Identification of ageing biomarkers in human dermis biopsies by thermal analysis (DSC) combined with Fourier transform infrared spectroscopy (FTIR/ATR)

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Identification of ageing biomarkers in human dermis

biopsies by thermal analysis (DSC) combined with

Fourier transform infrared spectroscopy (FTIR/ATR)

Rong Tang, Valérie Samouillan, Jany Dandurand, Colette Lacabanne,

Marie-Hélène Lacoste-Ferré, Patrick Bogdanowicz, P. Bianchi, Aurelie

Villaret, Florence Nadal-Wollbold

To cite this version:

Rong Tang, Valérie Samouillan, Jany Dandurand, Colette Lacabanne, Marie-Hélène Lacoste-Ferré, et

al.. Identification of ageing biomarkers in human dermis biopsies by thermal analysis (DSC) combined

with Fourier transform infrared spectroscopy (FTIR/ATR). Skin Research and Technology, Wiley,

2017, pp.1-8. �10.1111/srt.12373�. �hal-01538132�

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This is an author-deposited version published in :

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Eprints ID : 17892

To link to this article : DOI:10.1111/srt.12373

URL :

http://dx.doi.org/10.1111/srt.12373

To cite this version :

Tang, Rong and Samouillan, Valérie and

Dandurand, Jany and Lacabanne, Colette and Lacoste-Ferré,

Marie-Hélène and Bogdanowicz, Patrick and Bianchi, P. and Villaret,

Aurelie and Nadal-Wollbold, Florence Identification of ageing

biomarkers in human dermis biopsies by thermal analysis (DSC)

combined with Fourier transform infrared spectroscopy

(FTIR/ATR). (2017) Skin Research and Technology. pp. 1-8. ISSN

0909-752X

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Identification of ageing biomarkers in human dermis biopsies

by thermal analysis (DSC) combined with Fourier transform

infrared spectroscopy (FTIR/ATR)

R. Tang

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1_{CIRIMAT, Paul Sabatier University, Toulouse,}

France

2_{Pierre Fabre Dermo Cosmetique, Toulouse,}

France

Correspondence

Valarie Samouillan, CIRIMAT, Paul Sabatier University, Toulouse, France.

Email: valerie.samouillan@univ-tlse3.fr

=&3E>0(),/FG)0G(95H$The purpose of this clinical study was to identify suitable

biomarkers for a better understanding of the molecular and organizational changes in

human dermis during intrinsic and extrinsic ageing.

65:A(/9H$Sun- exposed and non- exposed skin biopsies were collected from twenty-

%#563!"'.%(!7%8#2%7!#(!3"'!5*'192!:;<=!><!+(7!?@<!&%+*2!'$7A,!B6%!6&7*#-!'*5+(#C+-tion and thermal transi%#563!"'.%(!7%8#2%7!#(!3"'!5*'192!:;<=!><!+(7!?@<!&%+*2!'$7A,!B6%!6&7*#-!'*5+(#C+-tions were determined by Differential Scanning Calorimetry

:DEFA,! G'1*#%*! B*+(2H'*.! I(H*+*%7! 29%-3*'2-'9&! :GBIJA! "+2! 12%7! 3'! #7%(3#H&! 36%!

absorption bands of the dermis and to quantify the different absorbance ratio.

I59)+:9H$The amounts of total, freezable and unfreezable water were determined. A

significant increasing amount of freezable water is evidenced in sun- exposed area skin

of aged group compared with young group (PK,<L;@A,! M('36%*! 2#5(#H#-+(3! %HH%-3! 'H!

extrinsic ageing (PK,<NOPA!#2!36%!7*+23#-!7%-*%+2%!'H!H#)*#$$+*&!-'$$+5%(Q!36%!.+#(!9*'3%#(!

component of dermis. The only significant effect of intrinsic ageing (PK,<LONA! #2! +(!

increase of the heat- stable fraction of collagens in dermis.

1(,3+)9*(,H$DSC and FTIR are well- suited techniques to characterize human skin,

giv-ing accurate results with a high reproducibility. The combination of these techniques

is useful for a better understanding of human skin modifications with intrinsic and

extrinsic ageing.

