Acute skin barrier disruption with repeated tape

stripping: an in vivo model for damage skin barrier

Yanrui Gao1,2, Xuemin Wang2, Shuangyu Chen3, Shuyuan Li4 and Xiaoping Liu1

1Shanghai Skin Disease Hospital, Clinical School of Anhui Medical University, Shanghai, China,2Skin & Cosmetics Research Dept, Shanghai Skin Disease Hospital, Shanghai, China,

3Shanghai Skin Disease Hospital, Clinical School of Xuzhou Medical University, Shanghai, China and 4Tongji Hospital of Tongji University,Shanghai, China

Purpose: To establish a model of standardized acute barrier

disruption, investigate the response of normal human to

repeated tape stripping, and analyze the change of damaged

skin with non-invasive examination techniques for skin, such

as TEWL and squamometry.

Methods: Repeated tape stripping with corneofix was applied

on three different anatomical sites; the measurement of TEWL

was performed on the baseline and after every 5 strips. Then,

the samples of corneofix were analyzed using Visioscan®

VC98 and squamometry.

Results: The parameter of TEWL and cohesion score show

stable change trend. TEWL increased with frequency of strip-

ping and were significantly higher compared with that of base-

line on three sites. The results of staining of corneofix showed

that the intercorneocyte cohesion is increased with the number

of strips, and the more the number of strips, the more the fixa-

tion of dye per cell.

Conclusion: The changes in the skin barrier function of differ-

ent sites were different after it accepted physical stimulation,

the process of damaging skin barrier could be divided into

three stages based on the △TEWL. In addition, through strip-

ping the skin of an adult, the in vivo model of damaged skin

barrier could be setup.

Key words: skin barrier – tape stripping – non-invasive –

transepidermal water loss – squamometry

� 2012 John Wiley & Sons A/S. Published by BlackwellPublishing LtdAccepted for publication 10 November 2012

THE STRATUM corneum (SC) is the outermostlayer of the skin. One of its most important

roles is as the limiting barrier to water loss byevaporation, as well as its role as a barrieragainst the penetration of foreign physical,chemical, and pathogenic components from theenvironment (1, 2). The barrier function of theskin resides primarily in the epidermis and islocalized in SC typically 10–20 lm thick (3).In daily life, our skin is often affected by

physical stimulation from outside, especiallyoccupational dermatosis; owing to often con-tacting mechanical stimulation, the barrier func-tion of skin is damaged, and so the diseases iscaused. Therefore, the study of skin response tomechanical stimulation is very important.In humans, the skin barrier is assessed by

non-invasive methods, such as the measure-ment of transepidermal water loss (TEWL).Although limitations have been reported (4, 5),measurement of TEWL has been demonstratedto be a suitable method to assess barrier func-tion indirectly.

Tape stripping, first described by Wolf(6), is arobust and simple method in SC physiologyresearch. Adhesive films are pressed onto thesurface of the skin and then removed, thesuperficial layers of the SC adhere to the filmand are accessible for further investigations. Atthe same time, repeated tape stripping maybean effective stimulation for impaired skin (7).The aim of our study was to establish a

model of standardized acute barrier disruption,investigate the response of normal human torepeated tape stripping, and analyze the changeof damaged skin with a non-invasive technique.

Materials and Methods

SubjectsThirty healthy volunteers of both genders with-out any disease, aged 20–45(32.1 � 5.8) years,participated in this study after giving theirinformed consent. Informed consent wasobtained from each subject after the study pro-tocol had been fully explained.

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Skin Research and Technology 2013; 19: 162–168Printed in Singapore � All rights reserveddoi: 10.1111/srt.12028

© 2012 John Wiley & Sons A/S.Published by Blackwell Publishing Ltd

Skin Research and Technology

MaterialsCorneofix (Courage & Khaza-ka ElectronicGmbH, Cologne, Germany) is a tailor-madepatch, with which we can obtain horniness cellsand quantitatively analyze the scales.Tewameter® (Tewameter TM210, Courage +

Khazaka, Cologne, Germany) was used to mea-sure TEWL.Visioscan® VC98 (CK Electronic GmbH, Koln

Germany) consists of a special b/w video sen-sor chip with a very high resolution, an objec-tive, and a UVA light source. The video sensorchip presents the skin image in 256 differentgray levels, which are used to calculate thefollowing skin roughness parameters: skinsmoothness (Sesm), skin roughness (Ser), scali-ness (Sesc), and wrinkles (Sew) (8).Toluidine blue and Basic fuchsin [Sangon bio-

tech (shanghai) Co.] were dissolved in 30% eth-anol and the concentration ratio was 0.75%: 0.5%.

