Document Ne189eQbBYEYL8k6Ke547DxLR
EFFICACY REPORT OXYGENATE THE SKIN TO REDUCE ACNE SCARS ON A CAUCASIAN PHOTOTYPE
Product: Fiflow AA Beginning of the study: 01/23/2020 End of the study: 03/12/2020
PURPOSE OF THE STUDY
Acne is a common skin disease that generally appears with puberty. The limited exposure of damaged tissues to oxygen (from air) leads to poor wound healing of acne scars. The severe sequel of acne scarring can lead to long-term psychological injury. There are a wide range of treatments for acne scars such as surgical techniques, chemical peels, ablative lasers, fractional lasers and collagen induction therapy. However, these solutions are often scarying and very intrusive for the patients. It is widely prefered to use topical applications.
Oxygen therapeutics are derived from hemoglobin or perfluorochemical-based compounds. Fully fluorinated perfluorocarbons (hydrogen atoms have been replaced by fluorine) are stable and inert due to the strong C-F bond. They have the key property for carrying gases, at 9 times superior to the function of respiratory gases in the human body. The solubility of the gases, notably oxygen and carbon dioxide, in Perfluorocarbons is a fully reversible phenomenon based on physical gas diffusion (Fick's law).
Dual Laser Confocal Microscopy is the new non-invasive examination technique for the skin in vivo in real time, which is useful to evaluate the effectiveness of this advanced therapeutic approach.
This study focuses on the treatment of acne scars using Perfluorocarbons and the follow-up observations with Dual Laser Confocal Microscopy. Our subject was a 21-years+ (non-smoker) woman with a fair phototype. Our subject showed a lot of acne scars on her face since she takes contraception. The testing had been performed according to a half-face method. The products (one serum and one cream) containing respectively 15 and 10% of Fiflow AA (Perfluorohexane (and) Perfluoroperhydrophenanthrene (and) Perfluorodecalin (and) Perfluorodimethylcyclohexane) have been applied twice a day (0.2g/0.15ml; morning and evening) on the left side of the face. Macroscopic pictures have been taken with two different macro-cameras (one is linked to the confocal microscope) before starting the study, then again after 1 week, 2 weeks, 4 weeks and 6 weeks. Hydration, skin elasticity, melanin index and ultrasounds (collagen density and skin thickness) measurements were taken using a multiparameter skin analyser. Three repeatability measurements have been done for each parameter. Reflectance Confocal Microscopy images and microcirculation videos have been recorded with the Reflectance Confocal Microscope, focusing on the Stratum Corneum, Dermal-Epidermal Junction and upper dermis.
ENVIRONMENTAL CONDITIONS
Time of measurement
Date of testing
W0 (Before starting products application) W1 W2 W4 W6
01/30/2020
02/06/2020 02/13/2020 02/27/2020 03/12/2020
Weather
Rainy
Cold Rainy Rainy Cloudy
Atmospherical pressure
Air Quality Index
1010 hPa
40
Outside temperature
7C
Outside relative humidity 95%
1031 hPa
60
3C
83%
1006 hPa
37
6C
87%
1000 hPa
39
5C
93%
1017 hPa
22
8C
85%
Room temperature
21.6C
Room relative humidity
33%
21.6C
29%
21.5C
34%
21.2C
28%
21.8C
41%
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TESTING EQUIPMENT Skin property to assess
Macroscopic aspect
Melanin content Erythema index Skin luminosity Skin hydration Sebum content Collagen density Skin thickness Skin elasticity Stratum Corneum structure Stratum Granulosum structure Dermal Epidermal Junction structure Dermis structure Microcirculation
Test location
Tool used
Defined scar on each side of the face Macro-camera
Entire face then each side of the face Dermatoscopy
Defined scar on each side of the face Skin colour probe
Defined scar on each side of the face Cheek of each side of the face Nostril of each side of the face
Defined scar on each side of the face Cheek of each side of the face
Hydration pin probe
Sebum collecting device Ultrasounds probe Microscope Ultrasounds probe Skin elasticity probe (suction cup)
Microscope
Microscope (20-s video)
Device used Canon EOS DermaLab SkinLab Combo Dual Laser Confocal Microscope (DLCM)
DermaLab SkinLab Combo
Dual Laser Confocal Microscope (DLCM) DermaLab SkinLab Combo
Dual Laser Confocal Microscope (DLCM)
SUBJECT
First Name Gender Birth date Phototype Origin Smoker Skin Type Work Cosmetic use Sun exposure Sunscreens use Acne
PRODUCTS AND APPLICATION
Product number Type of product Batch number Fiflow AA content Location of application Amount of product applied Time of application
Mlanie F 2/23/1999 II France No Mixte with acne
Inside Everyday Summer Yes For more than 2 years now / Began with contraception
1 Serum PFI3696CS 15% Left side of the face and neck 0.2g / 0.15 ml Every day / Twice a day (morning and evening)
2 Cream PFI3695CS 10% Left side of the face and neck 0.2g / 0.15 ml Every day / Twice a day (morning and evening)
CONCLUSIONS
The long-term study shows a reduced inflammation and redness of acne scars as soon as 2 weeks after having started the study. On the right side of the face (control area), acne scars are still present forming bumps.
