Carbohydrate Polymers 240 (2020) 116315

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

In vivo evaluation of the wound healing properties of bio-nanofiber T

chitosan/ polyvinyl alcohol incorporating honey and Nepeta dschuparensis
Atena Naeimi1,*, Maryam Payandeh2, Abdollah Ramzani Ghara2,*, Fereshteh Ezzati Ghadi2
1
Department of Chemistry, Faculty of Science, University of Jiroft, Jiroft 7867161167, Iran
2
Department of Biology, Faculty of Science, University of Jiroft, Jiroft, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Engineering bioscaffolds for improved cutaneous tissue regeneration remains a healthcare challenge. To help
Bio-nanofiber address this problem, we report on the fabrication and characterization of electrospun polyvinyl alcohol and
Chitosan chitosan (PVA/Chit) nanofiber mats loaded honey and Nepeta dschuparensis plant for faster wound healing ap-
Pharmaceutical industrials plications. The morphology of nanofiber mats was examined by SEM and TEM. The physicochemical and thermal
Wound healing
stability characterizations were done by FT-IR and TGA/DTA, which reveal the presence of honey and desired
plant into the nanofibers. PVA/Chit@Nep/Hon was investigated for wound healing therapy as a potential
therapeutic agent. The in vivo wound healing studies on the rats for 21 days revealed the wound healing faster
within three weeks by the incorporation of honey and plant into the nanofiber mats and hence these nanofiber
mats show great potential in acute and chronic wound healing applications.

1. Introduction environments to support wound healing and skin regeneration (Adamu,

Nowadays, a major public health concern along with the economic
burden is wound care (Ahn, Ardoña, Campbell, Gonzalez, & Parker,
2019; Basu, Uttamchand, & Inderchand, 2017; Fan, Yang, Yang, Peng,
& Hu, 2016). Currently a $25 billion healthcare cost is incurred for
around 6.5 million of patients affecting by chronic wounds every year
(Delli Santi & Borgognone, 2019; Enoch & Leaper, 2008). Removal of
nonviable tissue to promote cell proliferation, swabbing and cleaning
the wound area to treat infections and dressing to both protect the
wound from infections to enhance the healing process are components
of the standard of wound care (Kalantar et al., 2018; Wu et al., 2020).
To increase the wound healing process, design of nanofibrous mat/
wound dressing materials having antibiotic and antibacterial property
is extremely important (Han & Ceilley, 2017). In this regards, simpli-
city, flexibility, and capability to imitate the native skin extracellular
matrix structure, recapitulate the wound healing process, and provide
biomaterial tenability of electrospinning techniques causes that it has
become one of the most attractive to develop advanced bioactive
wound dressings.
Chitosan is a natural polymer having high biocompatibility; easily
stimulate cell growth and regulation (Darbasizadeh et al., 2019; Ding,
Deng, Du, Shi, & Wang, 2014; Huang et al., 2015). On the other hand,
PVA, as a synthetic polymer, provide mechanically durable and humid
Rahman, & Hamdan, 2019; Li et al., 2013; Wali, Gorain, Inamdar,
Kundu, & Badiger, 2019). These recently developed electrospun scaf-
folds including PVA and chitosan can fulfill most of the very essential
requirements for accelerated wound healing including minimized in-
fections (Abdeen, El Farargy, & Negm, 2018; Dubey & Gopinath, 2016;
Fathollahipour, Abouei Mehrizi, Ghaee, & Koosha, 2015; Nguyen et al.,
2019; Ruiz et al., 2019). Poor solubility, slower and uncontrollable
biodegradation rate, low strength and low thermal stability of these
kinds of nanofibers are the disadvantageous properties for wound
dressing application. Design of bio-nanocomposite and adding other
materials to solve these problems has attracted so much attention.
Hence, the high osmolarity, inhibine factor, acidic pH, high viscosity
and nutrient content of honey contribute to the inhibition of bacterial
growth and the promote wound healing (Subrahmanyam, Sahapure, &
Nagane, 2001). By incorporating the honey in nanofibers, the formation
of granulation tissue, activation of fibroblasts, thickness of epidermis
and collagen fibers, decrease infection, inflammation, edema and de-
hiscence are increased and thermal stability and strength of them are
enhanced (Ghaderi & Afshar, 2004; Subrahmanyam et al., 2001). On
the other hand, Nepeta dschuparensis Bornm is an herbaceous aromatic
plant, which belongs to the Lamiaceae family. It has been widely used
as a traditional herbal medicine due to its flavonoids compound. An-
tibacterial, antioxidant and anti-inflammatory of Nepeta spices attract

