2 nd International Congress of Plastic Dermatology. Vol. 4, n. 1, January-April Adele Sparavigna, Michele Setaro, Linda Frisenda

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1 Vol. 4, n. 1, January-April 2008 Physical and microbiological properties of a new nail protective medical device Adele Sparavigna, Michele Setaro, Linda Frisenda The use of antisense oligonucleotides in skin lightening products Melizza Bautista, Falen Lockett, Jaimie Mecca, Wanphimon Sawatdeekhachornphat, Angelica Castro, Sujani Yarlagadda, Salvador Gonzalez, Neena Philips Coating on micronized titanium dioxide increases safety and maintains efficacy as sunscreen filter Jayson Goodner, Uma Patil, Yousun Lim, Sujani Yarlagadda, Angelica Castro, Salvador Gonzalez, Neena Philips Study on the effectiveness of a silicone gel in treating surgical wounds Vincenzo De Giorgi, Serena Sestini, Barbara Alfaioli, Marta Grazzini, Agata Janowska, Andrea Saggini, Torello Lotti New way to protect the skin against sunlight damages Riccarda Serri Dark skin dermocosmetology Stefano Veraldi Cosmetic use of poly-l-lactic acid for skin rejuvenation: New indications Alessio Redaelli The reflectance confocal microscopy in the study of hair follicle pigmentary unit Fabio Rinaldi, Giammaria Giuliani Kaposi s sarcoma: Story of a 30-year clinical experience Lucia Brambilla, Vinicio Boneschi Anti-aging principles into cosmetic products. The challenges Piera Fileccia 2 nd International Congress of Plastic Dermatology Milan, March 6-8, 2008 A B S T R A C T S Indexed in: EMBASE, EMNursing, Compendex, GEOBASE

2 Cari Colleghi, c i rca un anno fa è partita la grande macchina organizzativa del 2 Congresso Internazionale di Dermatologia Plastica I mesi sono apparentemente volati ma sono stati pieni di idee che hanno dato vita ad un evento importante per la nostra storia. Il 2 Congresso ISPLAD sarà ricordato per l alto valore scientifico delle comunicazioni, per gli oltre duecento relatori, per la partecipazione di numerosi e validi colleghi stranieri, per gli oltre cinquanta giornalisti accreditati, per i numerosi work-shop, per i corsi formativi, per l entusiasmo e la professionalità di chi ha coordinato e organizzato gli eventi, per i numerosissimi dermatologi partecipanti. Non sarà dimenticata la stima e la fiducia dei numerosi sponsor che ci hanno permesso di realizzare tutto questo. Resterà nelle nostre mani un ricordo concreto di questi momenti vissuti: sarà questa nostra rivista, il vostro JPD che state per leggere. Un JPD speciale, non solo per la bella copertina color oro, ma per la presenza, oltre ad importanti articoli, degli abstract dei lavori congressuali, che potranno così essere letti e conosciuti da tutta la Comunità Scientifica Internazionale. Un grande impegno editoriale che dimostra il coraggio e la lungimiranza del nostro editore. Un particolare ringraziamento va ad Antonio Di Maio, Managing Editor e anima pulsante del JPD. Ci sorprende sempre la sua passione nel raggiungere gli obiettivi, la sua disponibilità ad ogni iniziativa e il suo gran cuore. Ma tornando all ISPLAD permettetemi di r i c o rd a re l importante riconoscimento che abbiamo ricevuto nell essere stati accolti ufficialmente dalla Lega Internazionale delle Società Scientifiche Dermatologiche (ILDS), fatto che ci permetterà di partecipare attivamente alle decisioni che r i g u a rde ranno il futuro della Dermatologia Internazionale. Buon Congresso e buona lettura. Dear Colleagues, Just over a year ago, the grand organizational plans for the 2nd International Congress of Plastic Dermatology were formed. The months seem to have flown by and they were full of creative ideas that gave life to this extremely important event in our history. The 2 nd ISPLAD Congress will be remembered for the quality and scientific value of the presentations, the impressive number of over 200 speakers, the participation of both locally-based dermatologists and respected colleagues from all over the world, as well as its workshops and training courses. I am quite sure that the enthusiasm and professionalism of the event s coordinators and the more-than-50 accredited journalists won t be forgotten for a long time to come either. We remain incredibly thankful for the trust and faith of our sponsors who aided us in accomplishing our goals. With our journal, we hold in our hands a concrete memory of all these shared moments: your JPD awaits you. This will be a special edition, not only because of its magnificent gold cover, but also for the presence of abstracts from the congressional research papers, in addition to other important articles. By these means, the fruits of labour of our congress will be read and made known to the international scientific community. This has been an intense editorial undertaking and clearly demonstrates the courage and foresight of our editor. I take this chance to extend special thanks to Antonio Di Maio, managing editor and lively soul behind the JPD. He never fails to surprise us with his passion in achieving objectives, his helpfulness in every initiative, and his big heart. Turning once more to ISPLAD, we are all very proud of the recognition we have received through our official acceptance to the International League of Dermatological Societies. This new status will allow us to participate in important decisions regarding the future of international dermatology. I hope you enjoy the congress and readings. Antonino Di Pietro 1

