a drastically reduced time, which would translate
to better patient care. From the perspective of clinical prac-
tice, hUCMSCs provide clear advantages over hBMSCs in
that hUCMSCs are easily obtained from a discarded tissue,
are in abundant supply, have no donor site morbidity, and
are highly expendable before senescence or differentiation.
Moreover, in the future, if parents cryogenically save part of
their child’s umbilical cord, these hUCMSCs would also be
available as an autologous cell source later in life with all of
the aforementioned advantages.
Based on these known advantages, and the results of this
study, we therefore conclude that hUCMSCs are a highly
desirable cell source for fibrocartilage tissue engineering and
have the potential to surpass the criterion standard of
hBMSCs in hyaline cartilage regeneration with further in-
vestigation of chondrogenic signals tailored to hUCMSCs.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Michael S. Detamore, Ph.D.
Department of Chemical and Petroleum Engineering
4132 Learned Hall, 1530 W 15
th
St
University of Kansas
Lawrence, KS 66045
E-mail: detamore@ku.edu
Received: July 13, 2008
Accepted: December 3, 2008
Online Publication Date: February 27, 2009
2266 WANG ET AL.
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The easily accessible mesenchymal stem cells in the Wharton's jelly of human umbilical cord tissue (hUCMSCs) have excellent proliferation and differentiation potential, but it remains unclear whether hUCMSCs can differentiate into odontoblasts. In this study, mesenchymal stem cells were isolated from the Wharton's jelly of human umbilical cord tissue using the simple method of tissue blocks culture attachment. UCMSC surface marker expression was then evaluated for the isolated cells using flow cytometry. The third-passage hUCMSCs induced by conditioned medium from developing tooth germ cells (TGC-CM) displayed high alkaline phosphatase (ALP) levels (P < 0.001), an enhanced ability to proliferate (P < 0.05), and the presence of mineralized nodules. These effects were not observed in cells treated with regular medium. After induction of hUCMSCs, the results of reverse transcriptional polymerase chain reaction (PCR) indicated that the dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) genes were significantly tested. Additionally, dentin sialoprotein (DSP) and DMP1 demonstrated significant levels of staining in an immunofluorescence analysis. In contrast, the control cells failed to display the characteristics of odontoblasts. Taken together, these results suggest that hUCMSCs can be induced to differentiate into odontoblast-like cells with TGC-CM and provide a novel strategy for tooth regeneration research.
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