Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 912458, 8 pages
http://dx.doi.org/10.1155/2013/912458
Research Article
New Guar Biopolymer Silver Nanocomposites for
Wound Healing Applications
Runa Ghosh Auddy, Md Farooque Abdullah, Suvadra Das, Partha Roy,
Sriparna Datta, and Arup Mukherjee
Department of Chemical Technology, University of Calcutta, 92 A.P.C. Road, Kolkata, West Bengal 700009, India
Correspondence should be addressed to Arup Mukherjee; arupm1234@gmail.com
Received 30 April 2013; Revised 10 August 2013; Accepted 18 August 2013
Academic Editor: Antonio Salgado
Copyright © 2013 Runa Ghosh Auddy et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Wound healing is an innate physiological response that helps restore cellular and anatomic continuity of a tissue. Selective
biodegradable and biocompatible polymer materials have provided useful scaffolds for wound healing and assisted cellular
messaging. In the present study, guar gum, a polymeric galactomannan, was intrinsically modified to a new cationic biopolymer
guar gum alkylamine (GGAA) for wound healing applications. Biologically synthesized silver nanoparticles (Agnp) were further
impregnated in GGAA for extended evaluations in punch wound models in rodents. SEM studies showed silver nanoparticles
well dispersed in the new guar matrix with a particle size of ∼18 nm. In wound healing experiments, faster healing and improved
cosmetic appearance were observed in the new nanobiomaterial treated group compared to commercially available silver alginate
cream. The total protein, DNA, and hydroxyproline contents of the wound tissues were also significantly higher in the treated
group as compared with the silver alginate cream (𝑃 < 0.05). Silver nanoparticles exerted positive effects because of their
antimicrobial properties. The nanobiomaterial was observed to promote wound closure by inducing proliferation and migration
of the keratinocytes at the wound site. The derivatized guar gum matrix additionally provided a hydrated surface necessary for cell
proliferation.
1. Introduction
Wounds inflict disruption of the cellular and anatomic
continuity of tissues and thus affect physiological functions. Wound healing therefore is a complex and important
biological response that helps in the restoration of tissue
integrity and body functions [1]. The process of wound
healing involves a highly integrated cascade of continuous
and overlapping biological events. Cellular discontinuity and
appearance of nascent cells also extend susceptible regions
for microbial infections. Coordinated completion of a series
of biochemical events and protection against invading organisms are therefore necessary for tissue rebuilding, haemostasis, and maturation [2]. The principle objective in wound
management is to heal the wound in the shortest possible
time, with minimal pain, discomfort, and scarring to the
patient, and must occur in a physiological environment conducive to tissue repair and regeneration [3]. Delayed healing
often results in bacterial infections, stress, and nutritional
deficiencies [4]. Currently, the demand for new wound healing materials with inherent antimicrobial properties is on the
rise. Polysaccharides associate low antigenicity and are often
a choice for wound management scaffolds [5, 6]. Chitosan
and different starch derivatives were experimented earlier
as wound healing materials. Polysaccharides are excellent
growth scaffolds, but their wound healing application is
limited as they are vulnerable to microbial contaminations.
Chitosan however is one exception. The cationic surface of
chitosan in effect provides a reasonable surface sterility for
tissue regeneration. This work therefore concentrates on
cationic modification of polysaccharide end groups so that
new material surfaces can be developed for facilitated tissue
regeneration during wound management. Guar gum is a
nontoxic biopolymer derived from the seeds of Cyamopsis tetragonoloba. Because of its high biocompatibility and
biodegradability, it is used extensively as a biomaterial in
2
a plethora of biological and technological processes [7]. Guar
and xanthan gums were patented earlier as bioabsorbable
materials for wound dressing [8]. Guar gum can be readily
modified by surface chemical functionalization to broaden its
application in different areas [9]. The intrinsic modifications
of the guar backbone are known to enhance stability and
water-absorbing capacity [10]. Metal nanoparticles on the
other hand are attractive tools in photothermal and cellular
drug-delivery applications [11]. Among the different metal
nanoparticles used, silver nanoparticles are gaining importance in biomedical applications due to their unique optical
properties related to surface plasmon resonance and antimicrobial properties [12, 13]. Wound healing is often delayed by
bacterial infestation at the wound site, and the inflammatory
phase becomes chronic suppressing the regenerative phase.