3 Journal of Plastic Dermatology Editor Antonino Di Pietro (Italy) Editor in Chief Francesco Bruno (Italy) Co-Editors Salvador Gonzalez (USA) Pedro Jaen (Spain) Associate Editors Francesco Antonaccio (Italy) Mariuccia Bucci (Italy) Franco Buttafarro (Italy) Ornella De Pità (Italy) Giulio Ferranti (Italy) Andrea Giacomelli (Italy) Alda Malasoma (Italy) Steven Nisticò (Italy) Elisabetta Perosino (Italy) Andrea Romani (Italy) Nerys Roberts (UK) Editorial Board Lucio Andreassi (Italy) Kenneth Arndt (USA) Bernd Rüdiger Balda (Austria) H.S. Black (USA) Lucia Brambilla (Italy) Günter Burg (Switzerland) Michele Carruba (Italy) Vincenzo De Sanctis (Italy) Aldo Di Carlo (Italy) Robin Eady AJ (UK) Paolo Fabbri (Italy) Ferdinando Ippolito (Italy) Giuseppe Micali (Italy) Martin Charles Jr Mihm (USA) Joe Pace (Malta) Lucio Pastore (Italy) Gerd Plewig (Germany) Riccarda Serri (Italy) Adele Sparavigna (Italy) Abel Torres (USA) Stefano Veraldi (Italy) Umberto Veronesi (Italy) Managing Editor Antonio Di Maio English editing Rewadee Anujapad Direttore Responsabile Direttore Generale Direttore Marketing Consulenza grafica Impaginazione Pietro Cazzola Armando Mazzù Antonio Di Maio Piero Merlini Clementina Pasina R e g i s t r. Tribunale di Milano n. 102 del 14/02/2005 Scripta Manent s.n.c. Via Bassini, Milano Tel / Fax scriman@tin.it Abbonamento annuale (3 numeri) Euro 39,00 Pagamento: conto corrente postale n intestato a: Edizioni Scripta Manent s.n.c., via Bassini Milano Stampa: Arti Grafiche Bazzi, Milano Sommario pag. 5 Physical and microbiological properties of a new nail protective medical device Adele Sparavigna, Michele Setaro, Linda Frisenda pag. 17 The use of antisense oligonucleotides in skin lightening products Melizza Bautista, Falen Lockett, Jaimie Mecca, Wanphimon Sawatdeekhachornphat, Angelica Castro, Sujani Yarlagadda, Salvador Gonzalez, Neena Philips pag. 21 Coating on micronized titanium dioxide increases safety and maintains efficacy as sunscreen filter Jayson Goodner, Uma Patil, Yousun Lim, Sujani Yarlagadda, Angelica Castro, Salvador Gonzalez, Neena Philips pag. 25 Studio prospettico sull evoluzione delle ferite chirurgiche trattate con gel al silicone Vincenzo De Giorgi, Serena Sestini, Barbara Alfaioli, Marta Grazzini, Agata Janowska, Andrea Saggini, Torello Lotti pag. 33 Incrementare la protezione cutanea da fotoinvecchiamento e danno solare Riccarda Serri pag. 37 Dermocosmetologia della pelle scura Stefano Veraldi pag. 41 Uso cosmetico dell acido L-polilattico per il ringiovanimento cutaneo: nuove indicazioni Alessio Redaelli pag. 49 The reflectance confocal microscopy in the study of hair follicle pigmentary unit Fabio Rinaldi, Giammaria Giuliani pag. 55 Ambulatorio sarcoma di Kaposi: racconto dell incontro con una patologia e di una esperienza medica durata 30 anni Lucia Brambilla, Vinicio Boneschi pag. 63 I principi attivi antiaging nei prodotti cosmetici. Le sfide (Seconda di due parti) Piera Fileccia pag nd International Congress of Plastic Dermatology Milan, March 6-8, 2008 A B S T R A C T S È vietata la riproduzione totale o parziale, con qualsiasi mezzo, di articoli, illustrazioni e fotografie senza l autorizzazione scritta dell Editore. L Editore non risponde dell opinione espressa dagli Autori degli articoli. Ai sensi della legge 675/96 è possibile in qualsiasi momento opporsi all invio della rivista comunicando per iscritto la propria decisione a: Edizioni Scripta Manent s.n.c. Via Bassini, Milano 3

4 Physical and microbiological properties of a new nail protective medical device Adele Sparavigna 1 Michele Setaro 2 Linda Fr i s e n d a 3 SU M M A R Y Physical and microbiological properties of a new nail protective medical device The study evaluated the physical and microbiological properties of a hydro-alcoholic, film-forming solution containing hydroxypropyl-chitosan (HPCH) at different concentrations, and that forms the basis of a new medical device, including also piroctone olamine as a preservative, with protective activity for both toenails and fingernails. The following physical properties of 1% hydroxypropyl-chitosan solution were investigated either in vitro by using bovine hoof slices (as a well recognized model of human nails) or in vivo on healthy human nails: the film-forming capability, the adhesion of the product on the nail surface, the protective properties against abrasion (mechanical aggression) and temperature (physical aggression). The application of HPCH solution on a bovine nail slice, after evaporation of the solvent, forms a thin film that is very evident when examining the surface of the slice with a microscope scanner. The surface covered by HPCH film appears smoother compared to the irregular, rough surface of the control nail plate. Hydroxypropyl chitosan film adheres well to the nail surface during the stripping test, while the same film does not adhere so well to glass. The test there f o re confirms the existence of the film-forming capacity of hydro x ypropylchitosan selective to nail tissue, unlike that observed with common cosmetic nail varnish or glue. The presence of the HPCH film is also demonstrated by the thermography test performed in vivo on the nails of a healthy volunteer, which found a reduction in the temperature of the nail surface as a result of the presence of the film. Lastly, the film protects the surface of the nail from mechanical damage caused by abrasion, as demonstrated by the abrasion test performed in vivo on a healthy volunteer, which found significantly less abrasion on the nail surface protected by the HPCH film. The paper also investigated the microbiological properties and protection against nail fungal colonization provided by hydroxypropyl-chitosan solutions, with or without the preservative agent. When some drops of the HPCH solutions were put on a Petri dish inoculated with T. mentagrophytes, the growth of the nail pathogen on the HPCH film was prevented by a physical mechanism. In a further in vitro experiment, the application of µl of the device (0.5% HPCH and 0.5% piroctone olamine) on a bovine nail slice put on a Petri dish inoculated with T. rubrum, prevented the growth of the pathogen within and around the nail, by forming dose-dependent inhibition rings. Finally, the device prevented the in vitro nail experimental infection by T. rubrum either when applied before or after the bovine nail slices contamination. No growth of the nail pathogen was observed after transplant of the nail fragments, treated with the device, in a new plate not inoculated with the fungus, while on the contrary a regular growth was recorded for control nail plates. In conclusion, our data show that the new medical device, when applied on the nails, is capable to form a film, that adheres and penetrates into the nail structure, by supporting it and forming a protective film against physical and microbiological agents. In particular, the device prevents the nail infections by common pathogens such as T. mentagrophytes and T. rubrum in experimental infection models. 1 DermIng s.r.l., Monza, Milano (Italy) 2 Tecnolab del Lago Maggiore s.r.l., Verbania Fondotoce (Italy) 3 Polichem SA, Lugano (Switzerland) KEY WORDS: Hydroxy-propyl chitosan (HPCH), Nail film-forming, Physical protection, Microbiological protection 5

