Wound Healing: The Effect of Tea Tree Essential Oil on Human Dermal Fibroblast Cell Expression of Intercellular Adhesion Molecule -1 (ICAM-1)
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Wound Healing: The Effect of Tea Tree
Essential Oil on Human Dermal Fibroblast
Cell Expression of Intercellular Adhesion
Molecule -1 (ICAM-1)
2018
Dissertation completed and presented in part fulfilment of BSc
Biomedical Science Degree.
Cardiff School of Health Science
Cardiff Metropolitan University
Llandaff Campus
Western Avenue
Cardiff
CF5 2YB
iDeclaration
Statement 1
This work has not previously been accepted in substance for any degree and is
not being concurrently submitted in candidature for any degree.
Signed: ……………………….……………………… (Candidate)
Dated: 14th March 2018
Statement 2
This dissertation is the result of my own investigations, except where otherwise
stated. Where corrections services have been used, the extent and nature of the
correction is clearly marked in a footnote. Other sources are acknowledged by
footnotes giving explicit references. A bibliography is appended.
Signed: ……………………………………………… (Candidate)
Dated: 14th March 2018
Statement 3
I hereby give consent for my dissertation, if accepted to be available for
photocopying and for inter library loan, and for the title and summary to be made
available to outside organizations.
Signed: ……………………….……………………… (Candidate)
Dated: 14th March 2018
iiAcknowledgments
I would like to thank all the laboratory technicians and the project supervisor for
their guidance and support with practical and theoretical elements throughout the
duration of this project.
iiiAbstract Introduction: Impaired wound healing places a socioeconomic stain on the population with the incidence of chronic non-healing wounds. The major characteristic associated with chronic wounds is the prolonged inflammation. Tea Tree oil is regarded for its antimicrobial and anti-inflammatory effect. The project investigated the effect of Tea Tree Oil on the gene expression of ICAM-1 on human dermal fibroblast cells (in vitro) at a young age and old age, categorised by passage number. Methods: The TTO was diluted with DMSO to target the human dermal fibroblast cells. Treatment with TTO determined by cell viability assay to investigate the effect of different concentrations. The fibroblast cells were treated with 0.02% and 0.1% TTO in DMSO under – and + TNFα conditions. Expression of ICAM-1 on fibroblast cells measured by antibody attachment using flow cytometry. Results: TTO found to have no significant effect on the viability of cells, with an increased viability following 48-hour treatment. The treatment of TTO found to have a significant effect (p
Table of Contents
Description Page Number
Declarations ii
Acknowledgments iii
Abstract iv
1. Introduction 1
2. Materials and Methods 3
2.1 Ethical Application 3
2.2 Fibroblast Cell Culture 3
2.3 Detachment of Fibroblast Cells 3
2.4 Cell Viability Assay 4
2.5 Flow Cytometry 6
2.6 Statistical Analysis 7
3. Results 8
3.1 Cell Viability Assay 8
3.2 Flow Cytometry 10
4. Discussion 20
4.1 The Effect of Tea Tree Oil on Dermal
Fibroblast Cell Viability 20
4.2 ICAM-1 Expression Measured by Flow
Cytometry 21
4.3 Wound Healing in Young and Old Cells 23
4.4 Signalling Pathways Associated with ICAM-1 24
5. References 26
v1.Introduction
Wound healing remains to be a topic attracting research due to the complexity
associated with healing, there is much left to still be determined to further
understand the mechanisms and improve treatment options. Currently, the
process of wound healing can be established into four overlapping phases;
hemostasis, inflammation, proliferation, and remodelling(1). The appropriate
progression of these phases results in resolution of the wound, however there
are cases where this does not happen. In the event of impaired healing a wound
may become categorised as a chronic non-healing wound(2). The timeline for
healing can vary depending on severity of the wound, generally where there has
been no progression in 3 months the wound is deemed to be chronic(3). The result
of a chronic wound can be associated with underlying comorbidities such as
obesity and diabetes, with the majority of chronic wounds being vascular ulcers
and diabetic ulcers(3).
The incidence of chronic wounds is largely associated with the ageing population,
as with increasing age there is an onset of disease states and the wound healing
ability declines(4). As the population ageing continues with many now living to a
much higher age there is also the issue of new treatment and care options to
accommodate for the health implications experienced with ageing. The
occurrence of chronic wounds within society impact a patient’s quality of life but
also place a strain upon the NHS and the associated economic burden that
comes with treatment(5, 6).
