Role of Mesenchymal Stem Cells on Cornea Wound Healing Induced by Acute Alkali Burn
Citation: Yao L, Li Z-r, Su W-r, Li Y-p, Lin M-l, et al. (
Role of Mesenchymal Stem Cells on Cornea Wound Healing Induced by Acute Alkali Burn
Lin Yao 0
Zhan-rong Li 0
Wen-ru Su 0
Yong-ping Li 0
Miao-li Lin 0
Wen-xin Zhang 0
Yi Liu 0
Qian Wan 0
Dan Liang 0
Arto Urtti, University of Helsinki, Finland
0 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University , Guangzhou , People's Republic of China
The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 26106 MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68+ cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1a), tumor necrosis factor-alpha (TNF-a), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68+ cells was significantly lower in the MSC group and the mRNA levels of MIP-1a, TNF-a, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68+ cells and the down-regulation of MIP-1a, TNF-a and VEGF.
Corneal chemical burn is a common ophthalmologic
emergency. In general, corneal chemical burn manifests in four phases,
including immediate, acute, early repair and late repair phases.
Treatment during the acute phase of corneal chemical burn is
crucial for its clinical management [1,2]. In the acute phase of a
corneal chemical burn, slow epithelialization, persistent ulceration,
corneal perforation and angiogenesis are the most common
complications [1,2]. These complications are closely associated
with inflammation, which is an important element during corneal
wound healing after a chemical burn [3,4]. Thus, in the acute
phase of a corneal chemical burn, treatments that are
antiinflammatory, anti-angiogenic and that enhance epithelial healing
are critical aspects of clinical treatment. A number of treatment
modalities have been undertaken to treat corneal chemical burn
. However, no coherent strategy with regard to the ideal
treatment of corneal chemical burn yet exists.
Mesenchymal stem cells (MSCs) are a type of multipotent cell
originally isolated from bone marrow that have subsequently been
isolated from other tissues, such as adipose tissue , heart tissue
, cord blood  and oral tissue . Recently, an increasing
body of evidence indicates that MSCs possess multifunctional
properties from tissue repair/regeneration to
immunomodulatory/anti-inflammatory functions . More recently, MSCs
have been studied for the treatment of corneal chemical burn with
encouraging results . However, for clinical applications,
further studies are necessary to confirm and elucidate the
therapeutic effects and mechanisms of MSCs in treating corneal
chemical burn, especially in the acute phase. In addition, neither
application with a special hollow plastic tube nor transplantation
with amniotic membrane can be easily carried out in clinical
[15,16]. However, subconjunctival injection is one of the most
common clinical administration routes, and it can be easily
performed by ophthalmologists clinically.
Thus, this study aimed to investigate the effects and to explore
underlying mechanisms of subconjunctival administration of
MSCs in the acute phase in a rat corneal alkali burn model.
All procedures used in this study were in accordance with the
principles of the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. The study was approved by the
Research Ethics Committee of the Zhongshan Ophthalmic
Center, Sun Yat-sen University (approval ID: 2010-010;
Six-week-old female Sprague-Dawley rats (Guangdong
Provincial Center for Animal Experiment, Guangzhou, China) weighing
180220 g were anesthetized by intraperitoneal injection of 4 ml/
kg of 10% chloral hydrate (Zhongshan Ophthalmic Center, Sun
Yat-sen University, Guangzhou, China). At the end of the
experiment, all rats were sacrificed with an overdose of 10%
chloral hydrate. The corneas of the rats were harvested, and only
the right eye of each rat was used.
