Received: 16/09/2024 Accepted: 02/01/2024 Published: 03/03/2025 1 of 7
https://doi.org/10.52973/rcfcv-e35517 RevistaCientíca,FCV-LUZ/Vol.XXXV
ABSTRACT
The aim of this study is to evaluate the treatment efcacy of
Platelet–Rich Plasma (PRP), silver sulfadiazine, and Wharton Jelly
Mesenchymal Stem Cell–Derived Conditioned Medium (WJ–MSC–
CM) on burn wounds using a rat model. The study included four
groups, each with 16 rats, and the groups were further divided
into two subgroups (n=8) for the 7
th
and 14
th
days of the treatment
process. Group 1 received no treatment after the burn. Group 2
received PRP (Platelet–Rich Plasma) treatment on the rst day
after the burn. Group 3 was treated with silver sulfadiazine on
the rst day after the burn. Group 4 received WJ–MSC–CM on
the rst day after the burn. In the current study, the expression
of Caspase–3, Bcl–2, TNF–α, p21, and Beclin–1 genes among the
groups was evaluated by Real–time PCR. The silver sulfadiazine and
WJ–MSC–CM treatment groups exhibited lower Bcl–2 expression
and higher Caspase–3 and Beclin–1 expression compared to the
other groups. TNF–α and p21 expression was high in the burn
control group and showed lower expression in the treated groups.
The current ndings demonstrate that WJ–MSC–CM exhibits
healing efcacy on burn wounds comparable to the reference
drug (silver sulfadiazine) by inducing apoptosis and autophagy
and reducing necroptosis and DNA damage. Additionally, PRP
provided some positive benets compared to the control group
but was less effective than the other treatments.
Key words: Apoptosis; autophagy; burn wounds; Wharton
jelly mesenchymal stem cell–derived conditioned
medium
RESUMEN
El objetivo de este estudio es evaluar la ecacia del tratamiento
con Plasma Rico en Plaquetas (PRP), sulfadiazina de plata y Medio
Condicionado Derivado de Células Madre Mesenquimales de
Gelatina de Wharton (WJ–MSC–CM) en heridas por quemaduras
utilizando un modelo animal. El estudio presentado consistió
en 4 grupos, cada uno con 16 ratas, y los grupos se dividieron
además en dos subgrupos (n=8) para los días 7 y 14 del proceso
de tratamiento. El Grupo 1 no recibió ningún tratamiento después
de la quemadura. El Grupo 2 recibió tratamiento con PRP (Plasma
Rico en Plaquetas) el primer día después de la quemadura. El
Grupo 3 fue tratado con sulfadiazina de plata el primer día después
de la quemadura. El Grupo 4 recibió WJ–MSC–CM el primer día
después de la quemadura. En el estudio actual, la expresión de
los genes Caspasa–3, Bcl–2, TNF–α, p21 y Beclin–1entre los
grupos se evaluó mediante PCR en tiempo real. Los grupos de
tratamiento con sulfadiazina de plata y WJ–MSC–CM mostraron
una menor expresión de Bcl–2 y una mayor expresión de Caspasa-3
y Beclin–1en comparación con los otros grupos. La expresión
de TNF–α y p21 fue alta en el grupo de control de quemaduras
y mostró una menor expresión en los grupos tratados. Los
hallazgos actuales demuestran que WJ–MSC–CM presenta una
ecacia de curación en las heridas por quemaduras comparable
al medicamento de referencia (sulfadiazina de plata) al inducir
apoptosis y autofagia y reducir la necroptosis y el daño del ADN.
Además, el PRP proporcionó algunos benecios positivos en
comparación con el grupo de control, pero fue menos ecaz que
los otros tratamientos.
Palabras clave: Apoptosis; autofagia; heridas por quemaduras;
medio condicionado derivado de células madre
mesenquimales de la gelatina de Wharton
Effectiveness of Wharton’s jelly mesenchymal stem cell medium on
burn wound healing: Focus on apoptosis, necrosis, and autophagy
Ecacia del medio de células madre mesenquimales de la gelatina de Wharton en
la cicatrización de quemaduras: Enfoque en apoptosis, necrosis y autofagia
Zeynep Çelik–Kenar
1
* , Mehmet Tuzcu
1
, Gökhan Akçakavak
2
, Nijat Majidov
3
, Muhammed Öner
1
,
Ayşenur Tural–Çifçi
1
, Rabia Şahin
1
1
Selcuk University, Faculty of Veterinary Medicine, Department of Pathology. Türkiye.