The antibacterial effect of silver was also applied earlier
in antimicrobial material coatings and biological material
decontamination [14, 15]. Silver nanoparticles impregnated in
biocompatible polymeric matrix were therefore conceived for
antimicrobial wound healing material. We intrinsically modified guar gum, galactomannan, into a new derivative guar
gum alkylamine (GGAA). This GGAA was further loaded
with silver nanoparticles (Agnp) and new nanocomposites
(NAg-GGAA) developed for skin wound healing evaluations.
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3. Characterization of the Silver Nanoparticles
3.1. UV-Vis Spectroscopy. Plasmon response was recorded in
a UV-vis spectrophotometer (Shimadzu UV-2550). The samples were dissolved in HPLC water and were scanned at slow
speed at a resolution of 0.5 nm.
3.2. X-Ray Diffraction Analysis. XRD was used to determine
material crystallinity and the characteristics of the silver
nanoparticle in the new nanobio material. Samples were
exposed to a generator voltage of 45 KV at 25 mA using Cu
K𝛼 radiation in a PANalytical model PW 3040/60 X’Pert Pro
defractometer. The diffraction angle (2𝜃) range of observation
was 0–100∘ in a continuous scan mode at a scan rate of
0.5∘ /min at constant temperature of 22 ± 1∘ C.
3.3. Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Device (EDX). The surface morphology of
samples was examined in a scanning electron microscope
(Philips, XL30) equipped with an energy dispersive X-ray
device (EDX) attachment. SEM was operated at 10 kV after
spattering the dried samples with gold. EDX spectrum was
recorded from the samples by focusing the electron beam at
specific regions of the nanomaterial.
2. Materials and Methods
3.4. Wound Healing Experiments
2.1. Materials. All reagents were of analytical grade. Silver
nitrate, dextrose, ammonium hydroxide, benzoyl chloride,
guar gum (GG), sodium hydroxide, acetone, hydrochloric
acid, and dimethyl sulfoxide were procured from Merck
(India). Solvents and epichlorohydrin were purchased from
Spectrochem (India).
3.4.1. Experimental Animals. Male Wistar rats 150–200 g
were used in the wound healing experiments. Animals were
acclimatized for 7 days prior to the start of experiments in
the laboratory housing conditions of 26∘ C ± 2∘ C, 60–70%
RH, and 12-hour light and dark cycle. All the experimental
procedures and protocols used in this study were prepared as
per the OECD Guidelines and Gaitonde Committee Guidelines and were approved by the Institutional Animal Ethical
Committee (IAEC, registration no. 506/05/b/CPSCA). Animals were maintained with pellet foods available from Lipton
India Pvt. Ltd, and water was allowed ad libitum.
2.2. Preparation of Guar Gum Alkylamine. A three neck
round bottom flask (500 mL), fitted with a condenser, a mechanical stirrer and a nitrogen gas inlet, was used for the
reaction. Typically, 25 g of GG was reacted with epichlorohydrin in liquor ammonia for 2 h at 55 ± 5∘ C under stirring
and nitrogen purging [10]. The resultant guar gum alkylamine
(GGAA) was filtered, washed in isopropanol, water and
ethanol before being dried at 60∘ C in vacuum desiccators. The
yield average recorded after six independent experiments was
19 g.
2.3. Preparation of Silver Nanocomposites In Situ. Silver nanoparticles stabilized in GGAA were prepared by reduction of
ionic silver in alkaline (Na2 CO3 ) dextrose. Twenty mM silver
nitrate solution in 40 mL of 0.5% w/v GGAA was warmed
(60∘ C), and a mixture of 2 mL, 25 mM dextrose in sodium
carbonate (2 mL, 4 mM) was added for the reduction over a
period of 10 min. The final straw yellow nanobio conjugate
(NAg-GGAA) was separated upon addition of 50 mL acetone
under stirring. The final products were nanoparticle embedded powders. The powdered product was washed thrice in
100 mL of 50% ethanol and subsequently dried in air oven at
60∘ C.
