One of the major long term consequences of chronic non extreme sun exposures is the development of photoaging. The recent definition of a standard daily ultraviolet radiation (DUVR) spectrum has allowed us to reproduce non-zenithal sun exposure conditions. Exposure to simulated DUVR induces biological damage in human skin, suggesting the need for an appropriate daily photoprotection. The reconstructed human skin in vitro, including a living dermal equivalent and a fully differentiated epidermis represents a predictive tool to study the effects of UV. In the present study, sunscreen products were evaluated after topical application on human reconstructed skin in vitro. Two commercial sunscreens (A and B) having similar SPF value (~15) but different profile of transmission over the UVA range were tested on skin models exposed to increasing doses of DUVR. Another pair of sunscreens was also tested. Product C had a SPF ~18 with a well-balanced UVB/UVA absorption profile and product D, with SPF ~27 and low UVA absorption. Biological parameters were assessed by (i) histology, (ii) immunostaining of dermal fibroblasts and (iii) MMP-1 production. Products A and C gave a better protection from DUVR with regard to fibroblast alterations and MMP-1 release compared to products B and D respectively. These results support that a well balanced profile of absorption and the level of UVA protection is of higher relevance than the SPF value to ensure an efficient daily photoprotection from DUVR with regard to end points evaluated in this study, especially the photoaging related biological markers.

1. EVALUATION OF DAILY
PHOTOPROTECTION USING
RECONSTRUCTED SKIN MODEL :
Relevance of UVA absorption profile
and SPF value
F Lejeune, F Christiaens and F Bernerd
L’Oré al Research & Innovation, Clichy, Chevilly-Larue, France

2. ABSTRACT
One of the major long term consequences of chronic non extreme sun exposures is the development
of photoaging. The recent definition of a standard daily ultraviolet radiation (DUVR) spectrum has
allowed us to reproduce non-zenithal sun exposure conditions.
Exposure to simulated DUVR induces biological damage in human skin, suggesting the need for an
appropriate daily photoprotection. The reconstructed human skin in vitro, including a living dermal
equivalent and a fully differentiated epidermis represents a predictive tool to study the effects of UV.
In the present study, sunscreen products were evaluated after topical application on human
reconstructed skin in vitro. Two commercial sunscreens (A and B) having similar SPF value (~15) but
different profile of transmission over the UVA range were tested on skin models exposed to increasing
doses of DUVR. Another pair of sunscreens was also tested. Product C had a SPF ~18 with a well-
balanced UVB-UVA absorption profile and product D, with SPF ~27 and low UVA absorption.
Biological parameters were assessed by (i) histology, (ii) immunostaining of dermal fibroblasts and (iii)
MMP-1 production.
Products A and C gave a better protection from DUVR with regard to fibroblast alterations and MMP-1
release compared to products B and D respectively. These results support that a well balanced profile
of absorption and the level of UVA protection is of higher relevance than the SPF value to ensure an
efficient daily photoprotection from DUVR with regard to end points evaluated in this study, especially
the photoaging related biological markers.

3. INTRODUCTION
One of the major long term consequences of chronic sun exposure is the development of photoaging clinical signs
including wrinkles and sagging. These deleterious events mostly result from alterations of extracellular matrix and
fibroblasts in the dermal compartment. It is now well recognized that UVA wavelengths play a major role in
photoaging process indicating a predominant involvement of sub-erythemal sun exposures. It is therefore of
importance to design the most efficient photoprotection for all kinds of solar exposure.
The reconstructed human skin in vitro which includes a living dermal equivalent and a fully differentiated epidermis
represents a highly reliable predictive model to characterize wavelength and cell type specific biological damage
together with tissue distribution. Using this 3D skin model, we previously showed that while UVB directly affected
epidermis with sunburn related biological damage (1), UVA radiation directly impacted the dermal compartment
through fibroblast impairment and extracellular matrix modifications, including MMP-1 production (2).
Based on the identification of these biological end-points, evaluation of sunscreen efficiency could be assessed.
Test products can be topically applied to the skin model alike in vivo due to the presence of horny layers (3).
OBJECTIVE
Here we studied the efficiency of two pairs of sunscreen products (A/B, and C/D) having different
characteristics.
o The pair A/B was composed of two commercial sunscreen products. They have the
same labeled SPF of 15 but different UVA absorption profile.
o The pair C/D was composed as follow: Suncreen C had a SPF of 15 and a UVA-PF of
10 as measured by the PPD method. Suncreen D had a SPF of 30 and a UVA-PF of 4.
Test product was applied to the surface of reconstructed skin which was then exposed to increasing doses
of daily UV radiation (DUVR).