2
Aksaray University, Faculty of Veterinary Medicine, Department of Pathology. Türkiye.
3
Selcuk University, Faculty of Medicine, Department of Plastic Surgery, Türkiye.
*Corresponding author: zeynep.celik@selcuk.edu.tr
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2 of 7 3 of 7
INTRODUCTION
Thermal burn injuries are wounds caused by heat that occur in
the skin. Burn injuries can signicantly affect the skin and lead
to severe complications that may result in death [1]. Burns are
associated with approximately 15% of global human deaths
annually [2]. In animals, the incidence of burn injuries has increased
in recent years due to factors such as forest res and farm res
[3, 4, 5]. Burns can lead to severe complications such as shock,
infections, electrolyte imbalances, and respiratory failure [6].
Burn treatment can be approached through various methods.
One of the most critical components of topical wound treatment
is the use of silver–containing applications. Silver–containing
topical treatments signicantly reduce the risk of sepsis and
death. Silver ions contribute signicantly to bactericidal activity by
binding to bacterial DNA, while the sulfadiazine component affects
bacterial metabolic processes. Consequently, such treatments are
considered the gold standard in burn management [7, 8]. Another
treatment option is Platelet–Rich Plasma (PRP), which accelerates
wound healing, reduces infection rates, and is easily accessible
[9]. The effects of PRP on burn healing have been demonstrated
in numerous studies [10, 11, 12].
Apoptosis, or programmed cell death, is the process by which
cells systematically disassemble into component parts that will
later be ingested and removed [13]. Caspase–3 is known as a
major effector caspase and is described as the primary mediator
of apoptosis. Caspase–3 can be activated by both extrinsic (death
ligands) and intrinsic (mitochondrial) signaling pathways. The
anti–apoptotic protein Bcl–2 is located on the outer mitochondrial
membrane and prevents the release of apoptogenic factors from
the mitochondria into the cytosol [14, 15, 16, 17].
Autophagy involves the sequestration of intracellular
macromolecules and organelles within a membrane, which then
fuses with lysosomal enzymes for degradation. The most distinct
morphological feature of this type of cell death is the presence of
vesicles containing cytoplasmic fragments or intracellular organelles
such as mitochondria and endoplasmic reticulum (ER), surrounded by
a double–membrane structure. Beclin–1, located in the endoplasmic
reticulum, mitochondria, and perinuclear space, plays a role in the
formation of autophagosomes essential for autophagy [13].
Necroptosis is a form of programmed cell death that
mechanistically resembles apoptosis but differs from necrosis and
apoptosis in terms of its mechanistic and biochemical pathways [18,
19]. Necroptosis can be induced by receptors that trigger apoptosis,
such as the TNF receptor superfamily (FasL, TNFα, TRAIL, etc.),
TLRs, and interferon receptors [20, 21]. Singer and McClain [22]
reported an increase in necrosis, particularly at the 24
th
and 48
th
hours following thermal burns in rats (Rattus norvegicus), and
suggested that necrosis plays a more signicant role than apoptosis
in the progression of thermal burn wounds.
P21, also known as Cyclin–Dependent Kinase Inhibitor 1, is a
protein that halts the cell cycle by inhibiting cyclin–dependent
kinases [23]. Initially discovered as a tumor suppressor and cyclin–
dependent kinase (CDK) inhibitor, p21 plays a crucial role in halting
cell division in response to cell–toxic agents like DOX that cause
DNA damage [24, 25]. Excessive expression of p21 has been shown
to result in the arrest of the cell cycle at the G1, G2, or S phases
[26, 27, 28, 29]. Conversely, cells lacking p21 do not pause the cell
cycle despite DNA damage [29]. p21 can induce extrinsic apoptosis
through activation of TNF death receptor pathways or intrinsic
apoptosis via upregulation of the pro–apoptotic gene BAX [30].
In the past 20 years, stem cells have been utilized in the
treatment of various diseases, including burns [31]. Mesenchymal
stem cells, which can be obtained from various tissues and possess
self–renewal and multipotent differentiation capacities, are a
signicant resource for tissue repair. Mesenchymal stem cells
(MSCs) are multipotent cells that can differentiate into various
mesodermal cells, including bone, cartilage, and adipose tissue.
One source of these cells is the Wharton’s Jelly of the umbilical
cord. Wharton’s Jelly–derived mesenchymal stem cells from
humans are often preferred due to their high proliferation capacity
and ease of availability [32]. The presented study evaluates
apoptosis, necrosis/necroptosis, autophagy, and DNA damage in
a rat model of experimental thermal burns following treatment
with PRP, silver sulfadiazine, and Wharton’s Jelly mesenchymal
stem cell–derived conditioned medium.