⁎
Corresponding authors.
E-mail addresses: a.naeimi@ujiroft.ac.ir (A. Naeimi), a.ramzani@ujiroft.ac.ir (A.R. Ghara).

https://doi.org/10.1016/j.carbpol.2020.116315
Received 30 November 2019; Received in revised form 31 March 2020; Accepted 13 April 2020
Available online 20 April 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
A. Naeimi, et al.
so much attention due to the high content of essential oil, flavonoids, β-
caryophyllene, 1.8 cineole, thujone, β- eudesmol and pinene which
makes it suitable for wound dressing materials (Bandh, Kamili, Ganai,
Lone, & Saleem, 2011; Bejestani, 2018; Salehi et al., 2018).
In continuing our works for development of the novel bio nano-
composites (Naeimi, Amiri, & Ghasemi, 2017; Naeimi, Honarmand, &
Sedri, 2019; Noghi, Naeimi, & Hamidian, 2018; Sedri, Naeimi, &
Mohammadi, 2018) for different applications, here, we report the first
bio-nanofiber including Nepeta dschuparensis plant on the burns treat-
ment. The main aim of this paper was the design of a PVA/Chit@Nep/
Hon bio-nanofiber using honey and its application was evaluated as a
potential scaffold for burn treatment. The efficiency of it was compared
with PVA/Chit and silver sulfadiazine cream, as a topical antibiotic
using in partial thickness and full thickness burns, to show the effect of
honey and desired plant.
2. Materials and methods
Honey and Nepeta dschuparensis were collected form Bahraseman
village, Jiroft, Iran. PVA and chitosan ([2-amino-2-deoxy-(1-4)-β-D-
glucopyranose], with medium molecular weight, 400000 Da) were
purchased from Alderich and Fluka Company, respectively. Ethanol,
formalin, ketamine, normal saline (0.9%), hematoxylin and eosin were
supplied from Merck Company. Silver sulfadiazine 1% cream was
purchased from Sina Darou, Iran. PVA/Chit@Nep/Hon bio-nanofiber
was synthesized by electrospinning instrument (Nanoazma Company,
Iran). The functional groups of this bio nanocomposite were considered
by FT-IR spectrophotometer (NICOLET iS10). SEM images were per-
formed by IE 300X, Oxford, UK to have the shape of this hybrid. The
real size of nanofiber was investigated by TEM instrument (Philips
CM30). Thermal stability of PVA/Chit@Nep/Hon was investigated by
NETZSCH instrument (PC Luxx 409).
3. Experimental
3.1. Preparation of Nepeta dschuparensis extraction solution
10 g of Nepeta dschuparensis plant powder was added to 100 ml
ethanol (50%) and stirred within 24 hours at room temperature. The
extract was filtered with Whatman No 1. filter paper. The obtained
suspension was evaporated at 40 °C in rotary evaporator. The dried
sample stored at 4 °C.
3.2. Electrospinning of PVA/Chit@Nep/Hon bio nanofiber
3.5 g of PVA (10% w/w in water), 1 g of chitosan (3% w/w in HCl
0.5 M) and 0.5 g of Nepeta dschuparensis and honey were irradiated
under ultrasonic for 30 min. The prepared solution was stirred for
7 hours at 80 °C. The electrospinning of the final prepared solution was
performed at an electrical voltage of 17 kV at room temperature under
atmospheric pressure. The polymer fibers were injected using a 5 ml
syringe needle having 1.23 mm outer diameter and 0.83 mm internal
diameter at a flow rate of 0.5 ml/h.
3.3. Animals
Twenty male Wistar rats with an approximate weight of 180-220 g
were obtained from animal house of University of Kerman, Iran. The
rats were kept in polypropylene cages, fed with standard pellet and
water ad libitum. Animals were housed under standard environmental
conditions of temperature (22 ± 2 °C) and 12 hours light/dark cycle.
The study was performed according to the animal ethics committee
guidelines for the use of experimental animals.
