TRANSLATIONAL AND CLINICAL RESEARCH

Mesenchymal Stem Cells Enhance Wound Healing Through

Differentiation and Angiogenesis
YAOJIONG WU, LIWEN CHEN, PAUL G. SCOTT, EDWARD E. TREDGET
Department of Surgery, University of Alberta, Edmonton, Alberta, Canada

Key Words. Angiogenesis Regeneration/repair Diabetic mice Vascular endothelial growth factor Ang-1

ABSTRACT
Although chronic wounds are common, treatment for these wounds exhibited significantly accelerated wound closure, with
disabling conditions remains limited and largely ineffective. increased re-epithelialization, cellularity, and angiogenesis. No-
In this study, we examined the benefit of bone marrow- tably, BM-MSCs, but not CD34 bone marrow cells in the
derived mesenchymal stem cells (BM-MSCs) in wound heal- wound, expressed the keratinocyte-specific protein keratin and
ing. Using an excisional wound splinting model, we showed formed glandular structures, suggesting a direct contribution
that injection around the wound and application to the of BM-MSCs to cutaneous regeneration. Moreover, BM-MSC-
wound bed of green fluorescence protein (GFP) allogeneic conditioned medium promoted endothelial cell tube formation.
BM-MSCs significantly enhanced wound healing in normal Real-time polymerase chain reaction and Western blot analysis
and diabetic mice compared with that of allogeneic neonatal revealed high levels of vascular endothelial growth factor and
dermal fibroblasts or vehicle control medium. Fluorescence- angiopoietin-1 in BM-MSCs and significantly greater amounts
activated cell sorting analysis of cells derived from the of the proteins in BM-MSC-treated wounds. Thus, our data
wound for GFP-expressing BM-MSCs indicated engraft- suggest that BM-MSCs promote wound healing through dif-
ments of 27% at 7 days, 7.6% at 14 days, and 2.5% at 28 ferentiation and release of proangiogenic factors. STEM CELLS
days of total BM-MSCs administered. BM-MSC-treated 2007;25:2648 2659
Disclosure of potential conflicts of interest is found at the end of this article.

infarcted left ventricular wall [10]. Intracerebral transplantation of

INTRODUCTION BM-MSCs into acid sphingomyelinase-deficient mice was shown
to delay the onset of neurological abnormalities and extend their
Optimum healing of a cutaneous wound requires a well-orches- survival [11]. Intravenous infusion of BM-MSCs after stroke in rats
trated integration of the complex biological and molecular events of reduced the size of infarcts in the brain, with a reduced number of
cell migration and proliferation and extracellular matrix (ECM) glial cells and enhanced axonal growth in the lesion [12]. Alloge-
deposition, angiogenesis, and remodeling [13]. However, this neic BM-MSCs derived from healthy donors have been used to
orderly progression of the healing process is impaired in many treat diseases in humans [13]; however, their contribution to wound
chronic diseases, including diabetes [2]. Of the 150 million people healing has not been fully understood.
with diabetes worldwide, 15% present suffer from foot ulcerations, In this study, we examined the benefit of BM-MSCs in
which often become nonhealing chronic wounds [4]. Over the past wound healing on an excisional wound splinting model. We
decades, little improvement has been shown in preventing morbid- implanted green fluorescence protein (GFP)-expressing alloge-
ity and disability from chronic wounds [4]. The best available neic BM-MSCs into excisional wounds in nondiabetic and di-
treatment for chronic wounds achieves only a 50% healing rate that abetic mice and examined their effect on wound healing com-
is often temporary. Among the many factors contributing to non- pared with allogeneic neonatal dermal fibroblasts or vehicle
healing wounds, impairment in the production of cytokines by local control medium. We assessed wound healing using parameters
inflammatory cells and fibroblasts and reduced angiogenesis are of re-epithelialization, cellularity, angiogenesis, and dermal
crucial [2]. structural regeneration. Finally, we examined angiogenic factors
Bone marrow-derived mesenchymal stem cells (BM-MSCs), in BM-MSCs and BM-MSC-treated wounds compared with
which are also referred to as stromal progenitor cells, are self- vehicle medium- or fibroblast-treated wounds to determine the
renewing and expandable stem cells. BM-MSCs are able to differ- paracrine effect of BM-MSC-mediated wound healing.
entiate into adipocytes, osteoblasts, and chondrocytes [5]. They
have also been detected to express phenotypic genes of hepatocytes
[6], cardiomyocytes [7], astrocytes, and neurons [8, 9]. Implanta- MATERIALS AND METHODS
tion of ex vivo-expanded allogeneic BM-MSCs into swine isch-
emic myocardium after myocardial infarction led to long-term All animal procedures were approved under the guidelines of the
engraftment, expression of muscle-specific proteins, attenuated Health Sciences Animal Policy and Welfare Committee of the
contractile dysfunction, and reduced pathologic scarring of the University of Alberta.

Correspondence: Yaojiong Wu, M.D., Ph.D., 161 HMRC, University of Alberta, 113 Street & 87 Avenue, Edmonton, Alberta T6G 2E1,
Canada. Telephone: 780-492-8603; Fax: 780-492-6361; e-mail: yaojiong@ualberta.ca or yjwu2005@yahoo.com; Edward E. Tredget, M.D.,
M.Sc., FRCSC, Department of Surgery, 2D3.81, 8440 112 Street, University of Alberta, Edmonton, Alberta T6G 2B7, Canada. Telephone:
780-407-6979; Fax: 780-407-7394; e-mail: etredget@gpu.srv.ualberta.ca Received May 28, 2007; accepted for publication June 19, 2007;
first published online in STEM CELLS EXPRESS July 5, 2007. AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0226