After 6 weeks of application, skin hydration, skin luminosity, skin thickness and collagen network density have significantly increased due to the oxygenating power of perfluorocarbons on the skin cells. It consequently improves cell breathing and moreover, it stimulates fibroblasts and collagen network density. These results improved week after week for the both sides of the face. It has also been noticed that the cycle period has a strong impact on the woman skin : more pimples and greasy skin at menstruations time. The Air Quality Index has a strong impact on skin properties mainly on skin surface properties such as hydration, skin colour and sebum. This is due to the pollutant gases containing in air that can
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be removed with the use of perfluorocarbons products.
The following table summarizes the results of the entire study after 6 weeks of products application :
Skin property
Control Skin Area (Right side of the face)
Testing Area (Left side of the face)
Macroscopic aspect
No significant change. Pimples and comedo growing up Inflamed and swollen skin.
Less inflamed and swollen skin. Less comedo.
Melanin content
Reduced by 10%
Reduced by 7%
Skin luminosity
Increased by 12%
Increased by 14%
Erythema index
Decreased by 14%
Decreased by 24%
Skin hydration
Decreased by 17%
Increased by 8%
Sebum content
Decreased by 15%
Decreased by 53%
Collagen density
Increased by 10%
Increased by 24%
Skin thickness
Increased by 60%
Increased by 99%
Skin elasticity
Decreased by 5%
Increased by 7%
Stratum Granulosum structure
Inflammatory cells have been reduced by 70%
Inflammatory cells have been reduced by 91%
Dermal Epidermal Junction structure No changes
Visibly more regular and restructured
Dermis structure
Elastic fibers density has been reduced by 10%
Elastic fibers density has been increased by 144%
Microcirculation
Increased by 2%
Increased by 45%
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I. PROTOCOLS Dual Laser Confocal Microscope (DLCM)
The Dual Laser Confocal Microscope (DLCM) is equipped with a laser sweep with an imaging software. This non- invasive device makes it possible to have in vivo measurements by optical biopsies and to observe internal skin layers up to the deep dermis. It has a laser probe for observing deep skin layers and blood microcirculation, a macro digital camera to capture macroscopic skin images and has a digital high-resolution screen. Reflectance Confocal Microscopy is the only technique that enables the en-face (horizontal plane) visualization of the skin with a resolution at the cellular level and therefore may provide an alternative to histopathology. The contrast in RCM images relies on the differences in the reflectivity of the tissue, which is depending on chemical and molecular structures. Due to these variations of the refractive index, only a certain portion of the light is reflected. Structures with a higher refractive index appear bright in RCM. Melanin and melanosomes are the strongest natural sources of contrast resulting white in RCM. The deeper the skin section is the more laser power is needed to penetrate the skin. The VivaScope 1500 uses a laser diode with a near-infrared wavelength of 830 nm. Laser power of 830 nm causes no damage to tissue, but limits the imaging depth to 200-300 m, which corresponds to the papillary dermis, depending on the skin site. Higher laser power would provide an increased signal and contrast but is hazardous for the skin or the eyes. Confocal observations are performed at each time of measurement according to this method : After the subject is ensconsed in the chair and is relaxed, the experimenter placed on the test area, the ring with the adhesive patch oiled
beforehand in the center of the side that will be in contact with the skin. The experimenter placed the macro camera onto the ring to take a picture of macroscopic skin aspect of the subject After removing the macro camera from the ring, the experimenter applied a gel on the patch center where the laser will be in contact with. The assessor then positioned the removable device of the microscope on the ring. When corneocytes are detected, the depth is set to 0 m. The assessor looked for an area where the blood flow is easily viewable (it is possible to adjust magnification). This zone is sometimes
identifiable in a tiny way. It must ensured that the flow is clear and definite. The Stack (optical biopsy in vivo) is then started. According to the programming software, the device will focus on the selected location and
take multiple pictures (around thirty) while gradually increasing its observation depth. Images can be observed like a video. The experimenter can then zoom again on one or the other of each image to take photos or videos the most highlighting the blood circulation. The assessor removed the device from the ring and patch of the skin. All of the previously steps are repeated to study. The microcirculation on the patient after applying the product. Macroscopic picture of a defined scar on each side of the face is taken. Confocal observations of skin structures (Stratum Corneum, Stratum Granulosum, Dermal Epidermal Junction and collagen network) have been performed at each time of measurement. Microcirculation has been quantified by means of a 20-s video to count the number of blood cells in a capillary.
