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Temperature-Induced Changes in Structural and

Physicochemical Properties of Vernix CaseosaRobert Rissmann1, Hendrik W.W. Groenink1, Gert S. Gooris1, Marion H.M. Oudshoorn2, Wim E. Hennink2,Maria Ponec1 and Joke A. Bouwstra1

The skin of the third trimester fetus and early newborn exhibits a complex, multifunctional, highly hydrated butviscous skin-surface biofilm called vernix caseosa (VC). During birth, VC undergoes a substantial change froman aqueous and warm surrounding into a gaseous and colder environment postnatally. The aim of this studywas to investigate the structural and physicochemical changes in VC, which accompany physiologically relevantvariations in environment parameters, such as temperature and humidity. A remarkable difference wasobserved in water release and uptake properties: dehydration and rehydration processes take place two to fourtimes faster at 371C than at room temperature (RT). The dehydration was irreversible; rehydration was only

possible to a final weight of 55% (371

C) and 46% (RT) of the pre-desiccation weight. Differential scanningcalorimetry showed two different overlapping phase transitions within physiological temperature range.Investigation of the lipid organization by Fourier transform infrared spectroscopy and small-angle X-raydiffraction revealed a more disordered state of lipids at 371C than at RT, which might explain the fasterdehydration and rehydration process at 371C as well as the changes in thermotropic rheological behavior. Inconclusion, we demonstrated that VC properties adjust to the fundamental change from the intrauterine to thepost-natal environment.

Journal of Investigative Dermatology (2008)128,292299; doi:10.1038/sj.jid.5701022; published online 2 August 2007

INTRODUCTIONThe skin constitutes the major barrier between our organismand the environment. During the development of the skin in

utero, the skin of the fetus is often covered by a creamy, whitebiofilm called vernix caseosa (VC). The formation of VCcommences in the beginning of the last trimester of gestationand after delivery, it dries spontaneously on the skin.

VC consists of detached corneocytes very similar to thoseof the underlying uppermost layer of the epidermis, thestratum corneum (SC). VC corneocytes are polygonal orovoid in shape with diameters of 1040 mm (Agorastoset al.,1988). The corneocytes are embedded in a lipid matrix andthus, VCs basic structure shows similarities to that of the SC.However, VC consists ofB80% water, B10% proteins, andB10% lipids (Pickenset al., 2000;Hoegeret al., 2002). Thelipid fraction is mainly composed of sebaceous-derived,

nonpolar lipid classes comprising squalene, sterol esters, waxesters, and triglycerides (Haahti et al., 1961; Kaerkkaeinenet al., 1965). Recent studies revealed that the barrier lipids

cholesterol, free fatty acids, and ceramides (Hoeger et al.,2002) and lipids bound to the corneocytes of VC are present(Rissmannet al., 2006) in composition similar to that of SC.The level of barrier lipids in VC is, however, much lower thanin SC. Protein analysis showed the presence of antibioticpolypeptides (Akinbi et al., 2004; Tollin et al., 2005) andpolypeptides with innate immunity functions (Tollin et al.,2006).

In utero, VC constitutes a unique barrier between theamniotic fluid and the skin of the fetus. It has been proposedto play an important role in barrier formation (Pickenset al.,2000), SC hydration and pH-regulation in the neonatal skinadaptation process (Visscher et al., 2005): VC increases SC

hydration and in addition, it seems to enhance the acidmantle development after birth. In other reports, it was shownthat VC acts as a skin cleanser (Morailleet al., 2005) and thatit has multiple protective functions such as that of moisturi-zer, anti-infective, and antioxidant (reviewed by Haubrich,2003). This multifunctional character of VC has beenemphasized byHoath et al. (2006) who concisely reviewedVC characteristics.

During birth, however, VC undergoes a substantial changeand is transferred from an aqueous, warm and sterileenvironment to a gaseous, colder, and xenobiotic-containingenvironment in the post-natal situation. In the prenatalperiod, VC might facilitate skin maturation and in the post-

ORIGINAL ARTICLE

292 Journal of Investigative Dermatology (2008), Volume 128 &2007 The Society for Investigative Dermatology

Received 28 February 2007; revised 30 May 2007; accepted 6 June 2007;published online 2 August 2007

1Department of Drug Delivery Technology, Leiden/Amsterdam Center forDrug Research (LACDR), Leiden University, Leiden, The Netherlands and2Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences(UIPS), Utrecht University, Utrecht, The Netherlands