STEM CELLS 2007;25:2648 2659 www.StemCells.com

Wu, Chen, Scott et al. 2649

Isolation and Purification of MSCs with an antibody reacting to cytokeratins (in molecular sizes of 58,
56, 52, 60, 51, and 48 kDa; DAKO, Glostrup, Denmark, http://
Bone marrow was collected from the femurs of 57-week-old male www.dako.com), and visualized under confocal microscope.
C57BL/6 or C57-GFP transgenic (C57BL/6 TgN[ACT6EGFP])
mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org).
The mononuclear fraction of the bone marrow was isolated with a Wound Healing Model and BM-MSC
Ficoll-paque density gradient. The nucleated cells were plated in Transplantation
plastic tissue culture dishes and incubated in minimal essential BALB/c mice (8 weeks old; female; body weight, 20 23 g), db/db
medium (-MEM; Gibco, Grand Island, NY, http://www. mice (BKS.Cg-m / Leprdb/J, db/db; 13 weeks old; female;
invitrogen.com) supplemented with 17% fetal bovine serum (FBS). body weight, 46.3 3.3 g; blood glucose, 44.0 7.7 mM; triglyc-
BM-MSCs were first selected by their adherent property preferen- eride, 1.4 0.6 mM; and cholesterol, 2.4 0.6 mM) and their
tially attaching to uncoated polystyrene tissue culture dishes [14] normal littermates (db/m; 13 weeks old; female; body weight,
and further purified by immunodepletion using magnetic mi- 22.4 1.6 g; blood glucose, 12.6 10.8 mM; triglyceride, 0.8
crobeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http:// 0.2 mM; and cholesterol, 1.5 0.2 mM; t test, p .01) were
www.miltenyibiotec.com) and monoclonal antibodies against obtained from Jackson Laboratory. The animals were randomly
CD34, CD14, Gr1, CD3, and CD19. Passage 35 cells were used for divided into three groups, and the excisional wound splinting model
the experiments. was generated as described previously [18]. In brief, after hair
removal from the dorsal surface and anesthesia, two 6-mm full-
Flow Cytometry thickness excisional skin wounds were created on each side of the
Passage 3 BM-MSCs were resuspended in phosphate-buffered sa- midline. Each wound received 1 million cells (GFP BM-MSCs or
line (PBS) containing 1% bovine serum albumin at 106 cells per neonatal dermal fibroblasts derived from C57BL/6 or C57BL/6-
milliliter. Cell aliquots (100 l) were first blocked with Mouse BD GFP mice): 0.7 106 in 60 l of PBS injected intradermally around
Fc Block and then incubated with fluorescein isothiocyanate the wound at four injection sites and 0.3 106 in 20 l of growth
(FITC)- or phycoerythrin-conjugated monoclonal antibodies spe- factor-reduced Matrigel (BD Biosciences) applied onto the wound
cific for Sca-1, CD105 (endoglin), CD29, CD44, CD90, CD45, bed. In some experiments, equal numbers of CD34 murine bone
CD14, CD3, CD19, and CD34 or control isotype IgG on ice for 30 marrow cells isolated with antibody-coated magnetic microbeads
minutes. All antibodies were purchased from BD Pharmingen (San (Miltenyi Biotec) were implanted into excisional wounds in athymic
Diego, http://www.bdbiosciences.com/pharmingen). For detection nude mice (8 weeks old; female; Jackson Laboratory). A donut-
of GFP cells in the wounded skin, excised wounds together with shaped silicone splint was placed so that the wound was centered
a small amount of surrounding skin were dispersed into single-cell within the splint. An immediate-bonding adhesive (Krazy Glue,
suspensions as previously described [15]. In brief, the tissue was Columbus, OH, http://www.krazyglue.com) was used to fix the
incubated with dispase I (Sigma-Aldrich, St. Louis, http://www. splint to the skin, followed by interrupted sutures to stabilize its
sigmaaldrich.com) at 1 mg/ml overnight at 4C, minced, and incu- position (Fig. 1A), and Tegaderm (3M, London, ON, Canada,
bated in a digestion buffer containing hyaluronidase (1 mg/ml), http://www.3m.com) was placed over the wounds. The animals
collagenase D (1 mg/ml), and DNase (150 units/ml) in a 37C were housed individually. We tested the adhesive on the skin in
shaking water bath for 2 hours. The dispase and the hyaluronidase mice prior to this experiment and did not observe any skin irritation
digests were pooled and filtered through a 70-m nylon cell or allergic reaction.
strainer. Cells were pelleted and resuspended in PBS containing 3%
FBS and analyzed for GFP-positive cells. Cells from sham wounds Wound Analysis
were used as negative controls. Ten thousand events were analyzed Digital photographs of wounds were taken at days 0, 3, 7, 10, 14,
by flow cytometry (Becton, Dickinson and Company, Franklin 21, and 28. Time to wound closure was defined as the time at which
Lakes, NJ, http://www.bd.com) using CellQuest software. the wound bed was completely re-epithelialized and filled with new
tissue. Wound area was measured by tracing the wound margin and
MSC Differentiation Assays calculated using an image analysis program (NIH Image). The
Passage 4 BM-MSCs were incubated to differentiate into adipo- investigators measuring samples were blinded to group and treat-
cytes, osteoblasts, and chondrocytes in corresponding induction ment. The percentage of wound closure was calculated as follows:
medium for 3 weeks [5, 14]. Adipogenic medium contained 106 M (Area of original wound Area of actual wound)/Area of original
dexamethasone, 10 g/ml insulin, and 100 g/ml 3-isobutyl-L- wound 100. The inside edge of the splint exactly matched the
edge of the wound, so that the splinted hole was used to represent
methylxantine (Sigma-Aldrich). Osteogenic medium contained
the original wound size. Mice were sacrificed at 7, 14, and 28 days,
107 M dexamethasone, 50 g/ml ascorbic acid, and 10 mM
at which times, skin samples including the wound and 4 mm of the
-glycerophosphate (Sigma-Aldrich). Cultures were stained for al-
surrounding skin were harvested using a 10-mm biopsy punch. For
kaline phosphatase (alkaline phosphatase detection kit; Sigma-Al-
whole skin mount, the entire wound and surrounding skin was
drich). For chondrocyte differentiation, MSC pellets were cultured
placed on plastic (tissue culture dish) with the dermis side down and
in Dulbeccos modified Eagles medium (high-glucose) containing
photographed immediately.
107 M dexamethasone, 50 g/ml ascorbate-2-phosphate, 100
g/ml pyruvate (Sigma-Aldrich), 10 ng/ml transforming growth
factor-1 (R&D Systems Inc., Minneapolis, http://www. Histologic Examination
rndsystems.com), and 50 mg/ml ITS Premix (6.25 g/ml insulin, Tissue specimens were fixed in 3% paraformaldehyde for 24
6.25 g/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml hours and embedded in OCT. Six-micron-thick sections were
bovine serum albumin, and 5.35 mg/ml linoleic acid; BD Bio- stained with H&E for light microscopy. Histological scoring was
sciences, San Diego, http://www.bdbiosciences.com). The cultures performed in a blinded fashion. Each slide was given a histo-
were fixed, sectioned, and stained for Alcian Blue (Sigma-Aldrich) logical score ranging from 1 to 10 according to the following
or subjected to RNA extraction and reverse transcription-poly- parameters, modified from previous reports [19, 20]: re-epithe-
merase chain reaction (RT-PCR) analysis for expression of genes lialization and regeneration, dermal cellularity, granulation tis-
characteristic of chondrocytes [16]. For differentiation of BM- sue formation, and angiogenesis. Capillary density was assessed
MSCs into keratinocytes, human primary keratinocytes were cul- morphometrically by examining three fields per section of the
tured on two-well chamber slides to 80% confluence in K-SFM (a wound between the edges in six successive sections after immu-
serum-free medium for keratinocyte culture; Gibco) and then irra- nofluorescence staining for endothelial cells with an anti-CD31
diated with 10 Gy from a 60Co source at a dose rate of 0.3 antibody [21, 22]. The criteria used for histological scores of
Gy/minute to stop cell proliferation [17]. Twenty-four hours later, wound healing are summarized in Table 1.
104 GFP BM-MSCs were seeded on the keratinocyte monolayer For immunofluorescence, tissue sections were preincubated
and maintained in K-SFM supplemented with 1% FBS for 1 week. with sodium borohydride (1 mg/ml in PBS) to reduce autofluores-
Medium was changed at day 3. Cells were fixed, immunostained cence. Endogenous biotin was blocked with streptavidin biotin
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2650 Mesenchymal Stem Cells Enhance Wound Healing

