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

chitosan/ polyvinyl alcohol incorporating honey and Nepeta
dschuparensis

Atena Naeimi (Conceptualization) (Methodology) (Writing - original

draft) (Project administration) (Writing - review and editing), Maryam
Payandeh (Visualization) (Investigation), Abdollah Ramzani Ghara
(Supervision) (Conceptualization)
(Methodology)<ce:contributor-role>Writing -original draft),
Fereshteh Ezzati Ghadi (Writing - original draft) (Resources)
(Software)

PII: S0144-8617(20)30489-6
DOI: https://doi.org/10.1016/j.carbpol.2020.116315
Reference: CARP 116315

To appear in: Carbohydrate Polymers

Received Date: 30 November 2019

Revised Date: 31 March 2020
Accepted Date: 13 April 2020

Please cite this article as: Naeimi A, Payandeh M, Ghara AR, Ghadi FE, In vivo evaluation of
the wound healing properties of bio-nanofiber chitosan/ polyvinyl alcohol incorporating honey
and Nepeta dschuparensis, Carbohydrate Polymers (2020),
doi: https://doi.org/10.1016/j.carbpol.2020.116315
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© 2020 Published by Elsevier.

 In vivo evaluation of the wound healing properties of bio-
nanofiber 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
Email: a.ramzani@ujiroft.ac.ir (Abdollah Ramzani Ghara); a.naeimi@ujiroft.ac.ir (Atena
Naeimi)

Graphical abstract

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Highlights
 Design of electrospun PVA/Chitosan incorporating honey and Nepeta
dschuparensis
 First bio nanocomposite for wound healing application
 Its in vivo wound healing activity was evaluated
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 It is much more effective than PVA/Chit nanofiber and silver sulfadiazine

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Abstract
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Engineering bioscaffolds for improved cutaneous tissue regeneration remains a

healthcare challenge. To help address this problem, we report on the fabrication and

characterization of electrospun polyvinyl alcohol and chitosan (PVA/Chit) nanofiber

mats loaded honey and Nepeta dschuparensis plant for faster wound healing

applications. The morphology of nanofiber mats was examined by SEM and TEM.
The physicochemical and thermal 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.

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Keywords: Bio-nanofiber; Chitosan; Pharmaceutical industrials;
Wound
healing

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Introduction -p
Nowadays, a major public health concern along with the economic burden is wound
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care (Basu, Uttamchand, & Inderchand, 2017; Fan, Yang, Yang, Peng, & Hu, 2016;

Ahn, Ardoña, Campbell, Gonzalez, & Parker, 2019). Currently a $25 billion
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healthcare cost is incurred for around 6.5 million of patients affecting by chronic

wounds every year (Enoch & Leaper, 2008; Delli Santi & Borgognone, 2019).
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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
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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,
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design of nanofibrous mat/wound dressing materials having antibiotic and

antibacterial property is extremely important (Han & Ceilley, 2017). In this regards,

simplicity, 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 (Huang et al., 2015; Ding, Deng, Du, Shi, & Wang, 2014;

Darbasizadeh et al., 2019). On the other hand, PVA, as a synthetic polymer, provide

mechanically durable and humid environments to support wound healing and skin

regeneration (W. Li et al., 2013; Wali, Gorain, Inamdar, Kundu, & Badiger, 2019;

Adamu, Rahman, & Hamdan, 2019). These recently developed electrospun scaffolds

including PVA and chitosan can fulfill most of the very essential requirements for

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accelerated wound healing including minimized infections (Fathollahipour, Abouei

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Mehrizi, Ghaee, & Koosha, 2015; Dubey & Gopinath, 2016; Abdeen, El Farargy, &

Negm, 2018; Ruiz et al., 2019; Nguyen et al., 2019). Poor solubility, slower and
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uncontrollable biodegradation rate, low strength and low thermal stability of these

kinds of nanofibers are the disadvantageous properties for wound dressing

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application. Design of bio-nanocomposite and adding other materials to solve these
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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, &
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Nagane, 2001). By incorporating the honey in nanofibers, the formation of

granulation tissue, activation of fibroblasts, thickness of epidermis and collagen

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fibers, decrease infection, inflammation, edema and dehiscence are increased and
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thermal stability and strength of them are enhanced (Ghaderi & Afshar, 2004;

Subrahmanyam, Sahapure, & Nagane, 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. Antibacterial, antioxidant and anti-inflammatory of Nepeta

spices attract 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 nanocomposites (Naeimi,

Amiri, & Ghasemi, 2017; Noghi, Naeimi, & Hamidian, 2018; Sedri, Naeimi, &

Mohammadi, 2018; Naeimi, Honarmand, & Sedri, 2019) for different applications,

here, we report the first bio-nanofiber including Nepeta dschuparensis plant on the

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burns treatment. The main aim of this paper was the design of a PVA/Chit@Nep/Hon

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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
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sulfadiazine cream, as a topical antibiotic using in partial thickness and full thickness

burns, to show the effect of honey and desired plant.