Isolation, culture and labeling of MSCs
Bone marrow cells were collected by flushing the femurs and
tibias of two-week-old female Sprague-Dawley rats with
Dulbeccos modified Eagles medium (DMEM, Gibco-BRL, Grand
Island, New York). The cells were cultivated in 75-cm2 cell
culture flasks in DMEM supplemented with 10% fetal bovine
serum (FBS, Gibco-BRL, Grand Island, New York) and
penicillin/gentamycin (10 mg/ml, Sigma-Aldrich, St. Louis,
MO). After 72 hours, nonadherent cells were removed by
changing the culture medium. The culture medium was changed
every 3 days, and the cells were transferred when they reached
80% to 90% confluence. Flow cytometry analysis of MSCs and the
differentiation of osteoblasts and adipocytes were performed as
previously described . MSCs from passages 26 were used for
all experiments described. On the day of transplantation, MSCs
were labeled with DiI (Sigma-Aldrich, St. Louis, MO) and washed
twice in phosphate-buffered saline (PBS). The DiI-labeled MSCs
were applied to the corneal alkali burn to detect the migration
pattern of MSCs in the cornea and conjunctiva by confocal laser
Immunological phenotype of MSCs
For flow cytometry, MSCs from the third passage were
trypsinized, resuspended in PBS containing 10% FBS, and
incubated with monoclonal antibodies conjugated with either
FITC or PE for 30 minutes at 4uC. FITC-conjugated antibodies
against CD90 and PE-conjugated antibodies against CD29,
CD44, CD34, CD45, and CD11b were purchased from Biolegend
(Biolegend, San Diego, CA). Appropriate isotype antibody controls
(Biolegend, San Diego, CA) were used in each fluorescence
analysis. Cells were analyzed with a BD FACS Calibur, and the
data were analyzed using Cell Quest software.
Differentiation of MSCs in vitro
Both osteogenic and adipogenic differentiation of MSCs were
assessed in this study. Briefly, for osteogenic differentiation, MSCs
expanded in vitro for 3 passages were cultured for 16 days at 100%
confluence in DMEM-high glucose supplemented with 10% FBS,
penicillin/gentamycin (10 mg/ml), ascorbic acid (0.2 mM,
SigmaAldrich, St. Louis, MO), b-glycerophosphate disodium salt
pentahydrate (10 mM, Sigma-Aldrich, St. Louis, MO), and
dexamethasone (0.1 mM, Sigma-Aldrich, St. Louis, MO). The
medium was changed every 3 days. Alizarin red S (2%,
SigmaAldrich, St. Louis, MO) was used to stain calcium deposits.
To induce adipogenic differentiation, MSCs expanded in vitro
for 3 passages were cultured at 100% confluence for 9 days in
DMEM-low glucose supplemented with 10% FBS, penicillin/
gentamycin (10 mg/ml), indomethacin (0.2 mM, Sigma-Aldrich,
St. Louis, MO), insulin (10 mg/ml, Sigma-Aldrich, St. Louis, MO),
IBMX (0.5 mM, Sigma-Aldrich, St. Louis, MO), and
dexamethasone (1 mM). The medium was changed every 3 days. Oil red O
(0.3%, Sigma-Aldrich, St. Louis, MO) was used to stain lipid
droplets, which can be easily visualized by light microscopy.
Animal model of corneal alkali burn
A corneal alkali burn was generated in the right eye of each rat
(48 rats). The rats were anesthetized by intraperitoneal injection of
10% chloral hydrate (4 ml/kg). A piece of Whatman #3 filter
paper (3-mm diameter) soaked in 4 ml NaOH (1 mol/l) was
applied to the center of the cornea for 40 seconds. The cornea was
then rinsed with 60 ml of saline for 1 minute.
Subconjunctival injection of DiI-labeled MSCs
After the corneal alkali burn, the 48 rats were randomly divided
into MSC (n = 24) and control (n = 24) groups. Rats in the MSC
group received a subconjunctival injection of 0.1 ml PBS
containing 26106 DiI-labeled MSCs immediately and 3 days
after the corneal alkali burn. In the control group, rats received a
subconjunctival injection of 0.1 ml PBS immediately and 3 days
after the corneal alkali burn. All rats in each group were clinically
Observation and examination
Corneal fluorescence staining (8 rats/group) was examined by
slit-lamp at 3 and 7 days after the corneal alkali burn (SL-120;
Zeiss, Jena, Germany). All observations were performed by a
single experienced ophthalmologist who was blind to the allocation
of the animals in each group.