Correspondence: Professor Dr Joke A. Bouwstra, Leiden/Amsterdam Centerfor Drug Research, PO Box 9502, Leiden, RA 2300, The Netherlands.E-mail:[email protected]

Abbreviations: Cryo-SEM, cryo-scanning electron microscopy;DSC, differential scanning calorimetry; FTIR, Fourier transform infraredspectroscopy; RT, room temperature; SAXD, small-angle X-ray diffraction;SC, stratum corneum; VC, vernix caseosa
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natal period it might affect SC hydration. Therefore, changesin physicochemical properties of VC during birth are of greatinterest in order to better understand its role after birth. It hasbeen reported that temperature is of importance for the yieldvalue of VC, that is the minimum required shear stress toinitiate flow (Narendranet al., 2000). As no additional data

on temperature- and hydration-induced changes in VC havebeen reported yet, in this study, the physicochemical andstructural properties of VC were investigated as function oftemperature. This information will certainly contribute to abetter understanding of the effect of the natural environmenton the properties of VC and the role VC plays after delivery.

Dehydration and rehydration of VC were monitored at371C and room temperature (RT). Moreover, the localizationof water in VC during dehydration and rehydration wasexamined by cryo-scanning electron microscopy (cryo-SEM).Temperature-induced phase transitions and their effect on VCproperties between 15 and 371C were explored by means ofFourier transform infrared spectroscopy (FTIR), differential

scanning calorimetry (DSC) and small-angle X-ray diffraction(SAXD). Changes in flow properties were monitoredby rheological studies. These combined techniques provi-ded insight into the structural and physicochemical changesof VC.

RESULTSIrreversible water holding properties of VC during adehydrationrehydration cycleDehydration and rehydration studies of VC were performedto obtain information about the water holding properties ofVC. First, the dehydration rate of VC was investigated at RTand 371C over P2O5, after which rehydration at 100%

relative humidity was also examined at both temperatures. Inorder to determine whether dehydration was reversible, therehydrated samples were dehydrated again. The results ofthese studies are shown in Figures 1 and 2. The followingobservations are of importance:

Firstly, at 371C dehydration of VC is characterized by avery constant water loss during the first 48 hours (Figure 1).Only in the initial hour, the dehydration rate is faster (insetFigure 1). After 72hours, the dehydration process iscomplete, resulting in a final weight of 18.672.5% of theoriginal sample. This corresponds to the composition of VCwith B10% lipids and B10% proteins (Pickenset al., 2000;Hoeger et al ., 2002). The dehydration at RT is also

characterized by a constant weight loss (Figure 1), exceptfor the first few hours. The dehydration is complete after240hours with a final weight of 19.872.6% of the origi-nal sample. The dehydration process at 371C, however, ismuch faster than at RT. This is illustrated by the linear slope,which correlates to a loss of 14.7 mg water per g vernixper hour (interval from 248 hours, R2 0.993) and a loss of3.8mg/g hour (3144hours, R20.985) at 371C and RT,respectively.

Secondly, rehydration is a slower process than dehydra-tion. InFigure 2a, the rehydration of the VC at RT and 371C isdepicted. At both temperatures, a gradual increase in thewater level in VC is observed. The water uptake was more

rapid at 371C than at RT. The entire rehydration processes atboth temperatures show a hyperbolic shape: the longerrehydration takes place the slower the water uptake of VC.Figure 2 shows that the rehydration rate at 371C isapproximately twice the rehydration rate observed at RT.Furthermore, comparingFigures 1 and 2, it can be seen thatthe rehydration rate is two to three times slower than thedehydration processes. The rehydration was conducted untila constant weight of VC was obtained. In the graph (Figure 2),this is indicated by a plateau. At 371C, the final weight withequilibrium water content of VC after de- and rehydration is55.473.4% (w/w), whereas at RT, the final weight is46.071.7% of the initial value. This indicates that after

100

80

60

40

20

00 50

RT 37C

Time (hours)

Percentoforiginalweight

Percent

oforiginalweight

100 150 200

020

40

60

80

100

5 10 15

Time (hours)

Dehydration RT

1st dehydration 37C

2nd dehydration 37C

20 25

250

Figure 1. Temperature-dependent dehydration of VC. The weight

percentage of VC is plotted as a function of dehydration period. VC samples

(n3) were dehydrated in a desiccator over P2O5 at RT (squares) and 371C

(triangles) for 240 hours. The first 25 hours of dehydration are presented

in the inset together with the dehydration after the rehydration period

(diamonds). Data are presented as mean w/w%7SD.

