Keywords
lemongrass - dressing - wound healing - gingivectomy
Introduction
Oral cavity is a habitat of millions of pathogenic and opportunistic bacteria that
are favorable for infections in the presence of any cut or wound in the epithelium.
Surgical therapy performed in the oral cavity will result in oral wound, and without
controlling the microbial activity, the wound healing delays and the treatment fails.
Periodontal pack and periodontal dressing are the terms used for periodontal bandages.
To cover and protect wound surfaces, it is used after oral surgery, frequently applied
around the necks of the teeth, and adjacent tissue. In earlier days, dressing acts
as a protective barrier and has no curative properties. It is suggested to be used,
as it helps in reducing discomfort and pain postoperatively by shielding the site
of surgery even without any therapeutic effects.[1] On the other hand, several studies showed that the use of periodontal dressing has
little beneficial effects, increases bacterial plaque accumulation around surgical
sites, irritates healthy tissue, increases chance of infection and inflammation, and
causes difficulty in eating.[2]
[3] Therefore, some of clinicians would prefer the wound site left without any dressings.
One of the surgical therapies frequently performed in dentistry is gingivectomy, defined
as surgical removal of the gingiva to the bottom of the pocket.[4] It is indicated in cases of gingival enlargement where curettage is not able to
reduce the excessive tissue. Nevertheless, gingivectomy using surgical blade or knife
may create an extensive gingival wound and after the surgery is done, no sutures are
placed, therefore the surgical sites should be covered to prevent postoperative infection
and to accelerate healing. In this case, the role of periodontal dressings on gingival
healing after gingivectomy is still of considerable interest to oral surgeons.
Many attempts have been made to enhance the properties of dressings, that is, incorporating
chlorhexidine or antibiotics as antibacterial properties, to improve healing process
and prevent infections.[5]
[6] Even though the addition of these agents is beneficial, possible problem may occur
when antibiotics are involved, namely, emergence of resistant bacteria, opportunistic
infections, the possibility of sensitization and allergy, and the potential development
of candidiasis.[7] Therefore, alternatives of active compound should be discovered as a substitute
and to avoid the adverse effect of antibiotics incorporated in dressings. Furthermore,
it would be more favorable if the dressing will not serve as bacterial plaque retentive
factor and do not interfere with mastication activity.
Among various types of periodontal pack, resorbable dressing offers advantages, since
it does not require removal and can be safely degraded itself. It is a soft and soluble
dressing that adheres to the wound surface, dissolves in approximately 30 hours and
is consisted mostly of cellulose as a base which is a hydrophilic, elastic material
with several active agents incorporated.[8] In this present study, we evaluate the effect of a resorbable pack with lemongrass
extract as active ingredient. Lemongrass (Cymbopogon citratus) is a perennial herb belongs to Poacea, generically named as grasses.[9] Originating from the Southwest Asia, now it grows spontaneously around the world,
especially in the tropical and savannah regions.[10] Lemongrass has been widely used as a traditional remedy by preparing the “tea” or
infusion from fresh or dry leaves in almost all the continents and it comprises a
wide range of indications, from mild conditions such as flu, fever, cuts, coughing,
and headaches to more severe illness such as rheumatic, bladder disorder, diabetes,
and malaria.[11]
[12]
[13]
[14] It is considered as a potent antibacterial, antitussive, antiseptic, analgesic,
and anti-inflammatory agent.[15]
[16]
[17]
[18] The lemon-scented leaves of lemongrass is also used for insect repellent.[19] Studies discovering the use of lemongrass in dentistry showed that lemongrass oil
prepared as mouthwash is effective in management of candidiasis, periodontitis, and
as antidental plaque agent, and in the form of gel, it had been shown to reduce pocket
in periodontitis patients.[20]
[21]
[22]
[23]
[24] However, little is known about the efficacy of lemongrass as active agent in periodontal
dressing. We hypothesized that lemongrass wound dressing may enhance the healing process
in terms of growth of fibroblasts and collagen deposition.
The present study aimed to evaluate the effect of lemongrass wound dressing on the
healing process microscopically after gingivectomy in rats.
