Cosmetic

A Prospective Study of Anti-Aging TopicalTherapies Using a Quantitative Methodof AssessmentCorrado Rubino, M.D., Dip.E.B.O.P.R.A.S., Francesco Farace, M.D., Luca A. Dessy, M.D.,Marco P. G. Sanna, M.D., and Vittorio Mazzarello, M.D.Sassari, Italy

Background: In the treatment of photo-aged skin, glycolic acid works by removingsuperficial portions of the epidermis andstimulating dermis regeneration. VitaminsA, C, and E should stimulate collagen pro-duction and antioxidants should preventfree radical damage and skin aging. How-ever, the effectiveness of different therapieshas often relied on subjective methods ofassessment. Histologic analysis has seldombeen used because of the drawback of per-manent scarring. In the literature, the use ofa quantitative method for the assessment offacial rejuvenation has been described: thesilicone replica technique. The authors’ aimwas to promote and recommend the use ofthis technique and, in particular, to test theeffect of glycolic acid and multivitamin- andantioxidant-based products on skin texture.

Methods: The authors performed a pro-spective, randomized, double-blind, con-trolled study on 30 women treated topicallyin the outer canthal region (crow’s-feetarea). Patients were divided into threegroups (groups A, B, and C); each groupconsisted of five patients between the ages of31 and 40 years and five patients betweenthe ages of 41 and 50 years. Group A wastreated by glycolic acid application, initiallyat home for 2 weeks, followed by a higherconcentration administered in the officeweekly for six applications. Group B wastreated by topical application at home of a

multivitamin product daily for 3 months.Group C was treated with a cream base (pla-cebo) for 3 months and represented thecontrol group. Skin areas under treatmentwere photographed and reproduced by thesilicone replica technique at baseline and atthe end of treatment. This technique repro-duces exactly the skin’s texture. Digital im-ages were obtained from skin replicas andanalyzed by specific software for differentparameters: roughness, microsulcus num-ber, and width. Pretreatment and posttreat-ment values were compared using the Wil-coxon signed-rank test.

Results: In group A, microsulcus num-ber and width were statistically decreased,but roughness was not. In groups B and C,parameters were not statistically modified.

Conclusions: The silicone replica tech-nique allowed a quantitative analysis of re-sults obtained with different topical thera-pies. In particular, it confirmed the efficacyof glycolic acid in skin rejuvenation. (Plast.Reconstr. Surg. 115: 1156, 2005.)

The surface of the skin presents a typicaltexture that varies on the basis of deeper epi-dermal layer; different anatomical areas; and dif-ferent forces such as aging, sun exposure, dis-eases or metabolism disorders, cosmetics, and/ordrug use. Certainly, the face has the most variable

From the Department of Plastic Surgery and the Department of Biomedical Sciences, Section of Human Anatomy, University of Sassari.Received for publication December 2, 2003; revised May 13, 2004.

DOI: 10.1097/01.PRS.0000156142.12128.81

1156

superficial texture, because of its position, ex-posed constantly to atmospheric agents.1

Skin aging is an unavoidable and progressiveprocess caused by physiologic tissue aging andvarious external factors such as natural or arti-ficial chemical and physical agents. This pro-cess manifests as multiple alterations of normalskin morphology, in particular, enhancementof wrinkles. The appearance of wrinkles re-ceives much attention from the communityand from those in the field of cosmetologybecause it is linked to apparent age, markingthe passage from youth to senescence. For thisreason, attempts are made to delay the appear-ance of rhytids by addressing the causes,namely, tissue aging, elastic and collagen fiberdegradation, muscle action, and gravity.

