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CHAPTER 6
In VITRO
WOUND HEALING
STUDY
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
School of Science, SVKM’s NMIMS (Deemed-to-be) University Page 172
6. CHAPTER 6: IN VITRO WOUND HEALING STUDY
6.1. Introduction
6.1.1. In vitro assays
The term ‗in vitro tests‘ frequently known as ‗bioassays‘ is used for experiments to
investigate the effect of compounds or extracts which do not involve either living
animal tissue or whole animals.
All over the globe researchers and organizations are encouraging use of in vitro tests
and methods, wherever possible in the laboratory involving the use of bacteria,
worms, etc. instead of mammals. Hence, more and more of the scientifically valid
research methodologies are being designed to reduce; replace and refine the need for
laboratory animals. Nowadays computer based models and simulator protocols are
also employed to predict the outcome of testing.
In vitro tests are carried out in current ethnopharmacological research, not only
because of the ethical and financial constraints of using animals or animal tissue, but
also because they facilitate bioassay-guided isolation of ‗active‘ compounds
responsible for any activity. One of the important aspects of the in vitro assays is the
use of much smaller amount of test material necessary for the assay. As knowledge of
the biochemical processes underlying cell function and disease states has advanced in
recent years, there has been a large increase in the number of small-scale bioassays
that have been developed, particularly to enable the pharmaceutical industry to carry
out high-throughput screening of libraries of compounds (Houghton et al., 2007).
6.1.2. In vitro wound healing assays
Wounds are physical injuries that result in an opening or breaking of the skin. Wound
healing is a complex multifactorial process. It is a product of the integrated response
of several cell types to injury. Upon injury of adult mammalian skin, complex and
intricate processes are initiated to restore the function and integrity of the damaged
tissues.
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Wound healing process is complex process incolving granulation, collagenation,
collagen maturation and scar maturation which are concurrent but independent of
each other. Proper healing of wounds is essential for the restoration of disrupted
anatomical continuity and the disturbed functional status of the skin.
A plant extract used for wound healing may affect one or more of these processes, so
in vitro tests being used include those for inhibition of inflammation, stimulation of
fibroblast growth, antibacterial activity and antioxidant and free radical scavenging
effects. The in vitro wound healing assays allow the researcher to study cell migration
and cell interactions. The predominant cell populations in mammalian skin are
fibroblasts and keratinocytes. Accordingly, the vast majority of in vitro wound
healing studies utilize either one or both of these cell types as effective tools to
directly visualize cellular interaction (Oberringer et al., 2007).
6.1.3. Different cell based in vitro assays for wound healing
Different in vitro assay formats can be used to investigate the behavior of cell types,
which are relevant for human wound and soft-tissue healing. Some of the important
in vitro wound healing assays that are being routinely used are in vitro scratch assay;
Electric Cell-substrate Impedance Sensing (ECIS®); microfluidic chambers; and
Boyden chamber based transmembrane assays. ECIS® is a real-time, label-free,
impedance-based method to study the activities of cells grown in tissue culture. Some
of the important activities that can be studied through this technique include
morphological changes, cell locomotion, and other behaviors directed by the cell‘s
cytoskeleton (Chun-Chi Liang et al., 2007). This impedance-based cell monitoring
technology is a proprietary trade mark of Applied BioPhysics, Inc.
For many years, the Boyden chamber based transmembrane assays and scratch wound
assays were the only widely available formats to study cell migration and invasion.
However, new technologies such as microfluidic chambers and exclusion zone assays
have recently emerged as alternative phenotypic screening assays that provide
additional or complementary information to researchers (Hulkower and Herber,
2011). The study of cellular behavior in a two-dimensional culture dish offers the
ability to investigate specific targets with minimal interference from external factors,
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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but critical in vivo cues (paracrine signaling, three dimensional cues, etc.) are missing
and thus limit the translational applicability of in vitro studies. In vitro co-culture
experiments partially address the importance of paracrine interactions between
different skin cell populations, and therefore serve to evaluate the influence of wound-
healing-related factors in vitro. However, these models are also limited in their
biological relevance to wound healing (Chun-Chi Liang et al., 2007).
Nonetheless each of these methods provides limited information and may not fully
anticipate the results as might occur in humans where its effects are to be evaluated
ultimately. Furthermore, it takes a considerable amount of time to develop and
standardize a new method that may be suitable to replace existing in vivo methods.
Besides, the use of only one bioassay gives a very incomplete picture of the effect of
the extract on the whole system involved.
The assessment of the significance of the results of in vitro tests in relation to the
in vivo situation presents another major problem. Other factors, more directly related
to chemical kinetics such as rates of absorption, biotransformation, distribution and
excretion, which influence the exposure at the level of target cells in vivo cannot, at
present, be adequately simulated in vitro. Furthermore, even when the appropriate cell
type is used, intrinsic cell sensitivity depends on a number of cell characteristics
which are likely to be preserved only in part in vitro; these include chemical
biotransformation and binding, membrane permeability characteristics and surface
determinants, intracellular synthetic pathways and adaptive and recovery mechanisms.
In brief, the major problems in the interpretation of results obtained through these
in vitro assays to identify cell specific effects are as follows:
(1) Since basal cell functions always support specific cell functions, chemicals that
are capable of affecting basal cell functions are also likely to affect the specialized
ones.