Within the UK the most recent data on the prevalence of chronic wounds is the
NHS 2012/2013 data stating the UK annual NHS cost between £4.5 billion and
£5.1 billion with the average age of patients at 69 years(7). The impact of chronic
wounds within Wales 2016 had the annual cost of £328.8 million(8). The treatment
of wounds therefore is an area in need of attention to find more viable options.
The therapeutic use of essential oils is gaining attention for their potential in
treatment due to their natural beneficial effects.
1Tea tree oil (TTO) or Melaleuca oil is steam distilled from the leaves of the
Melaleuca alternifolia (tea tree) native to Australia. Tea tree oil is popular for use
as an ingredient in cosmetics and also as therapeutic treatment by topical
application(9). For this project the interest of TTO surrounds its potential use in
wound healing. The therapeutic use of TTO stems from the antimicrobial and
anti-inflammatory ability associated with its application, providing a beneficial
effect to the user(10). In wound healing the effects of TTO use may be attributed
to the antimicrobial, anti-inflammatory and immunomodulatory capabilities(11).
The composition of TTO can vary with estimates of up to 100 compounds(12). To
regulate TTO the International Organization for Standardization (ISO) provide the
recommendation for a terpinen-4-ol type (ISO 4730:2017) to ensure quality(13, 14).
TTO is chemically structured by terpene hydrocarbons, with the main structuring
attributed to monoterpenes, sesquiterpenes and the alcohols associated with
them(12). The compound terpinen-4-ol has been determined to be largely
responsible for the anti-bacterial property of TTO(9). The compound terpinen-4-
ol is also associated with the immunomodulatory action of TTO along with α-
terpineol(15).
The research project focused on the expression of intercellular adhesion
molecule-1 (ICAM-1) on human dermal fibroblast cells and how this expression
may be affected by treatment with TTO in vitro. In addition, the project also looked
to find whether the expression of ICAM-1 varied between young and old fibroblast
cells and how the TTO treatment impacted this.
The research aims were to determine an appropriate concentration of TTO that
would have the maximum effect on dermal fibroblast cells without inducing cell
death. Once suitable concentrations had been confirmed the aim was to analyse
how the TTO would affect the expression of ICAM-1 on the dermal fibroblast cell
surface, measured using flow cytometry.
22. Materials and Methods 2.1 Ethical Approval Application for ethical approval made to the BMS Ethical Approval Panel with confirmation of approval given 5th October 2018. Copy of ethical approval provided in the lab book. 2.2 Fibroblast Cell Culture The cell lines used were adult human dermal fibroblasts (HDFa), to compare the effect of TTO on young cells and older cells, for this two different cell lines were required. The two cell lines lot #1631448 (old cells with a passage no. >24) and lot #1813885 (young cells with a passage no.
disposal into the waste bucket. For detaching the cells 0.25% Trypsin-EDTA (Life
Technologies, ThermoFisher UK) was used, 3ml pipetted into the culture vessel
then gently rocked to ensure complete coverage of the fibroblast cells. Culture
vessel then incubated at 37°C for 5 minutes before being removed for the addition
of 7ml of media, by pipetting into the vessel to deactivate the trypsin. The solution
was centrifuged at 250rpm using a Rotina 380 R (Hettich Zentrifugen) machine
for 7 minutes. Once centrifuged the supernatant (trypsin) was removed using a
pipette gun and disposed into the waste bucket. Inverted microscope used to
assess the cell pellet, the fibroblast cells become rounded where they had
successfully detached from the culture vessel.
Fibroblast cells were transferred into a Bijou bottle and 10µl of the cell culture
pipetted into the well chamber of an Improved Neubauer haemocytometer for a
cell count. The live and dead cells were distinguished between using trypan blue,
which was added in an equal volume of dye to the volume of cells as dead cells
will take up the dye. The cell counts averaged result (80 x 104 cells/ml) meant
that the cell seeding density (5000/well) could be achieved by a 1 in 16 dilution.
This required 1ml of the fibroblast cells in 15ml media HDFa (Human Dermal
Fibroblast, adult). From this dilution 100µl was pipetted into each well of columns
4, 5, 6, 10, 11, and 12, (see lab book for detailed labelled well plate structure).
Into the each well of columns 1, 2, 3, 7, 8, and 9, 100µl of media only was pipetted
and the 96-well plate was then incubated at 37°C in 5% CO2 overnight.
2.4 Cell Viability Assay
The cell viability assay involved a 24-hour assessment and a 48-hour assessment
to determine the optimum concentration of TTO (Sigma Aldrich, w390208) that
would produce a maximum effect on the fibroblast cells without resulting in
complete cell death. The TTO concentrations were prepared through doubling
dilutions to observe the effect of different concentrations on the fibroblast cells.