To evaluate the development of corneal neovascularization
(CNV), ink perfusion via the aorta was performed on days 3 (8
rats/group) and 7 (8 rats/group) as described previously . The
eyes were fixed in 10% neutralized buffered formaldehyde
overnight, and the corneas were dissected and flattened for image
capture. Quantitative measurements of the CNV area were
assessed with Image ProH Plus 5.1 image analysis software (Media
Cybernetics, Silver Spring, MD).
Histological evaluation and migration of MSCs
On day 7 after the corneal alkali burn, 3 eyeballs randomly
selected from each group were excised for histological evaluation
and the examination of MSCs migration after the rats were
sacrificed. Cryosections (6-mm thick) were obtained for
hematoxylin-eosin (H-E) and immunofluorescence staining. For
immunofluorescence staining, the cryosections were placed in acetone for
15 min at 220uC. The samples were blocked in 1% goat serum
albumin (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS for
30 min and subsequently incubated with primary antibody
(monoclonal mouse antibody against rat CD68; Santa Cruz
Biotechnology, Santa Cruz, CA) diluted in PBS overnight at 4uC.
Sections incubated without primary antibody served as negative
controls. After washing with PBS, the samples were incubated with
a FITC-conjugated secondary IgG antibody for 45 min at room
temperature and washed again with PBS. The sections were
counterstained with Hoechst (Sigma-Aldrich, St. Louis, MO) for
examination by confocal laser scanning microscopy (LSM 510
Meta; Zeiss, Yena, Germany). The numbers of infiltrated cells and
CD68+ cells in the corneal stroma were counted in five randomly
selected fields (6400) of the H-E and immunofluorescence stained
slides by an experienced and independent cell scientist in a blinded
condition. Additionally, the slides were monitored for DiI to
examine the migration of engrafted MSCs by confocal laser
mRNA expression analysis
The mRNA expression levels of macrophage inflammatory
protein-1 alpha (MIP-1a), monocyte chemotactic protein-1
(MCP1), tumor necrosis factor-alpha (TNF-a) and vascular endothelial
growth factor (VEGF) in the corneas were evaluated using
realtime polymerase chain reaction (real-time PCR). 7 days after the
alkaline burn, 5 corneas from each group were dissected after the
rats were sacrificed. Each cornea was cut in half; one half was
analyzed using real-time PCR, and the other half was analyzed
using an enzyme-linked immunosorbent assay (ELISA). Total
RNA was extracted from the harvested corneas with TRIzol
(Invitrogen, Carlsbad, CA). The concentration of the total RNA
was detected. Genomic DNA was removed by DNase I digestion
(Ambion, Austin, TX). Total RNA (1 mg) in a 20 ml reaction
volume was reverse transcribed into cDNA using the PrimeScript
RT reagent kit (Takara, Dalian, China). The synthesized cDNA
was aliquoted. Real-time PCR in 96-well optical plates was
performed and analyzed with an ABI PRISM 7000 sequence
detection system (Applied Biosystems Inc., Foster City, CA). The
relative gene expression levels were calculated according to Oh
. The reactions were performed in a 20 ml volume using a
SYBR Green reaction mix (Takara, Dalian, China) with 2 ml
cDNA. The sequences of the PCR primers are listed in Table 1.
The thermal cycling consisted of denaturation for 30 sec at 95uC
followed by 40 cycles of 5 sec at 95uC and 30 sec at 60uC. To
confirm constant housekeeping gene expression levels in the
extracted total RNA, real-time PCR for b-actin was also
performed. Real-time PCR was quantified by an SDS 7000
(Applied Biosystems) with rat b-actin as the endogenous control.