ProcedureThree test anatomic sites, such as backside, thevolar forearm, and the volar upper arm, wereselected for each person, and left or rightside was randomly chosen. Here, a template(Transpore® tape) with a hole (sized 3cm 9

3cm) was applied before tape stripping, toensure that the tape removed SC from thesame site. Before the start of the trial, every-one had a rest for 30 min at the non-invasiveskin testing laboratory (temperature 20 � 2°C,RH 50 � 10%), then a sequence of tape strip-ping with Corneofix® were performed at eachsites until 30 strippings had been finished. TheCorneofix® tape was applied to the markedskin surface, rubbed lightly three times toensure adhesion, and then pulled off with onefluent and decisive movement. Corneofix®

samples were obtained with standard pressureand the fixed pressure time of 10 sec frommarked sites in a standardized way, and aconstant velocity of stripping should beapplied. For meeting these requirements, thewhole experiment was completed by one oper-ator. During this trial, the measurement ofTEWL was performed before the stripping(base line) and after every five strippings.TEWL measurement was delayed for 2 minafter each 5 strips to allow SC water diffu-sion to equilibrate. The whole experiments

were performed under good non-invasive labcondition.

VisioscanThe images of each samples of corneofix® tapesafter sampling were taken by Visioscan® VC98and software SELS® 2000 was used for analyz-ing. The SELS® values of Visioscan VC98 con-tain four parameters.The value of Sesc showed the level of dry-

ness of SC. It is the number of pixels wherethe gray level is higher than the threshold ofSesc. It reflects the state of dehydration of theskin.The value of Sesm was calculated from the

average width and depth of the wrinkles. Itidentifies the smoothness of the skin.The value of Ser was a parameter in contrast

to Sesm. Ser is the roughness parameter andcalculates the gray levels above the threshold incomparison with the entire image. It reflects the‘asperity’ of the skin.The value of Sew was calculated from the

proportion of horizontal and vertical wrinkles.It identifies aging including wrinkles.

StainingAfter carefully removing the Corenofix® fromthe films, the Corneofix® was fixed on atransparent microscope slide and stained for2.5 min with a solution of toluidine blue andbasic fuchsine in 30% ethanol (PolychromeMultiple Stain, PMS) (9, 10), applied to thesurface, and gently rinsed in successive tapwater. A dry Corenofix® without stripping,but staining with PMS, served as reference.Next, the Corenofix® were air-dried andsealed with neutral balsam for observationunder microscope(9100), while visual scoringof fixation of dye per cell (SD) and the inter-corneocyte cohesion (SC) were performedusing the following scale system.The score of fixation of dye per cell is as fol-

low: 0 = no staining; l = staining between thecorneocytes, or slight staining in the cells;2 = moderate staining in the cells; 3 = largeamount of dye into the cells, but not uniform;4 = strong staining of all cells.The score of intercorneocyte cohesion is as

follows: 0 = large sheet of cells; l = large clus-ters, only a few isolated cells; 2 = small clusters,

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many isolated cells; 3 = small clusters in dis-ruption, most cells isolated; 4 = all cells iso-lated, often cases of cell lysis.

StatisticsThe data were analyzed by SPSS 13.0 for Win-dows (SPSS Inc., Chicago, IL, USA). For statisti-cal analysis, we utilized repeated measuresANOVA and the Pearson or Spearman correlationcoefficients. Whenever ANOVA showed signifi-cant differences, the LSD test was introduced.Data are expressed as mean � SD, withP < 0.05 considered significant.