5 A. Sparavigna, M. Setaro, L. Frisenda I ntroduction The strength and physical character of the nail is attributable to both its constituents and design. The nail tensile, flexure and tearing strength changes with age, sex and the digit from which the nail derived. The nail is 1000 times more permeable to water than the skin, and consequently the nail structure reacts to p rolonged or repeated contact with water. Immersion of the nail in water for an hour increases its weight by over 20%, moreover it renders the nail more flexible. 1 The aspect of the nails may be affected by: longitudinal grooves, that may re p resent physiological long-lasting conditions as shallow and delicate f u r rows, and that become more prominent with age and in certain pathological conditions; longitudinal ridges, i.e. small rectilinear pro j e c t i o n s that usually extend from the proximal nail fold until the free edge of the nail; oblique lines, more common in children than in adults; transverse lines in form of sulci, that reflect a temporary reduction in nail matrix activity and are considered as re t rospective indicators of a trauma or other pathological conditions. 1 Lamellar splitting (onychoschizia) is a condition found in 27-35% of normal adult women. The distal portion of the nail splits horizontally in this condition. It is common in people who carry out a great deal of housework, whose nails are repeatedly soaked in water and then dried. Changes in the fingernails of old people are mostly related to diminished tissue repair and inflammatory or degenerative changes of the distal interphalangeal joint. These influences are associated with reduced rate of longitudinal nail growth, thinning of the nail plate and accentuation of longitudinal ridges. 1 Variations in thickness and consistency of the toenails occur in the elderly and are mostly attributable to changes in peripheral circ u l a t i o n. Repetitive and prolonged wetting and drying of f i n g e rnails is the single most common cause of splitting and ridges of the nails. Splitting of the nails is rarely caused by internal disease or vitamin deficiency, nail polish remover causes onychoschizia (lamellar splitting), finally trauma to the fingers contributes to onychoschizia. Healthy looking nails should be smooth, curv e d, void of any spotting, and should not have any hollows or ridges. Nail polishes are used since centuries with the aim to beautify, colour or hidden defects of the nails. Basic ingredients of the nail polishes are film forming agents, resins and plasticizers, solvents, and colouring agents. In addition to the traditional products, other products with selective ingredients become available with the aim to re i n f o rce and protect the nails. The use of a nail varnish, normally a water insoluble polyvinyl resin film, has the disadvantage that the re m o v a l of the nail varnish by an organic solvent or by nail filing can further damage the nail structure, by i n c reasing brittleness and splitting, and re n d e r i n g the nail keratin less resistant to the fungal infections. Among the cosmetic damages of the nails, the following can be included: breaking, splitting, fracturing, brittleness, white spots or ridges, poor nail growth, color or shape changes. Nails in bad conditions can be very harmful for the personal image, if neglected can cause chronic infections, associated to long-lasting embarrassment and pain. Noteworthy, they may be considered a social p roblem and/or a professional illness. Medicated nail lacquers are also available, which contain monoester resins as film forming agents, and antifungal agents as active ingredients, intended to manage nail diseases such as onychomycosis, but less effective than expected on the basis of their in vitro antifungal activity. In fact, the c o m m e rcially available medicated nail lacquers have some limitations due to the characteristics of the film-forming agents, which have no affinity to keratin and act as occlusive medications. Those entrap the active ingredient and reduce its diff u- sion from subsequent applications, as a result of the formation of thick lacquer layers that tend to split easily. The insoluble films need solvents and nail-file to be removed weekly or even more freq u e n t l y, pro c e d u res that damage the nail structure and render it more prone to re i n f e c t i o n. 2 To overcome the a.m. problems of the nail lacquers, an innovative proprietary technology of hydrolacquer has been developed by the Swiss company Polichem, by employing chitin derived hydrosoluble amino-polysaccharides. The new technology is based on hydroalcoholic solutions of hydroxypropyl-chitosan (HPCH), a water soluble semi-synthetic derivative of chitosan, which acts as a film forming agent. HPCH dissolves in high percentage in water, has affinity to air, is a highly plastic substance and forms a highly elastic film, it increases the dispersion of other ingredients, and its safety profile is excellent. Hydroxypropyl-chitosan is endowed with adhesive properties towards different biological tissues due to their positive charge. Moreover, the free hydroxypropyl grou- 6

6 Physical and microbiological properties of a new nail protective medical device Figure 1. Scan electron microscopy of a nail slice surface untreated (left) or covered by the hydroxypropyl-chitosan film (right). ps of HPCH interact with keratin, by hydrogen binding and other weak interactions that contribute to the improved drug transport and release. 3 The nail application of hydrolacquers prepared according to the ONY-TEC technology, even for chronic treatments like in onychomycosis, is easy and accepted by the patients due to the simple (rinsing) removal procedure and no need of nail filing. The ONY- T E C technology proved capable to inc rease nail permeation of actives, 4 to support the nail structure and to protect it against extern a l agents. When used as the basis of compositions with antimycotic agents, the ONY- T E C t e c h n o l o - gy strengthened the efficacy of antimycotic agents. 5 A new medical device (Myfungar, Polichem SA) has recently been developed, based on the above innovative technology, for nail protection against e x t e rnal agents. The composition of the device includes hydro x y p ropyl-chitosan as a film forming agent, water and ethanol for a prompt evaporation after application on the toenails or fing e rnails, and piroctone olamine, a well known 6 p re s e rvative agent with a broad antimicro b i a l spectrum. Aim of this study was to investigate the p rotective activity of the device and of its main i n g redient against physical and micro b i o l o g i c a l agents by i n - v i t ro and i n - v i v o functionality tests. hysical properties and protection Pagainst physical agents The investigations were performed by using a hydro-alcoholic solution containing 1% hydroxypropyl-chitosan (HPCH). 1) Film forming effect Method The assessment of the film forming effect was done in 2 in vitro studies by applying the product on the surface of bovine hoof slices (cut by criotomy) to check the film thickness and the covering property. Two investigations were performed: a) In the first investigation, we took a silicon cast of an untreated bovine hoof slice. HPCH solution was then applied to this layer and the silicon cast was taken again. The casts w e re observed by the stereo microscope, and images were obtained for the optical pro f i l o- m e t ry assessment. The casts were then subjected to gold metallisation for scanning e l e c t ronic microscope (SEM) observ a t i o n. b) In the second investigation, HPCH solution was applied to hoof slices, which were observed directly by means of a reflected light stereo microscope, then prepared by means of the gold metallisation process and observed with an electronic microscope. For the most effective observation of the thickness of the product applied, transverse sections were examined at the level of the central portion of the layers. Results The analysis of the images shows that the HPCH film is relatively thin, with a polished appearance which reproduces the underlying weft and reduces the surface roughness, by filling the holes and the uneven surface of the nail slices (Figures 1 and 2). The profile measurement analysis carried out on the casts confirms an evident decrease of superficial roughness 7