Enzyme-linked immunosorbent assay (ELISA)
VEGF protein levels in the corneas were determined using an
ELISA kit (R&D Systems, Minneapolis, MN). The corneas were
cut into small pieces, and the tissue was homogenized with a
mortar. The samples were lysed in 500 ml of extraction buffer and
centrifuged at 10,000 rpm for 10 minutes. The supernatants were
used for ELISA. Measurements were conducted according to the
instructions of the kit.
The chi-squared test was used to assess the corneal fluorescein
staining. The other data are expressed as the mean 6 standard
deviation. Comparisons of the parameters between the two groups
were obtained using Students t-test for independent samples with
SPSS software (SPSS 13.0. Chicago). A p value less than 0.05 was
considered statistically significant.
Characterization of MSCs
MSCs were isolated according to their ability to adhere to cell
culture plastic. In vitro, these adherent cells were spindle-shaped
and proliferated with a well-spread attached morphology. The
MSCs expressed high levels of CD29, CD44 and CD90 but did
not express the hematopoietic markers CD34, CD45 or CD11b
Gene Forward (59-39)
(Fig. 1A). After induction of osteogenic and adipogenic
differentiation, alizarin red S and oil red O staining were consistent with
osteogenic and adipogenic precursors, respectively (Fig. 1B, 1C).
Effects of MSCs on epithelial recovery
Corneal epithelial staining with fluorescein is indicative of
epithelial defects. The recovery of the corneal surface was
significantly faster in the MSC group than in the control group.
On day 3 after the alkali burn, the corneal epithelium of 7 out of 8
eyes had recovered in the MSC group, whereas all 8 eyes in the
control group displayed fluorescent staining consistent with a
damaged corneal epithelium (p = 0.001; Fig. 2A, 2C, 2E). On day
7, no damaged epithelium was observed in the corneas of the
MSC group. However, 6 corneas in the control group exhibited
fluorescein staining, consistent with a damaged epithelium
(p = 0.007; Fig. 2B, 2D, 2E).
Anti-inflammatory effects of MSCs in the cornea
H-E staining of the eyeball sections revealed that the primary
infiltrating cells in the corneal stroma after the corneal alkali burn
were inflammatory cells. The numbers of infiltrated cells and
CD68+ macrophages in the corneal stroma were significantly
lower in the MSC group than in the control group (p = 0.000 for
both; Fig. 3AE). In the control group, the corneal epithelium was
not repaired, and the corneal stroma was thicker and more swollen
than that in the MSC group (Fig. 3A, 3B).
To investigate the possible mechanism by which MSCs
attenuate inflammation, we assessed the production of the
chemotactic factors MIP-1a and MCP-1. We found that the
levels of MIP-1a were significantly lower in the MSC group than
in the control group (p = 0.017; Fig. 3F). However, the expression
of MCP-1 was not different between the two groups (p = 0.082;
Fig. 3G). In addition to MIP-1a, the expression of the
immunostimulatory cytokine TNF-a was lower in the MSC group
than in the control group (p = 0.001; Fig. 3H).
Effects of MSCs on CNV
On days 3 and 7 after the corneal alkali burn, CNV occurred in
both the control and MSC groups (Fig. 4AD). Compared to the
corneas of the control group, corneas in the MSC group showed
less CNV. The amount of CNV was quantified by measuring the
area of neovascularization. We found that the area of CNV was
significantly smaller in the MSC group than in the control group
(p = 0.001 on day 3, p = 0.016 on day 7; Fig. 4E), suggesting an
inhibitory effect of MSCs on CNV.
We further investigated the expression of VEGF to determine
the possible effect of MSCs on corneal angiogenesis. Both the
mRNA and protein levels of VEGF were significantly lower in the
MSC group compared to the control group (p = 0.002 and
p = 0.035, respectively; Fig. 4F, 4G), indicating a decrease in
corneal angiogenesis caused by MSCs.