3.4.2. Punch Wound Model. Twenty-four hours before the
beginning of wound healing experiments, the dorsal skin
of the rats was shaved. The next day, full thickness wounds
of 8 mm diameter were inflicted on the back side of each
rat using a special type of sterile circular blade, Acu-Punch
(Acuderm Inc., FL, USA), under light ether anesthesia. The
animals were then divided into 4 groups (𝑛 = 6):
Group I: normal paraffin treated (negative) control,
Group II: guar gum alkylamine (GGAA) treated
group,
Group III: silver nano-GGAA (NAg-GGAA) treated
group,
Group IV: silver alginate cream (positive control).
Special care was taken for maintaining aseptic condition
throughout the experiments. Test samples were dispersed in
sterile water and were applied on the wounds topically at
a fixed time each day. Silver alginate was also administered
topically.
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3.4.3. Determination of Wound Healing Rate
Table 1: Arbitrary scoring system for the measurement of wound
index.
Wound Closure Measurements. The extent of wound closure
was estimated by tracing the wound margin on a transparent
graph paper in mm scale on days 0, 5, 7, and 10, respectively
[16, 17]. The readings were indicative of the area of wound
closure expressed in mm2 on respective days. The evaluated
surface area was further used to determine the percentage of
wound contraction on day 10. The following formula was used
to determine % wound contraction:
Original wound area on day 0 − wound area on day 10
Original wound area on day 0
× 100.
(1)
Measurement of Wound Index. Wound index was measured
daily by an arbitrary scoring system (Table 1) [17].
3.4.4. Measurement of Tensile Strength. Tensile strength of
wound indicates collagenesis of the healing process. The force
required for opening the healed skin area is used as an
indicator for completeness of healing. The tensile strength
of newly repaired tissue of the wounds was measured using
a tensiometer (M/S Excel Enterprises, Kolkata) after 10 days
and were expressed in gm [17].
3.4.5. Estimation of Biochemical Markers. Synthesis and deposition of proteins mark the initiation of the healing process
after wounding. The protein and DNA contents reflect the
process of cytokinesis during healing process [18]. The quality
and quantity of protein deposited during the healing process
significantly influence the strength of a scar. More than 50%
of the protein in the scar tissue is made up of collagen,
and collagenesis is essential for the healing process [19].
Hydroxyproline, the basic constituent of collagen, is an
important marker of collagen synthesis [20].
For estimations of total protein and DNA content, the
healed wound tissue, after complete healing (10th day), was
excised using the 8 mm acupunch to avoid contamination
with the host tissue. The designated tissues were then subjected to homogenization in 5% TCA and centrifuged [21].
The resultant pellet was washed with 10% TCA, resuspended
in 5% TCA, and kept for 15 min in a water bath maintained
at 90∘ C. The contents were centrifuged, and aliquots of
DNA were derived from the supernatant for estimation by
the method of Burton [22]. The precipitated proteins were
suspended in 0.1 M Tris-HCl, pH 7.4, and the protein content
was estimated by the method of Lowry et al. [23].
Tissues were dried in a hot-air oven at 60–70∘ C to get
constant weight for measurement of hydroxyproline content.
Weighed parts were digested in 6 N HCl at 130∘C for 4 h
in sealed tubes, and pH was adjusted to 7.0. Samples were
further subjected to chloramines-T oxidation for 20 min,
and the reaction was terminated by the addition of 0.4 M
perchloric acid. Ehrlich reagent was used to develop color at
60∘ C, and the absorbance was measured at 557 nm using a
spectrophotometer (Shimadzu UV-2550) [24].
Gross changes
Wound index
1
2
Complete healing of wounds
Delayed but healthy healing
No initiation of healing, but the
environment is healthy
Formation of pus: evidence of necrosis
4
Total
10
3
3.4.6. Histopathological Study of the Regenerated Tissues.
Granular tissue samples from all groups collected upon
sacrifice on day 10 were preserved immediately in 10%
formalin solution and were evaluated by routine hematoxylin
and eosin staining for histological criteria. The extent of reepithelialization, maturation, and organization of the epidermal
squamous cells, the thickness of the granular cell layer, matrix
organization, and cellular infiltration were observed.