(2) The effects of a test substance on a cell system may be different depending on the
conditions of incubation (e.g. incubation time and concentration of test substance).
Therefore, unless a set of favorable circumstances occurs and a well-planned
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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experimental design is adhered to, it may prove difficult to distinguish between basal
and organ-specific effects.
6.1.4. In vitro scratch assay for wound healing
The in vitro scratch assay is an easy, low-cost and well-developed method to measure
cell migration in vitro. This assay generally involves first growing a confluent cell
monolayer. A small area is then disrupted and a group of cells destroyed or displaced
by scratching a line through the layer with an object such as a toothpick, pipette tip, or
needles. Chun-Chi Liang et al., (2007) have reported that the cells on the edge of the
newly created gap will move in a directed manner towards the centre of the gap/
scratch until the monolayer is reformed, toward the opening to close the ‗‗scratch‘‘
until new cell–cell contacts are established again. The open gap is inspected
microscopically over time as the cells move in and fill the damaged area. This
"healing" can take from several hours to over a day depending on the cell type,
conditions and the extent of the "wounded" region. All the images are captured at the
beginning and at regular intervals during the cell migration due to which an attempt is
being made to heal the scratch, and comparing the images to quantify the migration
rate of the cells. This live-cell imaging system is well suited for making cell migration
measurements by imaging inside the special incubator system. The system is ideal for
assays that benefit from a longer term kinetic read-out, where maintenance of the cells
at optimum physiological conditions for the duration of the experiment is important
(Carl Zeiss MicroImaging GmbH).
6.1.4.1. Significance of the in vitro scratch assay
Wound healing assays to monitor cell migration have been carried out in tissue
culture for many years to estimate the migration and proliferation rates of different
cells and culture conditions (Houghton et al.,). The basis of the scratch assay is
intended to monitor the second phase of wound healing, which is characterized by
proliferation and migration of either keratinocytes or fibroblasts (Schafer and Werner,
2007; Gurtner et al. 2008); though the scratch assay cannot substitute for in vivo
studies as a final proof for promoting wound healing.
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Compared to the other in vitro cell based assay methods, the in vitro scratch assay is
particularly suitable for studies on the effects of cell–matrix and cell–cell interactions
on cell migration, mimic cell migration during wound healing (Chun-Chi Liang et al.,
2007).
Despite many limitations of the method, overall, in vitro scratch assay is still often the
method of choice to analyze cell migration in a laboratory because it is easy to set up,
does not require any specialized equipment and all materials required for the assay are
available in any laboratory that performs cell culture (Chun-Chi Liang et al., 2007).
Many researchers have reported wound healing efficacy of plant extracts by in vitro
scratch assay method. Yeo Dodeh et al., (2011) have used in vitro scratch assay
employing the H292 human lung cells for demonstrating the wound healing effect of
extracts from Heliotropium. Hostanska et al., (2012) have showed that the low
potency homeopathic remedy (0712–2) exerted in vitro wound closure potential in
NIH 3T3 fibroblasts which was resulted from stimulation of fibroblasts motility.
Sevimli-Gür et al., (2011) have studied the wound healing activity of the four chief
saponins of Astragalus species by using in vitro wound healing, proliferation and
migration scratch assay using Human keratinocyte, HS2.
6.1.4.2. Advantages of in vitro scratch assay
One of the major advantages of this simple method is that it mimics to some extent
migration of cells in vivo (Chun-Chi Liang et al., 2007). In addition, the in vitro
scratch assay is also compatible with microscopy including live cell imaging,
allowing analysis of intracellular signaling events (e.g., by visualization of green
fluorescent protein (GFP)-tagged proteins for sub-cellular localization or fluorescent
resonance energy transfer for protein–protein interactions) during cell migration. On
the other hand, it is also probably the simplest method to study cell migration in vitro
and only uses the common and inexpensive supplies found in most laboratories
capable of cell culturing (Chun-Chi Liang et al., 2007).
6.1.4.3. Limitations of in vitro scratch assay
There are a number of disadvantages and limitations of the in vitro scratch assay
compared to other available methods. It does not replace other well-established
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methods for chemotaxis such as the Boyden chamber assay, as no chemical gradient is
established. It takes a relatively longer time to perform than some other methods. One
to two days are needed for the formation of cell monolayer and then 8–18 h for cell
migration to close the scratch. Moreover, the main drawback is that the scratch itself
often varies and is not highly reproducible. The traditional scratch method almost
always "scrapes" off the cell's protein coat hence it may not be suited for certain
assays that need to monitor cell-cell interactions and signaling pathways. Furthermore
it is not a method of choice if the availability of cells (e.g., specialized primary cells
that are hard to get in sufficient amount) or chemicals (e.g., expensive reagents) is
limiting (Chun-Chi Liang et al., 2007).
6.1.4.4. In vitro activity is no guarantee of an in vivo effect
When some significant desired effect is observed with a particular extract or
compound during in vitro tests, it is likely that a similar effect may not be exhibited
when the extract is given in vivo to a test animal or to a human volunteer. More
importantly, factors such as adsorption and metabolism may be responsible for
discrepancies between in vitro and in vivo activity of the drugs (Houghton et al.,
2007).
