J. Biomedical Science and Engineering, 2013, 6, 109-115 JBiSE
http://dx.doi.org/10.4236/jbise.2013.62014 Published Online February 2013 (http://www.scirp.org/journal/jbise/)
A study on the activity of dermal multipotent stem cells in
initiation of wound repair
Jifu Qu1,2, Tianmin Cheng 2, Yongping Su2, Chunmeng Shi2, Wei Sun1
1Trauma Center, Department of Emergency Medicine, Southwest Hospital, Third Military Medical University, Chongqing, China
2State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Combined Injuries of Chinese PLA, Academy of Preven-
tive Medicine, Third Military Medical University, Chongqing, China
Received 17 December 2012; revised 16 January 2013; accepted 24 January 2013
ABSTRACT
Background: Wound healing is a process of cell-cell
interaction and cell-extracellular matrix interaction.
Dermal multipotent stem cells (dMSCs) have the
abilities to promote survival and wound healing, but
the potential function of dMSCs in wound healing,
particularly in the initiation of wound repair, has not
been fully understood. Methods: dMSCs and fibro-
blasts were isolated from neonatal rat dermis and
were further purified and expanded. The cell cycles
were determined with flow cytometry, while the ra-
diosensitivity was measured by MTT assay. Rats were
wounded with a 7-cm incision on the back skin and
the wound fluids were collected by inserting two
pieces of sterile polyvinyl alcohol sponge (1 cm in di-
ameter and 0.4 cm in thickness) subcutaneously into
the dorsum of each rat through the midline of incision
on the 1st, 2nd, 3rd and 4th day after incision. The ef-
fects of wound fluids on the proliferation of dMSCs
and fibroblasts were measured with MTT assays.
dMSC’s abilities of adhesion and attachment and its
migration in response to wound fluids collected on the
1st day after incision were explored by measuring the
percentage of floating cells and the cells migrated into
wounding area in vitro, respectively. Results: The iso-
lated dMSCs were morphologically homogenous and
highly proliferative. Most of the cultured dMSCs
were quiescent with few apoptotic cells. Compared
with fibroblasts, dMSCs were more sensitive to ra-
diation and more proliferative in response to wound
fluids, especially to the wound fluids collected on the
1st day after wounding. Moreover, their abilities to
attach, adhere and migrate were significantly en-
hanced with the early-phase wound fluids. Conclu-
sions: As primitive stem cells, dMSCs are very re-
sponsive to wound fluids, which suggests dMSCs’
important role in wound healing, especially in initiat-
ing wound repair.
Keywords: Dermal Multipotent Stem Cells; Initiation of
Wound Repair; Wound Healing; Wound Fluids
1. INTRODUCTION
Stem cells are cells with self-renewal capacity, multipo-
tentiality and unparalleled superiority in cell-replacement
therapy, gene therapy, developmental biology, pharma-
cology and toxicology [1,2]. Mesenchymal stem cells
have the potential to differentiate into fibroblasts, vascu-
lar endothelial cells and other tissue repairing cells, there-
fore playing important roles in tissue repair [3]. Recent
studies have suggested that stem cells with different de-
velopmental potentials exist in several adult mammalian
tissues, especially in newly formed tissues or tissues with
rapid renewal potentials [4-6]. Skin, which consists of
the epidermis and dermis, is one of the rapidly renewing
tissues in adults. Several studies from our group and two
other groups have indicated that multipotent cells can be
isolated from adult mammalian dermis [4,7,8]. Given
their easy accessibility, these cells could be a source of
stem cells for cell transplantation and tissue engineering.
Particularly, we have found that multipotent stem cells
derived from dermal mesenchymal tissue, namely dMSCs,
have the abilities to promote survival and wound healing
in rats subjected to radiation and wound injury, as well as
enhance hematopoietic recovery in sublethally irradiated
rats [9,10].
Wound healing is a process of cell-cell interaction and
cell-extracellular matrix interaction. Wound environments
exert significant effects on cell growth and differentiation
during the healing process. Different cell types are acti-
vated by the environment and subsequently migrate to
and proliferate at the wound site. Studies have indicated
that it is the specific tissue environment that determines
the plasticity of mesenchymal stem cells in vivo [11-14].