J K L D M I . %

collagen denaturation, differential scanning calorimetry, Fourier transform infrared spectroscopy, human skin ageing, hydration, secondary structures of proteins

N ! | ! O C P IM . Q 1 P O M C

Skin ageing is a combination of chronological processes and external H+-3'*2!:+2!21(!%R9'2#3#'(A,!S*#(T$%2!+*%!36%!.+0'*!.+-*'2-'9#-!2#5(!'H! skin ageing resulting from molecular modification of dermis.

Among the various components of the dermis, extracellular ma-trix consisting of collagens, elastic fibres and amorphous ground substances is strongly involved in the process of cutaneous ageing,1 in close correlation with changes in hydration.2,3 Collagen, the most

abundant component in the dermis, comprises 80% of the tissue total dry mass and 90% of all dermal proteins. Collagens I and III repre-sent close to 90% and 10%, respectively, in the composition of dermal collagen fibrils. Altogether, the fibrils with their associated proteins confer tensile strength to the skin and are pivotal for the general or-ganization and stability of the dermal extracellular matrix among other functions.4

A great number of clinical studies have been performed on skin biopsies to help the understanding on mechanisms involved

#(! -6*'('$'5#-+$! :#(3*#(2#-A! +(7! 96'3'! :%R3*#(2#-A! +5%#(5!UQ@ using histological and ultrastructural approaches. Unfortunately, the complexity and slowness of the ageing processes leading to sub-tle changes of biological function provide the ageing research with enormous difficulties.

Although new information on whole skin and dermis can be reached at the molecular level through near- infrared diffusive- *%H$%-3#'(! :VIJ=!DJAQ7 _{attenuated total reflectance Fourier transform} #(H*+*%7!29%-3*'2-'9&!:MBJ=!GBIJAQ7,8 Raman spectroscopy,9,10 nuclear .+5(%3#-!*%2'(+(-%/.+5(%3#-!*%2'(+(-%!#.+5#(5!:VWJ/WJIAQ8,11 dy-(+.#-!8+9'1*!2'*93#'(!:DXEAQ12 changes in dermis hydration with age yield somewhat conflicting results, which could be attributed to the preparation of samples.

F'.)#(%7! "#36! 36%*.'5*+8#.%3*#-! +(+$&2#2! :BYA! .%+21*%-.%(32Q! 7#HH%*%(3#+$! 2-+((#(5! -+$'*#.%3*&! :DEFA! #2! +! 23+(7+*7! method to evaluate freezable and unfreezable water.13 DSC is also an appropriate method for assessing protein thermal stability and conformational changes. It is particularly well- suited to evaluate the thermal stability of purified collagens in solution or in their aggregated form,14 or directly in native tissues.LUQL@ It has been successfully applied to characterize collagen both in animal and human skins.L@ZLP In our previous work 20 on skin explants, we val-idated the combined use of DSC and FTIR techniques to obtain suitable biomarkers of the hydric organization and biomacromol-ecules integrity.

The aim of this work was to evaluate, for the first time, chrono-logical and photo- ageing with these techniques in a clinical study on )#'92#%2!H*'.!3"'!+5%!5*'192!:;<=!><!+(7![@<!&%+*2!'$7A,

R ! | ! 6 BP K I O B 2 % $B C . $ 6 K P 8 M . %

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This monocentric, comparative, open, study was conducted at the \#%**%! G+)*%! ET#(! J%2%+*-6! F%(3*%]FJ\Q! B'1$'12%! :G*+(-%AQ! #(! +--cordance with the ethical principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The protocol was approved by the Sud- Ouest et Outre Mer III Committee for the Protection of Persons :^36#-2! F'..#33%%Q! V_! ID! JF`! ;<LN=!M<<@PO=!>PA! +(7! 36%! G*%(-6! a%+$36!\*'71-32!E+H%3&!M5%(-&!:MVEWA,!^+-6!8'$1(3%%*!2#5(%7!+!"*#3-ten informed consent.