DermaLab SkinLab Combo
The DermaLab SkinLab Combo (multiparameter skin analyzer) is a non-invasive method to perform in vivo measurements of skin properties by means of different probes : Macro camera magnifies (x50) and vizualizes the surface of the skin with a polarized light (in this case) Skin colour tool gives the spectrocolourimetric parameters L*a*b* and erythema-melanin indexes for skin application. Melanin index is
defined as the pigmentation index of the skin Ultrasounds probe is based on measuring the acoustic response from the skin acoustic pulse is sent into the skin. It determines skin thickness
and collagen density Hydration pin probe measures the conductance to assess the water binding capacity of the Stratum Corneum Skin elasticity is based on the suction cup method upon measuring the necessary force to lift the skin a certain distance using negative
pressure as well as the time it takes for the skin to retract, when the negative pressure is released Sebum surface quantity is measured by means of a sebum collecting device. The transparency of the film increases with sebum Hydration, skin elasticity, sebum, melanin index, skin luminosity and ultrasounds (collagen density and skin thickness) measurements were taken at each time of the study. Three repeatability measurements have been done for each parameter.
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II. RESULTS - MACROSCOPIC ASPECT AND SKIN COLOUR Camera - Canon EOS
Time of measureme
nt
Control Area (Right side of the face)
Before having started the study (W0)
Testing Area (Left side of the face)
After 6 weeks (W6)
On the control skin area, skin is still visibly red and inflamed. New acne spots are visible. On the testing area, the skin is visibly less inflamed and less red. Few acne spots and acne scars.
Macro-camera of the DermaLab SkinLab Combo
Time of measureme
nt
Control Area (Right side of the face)
Testing Area (Left side of the face)
Before having started the study (W0)
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After 6 weeks (W6)
Macro-camera of the Dual Laser Confocal Microscope
Time of measurement
Control Area (Right side of the face)
Before having started the study (W0)
Testing Area (Left side of the face)
After 6 weeks (W6)
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Skin Colour
Air Quality Index
Evolution of skin colour properties
In Vivo testing - DermaLab Skinlab Combo - Skin colour probe 1 subject - Fair phototype - 21-years old woman
70
50
45 60
40
50
35
40 30 25
30 20
20
15
10
10 5
025-Jan-2020 30-Jan-2020
4-Feb-2020
0 9-Feb-2020 14-Feb-2020 19-Feb-2020 24-Feb-2020 29-Feb-2020 5-Mar-2020 10-Mar-2020 15-Mar-2020 Date of study
Skin conductance de<ining skin hydration (S)
Air Quality Index (Paris)
Erythema Index - Control skin area
Erythema index - With AA Serum and Cream
Luminosity With AA Serum and Cream
Luminosity Control skin area
The previous graph shows the impact of AA serum and cream application on skin colour. Due to the reduction of skin inflammation, we can easily observe that skin luminosity is higher on the tested area of the face. Erythema index has also decreased due to the oxygenation of the skin cells. The Air Quality Index (AQI) impacts the results.
Skin luminosity has increased by 14% on the testing area vs. 12% on the control side. Erythema index decreased on both areas : by 14% on the control vs. by 24% on the testing area.