Many studies have been performed on thepathogenesis, histologic features, and possibletherapy of rhytids. Clinically, chronic photoag-ing may result in fine wrinkles, texture abnor-malities, pigment dyschromias, and actinic ker-atoses. In recent years, many new products andtechniques have arisen that are useful in therejuvenation of facial skin. Chemical peeling,together with dermabrasion and laser tech-niques, are established techniques in aestheticrejuvenation. Furthermore, the use of topicalvitamin C, retinol, and antioxidants is emerg-ing. Although a number of reports on differenttherapies are present in the literature, thesehave often relied on the assessment of preop-erative and postoperative photographs to eval-uate outcome. This evaluation technique hasthe potential drawback of being subjective andflawed, despite attempts to standardize imageacquisition.

A quantitative assessment of various param-eters of skin surface texture is important toassess the efficacy of rejuvenation therapies.This is possible by using the silicone replicatechnique, by which a permanent impressionof the skin is obtained.2 Silicone skin replicasare considered a noninvasive, sensitive processfor achieving a negative cast of superficial skintexture. Araldite contra-replicas permit a per-manent positive unalterable representation ofthe skin relief to be obtained from these sili-cone replicas. Mechanical and optical pro-filometry are the most common techniques forobtaining numerical and mathematical datafrom both negative and positive skin replicas.Shadows generated by the peaks and valleys ofthe skin contra-replica surface are studied by op-tical profilometry.3 Replica data can be achieved

with optical or scanning electron microscopy andthen analyzed using specific software on a com-puter. For our study, we used scanning electronmicroscopy optical profilometry because it ismore accurate than the stylus method.

PATIENTS AND METHODS

A prospective, randomized, double-blind,controlled study was performed. A total of 30patients living in the North Sardinia area, rang-ing in age from 31 to 50 years, were enrolled inthis study. Approval was requested and ob-tained from the local ethical committee, andall subjects gave written informed consent. Thearea subjected to treatment was the outer can-thal region (crow’s-feet area). A thorough his-tory was taken from each patient, using a pa-tient record form (NeoStrata Company, Inc.,Princeton, N.J.),4 excluding subjects with activeherpes simplex, recent dermabrasion or facialsurgery, chemicals peels or topical retinoid usein the previous 6 months, acquired immuno-deficiency syndrome, or pregnancy. Electroly-sis, shaving, depilatories, or waxing agents werestopped 1 week before any treatment. Othervariables recorded were Fitzpatrick skin type,smoking habit status, and postoperativecomplications.

Patients were randomly grouped into threegroups, and each group consisted of five pa-tients aged between 31 and 40 years and fivepatients aged between 41 and 50 years. GroupA was treated by glycolic acid, by use of theSkin Rejuvenation System (NeoStrata Com-pany, Inc., Princeton, N.J.). Initially, patientsstarted to apply 10% glycolic acid cream twicea day for 2 weeks at home. Then, in an outpa-tient clinic for a total of six treatments, 35%glycolic acid lotion was applied for 1 to 2 min-utes, until erythema appeared, increasing thecontact time during successive weekly applica-tions for a maximum of 5 minutes; 50% gly-colic acid was then used with the same schemeweekly. Glycolic acid peel instructions weregiven to patients before and after treatment.4

Group B was treated by topical application athome care of a product containing vitamins A(retinyl palmitate), C (ascorbyl palmitate), andE (tocopherol); coenzyme Q10 (ubide-carenone); and lipoic acid (thioctic acid) in aspray vehicle (VeaMix; Hulka s.r.l., Rovigo,Italy). They were instructed to apply it twice aday for 3 months. Patients were observedmonthly. Group C applied topically a placebocream (Bioclin cream base; Instituto Ganassini,

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Milan, Italy) following the same scheme asgroup B.

At the end of treatment, aesthetic resultswere evaluated by patient self-assessment, by ablinded panel of reviewers (none of whom per-formed the peeling or the clinical follow-up)using pretreatment and posttreatment photo-graphs, and by a computer analysis of scanningelectron microscopy digital images obtainedfrom skin replicas. Superficial skin texture wasanalyzed with a silicone replica technique. Be-fore taking the replica, the skin had beenwashed and dried to remove the dirt. The se-bum was wiped off with ether or acetone.