Materials and Methods
This study evaluated and compared the effects of lemongrass periodontal dressing,
povidone iodine gel, and a cellulose-based periodontal dressing with myrrh as active
ingredient already available in the market (Reso-pac, Hager & Werken GmbH & Co. KG,
Germany) on wound healing after gingivectomy in rats assessed by fibroblast proliferation
and collagen deposition.
Experimental Animals
In this study, 30 male Sprague–Dawley rats, weighing 250 to 350 g were recruited.
One week before the experimental procedures, the animals underwent adaptation at air-conditioned
animal laboratory room (22 ± 3°C) with 12-hour light and dark cycle. The rats were
fed with commercial normal rodent pellet and filtered water ad libitum. All of the
animals received humane care according to the criteria outlined in the Guide for the
Care and the Use of Laboratory Animals prepared by the National Academy of Science
and published by the National Institute of Health.[25] All of the experimental procedures were performed in Tropical Biopharmaca Research
Centre, Bogor Agriculture Institute, West Java, Indonesia.
The experimental design and protocols were reviewed and approved by the Ethics Committee
of the Experimental Animal Care Society, Biopharmaca Research Centre, Bogor Agriculture
Institute, West Java, Indonesia, under file number 004–2017 KEH TROP BRC.
Preparation of Lemongrass Ethanol Extract
Lemongrass was cultivated in Subang, West Java Province, Indonesia at 6° 34' 17.7204''
S 107° 45' 31.4496'' E, 700 m above sea level and collected in September 2016. Plant
authentication was performed by a plant taxonomist with collection number BMK0204092016
deposited in Herbarium Bogoriense, Department of Biology, Bogor, West Java, Indonesia.
Fresh lemongrass of 5 kg was washed, minced, and dried in an oven for 3 days at 50°C.
Dried leaves were then ground to powder. Maceration of lemongrass was performed by
soaking powder in 70% ethanol, with a ratio of 1:10 for 3 × 24 hours at room temperature
(±27°C) and stirred with a shaker at 150 rpm, then filtered and concentrated by rotary
evaporator at a temperature of 50 to 60°C. Ethanol extract obtained was dark green
in color and tasted bitter.
Preparation of Lemongrass Resorbable Dressing
Dressing was prepared using thick extract of lemongrass, propylene glycol, methyl
paraben, prophyl paraben, aquadest, and hydroxypropilmethyl cellulose (HPMC) as gelling
agent. HPMC was soaked in hot aquadest (80°C) for a period of 2 hours. The dispersion
was then allowed to hydrate and swelled for 15 minutes. Subsequently, methyl paraben
and prophyl paraben were dissolved in ethanol and were added to swollen HPMC. Thick
extract of lemongrass was then poured into the mixture, and then prophylene glycol
was added and stirred until a homogenous gel was achieved. Aquadest was added until
the mixture form a gel base.
Gingivectomy and Experimental Animal Groups
Sample size calculation was performed by using the following formula:
(r – 1) (t – 1) ≥ 15
Where, t = number of groups
r = number of samples
(3–1)(n – 1) ≥ 15
2n – 2 ≥ 15
n ≥ 8.5
The minimum sample size per group was 8.5 rounded to 9. To anticipate drop out, 9
was rounded to 10.
The animals were randomly divided into three experimental groups (n = 10 per group). These groups received treatments as follows:
-
Group P (positive control), rats underwent gingivectomy and were given povidone iodine
gel 10%.
-
Group L, rats underwent gingivectomy and were given lemongrass periodontal dressing.
-
Group M (positive control), rats underwent gingivectomy and were given a cellulose-based
periodontal dressing with myrrh as active agent.
The animals were anesthetized with an intraperitoneal injection of ketamine and xyla
mixture, then a gingivectomy with external bevel excision was made on anterior area
of lower jaw in the labial surface of central incisive, exposing a rectangular area
of 5 mm × 3 mm, using a sterile stainless steel surgical blade no 15 (Aesculap AG,
Tuttlingen, Germany), and subsequently gingiva was separated by periosteal elevator.