7 A. Sparavigna, M. Setaro, L. Frisenda index (RA), calculated as the absolute mean of all the deviations from the mean. 2) Adhesion Method In order to observe the adhesive capacity of the product, we adopted the methods set out in the EN ISO 2409: 1994 standard. This standard describes a test method for the in vitro assessment of the resistance of a coating against detachment from a support. To do this, a square mesh section is cut into the coating until the support is reached. The supports used were bovine hoof slices and a microscope slide. As they were not completely applicable, given the nature of the support and product, the terms of the standard were used as general guidelines. a) Application to the bovine hoof slices The product was first pigmented with a few drops of methylene blue, then observed with a stereo microscope to ensure that the colouring agent was evenly distributed over the sample surface. The product was applied by a brush. Once the product had dried on the surface, a square mesh section was cut into it using a scalpel. On completion of this operation, an adhesive tape in accordance with the specifications set out in the standard was applied to the cut section. The tape was then removed as described in the standard and applied to a slide to observe it under a transmitted light microscope in two different types of lighting, in order to analyse the material removed by the tape. b) Application to the microscope slide We followed the same procedure, with the exception of the pigmentation of the product, and used a single type of lighting. Results The analysis of the images shows that the product in the test a) has good cohesion and re s i- stance to detachment from the nail surface ( F i g u re 3); this is confirmed by the absence of any HPCH particles on the stripped tape. In the test b) there is a detachment of particles of HPCH film evident on the stripped tape (Figure 4). 3) Anti-abrasion effectiveness Method The anti-abrasion effectiveness test involved provoking a series of incisions on the surface of the test object. With a view to producing the same type of incision in the different test conditions, we used a Dermal Torque Meter (D i a S t ron UK), which is fitted with a special probe which terminates in a rotating disc. With this Figure 2. Scan electron microscopy of a nail slice covered by the hydroxypropyl-chitosan film (section magnification 1.5K x). Figure 3. Test of adhesiveness of the hydroxypropyl-chitosan film on nail slice surface. Figure 4. Test of adhesiveness of hydroxypropyl-chitosan film on glass slide surface. 8

8 Physical and microbiological properties of a new nail protective medical device Figure 5. Protection of nail abrasion by hydroxypropyl-chitosan film on in vivo human nails. instrument and its software, it is possible to programme the torsion torque applicable to the rotating disc, to which a circular section of type 150 abrasive paper is attached. The probe is then placed on the surface to be subjected to abrasion. To check the protective properties of HPCH film, we proceeded in two stages: a) In vivo study: the product was applied by means of a brush on fingernails of 3 healthy volunteers. Abrasion was then applied to the t reated and untreated nails. Silicon casts of t reated and untreated nails were taken after abrasion and analyzed by computerized profile measurement. Silicon casts were taken on t reated and abraded nails after washing. The casts were then pre p a red using the gold metallisation process for the scanning electronic microscope (SEM) pro c e d u re. b) In vitro study: The product was applied by means of a brush to a bovine hoof layer, which was then subjected to abrasion along with a n o t h e r, untreated layer. With this method, it was possible to directly observe the surface under examination. The layers were observ e d with the stereo microscope, then pre p a red by means of the gold metallisation process and o b s e rved by SEM. The product was then applied to another layer, which was subjected to abrasion, then washed with water. The samples were observed by means of a re f l e c t e d light stereo microscope. Results a) In vivo study: the analysis of the images shows that the untreated surface of human nails has very deep incisions with jagged edges and a significant quantity of material removed, while the treated surface has shallower incisions with regular edges and a minimum quantity of material removed. The analysis of the images of the layer which was washed following abrasion also confirms the p revious observations (Figure 5). b) In vitro study: the optical profilometry of the casts confirms these results, showing a more regular and less deep profile, and the analysis of the casts under the electronic microscope also shows a more regular surface. 4) Thermographic activity Method The product was applied by a brush to the thumb nail of a volunteer. The product was left to dry, and the thumb was then placed in front of a thermal video camera properly configured and calibrated to highlight the nail surface. In vivo images of the treated and the untre a t e d thumb nails were then obtained. Results The analysis of the images shows that, when the thumb nail is covered by HPCH film, the area with a higher temperature is smaller than in the untreated thumb nail (Figure 6). Figure 6. Effect of hydroxypropyl-chitosan film on in vivo thermography of human thumb nails. 9