Figure 1. Characterization of BM-MSCs. (A): Fluorescence-activated cell sorting (FACS) analysis of BM-MSCs. Passage 3 BM-MSCs were
analyzed by FACS after staining with FITC- or PE-conjugated control isotype IgG (gray peaks) or antibodies against indicated cell surface proteins.
(B): Differentiation of BM-MSCs. Cultured in appropriate differentiation media, BM-MSCs differentiated into adipocytes, which are indicated by
accumulation of lipid vesicles in the cells (B), osteoblasts, which expressed alkaline phosphatase, as indicated in red (C), and chondrocytes in pellet
culture, which were positive for proteoglycans, as indicated in blue after Alcian Blue stain (D) and expressed genes characteristic of chondrocytes
by reverse transcription-polymerase chain reaction (E). Primers for Col-II detected procollagen IIa (upper band) and IIb transcripts. Abbreviations:
Actin, -actin; Agr, aggrecan; Col, collagen; Col-II, procollagen II; FITC, fluorescein isothiocyanate; PE, phycoerythrin; wk, week(s).

blocking kit (Vector Laboratories, Burlingame, CA, http://www. bation with a biotinylated secondary antibody (Jackson Immuno-
vectorlabs.com). Keratinocytes were stained with an antibody Research, West Grove, PA, http://www.jacksonimmuno.com) and
against epidermal keratin subunits (DAKO). GFP was detected with visualized with Fluor 568-conjugated streptavidin (Invitrogen,
an antibody (USBiological, Swampscott, MA, http://www.usbio. Carlsbad, CA, http://www.invitrogen.com). Nuclear staining with
net) and visualized with a FITC-conjugated secondary antibody. Hoechst and Ki67 (DAKO) was performed as previously described
Endothelial cells were identified with an antibody against CD31 or [23]. Isotype control antibodies were used for negative controls.
von Willebrand factor (vWF; BD Biosciences) followed by incu- Sections were examined with a Zeiss LSM 510 confocal microscope
Wu, Chen, Scott et al. 2651

Table 1. Criteria for histological scores

Angiogenesis (day
Score Epidermal and dermal regeneration Cell infiltration Granulation tissue 14 wounds only)

13 Minimal to moderate re-epithelialization Wound covered with thin to Granulation around wound Capillary density
with or without minimal developing moderate cell layer edges only 400/mm2
glandular structure formation in the
wound
47 Complete re-epithelialization with Wound covered with thick cell Granulation around wound edge Capillary density
minimal developing glandular layer and in 30%50% of wound 400600/mm2
structure formation in the wound bed
810 Complete re-epithelialization with Wound covered with very thick Thick granulation around Capillary density
considerable developing glandular and densely populated cell wound edge and in 50% of 600/mm2
structure formation in the wound layer wound bed

(Carl Zeiss, Jena, Germany, http://www.zeiss.com). The percent- phosphate dehydrogenase (GAPDH), forward 5-ATCATCCCT-
ages of Ki67-positive nuclei were determined by counting Ki67- GCATCCACT-3, reverse 5-ATCCACGACGGACACATT-3.
positive nuclei and total nuclei in five random fields per section Reactions were performed using SYBR Green PCR master mix
between wound edges using an image analysis program (NIH Im- (Applied Biosystems, Foster City, CA, http://www.
age), and four successive sections per wound were analyzed. Ap- appliedbiosystems.com) in a Bio-Rad iCycler iQ Detection Sys-
pendage-like structures in each wound section between wound tem (Hercules, CA, http://www.bio-rad.com). As an internal
edges were photographed to determine the numbers of total append- control, levels of GAPDH were quantified in parallel with target
age-like structures and Ki67-positive appendage-like structures genes. Normalization and fold changes were calculated using the
(with more than 10% Ki67-postive nuclei) per section. Ct method [25].