III. RESULTS - SKIN HYDRATION
Skin conductance de<ining skin hydration (S)
Evolution of skin hydration properties along 6 weeks of study
In Vivo testing - DermaLab Skinlab Combo - Hydration pin probe
1 subject - Fair phototype - 21-years old woman
70
350
Air Quality
60
300
Index (Paris)
Air Quality Index
50
250
40
200
30
150
20
100
10
50
025-Jan-2020 30-Jan-2020 4-Feb-2020 0 9-Feb-2020 14-Feb-2020 19-Feb-2020 24-Feb-2020 29-Feb-2020 5-Mar-2020 10-Mar-2020 15-Mar-2020 Date of study
Control area without product
With AA Serum and Cream
The previous graph clearly shows that skin hydration is impacted by Air Quality Index (AQI). An increase of AQI seems to decrease skin hydration. This is due to the increase of pollutants affected the skin.
After 6 weeks of study, skin hydration has increased by 8% on the testing area compared to a reduction by 17% on the control area.
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IV. RESULTS - SEBUM
Evolution of sebum content along 6 weeks of study
In Vivo testing - DermaLab Skinlab Combo - Sebum tape
1 subject - Fair phototype - 21-years old woman
70
25
Transparency of the <ilm (sebum content - %)
Air Quality Index
60
Air Quality
20
Index (Paris)
50
40
15
Control area
without
product
30 10
With AA
20
Serum and
5
Cream
10
025-Jan-2020 30-Jan-2020
4-Feb-2020
9-Feb-2020
14-Feb-2020 19-Feb-2020 24-Feb-2020 29-Feb-2020
Date of study
5-Mar-2020
0 10-Mar-2020 15-Mar-2020
The previous graph shows the impact of AA serum and cream application on sebum reduction. This property is also drastically impacted by AQI. Higher the AQI, higher the sebum content. This is due to the increase of pollutants affected the skin. Sebum content varies a lot on the testing area.
V. RESULTS - MICROCIRCULATION
Number of blood cells moving in a capillary for 20s
Evolution of microcirculation along 6 weeks of study
In Vivo testing - Dual Laser Confocal Microscope - 20-s video
70
1 subject - Fair phototype - 21-years old woman
35
Air Quality
60
30
Index (Paris)
50
25
Air Quality Index
40
20
Control area
without
30
15
product
20
10
With AA
Serum and
10
5
Cream
025-Jan-2020 30-Jan-2020
4-Feb-2020
0 9-Feb-2020 14-Feb-2020 19-Feb-2020 24-Feb-2020 29-Feb-2020 5-Mar-2020 10-Mar-2020 15-Mar-2020 Date of study
The previous graph highlights the huge impact of oxygenating products on the increase of microcirculation. This is interesting to notice that the control area without product is depending on the AQI whereas the assessed area seems to not be affected by the AQI. The microcirculation increases weeks after weeks with the use of AA serum and cream up to an increase by 45% after 6 weeks.
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Air Quality Index Retraction time of skn elasticity measurement (ms)
VI. RESULTS - SKIN THICKNESS - ELASTICITY - SKIN LAYERS RESTRUCTURING -
Evolution of skin elasticity properties along 6 weeks of study
In Vivo testing - DermaLab Skinlab Combo - Elasticity probe - Suction cup method 1 subject - Fair phototype - 21-years old woman
70
180
160 60
140 50
120
40
100
30
80
60 20
40
10 20
025-Jan-2020 30-Jan-2020 4-Feb-2020 0 9-Feb-2020 14-Feb-2020 19-Feb-2020 24-Feb-2020 29-Feb-2020 5-Mar-2020 10-Mar-2020 15-Mar-2020 Date of study
Air Quality Index (Paris)
Control area without product
With AA Serum and Cream
The previous graph highlights the huge impact of oxygenating products on skin elasticity. We can notice that retraction time is reduced when using AA serum and cream. However, this properties is depending on the dermis fibers. It is a long-term reaction to observe modifications.
The skin elasticity results are taken by precising the skin thickness value. The skin thickness is determined by means of the ultrasounds measurement.
The following table shows the ultrasounds pictures of the both sides of the face on the acne scar/comedo previously defined. The lightest/yellow area represents the epidermis, the green to red spots stand for the dermis and the black area is the subcutis.