Although activation of epithelial cells, fibroblasts and
endothelial cells has been observed after wounding, little
has been described about the multipotent cells in the

J. F. Qu et al. / J. Biomedical Science and Engineering 6 (2013) 109-115 110
dermis [15-18]. In our previous study, we investigated
the biological effects of the acute wound environment
(wound fluids) on dMSCs isolated from newborn rats
(neonatal dMSCs), and showed that neonatal suspensions
enhanced the initial rate of wound contraction and some
cells differentiated from dMSCs were found in the re-
generative dermis. We also observed that treatment with
acute wound fluid promoted proliferation and migration
of dMSCs [4]. These results suggest that neonatal
dMSCs could participate in the regeneration of the in-
jured dermis and thus may be an alternative choice for
cell-based therapies for skin injuries.
As stem cells are primitive with self-renewal capacity,
it awaits clarification whether the dMSCs are primitive
stem cells. Moreover, the potential function of dMSCs in
wound healing has also not been fully understood. In the
present study, we further characterized the radiosensitiv-
ity of dMSCs, as well as their proliferation, attachment,
adhesion and migration in response to early-phase wound
fluids so as to explore their potential roles in wound
healing.
2. MATERIALS AND METHODS
2.1. Cell Culture
dMSCs and fibroblasts were isolated from neonatal rat
dermis, cultured in IMDM supplemented with 10% (v/v)
fetal bovine serum, 100 U/mL penicillin and 100 µg/mL
streptomycin (Hyclone). dMSCs were amplified and
identified as previously described [9]. Briefly, skin tissue
from 1-day-old rat was digested with 0.25% trypsin at
4˚C overnight. The dermal layer (confirmed with histo-
logical examination) was dissociated by flushing with
D-Hanks’ solution; the suspension was filtered through a
nylon mesh and centrifuged to remove cellular debris.
The cell pellet was resuspended and cultured in IMDM
containing 10% FBS, 100 U/mL penicillin and 100
µg/mL streptomycin at 37˚C in a humidified atmosphere
containing 5% CO2. Six hours later, the adherent cells
were subcultured at a low density of 5 cells/cm2. The
presence of separately adherent single cell was assessed
microscopically. Two weeks later, single colonies were
isolated by cloning rings under an inverted microscope
and expanded. The differentiation characteristics of dMSC
were further confirmed in an induction medium contain-
ing dexamethasone as reported previously [4].
2.2. Radiosensitivity Measurement by MTT
Assay
Radiosensitivities of fibroblasts and the 10th passage of
dMSCs were measured by MTT assay. 80% confluent
cells were detached with 0.25% trypsin. After washed
once with D-Hank’s solution, the cell concentration was
adjusted to 2 × 104/mL with IMDM supplemented with
10 ml/L fetal bovine serum. 0.2 ml cells were transferred
into sterile penicillin bottles and irradiated with 60Co γ
ray at 0, 2, 6, 10, 12 and 15 Gy, respectively. After
irradiation, the cells were added into 96-well plate and
the numbers of live cells were measured with MTT assay
in quadruplicate for each radiation dosage. Specifically,
20 μL 5 g/L MTT solution (Sigma) was added into each
well of the 96-well plate and incubated for 4 hours at
37˚C in a humidified atmosphere containing 5% CO2.
Then the culture solution was removed and 150 μL
DMSO was added into each well and oscillated for 10
minutes. The absorptions at 492 nm were measured with
an enzyme immunoassay analyzer (HTS 7000 plus, Perkin
Elmer, USA).
2.3. Measurement of Cell Cycle and Apoptotic
Rate
The changes in cell cycle progression and apoptotic rate
of dMSCs at the 3rd passage were assayed with flow cy-
tometry. 80% confluent cells were detached with 0.25%
trypsin. After washed once with D-Hank’s solution, the
cells were fixed for 30 min with 70% alcohol at 4˚C, and
then centrifuged at 1500 rpm for 5 min. After discarding
the fixing solution, the cells were washed once with cold
PBS solution. dMSC cells were incubated with 200 µl of
lysis buffer, stained with propidium iodide at a concen-
tration of 50 mg/mL for 15 min, and analyzed with the
Coulter Epic C flow cytometer to calculate the G0/G1
population and apoptotic rate.