RT

60

Percentoforiginalweight

50

40

30

20

10

00 50 100 150

Time (hours)

200 250

015

20

25

30

2 4Time (hours)

%of

originalweight

6 8

300 350

37C

Figure 2. Rehydration of VC does not reach the pre-desiccation

weight.Weight percentage of VC relative to predesiccation weight as a

function of rehydration period over MilliQ water (n3). The initial 8 hours

period of rehydration is presented in the inset. Data are presented as mean

w/w%7SD.

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rehydration, the equilibrium water content is lower than thewater content of VC before dehydration. This suggests thatdehydrationrehydration process is irreversible.

Thirdly, when after rehydration, a second dehydration isperformed at 371C, the dehydration occurs faster than thedehydration of the original samples, as depicted in Figure 1.

The dehydration rate in the linear part of the seconddehydration curve between 5 minutes and 2 hours is53.1 mg/g hour (R2 0.987). This constitutes an almost fourtimes higher water release rate as compared with thedehydration of fresh VC samples.

During dehydration, cryo-SEM reveals changes in theultrastructure of VC only at low water content in VC andrehydration results in a reduced amount of water in thecorneocytes and an increasing number of water domains in thelipid domainsIn order to determine the localization of the water in VCduring the dehydration process, samples were collected at

various time intervals and investigated by cryo-SEM. InFigure3ae, the ultrastructure of VC samples at various dehydrationstates are shown. Untreated VC exhibits corneocytes inwhich scaffolds of dense keratin fibers are visible (asterisk inFigure 3a). These cells are characterized by a high contrast,which is caused by water (dark regions) and the keratin (white

network) in the interior of the cells. The corneocytes areembedded in a matrix of the smooth appearing lipids and arehardly in contact with each other. In the lipid matrix, smallround structures are observed, which most likely representwater droplets (Rissmannet al., 2006). After partial dehydra-tion at RT (29 and 39% weight loss, Figure 3b and c,

respectively), the corneocytes exhibit the same appearance asobserved in fully hydrated VC with the water mainlylocalized in the corneocytes. The droplets observed in freshVC (Figure 3a) are visible as well. A further dehydration (60%weight loss) yielded regions with water containing corneo-cytes, which have almost the same appearance as at higherhydration states. They appear slightly less round in shape(Figure 3d). In the same sample, also large domainscharacterized by a very smooth appearance with no contrastwere observed, indicating areas where originally water waspresent. In fully dehydrated VC samples (80% weight loss),the SEM photomicrographs showed only a very smoothsurface (Figure 3e), indicating the absence of water domains.

After full dehydration, VC was rehydrated and subsequentlystudied with cryo-SEM. The appearance of rehydrated VC isvery different from the fresh samples. A large number ofwater-filled round structures is now observed (arrowheads inFigure 3f) within the lipid matrix. Corneocytes are also visible(*). However, the keratin filaments in the corneocytes are

a b

10 m 10 m 10m

10 m10 m 10 m

c

d e f

Figure 3. Ultrastructure of VC during the dehydration and rehydration process. VC visualized by cryo-SEM at various hydration levels expressed as weight

percentage of VC relative to the predesiccation weight: (a) 100, (b) 71, (c) 61, (d) 40, (e) 20% of original weight after dehydration and (f) 50% of original weight

after rehydration from completely dehydrated. Corneocytes (*) are characterized by dark regions (localization of water) and a white network representing

keratin. These cells are embedded in the smooth appearing lipid matrix. In the lipid matrix, round structures are present representing phase separated water. ( f)

The number of round structures (indicated by arrow heads) increased after rehydration. Bar 10 mm.

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much more densely packed than observed in the images ofthe fresh VC, suggesting that less water is present in thecorneocytes.

Thermotropic transitions occur in VC within physiologicaltemperature range

The thermotropic phase behavior of VC was investigated byDSC between 5 and 501C. Upon heating of VC samples, theDSC thermogram showed two overlapping peaks (Figure 4).The exothermic transition has an onset temperature of20.170.31C. This event was followed by an endothermictransition with an onset at 25.971.41C. Both transitions showsimilar enthalpies of transitions, namely 0.1870.06 J/g forthe exothermal transition and 0.2070.04 J/g for the endo-thermal transition. No transitions were observed during thecooling cycle performed immediately after heating. In orderto obtain information about the reversibility of the transitions,the heating and cooling cycle was followed by a secondheating run 20minutes after cooling the sample to 51C.