Migration of MSCs
To investigate whether the DiI-labeled MSCs were successfully
engrafted after the corneal alkali burn, we traced the migration of
MSCs in the host by labeling them with DiI and detecting them
via confocal laser scanning microscopy. A large number of
DiIlabeled MSCs remained subconjunctival as they were originally
engrafted; no MSCs had infiltrated into the cornea (Fig. 5A, 5B).
Corneal chemical burn is a common type of ocular injury. It
often results in extensive damage and permanent visual
impairment in severe cases and in cases lacking proper management.
Therefore, prompt and appropriate treatment is necessary,
especially in the acute phase of a corneal chemical burn. Recently,
MSCs, a type of multipotent cell, have become a promising
approach for the treatment of corneal chemical burn .
Studies have shown that MSCs treatment enhances wound healing
and reconstitutes the corneal surface due to MSCs differentiation
[17,18]. However, others have demonstrated that the MSCs may
exert their beneficial effects by inhibiting inflammation and
angiogenesis rather than by differentiation [15,16]. Thus, further
studies are needed to elucidate the mechanisms utilized or
influenced by MSCs. In our study, consistent with previous
studies, we showed that subconjunctival injection of MSCs
accelerated corneal wound healing [15,16]. Importantly, we found
that a large number of DiI-labeled MSCs remained
subconjunctival as they had originally been engrafted, and none of them
infiltrated into the injured cornea (Figure 5A, B). Our results also
showed that subconjunctival injection of MSCs attenuated the
inflammation of the locally burned cornea by inhibiting
inflammatory cell infiltration and proinflammatory cytokine production.
These results further support previous studies [15,16], and they
suggest that MSCs treatment can accelerate the wound healing of
a chemically burned cornea via anti-inflammatory and
antiangiogenic activity. In addition, some studies have shown
beneficial effects of MSCs in the treatment of corneal chemical
burn in the early repair phase [16,18]. However, for corneal
chemical burn, treatment in the acute phase is crucial. Thus, this
study focused on the therapeutic effects of MSCs in the acute
phase of corneal chemical burn.
The studies of Ma et al.  and Oh et al.  reported that
MSCs treatment enhanced wound healing and accelerated corneal
surface reconstruction via its anti-inflammatory effects. However,
the anti-inflammatory mechanism of MSCs in corneal chemical
burns remains elusive. The study of Ma et al.  speculated that
the therapeutic anti-inflammatory effects of MSCs may be
associated with their inhibition of the expression of CD45, IL-2
and MMP2. The study of Oh et al.  further indicated that
MSCs exert their anti-inflammatory effects by suppressing the
infiltration of adaptive CD4+ T cells and the expression of CD4+ T
cell-related cytokines (IL-2, IFN-c) and MMP2 possibly by
secreting soluble factors. However, in corneal chemical burn,
when the cornea is injured, corneal epithelial cells and other local
cells immediately release a large quantity of mediators, including
inflammatory mediators and chemotactic factors, to recruit
inflammatory cells into the injured cornea. At 1224 hours after
the original injury, innate immune cells (such as neutrophils and
macrophages) arrive at the cornea and release inflammatory
mediators that contribute to the development of corneal disorders
. Therefore, inhibiting the early inflammatory reaction of
corneal cells and innate immune cells is critical for the early
treatment of corneal chemical burn. In our study, we first showed
that MSCs treatment suppresses the infiltration of inflammatory
cells and CD68+ macrophages, which are closely related to the
degree of inflammation . Next, we found that MSCs may
inhibit macrophage infiltration maybe by suppressing the
expression of the macrophage chemokine MIP-1a . In
addition, previous studies have demonstrated that MSCs are
capable of inhibiting proinflammatory cytokine (TNF-a)
production by macrophages [24,25]. Thus, we also examined whether
MSCs treatment is capable of inhibiting TNF-a production in a
locally burned cornea. Our results showed that subconjunctival
MSCs significantly decrease the production of TNF-a in locally
Figure 2. Effects of MSCs on epithelial recovery. (AD) Representative images of fluorescein staining of the cornea on day 3 and day 7 after the
alkali burn. Control group on day 3 (A) and day 7 (B); MSC group on day 3 (C) and day 7 (D). (E) Statistical analysis of number of the fluorescein
staining of the cornea on day 3 and day 7. On both day 3 and day 7, the quantity of fluorescein staining of the cornea was significantly lower in the
MSC group compared with the control group.