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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

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molecular weight, 400000 Da) were purchased from Alderich and Fluka Company,

respectively. Ethanol, formalin, ketamine, normal saline (0.9%), hematoxylin and

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eosin were supplied from Merck Company. Silver sulfadiazine 1% cream was
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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 performed 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).

Experimental

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

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evaporator. The dried sample stored at 4 °C.

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Electrospinning of PVA/Chit@Nep/Hon bio nanofiber
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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
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prepared solution was stirred for 7 hours at 80 °C. The electrospinning of the final
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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
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flow rate of 0.5 ml/h.

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Animals
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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 ºC) and 12

hours light/dark cycle. The study was performed according to the animal ethics
committee guidelines for the use of experimental animals.

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 maintaining on the back of rats for 5 second (Walker & Mason Jr, 1968).

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Experimental animals were divided into four groups as follows: Group (I) the burned

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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
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and Group (IV) PVA/Chit@Nep/Hon nanofiber was applied daily. Silver sulfadiazine

and bio-nanofibers and were applied topically to cover the wound area every 24 hours
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for three times within 7, 14 and 21 days of experiment.
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Wound closure

In order to evaluate the wound closure, wound images were taken on day zero, 7th,
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14th and 21th day of treatment. The percentage of wound closure was calculated by the

following formula:
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Wound closure (%) =

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Where, A0 and At are the wound sizes at initial and time t, respectively.

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).

Statistical analysis

All data are expressed as Mean ± Standard Deviation (S.D.). A statistical software

package, SPSS version 19 was used to perform statistical analysis. Data were

analyzed by ANOVA, followed by post hoc LSD test. Statistical significance was

accepted at P<0.05.

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Results and discussion

PVA is used in a variety of medical applications because of its biocompatibility, low

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tendency for protein adhesion, and low toxicity. Hence, PVA, natural polymer

(chitosan), medicinal plant (Nepeta dschuparensis) and honey were mixed under
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ultrasonic irradiation and PVA/Chit@Nep/Hon bio-nanofibers was obtained using
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electrospinning instrument (Scheme I).

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Scheme I. Preparation and components of PVA/Chit@Nep/Hon bio-nanofiber
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To confirm shape and morphology of this fiber, SEM and TEM were investigated.
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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
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electrospun PVA/Chit nanofiber with 95-150 nm of sizes. Upon increasing the honey

and Nepeta dschuparensis, a granular morphology appeared on the surface of

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PVA/Chit fibers (Fig. 1(b)). It seems that by adding the honey and Nepeta

dschuparensis plant, solution viscosity and also the fiber diameters were enhanced.
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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.
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Fig.1. SEM images of electrospun PVA/Chit (a) and PVA/Chit@Nep/Hon (b) nanofibers

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Fig.2. TEM images of electrospun PVA/Chit (a) and PVA/Chit@Nep/Hon (b) nanofibers
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Chitosan as a hydroxyl-rich structure along with amine group could be act with

Nepeta dschuparensis plant and honey. The FT-IR of electrospun

PVA/Chit@Nep/Hon and PVA/Chit nanofibers were confirmed their purity, structure

and functional groups (Fig.3). The FT-IR 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 C-C and C-O 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.

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A good and effective interaction and more connection between the component of

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electrospun PVA/Chit@Nep/Hon nanofiber were made it strength (Jin & Bai, 2002;

Grande & Carvalho, 2011; Biranje, Madiwale, & Adivarekar, 2017).

