A study on the activity of dermal multipotent stem cells in initiation of wound repair

doi:10.4236/jbise.2013.62014

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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

Email: [email protected]

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

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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 (

± 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.

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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 (

± 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 (

± 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

(

± 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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