As the TTO needed to target the fibroblast cells in media the oil was diluted with
an equal volume of dimethyl sulfoxide (DMSO), a method used in similar research
of anti-inflammatory activity of clove oil, 2017(16). The mixture of DMSO:TTO was
produced by pipetting 0.5ml DMSO into a Bijou bottle then 0.5ml TTO into the
same Bijou bottle and mixed by pipetting up and down. The concentration range
4of TTO was 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, 0.003125%,
0.0015625%, and a control for comparison with no TTO treatment.
In a centrifuge tube 9980µl HDFa media was pipetted followed by 20µl of the
DMSO:TTO mixture providing the highest concentration (0.1%), 0.1% DMSO:
0.1% TTEO in media (v/v). The 0.1% concentration required a centrifuge tube as
it is made from high grade polypropylene and at this concentration it was found
the DMSO/TTO reacted with the plastic of universal tubes. The following
concentrations were made up in 6 universal tubes and labelled with the
appropriate concentration, into each of these tubes 4ml of HDFa media was
pipetted. From the centrifuge tube [0.1%] 4ml was removed and pipetted into the
next tube [0.05%], this method was carried out consecutively until the lowest
concentration was achieved [0.0015626%].
To prepare the plate for the addition of TTO the 96-well plate was removed from
the incubator to the laminar flow cabinet to remove the 100µl of media from the
wells A4 to H6 and A10 to H12. After the removal of media from a well 100µl of
the corresponding concentration of TTO was added. The plate also included
blank wells and negative control wells. Plate incubated at 37°C in 5% CO2
overnight.
Plate removed from incubation following 24 hours, addition of 20µl of CellTiter
Blue (CTB) reagent to the 24-hour assay wells (column 1 to 6) and the plate was
incubated at 37°C in 5% CO2 for 4 hours before reading on a fluorometer (Plate
Reader Infinite 200) the absorbance at 560nm. Plate put back into incubation at
37°C in 5% CO2 for another 24 hours for the 48hour assay.
Cell viability assay repeat at higher concentrations; 0.8%, 0.4%, 0.2%, 0.1%,
0.05%, 0.025%, and 0.0125%. As higher concentrations were being tested the
use of 15ml polypropylene tubes were required for the doubling dilutions to
reduce the risk of a reaction with the plastic that would impact results. In addition,
the serial dilutions were made in smaller volume from the DMSO:TTO mixture.
To make the highest concentration 0.8% 4920µl of media was pipetted into a
polypropylene tube followed by 40µl from the DMSO:TTO mixture (0.8%
DMSO/0.8% TTO). From this concentration, 6 polypropylene tubes were used
with 2ml of media pipetted into each tube. Concentrations then made up by
5doubling dilutions until the final lowest concentration was achieved (0.0125%).
Plate incubated at 37°C in 5% CO2 and the fluorometer readings (absorbance at
560nm) carried out at 24-hours and 48-hours.
2.5 Flow Cytometry
Preparation of the samples required the use of 12-well plates (x3) working with a
cell seeding density of 50,000 cells per well. The use of a primary antibody (1°
Ab) Mouse IgG1-k (BD Pharmingen™, BD Biosciences) and a secondary
antibody (2° Ab) Goat Ig (BD Pharmingen™, BD Biosciences) to measure ICAM-
1 expression on the fibroblast cells.
All three 12-well plates had been pre-prepared with the fibroblasts seeded and
the HDFa media, then incubated at 37°C in 5% CO2. The next step was to detach
the cells from the wells across the three plates through trypsinisation in a laminar
flow cabinet. From each well 1000µl of media was removed and disposed of into
the waste bucket (water and chlorine tablet). The wells were then each washed
with 1000µl of PBS and removed working down the columns. Next 500µl of 0.25%
Trypsin-EDTA solution was added to each well and incubated for 5 minutes at
37°C in 5% CO2. Plates removed from incubation and 1ml of media was added
to each well to deactivate the trypsin. The inverted microscope was used to
ensure detachment of cells.
The content of each well was then transferred to a round bottomed falcon tube,
and labelled to co-ordinate with well position to keep track of the samples. The
tubes were then centrifuged at 250 rpm for 5 minutes. Addition of 100µl FACS
buffer to each tube to resuspend the pellet and centrifuged at 250 rpm for 5
minutes. Aspirate FACS from tubes by tipping excess content into the waste
bucket (water and chlorine tablet) and lightly dabbing the tube onto absorbent
tissue to remove any excess remaining.