X and Y Chromosome Fluorescence In Situ Western Blotting

Hybridization Western blots were performed as previously described [26]. Briefly,
tissue proteins were extracted in a lysis buffer containing 1% Triton
X and Y chromosome fluorescence in situ hybridization (FISH) was X-100 and proteinase inhibitors (Sigma-Aldrich). Equal amounts of
performed as previously described with minor modification [6]. In total protein (100 g for lysates and 25 g for conditioned media)
brief, tissue sections 6 m in thickness were fixed three times in were separated on 12% SDS-polyacrylamide gel electrophoresis
Carnoy fixative and then incubated with an anti-pan-cytokeratin gels by electrophoresis and transferred to nitrocellulose membranes.
antibody (DAKO). Sections were digested with proteinase K (10 Membranes were incubated overnight at 4C with monoclonal an-
g/ml; Sigma-Aldrich) and denatured at 72C for 5 minutes in tibody against Ang-1, Ang-2 (Chemicon, Temecula, CA, http://
preheated 70% formamide in 2 standard saline citrate buffer (pH www.chemicon.com), or VEGF- (R&D Systems).
7.0). After dehydration, a mixture of FITC-labeled mouse X-chro-
mosome probe and Cy3-labeled Y-chromosome probe (STAR Statistical Analysis
FISH; Cambio, Dry Drayton, U.K., http://www.cambio.co.uk) were
incubated with tissue sections overnight at 42C. Sections were All values are expressed as mean SD. Students paired t test was
incubated with the anti-pan-cytokeratin antibody again followed by performed for comparison of data of paired samples, and analysis of
staining with a Cy5-conjugated secondary antibody. Nuclei were variance was used for multiple group comparisons, followed by
stained with Hoechst. Sections were examined with a confocal Friedmans post test. A probability (p) value .05 was considered
microscope. significant.

Endothelial Cell Network Formation Assay

RESULTS
Human umbilical vein endothelial cells (HUVECs) (2.5 104 cells
per well) were suspended in 0.4 ml of epithelial growth medium
(EGM)-2 basal medium supplemented with 2% FBS and a EGM-2 Characterization of BM-MSCs
SingleQuot kit (Cambrex, Walkersville, MD, http://www.cambrex. Fluorescence-activated cell sorting (FACS) analysis of our BM-
com), vehicle-, fibroblast-, or BM-MSC-conditioned EGM-2 basal
MSCs showed that they were negative for lineage cell markers such
medium supplemented with 0.75% FBS alone; seeded onto Matrigel
(BD Biosciences)-coated 24-well plates; and incubated at 37C/5% as CD34, CD45, CD14, CD3, and CD19 and strongly expressed
CO2 for 12 hours. After removal of the medium, the cells were typical surface antigens such as Sca-1, CD29, CD44, CD105, and
fixed, and images were captured. The total length of the tube-like CD90 (Fig. 1A). When cultured in adipogenic, osteogenic, or
structures was determined using NIH Image software [24]. Four chondrogenic medium, they differentiated into adipocytes (Fig.
random fields were measured for each well. 1B), osteoblasts (Fig. 1C), or chondrocytes (Fig. 1D, 1E).

Real-Time PCR Analysis BM-MSCs Enhance Wound Healing

Total RNA was extracted (RNeasy Mini Kit; Qiagen, Hilden, BM-MSC-treated wounds exhibited accelerated wound closure
Germany, http://www1.qiagen.com) from cultured BM-MSCs or in BALB/c mice (Fig. 2A, 2B) and genetically diabetic db/db
neonatal dermal fibroblasts that were 80% confluent and had mice (Fig. 2C) compared with fibroblast- or vehicle medium-
been treated in hypoxic conditions (1% O2) for 8 hours and treated wounds. The enhancement appeared early, at 3 days after
reverse transcribed using the SuperScript First-Strand Synthesis implantation in BALB/c mice, and became more evident after 7
kit (Invitrogen). The primers are as follows: vascular endothelial
growth factor (VEGF)-, forward 5-AGAGCAACATCAC-
days in both BALB/c and db/db mice. At 7 days, wound closure
CATGCAG-3, reverse 5-CAGTGAACGCTCCAGGATTT-3; in BM-MSC-treated db/db mice appeared similar to that in db/m
angiopoietin (Ang)-2, forward 5-GACTTCCAGAGGACGTG- mice. At 28 days, 4 of 12 wounds in BM-MSC-treated db/db
GAAAG-3, reverse 5-CTCATTGCCCAGCCAGTACTC-3; mice (n 6) achieved complete wound closure, but no com-
Ang-1, forward 5-TTGTGATTCTGGTGATTGTGG-3, reverse pletely closed wounds were seen in fibroblast-treated (n 6) or
5-CTTGTTTCGCTTTATTTTTGT-3; and glyceraldehyde-3- vehicle medium-treated (n 5) mice. As splints in some db/m
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2652 Mesenchymal Stem Cells Enhance Wound Healing

Figure 2. Effects of MSC on wound closure. (A): Images of wounds in BALB/c mice. Murine excisional wound splinting model (top panel), in which
wounds received implantation of GFP MSC, FB, or control vehicle medium (sham). Representative photographs of the wounds at day 7 (with
transparent Tegaderm dressing, middle panel) and day 14 (after removal of dressing, bottom panel). (B): Wound measurement of sham group (n
11 at days 3 and 7; n 6 at days 10 and 14), FB group (n 12 at days 3 and 7; n 7 at days 10 and 14), and MSC group (equal animal numbers
to FB group) in BALB/c mice. Analysis of variance (ANOVA), versus sham or FB, , p .001. (C): Wound measurement of sham group in db/m
mice at days 3 and 7 (n 10), sham group (n 6 at days 7 and 14; n 5 at days 21 and 28), FB group (n 7 at days 7 and 14; n 6 at days
21 and 28) and MSC group (equal animal numbers to FB group) in db/db mice. ANOVA, , p .01. Abbreviations: FB, fibroblasts; MSC, bone
marrow-derived MSCs; wk, week(s).