3.4.7. Toxicological Evaluation of NAg-GGAA
Primary Skin Irritation Experiment. A primary skin irritation
study was conducted with albino rabbit to determine the
irritation potential of the NAg-GGAA. Each animal was
treated with 500 𝜇L of NAg-GGAA and applied to the skin
of one flank using a gauze patch. The patch was held in
place with a semiocclusive bandage for 4 h, after which the
patch was removed and the skin was cleaned of residual test
drug. Skin reactions and irritation effects were assessed at
approximately 1, 24, 48, and 72 h after the removal of the
dressings. Adjacent areas of untreated skin from each animal
served as controls. Erythema and edema were scored on a
scale of 0–4, with 0 showing no effect and 4 representing
severe erythema or edema.
3.5. Statistical Analysis. All the results presented here are
shown as mean ± standard error of mean (SEM). The statistical significance was assessed using one-way analysis of variance (ANOVA) with post hoc pairwise comparisons between
groups using the Bonferroni method. For all analyses, 𝑃 <
0.05 was considered to be significant. Statistical analysis was
performed using the computer statistical package SPSS/10.0
(SPSS, Chicago, IL, USA).
4. Results and Discussion
Wound management in the shortest possible time is of utmost
importance, and the search for new biocompatible materials
has led scientists to utilize the potential of polysaccharides
derived from plants as wound management aids [5]. Polysaccharides or their derivatives can provide the ideal conditions for enhancing the wound healing process by actively
participating in the wound healing process by facilitating
cellular messaging and providing a hydrated surface. Silver
is known for its antimicrobial activity since antiquity. Silvercontaining drugs are also prevalent in markets and are applied
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Table 2: Physicochemical characterization of guar gum and its newer derivatives.
I
II
to treat different kinds of wounds. However, many of these
silver preparations cause cosmetic abnormality (argyria) and
delayed wound healing for ionic silver reactions in biological
fluids [25]. Silver nanoparticles associate strong antimicrobial
potentials and are gaining importance due to their high surface area to volume ratio and unique physical and chemical
properties [26]. We developed a newer preparation based on
silver nanoparticles matrixed in hydropolymer guar gum for
wound healing applications.
4.1. Physicochemical Characterization of Guar Gum Alkylamine. Guar gum is a galactomannan in which one unit
is substituted in every second galactose with a monomer
mannose arranged in a chain. The new cationic modification
is dependent upon incorporation of alkylamine groups on
exposed hydroxyls. GGAA also forms stable films by itself
or upon further hydrophobic substitution [27]. The new
biomaterial properties were therefore dependent upon the
number of head group substitutions on guar galactomannan
backbone.
4.1.1. CHN Analysis and Degree of Substitution. Biopolymers
were analyzed for C, H, N and percentage (w/w) composition
and the degree of substitution. GGAA composition observations were C, 48.57%, N, 3.28%, H, 6.59% as compared to
those of GG, C, 30.2%, and H, 4.79%. The degree of substitution for GGAA was calculated from nitrogen percentage
estimations and was recorded as 0.457 (Table 2).
4.2. Characterization of the Silver Nanoparticles
4.2.1. UV-Vis Spectroscopy. The UV-Vis absorption spectra
of the GGAA and NAg-GGAA were recorded in aqueous
solution and are shown in Figure 1. The 𝜆 max of silver
nanoparticles was observed at 415 nm. A remarkable broadening of peak at around 350 nm to 550 nm is indicative of
polydispersity of the particles in the polymeric matrix.
4.2.2. X-Ray Diffraction Pattern. The XRD patterns of silver
nanoparticles indicated a crystalline nature (Figure 2). Silver
oxide peak at 2𝜃 38 degrees was negligible indicating purity
of nanoparticles even after storage. This is likely due to
GGAA protective capping of silver nanoparticles. Hundredpercent crystalline peak was observed at 2𝜃, 72 degree and
corresponded with a near-spherical structure.
4.2.3. Scanning Electron Microscopy. Scanning electron microscope pictures exhibit the surface morphology of biomaterials. Biopolymer embedded nanoparticles were observed
Degree of substitution
—
0.457
0.300
Abs
Compound no.