Preparation of 1° Ab as a 1 in 50 dilution; 100µl 1° Ab into 5ml FACS buffer
(500µg/ml divided by 50 to achieve 10µg/test) and then 100µl pipetted into each
tube except the no Ab control and the 2° Ab control which instead 100 µl FACS
buffer is added. Tubes then incubated on ice (polystyrene ice box) in the light for
30 minutes and vortexed every 10 minutes. Following this incubation 500µl of
FACS buffer was added and tubes centrifuged at 220 rpm for 5 minutes. Aspirate
6excess from tubes by tipping into waste bucket and dabbing the tubes on absorbent tissue to remove any excess. Prepare 2° Ab as a 1 in 50 dilution; 100µl 2° Ab into 5ml FACS buffer and add 100µl to all tubes except the no Ab control and 1° Ab control. Into these control tubes add 100µl FACS buffer. Incubation of all tubes on ice in the dark for 30 minutes with tubes vortexed every 10 minutes. After incubation add 500µl FACS buffer to each tube and centrifuge at 220rpm for 5 minutes. Aspirate excess from tubes into waste and dab excess onto absorbent tissue. Resuspend pellet in 500µl FACS buffer and keep the tubes on ice before flow cytometry reading, using a BD Accuri C6 flow cytometer. 2.6 Statistical Analysis The samples for cell viability and flow cytometry were organised and tested in triplicate with results displayed as the mean of the triplicates. Although no complete plate repeats were conducted, this is taken into consideration when discussing the results and their significance. Additional consideration includes the cell count during flow cytometry FL1-A measure where cell count indicated cell death. The data analysis was conducted using statistical package on Microsoft Excel, cell viability assessed using single factor ANOVA and flow cytometry results assessed using the 2-sample t-test (p
3.Results
3.1 Cell Viability Assay
Cell viability assay conducted to determine the highest concentration of TTEO in
DMSO that would not result in cell death but produce a maximum effect. The
viability of treated fibroblast cells observed over a 24-hour and 48-hour period.
Cell Viability of Human Dermal Fibroblasts After 24 hour Tea Tree Oil
Treatment
120
Mean Revlative Cell Viability(%)
100
80
60
40
20
0
Control 0.0015625 0.003125 0.00625 0.0125 0.025 0.05 0.1
Concentration of Tea Tree Oil in DMSO (%)
Figure 1- The Mean Relative Cell Viability of Human Dermal Fibroblasts Treated
with Increasing Concentration of TTO in DMSO in a 24-Hour Assay.
Fibroblast cells incubated in media HDFa treated with 100µl of TTO in DMSO and
incubated for 24 hours (n=3). The mean viability of sample triplicates where
fluorescence absorbance measured at 560nm. No statistical significance in the
cell viability of the increasing concentrations.
8Cell Viability of Human Dermal Fibroblast Cells After 48 hour Tea Tree Oil
Treatment
140
120
Mean Relative Cell Viability (%)
100
80
60
40
20
0
Control 0.0015625 0.003125 0.00625 0.0125 0.025 0.05 0.1
Concentation of Tea Tree Oil in DMSO (%)
Figure 2 – The Mean Relative Cell Viability of Human Dermal Fibroblasts Treated
with Increasing Concentration of TTO in DMSO in a 48-Hour Assay.
Fibroblast cells incubated in media HDFa treated with 100µl of TTO in DMSO and
incubated for 48 hours (n=3). The mean viability of sample triplicates where
fluorescence absorbance measured at 560nm. No statistical significance in the
cell viability of the increasing concentrations.
93.2 Flow Cytometry
Method of indirect flow cytometry used to measure the level of ICAM-1 expression
on young and old human dermal fibroblasts where fluorescence is directly
proportional to the expression of ICAM-1.
Negative Control Mean Cellular Fluorescence of ICAM-1 by Young and Old
Fibroblast Cells
Mean Cellular Fluorescence of ICAM-1 (FL1-A)
6000
5000
4000
3000
2000
1000
0
Young (P8) Old (P26)
Fibroblast Cell Age (Passage no.)
Figure 3 – The Mean Cellular Fluorescence of ICAM-1 for Young and Old Human
Dermal Fibroblasts in media HDFa.
Young and old fibroblast expression of ICAM-1 with no TTO treatment (n=3),
where the mean of triplicate samples was calculated and displayed no statistically
significant difference in ICAM-1 expression.