Hence, as Houghton et al., (2007) have stated correctly that it is important to design
in vitro experiments to approximate as closely as possible to the disease. The in vitro
assay may also be difficult to set up and standardized as many diseases are caused due
to more than one factor. Hence one in vitro test alone is not sufficient to test the
usefulness of the extract against a particular disease
Ideally, as correctly stated by Houghton et al., (2007), in vitro tests should be backed
up with in vivo studies, and ultimately clinical, studies. In designing and choosing the
in vitro tests, the processes underlying disease states should be known, explored and
investigated to determine the best mix of tests, in order to lay a basis for taking an
extract or preparation into in vivo or clinical tests .
Animals will continue to be important research tools in toxicological studies for the
development of new chemical agents or drugs etc. Moreover to confirm and validate
the results of in vitro tests, animal models will be mandatory and still be needed at a
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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later stage of new drug development before exposure to humans and other animals to
predict the potential danger or risk of exposure. Research involving laboratory
animals is necessary to ensure and enhance human and animal health and protection
of the environment. In the absence of human data, research with experimental animals
is the most reliable means of detecting important toxic properties of the new drug or
agent or a chemical substance as well as it is useful for estimating the risks to human
health and the environment (SOT Animals in Research Public Policy Statement 1999;
www.toxicology.org).
6.1.4.5. Rationale for in vitro scratch assay for the present study
The in vitro wound healing efficacy testing by scratch assay was employed in the
present study at the earlier stage of the research work as a preliminary pilot screening
of extracts of the four selected plants. The main objective of this study was to carry
out initial screening of the test samples for evaluation of their wound healing
potential. Another important point of consideration for employing the in vitro assay
was that the results of the assay could be indicative of the molecular and cellular
mechanism involved and the mode of action of the plant extracts during the wound
healing process.
Moreover, the findings and results of this study would serve as an important evidence
of support to identify and establish at which stage of the wound healing process the
plant extracts are effective. It was felt that in vitro assay would give any positive
indications for promotion of wound healing efficacy for the four plants proposed to be
investigated for the present study, and subsequently it would be possible to limit the
total number of animals to be employed during the in vivo studies involving animals
as experimental models. In the light of the above, the present study was initiated and
undertaken so that it was intended to limit the total number of palnts from the four
plants to be subsequently tested by in vivo tests.
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6.2. Material and methods
6.2.1. In vitro wound healing by scratch assay
6.2.1.1. Chemicals and reagents
Dimethyl sulfoxide (DMSO); Bovine Serum Albumin (BSA); Penicillin;
Streptomycin; and Mitomycin C were obtained from Sigma Aldrich. Glacial acetic
acid; NaCl; KH2PO4; KCl and Na2HPO4, were from Qualigens, India. Recombinant
human platelet-derived growth factor-BB (PDGF) was obtained from Invitrogen,
Germany. HyClone fetal bovine serum (FBS) was from Thermo Scientific. Gibco®
Dulbecco‘s Modified Eagle Medium (DMEM) was from Life Technologies, India and
Trypsin was from HiMedia Laboratories, India.
6.2.1.2. Preparation of solution and reagents
For the present in vitro scratch assay, the method as described by Fronza et al., (2009)
with a few modifications was employed. The tissue culture medium, solutions and
reagents required for the same were prepared as described below:
1. Dulbecco’s Modified Eagle Medium (DMEM medium)
Heat inactivated DMEM dehydrated media was reconstituted in distilled water as per
the manufacturer‘s instruction and filter sterilized and used for all the tissue culture
experiments.
2. Phosphate-buffered saline, pH 7.4 (PBS)
For the preparation of phosphate-buffered saline, pH 7.4, 8.0 g of NaCl, 0.2 g
KH2PO4; 0.2 g of KCl; 2.18 g of Na2HPO4 were dissolved in 800 mL of distilled
water. After mixing, pH was adjusted to 7.4 and the volume of the solution was
adjusted to 1000 mL. The buffer was later autoclaved at 15 lbs. pressure at 1210
C for
15 minutes and used for all the tissue culture experiments.
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3. Recombinant human hlatelet-derived growth factor-BB (PDGF)
The content of the vial of PDGF BB (5µg) were reconstituted as per the
manufacturer‘s instruction in 1.0 mL of 100 mM acetic acid containing 0.1% BSA.
Later this solution was filter sterilized and aliquots were stored at -20 0C. PDGF BB
was used as positive control for the in vitro scratch assay for wound healing.
4. Pen Strep antibiotic mixture
Antibiotics penicillin and streptomycin at a final concentration of 100 I.U. /mL of
penicillin and 100 μg/mL of streptomycin were used for all the tissue culture
experiments to prevent microbial contamination of tissue culture media.
6.2.1.3. Preparation of NIH 3T3 mouse fibroblast cells
NIH 3T3 mouse embryonic fibroblast cells used in the present study were procured
from the lab of Dr. Sorab Dalal, ACTREC, Mumbai. All the tissue culture
experimental studies of the in vitro scratch assay for wound healing activity of the
methanolic extracts of the selected plants were carried out at Dr. Sorab Dalal‘s
laboratory, ACTREC, Mumbai.
6.2.1.4. Preparation of test samples
For this experimental study, plant methanolic extracts of the four selected plants viz.