No transitions were detectable. However, after 1 hour,equilibration at RT, heating of the sample showed the sametransitions as observed with the fresh sample. This reversiblenature suggests that the phase transitions originate fromlipids in VC.

Lipid organization is more disordered at elevated temperaturesIn order to obtain more details about the thermal-inducedphase transitions in VC, FTIR was employed. FTIR permits tostudy the conformational ordering and packing of the lipids inVC. In the FTIR spectrum of fresh VC samples, one of themajor absorption peaks can be ascribed to the asymmetricCH2 stretching vibration (B2,920 cm

1). This peak provides

information about the conformational orderdisorder transi-tions, such as a crystalline-liquid phase transition (Mantschand McElhaney, 1991). The CH2 scissoring mode located atB1,468 cm1 provides information about the packing of the

lipids: a splitting of the scissoring contour indicates thepresence of an orthorhombic lateral packing (Casal andMantsch, 1984). Therefore, the combination of the asym-metric stretching and scissoring modes provides informationabout the packing of the lipids in the sample.

The FTIR spectrum of VC shows that at 101C, the

asymmetric CH2-stretching vibration has a peak maximumat 2,918.2 cm1, indicating a conformational ordered state ofthe lipids. When increasing the temperature, the peakmaximum shifts gradually to 2,924.0 cm1 at 281C (Figure5a), which indicates a gradual change of the conformationallipid order from an ordered to a more disordered state. Thethermotropic response curve displays a plateau, which isreached at about 281C (Figure 5b).

As far as the scissoring mode is concerned, at 101C, nosplitting in the contour is observed at B1,468 cm1, whichindicates that no orthorhombic lateral packing is present inVC (not shown).

Long-range ordering in VC disappears at elevated temperatures,but the changes are reversibleIn our previous study, it was observed that a small populationof lipids forms a long-range ordering at RT, indicated by thepresence of one or two diffraction peaks in the diffractionpatterns of some VC samples (Rissmannet al., 2006). In thisstudy, the diffraction pattern was measured as function oftemperature. A typical example is shown inFigure 6. At 171C,one peak was observed at q1.39nm1 (d4.52 nm,bottom curve). Upon heating, the peak disappears at around331C indicating the absence of long-range ordering in thesample. Subsequently, the sample was cooled down from351C (dashed curve) to 171C (top curve). At around 271C, the

peak at q1.39nm1 reappears, which demonstrates thereversible nature of this transition.

Rheological characterization of VC shows constant values forviscosity and elasticity at elevated temperatures and areversible characterThe viscoelastic properties of VC were examined by means ofrheology. Elasticity (G0) and viscosity (G00) of fresh VCsamples were measured as function of temperature. Theresults are provided inFigure 7. VC showed a tand, that is thequotient of G00 and G0, between 0.12 and 0.4 underlining aviscoelastic flow behavior. The G0 and G00 values decline withincreasing temperature and a plateau with constant values

was reached at around 321C (arrow). During the coolingprocess to 101C, the viscosity and elasticity returned to theoriginal values indicating a reversible process (data notshown).

DISCUSSIONThe skin of the fetus develops in an aqueous, warm, andsterile environment, whereas the SC is still premature(reviewed by Chiou and Blume-Peytavi, 2004). During theintrauterine maturation of the skin barrier, VC forms anadditional barrier affecting the interaction between SC andthe amniotic fluid. The transition from prenatal to post-natalstate marks a fundamental change in environment for the skin

Heatflow

(W/g)

Exotherm

20.6C0.14 J/g

20.6C0.25 J/g

24.0C0.25 J/g

19.1C0.16 J/g

26.3C0.18 J/g

25.8C0.18 J/g

23.7C

28.8C

28.7C

23.0C

22.1C

25.7C

10 15 20

Temperature (C)

25 30 35 40

Figure 4. Thermotropic phase transitions of VC as observed by DSC.

DSC was employed to examine phase transitions in VC between 5 and 501C.

The heatflow of three different VC samples is provided as the function of

temperature during the heating process.

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of the baby from aqueous surrounding at 371C to a lowrelative humidity at reduced temperature. Our investigationsaimed to characterize properties of VC during this transitionand examine changes by biophysical and visualizationmethods.