The replica material is a polyvinylsiloxanederivative (Coltene President plus light body;Coltene/Whaledent, Inc., Cuyahoga Falls,Ohio) for dental application. It is composed oftwo substances, a base (polyvinylsiloxane) and acatalyst, which are mixed together directly beforeapplication onto the skin. Immediately after mix-ing the silicone-based material, it is spread overthe skin surface. Silicone solidifies 1 minute later,and the cast is removed gently from the skinsurface (Fig. 1).

The first cast was always obtained beforestarting treatment from the outer canthal re-gion; the second cast was taken 1 week after theend of treatment. After the silicone solidified, athree-dimensional positive contra-replica wasobtained with an epoxydic substance(araldite). Each positive cast was then spat-tered with palladium-gold and analyzed byscanning electron microscopy.

Four monochrome digital pictures for everycast were filed onto a computer hard disk dur-ing scanning electron microscopy observation.All images were processed with a specific soft-

ware based on a combination of algorithm,binary system, thinning, panning, and linear-ization.5 Computer analysis allowed a geomet-ric description of the superficial skin surfaceon the basis of different skin surface topo-graphic parameters, as described by Takahashi:roughness, skin microsulcus number, andwidth (Table 1).6 Roughness is defined by thestandard deviation of gray-level value of eachpixel in a video image and is expressed as anaverage. It tends to decrease if the image pixelshave a more similar gray level, and vice versa.Skin microsulcus number is defined by thenumber of straight lines in the linearized im-age and practically expresses the number ofskin furrows in each image. Skin microsulcuswidth was measured by taking a ratio of dotsbetween binary and thinned images and is alsoexpressed as an average.

Pretreatment and posttreatment quantitativevalues from a single patient constituted apaired data set. Data variations were comparedusing the Wilcoxon matched-pairs signed rank-sum test. Statistical significance was designatedas a value of p � 0.05.

RESULTS

Fitzpatrick skin type ranged from II to IV.Baseline and end-treatment photographs of apatient treated by glycolic acid are shown inFigure 2.

In group A, eight patients noticed an im-provement of wrinkles of the outer canthalarea, with more youthful and luminous skin,whereas two did not notice any improvements.The blinded panel of reviewers noticed an im-provement in all cases using pretreatment andposttreatment photographs, but it was not pos-

FIG. 1. (Left) The silicone-based material is spread over the skin. (Right) The replica after removal,reproducing superficial skin texture.

1158 PLASTIC AND RECONSTRUCTIVE SURGERY, April 1, 2005

sible to give a quantitative judgment. In allpatients, transient erythema and desquamationdissolved. No alteration of pigmentation wasobserved. After computer analysis of Taka-hashi’s parameters, a statistically significant de-crease of width and skin microsulcus numberwas noted, whereas roughness was not alteredsignificantly (Table II and Fig. 3).

In group B, five patients noticed an improve-ment of wrinkles and skin surface of the outercanthal area, whereas four did not notice anyimprovements and one noticed a worsening.Reviewers did not notice any modification inany of the cases, except for two with transientdesquamation. None of Takahashi’s parame-ters altered significantly (Table II and Fig. 4).

In group C, five patients noticed an improve-ment of wrinkles and skin surface of the outercanthal area, whereas others did not notice anyimprovements. Reviewers did not notice anymodification or side effect in any of the cases.None of Takahashi’s parameters altered signif-icantly (Table II and Fig. 5).

DISCUSSION

Today, rhytid development is the focus ofattention because it correlates with apparentage and signifies the passage from youth tosenescence. For this reason, attempts are madeto delay the appearance of rhytids by acting onthe causes responsible for their development,namely, tissue aging, elastic and collagen fiberdegradation, muscle action, and gravity.