Results

The TEWL of different stripsThe influence of the number of strips on TEWLmeasurements performed at the three anatomicsites is shown in Fig. 1 and Table 1. The resultsshowed that the TEWL was increased with fre-quency of stripping and were significantlyhigher compared with baseline (non-strippings)on both sites (repeated measures ANOVA,P < 0.05).The two TEWL curves of the volar forearm

and upper arm were with the same trend, buton the back, the TEWL curve trend was differ-ent. So the TEWL at the three sites of the samenumber of tape strippings was analyzed usingLSD test. From 0 strip (baseline) to 5 strips, thedifference in TEWL at the three sites was non-significant (P > 0.05), but from 10 to 30 strips,the difference in TEWL between back and thevolar forearm or upper arm was significant(P < 0.05); the difference between the volar

forearm and upper arm was insignificant(P > 0.05).To exactly describe the relationship between

the TEWL(y) and the number of strippings (x),curve estimation was performed with SPSS 13.0.The TEWL at three sites as a function of thenumber of tape stripings, and the curve areshown in chart 1, a: y= 2.362 + 0.462x�0.023x2+ 0.0005x3; R2 = 0.999; P < 0.0001; b: y =2.259 + 0.442x�0.021x2 + 0.0005x3; R2 = 0.997;P < 0.001; c: y= 3.094 + 0.587x�0.031x2+ 0.001x3;R2 = 0.999; P < 0.0001.

The DTEWL values of different stripsIn Fig. 2 and Table 2, the change in trendbetween every two successive TEWL is shown.For example, DTEWL at 5 strips equals TEWLat 5 strips minus that at baseline; DTEWL at10 strips equals TEWL at 10 strips minus thatat 5 strips, and so on. In the initial tape strip-ping, △TEWL of three sites was relatively closeand was about 2.0; after that, △TEWL gradu-ally reduced and maintained a relatively stablelevel; In the third stage, △TEWL gradually roseand there was an upward increase in trend atthe back in comparison with that of the volarforearm and upper arm, with an ongoingincrease up to 5.46.

The SELS values of Visioscan VC98The changes in the value of Sesc showed a pro-nounced increase after the first 5 strips(P < 0.05) with an ongoing increase up to the30th strip on three sites (shown in Fig. 3 andTable 3). At the back site, there was a lowerincrease in trend in comparison with that of thevolar forearm and upper arm. At the baseline(first stripping), the difference between eachother was insignificant, but after 5–30 strips,these changes reached significance. There wasno significance with regard to other values.

The result of visual scoring under microscopeThe results of dying and the standard visualscore are shown in chart 2. The results of visualscoring under microscope are shown in Figs. 4and 5. Scoring of intercorneocyte cohesionincreased with the number of strips, the morethe strips, the higher the scoring; comparedwith that of baseline, significant differencesFig. 1. The corelation between TEWL and the number of strips.

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were observed (P < 0.05), and so was the scor-ing of fixation of dye per cell.

Discussion

The epidermis of human serves as a barrier tothe outside world. The SC, as its external layer,plays an important mechanical protective role,and minimizes the exchange of materialsbetween our body and the environment.The process of multiple stripping on the same

subject at sites in the same general area of theskin caused different degrees of irritation, andremoved the superficial corneocytes by theadhesive force of tapes, through which we set amodel of skin barrier damage aimed at deter-mining skin’s response. This study mainlyfocused on TEWL to evaluate skin barrierchanges.

TEWL is an index of skin barrier function,and high TEWL correlates with low epidermalbarrier function. In our study, we found thatTEWL shows a significant increase after sequen-tial tape stripping, and that the tendency ofthree anatomic sites is different, and as the skinbarrier was disrupted, the TEWL increasedslowly at first, then dramatically.The Sesc value of VC98 shows the level of

dryness of the SC and indirectly reflects state ofroughness of the skin. In the damage progress,the value increases with the number of stripes,which reveals that after accepting the repeatedphysical stimulation, the skin becomes moreand more dry and rough, and the reaction ofthe different sites is different.At the same time, we observed the surface

cells from lesions through squamometry. The

TABLE 3. The Sesc value at the three sites with different strips

No. of strips 1 5 10 15 20 25 30

Volar forearm 1.00 � 0.25 1.51 � 0.30*† 1.82 � 0.37*† 2.04 � 0.36*† 2.20 � 0.39*† 2.18 � 0.40*† 2.17 � 0.36*†