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Fig.3. FT-IR of electrospun PVA/Chit and PVA/Chit@Nep/Hon nanofibers

Thermal characteristic of PVA/Chit nanocomposite and PVA/Chit@Nep/Hon

nanofibers were obtained using TGA (Fig. 4 (a) and 4 (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

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that looks like char. In fact, this char like a layer was acted as an effective barrier to

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the permeation of oxygen improving the char residue. The presence of crystalline

structure and great compactness between the honey and Nepeta dschuparensis plant
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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

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these results very well (Grande & Carvalho, 2011).
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Fig.4. Thermal properties of PVA/Chit nanocomposite (a) PVA/Chit@Nep/Hon nanofibers (b)

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Wound closure (%) analysis was performed to evaluate the wound healing properties
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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
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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
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percentage in group (IV) which treated with PVA/Chit@Nep/Hon (14.92± 0.008) was

significantly higher than control group (2.86± 0.034), silver sulfadiazine treated rats
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(4.74 ± 0.009) and PVA/Chit (11.42 ± 0.008) group (P<0.05 and P<0.001,

respectively). On the 14th day, the percentage of wound closure in the group (IV)
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(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 undesirable 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 development

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of systemic side effect (Atiyeh, Costagliola, Hayek, & Dibo, 2007). However, deep-

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dermal wounds heal slowly and also silver sulfadiazine may delay the healing process

due to its adverse effects (Hosseinimehr et al., 2010). It should be noted that natural
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product can accelerate wound healing (Shetty et al., 2008; Kim et al., 2009).

Therefore, there were significant differences regarding percentages of wound closure

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between days 7, 14 and 21 of experiments, in all treated groups. The obtained results
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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, & Opanasopit (2014) on electrospun chitosan/polyvinyl

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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
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honey and Nepeta dschuparensis.

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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 sulfadiazine, Group (III) showed animals
treated with PVA/Chit nanofiber and Group (IV) rats were received PVA/Chit@Nep/Hon. Each point
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represented Mean ± S.D. of the wound closure percentage in the related group. “a” shows P value less
than 0.05 between Group IV and Group I treated rats.
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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
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showed in Fig 6-8. Histopathological evaluation was used as general parameter for the

evaluation of wound healing process. Histopathological observations on day 7 were

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shown that sever inflammation and granulation tissue in all the experimental groups.

Moreover, results showed necrosis of superficial tissue and also widespread edema of
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burn wound area of group (I), (II) and (III) (Fig. 6(a), Fig. 6(b) and Fig. 6(c),
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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)).

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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
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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.
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On day 14, results showed sever inflammation include necrosis of superficial tissue

and edema in control, silver sulfadiazine and PVA/Chit treated group. While,
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PVA/Chit@Nep/Hon treated animals showed necrosis tissue, middle inflammation

and neovascularization with compare to group (II) and (III) (Fig. 7(d)).
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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, magnification× 10). Animals with
burn wound without treatment (a), animals treated with 1% silver sulfadiazine (b), animals
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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.
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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
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group (Fig. 8(a)). Silver sulfadiazine administration to Group (II) (Fig. 8(b)) shows

tissue necrosis, edema of burn wound area and severs inflammation in comparison
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with Group (IV) (Fig. 8(d)). Moreover, photomicrograph from group (III) shows that

its tissue necrosis is less than group (I) and (II), respectively (Fig. 8(c)).
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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
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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.
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In burn healing process, the proliferative phase typically demonstrates angiogenesis,

granulation and collagen deposition (Nayak, Pereira, & Maharaj, 2007). The
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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

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histopathological study also revealed that minimal inflammation, angiogenesis and

increased collagen deposition in rats treated with PVA/Chit@Nep/Hon. One of the

contradictory results of this study was that silver sulfadiazine 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-epithelialization occurs as results of keratinocyte

proliferation and migration 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

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nanocomposite showed the good potential for supporting cell attachment and

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proliferation for skin tissue engineering and it is much more effective than Chit/PVA

(this work), Honey (Mohamed et al., 2014), PVA/honey (Tavakoli & Tang, 2017),
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Chit/honey (Movaffagh et al., 2019) and PVA/chitosan along with other compounds

(Movaffagh et al., 2019). The morphology of this electrospun bio nanocomposite is

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similar to the natural extracellular matrix in skin promoting cell adhesion, migration
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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,

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biodegradable and good solubility are unique properties of this bio nanofiber for

wound healing applications. The electrospun PVA/Chit@Nep/Hon nanofiber showed

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a good potential for supporting cell attachment and proliferation for skin tissue
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engineering.

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. This study revealed

that topical application of this bio-nanofiber showed strong wound healing activities

in burn wound area.

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Acknowledgment

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We are thankful to University of Jiroft for their support on this work.

Author contributions
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Atena Naeimi: Conceptualization, Methodology, Writing-Original Draft, Project
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administration, Writing - Review & Editing, Supervision. Maryam Payandeh:
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Visualization, Investigation. Abdollah Ramzani Ghara: Supervision,

Conceptualization, Methodology, Writing-Original Draft. Fereshteh Ezzati Ghadi:

Writing-Original Draft, Resources, Software.

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