mice were not tightly adherent to the skin and failed to restrict examined; n 5) compared with fibroblast-treated wounds
skin contraction after 14 days because of movement and hair (complete re-epithelialization in 6 of 10 wounds; n 5) or
regrowth, data on wound closure in these mice after 14 days vehicle medium-treated wounds (complete re-epithelialization
were excluded. In contrast, splints remained firmly adherent to in 4 of 10 wounds; n 5). Analysis of day 7 and 14 wounds
the skin in db/db mice, as the animals had slower physical indicated that BM-MSC-treated wounds had enhanced cellular-
movement and hair regrowth. Fibroblast-treatment accelerated ity (Fig. 3A, 3B) and increased vasculature (also shown in Fig.
wound closure in db/db mice at 7, 14, and 21 days (p .01) but 5). In addition, granulation tissue in BM-MSC-treated wounds
not at 28 days and in BALB/c mice compared with vehicle appeared to be thicker and larger. Consistent with these find-
medium treatment. ings, the histological scores of BM-MSC-treated wounds of 7
Histological evaluation of wounds in BALB/c mice at 7 and 14 days were significantly higher (Fig. 3C; n 5; p
days disclosed enhanced re-epithelialization in BM-MSC- .001). In addition, BM-MSC-treated wounds appeared to have
treated wounds (complete epithelialization in all 10 wounds increased numbers of skin appendages (Fig. 3A), suggesting
Wu, Chen, Scott et al. 2653

Figure 3. Histological analysis of wounds in BALB/c mice. (A): Wound histological images (H&E stain). Wound edges are indicated by arrows.
(B): Wound cellularity was determined by counting the number of nuclei per high-power (400) field (n 5; , p .01). (C): Wound histological
scores (n 5 at days 7 and 14; analysis of variance [ANOVA], , p .001). (D): Confocal images show Ki67 and Hoechst stain of normal skin
or day 7 wounds treated with vehicle medium (sham), FB, and MSC. Ki67-positive skin appendages are indicated by arrows. The epidermis or wound
surfaces are indicated by arrowheads. Scale bar 20 m. (E): Percentages of Ki67-positive cells in day 7 wounds were counted after immunostaining
(n 5; ANOVA, , p .01 MSC or FB vs. sham; #, p .001 MSC vs. FB). (F): Numbers of Ki67-positive skin appendages per wound section
between wound edges in day 7 wounds (n 5; , p .001 vs. sham or FB). Abbreviations: Ep, epidermis; F, hair follicle; FB, fibroblasts; M, muscle;
MSC, bone marrow-derived MSCs; W, wound bed; wk, week(s).

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2654 Mesenchymal Stem Cells Enhance Wound Healing

Figure 4. Differentiation and engraftment of bone marrow-derived (BM)-MSCs. Tissue sections were immunostained with an anti-pan-cytokeratin
antibody. Nuclei (blue) were stained with Hoechst. (A): Confocal microscopy showed that cytokeratin-positive (red) BM-MSCs (green) appeared in
the dermis and epidermis (indicated by arrows) of a day 7 BM-MSC-treated wound but not vehicle medium- or FB-treated wound. Scale bar 50
m. (B, C): GFP BM-MSCs (green) expressing cytokeratin (red) formed a nodule that resembled early sweat or sebaceous glands in the dermis of
a day 7 wound (B), and such structures became more mature in wounds at 14 days (C). Scale bar 50 m. (D): Implanted DiI-labeled CD34 BM
cells (red) were shown in a day 10 wound. Keratinocytes were stained green, and nuclei were stained blue. Scale bar 20 m. (E): A representative
image of X and Y chromosome fluorescence in situ hybridization stain of a day 7 MSC-treated wound. Y-chromosome (red)-positive cells derived
from implanted MSCs contained no more than one X-chromosome (green). Cytokeratin was immunostained pink. Scale bar 20 m. (FH):
Confocal microscopic images showing GFP BM-MSCs (green) cocultured with preirradiated dermal keratinocytes for 1 wk after immunostaining
for cytokeratins (red). Nuclei were stained with Hoechst (blue). After merging (F) and (G), cells expressing GFP and cytokeratins are indicated in
yellow (arrowhead [H]). Scale bar 20 m. (I): The number of cells per wound was determined after digestion of the entire wound (n 5; t test,
, p .001). (J): One representative result of five fluorescence-activated cell sorting analyses of skin digests for GFP-positive cells at different times
after wounding. Cells from sham wounds were used for negative controls and gate setting. Values represent percentages of GFP-positive cells.
Abbreviations: FB, fibroblasts; GFP, green fluorescent protein; wk, week(s).
Wu, Chen, Scott et al. 2655

Figure 5. Effects of BM-MSC on wound

vascularity. (A): Representative images of
whole skin mounts of day 7 wounds in
BALB/c mice treated with vehicle medium
(sham), FB, or MSC. Vehicle- or FB-treated
wound appeared more transparent with de-
fects (holes), whereas BM-MSC-treated
wound shows blood vessels growing from
surrounding tissue. (B): Immunofluores-
cence for endothelial cells. Day 7 (upper
panel) or 14 (lower panel) wound sections
were stained with an anti-CD31 antibody
and detected with Fluor 568 (red). Nuclei
were stained with Hoechst. Arrowheads in-
dicate the epidermis. Scale bar 20 m.
(C): Capillary density in 2-wk-old wounds
was counted after CD31 staining (n 6; ,
p .0001). Abbreviations: FB, fibroblasts;
GFP, green fluorescent protein; MSC, bone
marrow-derived MSCs; wk, week(s).