Time of measureme
nt
Control Area (Right side of the face)
Testing Area (Left side of the face)
Before having started the study (W0)
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After 6 weeks (W6)
The results are comparable on the control skin area. However, the dermis fraction is denser and more intense on the testing area after 6 weeks of study. It means that dermis fibers such as collagen and elastin have been created ; skin thickness has been increased by 99%.
Stratum Corneum structure (0 micron depth)
Time of
Before having started the study
measurement
(W0)
After 4 weeks (W4)
After 6 weeks (W6)
Control Area (Right side of
the face)
Testing Area (Left side of
the face)
The face has a lack of hydration at the beginning of the study with desquamating corneocytes. Weeks after weeks, the skin layer is moisturized on the testing area: no more desquamating corneocytes, regular layer and thinner skin furrows. Results are comparable on the control skin area.
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Stratum Granulosum structure (50 microns depth)
Time of
Before having started the study
measurement
(W0)
Control Area (Right side of
the face)
After 4 weeks (W4)
After 6 weeks (W6)
Testing Area (Left side of
the face)
Percentage of reduction of in<lammatory cells
Evolution of in<lammatory cells of Stratum Granulosum along 6 weeks of study In vitro testing - Dual Laser Confocal Microscope - Image analysis 1 subject - Fair phototype - 21-years old woman
120
100
80
Control Skin Area
60
With AA Serum and AA
Cream
40
20
0 After 1 week After 2 weeks After 4 weeks After 6 weeks
Before starting the study, Stratum Granulosum was not well structured with amorphous keratinocytes although they were already organised in honeycomb pattern. With the use of serum and cream containing perfluorocarbons, the skin layer became well-defined with a visible honeycomb pattern of regular keratinocytes. Moreover, at the begenning of the study, we can notice bright inflammatory cells between keratinocytes that are no more visible after 6 weeks of products application. This reduction of inflammatory cells is visible on both sides of the face. This is probably due to the cycle period of the subject. On the control area, inflammatory cells have been reduced by 70% vs. 91% for the testing area.
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Dermal Epidermal Junction structure (96 microns depth)
Time of
Before having started the study
measurement
(W0)
After 4 weeks (W4)
Control Area (Right side of
the face)
After 6 weeks (W6)
Testing Area (Left side of
the face)
On the control area, few dermal papillae are visible. They are small and not well-defined. On the testing area, the skin layer is amorphous before applying the products. Few dermal papillae are visible. After 4 weeks, the layer was restructured and there are much more dermal papillae visible and clearly defined. This was enhanced after 6 weeks of study with round and clear papillae with more blood cells inside.
Time of measurement
Before having started the study (W0)
After 4 weeks (W4)
After 6 weeks (W6)
Control Area (Right side of
the face)
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Testing Area (Left side of
the face)
The previous mozaics show the structure of Dermal-Epidermal junction on a wide area. Black area are visible for the control skin area. They are due to the irregular skin surface because of the inflammation of acne spots. On the testing area, the layer was not regular before starting the study. We can already see fibers structures on the right part of the image because the skin is inflamed. Weeks after weeks, the layer is more defined and regular because the inflammation is reduced.
Density of dermis fibers (134 microns depth)
Time of
Before having started the study
measurement
(W0)
After 4 weeks (W4)
After 6 weeks (W6)
Control Area (Right side of
the face)
Testing Area (Left side of
the face)
Before starting the study, both sides do not show any clearly defined elastic fibers. The skin is bumped and inflamed so we mainly see the end of the dermal-epidermal junction. No bright structures. The results are not significant on the control skin area even if some blurry elastic fibers are visible after 6 weeks of study. On the testing area, we clearly see a restructuring step weeks after weeks until obtain a dense and strong network of elastic fibers, long and bright.
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Percentage of increase of the mean area of dermis <ibers
Evolution of the density of dermis <ibers along 6 weeks of study In vitro testing - Dual Laser Confocal Microscope - Image analysis 1 subject - Fair phototype - 21-years old woman
200 150
Control Skin Area 100
With AA Serum and AA Cream 50 0
After 1 week After 2 weeks After 4 weeks After 6 weeks
-50 The previous graph shows the quantification of the density of dermis fibers on 6 weeks. We can easily notice that there is no difference on the control skin area, the results are not significant. On the testing area, the results are comparable after 1 week. As soon as 2 weeks after products application, elastic fibers are created and their density is increased weeks after weeks up to 144% after 6 weeks.
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