Volar upper arm 1.04 � 0.22 1.59 � 0.37*† 1.90 � 0.41*† 2.00 � 0.34*† 2.14 � 0.35*† 2.21 � 0.44*† 2.33 � 0.43*†

Backside 0.99 � 0.32 1.21 � 0.30* 1.45 � 0.31* 1.51 � 0.35* 1.57 � 0.34* 1.55 � 0.31* 1.71 � 0.30*

*indicates significant difference compared with baseline(P < 0.05)†indicate significant difference compared with backside(P < 0.05).

TABLE 1. The TEWL value at the three sites with different strips

No. of strips 0 5 10 15 20 25 30

Volar forearm 2.26 � 1.36 4.44 � 1.57* 4.87 � 1.76*† 5.57 � 1.99*† 5.93 � 2.21*† 6.56 � 2.97*† 7.50 � 4.62*†

Volar upper arm 2.19 � 1.10 4.22 � 1.30* 4.87 � 1.61*† 5.76 � 1.82*† 6.47 � 2.50*† 7.42 � 3.50*† 8.99 � 4.87*†

Backside 3.04 � 2.29 5.46 � 3.09* 6.94 � 4.63* 8.27 � 5.63* 10.24 � 6.16* 14.60 � 10.84* 20.06 � 12.72*

*indicates significant difference compared with baseline(P < 0.05)†indicates significant difference compared with backside(P < 0.05).

Fig. 2. △TEWL at different stripes and location. Fig. 3. The SESC value of VC98.

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visual scoring of fixation of dye per cell showedthat at the baseline, the corneocytes were stain-ing slightly and the average SD was 1 score,which noted that the corneocytes were normalor not injured. With the strips, the corneocyteswere the sub-healthy or moderately damaged,and the cells were moderately staining. At last,the more the number of strips, the more wasthe damage, the corneocytes were morestrongly staining, large amount of dye found inthe cells, which indicated that the cells were lar-gely destroyed. In addition, the cohesion scoreshowed that the intercorneocyte cohesionincreases with the depth of the horny layer. Theamount of SC removed is an interactionbetween the adhesive force of the stripping andthe cohesive force of the SC, the amount ofremoved SC/(adhesive force – cohesive force).While one strips the SC, the adhesive propertiesof the tape were assumed constant, but thecohesive properties of the SC increase. There-fore, when the SC is progressively stripped, theamount of SC that is removed with each stripdecreases. If △TEWL were in proportion to theamount of stripped horny layer, △TEWL shouldalso decline, but our result is not so; therefore,we forecast that more important factors exist in

the deep layer in maintaining the skin barrierfunction, that is, deep layer plays a moreimportant role in maintaining the skin barrierthan the shallow layer. Of course, the role ofthe shallow layer is also very important.In addition, in our model, combined with the

information of △TEWL, we divided skin barrierdamage process into three stages. The first stage(from first tape to 10th tape), because of the lossof superficial stratum corneum cell, the skinbarrier began to be destroyed, TEWL increasedobviously, but the △TEWL gradually declined.In the second phase (from 10th tape to 20thtape), SC continued to be stripped, the skin bar-rier continued to be damaged, but may be dueto the body self-balance (self-repair) effect,△TEWL became smaller and stable. The thirdstage (from 20th tape to last tape), the damagedskin barrier continued to be aggravated,

TABLE 2. The △TEWL value at the three sites with different strips

No. of strips 5 10 15 20 25 30

Volar forearm 2.18 � 1.56* 0.43 � 1.33 0.70 � 0.97 0.36 � 0.97 0.63 � 1.35 0.94 � 1.90†

Volar upper arm 2.03 � 1.08* 0.64 � 1.13 0.89 � 0.81 0.71 � 1.24 0.96 � 1.57 1.57 � 1.82†

Backside 2.42 � 1.78* 1.47 � 2.30 1.34 � 1.74 1.97 � 2.02 4.36 � 5.74† 5.46 � 5.91†

*indicates significant difference compared with 10 strip(P < 0.05)†indicate significate difference compared with 20 strip(P < 0.05).