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Carbohydrate Polymers 240 (2020) 116315
3.4. Burn wound model
Rats were anaesthetized by giving intra-peritoneal (IP) injection
with 5% ketamine (50 mg/kg). Thereafter, a shaver was used to remove
hair from the dorsal area and skin was sterilized using 96% ethanol.
Second degree burn wounds were created using a metal cylinder
(2.2 cm diameter) immersing in boiling water for 3 minutes and main-
taining on the back of rats for 5 second (Walker & Mason, 1968). Ex-
perimental animals were divided into four groups as follows: Group (I)
the burned area in this group remained without any treatments, Group
(II) rats were received 1% silver sulfadiazine on burn area, Group (III)
animals were treated with PVA/Chit daily and Group (IV) PVA/Chit@
Nep/Hon nanofiber was applied daily. Silver sulfadiazine and bio-na-
nofibers and were applied topically to cover the wound area every
24 hours for three times within 7, 14 and 21 days of experiment.
3.5. Wound closure
In order to evaluate the wound closure, wound images were taken
on day zero, 7th, 14th and 21th day of treatment. The percentage of
wound closure was calculated by the following formula:
A0 − At
Wound closure (%) = × 100
A0
Where, A0 and At are the wound sizes at initial and time t, respectively.
3.6. Histopathological study
The rats were sacrificed on the day 7th, 14th and 21th days and the
skin tissues of the burnt area were collected. All samples were fixed in
10% formalin. After fixation in formalin, the skin tissues embedded in
paraffin, cut into 5 μm sections. Hematoxylin and eosin were used to
stain tissue sections (Li et al., 2018).
3.7. Statistical analysis
All data are expressed as Mean ± Standard Deviation (S.D.). A
statistical software package, SPSS version 19 was used to perform sta-
tistical analysis. Data were analyzed by ANOVA, followed by post hoc
LSD test. Statistical significance was accepted at P < 0.05.
4. Results and discussion
PVA is used in a variety of medical applications because of its bio-
compatibility, low tendency for protein adhesion, and low toxicity.
Hence, PVA, natural polymer (chitosan), medicinal plant (Nepeta
dschuparensis) and honey were mixed under ultrasonic irradiation and
PVA/Chit@Nep/Hon bio-nanofibers was obtained using electrospin-
ning instrument (Scheme 1).
To confirm shape and morphology of this fiber, SEM and TEM were
investigated. SEM images of electrospun PVA/Chit (a) and PVA/Chit@
Nep/Hon (b) nanofibers were illustrated at Fig. 1. Smooth surface and
uniaxially aligned were shown for electrospun PVA/Chit nanofiber with
95-150 nm of sizes. Upon increasing the honey and Nepeta dschupar-
ensis, a granular morphology appeared on the surface of PVA/Chit fi-
bers (Fig. 1(b)). It seems that by adding the honey and Nepeta dschu-
parensis plant, solution viscosity and also the fiber diameters were
enhanced. By comparing the TEM images of PVA/Chit and PVA/Chit@
Nep/Hon in Fig. 2, the distribution of spherical particles of honey and
plant (dark grain) in the PVA/Chit polymeric matrices was observed
very well. The randomly distributed particles affected the surface
roughness and produced stress concentrations in the nanofibers.
Chitosan as a hydroxyl-rich structure along with amine group could
be act with Nepeta dschuparensis plant and honey. The FT-IR of elec-
trospun PVA/Chit@Nep/Hon and PVA/Chit nanofibers were confirmed
their purity, structure and functional groups (Fig. 3). The FT-IR
A. Naeimi, et al. Carbohydrate Polymers 240 (2020) 116315

Scheme 1. Preparation and components of PVA/Chit@Nep/Hon bio-nanofiber.

