Keywords reactive oxygen species - antioxidant - superoxide dismutase - wound healing
The use of flap is a common procedure in plastic surgery.[1 ] Flap is used to repair skin defects[2 ] but in this surgery, restoring blood flow to ischemic tissue leads to tissue damage.
This damage is ischemia–reperfusion (IR) injury.[3 ] Following this damage, tissue necrosis and cell death occur. One of the mechanisms
of this damage is oxidative stress, which damages cells and tissues. As blood returns
to the tissue, neutrophils accumulate in the tissue. Neutrophils produce reactive
oxygen species (ROS). This activated oxygen has detrimental effects on the mitochondrial
membrane. As a result of a series of interactions, the mitochondrial membrane ruptures
and eventually leads to cell necrosis. In addition, preapoptotic agents are released
into the cytosol after the mitochondrial membrane ruptures, leading to cell apoptosis.[4 ]
[5 ]
[6 ] Many environmental stimuli catalyze the production of ROS, which may be involved
in the pathogenesis of several skin disorders including flap necrosis.[7 ] ROS induces several deleterious effects, such as deoxyribonucleic acid damage, inflammatory
responses, and damage to the integrity of the extracellular matrix (ECM). Therefore,
ROS production plays an important role in skin damage.[8 ] Numerous studies have been performed to evaluate the effect of ROS using different
therapeutic agents on IR phenomena, inflammatory reactions, skin diseases, and survival
of skin flaps.[9 ] These studies have shown that by reducing the rate of oxidative stress, both the
rate of tissue necrosis and direct cell damage are reduced. Also, the rate of apoptosis
(a type of cellular death) and apoptosis-promoting mediators is reduced.
At inflammation conditions, ROS enhances the secretion of cytokines[10 ] and induction of matrix metalloproteinases (MMPs). MMPs are enzymes that destroy
the ECM and the connections between endothelial cells.[11 ] Also, ROS can directly or indirectly (activated by ECM proteolysis) alter or destroy
proteins as well as disrupt skin fibroblasts and keratinocyte function.[12 ] Oxidative stress and the production of ROS in tissues lead to tissue damage. The
skin is no exception and if a way can be found to reduce the oxidative damage following
(tissue loss of) the flap surgery, it can increase the survival rate of the tissue.
Using strategies such as preventing tissue necrosis can cause to reduce the production
or elimination of ROSs as a cause of tissue damage and death. Managing ROS production
by inflammatory mediators and increasing the antioxidant capacity of the tissue, and
even the use of external antioxidants, can increase the survival of skin flaps.[13 ]
One of the novel treatments for reducing the level of necrosis and improving the survival
of flap is an application of stem cells.[14 ] Bone marrow mesenchymal stem cells (BMMSCs) have a very high capacity and their
transplantation in the wound matrix promotes angiogenesis and progress in the wound-healing
process.[15 ]
Different types of cells are involved in wound healing. One type of these cells is
the mast cell (MC).
MCs are specialized secretory cells of the immune system that originate from cluster
of differentiation (CD)34 +/CD117+ bone marrow cell, and are present in large numbers
in the skin.[16 ] MCs are responsible for producing different intermediates. These intermediates are
stored as premade in intracellular granules. The contents of these granules affect
the repair stages of skin wounds as inflammatory intermediates.[17 ] So, it is likely that if we can reduce necrosis and ROS by using MCs and BMMSCs,
the survival of the skin flap will probably increase. On the other hand, studies have
shown that due to oxidative stress conditions and reduced stem cell life, the focus
on MC characteristics is increased. These cells are able to act under stress conditions
as a regulator of the environment; mutations have been observed in some MC genes that
damage due to oxidative stress and cannot be controlled and regulated. Therefore,
the presence of two skin-tissue-supporting cells in the healing process promotes the
wound-healing process, both of which are sensitive and vulnerable to oxidative stress.
MCs release polypeptide growth factors including basic fibroblast growth factor (FGF-2),
vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), transforming
growth factor beta (TGF-β), and interleukin (IL)-8. These cytokines are involved in
normal as well as tumor-associated angiogenesis.[18 ]
In this paper we assess the cellular and molecular role of MCs in the three major
stages of wound healing (inflammation, proliferation, and regeneration). Also, we
assess therapeutic strategies for improving wound healing by using the effects of
MCs and BMMSCs on oxidative stress and necrosis of flap tissue. The present study
showed the protective effects of stem cells alongside MCs on oxidative damages and
histopathological changes induced by flap surgery in rat's skin tissue.