Percentage composition CHN analysis in combustion technique
Calculated %
Observed %
Code
Compound name
C
H
N
C
H
N
GG
Guar gum
32.9
4.57
—
30.2
4.79
—
GGAA
Guar gum alkylamine
45.56
5.60
2.53
48.57
6.59
3.28
0.150
0.000
200.00
500.00
(nm)
800.00
Figure 1: UV-spectra of GGAA and NAg-GGAA. Colour codes:
GGAA (purple) and NAg-GGAA (yellow).
Iobs (cts)
450
400
350
300
250
200
150
100
50
0
0
20
40
60
Position (2𝜃)
80
100
120
Figure 2: X-ray diffraction pattern of NAg-GGAA.
in SEM studies and EDX studies (Figures 3(a) and 3(b)).
The particles appeared dispersed uniformly in the polymeric matrix. The EDX spectra of the nanocomposite
material showed the presence of characteristic silver peaks
(Figure 3(b)) which confirms that reduction of Ag+ ions to
nanocrystalline elemental silver.
4.3. Wound Healing Properties of NAg-GGAA
4.3.1. Effect on Wound Dimensions. The extent of wound closure was estimated by tracing the wound margin on
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C
Spectrum 8
N
Ag
O
Ag
Ag
Ag
0
0.5
1
1.5
2
2.5
3
Full scale 168 cts cursor: −0.026 (2652 cts)
(keV)
(a)
3.5
4
5
4.5
(b)
a transparent graph paper in mm scale on days 0, 5, 7, and
10, respectively. The readings were indicative of the area
of wound closure expressed in mm2 . The evaluated surface
area was further used to determine the percentage of wound
contraction on day 10 [28]. On the 5th day, the test group
showed a significant reduction of wound size (13.8 mm2 ) in
comparison to that of the untreated control (45.25 mm2 ).
Complete closure was observed on day 10 (Figure 4).
Similarly, on comparing the percentage of wound contraction, it was observed that the extent of healing was higher
in NAg-GGAA group (∼98%) in comparison to the silver
alginate cream (∼90%) with respect to day 0 wound size (𝑃 <
0.05). GGAA also showed about 69% reduction in wound size
on day 10 compared to day 0 (Table 3). On day 10, however,
only ∼54% of the wound was closed in the untreated control
group.
4.3.2. Effect on Wound Index. The mean wound indices on
days 3, 5, 7, and 10 of each of the treatment groups were
considered for evaluation. On day 10, the NAg-GGAA and
silver alginate treated groups showed mean wound index of
1.35 and 1.56, respectively (𝑃 < 0.05), compared to untreated
control. Mean wound indices of GGAA treated group also
showed significant reduction (𝑃 < 0.05) as compared to
control.
4.3.3. Effect on Healing Period. On evaluating the number
of days required for complete healing it was observed that
the NAg-GGAA treated group showed complete healing on
the 10th day. Silver alginate group took a healing time of 12
days for complete healing. Both are highly significant (𝑃 <
0.001) in comparison to the untreated control group which
took 18 days to heal. Statistically significant faster healing was
observed in NAg-GGAA treated group as compared to silver
alginate group (𝑃 < 0.05). GGAA treated group also showed
faster healing time (15 days, 𝑃 < 0.05) as compared to control
(Table 3).
Open wounds are vulnerable to bacterial infections which
interfere with the healing process and cause delayed healing.
Silver nanoparticles exhibit enhanced rate of antimicrobial
activity due to their unique physical properties such as high
Wound area (mm2 )
Figure 3: (a) SEM study of NAg-GGAA and (b) EDX spectra of NAg-GGAA showing the presence of silver peaks.