MATERIAL AND METHODS
The tissues used in this study were obtained from the project
titled “Investigation of the Effectiveness of Wharton’s Jelly
Mesenchymal Stem Cell–Derived Conditioning Medium on Wound
Healing in a Burn Model,” which was approved by the Selçuk
University Experimental Medicine Application and Research Center
Ethics Committee under decision number 2022-24 dated June
24, 2022. The study, identied by project number 22122017,
was conducted at the Selçuk University Experimental Medicine
Research and Application Center with the ethics approval dated
June 24, 2022.
Animal material
A total of 64 Wistar Albino rats (Rattus norvegicus), comprising
32 females and 32 males, each weighing between 300–350 g
(Mettler Toledo, Balance XPR204S, Switzerland) were used in
the experiment. All procedures were performed under general
anesthesia. The rats were acclimatized to the environment for
one week under standard laboratory conditions: standard diet,
a 12–hour light–dark cycle, and appropriate temperature and
humidity levels were maintained.
Determination of experimental groups and burn application
After shaving the dorsal areas of the rats under general
anesthesia, a second–degree deep burn model was applied
[33]. The homogeneity of the burn wound was ensured using a
device that measured and maintained a constant temperature.
This allowed burns to be induced in each rat by applying heat
at the same temperature. No pressure was applied during the
heat application to create the burn. Each rat was placed in a
separate cage. For pain management, 2 mg·ml
-1
acetaminophen
was administered via drinking water. The rats were monitored
daily post–surgery for overall condition, body weight, signs of
malnutrition, and infection. A total of 64 rats were randomly divided
into 4 groups: Group 1 (n=16), Group 2 (n=16), Group 3 (n=16),
and Group 4 (n=16). Each group consisted of 8 females and 8
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males. Each group was further divided into two subgroups: Group
1a (n=8), Group 1b (n=8), Group 2a (n=8), Group 2b (n=8), Group
3a (n=8), Group 3b (n=8), Group 4a (n=8), and Group 4b (n=8).
Each subgroup contained 4 males and 4 females. Group 1; received
no treatment post–burn. Group 2; received PRP (Platelet–Rich
Plasma) treatment on the rst day (d) post–burn. Group 3; was
treated with Silver Sulfadiazine (1%) on the rst d post–burn.
Group 4; received Wharton’s Jelly Mesenchymal Stem Cell–Derived
Conditioned Medium on the rst d post–burn. Samples were
collected from rats in Groups 1a, 2a, 3a, and 4a seven d after
treatment, and from rats in Groups 1b, 2b, 3b, and 4b fourteen d
after treatment. Euthanasia was performed via cervical dislocation
under general anesthesia on the 7
th
d for 32 rats and on the 14
th
d
for the remaining 32 rats. The tissues collected from the lesioned
areas were preserved at -80°C (ZK Meiling, DW–HL–398S, China)
until molecular analysis was conducted.
Real–time PCR analysis
RNA isolation was performed from the samples collected during
necropsy and stored at -80°C using the SanPrep Column microRNA
Miniprep Kit (BIO BASIC, USA, Catalog No.: SK881), following the
manufacturer’s recommendations. cDNA was synthesized from
the isolated RNA using the OneScript Plus Synthesis Kit (ABM,
Canada, G236, Catalog No.: G236), according to the manufacturer’s
instructions. Real–time PCR was conducted using the Roche
LightCycler 96 (Roche, Switzerland) instrument. For this purpose,
ABM BlasTaq™ 2× PCR MasterMix (ABM, Canada, Catalog No: G891)
was used. The primer sequences utilized in the study are provided
in TABLE I. Separate reaction mixtures were prepared for each
gene of interest. For each sample, 15 μl of reaction mixture and
5 μl of cDNA sample were added to the capillaries. The real–time
PCR conditions included: 180 s of pre–incubation at 95°C, 15 s of
denaturation at 95°C, 60 s of annealing at 60°C, 3 s of extension
at 72°C, and 30 s of cooling at 40°C. The expression levels of the
genes investigated were calculated using the 2ΔΔ
Ct
(Delta Delta
Ct) method [34, 35].
Statistical analysis
Parametric data obtained from molecular analyses were evaluated
using the SPSS 22.0 (IBM SPSS Statistics 22, USA) statistical program
with One–Way ANOVA and post hoc Duncan test. The data were
presented as mean ± standard error (mean±SE). A P–value of less
than 0.05 was considered the threshold for statistical signicance.