10ICAM-1 Expression by Young Fibroblast Cells Treated with a Low
Concentration of Tea Tree Oil
Mean Cellular Fluorescence of ICAM-1 (FL1-A)
6,000
5,000
4,000
3,000
2,000
1,000
0
Vehicle Control 0.02
Concentration of Tea Tree Oil in DMSO (%)
Figure 4 – The Mean Fluorescence of ICAM-1 for Young Human Dermal
Fibroblast cells (Passage 8) Treated at Low Concentration of TTO in DMSO.
Fibroblast cells in media HDFa treated with 0.02% TTO and vehicle control (n=3)
indicated no statistical significance in the difference between low concentration
and vehicle control containing media HDFa and DMSO only.
11ICAM-1 Expression by Young Dermal Fibroblast Cells Treated with a
Mean Cellular Fluorescence of ICAM-1 (FL1-A) High Concentration of Tea Tree Oil
25,000
*
20,000
15,000
10,000
5,000
0
Vehicle Control 0.1
Concentration of Tea Tree Oil in DMSO (%)
Figure 5 – The Mean Cellular Fluorescence of ICAM-1 for Young Human Dermal
Fibroblast Cells (Passage 8) Treated with 0.1% TTO in DMSO.
Fibroblast cells in media HDFa treated with 0.1% TTO and vehicle control (n=3)
indicated increased fluorescence with high concentration treatment. Vehicle
control containing media HDFa and DMSO only. * pICAM-1 Expression by Old Dermal Fibroblast Cells Treated with a
Low Concentration of Tea Tree Oil
9,000
Mean Cellular Fluorescence of ICAM-1 (FL1-A)
*
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
Vehicle Control 0.02
Concentration of Tea Tree Oil in DMSO (%)
Figure 6 – The Mean Cellular Fluorescence of ICAM-1 for Old Human Dermal
Fibroblasts (Passage 26) Treated with 0.02% TTO in DMSO.
Fibroblast cells in media HDFa treated with 0.02% TTO and vehicle control (n=3)
indicated increased fluorescence with low concentration treatment. Vehicle
control containing media HDFa and DMSO only. * pICAM-1Expression by Old Dermal Fibroblast Cells Treated with a
Mean Cellular Fluorescence of ICAM-1 (FL1-A) High Concentration of Tea Tree Oil
16,000 *
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
Vehicle Control 0.1
Concentration of Tea Tree Oil in DMSO (%)
Figure 7 – The Mean Cellular Fluorescence of ICAM-1 for Old Human Dermal
Fibroblast Cells (Passage 26) Treated with 0.1% Tea Tree Oil in DMSO.
Fibroblast cells in media HDFa treated with 0.1% TTO and vehicle control (n=3)
indicated increased fluorescence with a high concentration treatment. Vehicle
control containing media HDFa and DMSO only. * pNegative Control ICAM-1 Expression by Young and Old Dermal
Mean Cellular Fluorescence of Icam-1 (FL1-A) Fibroblast Cells with TNFα
600,000
*
500,000
400,000
300,000
200,000
100,000
0
Young (P8) Old (P26)
Fibroblast Cell Age (Passage No.)
Figure 8 – The Mean Cellular Fluorescence of ICAM-1 for Young (Passage 8)
and Old (Passage 26) Human Dermal Fibroblast Cells Under Negative Control
Conditions.
Fibroblast cells in media HDFa supplemented with 10µl TNFα (n=3) where mean
fluorescence of triplicates displayed significant difference of ICAM-1 increased
expression, * p < 0.05 vs vehicle control.
15ICAM-1 Expression by Young Fibroblast Cells + TNFα and Treated at a Low
Mean Cellular Fluorescence of ICAM-1 (FL1-A)
Concentration of Tea Tree Oil
600,000
500,000
400,000
300,000
200,000
100,000
0
Vehicle Control 0.02
Concentration of Tea Tree Oil in DMSO (%)
Figure 9 – The Mean Cellular Fluorescence of ICAM-1 for Young Human Dermal
Fibroblast Cells (Passage 8) Treated with Low TTO Concentration.
Fibroblast cells in media HDFa supplemented with 10µl TNFα and treated with
0.02% TTO in DMSO (n=3) resulted in no significant difference between treated
cells and the vehicle control.
16ICAM-1 Expression by Young Dermal Fibroblast Cells + TNFα and
Mean Cellular Fluorescence of ICAM-1 (FL1-A) Treated at a High Concentration of Tea Tree Oil
500,000 *
450,000
400,000
350,000
300,000
250,000
200,000
150,000
100,000
50,000
0
Vehicle Control 0.1
Concentration of Tea Tree Oil in DMSO (%)
Figure 10 – The Mean Cellular Fluorescence of ICAM-1 for Young Human Dermal
Fibroblast Cells (Passage 8) Treated with High Concentration TTO in DMSO.