Each group received allocated regimen immediately after the wounding procedures by
researcher's assistant. The animals were sacrificed with euthanasia and exsanguination,
three rats for each stage of assessments, that is, at days 4, 7, and 14 after gingivectomy.
Histological Assay
Tissue specimen were excised and immersed at days 4, 7, and 14 after gingivectomy
in alcohol (70, 80, 90, 95, and 100%, respectively) and cleared three times in xylol,
for 1 hour for each cycle. Paraffin was infiltrated to the specimen in three cycles,
each cycle was performed for 1 hour. Specimens were embedded in liquid paraffin, blocked
and sectioned with 5 μm using a microtome. These sections were then stained with hematoxylin
and eosin (H&E) for fibroblast assessment, while Masson's trichrome staining was employed
to reveal collagen deposition, then mounted with cover glass and glued with Entellan.
The stained samples were viewed under electron microscope (Nikon Eclipse 80i) at ×40
magnification in five fields of view.
Statistical Analysis
All data were subjected to statistical analysis using SPSS 20.0 (SPSS, IBM, New York,
New York, United States). To test the normality of the data, Shapiro–Wilk test was
employed, and all data were normally distributed. All values were represented as means ± standard
deviation (SD) and were analyzed using one-way analysis of variance (ANOVA) followed
by Tukey's post hoc for multiple comparisons to measure specific differences between
pairs of means. A p-value of ≤0.05 was considered statistically significant.
Results
All 30 rats survived the surgical procedures with no complications. Findings on each
group were evaluated and histological differences were compared between control and
experimental groups of section.
The growth of fibroblasts and collagen deposition were calculated in days 4, 7, and
14 after gingivectomy. On day 4 following gingivectomy, the comparison of the histological
findings demonstrated that in the group L, fibroblast activity was better expressed
than in groups M and P (p ≤ 0.05; [Table 1]). Tukey's post hoc analysis revealed that there were significant differences in
fibroblast between groups L and P, and between groups L and M with no significant
difference between groups P and M ([Table 2]). Histological assessment showed fibroblast density among groups at day 4 ([Fig. 1]).
Table 1
Fibroblast proliferation on day 4
|
Group
|
Number of fibroblast
|
p-Value
|
|
Mean
|
SD (±)
|
|
P
|
72.4
|
5.6
|
|
|
L
|
105.8
|
17.8
|
0.038[a]
|
|
M
|
72.5
|
14.5
|
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SD, standard deviation.
a Statistically significant, analysis of variance.
Table 2
Tukey's post hoc analysis on fibroblast on day 4
|
Group
|
Group
|
Mean difference
|
SE
|
p-Value
|
|
P
|
L
|
33.40000
|
11.16980
|
0.05[a]
|
|
L
|
M
|
33.3333
|
11.16980
|
0.05[a]
|
|
M
|
P
|
0.06667
|
11.16980
|
1.000
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SE, standard error.
a Statistically significant.
Fig. 1 Histological assessment of fibroblast density among groups at day 4 (hematoxylin
and eosin [H&E], ×40). Fibroblast activity was better expressed in lemongrass group
than in myrrh and povidone iodine group (p < 0.05).
On day 7 following gingivectomy, group L has the most fibroblast proliferation compared
with groups P and M (p < 0.00). The number of fibroblasts in group P was fewer, whereas in group M was moderate
and group L had the greatest ([Table 3]). The Tukey post hoc showed that fibroblast in group L was greater compared with
groups P and to M ([Table 4]). [Fig. 2] showed histological assessment of fibroblast density among groups at day 7.
Table 3
Fibroblast proliferation on day 7
|
Group
|
Number of fibroblasts
|
p-Value
|
|
Mean
|
SD (±)
|
|
P
|
51.6
|
26.9
|
|
|
L
|
281.5
|
11.9
|
0.000[a]
|
|
M
|
116.6
|
22.7
|
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SD, standard deviation.
a Statistically significant, analysis of variance.