Methods
Preparation of Animals
This study was performed according to the “guidelines for taking care of and using
laboratory animals” prepared by faculty of medicine at Lorestan University of Medical
Sciences (LUMS.REC.1396.254).
In this research, 54 albino Wistar male rats weighing 250 to 300 g were prepared from
Elmbavaran Aftab Co., Lorestan, and tested. All animals had free access to vital facilities
as well as food and water throughout the entire experiment. They were then categorized
into three groups: (1) nonsurgery, (2) surgery, and (3) surgery + BMMSCs. Groups 2
and 3 were operated with a 3 × 8 cm flap and in group 3, cell injections (7 × 109 cell injection at the time of surgery) were performed. Animals in group 3 receiving
cell: wound-healing access by flap surgical induction +cells (WH+ C), rats with wound-healing access (WH) through flap surgical induction, and rats
with intact skin (IS) without surgery or damage to the skin as the control group.
On the surgery day (day zero), the animals in all experiments were anesthetized using
ketamine 50 mg/kg and xylazine 5 mg/kg. Then the site of surgery was shaved using
a Moser and Gillette before creating the flap. Next, in the WH+ C and WH groups, and using scalpel, random skin flap with dimensions of 3 × 8 cm was
created on the back of animals, whose base was proximal to the animals' tail. In creating
the wound, all skin layers had been elevated with panniculus carnosus muscle, such
that there would be no attachment, even vascular attachment, except in the base region.
Next, the tissue was elevated and returned to its original position and sutured ([Fig. 1 ]). Before flap tissue placement in the WH+ C group, 7 × 109 cells with a volume of 0.5 mL were injected at several points in the wound. After
the surgery, the animals were monitored and controlled clinically and for any evidence
of inflammation or bleeding. Then, the animals in all groups were sacrificed on days
4, 7, and 15 through inhaling chloroform during deep anesthesia, whereby biopsy was
taken from the region of the flap between necrotic (limited by the yellow line) and
survival on the red arrow for histology study (six samples at every time point).[19 ]
Fig. 1 Representative gross view of surviving area and statistical analysis. (A) The yellow lines show limiting of the necrotic area in the three groups on surgery
day (day zero) and postsurgery days (days 4, 7, and 15). (B) Image indicates that the survival percentage of WH group and WH+ C group was higher than that of the control group 4, 7, and 15 days postoperatively;
*p < 0.05. WH, rats with wound-healing access through flap surgical induction; WH+ C, rats with wound-healing access by flap surgical induction + cells.
Clinical Investigation
On days 4, 7, and 15 postoperation, some images were taken using digital camera at
the site of flap. The survival of the improved region, which was free of inflammation
and necrosis, was investigated and further controlled and evaluated against the characteristics
of the control group flap. At every time point of imaging, Image software (St. Peterborough,
NH) was used for measuring the percentage of survived regions in relation to the entire
surface of flaps.[17 ]
Evaluation by H&E Staining
For histopathological evaluation on days 4, 7, and 15, a 1.0 × 1.0 cm sample of tissue
was obtained from intermediate zone of each flap. The specimen was immersed in 4%
paraformaldehyde, and after ∼24 hours the tissue was completely wrapped in paraffin
and by microtome cut into 5-μm-thick sections for detection of histological indicators.