Irreversibility of water holding properties of the VCVC dehydration. With B80%, water constitutes the maincomponent of VC (Pickens et al., 2000) immediately afterdelivery. Being at the interface between the babys skin(371C, Visscher et al., 2005) and the environment(RTB221C), VC is exposed to a substantial temperature

Intensity

0 1

4.52

4.52

17C

21C

25C

29C

33C35C33C

29C

25C

21C

17C

2 3

q(nm1)

Figure 6. Thermotropic SAXD-patterns of VC. Scattered intensity

(arbitrary units) plotted as function of scattering vector (q). Sequential SAXD

patterns are plotted of a VC sample during heating from 17 (bottom curve) to

351C (dashed curve) and cooling down to 171C (top curve).

100,000

Elasticity(Pa)and

viscosity(Pa.s

)

10,000

1,00010 20 30

Temperature (C)

40 50

Elasticity

Viscosity

Figure 7. Temperature-dependent rheological characterization of VC.

Rheological properties of VC were measured as a function of temperature.

Elasticity (G0, squares) and viscosity (G00, circles) decrease upon heating until a

plateau is reached (arrow). Data are presented as mean7SD (n7).

50C

a

b

40C

30C

20C

10C

2,910

2,926

2,924

2,922

2,920

2,918

0 10 20

Temperature (C)

30 40 50

2,920

Wavenumber (cm1)

Waven

umber(cm1)

Absorbance

2,930 2,940

Figure 5. Thermotropic behavior of VC lipids as monitored by FTIR.

The FTIR spectrum of VC samples shows the asymmetric CH 2stretching

vibration. (a) The spectra are depicted from 10 to 501C from bottom to top

with 21C interval. (b) The wave number of the peak maximum of the CH2stretching vibration is plotted against the temperature. Data are presented as

mean7SD (n3).



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gradient. We therefore studied the dehydration behavior ofVC at both RT and 371C. Figure 1 shows that at 371C, thedehydration is about four times faster than at 221C. Thissuggests that owing to a temperature gradient in VC afterdelivery, a remarkable difference in the dehydration behaviormight occur within VC, when comparing the superficial layer

of VC to the inner layer close to the babys skin. Interestingly,no ultrastructural change in VC is observed by cryo-SEMduring the dehydration process, even down to 60% of itsoriginal weight (Figure 3c). Subsequent dehydration to 40%of the original weight led to formation of two types ofdomains, namely regions with water-containing corneocytesand regions with a smooth appearance, indicating thepresence of water-free domains (Figure 3d). However,bound-water might still be present in these regions. As faras the dehydration rate is concerned, this is significantlyhigher in the first hour(s) of dehydration, followed by aconstant weight loss in time. This decrease indicates a rapidwater release in the beginning, but also a sustained release

during the remaining period, the latter being substantiallyextended at RT.Pickenset al. (2000)speculated that this firstrelease might be caused by evaporation from the small roundwater pools, which is then followed by the constant waterrelease from the corneocytes. Our ultrastructural investiga-tion shows, however, that the water pools within the lipidsare still present after 29 and 39% water loss, respectively(Figure 3b and c). An alternative explanation is a rapiddehydration in the initial period leading to the formation ofa thin non-hydrated layer at the VCair interface that acts as abarrier for water transport from the interior of VC. Witha constant layer thickness and a constant water activity, aconstant water flux (and thus a zero order decrease in weight)

can be expected.

VC rehydration. After complete dehydration, VC wasrehydrated. In contrast to the finding of Pickens et al.(2000)who submersed their specimen, a complete rehydra-tion of VC at 100% relative humidity was not observed.Compared with the pre-desiccation weight, water levelsup to around 35% (w/w, 371C) and 26% (w/w, RT)were obtained, which is much lower than the originalwater level of about 80% (w/w). This might be due tochanges in the cornified envelope or keratin during the dryingprocess. VCs behavior is different from the dehydration andrehydration of SC, which is a reversible process (Anderson

et al., 1973).Rehydrated VC samples revealed major ultrastructural

differences in water localization and appearance of thekeratin filaments of the corneocytes (Figure 3f) as comparedwith untreated VC samples (Figure 3a): (i) less water is presentin the corneocytes indicating limited water uptake duringrehydration; (ii) the presence of large number of water-filledround structures in the extracellular lipid matrix indicates thatduring rehydration water does not cross the densely cross-linked cornified envelope. The observed difference in waterlocalization in rehydrated VC might also explain theincreased dehydration rate from the rehydrated VC ascompared to fresh VC (Figure 1b).