2.4. Collection of Rat Wound Fluids
Wistar rats were bred and fed in Animal Center of our
university with first class standard. All animal proce-
dures were approved by the institution’s animal-use
committee and by the Ministry of Justice. 15-week old
Wistar rats of mixed sex were wounded with a 7-cm in-
cision on the back skin and the wound fluids were col-
lected by inserting two pieces of sterile polyvinyl alcohol
sponges (1cm in diameter and 0.4 cm in thickness) sub-
cutaneously in the dorsum of each animal through the
midline incision [10]. The rats were then sacrificed on
the 1st, 2nd, 3rd and 4th day after wounding. The wound
fluids were collected from the sponge after the cellular
components were removed by centrifugation and sterilized
with a 0.2 µm filter and then stored at −20˚C until use.
2.5. Effect of Wound Fluids on Proliferation of
dMSCs
After 48-hour culture in serum-free IMDM [19], cells
were plated at 2 × 104/mL to 96-well plate. The wound
fluids were added at various concentrations of 10ml/L,

J. F. Qu et al. / J. Biomedical Science and Engineering 6 (2013) 109-115
111
20 ml/L and 30 ml/L and the cells were further incubated
for 24 hours. The control group cells were not treated
with wound fluids (0 ml/L). The numbers of live cells
were then measured by MTT assay. Each experiment
was performed 3 times in quadruplicate.
experimental group. After incubated for 24 hours, the
cell monolayer was scratched to form a 1 mm-wide clear
area using a sterile needle. 8 hours later, the number of
cells migrated into the wounded area was measured with
an inverted light microscope. The migration rates were
expressed as the number of migrated cells per field and
the percentage of the shortened wound width [4].
2.6. Effect of Wound Fluids on dMSC
Attachment 2.9. Statistical Analysis
dMSCs were cultured in flasks and maintained in se-
rum-free IMDM for 48 hours. Then the cells were incu-
bated with either IMDM plus 10 ml/L wound fluids col-
lected on the first day after rats were wounded or IMDM
alone for 6 hours. The cells were then detached with
0.25% trypsin and 2 × 105 cells were inoculated into
culture flasks. After incubation for 6 hours, cells re-
mained floating in the flasks were then collected and
counted. The percentages of attached cells were calcu-
lated [20].
Data were analyzed using paired Student’s t-test with
SPSS11.0 statistical software. Results were expressed as
mean ± SD (
x
± s). p < 0.05 was considered statistic-
cally significant.
3. RESULTS
3.1. Effect of Radiation on Survival and Cell
Cycle Progression of dMSCs
As previously described, the isolated dMSCs were smooth
and homogenous in morphology in culture, and they
maintained strong proliferative activity after being sub-
cultured for more than ten passages. The survival rate of
dMSCs in response to different dosages of γ irradiation
was measured by MTT assay and compared to fibroblasts.
As shown in Table 1, dMSCs tolerated irradiation at a
dose of 2 Gy, but their survival rate was significantly
decreased by increasing dose of irradiation from 6 Gy to
15 Gy. By contrast, fibroblasts were less sensitive to ir-
radiation, showing good tolerance to irradiation up to the
dose of 10 Gy (Ta b le 2) and only susceptible to higher
dosages of irradiation (12 Gy to 15 Gy). As higher sensi-
tivity generally reflects stronger primitiveness of the
cells, our results here suggest that dMSCs are more
primitive cells compared to fibroblasts. Further support-
ing this, flow cytometry analysis indicated that most of
the dMSCs cultured in vitro were in quiescent state with
95% population in G0/G1 phase and only 0.34% apop-
totic cells (Figure 1). After radiation with 15Gy 60Co γ
ray, the number of apoptotic cells increased by 20%
(Figure 2).
2.7. Effect of Wound Fluids on dMSC Adhesion
dMSCs were cultured in flask and maintained in serum-
free IMDM for 48 hours. Then the cells were further
cultured in IMDM alone or IMDM plus 10 ml/L wound
fluids, which were collected on the first day after rats
were wounded. 24 hours later, the dMSCs were digested
with 0.25% trypsin for 2 minutes to lift cells that loosely
adhered to the flask and the digestion was stopped with
serum. The suspended cells were collected and counted.
The cells remained adhered were scraped, collected and
counted. The adhesion ability of cells was calculated as
percentages of suspended cells in the total of suspended
and adhered cells [20].