Fibroblast cells in media HDFa supplemented with 10µl TNFα and treated with
0.1% TTO in DMSO (n=3) resulted in a significant difference in the expression of
ICAM-1 between treated cells and vehicle control (cells in media HDFa and
DMSO). * PICAM-1 Expression by Old Dermal Fibroblast Cells + TNFα and Treated with
a Low Concentration of Tea Tree Oil
Mean Cellular Fluorescence of ICAM-1 (FL1-A)
350,000
300,000
250,000
200,000
150,000
100,000
50,000
0
Vehicle Control 0.02
Concentration of Tea Tree Oil in DMSO (%)
Figure 11 – The Mean Cellular Fluorescence of ICAM-1 for Old Human Dermal
Fibroblast Cells (Passage 26).
Fibroblast cells in media HDFa supplemented with 10µl TNFα and treated with
0.02% TTO in DMSO (n=3) resulted in no significant difference in the expression
of ICAM-1 between treated cells and vehicle control (cells in media HDFa and
DMSO).
18ICAM-1 Expression by Old Dermal Fibroblast Cells + TNFα and
Treated with a High Concentration of Tea Tree Oil
Mean Cellular Fluorescence of ICAM-1 (FL1-A)
350,000
*
300,000
250,000
200,000
150,000
100,000
50,000
0
Vehicle Control 0.1
Concentration of Tea Tree Oil in DMSO (%)
Figure 12 – The Mean Cellular Fluorescence of ICAM-1 for Old Human Dermal
Fibroblasts Cells (Passage 26).
Fibroblast cells in media HDFa supplemented with 10µl TNFα and treated with
0.1% TTO in DMSO (n=3) resulted in a significant difference in the expression of
ICAM-1 between treated cells and vehicle control (cells in media HDFa and
DMSO). * P4.Discussion
Wound healing is a complex cascade of cellular interactions and in some cases
these components become inhibited or impaired resulting in chronic wounds.
Chronic wounds are often associated with a prolonged period in the inflammatory
phase preventing proper healing(17). The expression of ICAM-1 can be
upregulated by the cytokine TNFα via signalling pathways, which has a key role
in the onset of inflammation(18). There are several studies that identify the role of
ICAM-1 in wound healing and the importance of this molecule in inflammation
and vascular permeability(19, 20). Therefore, TNFα was used in treating cells to
provide a positive control as it is known this cytokine upregulates the expression
of ICAM-1. The project looked to assess how the treatment of fibroblasts with
TTO would affect the expression of ICAM-1 on young and old cells in vitro.
4.1 The Effect of Tea Tree Oil on Dermal Fibroblast Cell Viability
The treatment of fibroblast cells with TTO required identifying a suitable
concentration that would not result in cell death. In order to determine a suitable
concentration a 24-hour and 48-hour viability assay was carried out. The
concentrations of TTO in DMSO used were within the concentrations previous
studies found to be safe for use, as DMSO concentration above 1% is toxic to
cells(21).
The 24-hour and 48-hour cell viability assay displayed no statistically significant
difference between fibroblast response at each concentration, although there was
an observed effect. The 24-hour assay results indicated a small decrease in
viability (no more than 7%) with the most notable decrease being the sample
treated with 0.1% (Fig 1). In comparison, the observation of results for the 48-
hour viability suggest prolonged treatment with TTO in DMSO had a mitogenic
effect with a 11-16% increase in viability (Fig 2). In consideration of the viability
results the 48-hour assay indicated that the 0.1% concentration would be safe for
use and provide the maximum effect on cells.
However, ideally the cell viability assay would have been repeated with more time
to allow for an increased confidence in the results produced and to ascertain
whether repeats would have provided the same or different result. In reference to
20issues encountered with the flow cytometry, it may have proven beneficial to have
tested the concentration 0.05 and to have based the concentration decision on
the 24-hour assay as this was the incubation time before carrying out flow
cytometry. Moreover, the cell viability was carried out on only young fibroblast
cells (Passage 6) which left a gap in the knowledge of how the old cells may have
reacted to the TTO concentrations and whether there would have been a
significant difference in response.