Twenty eight healthy female volunteers divided in two groups: +5%7!;<Z><!&%+*2! +(7!?@<!&%+*2!'$7!"#36! +!EFIV^bM!2-'*%Q!*%29%--3#8%$&Q!c!+(7!?!+3!;,!EFIV^bM!:EF'*%!H'*!IV3*#(2#-!+(7!^b3*#(2#-!2T#(! M5%#(5A!#2!+!9+*+.%3%*!"6#-6!%8+$1+3%!+(7!7#HH%*%(3#+3%!#(3*#(2#-!:c;A! +(7!%R3*#(2#-!:?;A!+5%#(5,

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Two four mm punch biopsies were taken from each volunteer: on sun- protected left buttock and left dorsal forearm resulting in four 2%*#%2d!e'1(5!21(=!\*'3%-3%7!:e\AQ!M5%7!21(=!\*'3%-3%7!:M\AQ!e'1(5! 21(=!^R9'2%7! :e^A! +(7! M5%7! 21(=!^R9'2%7! :M^A,! B6%! 2T#(! )#'92&!

procedure was performed by a suitably qualified medical specialist. Samples were immediately rinsed in Phosphate Buffered Saline so-$13#'(Q!H*'C%(!#(!$#f1#7!(#3*'5%(!+(7!23'*%7!+3!g;<_F,!S%!9%*H'*.%7! stepwise thawing of the explants, (1st _{stage at 5°C for 24 hours, and} 2nd!23+5%!+3!;<_F!H'*!L<!.#(13%2A!1(3#$!DEF!+(7!GBIJ!+(+$&2#2,

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D#HH%*%(3#+$! 2-+((#(5! -+$'*#.%3*&! :DEFA! .%+21*%.%(32! "%*%! 9%*-H'*.%7! "#36! +! DEF! \&*#2! -+$'*#.%3%*! :\%*T#(! ^$.%*Q! S+$36+.Q! WMQ! hEMA!12#(5!+(!%.93&!9+(!+2!*%H%*%(-%,!B6%!-+$'*#.%3%*!"+2!-+$#)*+3%7! using the manufacturer’s instructions with pure water, cyclohex-ane and Indium as standards, resulting in a temperature accuracy of ±0.1°C and an enthalpy accuracy of ±0.2 J ggL. Temperature calibra-tion was undertaken at each scanning rate. Skin biopsies (5- 8 mg by .+22A!"%*%!2%+$%7!#(!6%*.%3#-!+$1.#(#1.!9+(,!E+.9$%2!"%*%!-''$%7! at 10°C mingL! 3'! g><_F! :L23! -''$#(5! 2-+(AQ! 36%(! 6%$7! L<!.#(13%2! 3'! freeze water.

To determine freezable water amount and intrinsic thermal tran-sitions, the samples were heated at 10°C mingL to 85°C (1st heat-#(5! 2-+(AQ! 36%(! -''$%7! +3! gL<_F!.#(gL_{! 3'! g><_F! +(7! 36%(! 6%+3%7! +3!} 10°C mingL to 85°C (2nd!6%+3#(5!2-+(A,

After completing the DSC measurements, pans were reweighted to check that they had been correctly sealed. The sample pans were pierced and dried to constant mass at 195°C for 10 minutes to deter-mine the sample dry mass and the total water mass.

R#V!|!;()0*50$P0&,9U(0'$O,U0&05/$&,&+S9*9$W;POIX

FTIR/ATR spectra were acquired using a Nicolet 5700 FTIR (Thermo G#26%*! E-#%(3#H#-Q! S+$36+.Q! WMQ! hEMA! %f1#99%7! #(! MBJ! 7%8#-%! "#36! +! i`*! )%+.! 29$#33%*! +(7! +! WFB/`! 7%3%-3'*,! ^R9$+(32! :7%*.#2! H+-%A! were directly laid on the ATR accessory (Smart Orbit with a type IIA 7#+.'(7!-*&23+$A!+(7!-'8%*%7!)&!+!6%*.%3#-!-+9!"#36!+(!jkl!*#(5!3'! avoid dehydration of the sample during spectra acquisition. Spectra were recorded over the region of 4000–450 cmgL with a spectral resolution of 1 cmgL _{and 80 accumulations. Spectral data were} col-$%-3%7!12#(5!k.(#-!O,<!:B6%*.'!G#26%*!E-#%(3#H#-A,!MH3%*!)+-T5*'1(7! subtraction and baseline correction achieved on Omnic 8.0 (Thermo G#26%*! E-#%(3#H#-AQ! 29%-3*+$! 7+3+! "%*%! ('*.+$#C%7! #(! 36%! +.#7%! II! *%-gion. For each series, a mean representative spectrum was computed. E%-'(7!7%*#8+3#8%2!+(7!G'1*#%*=!E%$H=!D%-'(8'$13#'(!:GEDA!"%*%!12%7! to enhance the chemical information present in overlapping infrared absorption bands.