Temperature-induced changes in the VC lipid organizationare reversible.As pointed out above, the dehydration rate ofVC samples at 371C and RT is very different. Thermotropicbehavior of VC was investigated by DSC to unravel whetherthese differences could be related to structural changes in VCbetween RT and 371C. Upon heating of VC samples, two

overlapping transitions were observed: an exothermic andendothermic one with onset temperatures at B20 and 261C,respectively. Owing to its reversible nature, these events wereassigned to the changes in the organization of the inter-cellular lipids. In order to investigate this, the lateral packingand long-range ordering in VC were examined as function oftemperature with FTIR and SAXD. Combination of resultsfrom the scissoring and asymmetric stretching vibrationsrevealed that at RT, the lipids are partially in an ordered state,but not in the orthorhombic lateral packing. Upon increasingthe temperature, the conformational disorder of the lipids,monitored by the asymmetric stretching mode, increased.This represents a phase change of the lipids from an ordered

to disordered (liquid) state. The increase of disordering wasalso observed in the long-range ordering of the lipids. SAXDpatterns showed peaks at 4.5 nm representing the presence oflipids in a long-range ordering. Upon heating, the peakdisappeared around 331C (Figure 6), which also correlateswith the offset temperature of the endothermic peakmeasured by DSC. This transition could therefore representa loss of the long-range ordering. The reversible nature of thislong-range ordering was confirmed by the reappearance ofthe peak during the cooling of the sample. When comparingthe temperature and nature of the transitions of VC with thethree major endothermic lipid phase transitions observed inhuman SC (Golden et al., 1986;Bouwstra et al., 1989), the

transitions observed in VC are very different.

Dehydration and lipid organization.The increased dehydra-tion rate at 371C can be partially ascribed to the higher vaporpressure of water at the elevated temperature. Pavlenko andBasok (2005) showed that the dehydration from a technicalwater-in-oil emulsion is about two times higher at 401C thanat 201C. Our results from VC indicate that the dehydrationrate is up to 3.8 times higher at 371C than at RT. The higherstate of disorder in the lipid organization at 371C mighttherefore have a substantial contribution to the increaseddehydration rate and to the thermotropic changes in elasticityand viscosity (Figure 7). An increase in lipid disorder was also

shown to increase the diffusion rate across SC (Ogisoet al.,1996). The phase changes might therefore be responsible foran improved function of VC, as waterproofing film duringthe prenatal period, and might facilitate skin maturation inutero. After birth, a substantial temperature gradient existsbetween the newborns skin and its environment. This mighthave a major impact on VCs function. Close to the interfaceVC/SC, where the temperature is still 371C, a high waterrelease towards the skin is possible due to the lower degree oflipid order. VC in direct contact with the environment (221C)is characterized by a higher degree of lipid order. This mightaccount for the reduced water release from VC to theenvironment. Moreover, with the higher degree of lipid order,

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VC forms an improved barrier to xenobiotics, which mayinfect the neonate through the skin. Therefore, the changes inlipid organization in VC result in both a better protection ofthe newborns skin and a prolongation of SC hydration.Furthermore, the decreased dehydration rate at lowertemperatures is thermally advantageous to the newborn

infant, because evaporative water and heat loss after birthcan be minimized, whereas a flexible and hydrated skinsurface mantle is retained.

For VC, the skin surface of the neonate constitutes a bigchallenge in terms of the tremendous temperature gradient.However, our studies show that the VC thermal transitionsare of reversible nature (DSC, SAXD). This suggests that VCcan postnatally undergo several transitions at the interfacebetween skin and environment. Unlike the reversibletemperature-dependent features, the water-dependent prop-erties, that is, dehydration and rehydration, are irreversible.

In conclusion, our results clearly show (1) the irrever-sibility of water holding properties of the VC and their

ultrastructural changes and (2) reversibility of the lipidorganization after birth. This demonstrates the presence oftemperature-dependent changes in properties of VC withinthe physiological temperature range, for example lipidorganization and water-holding properties. This suggests thatVC adjusts to the fundamental change from the intrauterine tothe post-natal environment.

MATERIALS AND METHODSCollection and preparation of VCVC was scraped off gently with a sterile plastic spoon immediately

after vaginal delivery or cesarean section of healthy term neonates.