(i) Epipremnum aureum; (ii) Hibiscus rosa-sinensis; (iii) Tabernaemontana
divaricata; and (iv) Polyalthia longifolia at a final concentration of 10 µg/mL were
prepared in 0.1% DMSO and evaluated for in vitro wound healing activity by the
scratch assay. PDGF at final concentration of 2 ng/mL was used as a positive control
while 1% DMSO was used as the negative control.
6.2.1.5. Experimental procedure for the in vitro scratch assay
In vitro wound healing activity of the methanolic extracts of all the four selected
plants was studied by the scratch assay using 3T3 fibroblasts according to the method
of Fronza et al., (2009) with a few modifications. The schematic representation of the
experiments undertaken is given in Figure 6.1. For this study, the effect of plant
methanolic extracts on the migration of NIH 3T3 mouse fibroblast cells was evaluated
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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and compared with the migration response of the cells when treated with PDGF
(positive control) and DMSO (vehicle control). In brief, NIH 3T3 mouse fibroblast
cells were allowed to grow till confluent in DMEM medium with antibiotics (100
I.U./mL penicillin, 100 μg /mL streptomycin) and 10 % FBS. Then the cells were split
in a 6 well tissue culture plate and allowed to grow till they formed a confluent
monolayer. The monolayered cells were then treated with Mitomycin C for 3 h at
37 0C in a CO2 incubator. Mitomycin C inhibits DNA synthesis, hence, its treatment
prior to the plant extract treatment rules out multiplication of the fibroblast cells and
thus confirming whatever gap filling/wound healing occurs is only due to migration
and proliferation of the fibroblast cells. After 3 h, Mitomycin C was washed off with
PBS and the wells were replenished with fresh medium. A scratch/ wound was made
in the monolayer of the cells with the help of sterile Eppendroff pipette tip in each
well. After the formation of the wound, cell debris was removed by discarding the
medium and washing the wells with PBS and the wells were again replenished with
fresh DMEM medium. The cell monolayer was then treated with plant extracts;
DMSO; and PDGF in the respective wells. Later this treated 6 well plate was
incubated for 20 h at 37 0C in a CO2 chamber arranged in an assembly with a
microscope, (Axiovert 200M, Carl Zeiss, Germany) equipped with a high resolution
digital camera, (AxioMRm, Carl Zeiss, Germany) for imaging and recording of
migration of fibroblasts. The distance migrated by the fibroblast cells in each well was
monitored and recorded every hour and later the distance covered by the fibroblasts
and subsequent closure of the scratch were measured and analyzed by using
Axiovision Rel. V: 4·6 MetaMorph software, version: 7.1.0.0 (Carl Zeiss, Germany).
The observations and results for the percent migration of the fibroblast cells were
calculated and compared with controls. All the experiments were performed in
triplicates.
6.2.1.6. Statistical analysis
The data of the percent migration of the NIH 3T3 mouse fibroblasts obtained after the
in vitro scratch assay study were analyzed by one way ANOVA and the results were
considered significant when p< 0.05. A statistical analysis employing 2 way ANOVA
was carried out for the same data by GraphPad Prism software, Version 5.0
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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(GraphPad Software Inc., USA). All the experiments were done in triplicates and the
results are described as mean ± SEM (n=3).
Figure 6.1: Diagrammatic representation of in vitro wound healing by scratch
assay.
6.3. Results and discussion
The plant methanolic extracts of all the four selected plants for the present study, viz.
(i) E. aureum; (ii) H. rosa-sinensis; (iii) T. divaricata; and (iv) P. longifolia at the
final concentration of 10µg/mL were evaluated by in vitro scratch assay for their
effect on the migration of 3T3 mouse fibroblasts. The cellular migration was observed
continuously for 20 h of the study period.
Key: - DMSO: - Dimethyl sulphoxide; PDGF BB: - Recombinant human
platelet derived growth factor-BB; HMEA: - Hot methanolic extract of leaves of
E. aureum; HMHRS: - Hot methanolic extract of leaves of H. rosa-sinensis;
HMTD: - Hot methanolic extract of leaves of T. divaricata; HMPL: - Hot
methanolic extract of leaves of P. longifolia.
Experimental study design for in vitro wound healing by scratch assay
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6.3.1. In vitro wound healing efficacy of E. aureum
The methanolic extract of the leaves of the plant E. aureum was evaluated by in vitro
scratch assay for its effect on the migration of 3T3 mouse fibroblasts.
The observations are represented in Table 6.1; and Figures 6.2 and 6.3. From the
experimental observation, it was found that the percent migration of 3T3 mouse
fibroblasts at the end of 10 h was 38.11 ± 10.17 % in response to the treatment with
DMSO (vehicle control); 56.38± 6.93 % migration was the response when cells were
treated with PDGF (positive control); and 23.98 ± 1.41 % was percent migration of
cells when treated with plant methanolic extract of E. aureum (HMEA). Whereas at
the end of experimental observation period of 20 h, the percent migration of 3T3
mouse fibroblasts was 70.7 ± 11.49 % after treatment with DMSO (vehicle control);
93.16 ± 2.23 % after treatment with PDGF (positive control); and 58.28 ± 3.85 %
after treatment with HMEA. From these results, it was concluded that the methanolic
extract of the plant E. aureum, at a final concentration of 10µg/mL was ineffective in
promotion of migration of 3T3 fibroblasts at the end of the incubation time period of
20 h. As per the graphical results of the statistical analysis, as described in Figures 6.2
and 6.3, it was observed that the plant extract treated cells exhibited statistically
significant inhibition of fibroblast migration at the end of 20 h (p < 0.05) when
compared with that of the negative control (DMSO).