enhanced cutaneous regeneration. Indeed, BM-MSC-treated BM-MSCs Contribute to Dermal Keratinocytes and
wounds exhibited increased numbers of appendage-like struc- Appendages
tures at 7 days compared with vehicle medium- or fibroblast- To determine whether BM-MSCs in the wounded skin could
treated wounds (sham, 4.5 0.42; fibroblast, 4.7 0.53; MSC, contribute to keratinocytes, we performed immunostaining for
10.2 0.79 per wound section; n 5; MSC vs. sham or GFP and epithelial protein cytokeratin. We used an anti-cyto-
fibroblast, p .001). We then measured the fractions of divid- keratin antibody that reacted to several keratin subunits (K4, K5,
ing cells in the wound sections after staining for Ki67 [27]. As K6, K8, K14, and K16). In the day 7 wound, GFP and cytoker-
shown in Figure 3D and 3E, BM-MSC-treated wounds exhibited atin double-positive cells were found especially in the dermis
significantly increased proportions of Ki67-positive cells, along adjacent to the epidermis, and some appeared in the epidermis
with increased numbers of Ki67-positive hair follicle- or sweat/ (Fig. 4A). Some BM-MSCs positive for GFP and cytokeratin in
sebaceous gland-like structures (with more than 10% Ki67- the dermis of the wounded skin formed structures that resem-
positive nuclei) compared with vehicle medium- or fibro- bled developing sweat or sebaceous glands (Fig. 4B). Such
blast-treated wounds (Fig. 3E, 3F; n 5; p .001). structures appeared more mature at 14 days (Fig. 4C). GFP
Fibroblast-treated wounds showed increased numbers of cells were not detected in GFP-negative fibroblast- or vehicle
Ki67-positive cells (Fig. 3E; p .01) but not Ki67-positive medium-treated wounds (Fig. 4A), indicating specificity of the
appendages (Fig. 3F). immunostaining. To determine the fractions of BM-MSCs ex-
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2656 Mesenchymal Stem Cells Enhance Wound Healing

Figure 6. Effects of bone marrow-derived MSC (BM-MSC)-conditioned medium on human umbilical vein endothelial cells (HUVECs). (A):
Endothelial cell tube formation. HUVECs were suspended in vehicle-, MSC-, or FB-conditioned basal EGM-2 supplemented with 0.75% fetal bovine
serum (FBS) or complete EGM-2, which contained growth factor cocktail and 2% FBS, and incubated for 24 hours. Representative fields are shown.
The total length of the tube network per field was quantified. Experiments were performed in triplicate wells (n 4; , p .05; , p .001). (B):
Real-time polymerase chain reaction analysis shows expression levels of VEGF-, Ang-1, and Ang-2 (relative to glyceraldehyde-3-phosphate
dehydrogenase) in BM-MSCs and FBs. (C): Western blot assay shows expression of VEGF-, Ang-1, and Ang-2 in FB- or BM-MSC-conditioned
medium and vehicle medium (sham)-, FB-, or BM-MSC-treated wounds. The experiment was repeated three times, and one representative result is
shown. Abbreviations: Ang, angiopoietin; EGM, epithelial growth medium; FB, fibroblast; FB-M, fibroblast medium; MSC-M, bone marrow-derived
MSC medium; VEGF, vascular endothelial growth factor; vehicle-M, vehicle medium; wk, week.

pressing cytokeratin, we analyzed cells derived from digestion nostaining for cytokeratins indicated that 17.2% 4.3% of
of wounds. Slides were prepared on a cytospin and immuno- BM-MSCs became cytokeratin-positive (Fig. 4F 4H).
stained for GFP and cytokeratin. Analysis of 200 GFP-express-
ing cells per sample indicated that 15.3% 2.1% of them in day Engraftment of BM-MSCs into the Wounded Skin
7 wounds (n 5) and 47.6% 6.3% of them in day 14 wounds Immunostaining of wound sections for GFP showed a large
(n 5) were cytokeratin-positive. To determine whether number of GFP BM-MSCs in BM-MSC-treated wounds at 7
CD34 cells contribute to keratinocytes, we implanted equal days. The cells were mostly found in the newly formed dermis,
numbers of DiI-labeled CD34 murine bone marrow cells to but some appeared in the epidermis (Fig. 4A). GFP cells were
excisional wounds in nude mice (n 3) [28]. Immunofluores- not detected in the surrounding normal skin. Decreased numbers
cence analysis of wound sections at 10 days for cytokeratin of GFP BM-MSCs were localized to the centers of wounds and
expression using an anti-pan-cytokeratin antibody (the same skin appendages at 14 days; in addition, markedly fewer GFP
antibody as used in Fig. 4A 4C) showed engraftment of DiI- BM-MSCs were detected in the wound at 28 days. To determine
CD34 cells, but none of them expressed cytokeratin or were the numbers of GFP BM-MSCs engrafted into the wounded
localized with appendages (Fig. 4D). To determine whether the skin at different stages, we excised the entire wound along with
GFP and cytokeratin double-positive cells were derived from a small amount of the surrounding skin and dispersed it into a
cell fusion, we performed X and Y chromosome FISH using single cell suspension. Counting of cells in the suspension with
FITC-labeled X-chromosome probe and Cy3-labeled Y-chro- a cytometer resulted in the number of total cells per wound: in
mosome. BM-MSCs were derived from male mice, whereas the the sham group, 1.86 0.22 106 at 7 days, 1.51 0.14 106
recipient mice were female. Cytokeratin was counterstained and at 14 days, and 1.42 0.19 106 at 28 days; in BM-MSC-
detected with a Cy5-labled secondary antibody. Y-chromosome- treated mice, 2.67 0.27 106 at 7 days, 2.16 0.18 106
positive cells were detected in the dermis and epidermis (Fig. at 14 days, and 1.44 0.20 106 at 28 days (Fig. 4I). FACS
4E). Some Y-chromosome-positive cells were also positive for analysis of the skin digests for GFP-positive cells indicated that
cytokeratin but contained no more than one X-chromosome, 10.1% 1.7% at 7 days, 3.5% 0.48% at 14 days (Fig. 4J; n
indicating that the cells were not derived from fusion of MSCs 5), and 1.8% 0.21% at 28 days of the total cells were
with keratinocytes. To examine whether BM-MSCs differentiate GFP-positive in BM-MSC-treated wounds. Taking the initially
into keratinocytes in vitro, we cultured GFP BM-MSCs (neg- implanted 1 million BM-MSCs per wound as 100%, after cal-
ative for cytokeratins) with preirradiated keratinocytes in a culation, BM-MSC engraftment to the wound was 27% at 7
medium favoring keratinocyte growth. Seven days later, immu- days, 7.6% at 14 days, and 2.5% at 28 days.
Wu, Chen, Scott et al. 2657