burned cornea. These findings, combined with the
aforementioned pathological progress of corneal chemical burn, indicate
that the anti-inflammatory mechanism of MSCs in the acute phase
may be that they first inhibit the release of MIP-1a by local
corneal cells and that subsequently, they decrease the infiltration of
macrophages and TNF-a production.
Currently, it is thought that MSCs provide therapeutic effects
through both cell-membrane contact and soluble factors .
In this study, we found that few MSCs could infiltrate into the
wounded cornea. However, on day 7 after the corneal alkali burn,
a large number of DiI-labeled MSCs had accumulated in the
subconjunctival tissue where the MSCs were injected. Therefore,
we speculate that soluble factors secreted by the MSCs rather than
cell-membrane contact played an important role in this model.
Limbal stem cell (LSC) transplantation has been widely used
clinically for ocular surface reconstruction in corneal chemical
burns. However, it is generally accepted that LSC transplantation
should be used in the early or late repair phase of a corneal
chemical burn rather than in the acute phase . The current
treatment options for corneal chemical burn are prompt
antiinflammatory therapy in the early phase and the provision of LSC
in the late stage after the inflammation has subsided [32,33].
Unlike LSC transplantation, in our study, MSCs treatment could
be applied repeatedly in the acute phase of a corneal chemical
burn. It is also expected that in future clinical settings, MSCs will
be used first to inhibit inflammation and angiogenesis, followed by
LSC transplantation to reconstruct the ocular surface.
In addition, LSC transplantation has its limitations in clinical
applications. First, for autotransplantation, it is not suitable for
binoculus sufferers because it risks damaging the comparatively
healthy eye. Second, for allotransplantation, the risk of significant
side effects from long-term immunosuppression is a major
drawback. Unlike LSC, MSCs are easy to isolate in sufficient
numbers for clinical autotransplantation or allotransplantation.
Moreover, our study and other studies have shown encouraging
results of MSCs allotransplantation in both the acute phase
(inhibiting inflammation and angiogenesis) and the early repair
phase (reconstructing ocular surface) of corneal chemical burn
. Taken together, these findings show the great superiority
of the MSCs application in the clinical setting.
To administer MSCs to the injured cornea of rats, previous
studies have used intravenous infusion, amniotic membrane as a
carrier or a special hollow plastic tube . However, for
clinical applications, these methods are difficult to perform and are
not accepted by ophthalmologists for treating corneal disorders.
Unlike these methods, subconjunctival injection is a common and
easy clinical administration route for the treatment of
ophthalmological disorders. Thus, in this study, we used subconjunctival
injection as a route to administer MSCs. Consistent with
previous studies [15,16], our study showed similartherapeutic effects on
Figure 3. Anti-inflammatory effects of MSCs in the cornea. (A B) H-E staining on day 7 (Magnification, 6200); (C D) Immunofluorescence
staining of CD68 on day 7 (Magnification, 6400); (A C) Control group; (B D) MSC group. (E) Statistical analysis of infiltrated cells and infiltrated CD68+
cells in the corneal stroma on day 7 after the corneal alkali burn. (FH) Statistical analysis of real-time PCR results of MIP-1a (F), MCP-1 (G) and TNF-a
(H) on day 7 (* p,0.05; ** p,0.01; *** p,0.001).
Figure 4. Effects of MSCs on CNV. (AD) Representative images of CNV on day 3 and day 7 after the alkali burn. Control group on day 3 (A) and
day 7 (B); MSC group on day 3 (C) and day 7 (D). (E) Area of CNV on day 3 and day 7 in both groups. (F G) Statistical analysis of real-time PCR and
ELISA results of VEGF on day 7 (* p,0.05; ** p,0.01).
corneal chemical burn by subconjunctival injection of MSCs. This
finding suggests that subconjuctival injection is a good alternative
for the clinical application of MSCs to corneal chemical burn.