spectrum of Chit/PVA nanocomposite showed the characteristics peaks
at 3427 cm−1 and 1741 cm−1 relating to the hydroxyl groups that
overlapped with –NH2 vibration of chitosan. The band around 3280 cm-
1
and 1444 cm-1 were corresponded to stretching and bending vibration
of OH groups, respectively. The peak at 2945 cm-1 was related to
asymmetric CH2 group stretching vibration. The peaks at 1541 and
1699 cm-1 are attributed to C = C vibration of PVA. The corresponding
peaks to CeC and CeO were observed at 848 and 1099 cm-1,
respectively. By incorporating honey and Nepeta dschuparensis plant in
Chit/PVA nanocomposite, hydrogen bonds between functional groups
of honey and Nepeta dschuparensis with hydroxyl and amine groups
from PVA and chitosan were formed. A good and effective interaction
and more connection between the component of electrospun PVA/
Chit@Nep/Hon nanofiber were made it strength (Biranje, Madiwale, &
Adivarekar, 2017; Grande & Carvalho, 2011; Jin & Bai, 2002).
Thermal characteristic of PVA/Chit nanocomposite and PVA/Chit@

Fig. 1. SEM images of electrospun PVA/Chit (a) and PVA/Chit@Nep/Hon (b) nanofibers.

3
A. Naeimi, et al.
Fig. 2. TEM images of electrospun PVA/Chit (a) and PVA/Chit@Nep/Hon (b) nanofibers.
Nep/Hon nanofibers were obtained using TGA (Fig. 4(a) and (b)). In
TGA cure of PVA/Chit nanocomposite, there are two degradation stages
(Fig. 4(a)). The first weight-loss was started at 250 °C of temperature
onset relating to melting point of PVA. The second degradation occurs
at 350 °C which is corresponded to chitosan. On the other hand, there
are these two steps and in TGA of PVA/Chit@Nep/Hon and considering
of char residue showed that the honey component was acted as a good
char insulator in the polymers matrix. In TGA of PVA/Chit@Nep/Hon
nanofibers, it seems that carbon particle formation of honey layered
structure in the PVA and chitosan network that looks like char. In fact,
this char like a layer was acted as an effective barrier to the permeation
of oxygen improving the char residue. The presence of crystalline
structure and great compactness between the honey and Nepeta dschu-
parensis plant and PVA/Chit matrix lead to high thermal stability of this
bio nanocomposite. DTG and DTA cures of PVA/Chit and PVA/Chit@
Nep/Hon nanofibers were confirmed these results very well (Grande &
Carvalho, 2011).
Wound closure (%) analysis was performed to evaluate the wound
healing properties of bio-nanofiber (PVA/chit and PVA/chit@Nep/
Hon) on second degree of burn skin (Fig. 5). The percentage of wound
closure on the first day did not differ among the four groups. The results
of calculating the wound closure percentage with respect to the first day
were demonstrated that in day 7 of experiments, the wound closure
percentage in group (IV) which treated with PVA/Chit@Nep/Hon
(14.92 ± 0.008) was significantly higher than control group
Fig. 3. FT-IR of electrospun PVA/Chit and PVA/Chit@Nep/Hon nanofibers.
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Carbohydrate Polymers 240 (2020) 116315
(2.86 ± 0.034), silver sulfadiazine treated rats (4.74 ± 0.009) and
PVA/Chit (11.42 ± 0.008) group (P < 0.05 and P < 0.001, respec-
tively). On the 14th day, the percentage of wound closure in the group
(IV) (34.78 ± 0.014) was much more than control group
(17.21 ± 0.008) and silver sulfadiazine treated animals
(18.41 ± 0.009) (P < 0.01 and P < 0.001). Moreover, group (III)
were increased the percentage of wound closure (21.42 ± 0.008) when
compare with silver sulfadiazine group (18.41 ± 0.009). However, on