BM-MSCs Enhance Angiogenesis and hyperlipidemia, symptoms similar to type II diabetes. Our
The BALB/c mouse skin is thin and semitransparent, which results indicate that wound closure is significantly delayed in the
allows macroscopic visualization of blood vessels. In wounds of mice and implantation of BM-MSCs significantly promotes the
sham and fibroblast groups at 7 days in BALB/c mice, blood wound healing process. Consistent with our findings, BM-
vessels were seen clearly in the skin surrounding the wounds but MSCs was recently shown to increase the tensile strength of
were limited in the wounds. In contrast, in wounds of MSC incisional wounds in nondiabetic rats [30].
group, vessels and their fine branches extended into the wounds, We have shown that a substantial fraction of GFP-express-
forming networks (Fig. 5A). Immunohistological staining of ing BM-MSCs engrafted in the wound appeared to coexpress
tissue sections for endothelial protein CD31 (Fig. 5B) or vWF cytokeratin subunits and therefore may adopt an epithelial-like
phenotype. Moreover, some GFP-expressing BM-MSCs were
(data not shown) showed increased vasculature in BM-MSC-
found to form sweat or sebaceous gland-like structures. Our
treated wounds at 7 and 14 days compared with vehicle medi-
finding is consistent with a recent study in which intravenously
um- or fibroblast-treated wounds. Capillary densities in wounds
infused 5-bromo-2-deoxyuridine-labeled ex vivo-expanded
at 14 days were assessed morphometrically after immunohisto-
bone marrow adherent cells containing MSCs and CD34 cells
chemical staining for CD31 [21, 22]. As shown in Figure 6C,
were found to contribute to cells in hair follicles, sebaceous
capillary density was significantly higher in BM-MSC-treated
glands, and blood vessels in the dermis of full-thickness skin
wounds (771 55/mm2) than in vehicle medium-treated
wounds [31]. Notably, in our study, similarly implanted CD34
(357 51/mm2) or fibroblast-treated (398 44/mm2) wounds
bone marrow cells did not express cytokeratin or incorporate
(n 5; p .001). BM-MSCs appeared close to vasculature but
into skin epidermis and appendages in the wound. Our results
were not in the walls of blood vessels. To determine whether
indicate that BM-MSCs but not CD34 cells can contribute to
BM-MSCs could enhance angiogenesis through a paracrine
cutaneous structures. In a previous study, BM-MSCs were
effect, we cultured HUVECs in BM-MSC-conditioned medium
found to repair epithelium in vitro through differentiation and
derived from culturing of BM-MSCs under hypoxic conditions
fusion [32]. Our X and Y chromosome FISH data suggest that
(1% O2) for 24 hours and found that the medium significantly
the cytokeratin-expressing MSCs in the wound are formed via
enhanced HUVEC tube formation on Matrigel compared with
differentiation but not cell fusion. In a recent study, similarly
vehicle control medium or fibroblast-conditioned medium (Fig.
transplanted ex vivo-expanded BM-MSCs were found to differ-
6A). Real-time PCR analysis showed that VEGF- was highly
entiate into hepatocytes without evidence of cell fusion [6]. In
expressed in BM-MSCs and fibroblasts, but the Ang-1/Ang-2
postpartum humans, injury to the skin and other tissues heals not
ratio was much greater in BM-MSCs (5.7) than in fibroblasts
by the regeneration of the tissue to the preinjured form but by
(1.07) (Fig. 6B). To examine the protein expression levels of
the formation of scar tissue. Our data and those of others suggest
these genes, we performed Western blot analysis of concen-
that BM-MSCs engrafted in the wound may contribute to cells
trated BM-MSC- or fibroblast-conditioned medium under hy- in the skin epidermis and appendages, thus mediating dermal
poxic conditions and lysate derived from vehicle medium regeneration. Of note, our data showed limited GFP-expressing
(sham)-, fibroblast- or BM-MSC-treated wounds. Our data epithelial cells in the wound by 4 weeks when the wound was
showed a greater amount of Ang-1 protein in BM-MSC-condi- closed. This is probably due to rapid turnover of epithelial cells
tioned medium and higher levels of Ang-1 in BM-MSC-treated during wound healing. Consistent with our findings, epithelial
wounds at 7 and 14 days but unchanged amounts of Ang-2 (Fig. cells derived from endogenous bone marrow cells were found
6C). Under reducing conditions, the anti-VEGF- antibody de- transiently during wound healing [23]. These data suggest that
tected a major band of approximately 22 kDa, which corre- the bone marrow may not provide long-term self-renewal stem
sponds to the molecular size of VEGF164 [29]. Of note, larger cells for dermal keratinocytes. In this study, we found that
amounts of VEGF- were detected in BM-MSC-treated wounds BM-MSC-treated wounds exhibited a significantly increased
compared with vehicle medium- or fibroblast-treated wounds at number of regenerating appendage-like structures rich in Ki67-
7 and 14 days (Fig. 6C). positive dividing cells. Interestingly, these structures were
largely formed by endogenous keratinocytes, suggesting that
paracrine factors released by engrafted BM-MSCs in the wound
DISCUSSION also play an important role in cutaneous regeneration.
We determined the numbers of GFP-positive BM-MSCs in
Diabetic ulcers and other chronic wounds are difficult to heal, the wound at various stages of wound healing by FACS analysis
and little improvement has been shown in preventing the asso- coupled with immunohistochemical assessment. Our data indi-
ciated morbidity and disability in the past few decades [4]. The cate a substantial engraftment of BM-MSCs in the day 7 wounds
best available treatment for chronic wounds achieves only a and a rapid reduction of the cells in the day 14 wounds and
50% healing rate that is often temporary. Innovative treatments thereafter. Consistent with our findings, a recent study showed
to enhance wound healing and regeneration are needed. Here, a dramatic decline of engrafted BM-MSCs in the acutely in-
we show that BM-MSCs enhance wound healing in nondiabetic farcted myocardium after intramyocardial injection [33]. The
and diabetic mice by promoting re-epithelialization, cell infil- mechanisms involved with the decline of implanted MSCs are
tration, and angiogenesis. not fully understood. It is likely that with progression of wound
We used an excisional wound splinting model and found healing process, cytokines and ECM molecules favorable to
that splinting prevented skin contraction and allowed wounds to MSC survival and engraftment decrease. It is known that en-
heal through granulation and re-epithelialization, as reported graftment of BM-MSCs to normal nonhematopoietic tissues is
previously [18]. In addition, the model resulted in uniform extremely low.
wound closure due to minimization of variations caused by skin Neovascularization is a crucial step in the wound healing
contraction and wound dressings. To assess the effect of BM- process [13, 34]. The formation of new blood vessels is nec-
MSCs in healing diabetic wounds, we used genetically diabetic essary to sustain the newly formed granulation tissue and the
db/db mice, which have been known to have markedly impaired survival of keratinocytes. In this study, we demonstrated that
wound healing and have been used extensively to study the BM-MSC-treated wounds had enhanced capillary density, sug-
effect of therapeutic reagents on wound healing [19, 20]. Indeed, gesting that BM-MSCs promote angiogenesis; however, BM-
our db/db mice exhibited significant obesity, hyperglycemia, MSCs were not found in the vascular structures but in close
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2658 Mesenchymal Stem Cells Enhance Wound Healing