9 A. Sparavigna, M. Setaro, L. Frisenda icrobiological properties M and protection against fungal colonization The investigations were performed by using solutions containing different concentrations of HPCH and eventually a preservative agent as specified. 5) Protective film against the growth of T. mentagrophytes Method Sabouraud Dextrose Agar (SDA) square plates were prepared by inoculating x 10 3 T. mentagrophytes (DSMZ) CFU/mL of agar, according to standard pro c e d u res. Two hydro - alcoholic solutions containing 0.3% and 1.0% hydroxypropyl-chitosan respectively, were put on the surface of the inoculated plates. The growth of the pathogenic agent was observed after 5 days of incubation. Results There was a full growth of T. mentagrophytes in the whole plate, with the exception of the place where HPCH formed a protective film on agar surface. In fact, the hydroxypropyl-chitosan film prevented the fungus hyphae from penetrating the film and growing on the same (Figure 7). The study concluded that the device does form a physical protection against the microbiological agent. 6) Prevention of in vitro growth of T. rubrum on bovine nail slices Method SDA square plates were prepared by inoculating x 10 3 T. rubrum (DSMZ) CFU/mL of agar, according to standard procedures. The device (Myfungar, Polichem SA, containing 0.5% HPCH and 0.5% of piroctone olamine as a preservative) was added on the surface of the agar plate either by adsorbing 10 µl of the solution on a 10 mm neutral disk or by placing 10 and 20 µl of the solution on 10 x 20 mm 75 µm thickness nail slices obtained from bovine hooves. A 0.5% HPCH (10 µl) solution on a disk was used as a control. The plates were then incubated at 32 ± 1 C for 5 days. Results The HPCH control area showed an abundant growth of T. rubrum. The areas inoculated with the Myfungar device, placed on nail slices, showed dose-dependent inhibition rings at the 2 doses tested. The results are summarized in Figure 8. Inhibition rings of T. rubrum growth were visually evaluated as a consequence of protective activity of the device against pathogen growing onto the nail plates. 7) Prevention of in vitro nail experimental infection by T. rubrum Method a) Application of test products before nail contamination: SDA square plates were prepared by inoculating x 10 3 T. rubrum (DSMZ) CFU/mL of agar, according to standard procedures. Three and four bovine nail slices with a µm thickness were inserted vertically and at an equal distances into the agar until they touched the base of the plate, in a way that each nail protruded from the agar by 4-5 mm. All the nail fragments had previously been immersed in the test preparations of the device (Myfungar ) and left to dry. The tests were done in duplicate. The plate with 4 nail fragments was used to assess the fungal growth and the presence of inhibition rings after 7, 14 and 21 days of incubation. The plate, containing 3 fragments, was used for the withdrawal of Figure 7. Growth of T. mentagrophytes after application of 0.3 (left) or 1% (right) hydro alcoholic HPCH solution. No growth is observed over the HPCH film. Figure 8. Inhibition rings of T. rubrum growth by application of 10 or 20 µl octopirox (Myfungar ) device solution on bovine nail slices or on disk. Control: 10 µl HPCH solution. 10

10 Physical and microbiological properties of a new nail protective medical device an entire nail fragment after one, two, three weeks respectively. Each withdrawn nail fragment was inserted singularly in a sterile SDA plate (not infected), then incubated and examined after three weeks to check, through the presence or absence of fungal growth, whether the preventive treatment had brought about a definitive inhibition of the fungus. An identical experimental procedure to that above described was carried out with untreated nails (control) and nails treated with HPCH solution. b) Application of test product after nail contamination: SDA square plates were pre p a re d by inoculating x 10 3 T. ru b ru m (DSMZ) CFU/mL of agar, according to stand a rd pro c e d u res. Numerous plates of untre a- ted bovine nail slices with a thickness of µm were then placed on each plate, as described above. Thicker fragments were used in this experiment, in order to assess the activity with the mycelium embedded m o re deeply in the nail. The plates were then incubated at 26 C. After one week, the entire surface of the plates and the nails inserted in them were evenly covered by the mycelium. Seven, 14 and 21 days after the sowing of the dermatophytes, a nail fragment completely covere d in the mycelium was withdrawn and brushed with the device (Myfungar ), HPCH solution, or without treatment (contro l ). After drying the nail was inserted in the agar layer of new plates in which the dermatophytes had not been sown. The plate was then incubated and examined once a week for 21 days to assess the fungal gro w t h. Results a) The fungal growth inhibition rings produced by spreading the device in the agar is summarized in Table 1. The control nails (no treatment) and those treated with HPCH solution showed no inhibition rings and the small fungal colonies present when the nail was inserted in the agar rapidly invaded the entire surface of the dish. The nails treated with the device produced small inhibition rings. The table also contains the results obtained with the transplant of the treated or control nails, withdrawn after 7, 14 and 21 days of insertion in the agar already containing the fungal colonies, into new dishes containing nutritional medium only. The growth of T. rubrum was inhibited by the device at all time points. b) Data on fungal growth produced by nail fragments deeply contaminated by the mycelium of T. rubrum and subsequently treated with the device are reported in the Table 2. T. rubrum strain was eradicated, with absence of growth after 3 weeks. HPCH solution was devoid of any effect in this experiment, as the growth of T. rubrum was similar to that of untreated controls. Test product MEAN RING* (mm) AFTER DAYS Growth after transplant on day ** 14** 21** Myfungar Control HPCH solution * = mean of 4 values + = growth; = no growth ** the presence or absence of fungal growth was assessed 3 weeks after transplant Table 1. Prevention of in vitro nail experimental infection by T. rubrum. Test a) application of test products before nail contamination. Weeks after contamination Growth* after transplant days Myfungar Control HPCH solution *fungal growth in the transplanted Petri dish Table 2. Prevention of in vitro nail experimental infection by T. rubrum. Test b) application of test products 1, 2 or 3 weeks after nail contamination. 11