In summary, our study demonstrated that topical
subconjunctival injection of MSCs accelerates corneal epithelial recovery and
inhibits neovascularization in a corneal alkali burn rat model.
Mechanistically, our results found that MSCs may exert their
therapeutic effects by suppressing the early inflammatory reaction
of local corneal cells and innate immune cells via paracrine
mechanisms. These findings further confirm and elucidate the
anti-inflammatory mechanism of MSCs in the acute phase of a
corneal chemical burn, and they suggest that the subconjunctival
administration of MSCs could be a good alternative treatment for
the clinical management of corneal chemical burn.
Conceived and designed the experiments: DL LY ZRL YPL. Performed
the experiments: LY ZRL WXZ MLL YL QW. Analyzed the data: LY
ZRL WRS. Contributed reagents/materials/analysis tools: DL YPL.
Wrote the paper: LY. Paper revision: DL WRS ZRL YPL.
1. Wagoner MD ( 1997 ) Chemical injuries of the eye: current concepts in pathophysiology and therapy . Surv Ophthalmol 41 : 275 - 313 .
2. Brodovsky SC , McCarty CA , Snibson G , Loughnan M , Sullivan L , et al. ( 2000 ) Management of alkali burns: an 11-year retrospective review . Ophthalmology 107 : 1829 - 1835 .
3. Kuo IC ( 2004 ) Corneal wound healing . Curr Opin Ophthalmol 15 : 311 - 315 .
4. Adamis AP , Aiello LP , D' Amato RA ( 1999 ) Angiogenesis and ophthalmic disease . Angiogenesis 3 : 9 - 14 .
5. He J , Bazan NG , Bazan HE ( 2006 ) Alkali-induced corneal stromal melting prevention by a novel platelet-activating factor receptor antagonist . Arch Ophthalmol 124 : 70 - 78 .
6. Zannettino AC , Paton S , Arthur A , Khor F , Itescu S , et al. ( 2008 ) Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo . J Cell Physiol 214 : 413 - 421 .
7. Hoogduijin MJ , Crop MJ , Peeters AM , Van Osch GJ , Balk AH , et al. ( 2007 ) Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities . Stem Cells Dev 16 : 597 - 604 .
8. Oh W , Kim DS , Yang YS , Lee JK ( 2008 ) Immunological properties of umbilical cord blood-derived mesenchymal stromal cells . Cell Immunol 25 : 116 - 123 .
9. Zhang Q , Shi S , Liu Y , Uyanne J , Shi Y , et al. ( 2009 ) Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis . J Immunol 183 : 7787 - 7798 .
10. Zhang QZ , Su WR , Shi SH , Wilder-Smith P , Xiang AP , et al. ( 2010 ) Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing . Stem Cells 28 : 1856 - 1868 .
11. Su WR , Zhang QZ , Shi SH , Nguyen AL , Le AD ( 2011 ) Human gingiva-derived mesenchymal stromal cells attenuate contact hypersensitivity via prostaglandin E(2) -dependent mechanisms . Stem Cells 29 : 1849 - 1860 .
12. Nauta AJ , Fibbe WE ( 2007 ) Immunomodulatory properties of mesenchymal stromal cells . Blood 110 : 3499 - 3506 .
13. Zhao S , Wehner R , Bornhauser M , Wassmuth R , Bachmann M , et al. ( 2010 ) Immunomodulatory properties of mesenchymal stromal cells and their therapeutic consequences for immune-mediated disorders . Stem Cells Dev 19 : 607 - 614 .
14. Uccelli A , Moretta L , Pistoia V ( 2008 ) Mesenchymal stem cells in health and disease . Nat Rev Immunol 8 : 726 - 736 .