R#Y!|!%:&:*9:*3&+$&,&+S9*9

Quantitative values are shown as mean±SEM. Statistical analysis have )%%(!7'(%!"#36!EM!2'H3"+*%,!S#$-'R'(=!W+((=!S6#3(%&!3%23!6+2!)%%(! 12%7!3'!-'.9+*%!7+3+!)%3"%%(!5*'192!+(7!S#$-'R'(!2#5(%7=!*+(T!3%23! 3'! -'.9+*%! 7+3+! )%3"%%(! +*%+2! :21(=!9*'3%-3%7! +(7! 21(=!%R9'2%7A! from the same group. It was considered statistically significant thresh-old of P- value less than .05.

T ! | ! I K % Q 2P % $B C . $ . O % 1 Q % % O M C

T#N!|!PA50'&+$3A&0&3:50*@&:*(,

In Figure 1 are reported the DSC curves (normalized to the initial .+22A!'H!+!2T#(!)#'92&!#(!36%!21(=!9*'3%-3%7!C'(%!'H!+!&'1(5!21)0%-3,

T#R!|!D&:50$Z)&,:*U*3&:*(,

B6%!%(7'36%*.#-!9%+T2!*%-'*7%7!#(!36%!mg;<n!;<_Fo!C'(%!-'**%29'(7! to the melting of freezable water. This extrinsic transition is widely used to quantify the amount of total freezable water in hydrated pro-teins and tissues (by dividing the area of the measured endothermic peak by 334 J ggL, corresponding to the melting enthalpy of pure ice +3!<_FAQ21 and completes thermogravimetric analyses giving the total amount of water. The amount of unfreezable water can be calculated by a simple difference. These quantifications were performed for all the samples and reported in Table 1.

The total hydration of human skin biopsies is consistent with litera-ture data,22 giving an average value of 70% for the hydration of human dermis and epidermis. This remarkably high value is due to a combina-tion of physical and chemical factors including the presence of inter-connected gaps, the particularly hygroscopic nature of hyaluronic acid and the hydrophilicity of collagen. If a well- documented literature is available for the total hydration of stratum corneum, epidermis and dermis,3 few data, obtained from Raman spectroscopy,10 DVS,12 NMR 11 _{are reported on the indirect quantification of the different kinds of} "+3%*!:(+.%$&!H*%%!+(7!)'1(7!"+3%*A!#(!61.+(!2T#(,!I(!+!9*%$#.#(+*&! study on human abdominal skin biopsies 20 _{we justified the use of the} terms freezable/unfreezable water instead of free/bound water rather reserved to vibrational or relaxational techniques. The present DSC

study shows that the freezable water (that covers bulk water in excess )13!+$2'!-'(H#(%7!"+3%*!#(!.%2'9'*%2A!*'156$&!*%9*%2%(32!3"'=!36#*7!'H! the total water in human skin in accordance with previous NMR, DVS, and DSC results.11,12,20,23 _{One- third of the total water of human skin} is unfreezable water, corresponding to the filling of the first hydration shell of proteins and other hydrophilic components.

There is a significant increasing amount of freezable water in AE 2%*#%2! -'.9+*%7! "#36! e^! 5*'19! :U@,;p! 82! NO,NpQ! PK,<L;@AQ! +22'-#-ated with a decrease amount of unfreezable water (21.0% vs 23.7%,

PK,<UPPAQ!-+12#(5!+!8%*&!2#5(#H#-+(3!7%-*%+2%!:<,>qN!82!<,NP<Q!PK,<<>PA!

of the ratio of unfreezable water to freezable water.

Between two areas of the aged groups, we note a similar tendency for the different water structures, highlighting the preponderant effect of extrinsic ageing on the hydric organization of the skin.