The samples were transferred in sterile plastic tubes, and stored at

41C until use. For the experiments, various samples from differentdonors were used. The collection of VC was approved by the ethical

committee of the Leiden University Medical Center and informed

consent was given by the parents. The samples were taken in

adherence to the Declaration of Helsinki Principles.

Dehydration and rehydrationWeighing boats with a well-defined surface area (30 mm2) and depth

(1 mm) were designed in such a manner that the boats were filled

with VC having a flat surface and a constant thickness. The VC

samples (three donors, each with three replicates) were transferred

into P2O5-containing desiccators and dehydrated at 22241C (RT)

and at 371C, respectively. During the dehydration process, the

samples were weighed (Microbalance, Mettler TG 50, Switzerland)at various time intervals. After dehydration, the samples were

rehydrated at 100% relative humidity over MilliQ water until a

constant weight value was reached. In order to study reversibility of

water-holding properties, the rehydrated samples were dehydrated

again. Weight percentage (t0, 100%) was plotted against time.

In order to visualize ultrastructural changes during the dehydra-

tion and rehydration process, VC samples were also collected to

examine the water distribution by cryo-SEM technique as described

in more detail in a previous study (Rissmannet al., 2006). Briefly, a

small amount of VC (B1 mg) was placed in a small cylindrical

sample holder and cryo-fixed in liquid propane (KF80, Reichert-

Jung, Vienna, Austria). The cryo-fixed samples were sliced at 901C

and transferred into the cryo-scanning electron microscope (6300

FESEM, Yeol, Tokyo, Japan). The samples were freeze dried for

1minute at 901C at 0.1Pa and subsequently coated with 5nm

platinum. The samples were examined in the electron microscope at

1901C. With this method free water can be detected in the

structure.

Differential scanning calorimetryDSC analysis was performed to study the thermotropic behavior of

VC. DSC measurements were performed on a Q-1000 calorimeter

(TA Instruments, New Castle, DE). The fresh VC (510 mg) was

transferred into an aluminum pan, which was hermetically sealed in

order to prevent evaporation of water. After 5 minutes equilibration

at 51C, DSC was performed with a heating rate of 21C/minute and a

modulation of 711C/minute up to 501C. The reversibility of the

events was studied by cooling and heating cycles between 5 and

501C.

Fourier transform infrared spectroscopy

Fresh VC samples were sandwiched with a distinct thickness of12 mm between two ZnSe windows. Subsequently, the windows

were mounted into a special designed heating/cooling cell. FTIR

spectra were acquired on a Bio-Rad Excalibur FTS 4000 XM (Biorad

Laboratories Inc., Cambridge, MA), equipped with a SHA 10 FTIR air

purifying system (Hitma BV, Uithoorn, The Netherlands) and a

mercurycadmiumtelluride detector, which was cooled with liquid

nitrogen. The infrared spectra in the frequency range of

4004,000 cm1 were collected during 8 minutes at 21C intervals

between 10 and 501C as a function of temperature (heating rate of

0.251C/minute). Each spectrum resulted from the co-addition of 128

scans with a nominal resolution of 1 cm1.

Small-angle X-ray diffractionThe SAXD measurements were conducted at station BM26B at the

European Synchrotron Radiation Facility in Grenoble, France (Bras,

1998). Fresh VC samples (B3 mg) were applied onto a mica

window, transferred into a special sample holder, which was

subsequently mounted in the X-ray beam. The diffraction data were

collected by a two-dimensional gas-filled area detector with a 1.5 m

sample-detector distance. The X-ray wavelength was 1.24A.

Sequential diffraction patterns were acquired during heating and

subsequent cooling between 15 and 351C for 2 minutes/1C.

Diffraction data were collected on a two-dimensional multiwire

gas-filled area detector. The spatial calibration of this detector was

performed using silver behenate and cholesterol.

RheologyFlow properties of VC were studied on a rheometer (AR1000-N, TA

instruments, Etten-Leur, The Netherlands) with a steel cone (11,

20 mm diameter). In order to prevent the evaporation of water, a

solvent trap was installed above the cone. Viscosity and elasticity

were recorded in the oscillation mode with a controlled strain

of 0.1% at a frequency of 1 Hz. The temperature-induced changes

were studied between 10 and 451C with a heating/cooling rate of

21C/minute.

CONFLICT OF INTERESTThe authors state no conflict of interest.