proximity. The discrepancy led us to study the paracrine effect Freshly isolated BM cells enriched for hematopoietic progenitor
of BM-MSCs in angiogenesis. Indeed, we found that BM-MSC- cells from nondiabetic mice were found to promote wound
conditioned medium promoted endothelial tube formation and healing in diabetic db/db mice, but impaired functions were
that BM-MSCs expressed high levels of VEGF- and Ang-1 but found with the cells derived from db/db mice [40]. Application
not Ang-2. Notably, BM-MSC treatment resulted in signifi- of macrophages derived from healthy donors was found to
cantly increased amounts of Ang-1 and VEGF- in the wounds. promote healing of chronic ulcers in patients [41].
VEGF plays a key role in angiogenesis by stimulating endothe- Our results did not show enhancement of wound healing in
lial cell proliferation, migration, and organization into tubules allogeneic fibroblast-treated nondiabetic mice, although modest
[34, 35]. Moreover, VEGF increases circulating endothelial improvement was observed in the early stages in db/db mice.
progenitor cells [35]. The angiopoietins represent another major Previously, patients with diabetic or venous skin ulcers have
family of angiogenic factors. Ang-1 and Ang-2 are ligands for been treated with two FDA-approved engineered living skin
the Tie2 receptor tyrosine kinase that is present on endothelial products containing allogeneic fibroblasts [42]; however, the
cells and endothelial progenitor cells. Ang-1-Tie2 interactions benefit of allogeneic fibroblasts in wound healing remains con-
mediate neovessel maturation into more complex and larger troversial [43, 44]. Our study showed a lower number of allo-
vascular structures and maintain vessel integrity through the geneic fibroblasts engrafted into the wound compared with
recruitment of periendothelial cells and the reestablishment of allogeneic BM-MSCs. In a previous study, allogeneic fibro-
basement membrane. Ang-2, as an endogenous antagonist of blasts was found to cause increased inflammation and scar
Tie2, functions to block this Ang1-Tie2 signaling [34, 35]. formation [44].
Angiogenesis is a complex process controlled by the balance of In conclusion, this study demonstrates systemically the ben-
proangiogenic and antiangiogenic factors [35]. Our results sug- eficial effect of BM-MSCs in cutaneous regeneration and
gest that BM-MSCs engrafted in the wound release proangio- wound healing in nondiabetic and diabetic mice through differ-
genic factors, which may be partially responsible for MSC- entiation and paracrine effects. Administration of allogeneic
mediated enhanced angiogenesis. BM-MSCs or BM-MSC-derived molecules may represent novel
Low incidence of differentiation of MSCs into cardiomyo- therapeutic approaches in the treatment of chronic wounds and
cytes was reported recently [33], and procrine mechanism was other conditions.
suggested to play a major role in MSC-mediated myocardial
repair [36, 37]. However, the incidences of differentiation of
MSCs into keratinocytes found in this study and others are much ACKNOWLEDGMENTS
higher [38, 39]. In our recent study, we found that injection of
MSC-conditioned medium could accelerate wound closure, but We gratefully acknowledge H.A. Shankowsky for excellent
the enhancement was less dramatic and the wound quality was assistance with animal protocols and secretary and M. Cao for
less evidently improved compared with cell implantation (data technical support. This work was supported in part by Firefight-
not shown). Theses results suggest that differentiation of MSCs ers Burn Trust Fund and the University of Alberta Hospital
may be indispensable in MSC-mediated cutaneous repair/regen- Foundation.
eration, although paracrine factors are important.
In this study, we used purified BM-MSCs, which eliminated
blood lineage cells, aiming to verify the bone marrow cells DISCLOSURE OF POTENTIAL CONFLICTS
accounting for cutaneous regeneration and enhanced wound OF INTEREST
healing. Previous studies suggest that other populations of BM
cells may also be beneficial to the healing of chronic wounds. The authors indicate no potential conflicts of interest.

11 Jin HK, Carter JE, Huntley GW et al. Intracerebral transplantation of

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