Table 4
Tukey's post hoc analysis on fibroblast on day 7
|
Group
|
Group
|
Mean difference
|
SE
|
p-Value
|
|
P
|
L
|
229.9
|
17.55052
|
0.000[a]
|
|
L
|
M
|
164.9
|
17.55052
|
0.000[a]
|
|
M
|
P
|
65.0
|
17.55052
|
0.023[a]
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SE, standard error.
a Statistically significant.
Fig. 2 Histological assessment of fibroblast density among groups at day 7 (hematoxylin
and eosin [H&E], ×40). Fibroblast in lemongrass group was greater compared to myrrh
and povidone iodine group (p < 0.05).
On day 14 following gingivectomy, the comparison of the histological findings demonstrated
that epithelium in group P had started to form, while in groups L and M, the epithelium
was better developed with a greater number of layers and even slight keratinization
([Fig. 3]). Fibroblast number in group P became the highest ([Table 5]), the Tukey post hoc was then employed to find the comparison between pair groups,
and it was shown that no significant differences between groups L and M ([Table 6]). Means of fibroblasts among groups on days 4, 7, and 14 were significantly different
([Table 7]). Increased formations of new collagen were observed in all groups, although the
differences between groups were not statistically significant (p = 0.868; [Table 8]). [Fig. 4] displayed histological assessment of collagen deposition among groups at day 14.
Fig. 3 Histological assessment of fibroblast density among groups at day 14 (hematoxylin
and eosin [H&E], ×40). It was shown that epithelium in povidone iodine group had started
to form, while in myrrh and lemongrass group, the epithelium was better developed,
with a greater number of layers, and even slight keratinization.
Table 5
Fibroblasts proliferation on day 14
|
Group
|
Number of fibroblast
|
p-Value
|
|
Mean
|
SD (±)
|
|
P
|
187.5
|
11.5
|
|
|
L
|
62.6
|
27.7
|
0.001[a]
|
|
M
|
102.2
|
14.2
|
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SD, standard deviation.
a Statistically significant, analysis of variance.
Table 6
Tukey's post hoc analysis on fibroblast on day 14
|
Group
|
Group
|
Mean difference
|
SE
|
p-Value
|
|
P
|
L
|
124.9
|
15.65016
|
0.001[a]
|
|
L
|
M
|
39.6
|
15.65016
|
0.098
|
|
M
|
P
|
85.3
|
15.65016
|
0.004[a]
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SE, standard error.
a Statistically significant.
Table 7
Mean of fibroblast among groups on days 4, 7, and 14
|
P
|
L
|
M
|
p-Value
|
|
Day 4
|
72.5
|
105.8
|
72.5
|
0.038[a]
|
|
Day 7
|
51.6
|
281.5
|
116.6
|
0.000[a]
|
|
Day 14
|
187.5
|
62.6
|
102.2
|
0.001[a]
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone.
a Statistically significant, analysis of variance.
Table 8
Collagen deposition on day 14
|
Group
|
Number of collagen
|
p-Value
|
|
Mean
|
SD (±)
|
|
P
|
58,741.3
|
36,098.3
|
|
|
L
|
71,845.3
|
19,414.1
|
0.868
|
|
M
|
68,916.7
|
35,370.0
|
|
Abbreviations: L, lemongrass; M, myrrh; P, povidone; SD, standard deviation.
Note: Analysis of variance, not significant.
Fig. 4 Histological assessment of collagen deposition among groups at day 14 (Masson's trichrome,
×40). Increased formations of new collagen was observed in all groups although the
differences between groups were not statistically significant (p = 0.868).