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ACKNOWLEDGMENTSWe thank the Department of Obstetrics from the Leiden University MedicalCenter and especially Dr Sicco Scherjon for the provision of VC. The supportof Dr Wim Bras and Dr Kristina Kvashnina at the ESRF in Grenoble and theskillful help of Raphael Zwier were highly appreciated. This work wasfinancially supported by the Dutch Technology Foundation STW, Grant no.LGT 6117.

REFERENCES

Agorastos T, Hollweg G, Grussendorf EI, Papaloucas A (1988) Features ofvernix caseosa cells. Am J Perinatol5:2539

Akinbi HT, Narendran V, Pass AK, Markart P, Hoath SB (2004) Host defenseproteins in vernix caseosa and amniotic fluid. Am J Obstet Gynecol191:20906

Anderson RL, Cassidy JM, Hansen JR, Yellin W (1973) Hydration of stratumcorneum. Biopolymers12:2789802

Bouwstra JA, Peschier LJC, Brussee J, Bodde HE (1989) Effect of N-alkyl-azocycloheptan-2-ones including azone on the thermal-behavior ofhuman stratum-corneum. IntJ Pharm 52:4754

Bras W (1998) A SAXS/WAXS beamline at the ESRF and future experiments.J Macromol Sci Phys B37:55766

Casal HL, Mantsch HH (1984) Polymorphic phase behaviour of phospholipidmembranes studied by infrared spectroscopy. Biochim Biophys Acta779:381401

Chiou YB, Blume-Peytavi U (2004) Stratum corneum maturation. A review ofneonatal skin function. Skin Pharmacol Physiol 17:5766

Golden GM, Guzek DB, Harris RR, McKie JE, Potts RO (1986) Lipidthermotropic transitions in human stratum corneum. J Invest Dermatol86:2559

Haahti E, Nikkari T, Salmi AM, Laaksonen AL (1961) Fatty acids of vernixcaseosa. Scand J Clin Lab Invest13:703

Haubrich KA (2003) Role of Vernix caseosa in the neonate: potentialapplication in the adult population. AACN Clin Issues14:45764

Hoath SB, Pickens WL, Visscher MO (2006) The biology of vernix caseosa.IntJ Cosmet Sci28:31933

Hoeger PH, Schreiner V, Klaassen IA, Enzmann CC, Friedrichs K, Bleck O(2002) Epidermal barrier lipids in human vernix caseosa: correspondingceramide pattern in vernix and fetal skin. Br J Dermatol146:194201

Kaerkkaeinen J, Nikkari T, Ruponen S, Haahti E (1965) Lipids of vernixcaseosa. J Invest Dermatol 44:3338

Mantsch HH, McElhaney RN (1991) Phospholipid phase transitions in modeland biological membranes as studied by infrared spectroscopy. ChemPhys Lipids57:21326

Moraille R, Pickens WL, Visscher MO, Hoath SB (2005) A novel role forvernix caseosa as a skin cleanser. Biol Neonate 87:814

Narendran V, Wickett RR, Pickens WL, Hoath SB (2000) Interaction betweenpulmonary surfactant and vernix: a potential mechanism for induction ofamniotic fluid turbidity. Pediatr Res 48:1204

Ogiso T, Ogiso H, Paku T, Iwaki M (1996) Phase transitions of rat stratumcorneum lipids by an electron paramagnetic resonance study andrelationship of phase states to drug penetration. Biochim Biophys Acta1301:97104

Pavlenko AM, Basok BI (2005) Kinetics of water evaporation from emulsions.Heat Transfer Res36:42530

Pickens WL, Warner RR, Boissy YL, Boissy RE, Hoath SB (2000)Characterization of vernix caseosa: water content, morphology, andelemental analysis. J Invest Dermatol115:87581

Rissmann R, Groenink HW, Weerheim AM, Hoath SB, Ponec M, Bouwstra JA(2006) New insights into ultrastructure, lipid composition and organiza-tion of vernix caseosa. J Invest Dermatol126:182333

Tollin M, Bergsson G, Kai-Larsen Y et al. (2005) Vernix caseosa as a multi-component defence system based on polypeptides, lipids and theirinteractions.Cell Mol Life Sci62:23909

Tollin M, Jagerbrink T, Haraldsson A, Agerberth B, Jornvall H (2006)Proteome analysis of vernix caseosa. Pediatr Res6:4304

Visscher MO, Narendran V, Pickens WL, LaRuffa AA, Meinzen-Derr J, Allen Ket al.(2005) Vernix caseosa in neonatal adaptation. J Perinatol25:4406

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