The physician involved in the assessmentand treatment of facial skin aging should beinformed about different therapeutic optionsregarding skin type and aging grade. Varioustopical therapies are used in the treatment ofphotoaged skin. The economical and commer-cial interest in this field is important. Newproducts are proposed daily. Products basedon glycolic acid and multivitamin and antioxi-dant components are readily available on themarket.

Van Scott and Yu reported on the effects ofalpha-hydroxy acids on keratinization disor-ders and on wrinkled skin.4,7 Low concentra-

TABLE IIImage Analysis Results

Before Treatment(mean � SD)

After Treatment(mean � SD) p*

Group ANUM 287 � 83 249 � 69 �0.0001WD 1.624 � 0.185 1.393 � 0.150 �0.0001KSD 7.07 � 0.746 7.135 � 0.833 0.126

Group BNUM 304 � 48 301 � 50 0.3173WD 1.349 � 0.057 1.333 � 0.068 0.093KSD 5.887 � 0.495 5.78 � 0.438 0.1052

Group CNUM 222 � 49 220 � 51 0.5552WD 1.599 � 0.147 1.579 � 0.147 0.131KSD 4.56 � 0.648 4.63 � 0.835 0.8337

* Two-tailed probability.

TABLE IParameters Used for Image Analysis of Skin Surface Replica

Index Definition Meaning

KSD Standard deviation of gray-level value of eachpixel in video image

Average of skin roughness

NUM No. of straight lines in linearized image No. of skin furrowsWD Ratio of dots between binary and thinned images Average width of skin furrows

FIG. 2. Patient treated by glycolic acid. (Above) Beforetreatment. (Below) After treatment, a slight improvement canbe noted.

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tions of any alpha-hydroxy acids diminish cor-neocyte cohesion, clinically seen as a stratumcorneum separation in sheetlike fragments.Histologically, separation is found to occur inthe innermost level of stratum corneum, result-ing in a temporary thinning of it. However,stratum corneum acquires a normal thicknesswith sustained application. Dermal responsesappear within 2 to 3 months, characterized byskin thickness, associated with increased accu-mulation of dermal glycosaminoglycans. Clini-cally, increased skin plumpness occurs. Thestructure of alpha-hydroxy acids is a terminalcarboxyl group on the second or alpha carbonand a variable length carbon chain. Thestrength of the acid derives from its ability todonate a proton. The pH of a solution of al-pha-hydroxy acids varies with both the concen-

tration of the specific acid and the amount ofbuffering or neutralizing agent used. Glycolicacid has the smallest molecule of all alpha-hydroxy acids with a single carbon chain and,for this reason, easily penetrates into the layersof the epidermis. The effectiveness of glycolicacid in the treatment of rhytids has been de-scribed in various studies.8,9

Vitamin C has been recruited in the struggleagainst photoaging as a result of its role incollagen synthesis and its potent antioxidantstrength. The ability of topical vitamin C tostimulate collagen synthesis in the skin hasbeen proven only in vitro.10 Furthermore, vita-min C has been shown to be the main water-soluble nonenzymatic antioxidant.11

Effects of retinoid on photoaged skin arewell documented in the literature.12 Tretinoin

FIG. 3. Scanning electron microscope images of a patienttreated by glycolic acid application. (Above) Before treatment.(Below) After treatment.

FIG. 4. Scanning electron microscope images of a patienttreated by multivitamin and antioxidant products. (Above)Before treatment. (Below) After treatment.

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improves fine wrinkling, texture, and color ofskin. Histologic effects are thinning of the stra-tum corneum, thickening of the epidermis,increased collagen synthesis, and angiogenesisin the dermis. Several dose-response studieshave been conducted showing that higher con-centrations are correlated with improved clin-ical response.