Discussion
The present study was conducted to evaluate the effect of lemongrass wound dressing
on the healing process after gingivectomy in rats at histological level. In wound
healing, good tissue growth was defined as a tissue rich in fibroblasts and dense
in newly synthesized collagen determined by Masson's trichrome.[26]
To the best of our knowledge, no studies have been performed to evaluate the effect
of lemongrass extract incorporated in wound/periodontal dressings. The findings of
this present study revealed that on days 4 and 7, the rate of fibroblast proliferation
of the wound was significantly superior following the application of lemongrass periodontal
dressing compared with other groups. Fibroblasts are the main source of extracellular
matrix protein, mostly collagen and fibronectin, that constitutes the newly formed
granulation tissue, thereby providing structural integrity to the wound.[27] In wound healing, an inflammatory phase initiates the process, where platelets,
neutrophils, macrophages, and lymphocytes migrate to a wound. Subsequently, the proliferative
phase shows an increase in fibroblasts and macrophages with a decrease in the acute-phase
reactants. The final phase is a remodeling phase where fibroblasts assist in the reconstruction
of the extracellular matrix and deposit collagen. It can be observed that epithelial
cells start to migrate to the borders of the lesion, and to form the new epithelium,
while fibroblasts continue to proliferate during the first few days postsurgery.[28]
[29] Therefore, fibroblasts play a significant role in the healing of tissue trauma,
including surgical wounds, as well as in epithelization and collagen synthesis.[30]
[31] Our findings showed that fibroblast activity was greater in the lemongrass group,
suggesting that lemongrass pack has a positive effect on wound healing. Furthermore,
in lemongrass and cellulose-based pack groups, epithelium was better developed, indicating
that the proliferation phase characterized by epithelial proliferation had occurred.
This result is in agreement with a study conducted by Primasari and Sinulingga which
observed an increase in the thickness of the oral mucosal epithelium after administration
of lemongrass extract in buccal part of rats following incisions.[32]
We used povidone iodine gel and a cellulose-based dressing containing myrrh as control,
since povidone iodine is considered to have the broadest spectrum of antimicrobial
action and rapid antiviral and antifungal activity. The mechanism of action of povidone
iodine is attributable by the active moiety (non-Polyvinylpyrrolidone [PVP]-bound
“free” iodine) being released into solution from the PVP-I complex, penetrating the
cell wall, and inactivating cells by forming complexes with amino acids and unsaturated
fatty acids, resulting in impaired protein synthesis, and alteration of cell membranes.[33] While myrrh (a mixture of volatile oil, gum, and resin) is a popular herbal compound
that has been commonly used to treat a variety of inflammatory conditions for centuries,
it has been shown to be biocompatible with oral tissues and promotes healing by promoting
earlier remodeling.[34] The healing properties of myrrh may be explained by its induction of maturation
and activation of leucocytes.[35]
Periodontal dressing is placed to protect oral wound against mechanical injuries during
mastication and against bacterial invasion into the tissue, with the aim to decrease
pain and facilitate healing, without any adverse effects such as increase formation
of bacterial plaque, bacterial resistance, cytotoxicity, delayed wound healing, or
disturbed occlusion. Furthermore, the ease of use is also one consideration of choice.
Cellulose-based periodontal pack is soft and unlike conventional periodontal dressings,
it remains elastic at all times, it needs no mixing in preparation and slowly gets
dissolved over a period of 2 to 3 days, hence no removal is needed. It is suggested
that incorporating lemongrass extract in a cellulose-based matrix, may enhance the
biological properties of this resorbable pack.
Several studies had evaluated the effect of herbal active compounds incorporated in
wound dressing. Faiga et al studied the effect of a mixture of Curcuma longa rhizome extract and zinc oxide–eugenol (ZOE) dressing on angiogenesis, compared with
ZOE dressing alone. The addition of C. longa rhizome extract showed added value in the wound healing process in term of the number
of neovascular.[36] Nevertheless, the wound sites examined were in the dorsal part of rats which had
different environment and circumstances from oral sites. In the present study, wounds
were created in the gingival tissue surrounding by oral environment. Unlike skin surface,
wound healing in the oral cavity occurs in the presence of many challenges, including
a high bacterial and viral load and cannot be sterilized from bacterial plaque formation.
Therefore, wound surface must be protected from the external environment or infection
after oral surgery since infection keeps a wound in an inflammatory state and may
lead to a delayed healing.
Within the wound healing process, the inflammatory phase involves homeostasis and
inflammation that start at the moment of injury and continue for up to 4 to 6 days.
The proliferation phase involves epithelialization, angiogenesis, granulation tissue
formation, and collagen deposition, and takes place from days 4 to 14 after injury.