2.8. Effect of Wound Fluids on dMSC Migration
Cell motility was analyzed using an in vitro wound
model of cell monolayer. Synchronized dMSCs were
inoculated at 2 × 105/well into 24-well plate and cultured
for three days to reach 80% confluency. The cells were
then cultured in IMDM alone in the control group or
IMDM supplemented with 10 ml/L wound fluids in the
Table 1. Dosage effect of γ radiation on the growth of dMSCs.
60Co dosage 0 Gy 2 Gy 6 Gy 10 Gy 12 Gy 15 Gy
A 490 nm 1.095 ± 0.084 1.303 ± 0.071* 1.177 ± 0.015 1.030 ± 0.026 0.877 ± 0.031* 0.821 ± 0.073#
Cell growth was measured by MTT assay and presented as absorption values at 490 nm wavelength. Data represented four independent experiments. *p < 0.05,
#p < 0.01 compared with no radiation group.
Table 2. Dosage effect of γ radiation on the growth of fibroblasts.
60Co dosage 0 Gy 2 Gy 6 Gy 10 Gy 12 Gy 15 Gy
A 490 nm 0.238 ± 0.041 0.260 ± 0.032 0.166 ± 0.012* 0.074 ± 0.006* 0.060 ± 0.007* 0.059 ± 0.005*
Cell growth was measured by MTT assay and presented as absorption values at 490nm wavelength. Data represented four independent experiments. *p < 0.05
compared with no radiation group.

J. F. Qu et al. / J. Biomedical Science and Engineering 6 (2013) 109-115 112
Figure 1. Flow cytometry of dMSCs cultured in normal condi-
tions.
Figure 2. Flow cytometryof dMSCs irradiated by 15 Gy 60Co γ
ray.
3.2. Effect of Wound Fluids on dMSC
Proliferation
We next examined the proliferation of dMSCs in
response to wound fluids collected on the 1st, 2nd, 3rd and
4th day after the rats were wounded. As shown in Tab le 3,
would fluids of different concentrations and from various
time points all significantly stimulated the proliferation
of dMSCs. Notably, wound fluids collected on the 1st and
2nd day after wounding showed a greater stimulatory
effect on dMSCs proliferation than those collected on the
3rd and 4th day. In addition, the wound fluids applied at
lower concentration (10 ml/L) more strongly augmented
Table 3. Effect of wound fluids on dMSC proliferation (
x
± s).
Time post-injury(d)
Concentration
(mL/L) 1 2 3 4
10 1.30 ± 0.181.30 ± 0.06 0.93 ± 0.11b 1.01 ± 0.25b
20 1.15 ± 0.241.05 ± 0.17a 0.77 ± 0.07b 0.90 ± 0.09b
30 0.98 ± 0.14a0.78 ± 0.06a 0.76 ± 0.16 0.76 ± 0.04a
Data represented four independent experiments. Values of the control group:
0.33 ± 0.02; a: p < 0.05 vs 10 ml/L group of the same time; b: p < 0.05 vs 1st
day group of the same concentration.
the proliferation of dMSCs as compared to would fluids
diluted at 20 ml/L or 30 ml/L.
3.3. Effect of Early-Stage Wound Fluids on
dMSC Attachment, Adhesion and Migration
Since the wound fluid collected on the first day after the
rats were wounded showed the most significant effect on
cell growth, we further examined its effect on the at-
tachment, adhesion and migration of dMSCs. Table 4
showed that stimulation with the 1st day wound fluid
significantly enhanced the attachment ability of dMSCs,
as indicated by the increase in the percentage of cells
attached to culture flasks from 58.84% ± 6.91% in the
control group to 80.42% ± 8.52% in the wound fluid-
treated group. Similarly, the percentage of adherent
dMSCs also increased from 45.56% ± 4.63% in the con-
trol group to 74.95% ± 7.67% in the wound fluid-treated
group. Using the in vitro wound repair model, we further
found that treatment with early-stage wound fluid sig-
nificantly increased the number of dMSCs migrated into
the wounded area (120.84 ± 13.31 per field) compared to
the control group (60.78 ± 4.28 per field), and increased
the percentage of recovered wound area from 54.28% ±
3.63% to 85.47% ± 8.53%, suggesting that the wound-
fluid stimulation enhanced dMSCs migration (Figure 3,
Table 5).