No significant differences are found between the amounts of total, freezable and unfreezable water of YP and AP skins, what also implies +!2#.#$+*!*+3#'!'H!1(H*%%C+)$%!"+3%*!3'!H*%%C+)$%!"+3%*,!S%!-+(!+221.%! that chronological intrinsic ageing only does not give rise to the alter-ation of the hydric structures.

It can be noticed that no significant difference are found between the hydric organization of YP and YE groups. The comparison is less straightforward in this case, since it deals with two anatomically differ-ent zones—buttocks and forearms—that may bias the final data.

Our results can be connected with previous Raman studies 10 evi-7%(-#(5!:LA!"+3%*!23*1-31*%2!+(7!+.'1(3!"%*%!2#.#$+*!#(!21(=!%R9'2%7! +(7!21(=!9*'3%-3%7!*%5#'(2!'H!&'1(5!2T#(!+(7!:;A!"+3%*!23*1-31*%2!+(7! amount were similar in the chronologically aged skin compared with young skin. Last but not least, our results showed an increased content of total and non- bounded water in the photoaged skin.

Gniadecka et al. highlighted a decrease on echogenicity in the upper dermis of sun- exposed region in photoaged skin, that could be

! " # $ % & ' ( )DSC curves of a skin biopsy from the 20- 30 years old group, sun- protected zone (first and second heating, scanning rate 10°C mingL, and enlargement #(!36%!m@<=!OU_Fo!"#(7'"A

due to a degradation of collagen, accumulation of GAGs and water in the upper dermis leading to an increased content of total and non- bounded water.10,24

It must be reminded that the collagen secondary structure is sta-bilized by both intra- and inter- chain water bridges with carbonyl and hydroxyprolyl groups.25 Thus, despite an increase of the total water content, the decrease in the bounded- water content (protein- water #(3%*+-3#'(2A!"#$$!$%+7!3'!36%!#(23+)#$#3&!+(7!H*+5.%(3+3#'(!'H!-'$$+5%(! fibrils.;@

T#T!|!1(++&>5,$/5,&:)0&:*(,

Enlargement of Figure 1 shows the DSC curves of a sun- protected 2T#(!)#'92&!'H!+!&'1(5!21)0%-3!#(!36%!@<=!OU_F!C'(%,!B6%!%(7'36%*.#-! phenomenon recorded in this temperature zone during the first heat-ing is associated with the irreversible thermal denaturation of type I and type III collagens.20 _{The collagen denaturation—distinct from} degradation—is a thermally activated process that involves rupture of hydrogen bonds coupling the three α- chains and a rearrangement of the triple helix into a random chain configuration.14

As shown on Figure 1, the main characteristics of the denaturation peak are T_onset, T_max, T_mid, T_end and the area under the endotherm peak.

T_mid represents the temperature at which 50% of collagen is denatured and it is computed from the integral of the denaturation endotherm.

T_onset and Tend are computed from a fixed threshold value of the signal first derivative. T_onset, T_max, T_mid are alternatively taken as the denatur-ation temperature according to the previous studies 15,17,19 while Tend, +$2'!T('"(!+2!36%!j*%-'8%*&!3%.9%*+31*%l!-+(!)%!12%7!3'!3+T%!#(3'!+--count the widening of the collagen thermal denaturation under certain conditions.17 ΔH, once normalized to the dry mass, gives a measure of 36%! 29%-#H#-! 6%+3/%(36+$9&! 'H! 7%(+31*+3#'(,! S%! *%9'*3%7! #(! G#51*%!;! the mean values of T_mid!:;MA!+(7!T_end !:;`A!H'*!36%!7#HH%*%(3!2%*#%2!'H!)#-opsies. The Tmid!3%.9%*+31*%!#2!$'-+3%7!+3!@q_F!H'*!36%!21(=!9*'3%-3%7! )#'92#%2!'H!&'1(5!21)0%-32!:e\A,!I3!H#32!"#36!36%!3%.9%*+31*%!7%(+31*-ation range of skins from animals L@ or abdominal human skins.20 It is known that the denaturation temperature drastically increases as the hydration decreases,15 _{due to the decrease of intrafibrillar water} and to the replacement of protein- protein hydrogen bonds by protein- water hydrogen bonds. This temperature becomes independent upon hydration above 1 kg of water per kg of dry tissue,L@ what is checked for the studied samples, with a total amount of water of 3 kg per kg of dry mass. In this case, the denaturation temperature can be considered as an intrinsic characteristic of collagens in the skin and can be com-pared to each other regardless of hydration.