Fig. 4. Amount of dye in cells.

TABLE 4. Different characteristics of three stages of damaged skin

Stage First stage Second stage Third stage

TEWL Low Middle High

△TEWL Reduce gradually Relatively stable Increase gradually

Sesc Low Middle High

SD Shallower Moderate Deeper

SC Smaller Moderate Larger

Fig. 5. Intercorneocyte cohesion.

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breaking the limit of the skin TEWL, and△TEWL kept rising sharply. The three stages inthe skin barrier damage process appeared oneby one, and both of them is essential. But a pre-vious study (5) pointed out that some individu-als do not demonstrate increased TEWL despitean equivalent mass of SC being removed com-pared with those who do show a response. Thethree stages in the skin barrier damage indicatethat different treatments should be adaptedaccording to different degrees of damage inclinical practice. For example, if the damage islimited in the first stage which is slightly dis-rupted, some moisturizers can be used forrelieving disruption of skin barrier; but, if inthe third stage, because the superficial corneumwas seriously damaged, a large amount of cor-neocytes and lipids were lost; therefore, asidefrom simple skin moisturizers, increase in theuse of physiological lipid creams (11) and theskin hyperplasia agents to promote the skinbarrier self-repairing maybe a reasonablechoice.We can get that the skin barrier damage pro-

gress can be divided into three phases with

their own characters (Table 4), and the threestages are successively evolving. At the sametime, through this method, we can establish askin model with damaged barrier function,which can be used for evaluation of the self-repaired capability of skin and the efficiency ofbabies’ creams or cosmetics. Other investigatorshave developed their own models for mechani-cal irritation, such as percussive (12) and abra-sive trauma (13). Compared with others, ourmethod is convenient, minimally invasive,quantifiable and combined with Corneofix®

dying, we can observe characteristic surfacecells form lesions. Of course, the baby’s skin,different from the adults’, is very sensitive, thin,and fragile (14) Some scholars reported that theaverage TEWL for infants ranges from 15 to30 g/m2/h (15). These data show that infantskin barrier is not the same as in adults, andcontinues to develop during the first year oflife. In 1987, Barker evoked the idea that thepreterm infant’s epidermis resembles thestripped skin of an adult(16). Fifteen years later,Sekkat developed an in vitro model for thedeveloping skin of the premature neonate with

(a) (b) (c)

chart 1. The curve of TEWL value and the number of stripps

chart 2. The Chart for visual scoring of dying.

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tape stripping on porcine (17). Now, on healthysubjects, using the curvilinear equation, we canweaken adults’ skin barrier to a degree, whichparallels that of infants.In a conclusion, this study only qualitatively

demonstrated the process of skin lesions. Asregards the specific molecular mechanism, thenew quantitative technology should be adaptedin a further study.

Conclusion

The skin barrier function is one of the mostbasic problems in the dermatology. In clinicalskin diseases, the disturbed skin barrier runsthrough the whole process of the etiology andpathology of skin diseases. This article describesthe change in the skin barrier function acceptedphysical stimulation, and evaluation with non-invasive detection methods, and also objectively

demonstrates the damaged process of the skinbarrier function and divides this process intodifferent stages based on the △TEWL, whichhelp us more profoundly to understand theskin barrier function, so as to understand otherrelated diseases. This trial, at the same time,provides a new experience of non-invasivemethod for the basic research of the dermatol-ogy field.

Acknowledgement

The authors thank all colleagues in the Skinand Cosmetic Research Center for their helpand acknowledge doctors for reviewing, critiqu-ing, and otherwise improving the manuscript.

Declaration of interest

The authors declare no conflicts of interest.

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Address:Xuemin WangSkin & Cosmetics Research DepartmentShanghai Skin Disease Hospital1278 Baode RoadShanghai 200443ChinaTel: +086-21-61833060Fax: +086-21-61833062e-mail: gyanrui@126.com

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