Epithelial-cell migration starts after 24 hours.
We had also observed a beneficial effects of Moringa oleifera Lam leaves extract incorporated in a cellulose-based wound dressing on wound healing
in palatal rats, but the applications of the dressing were more frequent (once a day
in 14 days) which clinicians or patients were not in favor of that.[37] In the present study, the application of lemongrass periodontal pack was only once
immediately after wounding and no repetition was done which is easier and favorable,
and we found that one time application is able to accelerate wound healing following
rat's gingivectomy.
Lemongrass has already known for its beneficial effects in oral health. As a folk
medicine, based on empirical studies, it is commonly used as remedies for gum swelling,
periodontitis (inflammation of tooth supporting tissue), and tooth ache by removing
bacteria from the oral cavity.[38] It is interesting to note that in vitro study showed that lemongrass indeed possess antimicrobial activity against several
oral pathogens. Rêgo et al investigated that essential oil of lemongrass was effective
in controlling bacterial growth in biofilms of Streptococcus mutans, the main causative agent of tooth decay.[39] Furthermore, it was shown that lemongrass essential oil has remarkable antimicrobial
and antibiofilm activities against the dental plaque bacteria.[40] The oral cavity harbors over 700 different species of bacteria and is described
as one of the most intricate ecosystem. Though the majority of them are considered
as commensally, some of them are responsible for oral infections ranging from tooth
decay to periodontal diseases and gingiva related infections.[41] That is why wound healing located in oral sites will always have sustainable challenge
from bacteria residing in this environment. In another study, lemongrass essential
oil was able to inhibit the growth of putative periodontal pathogens, especially Aggregatibacter actinomycetemcomitans, Actinomyces naeslundii, and Porphyromonas gingivalis, as well as the tetracycline-resistant strains. The results showed the superior properties
of the essential oil and suggested the use of the oil with other antibiotics against
resistant bacteria.[42]
[43]
[44] The mechanism of action suggested is that lemongrass oil and its components induced
intracellular leakage, as well as ultrastructural changes in bacterial cells, as it
was shown by electron micrographs that a hole occurring on the surface of bacterial
cell treated with citral, a main component of lemongrass, at a concentration of 4
μL/mL.[45]
Another beneficial effect of lemongrass essential oil is its anti-inflammatory, as
well as antifungal, properties. The exact mechanism of the anti-inflammatory effect
of lemongrass is unclear. However, it has been suggested that several molecules contribute
to the partial inhibition of the release of inflammation mediator substances.[46] Citral, geranial, neral, and carvone, the main components of lemongrass, have been
shown to inhibit the production of interleukin (IL)-1b, IL-6, and tumor necrosis factor
(TNF)-α.[47]
[48] Moreover, lemongrass was shown to have antioxidant activity and is able to scavenge
free-radical molecules.[49] In a study evaluating lemongrass aqueous extract, pholyphenolic compounds found
in lemongrass, that is, rutin, isoquercetin, catechin, and quercetin were able to
reduce the atherogenic index and enhances the serum antioxidant capacity in rats.[50] Wound healing implicates inflammation in the phases. Inflammation is a double-edged
sword, having both favorable and unfavorable consequences and during inflammation
phase, leucocytes produces oxygen radicals as oxygen-dependent mechanisms. These free
radicals are excreted as a weapon to kill bacteria but also may have detrimental effect
to the body cells. An excess of free radicals and/or a depletion of antioxidants may
result in oxidative stress leading to cellular dysfunction and sometimes cells' death.
In this circumstance, antioxidants help to protect cells from damage which is caused
by free radicals produced during inflammation.[51]
Conclusion
In conclusions, within the limitations of the present study, the following inferences
can be drawn: (1) lemongrass extract incorporated in resorbable periodontal dressing
was able to stimulate fibroblast and collagen deposition during the initial phase
of wound healing after gingivectomy in rats, and (2) one-time application of lemongrass
pack immediately after gingivectomy was sufficient to protect wound site and accelerate
healing. However, further studies are required to clarify the optimal concentration
and physical stability before its clinical application.