From these results it was thus evident that the phytochemicals present in the
methanolic extract of the leaves of the plant E. aureum were causing the suppression
of 3T3 fibroblast cells‘ migration.
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Table 6.1: Would healing effect of E. aureum by in vitro scratch assay
Time % Migration of NIH 3T3 mouse fibroblasts
DMSO PDGF HMEA
1 h 2.19 ± 0.45 1.37 ± 0.29
2 h 4.96 ±3.85 4.01 ± 2.08 3.15 ± 0.51
3 h 9.36 ± 5.01 5.47 ± 3.74 4.12 ± 0.14
4 h 12.35 ± 6.07 14.79 ± 3.2 8.05 ± 0.85
5 h 15.99 ± 5.57 20.41 ± 6.73 10.21 ± 0.42
6 h 19.43 ± 6.88 27.03 ± 6.97 12.58 ± 0.71
7 h 23.48 ± 8.4 36.36 ± 6.94 15.51 ± 0.72
8 h 27.48 ± 7.74 43.55 ± 5.24 17.79 ± 0.85
9 h 32.79 ± 11.23 49 ± 8.57 20.88 ± 0.31
10 h 38.11 ± 10.17 56.38± 6.93 23.98 ± 1.41
11 h 41.85 ± 11.18 60.26 ± 4.95 26.44 ± 2.56
12 h 45.09 ± 11.18 61.6 ± 8.31 28.43 ± 2.2
13 h 48.18 ± 10.93 67.18 ± 7.96 30.53 ± 2.14
14 h 53.39 ± 10.27 70.01 ± 8.59 36.83 ± 3.01
15 h 56.33 ± 12.2 75.11 ± 6.02 39.49 ± 3.05
16 h 59.97 ± 12.2 79.71 ± 5.34 44 ± 4.03
17 h 62.7 ± 12.5 81.85 ± 5.28 47.24 ± 4.07
18 h 62.5 ± 11.59 85.74 ± 5.31 50.33 ± 3.91
19 h 66.55 ± 11.49 89.24 ± 5.72 54.4 ± 3.99
20 h 70.7 ± 11.49 93.16 ± 2.23 58.28 ± 3.85
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMEA: - Hot methanolic extract of leaves of E. aureum. Values
are mean ± SEM (n=3)
In vitro scratch assay for E. aureum
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Figure 6.2: In vitro scratch assay for E. aureum - Graphical representation
0
20
40
60
80
100
120
0 5 10 15 20 25
% M
igra
tion
Time in hour
In vitro wound healing assay
DMSO
PDGF
HMEA
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet
derived growth factor-BB; HMEA: - Hot methanolic extract of leaves of
E. aureum. Values are mean ± SEM (n=3)
In vitro scratch assay for E. aureum
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Figure 6.3: In vitro scratch assay for E. aureum-Phase contrast microscopic
images
Representative microphotographs are for the cell migration into the experimentally
created scratch/gap in response to treatments.
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMEA: - Hot methanolic extract of leaves of E. aureum.
(A):- DMSO at time 0 h, 10 h, 20 h; (B):- PDGF at time 0 h, 10 h, 20 h; (C) :- HMEA
at time 0 h, 10 h, 20 h
In vitro scratch assay for E. aureum
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6.3.2. In vitro wound healing efficacy of H. rosa-sinensis
The results of the study for the evaluation of effect of phytoconstituents of the
methanolic extract of the leaves of the plant H. rosa-sinensis on the migration of 3T3
mouse fibroblasts by in vitro scratch assay are described in Table 6.2; Figures 6.4 and
6.5. From the experimental observations, it was found that the percent migration of
3T3 mouse fibroblasts was 38.11±10.17% in response to the treatment with DMSO
(vehicle control); 56.38± 6.93% in response to the treatment with PDGF (positive
control); and 45.15± 11.09 % in response to the treatment with the plant extract at the
end of 10 h. Whereas at the end of experimental observation period of 20 h, the
percent migration of 3T3 mouse fibroblasts was 70.7 ± 11.49 % in response to the
treatment with DMSO; 93.16 ± 2.23 % in response to the treatment with PDGF; and
78.39 ± 19.92 % in response to the treatment with H. rosa-sinensis extract.
From these results, it can be concluded that the methanolic extract of the plant
H. rosa-sinensis (HMHRS) at a final concentration of 10µg/mL was not as effective
in promoting 3T3 fibroblasts migration at the end of the incubation time period of
20 h as compared to that for the positive control after treatment with PDGF. Statistical
analysis of the data indicated that value of the percent migration of the fibroblasts in
response to the plant extract was statistically not significant (where
p< 0.05) when compared with that of the control cells.