90
80
70
60
50
40
30
20
10
0
Day 0
NAg-GGAA
Silver alginate
Day 5
Day 7
Days of healing
Day 10
GGAA
Untreated control
Figure 4: Area of wound closure in mm2 at 0th, 5th, 7th and 10th
day. Results are mean ± SEM, 𝑛 = 6 in each group, a 𝑃 < 0.05
compared to control group, b 𝑃 < 0.05 compared to GGAA treated
group, c 𝑃 < 0.05 compared to silver alginate group.
surface area to volume ratio and plasmon response. The
antimicrobial activity of silver nanoparticles can be exerted
by their interference with the cell membrane permeability
causing degradation of the bacterial cell [29]. Additionally,
silver nanoparticles can promote the rate of wound closure by
promoting proliferation and migration of the keratinocytes
at the wound site [30]. Also, silver nanoparticles can induce
the differentiation of fibroblasts to myofibroblasts resulting in
faster wound contraction.
Silver nanoparticles however tend to agglomerate, and
they are short-lived in the circulation. As a result, silver
nanoparticles are incorporated in suitable polymer matrix
for the production of highly stable and uniformly distributed
silver nanoparticles. The silver impregnated guar matrix
provides a sustained release of silver while exerting its antimicrobial activity [31]. The polymeric matrix also provides a
hydrated surface for wound healing. Thus, in most of the
parameters the GGAA treated group showed positive result
with respect to the control.
4.3.4. Tensile Strength in the Healing Wounds. A key parameter in healing involves regaining strength of the regenerated
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Table 3: Changes in physical characteristics of wounds in different treatment groups.
Treatment groups
Untreated control
GGAA treated
NAg-GGAA treated
Silver alginate
Percentage wound contraction on the 10th day
54.58 ± 3.45
69.64 ± 4.86a
98.52 ± 7.54a,b,c
89.15 ± 6.96a,b
Wound index
2.69 ± 0.08
1.94 ± 0.06a
1.35 ± 0.05a,b
1.56 ± 0.05a,b
Healing period (days)
18.83 ± 0.48
15.50 ± 0.43a
9.83 ± 0.31a,b,c
12.5 ± 0.43a,b
Tensile strength (g)
257.2 ± 10.73
356.8 ± 9.88a
522.5 ± 12.9a,b, c
460.5 ± 11.66a,b
Values were mean ± SEM, 𝑛 = 6 in each group. 𝑃 < 0.05 compared to control group, 𝑃 < 0.05 compared to GGAA treated group, 𝑃 < 0.05 compared to
silver alginate group.
c
b
a
Table 4: Changes in biochemical parameters of wound tissues in different treatment groups.
Treatment groups
Untreated control
GGAA treated
NAg-GGAA treated
Silver alginate
DNA (mg/g tissue)
1.02 ± 0.06
1.30 ± 0.03a
2.36 ± 0.04a,b,c
2.04 ± 0.09a,b
Total protein (mg/g tissue)
15.13 ± 0.18
16.98 ± 0.18a
23.91 ± 0.19a,b,c
21.27 ± 0.37a,b
Hydroxyproline (𝜇g/mg tissue)
0.81 ± 0.02
1.30 ± 0.03a
2.36 ± 0.04a,b,c
2.04 ± 0.09a,b
Values were mean ± SEM, 𝑛 = 8 in each group. 𝑃 < 0.05 compared to control group, 𝑃 < 0.05 compared to GGAA treated group, 𝑃 < 0.05 compared to
silver alginate group.
a
dermal matrix. Tensile strength reflects the quality and speed
of tissue regeneration. Tensile strength is directly related
to collagen content of wounds [32]. Collagen is the main
element responsible for tissue integrity and provides a platform for reepithelialization and is essential for restoring skin
functionality during injury. The mean tensile strength of the
NAg-GGAA treated group (522 g) was significantly greater
(𝑃 < 0.05) than that of the untreated control group (257 g)
and silver alginate group (460 g) (Table 3). Higher tensile
strength of GGAA-silver-nanocomposite treated wounds
indicates better quality healing than the silver alginate treated
wounds. The increase in tensile strength of treated wounds
may be due to the increase in collagen concentration and
twisting of the collagen fibers. The greater the tensile strength,
the better the healing. The NAg-GGAA particles possibly
modulate collagen deposition at the site of the wound during
the healing process and promote a regulated differentiation
of fibroblasts [33]. GGAA treated group also showed higher
tensile strength (357 g) as compared to control (Table 3).