the day 21 post-burn, PVA/Chit@Nep/Hon was enhanced very well
(P < 0.001) the percentages of wound closure (59.85 ± 0.14) with
compare to control group (40.45 ± 0.008) whereas, the percentage of
wound closure in group (II) (37.42 ± 0.008) was less than group (III)
(44.28 ± 0.009) and (IV) (Fig. 5). Moreover, percentage of wound
closure was diminished in silver sulfadiazine topical treatments to burn
skin by day 21 when compared to untreated rats. Silver sulfadiazine is
an antibacterial agent for topical treatment of burn skin, but undesir-
able characteristics are associated with its clinical use. It seems that its
delayed wound healing is claimed to be the emergence of resistant
strains of microbial species, retardation of wound healing and devel-
opment of systemic side effect (Atiyeh, Costagliola, Hayek, & Dibo,
2007). However, deep-dermal wounds heal slowly and also silver sul-
fadiazine may delay the healing process due to its adverse effects
(Hosseinimehr et al., 2010). It should be noted that natural product can
accelerate wound healing (Kim, Park, & Sung, 2009; Shetty, Udupa, &
Udupa, 2008). Therefore, there were significant differences regarding
A. Naeimi, et al.
Fig. 4. Thermal properties of PVA/Chit nanocomposite (a) PVA/Chit@Nep/Hon nanofibers (b).
percentages of wound closure between days 7, 14 and 21 of experi-
ments, in all treated groups. The obtained results were shown that PVA/
Chit@Nep/Hon and PVA/Chit could promote the wound closure. These
results are in agreement with a study by Charernsriwilaiwat,
Rojanarata, Ngawhirunpat, and Opanasopit (2014) on electrospun
chitosan/polyvinyl alcohol nanofiber mats for wound. However, PVA/
Chit@Nep/Hon was more effective than PVA/Chit treated group due to
antioxidant and antibacterial activity of honey and Nepeta dschupar-
ensis.
The histopathological studies of burn wound area were performed
on the 7th, 14th and 21th day of experiment. The histopathological
features of the skin tissue of all groups showed in Figs. 6–8. Histo-
pathological evaluation was used as general parameter for the evalua-
tion of wound healing process. Histopathological observations on day 7
were shown that sever inflammation and granulation tissue in all the
experimental groups. Moreover, results showed necrosis of superficial
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Carbohydrate Polymers 240 (2020) 116315
tissue and also widespread edema of burn wound area of group (I), (II)
and (III) (Fig. 6(a)–(c), respectively. In addition, PVA/Chit@Nep/Hon
treated groups have shown re-epithelialization as well as increased
angiogenesis of wound compared to other treatment groups (Fig. 6(d)).
On day 14, results showed sever inflammation include necrosis of
superficial tissue and edema in control, silver sulfadiazine and PVA/
Chit treated group. While, PVA/Chit@Nep/Hon treated animals
showed necrosis tissue, middle inflammation and neovascularization
with compare to group (II) and (III) (Fig. 7(d)).
On day 21, rats treated with PVA/Chit@Nep/Hon showed the
complete formation of epidermis and dermis layers which clearly were
visible when compared with control group (Fig. 8(a)). Silver sulfadia-
zine administration to Group (II) (Fig. 8(b)) shows tissue necrosis,
edema of burn wound area and severs inflammation in comparison with
Group (IV) (Fig. 8(d)). Moreover, photomicrograph from group (III)
shows that its tissue necrosis is less than group (I) and (II), respectively
A. Naeimi, et al. Carbohydrate Polymers 240 (2020) 116315