Even if the mean value of T_mid is slightly depressed in the sun- exposed aged skin (AE, T_midK@@,U_FA! ('! 2#5(#H#-+(3! 7#HH%*%(-%! (PK,<O>@A!#2!H'1(7!"#36!36%!21(=!%R9'2%7!&'1(5!2T#(!:e^Q!T_midK@O,<_FA,! Nevertheless, the decrease of T_mid is significant between the

Skin samples % Total water [$;055@&4+5$?&:50 % Q,U055@&4+5$ water I&:*($),U055@-&4+5FU055@&4+5$ water YP 75.2±1.7 51.4±1.3 ;>,OrL,@ <,N@@r<,<>O YE 72.1±1.5 48.4±1.2a _{23.7±0.7 0.490±0.017}d AP q@,>rL,L 52.7±0.9b _;>,@r<,@c _0.448±0.013e

AE qq,;r<,@ U@,;r<,qa,b _21.0±0.7c _<,>qNr<,<L@d,e

* + , - & ' ( )_{Amounts of total, freezable} and unfreezable water in four skin series: e'1(5!21(=!\*'3%-3%7!:e\AQ!M5%7!21(=! \*'3%-3%7!:M\AQ!e'1(5!21(=!^R9'2%7!:e^A! +(7!M5%7!21(=!^R9'2%7!:M^A,!E#5(#H#-+(3! difference was noted as follows: a_{YE vs AE} (PK,<L;@AQ!bAP vs AE (PK,<LU@AQ!cAP vs AE (PK,<N@PAQ!d_{YE vs AE (PK,<<>PA!+(7!}e_{AP vs} AE (PK,<LL@A

! " # $ % & ' . )Main thermal characteristics of the denaturation endotherm in skin biopsies: A, T_mid and B, T_end

non- exposed aged skin (AP, TmidK@q,N_FQ! PK,<>L>A! +(7! 36%! 21(=! %R9'2%7!+5%7!2T#(!:M^A,

These results could be in favour of a slight fragmentation of the collagen fibres with the cumulative UV exposition during life, which can be attributed to the decrease of unfreezable water observed in sun- exposed skin of aged subjects inducing collagen destabilization as it has been described.L<Q;UQ;@

On the contrary, ΔH is similar in the four series of biopsies with the 8+$1%2!'H!LL,;!:e\AQ!LL,<!:e^AQ!L;,>!:M\A!+(7!LL,;!s!5gL!:M^A,!M!9*%8#'12!

DSC study on collagen rat skin also evidenced that the total denatur-ation enthalpy did not change with age.17

The Tend temperature is significantly increased (PK,<LONA!)%3"%%(! the sun- protected skin of young subjects (YP, T_endK@P,P_FQ!PK,<LONA! and the sun- protected skin of elderly subjects (AP, TendKq;,>_FA,!k(! the contrary, T_end is constant for the sun- exposed young and aged skins. Such an increase of the Tend temperature has been already evidenced with age in rat skin,17 and associated with the increase of heat- stable crosslinks in collagens. In our study, this increase of ! " # $ % & ' / )M8%*+5%7!('*.+$#C%7!GBIJ!29%-3*+!#(!36%!:N<<<=!;O<<AQ!:LqU<=!LU<<A!+(7!:LU<<=!L<<<A!-.gL regions for a matched set of skin biopsies

* + , - & ' . )FTIR assignment and approximate descriptions of vibrational modes for human reticular dermis

=&,/$

position/cm\N Assignment

3375- 3289 Amide A: mainly ν:V=!aA!.'7%!'H!9*'3%#(!"#36!36%!-'(3*#)13#'(!'H!36%!ν:k=!aA!23*%3-6#(5!.'7%!#(!a₂O and polysaccharides

3073 ν

:FaA!+*'.+3#-3011 ν:KFaA!'H!1(2+31*+3%7!$#9#72Q!3*#5$&-%*#7%2Q!H+33&!+-#72 2957, 2924,