4.3.5. Changes in the Biochemical Markers of the Healed
Wounds. DNA and total protein content are indicative markers of cell growth after tissue injury [34, 35]. The hydroxyproline content, DNA, and total protein contents of granulation
tissues after complete healing are given in Table 4. The mean
hydroxyproline content was found to be significantly higher
in all treatment groups when compared to the untreated
control (𝑃 < 0.01). Thus, high levels of hydroxyproline
indicate higher collagen production which is essential for the
healing process [17]. The DNA content in wounds treated
with NAg-GGAA and silver alginate treated groups was
significantly higher than in the untreated control group
(𝑃 < 0.001). However, DNA content of wound tissue of NAgGGAA treated group was statistically higher (𝑃 < 0.05) than
that of silver alginate treated group. Similar phenomenon was
noted in case of total protein content of wound tissues where
b
c
NAg-GGAA and silver alginate treated groups showed higher
protein contents (23.9 and 21.3 mg/gm wet tissue, 𝑃 < 0.001)
when compared to the untreated (paraffin treated) control
group (15.1 mg/gm wet tissue) but total protein content of
NAg-GGAA treated wound tissue was statistically higher
(𝑃 < 0.05) than that of silver alginate treated group. Increased
DNA content may be related to the upregulation of zinc
metabolism which leads to enhanced production of RNA and
DNA synthetases [36].
Thus, the higher levels of the major biochemical markers (compared to the untreated control) indicate cellular
proliferation at the wound site and thereby faster healing
of wound. Interestingly, the levels of all of the selected
biochemical markers were statistically higher (𝑃 < 0.05) in
NAg-GGAA treated group than that of silver alginate treated
group indicating faster and quality healing (Table 4).
4.3.6. Histopathological Study of the Regenerated Tissues.
Hematoxylin and eosin (H&E) stained sections of granulations tissue on day 10 of the untreated control animals
(Figure 5(a)) showed the presence of acute inflammatory cells
and very few blood vessels which were prominent and dilated.
It also showed lesser epithelialization and lesser collagen
formation. In contrast, a well organized granulation tissue
was observed in the NAg-GGAA treated group (Figure 5(d)).
New blood vessel formation, epithelialization, and increase
in fibroblast cells were observed in the silver nano-GGAA
treated group. The silver alginate treated group showed granular tissue regeneration and new blood vessel formation but
poor epithelialization (Figure 5(c)). The GGAA (Figure 5(b))
showed regenerative changes with poor epithelial layer formation. These results indicate that the tissue architecture
was well advanced on day 10 in the Ag-nano-GGAA treated
group compared to the other groups. Further, the cosmetic
appearance was much improved in the NAg-GGAA treated
group.
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(a)
(b)
(c)
(d)
Figure 5: Hematoxylin and eosin stained granulation tissue at day 10. (a) Untreated control group, (b) GGAA treated group, (c) silver alginate
cream with thin layer of epithelialization, and (d) NAg-GGAA treated group showing well organized granulation tissue and epithelialization
(magnification 40x).
4.3.7. Dermal Toxicity Study. No irritation was observed following the 4 h dermal exposure of NAg-GGAA over the skin
of the test animals.
5. Conclusion
We have developed silver nanocomposites embedded in
cationic guar gum polymeric matrix. The new nanobiocomposite was further evaluated for wound healing application
in rat punch wound model. The new GGAA matrix provided
stabilization of silver nanoparticles. The embedded spherical
silver nanoparticles were well dispersed in the polymeric
matrix. Wound healing experiments with NAg-GGAA have
demonstrated the superior wound healing efficacy of the new
nanocomposite as compared to that of silver alginate cream
in parallel runs. The nanobiocomposite promote wound
healing by modulation of collagen deposition and regulation
of keratinocytes and support the essential re-epithelialization
process.
Acknowledgments
The authors would like to thank the Centre for Research in
Nanoscience and Nanotechnology, University of Calcutta, for
extending financial assistance. The Research Associateship to
Partha Roy and Senior Research Fellowship to Suvadra Das
from the Council of Scientific and Industrial Research are
gratefully acknowledged. Md Farooque Abdullah is thankful
to the University Grants Commission (India) for the award of
Maulana Azad fellowship.
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