4. MATERIAL AND METHODS
1 Cells : Human fibroblasts and keratinocytes were obtained from normal human breast skin
2 Reconstructed human skin in vitro
Dermal equivalents were prepared with 106 dermal fibroblasts and bovine type I collagen.
Epidermal keratinocytes were seeded at 33000/cm². The culture was performed during 7 days in submerged
conditions, then raised on a grid at the air liquid interface for subsequent 7 days of culture. Culture medium
was changed every 2 days.
KERATINOCYTES
3 days
7days 7d
Collagen, Fibroblasts Dermal equivalent Seeding of Normal human skin
« Monolayered Epidermis » Air-liquid interface
Medium Keratinocytes Epidermal differentiation
Human skin in vitro
0,5
SSR
0,4 Daily UV (DUVR)
Experimental source for Daily UV (ref 4)
0,3
3 UV source 0,2
SSR (Solar Simulated Radiation)
( UVA/UVB = 12 ) Erythemal exposure
DUVR (Daily UV) UVA/UVB = 27. Non erythemal
0,1
exposure
290 300 310 320 330 340 350 360 370 380 390 400
Wavelength (nm)

5. MATERIAL AND METHODS
4 Sunscreen products and treatment :
Products C and D were prepared in the same vehicle (oil in water emulsion).
Sunscreen products or vehicle were applied topically at ~ 2mg/cm² 10 minutes prior UV exposure. Samples
were then rinsed and analyzed 48 hours after UV exposure for classical histology. Culture medium were
collected and MMP1 protein level was assessed using ELISA (Amersham).
Product A Product B Product A
0.5
Product B
Avobenzone : 2% Octinoxate : 6% 0.4
Terephthalylidene Dicamphor Sulfonic Acid : 2% Zinc Oxide : 3%
Absorbance
(Mexoryl SX ™) 0.3
(AU)
Octrocrylene 10 % 0.2
Drometrizole Trisiloxane 1%
0.1
0
300 320 340 360 380
Wavelength (nm) 400
1
Product C Product D Product C
0,8
Butyl Methoxydibenzoylmethane: 3% Octocrylene : 2,5% Product D
Absorbance
Terephthalylidene Dicamphor Sulfonic Acid : 3% Ethylhexyl Methoxycinnamate 0,6
Octrocrylene 5% : 7,5%
Drometrizole Trisiloxane 1% Ethylhexyl Salicylate: 2,5% 0,4
Zinc Oxide : 6%
0,2
SPF 18 ± 2 27 ± 2
0
290 300 310 320 330 340 350 360 370 380 390 400
UVA PF (PPD) 10 ± 2 4±1
Wavelength (nm)

6. RESULTS
1 Effects of DUVR exposure
Histology Control 12 J/cm² DUVR 30 J/cm² DUVR
A B C
Vimentin
D E F
Visualization of DUVR induced damage in human reconstructed skin
Histological section and vimentin immunostaining allowed to revealed that DUVR exposure induced
the disappearance of dermal fibroblasts and alterations of epidermal granular layers at 48 h.

7. RESULTS
2 Evaluation of products A and B
Histology of reconstructed skins treated Vimentin staining of reconstructed
with sunscreen products A and B skins treated with sunscreen
products A and B
48 hours post exposure to DUVR, topical application of sunscreen product A could prevent morphological
alterations induced by exposure to increasing doses of DUVR, i.e. disappearance of dermal fibroblasts and
modifications of epidermal granular layers, while this protection was not afforded by sunscreen product B.

8. RESULTS
3 Evaluation of products A and B
50
40
MMP1 ng/ml
30
20
10
0
0J/cm² 12J/cm² 0J/cm² 30J/cm² 50J/cm² 0J/cm² 30J/cm² 50J/cm²
No product Sunscreen A Sunscreen B
MMP1 production

9. RESULTS
4 Evaluation of products C and D
Histology of reconstructed skins treated with sunscreen
products C and D
48 hours post exposure to DUVR, topical application of sunscreen product C could prevent morphological
alterations induced by exposure to increasing doses of DUVR, i.e. disappearance of dermal fibroblasts and
modifications of epidermal granular layers, while this protection was not afforded by sunscreen product D.

10. RESULTS
5 Evaluation of products C and D
MMP1 ng/ml
MMP1 ng/ml
40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0
0
0J/cm² 30J/cm² 0J/cm² 30J/cm² 0J/cm² 30J/cm² 0J/cm² 30J/cm²
Vehicle Product C Vehicle Product D
MMP1 production

11. CONCLUSIONS
Our results showed that product A afforded a better protection against biological damage
mostly located within the dermal equivalent and previously identified after UVA exposure
compared to product B.
 These results indicate that the SPF value is not sufficient for evaluation of
daily UV protection and emphasize the need for a high UVA absorption with
regard to the prevention of photoaging-related biological end-points.
Our results showed that product C afforded a better protection against biological
damage mostly located within the dermal equivalent compared to product D.
These results indicate that absorption profile (well balanced UVB/UVA level
of protection) is of higher relevance than SPF value for daily UV
photoprotection as regards the prevention of UV-induced photodamage.
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connective tissue repair and implications in photoaging. Cell Death Diff., 1998, 5:792-802
3- Bernerd F, Vioux C, Lejeune F. and Asselineau D. The sun protection factor (SPF) inadequately defines broad spectrum
photoprotection : demonstration using skin reconstructed in vitro exposed to UVA, UVB or UV-solar simulated radiation. Eur. J.
Dermatol. 2003, 13: 242-249
4- Christiaens F, Chardon A, Fourtanier A, Frederick JE. Standard ultraviolet daylight for non-extreme exposure conditions. Photochem
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