11 A. Sparavigna, M. Setaro, L. Frisenda D iscussion Common cosmetic or medicated nail varnishes based on water insoluble polyvinyl or monoester resin films have the disadvantage that the removal of the nail varnish by an organic solvent or by nail filing further damages the nail structure and renders the nail keratin less resistant to the fungal infections. This may be one of the reasons for a lower than expected efficacy rate of traditional antifungal nail varnishes in the management of onychomycosis. The new hydrolacquer technology developed by Polichem overcomes the problem evidenced by the polyvinyl resin film, by employing an innovative film forming agent, hydroxypropylchitosan (HPCH), which has the characteristic of being an hydrosoluble amino-polysaccharide. Applied as an hydroalcoholic solution in a series of in vitro and in vivo investigational studies, the solution quickly evaporates, by forming a thin film that demonstrates unique properties of affinity and selective adhesiveness to the keratin structure of the nail. The film appears to penetrate into the keratin holes and to smoothen the uneven keratin surface, by physically supporting the nail and by protecting it against mechanical and other physical agents. The physical support and the protective activity of HPCH stays in place and it is not vanished by the removal procedures. In fact, contrary to the traditional nail laquers, the HPCH film can be removed just by water and care should be used to leave it on the nails long enough to allow its action (for example, by applying it in the evening before bedding or after shower on dry nails and avoiding washing for 6 hours). M o re o v e r, in our experience, the film formed after the evaporation of the medical device was able to prevent the growth of the most common nail pathogens within and around the nail in experimental infection models. According to liter a t u re data, 7 the selective affinity for keratin results in an intimate contact of HPCH with the nail surface that allows a better passive diff u s i o n of ingredients to the nail compared to common medicated nail lacquers. Our data on the microbiological properties of the new medical device a re in full agreement with the a.m. report. In conclusion, our data show that the new medical device, when applied on the nails, is capable to form a film, that adheres and penetrates into the nail structure, by supporting it and forming a p rotective film against physical and micro b i o l o g i- cal agents. In particular, the device may pre v e n t the nail infections by common pathogens such as T. mentagro p h y t e s and T. ru b ru m. Furthermore, it is easy to apply and does not re q u i re specific removal, characteristic that may improve the p a t i e n t s compliance to tre a t m e n t. R eferences 1. Dawber RPR, de Berker DAR, Baran R. Science of the nail apparatus. In: Baran and Dawber s Disease of the nails and their management, 3 rd E d. Blackwell Sciencie Tosti A, Baran R., et al. Onychomycosis and its treatment. In: Baran, R. et al., editors. A text atlas of nail disorders. Techniques in investigation and diagnosis, 3 rd Edn. London: Martin Dunitz 2003, p Legora M, Mailland F, Mechanism of adherence to the nail surface of a film formed by water soluble chitosan. Proc. 16 th Congress EADV, Vienna, May Monti D, Saccomani L, Chetoni P, Burgalassi S, Saettone M F, Mailland F. In vitro transungual permeation of ciclopirox from a hydroxypropyl-chitosan-based, water-soluble nail lacquer. Drug development and Industrial Pharmacy 2005; 31:11 5. Baran R, Mailland F. Transungual delivery of drugs: new perspectives. Proc. 34th Annual ESDR Meeting, Vienna, September 9-11 t h 2004, in J Invest Dermatol 2004; 123:A74 6. INCI - International Nomenclature Cosmetic Ingredient. Monograph of Octopirox, Monti D, Saccomani L, Chetoni P, Burgalassi S, Mailland F. HPCH-based nail lacquers: ex vivo study on permeation of three antimycotics through bovine hoof membranes Proc. 5 th World Meeting on Pharmaceutics and Pharmaceutical Technology, Geneva (Switzerland), March 2006 A cknowledgements We gratefully acknowledge the cooperation of the following Scientists: Dr. Daniela Monti, Dr. Luigi Saccomani - Dept. Biorganic Chemistry, Univ. Pisa (Italy) - for kindly providing the bovine nail slices; Prof. Francesco Dubini and Dr. Maria Grazia Bellotti - Institute of Microbiology - Univ. Milan (Italy) - for the investigations on experimental onychomycosis; Dr. Alessandra Frangi - Microbiological Lab. IPAS Ligornetto (Switzerland) - for the other microbiological investigations. 12

12 The use of antisense oligonucleotides in skin lightening products Melizza Bautista 1 Falen Lockett 1 Jaimie Mecca 1 Wanphimon Sawatdeekhachornphat 1 Angelica Castro 1 Sujani Ya r l a g a d d a 1 Salvador Gonzalez 2 Neena Philips 1 SU M M A R Y The use of antisense oligonucleotides in skin lightening products Developments in gene sequencing, safety, specificity and simplicity of the concept have resulted in the investigation and development of antisense oligonucleotides as therapeutic agents. Most current skin lighteners work to inhibit tyrosinase to deactivate melanin synthesis, leading to lighter and brighter skin. Antisense oligonucleotides work by interfering with the gene expression of tyrosinase, and the production of melanin ceases as a result. Topical application of antisense oligonucleotides is effective on pigmented spots and non-pigmented skin. Antisense technology is in its early stages and additional trials with proper controls should be conducted to ensure efficacy, proper specificity, and safety. KE Y W O R D S: Melanogenesis, Anti-sense oligonucleotides, Brightening, Cosmetics 1 School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, USA 2 Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA B ackground Many women who seek affordable and safer cosmetic remedies for skin lightening look to topical skin lightening creams and soaps as alternates to invasive laser and chemical peel treatments. These products have become increasingly popular amongst women of color, particularly in Asian countries, where those with lighter skin are perceived as more youthful, successful and attractive. Many relate this shared cultural ideology to historical times when darker skin was associated with being a laborer, who worked outdoors in fields or farms, and lighter skin to upper classmen who enjoyed their privileges indoors. 1 Women not only look to skin lighteners for a brighter, youthful appearance, but also to correct the uneven appearance of dark spots or blotches that occur on the skin. Hyperpigmentation can be triggered by an array of factors that include genetic predisposition (as in freckles), environmental stress (as in solar lentigines, age spots caused by UV exposure), and hormonal fluctuations as melasma. 5 Skin lighteners help to even skin appearance and improve self-perception. Skin color is primarily due to the pigment, melanin, in the skin. Yet, the determinant of skin color also considers factors such as the thickness of the skin and the presence of pigments such as carotene which, in excess, contributes an orange-yellow tint. The density and dilation of blood vessels as well as the oxygen content in the blood also play a role in how pink or red the skin appears in contrast to a bluish hint when oxygen levels are deficient. Ultimately, it is the active degree of melanogenesis that determines how dark or light skin becomes. Melanin exists as two main types in the skin, eumelanin, which is responsible for the black or brown pigmentation, and pheomelanin, which gives yellow or red hues seen as red heads. Dark skinned people produce more melanin than do light skinned people. 5 Synthesis of melanin occurs within melanocytes in the stratum basale of the epidermis. Melanocytes are stimulated to produce melanin in response to UV radiation. Melanin is stored within melanosomes that are transferred to other cells (keratinocytes) via microtubules and actin filaments to protect the nucleus of other cells from UV damage. 3 Melanogenesis is regulated by the enzyme tyro s i- nase, tyro s i n a s e - related protein TRP1 and TRP-2. Ty rosinase regulates the hydrolysis of tyrosine to form L-dihydroxyphenylalanine (L-DOPA ) a n d further dehydroxylation to dopaquinione. 17