2873, 2852

νas(CH₃AQ!νas(CH₂AQ!νs(CH₃AQνs(CH₂AQ!:Y$&Q!\*'Q!a&9Q!M$+A!'H!9*'3%#(2!t!96'296'$#9#72Q!3*#5$&-%*#7%2 Most representative of proteins: νs(CH₃A

Most representative of lipids: νas(CH₂A!+(7!νs(CH₂A 1744 ν:FKkA!'H!3*#5$&-%*#7%2Q!-6'$%23%*'$!%23%*2Q!96'296'$#9#72 L@PN=!L@>< Amide I: ν:FKkA!t!δ:k=!aA!"+3%*

1558- 1542 Amide II: ν:F=!VAQ!δ!:V=!aA 1520, 1515, 1507 E#7%!-6+#(2!:B&*'2#(%Q!\6%(&$+$+(#(%A 1452 δ(CH₂A!2-#22'*#(5Q!δ(CH₃A!)%(7#(5!'H!$#9#72!:.+#($&AQ!9*'3%#(2 1401- 1393 ν s:Fkk=!A!'H!H*%%!+.#('!+-#72Q!H+33&!+-#7Q!:YMYA2 1338 δ(CH₂A!"+55#(5!'H!9*'$#(%!-6+#( E9%-#H#-!)+(7!'H!-'$$+5%(!:+(7!%$+23#(!"#36!+!.#('*!-'(3*#)13#'(A 1310- 1202 Amide III δ_plan!:V=!aA!+(7!ν:F=!VA!'H!9*'3%#(2

1238 cmgLd!29%-#H#-!'H!^FW!:-'$$+5%(A 1205 cmgL_{d!29%-#H#-!'H!^FW!:-'$$+5%(A}

L;N@=!L;>U! νas(PO₂gA!23*%3-6#(5!'H!96'296'$#9#72Q!(1-$%#-!+-#72! 1250- 1210 νas(SO₃gA!21$H+3%7!:YMYA2 LL@q=!LLU@ ν as:Fk=!k=!FA!-6'$%23%*'$!%23%*2Q!96'296'$#9#72 1200- 1000 ν:F=!kAQ!ν:F=!FAQ!ν:F=!kaAQ!ν:F=!k=!FA!'H!9*'3%#(2Q!'$#5'2+--6+*#7%2Q!5$&-'$#9#72 1171 cmgL_{: ν+2:Fk=!k=!FA!'H!-6'$%23%*'$!%23%*2} 1159 cmgL_{: ν:F=!kaA!'H!a&9!+(7!(1-$%#-!+-#72} 1115 cmgL: ν!:F=!kA!-+*)'6&7*+3%2 1081 cmgL: ν:F=!k=!FA!-'$$+5%(Q!5$&-'5%(Q!'$#5'2+--6+*#7%2Q!5$&-'$#9#72Q!9*'3%'5$&-+(2Q!+(7!νs(PO₂gA!(1-$%#-!+-#72Q!96'296'$#9#72 1032 cmgL_{: ν2:Fk=!k=!FA!'H!-+*)'6&7*+3%2!*%2#71%2!'H!-'$$+5%(Q!:YMYA2Q!νs(SO} 3 g_A!:YMYA2

heat- stable crosslinks seems to be achieved mainly by the chronolog-ical ageing. From these results, we can hypothesize that age- induced glycation products and/or carbonyl changes could be one of the post- translational modifications of chronological ageing.27,28

T#V!|!"*40&:*(,&+$3A&0&3:50*@&:*(,

Native biological tissues at a constant hydration can be directly ana-lysed by FTIR in the ATR mode without any other preparation. This technique is widely applied to investigate in vivo or in vitro the out-ermost layer of human skin, that is, the stratum corneum,29–32 but few data are available on the internal layer of human skin, that is, the reticular dermis. The measuring depth of ATR- FTIR in the skin is typi--+$$&!+!H%"!.#-*'(2!'8%*!36%!"+8%!(1.)%*!"#(7'"!N<<<Z@U<!-.gL.32