4.2 ICAM-1 Expression Measured by Flow Cytometry
The expression of ICAM-1 was measured by flow cytometry for both young and
old fibroblast cells. Fibroblast cells were treated at low and high TTO in DMSO
concentrations (0.02% and 0.1%) under the conditions -TNFα and +TNFα. In
consideration of the results it should be noted that the cell count measure for flow
cytometry differed between young (5000 count) and old (2500 count) cells. Cell
count differed due to encountered problems with the flow cytometry machine.
When reading the samples from young cells the machine became blocked and
required to be restarted and filters cleaned.
This delay may have affected the results although samples remained on ice
throughout this period, samples were again vortexed before reading in attempt to
minimise clumping. This was due to finding the fibroblast and TTO to be sticky in
nature resulting in the clogging of the reader. Therefore, when measuring the old
cell sample the count was reduced to 2500 so to avoid blocking the machine with
the sample. This is a variable that should be considered with the results, however,
the cell count also suffered due to cell death resulting from the 0.1% treatment.
The negative control of young and old fibroblast cells incubated in media HDFa
indicated the level of ICAM-1 expression was not statistically significant between
the age of cells, with an observation of slightly less expression of ICAM-1 on the
older fibroblast cells when untreated (Fig 3). The treatment of young cells (P8)
with TTO in DMSO (v/v) displayed a significant difference in the response
between low and high TTO concentration. At 0.02% concentration the expression
of ICAM-1 on young fibroblast cells showed a small increase indicating the
potential influence of TTO in expression (Fig 4). Whereas the response of the
young fibroblast cells treated with 0.1% concentration showed an approximate 4-
21fold increase in ICAM-1 expression compared to that at low concentration (Fig 5).
This would denote a significant influence of the 0.1% TTO in fibroblast cell
expression.
In contrast, the old fibroblast cells (P26) showed a significant response to
both the low and high concentration of TTO in DMSO (v/v) treatment. The
treatment with 0.02% displayed a 2-fold increase above the vehicle control
denoting the effect of TTO (Fig 6). An increased response in expression was
recorded with 0.1% TTO treatment of old fibroblast cells at a 3.5-fold increase in
ICAM-1 expression despite being affected by cell death (Fig 7). The significance
in increase of ICAM-1 expression on older cells implies there is a greater
response to the TTO in comparison to the young cells in stimulating ICAM-1
expression. Under these conditions the TTO is producing a pro-inflammatory
response on the cells.
As a positive control both young and old fibroblast cells were incubated in media
HDFa supplemented with TNFα. The expression of ICAM-1 was measured under
these conditions and for both young and old fibroblast cells finding a significant
difference in the expression of ICAM-1. The response to TNFα resulted in
significant differences. Although both young and old cells were induced to up-
regulate ICAM-1 expression the response was greater in young cells (Fig 8).
The treatment of young cells with 0.02% TTEO in DMSO (v/v) +TNFα
resulted in no significant difference in ICAM-1 expression compared to the vehicle
control (Fig 9). This correlates with the finding (-TNFα) where at a low
concentration young cells were not significantly stimulated by TTO to increase
ICAM-1. Furthermore, the treatment at 0.1% resulted in the cellular response of
down-regulation of ICAM-1 (Fig 10). This result correlates with the findings of -
TNFα that the higher concentration had an increased effect on cells. Although in
this case in the presence of TNFα the down-regulation may indicate the anti-
inflammatory effect of TTO. However, this result may not only be attributed to
anti-inflammatory action but to the finding of high cell death occurring with this
treatment. The event of cell death may be a result of the manipulation and
physical stress cells endured during the flow cytometry procedure, making them
more vulnerable to the TTO.
22Similarly, the treatment of old fibroblast cells with TTO at 0.02% and 0.1%
+ TNFα reflected the trend of the young cells treated in this way. The old fibroblast
cells treated with 0.02% TTO in DMSO displayed no significant difference to the
expression of ICAM-1 by the vehicle control with only a small observable increase
suggesting a small influence of TTO (Fig 11). The treatment with 0.1% TTO in
DMSO + TNFα indicated a significant difference in response compared to vehicle
control denoting the recognisable effect of TTO on old fibroblast cells (Fig 12).
The results presented a decrease in the ICAM-1 expression which again could
be attributed to the high cell death occurring at this concentration which was not
anticipated by the cell viability, and it would have been beneficial to repeat both
viability and flow cytometry elements.