The paraffin sections were stained with hematoxylin and eosin (H&E). The histological
characteristics of the flaps were examined under an optical microscope. Skin flap
capillary angiogenesis was assessed. The number of new blood vessels was counted in
six randomly selected high-power fields (200 × ). Then, we counted the number of microvessels
per unit area (/mm2 ), which was taken as the microvessel density.[20 ]
Evaluation of Platelet Endothelial Cell Adhesion Molecule (Cluster of Differentiation
31, CD31) by Immunohistochemistry Technique
We used the same H&E dyeing method to make the slices, baked them in the oven at a
constant temperature of 60°C, and then removed the slices from the oven and placed
them in xylene and then in ethanol with a gradient concentration to hydration, digestion,
and repair of antigen. After cooling naturally at room temperature, according to the
instructions of the immunohistochemistry kit, we added antibodies to the slices and
then observed the slices under a microscope.[20 ]
Determining the Number and Extent of Activity of MCs
To assess the number of MCs and the extent of activity of these cells (degranulation),
a semi-quantitative analysis was performed. When counting, the total number of MCs
that were observable in different forms was determined on the skin cut. Next, the
ratio of granulated MCs and degranulated MCs was counted in each of the different
views through 200 µm magnification. (All images have been recorded using Leica microscope
in the major magnification of 100, 200, or 400 µm.)[20 ]
Tracking Cells In Vivo
For short-term tracking of the cells transplanted inside the body, BMMSCs (IBRC-C10163),
purchased from Genetics Reserve Center of Iran), were used. For the tracking, after
transplantation in the tissue, the cells were labeled through CM-Dil (Invitrogen,
Carlsbad, CA) based on the manufacturer's instructions. Specifically, BMMSCs were
incubated for 2 hours and injected in the flap on day zero of surgery. They were then
observed under fluorescent microscope after sampling on predetermined days.[20 ]
Biochemical Analysis
The frozen samples from flap tissue at −80°C were homogenized in phosphate-buffered
saline and centrifuged at 5°C. The supernatant was used for the analysis of malondialdehyde
(MDA) and superoxide dismutase (SOD). The amount of flap tissue SOD enzyme was determined
according to the method of Kakkar et al.[21 ] To evaluate lipid peroxidation, a commercial kit for MDA was used (Asan, Khorramabad,
Iran).[22 ]
Statistical Analysis
For statistical analysis of the data, Statistical Package for the Social Sciences , version 21, was used. Based on analysis of variance (ANOVA), the difference between
the means of the study groups was evaluated. Also, post hoc test (Tukey) indicated
the place of difference between the study groups, with p < 0.05 considered statistically significant.
Results
The Therapeutic Effects of Stem Cells for the Skin Flaps
The survival of skin flap was investigated in the rats undergoing treatment with BMMSCs
or without treatment on predetermined days. The images of the skin flaps at different
time points are shown in [Fig. 1A ]. In WH group, in the initial stages postsurgery, the skin had unsuitable status,
and the level of skin survival calculated as the percentage of the survived region
in relation to the entire surface of the skin flap showed low level of survival. This
could suggest severe ischemia that had resulted in necrosis of the skin flap tissue
and was associated with a poor clinical outcome. In the WH+ C group, with BMMSCs, a better therapeutic outcome was observed for the skin flap
tissue survival. Eventually, in all of the three phases of wound survival—inflammation
(day 4), proliferation (day 7), and regeneration (day 15)—a significant difference
was observed in the percentage of flap survival between WH+ C and WH groups, where the p -values were as follows: p = 0.04, p = 0.02, and p = 0.04, respectively ([Fig. 1B ]). Flap survival rates were as follows—day 4: 56.312 ± 1.98 and 68.215 ± 2.07; day
7: 76.812 ± 2.528 and 83.567 ± 1.3; and day 15: 78 ± 1.405 and 88.731 ± 1.35 in WH
and WH+ C groups, orderly.
Evaluation of H&E Staining Results
According to the results, the number of vessels in the inflammation phase on day 4
differed significantly between the study groups (p = 0.004), as found in the Tukey post hoc test ([Fig. 2 ]). Also, the difference in the number of vessels in the proliferation phase on day
7 was significant between the WH+ C and WH groups (p = 0.03). Further, for the number of vessels in the regeneration phase, day 15, the
difference between WH+ C and WH groups was significant (p = 0.04). With these results, it can be stated that regeneration of vessels in both
WH and WH+ C groups was observed in the middle of the flap on day 7. The number of vessels in
the wound on day 7 reached its maximum in WH+ C rats, and then diminished considerably on day 15. On the other hand, the number
of vessels in WH rats increased constantly. This increase for WH rats was sustained
until the end of experiment on day 15. In both groups, angiogenesis occurred in the
proliferation stage, but this value was lower in WH rats on day 7 ([Fig. 2 ]).