Mean FTIR spectra of the skin biopsies are collected in Figure 3. Table 2 summarizes IR bands present in the reticular dermis of the four types of biopsies. Peaks assignments were realized according to liter-ature data on proteins 33 _{such as collagen} >NZ>@ _{and biological tissues} 37

such as dermis.38

The classical absorption bands of proteins (amide A, amide I, II, IIIA! +*%! H'1(7! '(! 36%! 29%-3*+! 'H! 36%! H'1*! 2%*#%2! 'H! )#'92#%2! +(7! 36%#*! positions are very close to the absorption bands of pure type I col-lagen. Notably, collagen absorption features in the fingerprint region (the specific triplet of bands at 1204, 1239 and 1282 cmgL37 _{as well} as the specific band at 1338 cmgL8,37 are found in dermis samples, and the correlation coefficient between collagen and dermis spectra is more than 0.95 in this region. It must be pointed out that the Amide II is slightly shifted towards low wave numbers (2 cmgL A!#(!36%!21(!%R-posed skin of elderly subject. This shift is associated with a change in intensity of the different components of the amide II as seen in the GED!29%-3*+!:G#51*%!NA,

In a previous work on human skin biopsies we evidenced a pro-nounced shift of the Amide II band towards low wavenumber in heat- denatured derma 20 and it was attributed to the depletion of the triple helical structure of collagen and the enhancement of disordered struc-tures.>@ In sun- exposed skin biopsies, this slight shift of the amide II must be correlated to the thermal evolution of the collagen denatur-ation evidencing a destabilizdenatur-ation of the triple helix domain of collagen. In order to quantify the variation of the vibrational answer of the different skin biopsies, the area of the different absorption bands were computed from the individual spectrum of each tissue and the appropriate ratio of areas were performed according to the literature data.37,39,40 _{The most relevant indicators are displayed in Figure 5.}

M2! 26'"(! #(! G#51*%!UMQ! +! 2#5(#H#-+(3! 7*+23#-! 7%-*%+2%! :gL@pQ!

PK,<NOPA! 'H! 36%! +*%+! *+3#'! :L>>O!-.gLA/:LqU<=!L<<<!-.gL_{AQ! #2!} %8#-7%(-%7! #(! 21(=!%R9'2%7! +5%7! 2T#(! :M^A! -'.9+*%7! "#36! 21(=!%R9'2%7! ! " # $ % & ' 0 )Averaged FSD spectra computed from the individual

GBIJ!29%-3*+!'H!2T#(!)#'92#%2!#(!36%!:L@<<=!LU;<A!-.gL region

! " # $ % & ' 1 )Area ratios of the different absorption bands from FTIR spectra: A, Area ratio (1338 cmgL_{A/:LqU<=!L<<<!-.}gL_A! and B, Area ratio (1204 cmgLA/

&'1(5!2T#(!:e^A,!B6#2!#(7#-+3'*!#2!9*'9'*3#'(+$!3'!H#)*#$$+*&!-'$$+5%(!-'(-tent, the main protein component of dermis. However, with age no significant difference was observed on non- exposed area. This is cor-roborated by a similar evolution of the area ratio (1204 cmgL_A/:LqU<=! 1000 cmgLAQ!+('36%*!.+*T%*!'H!36%!^FW!9*'3%#(2!+.'1(3!:G#51*%!U`A! :gLOpQ!PK,<;>PA,!B6%2%!+*%+!*+3#'!%8'$13#'(2!+*%!-'(2#23%(3!"#36!36%! literature data, showing alterations in the amount and architecture of collagen content in human dermis under extrinsic ageing.;@

V ! | ! 1M C 1 2Q % O M C

DSC is a powerful technique to evaluate the hydric organization of human dermis and the collagen stability. For the first time, a direct quantification of freezable and unfreezable water in human skin bi-opsies from two age groups has been achieved. Photoageing induces a decrease of unfreezable water and an increase of freezable water, highlighting the predominant role of extrinsic ageing on water or-ganization in human skin. Moreover, photoageing- induced decrease collagen stability could be explained by the detriment of interaction bounded- water to protein and/or collagen fragmentation. In contrast, chronological ageing- increased collagen stability could be due to age- induced glycation and carbonyl modifications. These alterations of the collagenic fraction with extrinsic ageing are confirmed by the decrease of the specific vibrational signature of fibrillar collagens in dermis as shown by FTIR analysis. These results are promising for the identification of new biomarkers of ageing in a future work exploring the skin at different depth levels with Raman confocal microscopy, a technique which becomes more frequent for biological analysis.

I K ; K I K C 1 K %

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