This study, by demonstrating the effectiveness of both current
methods used in burn treatment and innovative biological
treatment options, may contribute to the development of more
effective treatment strategies in clinical applications.
RESULTS AND DISCUSSION
In the statistical analysis of Bcl–2 levels, no signicant differences
were observed between the burn control group at the 1
st
week PRP
and the 2
nd
week PRP. However, signicant differences were found
between these groups and the other groups. The lowest Bcl–2
expression levels were observed in the group treated with silver
sulfadiazine at the 1
st
week. The results are presented in FIG. 1.
This results suggests that silver sulfadiazine and Wharton jelly
may induce apoptosis, contributing to the reduction of burn–related
tissue damage. Similarly, the lowest levels of Caspase–3 expression
were found in the burn control group, while the highest levels
were in the silver sulfadiazine and Wharton jelly–treated groups,
indicating that these treatments utilize apoptosis to address burn–
induced tissue damage.
In the statistical evaluation of Caspase–3, differences were
observed between the burn control group at the 1
st
week and
the groups treated with silver sulfadiazine, as well as between
the groups treated with Wharton jelly. However, no signicant
differences were found between the burn control group and the
other groups at the 2
nd
week. The results are presented in FIG. 2.
Although the Caspase–3 expression levels in the burn control
group at the 2
nd
week were about half those in the Wharton
jelly–treated 2
nd
week group, no signicant statistical difference
TABLE I
Sequences of primers used
Primer Primer Sequence 5′-3′
Bcl–2
F:5’–GCAGCTTCTTTCCCCGGAAGGA–3’
R:5’–AGGTGCAGCTGACTGGACATCT–3’
Caspase–3
F:5’–GGTATTGAGACAGACAGTGG–3’
R:5’–CATGGGATCTGTTTCTTTGC–3’
TNF–α
F:5’–GTCGTAGCAAACCACCAAGC–3’
R:5’–TGTGGGTGAGGAGCACATAG–3’
p21
F:5′–TTGCACTCTGGTGTCTGAGC–3′
R:5′–AATCTGTCAGGCTGGTCTGC–3′
Beclin–1
F:5’–AGCACGCCATGTATAGCAAAGA–3’
R:5’–GGAAGAGGGAAAGGACAGCAT–3’
β–actin
F:5’–GGCCACAATGGCTGACCATTC–3’
R:5’–AAGGTGACAGCATTGCTTC–3’
327.78
196.9
117.42
178.51
331.26
268.65
132.26
182.14
Control PRP Silver Wharton Control PRP Silver Wharton
Day 7 Day14
0
100
200
300
400
Control PRP Silver sulfadiazine Wharton's jelly
Bcl-2
a
abc
c
bc
a
ab
bc
bc
FIGURE 1. Distribution of Bcl–2 among groups according to Real Time PCR
results.
a–c
Dierent superscripts indicate statistical signicance (P<0.05)
Evaluation of the eectiveness of Wharton's jelly mesenchymal stem cell / Çelik-Kenar et al._____________________________________
4 of 7 5 of 7
was found, suggesting that apoptosis begins to increase after
the second week post–burn. Tan and Zhang [36] reported that
apoptosis increased after the rst 6 h in a thermal burn model in
rats, while Xiao and Li [33] noted an increase in apoptosis within
the rst 48 h, followed by a decrease. The present study’s ndings
are consistent with these studies. Additionally, unlike previous
studies, this study assessed apoptosis rates on the 7
th
and 14
th
days post–burn and found that apoptosis did not decrease after 48
h but rather increased. Gravante and Palmieri [37] noted that the
presence and increase of apoptosis could lead to growth in the burn
wound. However, in this study, increased apoptosis rates in the burn
wounds, following the application of silver sulfadiazine, PRP, and
Wharton jelly, suggest that apoptosis plays a crucial role in tissue
repair rather than deepening the burn wound. Furthermore, the
greater Caspase–3 expression observed in the burn control group
at the 2
nd
week compared to the 1
st
week suggests that apoptosis is
involved in wound healing even in the absence of treatment agents.
In the analysis of Beclin–1, no signicant differences were found
between the burn control group and the PRP–treated groups.
However, differences were observed between the groups treated
with silver sulfadiazine and Wharton jelly. The highest Beclin–1levels
were observed in the Wharton jelly–treated groups, particularly in
the 1
st
week group, while the lowest Beclin–1levels were found in
the burn control group. The results are presented in FIG. 3.
Based on these results, it can be concluded that silver
sulfadiazine and Wharton jelly applications might enhance
autophagy to address tissue damage and promote regeneration.