13 M. Bautista, F. Lockett, J. Mecca, W. Sawatdeekhachornphat, A.Castro, S. Yarlagadda, S. Gonzalez, N. Philips Dopaquinone combines with cysteine to produce pheomelanins or is converted to dopachrome, which, through TRP-1 and TRP-2, produce eumelanins that leads to tanning. 2,4 Current methods of skin whitening, via tyrosinase inhibition, include the use of hydroquinone (HQ), natural extracts such as arbutin, exfoliants such as AHA s, and intense pulse light (IPL). 6,7 HQ is found in common foods such as wheat, berries, coffee and tea. HQ inhibits tyrosinase, by interfering with copper binding and other possible mechanisms include selective cytotoxicity of melanocytes with melanosome degradation, inhibition of melanin synthesis. Information is available on the reproductive, development or carcinogenic effects of HQ in humans. EPA however has not classified HQ for carcinogenicity. Common side effects are redness, mild burning, and itching. Arbutin is an extract from cranberries, blueberries, and bearberry plant. Arbutin competitively and reversibly inhibits tyrosinase without affecting RNA melanin synthesis. Also, Arbutin inhibits melanosome maturation and is less cytotoxic to melanocytes in comparison to HQ. It is a very safe skin agent for external use without unpleasant odor or side effects. AHA preparations are commonly found with glycolic acid (GA) 5%- 20% and lactic acid (LA) 8%-12%. Both are effective and safe peeling agents in epidermal melasma 6. AHAs have been used for centuries to treat dry skin, acne, actinic damage, and improve skin texture, color, and wrinkles. AHA suppresses melanin activity by directly inhibiting tyrosinase without affecting mrna and protein expression. IPL photo facial is a new way to improve skin. Unlike a laser which emits one specific wavelength of light, IPL emits a broad spectrum of light with each pulse. The broad spectrum of light in each pulse allows it to treat a variety of skin imperfections simultaneously. IPL penetrates skin and attacks the root of skin blemishes and imperfections. The treatment side effects are mild swelling after treatment. 7 rawbacks of current topical D depigmenting agents Antisense oligonucleotides are small and welldefined synthetic single-stranded nucleic acid fragments which are synthesized to bind to the messenger RNA (mrna) of a targeted gene. When the antisense sequence binds to the mrna sequence, it prevents synthesis of a protein. 4 This synthesized nucleic acid is termed an anti-sense oligonucleotide because its base sequence is complementary to the gene s messenger RNA (mrna), which is called the sense sequence. They are different from conventional drugs in the respect that they are designed to act upstream by preventing the translation of mrna into proteins instead of interacting with protein molecules after they are produced. 8 The use of antisense oligonucleotides as therapy was first introduced by Z a m e c n i k and S t e p h e n s o n in 1978 who synthesized a 13-nucleotide oligonucleotide complement to the terminal sequences of Rous sarcoma virus 35S RNA, which i n t e r f e red with viral pro d u c t i o n. 4 As a result of the specificity to a targeted gene that can be c reated antisense therapy has presented itself to being applicable in various fields including the cosmetic products and specifically skin lightening where effective actives are few. 9 Most skin-lightening or depigmenting agents such as kojic acid, arbutin, ferulic acid, hydroquinone, guaiacol, and resorcinol reduce or block melanin production by inhibiting tyrosinase, which catalyzes the production of melanin by oxidation. Antisense oligonucleotides inactivate the gene information by binding to the messenger RNA so that translation cannot occur, halting the production of tyrosinase formation. 10 Also many of the skin lightening agents are unstable, moderately irritating to the skin, and potentially toxic because a high concentration must be used for perceptible eff e c t i- veness. Antisense oligonucleotides offer unprecedented specificity, biological stability, eff i c i e n t uptake and accumulation in cells by liposome encapsulation, for skin lightening cosmetic prod u c ts 4. The FDA has approved the sale and distribution of the first antisense oligonucleotides to treat cytomegalovirus retinitis, Vitravene. 9 The approval of Vitravene, no doubt, provides encouragement to extend the antisense technology to cosmetic products such as skin lightening products. LVMH Recherche of Christian Dior Parfums has conducted in vitro and in vivo clinical studies of a cosmetic product to evaluate the effect of antisense oligonucleotides on human melanogenesis. LVMH claims this report to be the first with a positive result in a cosmetic product based on antisense therapy. 4 The synergistic combination of TRP-1 and 18