Fig. 2 H&E staining of the flap tissue. Flap tissue fixed for H&E staining to detect subcutaneous
capillaries in the three groups. The results showed that the capillary density in
WH group (B) and WH+ C group (C) was significantly higher than that in the control group (A); *p < 0.05. Each arrowhead indicates the capillaries in the subcutaneous tissue. (D) Analysis of the number of vascular sections. Immunohistochemical staining of the
flap tissue. Flap tissue fixed for CD31 staining to detect subcutaneous capillaries
in the three groups. The results showed that the capillary density in WH group (F) and WH+ C group (G) was significantly higher than that in the control group (E); *p < 0.05. Each arrowhead indicates the capillaries in the subcutaneous tissue. (H) Analysis of the number of vascular sections; p < 0.05*, **p > 0.001; p < 0.05*, **p > 0.001. H&E, hematoxylin and eosin; WH, rats with wound-healing access through flap
surgical induction; WH+ C, rats with wound-healing access by flap surgical induction + cells.
Evaluation of Immunohistochemical Results
On day 4 of the observation, we randomly selected six slices of the flap samples in
each rat group for immunohistochemical staining, and measured the content of CD31
in the peripheral areas of the blood vessels, calculated the integral optical density
of each slice, and then finally obtained the average values. The results showed that
the average integral optical density of WH+ C group was higher than that of the other two groups (WH+ C group compared with the IS and WH groups), that is, p < 0.01, with statistical significance; and when WH+ C was compared with WH group, p < 0.001, with higher statistical significance ([Fig. 2 ]).
The Number and Extent of Activity of MCs
Comparing the total number of MCs on day 4, according to the results of ANOVA, a significant
difference was observed between the study groups on this day (p = 0.004). Comparison of the total number of MCs between the intact tissue (IS group)
and WH (p = 0.004), and between the WH+ C and WH (p = 0.003) groups showed a significant difference.
Comparing the extent of activity as degranulation of MCs on day 4, based on the results
of the ANOVA, a significant difference was observed in the activity number of MCs
between the study groups on this day (p = 0.001). Comparison of the extent of activity of MCs in the healthy tissue of IS
and WH (p = 0.001) and between the WH+ C and WH (p = 0.002) groups showed a significant difference.
In the flap wound in WH+ C rats, the total number of MCs decreased significantly on day 4, and began to rise
on day 7 deep in the dermis. On day 15, reduction in this group was observed. In the
flap wound in WH rats, the total number of MCs increased on day 4, but it diminished
on day 7. Finally, on day 15, it was close to the level of inflammation phase, day
4. A relatively low number of MCs was observed in the healthy tissues of IS rats ([Fig. 3D ]).
Fig. 3
(A) Presence of mast cells (MCs) and activity (degranulation) of these cells alongside
blood vessels. (B) Presence of MCs in the dermis from left to right in intact skin, skin with flap wound,
and skin with flap wound plus stem cells according to Toluidine blue staining. Toluidine
blue staining showed the flap wound on day 15 postsurgery (scale bar = 100 µm). (C) Statistical analysis of the number of MCs in the flap wound in all of the three study
groups. (D) Statistical analysis of determining the number of MCs activated in the middle of
the flap wound. ***p < 0.001, **p < 0.01, *p < 0.05.
The activity of MCs evaluated as degranulation of these cells increased throughout
the entire flap wound of WH+ C rats on days 7 and 15. However, on day 15, the regeneration and vascular deterioration
increased. Meanwhile, in WH rats, the extent of activity on day 7 remained very low
throughout the experiment, and this value was lower than days 4 and 15, representing
the inflammation and regeneration phases for this group ([Fig. 3A, B ]). Also, in comparing the extent of activity of WH and WH+ C groups, on days 7 and 15, a significant difference was observed, where the p -value on days 7 and 15 was reported as 0.02 and 0.01, respectively ([Fig. 3C ]).
Tracking the Cell Inside the Body
On day 7 post-BMMSC transplantation, we were able to track the transplanted cells
in the skin flap animal model. Fluorescent microscopy investigation of BMMSCs showed
CM-Dil label alongside blood vessels of the flap tissue ([Fig. 4 ]).
Fig. 4 Presence of stem cells labeled with CM-DiI, alongside the blood vessels on day 7
following flap wound surgery (scale bar = 50 µm).