Autophagy can have dual effects, either reducing or increasing
tissue damage. However, in light of other parameters, the higher
Beclin–1 levels in the silver sulfadiazine and Wharton jelly–treated
groups and the lower levels in the burn control group suggest
that autophagy favors cell regeneration and tissue repair. Tan and
Zhang [36] reported that autophagy peaked 12 h after a thermal
burn in a rat model, while Xiao and Li [33] noted it peaked at 72 h.
Similar results were obtained in this study. Additionally, while Bcl–2
expression levels were higher in the 1
st
week groups compared to
the 2
nd
week groups, this decrease in Bcl–2 levels over time was
interpreted as a result of apoptosis.
In the molecular and statistical analyses of TNF–α, it was observed
that both the 1
st
week burn group and the 2
nd
week burn group were
different from all other groups, with the highest TNF–α levels found
in the burn control group. The lowest TNF–α levels were observed in
the 2
nd
week group treated with silver sulfadiazine and the 1
st
week
group treated with Wharton jelly. The results are presented in FIG. 4.
20.67
44.57
53.69 53.68
30.94
45.86
51.55
58.06
Control PRP Silver Wharton Control PRP Silver Wharton
Day 7 Day14
0
20
40
60
Control PRP Silver sulfadiazine Wharton's jelly
Caspase-3
a
ab
b
b
ab
ab
b
b
41.29
76.66
142.25
173.59
31.4
89.74
111.1
164.74
Control PRP Silver Wharton Control PRP Silver Wharton
Day 7 Day14
0
50
100
150
200
Control PRP Silver sulfadiazine Wharton's jelly
a
a
ab
abc
bcd
de
cde
e
Beclin-1
264.23
128.57
142.25
123.29
264.39
133.84
111.1
163.91
Control PRP Silver Wharton Control PRP Silver Wharton
Day 7 Day14
0
100
200
300
Control PRP Silver sulfadiazine Wharton's jelly
TNF-
α
a
a
b
b
b
b
b
b
FIGURE 2. Distribution of Caspase–3 among groups according to Real Time PCR
results.
a–b
Dierent superscripts indicate statistical signicance (P<0.05)
FIGURE 3. Distribution of Beclin–1among groups according to Real Time PCR
results.
a–d
Dierent superscripts indicate statistical signicance (P<0.05)
FIGURE 4. Distribution of TNF–α among groups according to Real Time PCR
results.
a–b
Dierent superscripts indicate statistical signicance (P<0.05)
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5 of 7
In the investigation of necrosis/necroptosis, Real–Time PCR
analysis revealed that TNF–α expression levels were signicantly
higher in the burn control group compared to other groups, with
statistical analysis conrming differences between burn control
group and all other groups. Considering the destruction and repair
reactions in the burn control, silver sulfadiazine–treated, and
Wharton jelly–treated groups, these results are associated with
the role of TNF–α in inflammation.
The analyses revealed that the highest p21 expression levels
were observed in the burn control group. Differences were noted
between the burn control group and the 1
st
week PRP–treated
group, as well as between the burn control group and the silver
sulfadiazine–treated groups. The lowest p21 expression levels
were detected in the groups treated with silver sulfadiazine. The
results are presented in FIG. 5.
response make silver sulfadiazine a more successful option for
thermal burn injuries.
ACKNOWLEDGMENTS
The authors express their gratitude to the Selçuk University
Scientic Research Projects (BAP) Coordination Ofce for funding
this study (Project No: 23401097).
Conflict of Interests
The authors declare that there is no conflict of interest.
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in the burn control group, while it was signicantly reduced in the
silver sulfadiazine groups. Although p21 expression was lower in
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CONCLUSION
According to the results of the study, groups treated with silver
sulfadiazine and Wharton jelly exhibited higher levels of apoptosis
and autophagy, while showing less necroptosis and DNA damage.
Although the PRP–treated groups yielded better results compared
to the burn control group, no statistically signicant differences
were observed in apoptosis and autophagy. Although Wharton
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silver sulfadiazine, its limited practical use and slower treatment
311.17
139.97
105.96
178.51
310.77
247.98
111.1
185.4
Control PRP Silver Wharton Control PRP Silver Wharton
Day 7 Day14
0
100
200
300
Control PRP Silver sulfadiazine Wharton's jelly
p21
a
a
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abc
ab
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c
c
FIGURE 5. Distribution of p21 among groups according to Real Time PCR results.
a–c
Dierent superscripts indicate statistical signicance (P<0.05)
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