CHAPTER 6shodhganga.inflibnet.ac.in/bitstream/10603/73023/13/12. chapter 6.pdf · CHAPTER 6 | IN...

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.



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,



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



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.



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



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



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.

http://www.toxicology.org/



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.



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



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



(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



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.



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



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)




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.


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




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.



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


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



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


DMSO

PDGF

HMHRS


derived growth factor-BB; HMHRS: - Hot methanolic extract of leaves of

H. rosa-sinensis. Values are mean ± SEM (n=3)




Figure 6.5: In vitro scratch assay for H. rosa-sinensis-Phase contrast microscopic

images





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



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.



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


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



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


DMSO

PDGF

HMTD


derived growth factor-BB; HMTD: - Hot methanolic extract of leaves of

T. divaricata. Values are mean ± SEM (n=3)




Figure 6.7: In vitro scratch assay for T. divaricata-Phase contrast microscopic

images





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



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.



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


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



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


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)




Figure 6.9: In vitro scratch assay for P. longifolia-Phase contrast microscopic

images





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



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


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)



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



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.

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