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Table 6.2: Wound healing effect of H. rosa-sinensis by in vitro scratch assay
Time % Migration of NIH 3T3 mouse fibroblasts
DMSO PDGF HMHRS
1 h 2.19 ± 0.45 4.16 ± 0.79
2 h 4.96 ±3.85 4.01 ± 2.08 6.92 ± 1.1
3 h 9.36 ± 5.01 5.47 ± 3.74 11.54 ± 0.34
4 h 12.35 ± 6.07 14.79 ± 3.2 14.9 ± 2.43
5 h 15.99 ± 5.57 20.41 ± 6.73 21.02 ± 4.43
6 h 19.43 ± 6.88 27.03 ± 6.97 26.33 ± 6.44
7 h 23.48 ± 8.4 36.36 ± 6.94 30.77 ± 8.44
8 h 27.48 ± 7.74 43.55 ± 5.24 37.60 ± 12.50
9 h 32.79 ± 11.23 49 ± 8.57 41.48 ± 10.20
10 h 38.11 ± 10.17 56.38± 6.93 45.15 ± 11.09
11 h 41.85 ± 11.18 60.26 ± 4.95 48.47 ± 13.11
12 h 45.09 ± 11.18 61.6 ± 8.31 52.74 ± 11.82
13 h 48.18 ± 10.93 67.18 ± 7.96 57.39 ± 15.17
14 h 53.39 ± 10.27 70.01 ± 8.59 61.32 ± 17.88
15 h 56.33 ± 12.2 75.11 ± 6.02 66.68 ± 18.85
16 h 59.97 ± 12.2 79.71 ± 5.34 69.54 ± 21.15
17 h 62.7 ± 12.5 81.85 ± 5.28 71.30 ± 21.08
18 h 62.5 ± 11.59 85.74 ± 5.31 74.66 ± 20.88
19 h 66.55 ± 11.49 89.24 ± 5.72 76.74 ± 20.09
20 h 70.7 ± 11.49 93.16 ± 2.23 78.39 ± 19.92
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMHRS: - Hot methanolic extract of leaves of H. rosa-sinensis.
Values are mean ± SEM (n=3),
In vitro scratch assay for H. rosa-sinensis
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Figure 6.4: In vitro scratch assay for H. rosa-sinensis- Graphical representation
0
20
40
60
80
100
120
0 5 10 15 20 25
% M
igra
tion
Time in hour
In vitro wound healing assay
DMSO
PDGF
HMHRS
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet
derived growth factor-BB; HMHRS: - Hot methanolic extract of leaves of
H. rosa-sinensis. Values are mean ± SEM (n=3)
In vitro scratch assay for H. rosa-sinensis
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Figure 6.5: In vitro scratch assay for H. rosa-sinensis-Phase contrast microscopic
images
In vitro scratch assay for H. rosa-sinensis
Representative microphotographs are for the cell migration into the experimentally
created scratch/gap in response to treatments.
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMHRS: - Hot methanolic extract of leaves of H. rosa-sinensis.
(A):- DMSO at time 0 h, 10 h, 20 h; (B):- PDGF at time 0 h, 10 h, 20 h;
(C) :- HMHRS at time 0 h, 10 h, 20 h
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6.3.3. In vitro wound healing efficacy of T. divaricata
The effect of the methanolic extract of the plant T. divaricata on the migration of 3T3
mouse fibroblasts by in vitro scratch assay are represented in Table 6.3; and Figures
6.6 and 6.7. From the experimental findings, it was observed that the percent
migration of 3T3 mouse fibroblasts at the end of 10 h was 38.11 ± 10.17 % after
treatment with DMSO (vehicle control); 56.38± 6.93% after treatment with PDGF
(positive control); and 42.88 ± 8.45 % after treatment with plant extract of
T. divaricata. At the end of 20 h of the experimental observation period,
corresponding values for the percent migration of 3T3 mouse fibroblasts was found to
be 70.7 ± 11.49 % after treatment with DMSO; 93.16 ± 2.23 % after treatment with
PDGF; and 78.53 ± 13.57 % after treatment with the plant extract.
These all results collectively indicated that the phytochemicals present in the
methanolic extract of the plant T. divaricata (HMTD), when tested at a final
concentration of 10µg/mL was ineffective in promotion of migration of 3T3
fibroblasts. The statistical analysis of the experimental data of this study also
confirmed that the values for the percent migration of HMTD treated fibroblasts was
statistically not significant (where p<0.05) when compared with that of the control
cells.