Biochemical Findings
The MDA values were significantly reduced in the WH+ C group in comparison with WH and IS groups on day 7 (p = 0.0043). On day 15, the MDA levels in WH+ C group significantly decreased compared with WH and IS groups (p = 0.002). The SOD levels were significantly higher in the WH+ C animals compared with WH and IS groups on day 7 (p = 0.002). The SOD levels in WH+ C group significantly decreased compared with WH and IS groups on day 15 (p = 0.0045; [Table 1 ]).
Table 1
Comparison of the activities of MDA and SOD (mean ± SEM) in the tissue samples taken
from three groups (six samples from each group) on days 7 and 15
Variable name
MDA (nmol/mg protein): Day 7
SOD (U/mg protein): Day 7
MDA (nmol/mg protein): Day 15
SOD (U/mg protein): Day 15
p -Value
IS
0.086 ± 0.020
0.39 ± 0.06
0.09 3 ± 0.15
0.73 ± 0.04
0.0043
WH
0.079 ± 0.026
0.93 ± 0.08
0.084 ± 0.022
0.065 ± 0.029
0.002
WH+ C
0.073 ± 0.026
6.94 ± 0.20
0.76 ± 0.05
2.34 ± 0.23
0.0045
Abbreviations: IS; rats with intact skin; MDA, malondialdehyde; SEM, standard error
of mean; SOD, superoxide dismutase; WH, rats with wound-healing access through flap
surgical induction; WH+ C, rats with wound-healing access by flap surgical induction + cells.
Discussion
In biological systems, the normal processes of oxidation produce highly reactive free
radicals, which may continue to damage cells. Antioxidants play a housekeeping role,
scavenging free radicals before they get a chance to do harm to the body. Increased
ROS and oxidative stress occur frequently in malignancies and myeloproliferative diseases.
PARK7, a multifunctional protein with both antioxidant function and cytoprotective
activity against oxidative stress, increases with cancer progression, although the
mode of PARK7 regulation has not been studied in detail. Here, we investigated the
regulation of type of MCs in relation with ROS and wound healing. PARK7 levels were
reduced in systematic mastocytosis with low MC burden but, in marked contrast, increased
in patients with high MC burden or with advanced disease.[18 ]
In addition, subtypes of SOD are expressed and secreted from stem cells.[23 ] BMMSCs have a very high capacity and their transplantation in the wound matrix improves
wound healing.[15 ] Recent evidence has supported the protective role of BMMSCs against skin oxidative
damage, most of which is mediated by secretory factors.[24 ] The antioxidant function of mesenchymal stem cells was first demonstrated in dermal
fibroblasts after inducing chemical oxidative stress by tert -butyl hydroperoxide.[25 ] Flaps are commonly used in plastic surgery and for repair of skin defects,[1 ]
[2 ] but in this surgery, restoring blood flow to ischemic tissue leads to tissue damage
(IR injury) and necrosis.[3 ] One of the mechanisms of this damage is oxidative stress.[4 ]
[5 ]
[6 ] Therefore, stem cells can be used to increase survival in a skin flap. This study
shows an increase in flap survival with: (1) the presence of MCs with stem cells,
(2) improvement in angiogenesis, and (3) reduction in oxidative stress.
MCs secrete substances that can inhibit the growth of keratinocytes and re-epithelialization
in vitro and play a role in the homeostatic regulation of the epidermis.[26 ] Therefore, we expect that with less total number of MCs in the WH+ C group, the survival of the flap will be higher than in the WH group. Therefore,
we counted the number of MCs in the flap using toluidine blue staining. This study
showed that wound healing and re-epithelialization in the middle of the wound in the
flap were significantly different between WH and WH+ C groups. Specifically, the number of MCs increased significantly in the flap wound
in WH+ C rats in the proliferation stage. This suggested that MCs are involved in improving
wounds at the proliferation stage. Also, the number of MCs in the middle of the flap
wound diminished in WH+ C rats on day 4, and it was lower than the control groups. Although the mechanism
is unknown, application of MC precursors to wounded regions may be activated in the
late stage of wound healing after skin surgery.[27 ] The notable point is that the number of MCs in the damaged skin decreases in the
primary stage, and falls after improvement in the late stage of wound healing.[28 ] MCs are known to participate in three phases of wound healing: the inflammatory
reaction, angiogenesis, and ECM reabsorption. The inflammatory reaction is mediated
by released histamine and arachidonic acid metabolites. Growing evidence on wound
healing indicates that wound repair is associated with an increased number of MCs
purposefully located around blood vessels. Activated MCs control the key events of
the healing phases: triggering and modulation of the inflammatory stage, proliferation
of connective cellular elements, and final remodeling of the newly formed connective
tissue matrix. MC hyperplasia is found in different pathologies such as chronic inflammatory
processes, fibrotic disorders, wound healing, or neoplastic tissue transformation.