15. Oh JY , Kim MK , Shin MS , Lee HJ , Ko JH , et al. ( 2008 ) The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury . Stem Cell 26 : 1047 - 1055 .
16. Ma Y , Xu Y , Xiao Z , Yang W , Zhang C , et al. ( 2005 ) Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells . Stem Cells 24 : 315 - 321 .
17. Ye J , Yao K , Kim JC ( 2006 ) Mesenchymal stem cell transplantation in a rabbit corneal alkali burn model: engraftment and involvement in wound healing . Eye (Lond) 20 : 482 - 490 .
18. Jiang TS , Cai L , Ji WY , Hui YN , Wang YS , et al. ( 2010 ) Reconstruction of the corneal epithelium with induced marrow mesenchymal stem cells in rats . Mol Vis 16 : 1304 - 1316 .
19. Popp FC , Slowik P , Eggenhofer E , Renner P , Lang SA , et al. ( 2007 ) No contribution of multipotent mesenchymal stromal cells to liver regeneration in a rat model of prolonged hepatic injury . Stem Cells 25 : 639 - 645 .
20. Su W , Li Z , Lin M , Li Y , He Z , et al. ( 2011 ) The effect of doxycycline temperature-sensitive hydrogel on inhibiting the corneal neovascularization induced by BFGF in rats . Graefes Arch Clin Exp Ophthalmol 249 : 421 - 427 .
21. Wilson SE , Mohan RR , Mohan RR , Ambrosio R , Jr., Hong J ( 2001 ) The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells . Prog Retin Eye Res 20 : 625 - 637 .
22. Choi JA , Choi JS , Joo CK ( 2011 ) Effects of amniotic membrane suspension in the rat alkali burn model . Mol Vis 17 : 404 - 412 .
23. DiPietro LA , Burdick M , Low QE , Kunkel SL , Strieter RM ( 1998 ) MIP-1alpha as a critical macrophage chemoattractant in murine wound repair . J Clin Invest 15 : 1693 - 1698 .
24. Maggini J , Mirkin G , Bognanni I , Holmberg J , Piazzon IM , et al. ( 2010 ) Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile . PLoS One 5 : e9252 .
25. Aggarwal S , Pittenger MF ( 2005 ) Human mesenchymal stem cells modulate allogeneic immune cell responses . Blood 105 : 1815 - 1822 .
26. Lee JW , Fang X , Krasnodembskaya A , Howard JP , Matthay MA ( 2011 ) Mesenchymal stem cells for acute lung injury: role of paracrine soluble factors . Stem Cells 29 : 913 - 919 .
27. Krampera M , Glennie S , Dyson J , Scott D , Laylor R , et al. ( 2003 ) Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigenspecific T cells to their cognate peptide . Blood 101 : 3722 - 3729 .
28. Groh ME , Maitra B , Szekely E , Koc ON ( 2005 ) Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells . Exp Hematol 33 : 928 - 934 .
29. Shi W , Wang T , Gao H , Xie L ( 2009 ) Management of severe ocular burns with symblepharon . Graefes Arch Clin Exp Ophthalmol 247 : 101 - 106 .
30. Fogla R , Padmanabhan P ( 2005 ) Deep anterior lamellar keratoplasty combined with autologous limbal stem cell transplantation in unilateral severe chemical injury . Cornea 24 : 421 - 425 .
31. Borderie V , Touzeau O , Bourcier T , Allouch C , Scheer S , et al. ( 2003 ) Treatment of the sequelae of ocular burns using limbal transplantation . J Fr Ophtalmol 26 : 710 - 716 .
32. Kuckelkorn R , Schrage N , Keller G , Redbrake C ( 2002 ) Emergency treatment of chemical and thermal eye burns . Acta Ophthalmol Scand 80 : 4 - 10 .
33. Fish R , Davidson RS ( 2010 ) Management of ocular thermal and chemical injuries, including amniotic membrane therapy . Curr Opin Ophthalmol 21 : 317 - 321 .