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Table 6.3: Wound healing effect of T. divaricata by in vitro scratch assay
Time % Migration of NIH 3T3 fibroblasts
DMSO PDGF HMTD
1 h
2.19 ± 0.45 3.35 ± 2.46
2 h 4.96 ±3.85 4.01 ± 2.08 6.39 ± 2.94
3 h 9.36 ± 5.01 5.47 ± 3.74 10.36 ± 3.64
4 h 12.35 ± 6.07 14.79 ± 3.2 14.51 ± 4.16
5 h 15.99 ± 5.57 20.41 ± 6.73 20.54 ± 4.61
6 h 19.43 ± 6.88 27.03 ± 6.97 25.21 ± 4.50
7 h 23.48 ± 8.4 36.36 ± 6.94 28.68 ± 6.47
8 h 27.48 ± 7.74 43.55 ± 5.24 34.88 ± 6.65
9 h 32.79 ± 11.23 49 ± 8.57 39.638 ± 7.51
10 h 38.11 ± 10.17 56.38± 6.93 42.88 ± 8.45
11 h 41.85 ± 11.18 60.26 ± 4.95 46.44 ± 9.36
12 h 45.09 ± 11.18 61.6 ± 8.31 51.38 ± 10.05
13 h 48.18 ± 10.93 67.18 ± 7.96 55.83 ± 8.84
14 h 53.39 ± 10.27 70.01 ± 8.59 59.23 ± 9.05
15 h 56.33 ± 12.2 75.11 ± 6.02 60.98 ± 10.54
16 h 59.97 ± 12.2 79.71 ± 5.34 64.99 ± 11.10
17 h 62.7 ± 12.5 81.85 ± 5.28 70.61 ± 14.06
18 h 62.5 ± 11.59 85.74 ± 5.31 73.68 ± 15.18
19 h 66.55 ± 11.49 89.24 ± 5.72 76.36 ± 14.59
20 h 70.7 ± 11.49 93.16 ± 2.23 78.53 ± 13.57
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMTD: - Hot methanolic extract of leaves of T. divaricata. Values
are mean ± SEM (n=3),
In vitro scratch assay for T. divaricata
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Figure 6.6: In vitro scratch assay for T. divaricata- Graphical representation
0
20
40
60
80
100
120
0 5 10 15 20 25
% M
igra
tion
Time in hour
In vitro wound healing assay
DMSO
PDGF
HMTD
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet
derived growth factor-BB; HMTD: - Hot methanolic extract of leaves of
T. divaricata. Values are mean ± SEM (n=3)
In vitro scratch assay for T. divaricata
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Figure 6.7: In vitro scratch assay for T. divaricata-Phase contrast microscopic
images
In vitro scratch assay for T. divaricata
Representative microphotographs are for the cell migration into the experimentally
created scratch/gap in response to treatments.
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMTD: - Hot methanolic extract of leaves of T. divaricata.
(A):- DMSO at time 0 h, 10 h, 20 h; (B):- PDGF at time 0 h, 10 h, 20 h;
(C) :- HMTD at time 0 h, 10 h, 20 h
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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6.3.4. In vitro wound healing efficacy of P. longifolia
Similarly, the methanolic extract of the leaves of the plant P. longifolia was evaluated
by in vitro scratch assay for its effect on the migration of 3T3 mouse fibroblasts. The
cellular migration was observed continuously for 20 h. The results are described in
Table 6.4; and Figures 6.8 and 6.9. The percent migration of 3T3 mouse fibroblasts at
the end of 10 h was 38.11 ± 10.17 % in response to the treatment with DMSO
(vehicle control); 56.38± 6.93% in response to the treatment with PDGF (positive
control); and 43.07 ± 7.10 % in response to the treatment with the plant extract of
P. longifolia (HMPL). At the end of experimental observation period of 20 h, these
values were recorded at 70.7 ± 11.49 % in response to the treatment with DMSO;
93.16 ± 2.23 % in response to the treatment with PDGF; and 86.15 ± 11.76 % in
response to the treatment with HMPL.
Again there was a clear indication that the methanolic extract of the plant P. longifolia
(HMPL) at a concentration of 10µg/mL was also ineffective in promoting the
migration of 3T3 fibroblasts. Further, the values of the percent migration of the plant
extract treated fibroblasts was statistically not significant (where p<0.05) when
compared with that of the control cells.
However, from the photomicrographs, it can be seen that the gap/scratch is filled with
the migrated fibroblasts as seen with that when treated with PDGF treated cells.
Overall, taking into consideration the statistical analysis results, it was concluded that
that the phytoconstituents of the methanolic extract of the plant P. longifolia were
ineffective to promote the cellular migration of the fibroblast cells at the concentration
tested.
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Table 6.4: Wound healing effect of P. longifolia by in vitro scratch assay
Time % Migration of NIH 3T3 fibroblasts
DMSO PDGF HMPL
1 h 2.19 ± 0.45 3.36 ± 2.37
2 h 4.96 ±3.85 4.01 ± 2.08 5.27 ± 2.20
3 h 9.36 ± 5.01 5.47 ± 3.74 10.55 ± 4.69
4 h 12.35 ± 6.07 14.79 ± 3.2 11.85 ± 5.91
5 h 15.99 ± 5.57 20.41 ± 6.73 18.05 ± 2.91
6 h 19.43 ± 6.88 27.03 ± 6.97 24.14 ± 4.39
7 h 23.48 ± 8.4 36.36 ± 6.94 27.48 ± 3.86
8 h 27.48 ± 7.74 43.55 ± 5.24 29.82 ± 1.95
9 h 32.79 ± 11.23 49 ± 8.57 37.52 ± 6.14
10 h 38.11 ± 10.17 56.38± 6.93 43.07 ± 7.10
11 h 41.85 ± 11.18 60.26 ± 4.95 51.28 ± 8.99
12 h 45.09 ± 11.18 61.6 ± 8.31 58.93 ± 11.32
13 h 48.18 ± 10.93 67.18 ± 7.96 62.34 ± 12.49
14 h 53.39 ± 10.27 70.01 ± 8.59 67.58 ± 15.19
15 h 56.33 ± 12.2 75.11 ± 6.02 71.36 ± 17.53
16 h 59.97 ± 12.2 79.71 ± 5.34 73.82 ± 16.92
17 h 62.7 ± 12.5 81.85 ± 5.28 78.47 ± 16.34
18 h 62.5 ± 11.59 85.74 ± 5.31 81.38 ± 14.30
19 h 66.55 ± 11.49 89.24 ± 5.72 83.23 ± 13.52
20 h 70.7 ± 11.49 93.16 ± 2.23 86.15 ± 11.76
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet derived
growth factor-BB; HMPL: - Hot methanolic extract of leaves of P. longifolia.