BMMSC paracrine factors together with the number and periodic activity of MCs seem
to be effective in the three main stages of skin wound healing. Specifically, angiogenesis,
in addition to the effect of the growth factor released such as TGF-β1 from granules
of MCs in a site of the wound, alongside exogenous paracrine factors such as BMMSCs'
transplantation can better represent the granulated tissue. These conditions are important
in the proliferation stage and vascular deterioration in the regeneration phase.[29 ]
[30 ]
[31 ] In our study, the number of MCs was lower in the inflammation phase in the group
receiving cell, while in the proliferation phase, this number increased for stimulating
angiogenesis alongside BMMSCs. However, the number and extent of MCs in the regeneration
phase (day 15) of the wound-healing process decreased. Also, along with the deterioration
of blood vessels, overcomes angiogenesis and causes transition of the tissue from
granulated tissue to a tissue without vessels and rich in collagen. New blood vessels
are required at the site of wound to increase blood perfusion and promote tissue healing.
The course of re-epithelialization and survival of flap area were different in our
study between WH and WH+ C rats. Since MCs increase considerably in the inflammation and regeneration stages
after damage, studying the role of MCs in this model of skin wounds has many advantages.
Also, the activity of MCs in the damaged skin increased constantly in the WH+ C rats.
Since MCs aggregate in the vicinity of blood vessels and produce several strong angiogenesis
stimulating factors such as FGF2, VEGF, and TGF-β1, hence MCs are involved in angiogenesis.
Angiogenesis is an important factor in the survival of the skin flap, so we counted
the number of vessels using H&E staining. The present study indicated angiogenic response
to the wound damage in the proliferation stage of wound healing was weaker in WH compared
with WH+ C rats. The activity of releasing their granulation contents peaked on day 15 postsurgery
in the wound tissues of WH+ C rats. This finding suggested that factors such as TGF-β1, which is active in the
skin wound of WH+ C rats, may reach an adequately high concentration and suffice to inhibit angiogenesis
during late regeneration. In addition, MCs are a powerful modifier for ECM; on the
other hand, the compounds and environmental conditions of ECM are important factors
for regulating angiogenesis. Overall, these findings suggested that MCs can play a
key role in regulating the matrix reorganization and affect the delicate balance between
proangiogenic and antiangiogenic factors in the wound tissue.
Antioxidants are one of the factors in advancing the wound-healing process that play
a protective role against oxidative damage. The wound-healing process can be affected
by increased oxidative damage. Due to the activity of antioxidants at the site of
injury, it creates the right conditions for the wound to continue to heal. The return
of blood and oxygen to ischemic tissue leads to IR damage. The production of oxygen-free
radicals plays a role in causing this damage. During this oxidative stress, oxygen
destroys lipids in cell membranes, resulting in cell death. The level of oxidative
stress is determined by MDA (the product of lipid peroxidation). The body produces
antioxidants such as SOD to repair damage when responding to oxidative stress.[13 ] So, we measured the levels of MDA and SOD. In a recent study, the enzyme level of
SOD in the cell-receiving groups was higher than the other groups tested, which confirms
the antioxidant effect of BMMSCs. These findings indicate that the level of SOD is
increased in the cell-receiving groups, which is due to the stimulation of SOD production
due to the presence and antioxidant properties of BMMSCs. Moreover, another protein
factor, MDA, which is an important cause of tissue stress, was found to be lower in
cell-receiving animals. In several studies, different cells have been used to treat
wounds. In these studies, MDA and TNF-α were significantly reduced, indicating an
increase in SOD and CT activity in response to oxidative stress.[22 ] Although the present study showed a promising effect of BMMSCs with MCs, through
total antioxidant capacity modification, on wound healing in rats, it is necessary
to examine the data on the molecular mechanism finding from this result. The function
of MCs is not provided, which can be considered as a limitation of this research.