Low-molecular-weight antioxidants, espe-cially vitamins C and E (ascorbate and tocoph-erol, respectively) and lipoic acid, exert protec-tive effects against oxidative stress. However,controlled long-term studies on the efficacy oflow-molecular-weight antioxidants in the pre-vention or treatment of skin aging in humansare still lacking.13

The effectiveness of different therapies hasoften relied on subjective methods of assess-

ment based on preoperative and postoperativephotographs to evaluate outcome. Histologicanalyses have been seldom used because of thedrawback of permanent scarring, which is notacceptable in visible areas such as the face. Inthe literature, a quantitative method of assess-ment to evaluate superficial texture that hasbeen technically validated has been described.3This technique presents different fields of ap-plication. It has been used to analyze skin tex-ture,1 to analyze mammary capsule features af-ter implant reconstruction,14 and to study grafttexture modifications after transplantation.15

Furthermore, it has been reported to be used toevaluate skin rejuvenation therapies. Caputo etal.2 and others16,17 have used skin replicas to eval-uate antiphotoaging efficacy of topical retinoids.The effects of ascorbic acid on photodamagedskin have been studied with this technique.18

Grover et al. investigated the results of facial re-juvenation by carbon dioxide laser resurfac-ing.19,20 Recently, a new analytical system hasbeen proposed to directly evaluate skin surfacemorphology without using skin replicas.21 How-ever, this method needs further validation.

The aim of our study was to promote andpopularize the use of a quantitative and non-invasive method of assessment of skin topogra-phy. We chose to treat the crow’s-feet area,because it represents a barometer of the chro-nologic and environmental age of skin. This isbecause fine wrinkles appear first in this area.The topical therapies analyzed were chosenbecause they are readily available in the marketand are easy to perform.

We confirmed the efficacy of the topical ap-plication of glycolic acid in the treatment ofphotoaged skin. The patient self-assessmentand the subjective analysis of photographsproved to be flawed and not comparable,whereas scanning electron microscopy imageanalysis of skin replicas showed macroscopicand microscopic modifications of skin texture,allowing an accurate qualitative evaluation.Specific software analysis gave different param-eters, as previously described, that allowed astatistical analysis of their variation to prove theeffectiveness of therapies under examination.

To our knowledge, this report is the firstdescribing effects of glycolic acid peeling onskin texture of the outer canthal area using thesilicone skin replica technique. In patientstreated with glycolic acid, microsulcus numberand width decreased significantly. These datacorrelate with the clinical observation of a less

FIG. 5. Scanning electron microscope images of a patienttreated by a cream base. (Above) Before treatment. (Below)After treatment.

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wrinkled, more youthful, and bright skin in thecrow’s-feet area. The only parameter that didnot change significantly was roughness. This isdescribed as a standard deviation of gray-levelvalue of each pixel in the video image. Thewidespread desquamation at the end of treat-ment probably contributed to maintain a highvalue of this parameter.

Moreover, we proved the absence of objec-tive effects on skin texture of the multivitamin,antioxidant, and cream base products in theperiod of treatment. Several articles have re-ported the efficacy of products based on ascor-bic acid alone or retinaldehyde or retinolalone. The inefficacy of the multivitamin prod-uct tested could be explained by the shortnessof the treatment period and the low concen-tration of components present in the product.Furthermore, it is possible that larger samplesand/or a longer period of therapy might yielddifferent results.

CONCLUSIONS

The silicone replica technique allowed aquantitative analysis of results obtained withdifferent topical therapies and, in particular,confirmed the efficacy of glycolic acid in skinrejuvenation during the period of observation.We advocate the use of a quantitative and non-invasive method of assessment, such as the sil-icone replica technique, every time a new anti-aging product or technique needs to be testedand validated.

Corrado Rubino, M.D.Via Besta 16 G07100 Sassari, Italycorubino@uniss.it

ACKNOWLEDGMENT

The authors thank N. Spissu, M.D., for assistance withultrastructural evaluation.

REFERENCES

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19. Grover, R., Grobbelaar, A. O., Morgan, B. D., and Gault,D. T. A quantitative method for the assessment offacial rejuvenation: A prospective study investigatingthe carbon dioxide laser. Br. J. Plast. Surg. 51: 8, 1998.

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