Values are mean ± SEM (n=3),
In vitro scratch assay for P. longifolia
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Figure 6.8: In vitro scratch assay for P. longifolia- Graphical representation
0
20
40
60
80
100
120
0 5 10 15 20 25
% M
igra
tion
Time in hour
In vitro wound healing assay
DMSO
PDGF
HMPL
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - recombinant human platelet
derived growth factor-BB; HMPL: - Hot methanolic extract of leaves of
P. longifolia. Values are mean ± SEM (n=3)
In vitro scratch assay for P. longifolia
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Figure 6.9: In vitro scratch assay for P. longifolia-Phase contrast microscopic
images
In vitro scratch assay for P. longifolia
Representative microphotographs are for the cell migration into the experimentally
created scratch/gap in response to treatments.
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet
derived growth factor-BB; HMPL: - Hot methanolic extract of leaves of
P. longifolia. (A):- DMSO at time 0 h, 10 h, 20 h; (B):- PDGF at time 0 h, 10 h, 20 h;
(C) :- HMPL at time 0 h, 10 h, 20 h
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Wound healing effect of plants on migration of 3T3 fibroblasts
by in vitro scratch assay
4 8 12 16 200
50
100
150
DMSO
PDGF
HMEA
HMHRS
HMTD
HMPL
Time in hour
% M
lgra
tio
n o
f 3
T3
fib
ro
bla
sts
Figure 6.10: In vitro wound healing by scratch assay - 2 way ANOVA analysis
In vitro wound healing by in vitro scratch assay - 2 way ANOVA analysis
Key: - DMSO: - Dimethyl sulphoxide; PDGF: - Recombinant human platelet
derived growth factor-BB; HMEA: - Hot methanolic extract of leaves of
E. aureum; HMHRS: - Hot methanolic extract of leaves of H. rosa-sinensis;
HMTD: - Hot methanolic extract of leaves of T. divaricata; HMPL: - Hot
methanolic extract of leaves of P. longifolia. Values are mean ± SEM (n=3)
CHAPTER 6 | IN VITRO WOUND HEALING ASSAY
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Discussion
Cell migration and proliferation plays a vital role in the wound healing process.
The scratch assay is widely and frequently used assay for detection of in vitro wound
healing activity. Fibroblasts are the connective tissue cells which are responsible for
collagen deposition that is needed to repair tissue injury. In normal tissues, collagen
provides strength, integrity and structure. When tissues are disrupted following an
injury, collagen is needed to repair the defect and restore the anatomical structure and
function. Early in the proliferation phase, fibroblast activity is limited to cellular
replication and migration. Around the third day after wounding, the growing mass of
fibroblast cells begins to synthesize and secrete measurable amount of collagen.
Collagen levels rise continually for approximately three weeks. The amount of
collagen secreted during this period determines the tensile strength of the wound. An
increase in collagen production is an important factor for wound healing (Diegelmann
and Evans, 2004).
In the present study, amongst the four plants evaluated by this assay, it was observed
that the extract of the plant P. longifolia showed the highest percent migration of the
fibroblasts amongst the four plant extracts tested, While the migration observed in
response to the treatment with the plant extracts of H. rosa-sinensis and T. divaricata
were of moderate whereas the plant extract of E. aureum, in fact, suppressed the
migration of the fibroblasts.
In contrast to these findings, the observations of the pilot screening undertaken for the
same four plant species employing the in vivo wound healing efficacy by excision
model, indicated that two of these plants, viz. E. aureum and H. rosa-sinensis showed
potentially good wound healing efficacy. These aspects are discussed in detail in the
next chapter.
Wound healing is a complex process. It involves interactions of multiple cell types,
various cytokines, growth factors, their mediators, and the extracellular matrix
proteins (Werner and Grose, 2003). The fibroblasts are not the only type of cells
involved in the wound healing process as there are many different cell types and
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cytokines and growth factors involved in the wound healing process (Schäfer, M. and
Werner, 2007).
Hence, the migration and proliferation of the fibroblasts is not the only parameter to
be judged for drawing final conclusions regarding the wound healing efficacy of the
plant extracts. Considering all these facts, it was concluded that the observations
derived from this single experimental study of the in vitro scratch assay alone may not
be sufficient enough to furnish conclusive results. Ideally, for such purposes for
establishing the pharmacological efficacy of a test drug, the in vitro tests should be
backed up with suitable in vivo studies. Moreover, in designing the in vitro study for
aforementioned purposes, the process underlying the disease condition must be well
understood and taken into consideration before the commencement of the study.
In summary, the results of the in vitro scratch assay for evaluation of wound healing
efficacy of the plant extracts need further studies to be undertaken for uncovering of
the exact molecular mechanism and mode of action of the bioactive compounds in the
methanolic extract of the plant E. aureum and H. rosa-sinensis.
In conclusion, none of the four plant methanolic extracts evaluated by the
in vitro scratch assay exhibited statistically significant migration of fibroblasts.
Hence it was concluded that the in vitro scratch assay was not furnishing
conclusive results and hence it could not completely replace the in vivo studies
for providing a final proof and evidences in evaluating the wound healing
potential of the plant extracts.