The young and old fibroblasts indicated there was some differences in response
that may be attributed to the age of the cell. Where the cells were treated in the
absence of TNFα both young and old fibroblasts displayed increased ICAM-1
expression with 0.1% treatment indicating TTO may influence regulation of this
gene expression. The old fibroblast cells also showed significant response to
0.02% treatment, due to aging and impairment TTO may provide a stimulatory
effect in reactivating the ability of cells although this cannot be determined from
this project. Comparatively, in the presence of TNFα the response to TTO
indicated an anti-inflammatory effect. However, determining the beneficial effect
of TTO is difficult as at 0.1% there were varying levels of cell death between
young and old fibroblasts. Were repeats taken the cause may have been
established.
4.3 Wound Healing in Young and Old Cells
Wound healing and ageing are natural processes that occur over the course of a
life. As cells age their efficiency begins to decline and processes such as wound
healing are impacted by this deterioration in cellular ability(22). The complexity of
wound healing could be the result of impairment in numerous phases/processes
with different contributing factors.
23Furthermore, the capacity of fibroblast cells to deal with the stresses of wound
healing declines with age. The decline in fibroblast cell ability in wound healing
has been attributed to a reduced proliferative action hindering the remodelling
phase through reduced collagen production(23, 24). Further dysfunction of ageing
fibroblast cells has been identified in the regulation of reactive oxygen species
(ROS) due to a decline in superoxide dismutase (SOD1) expression(25). The
decline in fibroblast ability may fit with the increased response to TTO observed
in old fibroblasts in comparison to the young. The TTO may have had a
stimulatory effect in engaging signalling and response in old cells.
A recent study has highlighted an area of impact associated with aging
keratinocytes where intrinsic defects hinder communication with immune cells(26).
This could be a potential theory applied to research to determine any changes in
signalling of fibroblasts. The ability to stimulate aged cells where communication
is impaired may be a viable target for future treatment to enhance the healing
ability. The innate immune response during healing associated with inflammation
places importance on the migration of leucocytes to the wound bed(27). This
migration of leucocytes from blood vessels to the wound bed is facilitated by
ICAM-1(20). The regulation of ICAM-1 expression on cell surface showed
significant impact by TTO. This had potential to be a target in treatment as it is
established that in cases of ICAM-1 depletion wound healing is delayed in
mice(28). The necessity for ICAM-1 in healing may provide a future avenue for
research in further determining how the expression for this gene could be up-
regulated to aid wound healing.
4.4 Signalling Pathways Associated with ICAM-1
The actions involved in wound healing are coordinated by intricate
communication between cells. There is an understanding for the importance of
ICAM-1 expression on endothelial, lymphocyte, monocyte and fibroblast cells in
wound healing(29). The importance stems from the influence on immune cell
migration required during the inflammatory phase. Although the up-regulated
expression of ICAM-1 can be associated with rheumatic disease leading tissue
damage (30). In the case of underlying disease or early onset of disease the
regulation of ICAM-1 should come under consideration to refine treatment
24options. Therefore, ICAM-1 as a potential biomarker to monitor inflammatory
progression in chronic wounds and assess effectiveness of treatment could be
further researched.
The ICAM-1 gene is located on chromosome 19. It is associated with a variety of
signalling pathways in its regulation of expression on different cell types across
tissues throughout the body. The gene expression of ICAM-1 influences the
degree of adhesion that takes place(31). The mechanisms by which signalling
pathways activate the expression of ICAM-1 in fibroblasts lacks research but the
major influence of TNFα on ICAM-1 has been established(32) . In addition to TNFα
the following are associated with the ICAM-1 gene expression; IFN-γ, PMA, and
Interleukin molecules(33).
There are proposed pathways associated with other cell types such as in human
airway epithelial cells the regulation of ICAM-1 expression is controlled by TNFα
through the Nuclear factor (NF- kB) protein with mediation by the Rac1-ROS
cascade(34). Additionally, the expression of ICAM-1 on human epithelial cells has
been linked to the adenylate-cyclase dependent pathway(35) Research involving
the expression of ICAM-1 on T-cells found the contribution in regulation by
phosphotyrosyl phosphatase via NF-kB, Ets, STAT-1-dependent signalling(31).
There is also little research in the mechanism by which TTO exhibits an effect on
cells. There are findings on the immunomodulatory action of TTO that indicate
the mechanism of action through the inhibiting of both NF-kB signalling and
macrophage-type cell cytokine production(36).
In conclusion, there is a gap in knowledge surrounding the various components
that contribute to the signalling of transcription factors and cytokines in the
expression of ICAM-1 in fibroblast cells. The established knowledge of the role of
TNFα provides a standard by which to base further research in testing pathways
through enhancing or blocking by TTO to identify and measure specific
components of interaction.
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