4. DISCUSSIONS
In the present study, we demonstrate that dMSC are
primitive cells based on their high sensitivity to irradia-
tion and low apoptotic rate in steady state. Our previous
study showed that dMSCs were able to not only maintain
a strong proliferative capacity after being sub-cultured
for more than ten passages with uniformed morphology,
but also differentiate into osteoblasts and lipocytes. Col-
lectively, these data demonstrate that dMSCs are rela-
tively primitive mesenchymal multipotent stem cells with
the capacity of self-renewal and multi-directional differ-
entiation.
Wound healing is a complex process requiring the col-
laborative efforts of different cell types and the local
environment [21]. Most tissue-repairing cells are in qui-

J. F. Qu et al. / J. Biomedical Science and Engineering 6 (2013) 109-115 113
Table 4. Effect of 1st-day wound fluid on attachment and
adhesion of dMSCs (
x
± s).
Experimental group Control group
Attachment (%) 80.42 ± 8.52* 58.84 ± 6.91
Adhesion (%) 74.95 ± 7.67* 45.56 ± 4.63
Data represented four independent experiments. *: p < 0.05 vs the control
group.
Figure 3. Representative microscopic images of dMSCs in
the in vitro wound repair assay. (A) Immediately after
wounding of the monolayer 200×; (B) 8 hours after wound-
ing, without treatment 200×; (C) 8 hours after wounding,
treated with wound fluids 200×.
Table 5. Effect of 1st-day wound fluid on dMSC migration
(
x
± s).
Cell number (between the
scratch lines /per field)
Percent of shrinking
area (%)
Experimental group120.84 ± 13.31* 85.47 ± 8.53*
Control group 60.78 ± 4.28 54.28 ± 3.63
Data represented four independent experiments. *: p < 0.05 vs the control
group.
escent state until stimulated by wound fluids to express
specific genes and then initiate the tissue repair process.
Migration and proliferation of these cells at the wound
site is important for tissue granulation and repair, whereas
their attachment and adhesion abilities are essential to
prevent external injuries [20]. Our present study shows
that dMSCs are in quiescent G0/G1 phase without stimu-
lation, and their proliferation, migration, attachment and
adhesion were significantly enhanced in response to
wound fluids, suggesting the important roles of these
cells in wound healing and tissue repairing. Specifically,
the stronger responsiveness of dMSCs to the wound flu-
ids collected on the 1st and 2nd day after wounding than
those collected on the 3rd and 4th day after wounding in-
dicate their potential role in the initiation of wound repair.
Wound fluids are known to contain a variety of cytokines
and growth factors such as TGF-alpha, TGF-beta1, TNF-
alpha, PDGF-AA and IGF, as well as lysophospholipids
such as S1P, LPA and LPCs which regulate wound heal-
ing via activation of tissue-repairing cells [19,22-24].
Our previous study found that the levels of TNF-alpha,
bFGF and nerve growth factor (NGF) were significantly
increased in the wound fluids or wound sites during the
early stage after wounding [25,26]. Marikovsky et al.
reported that the activity of growth factors such as IGF-1
and HB-EGF, appeared 1 day after injury, reached maxi-
mal in 2-3 days and disappeared by 6 days after injury
[23]. Dvonch et al. showed that both the concentration
and the biologic activity of PDGF AA and mono-
cyte/macrophage-derived growth factor (MDGF) were
highest in the immediate postoperative period and de-
clined to negligible levels by 24 hours after surgery. Such
alterations in cytokines and growth factors might explain
why wound fluids collected from early stage (1 - 2 days)
are more effective in stimulating dMSCs. Further study
is necessary to reveal the molecular mechanisms by
which these cytokines and growth factors in wound flu-
ids regulate dMSCs.
In conclusion, dMSCs are relatively primitive mesen-
chymal stem cells that are highly responsive to wound
fluids and likely to play important roles in the initiation
of wound repair. The easy accessibility of dMSCs via
skin biopsy makes them a particularly attractive source
of dermis-derived multipotent cells for cell replacement

J. F. Qu et al. / J. Biomedical Science and Engineering 6 (2013) 109-115 114
therapies or for treatment of skin injury.
5. ACKNOWLEDGEMENTS
This study was supported by National Science Foundation of China
(30370562，81071562), the 973 Project of National Basic Research
Program of China (G1999054205 and 2005CB522605).
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