Fig. 5. Effect of different treatments on

wound closure rate of the rats with
second degree burn injury on days 7th,
14th and 21th of experiment. Group (I)
presented as untreated animals with
burn wound, Group (II) demonstrated
the animals treated with 1% silver sul-
fadiazine, Group (III) showed animals
treated with PVA/Chit nanofiber and
Group (IV) rats were received PVA/
Chit@Nep/Hon. Each point re-
presented Mean ± S.D. of the wound
closure percentage in the related group.
“a” shows P value less than 0.05 be-
tween Group IV and Group I treated
rats.

Fig. 6. Histopathological studies of burn wound in different groups on day 7 of the experiment. Hematoxylin and eosin (H& E) staining of skin tissue, magnification,
× 10. Animals with burn wound without treatment (a), animals treated with 1% silver sulfadiazine (b), animals treated with PVA/Chit (c) and PVA/Chit@Nep/Hon
treated rats (d). In these figures, the arrows1 are necrotic superficial tissue, the arrows 2 indicate edema and the arrows 3 show neovascularization.

6
A. Naeimi, et al.
Fig. 7. Histopathological observation of burn area of skin related to all treated groups on day 14 of the experiment (stained with hematoxylin and eosin, magni-
fication× 10). Animals with burn wound without treatment (a), animals treated with 1% silver sulfadiazine (b), animals treated with PVA/Chit (c) and PVA/Chit@
Nep/Hon treated rats (d). In these figures, the arrows 1 are necrotic of superficial tissue; the arrows 2 show edema and the arrows. 3 indicate necrosis tissue.
(Fig. 8(c)).
In burn healing process, the proliferative phase typically demon-
strates angiogenesis, granulation and collagen deposition (Nayak,
Pereira, & Maharaj, 2007). The granulation tissue is characterized by a
high density of new vessels, which is observed in PVA/Chit@Nep/Hon
treated group. According to obtained results from histopathological
study also revealed that minimal inflammation, angiogenesis and in-
creased collagen deposition in rats treated with PVA/Chit@Nep/Hon.
One of the contradictory results of this study was that silver sulfadia-
zine was less effective in burn wound healing when compared to the
PVA/chit group as vehicle control. In fact, the cytotoxic effect of silver
sulfadiazine on long period treatment causes that adverse reactions and
side effect were observed along with increasing resistance to silver
sulfadiazine (Chaby et al., 2005; Dunn & Edwards-Jones, 2004;
Mehrabani et al., 2016). Present study revealed that bio-nanofiber of
PVA/Chit@Nep/Hon is more effective than silver sulfadiazine. Re-epi-
thelialization occurs as results of keratinocyte proliferation and mi-
gration from the wound edges and skin appendages toward the center
of wound (Coelho et al., 2018). According to results from PVA/Chit@
Nep/Hon group, re-epithelialization was observed on burn wound area.
Overall, the use of natural compounds in electrospun PVA/Chit@
Nep/Hon nanofiber nanocomposite showed the good potential for
supporting cell attachment and proliferation for skin tissue engineering
and it is much more effective than Chit/PVA (this work), Honey
7
Carbohydrate Polymers 240 (2020) 116315
(Mohamed et al., 2014), PVA/honey (Tavakoli & Tang, 2017), Chit/
honey (Movaffagh et al., 2019) and PVA/chitosan along with other
compounds (Movaffagh et al., 2019). The morphology of this electro-
spun bio nanocomposite is similar to the natural extracellular matrix in
skin promoting cell adhesion, migration and proliferation
(Charernsriwilaiwat, Rojanarata, Ngawhirunpat, & Opanasopit, 2014).
Hence, this material can deliver various effective agents at the wound
site (Gizaw et al., 2018). Biocompatible, flexibility, ability of retain
moist environment, biodegradable and good solubility are unique
properties of this bio nanofiber for wound healing applications. The
electrospun PVA/Chit@Nep/Hon nanofiber showed a good potential
for supporting cell attachment and proliferation for skin tissue en-
gineering.
5. Conclusion
In the current study, honey and Nepeta dschuparensis plant was
added to PVA/Chit polymer matrix using electrospinning to have an
electrospun PVA/Chit@Nep/Hon bio-nanofiber. Smooth surface and
uniaxially aligned for PVA/Chit and spherical particles of honey and
plant particles were observed in polymeric matrices. On the other hand,
the percentage of wound closure and histopathological assay were
evaluated for wound dressing ability of nanofibers. The results suggest
that PVA/Chit@Nep/Hon has beneficial effect in burn wound healing.
A. Naeimi, et al.
Fig. 8. Histopathological studies of burn wounds at day 21 of study. Sections stained with H& E (× 10). Animals with burn wound without treatment (a), rats treated
with silver sulfadiazine (b), animals treated with PVA/Chit (c) and PVA/Chit@Nep/Hon treated rats (d). In these figures, the arrows 1 are necrosis tissue; the arrows
2 indicate neovascularization and the arrows 3 show re-epithelization.
This study revealed that topical application of this bio-nanofiber
showed strong wound healing activities in burn wound area.
CRediT authorship contribution statement
Atena Naeimi: Conceptualization, Methodology, Writing - original
draft, Project administration, Writing - review & editing. Maryam
Payandeh: Visualization, Investigation. Abdollah Ramzani Ghara:
Supervision, Conceptualization, Methodology, Writing - original draft.
Fereshteh Ezzati Ghadi: Writing - original draft, Resources, Software.
Acknowledgment
We are thankful to University of Jiroft for their support on this
work.
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