14 The use of antisense oligonucleotides in skin lightening products PKC-[beta]I antisense oligonucleotides led to increased inhibition of the tyrosinase enzyme s activity on human melanocytes and an observed in vivo skin lightening effect in both pigmented spots and nonpigmented areas. 4,11 This result is encouraging for the expanded use of antisense technology in cosmetics. 4 iscussion/future directions D of antisense technology The challenge to antisense technology is that it is still in the early stages and additional trials are needed to ensure safety, efficacy and specificity. The clinical data obtained is promising. The use of phosphorothioates and the therapeutic effects they are thought to induce should be examined critically because their backbone can cause sequence-independent effects. New chemical modifications of oligonucleotides are being developed that address the issue of degradation by nucleases and would prevent the formation of degradation products with cytotoxic potential. 9 A cknowledgement The paper composes Biochemistry/ Microtoxicity course in the Cosmetic science program. R eferences 1. Wadyka S. Trouble Spots Got You Down. Lighten Up. The New York Times. 2005; (www. n y t i m e s. c o m. Accessed on 2007 Dec 12) 2. Behrooz K. Melanin Biosynthesis Pathway and the Depigmenting Effect of Retinoids. Jahrom Univ of Med Sciences. 2005; W8.4:1( accessed on 2007 Dec 12) 3. Mitsunori F. Elucidation of Melanin Tr a n s p o r t Mechanism, A Fresh Tu rn in Membrane Tr a ff i c k i n g Research. 2005; 292. ( accessed on 2007 Dec 12) 4. Lazou K, Sadick NS, Kurfurst R, Bonnet-Duquennoy M., Neveu M., Nizard C, Heusele C, Schnebert S, Perrier E. The use of antisense strategy to modulate human melanogenesis, J of Drugs in Derm. 2007; 1-6. ( accessed on 2007 Dec 12) 5. Shai A, Maibach H, Baran R. Handbook of Cosmetic Skin Care. 1. Ed1: Martin Dunitz Ltd Bansal SB, Draelos ZD. Insight into skin lightening cosmeceuticals for women of color. J. of Drugs in Derm. 2007; 6: Purcell E, Condon C. Intense pulsed light therapy in the management of hereditary benign telangiectasia. Br J Plast Surg. 2004; 57: Weiss B. Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharma and Thera Agents, CRC Press Dias N, Stein CA. Antisense Oligonucleotides: Basic Concepts and Mechanisms. Mole Cancer Thera. 2002; 1: Uwe S, Max H, Hearing VJ. Cosmetic or derm a t o l o g i c a l p reparations comprising oligopeptides for lightening the skin of age marks and/or for preventing tanning of the skin, in particular tanning of the skin caused by UV radiation. 2001; ( h t t p : / / w w w. p a t e n t s t o rm.us. accessed on 2007 Dec 12) 11. Kurfurst R, Duquennoy MB, Lazou K, Decup L, Nizard.C, Schnebert S. Role and modulation of tyrosinase/tyrosinase related protein-1 complex and PKC beta-i in melanogenesis. Intern J of Cosmetics, 2005; 27:

15 Coating on micronized titanium dioxide increases safety and maintains efficacy as sunscreen filter Jayson Goodner 1 Uma Pa t i l 1 Yousun Lim 1 Sujani Ya r l a g a d d a 1 Angelica Castro 1 Salvador Gonzalez 2 Neena Philips 1 SU M M A R Y Coating on micronized titanium dioxide increases safety and maintains efficacy as sunscreen filter M i c ronized titanium dioxide (Ti O 2 ) is a widely used sunscreen ultraviolet (UV) radiation filter. The penetration of micronized Ti O 2 into the dermis, and its photocatalytic activity leading to generation of reactive oxygen species is a widespread concern. The coating of Ti O 2 with antioxidants and polymers reduces or eliminates photocatalytic activity. Further, chemical grafting of anti-oxidant molecules with an additional hydrophobic polymer coating directly onto Ti O 2 particle surfaces eliminates photocatalytic degradation while maintaining effective screen against UV radiation. KE Y W O R D S: M i c ronized titanium dioxide, Sunscreen filter, UV radiation itanium nanotechnology T for UV radiation protection 1 School of Natural Sciences, University College, Fairleigh Dickinson University, Teaneck, NJ, USA 2 Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA Consumers that use sunscre e n s without titanium dioxide (TiO 2 ) are exposed to more UV radiation than consumers relying on titanium products for sun protection. Consumers using sunscreens without titanium are exposed to an average of 20% more UVA radiation and increased risks for UVA radiationinduced skin damage, premature aging, wrinkling, and immune system damage. Despite the benefits of using titanium based sunscreens, there is concern regarding the possible absorption of micronized titanium particles into the dermal layer of the skin. The technology for micronzing titanium is a recent development and the health risks associated with them have not been fully researched. The benefit from TiO 2 for UV radiation protection needs to be balanced against concerns that nanoparticles may be unusually toxic to body systems. The risks of Ti O 2 a re based on its sunscre e n p roperties: the high surface reactivity of tiny particles and their ability to penetrate body tissues. The potential risks raise two key questions: (1) Does micronized titanium dioxide penetrate the dermis; (2) does it damage cells due to its photocatalytic activity. Prior to the 1990, larger titanium particles were used that left white tints/residues and did not adhere to skin. The development of nano-sized TiO 2 was primarily based on consumer feedback on the whitening effect that conventional titanium particles produced. Curre n t l y, the typical size range for titanium in sunscreens is nm. At these sizes titanium leaves a lesser whitish tint and forms a smoother barrier on skin. 1 Nanoparticles are currently widely used in sunscreens but they are rarely noted on product labels. There is evidence that the smaller particle titanium offers improved UV protection compared to conventional-sized counterparts as well 2. An estimated 1,000 tons of nanoparticles were used in sunscreen worldwide during Alternate UVA radiation sunscreen chemicals are zinc oxide, avobenzone and Mexoryl SX. Of all current sunscreen chemicals, 21

16 J. Goodner, U. Patil, Y. Lim, S. Yarlagadda, A. Castro, S. Gonzalez, N. Philips titanium dioxide offers the best UVA radiation protection. itanium toxicity T and skin penetration The primary toxicity concern of nanotitanium particles is free radical generation leading to oxidative stress and inflammation; that damages proteins, lipids and DNA. 4, 5 Ti t a n i u m has also been shown to induce oxidative stress in tissues, especially when catalyzed by UV light. In addition, nano-titanium particles, extracted fro m s u n s c reens, on skin are activated by UV light to generate reactive oxygen species damaging skin DNA and cell structure s. 6 Titanium hydro x y l radicals produced by UV radiation facilitate DNA and cell damage Diverse coatings, such as magnesium and various polymers, greatly re d u c e UV radiation reactivity of nano titanium 11, with m o re recent technology showing that chemical grafting of anti-oxidant molecules and polymers d i rectly onto titanium particles eliminates its photocatalytic degradation. 12 T h e re are no reports on the absorption of small-scale titanium s u n s c reen ingredients through healthy/intact skin or damaged skin In contrast, traditional suns c reens like oxybenzone and octinoxate absorb into healthy skin, and by acting like estro g e n s raise risks for breast cancer, and hormone-driven uterine damage. 17 C onclusion The current weight of evidence suggests that nano titanium does not penetrate the skin. The advancements and additions to nanotechnology makes titanium-based formulations among the safest, most effective sunscreens on the market. A cknowledgement The paper composes Biochemistry/Microtoxicity course in the Cosmetic Science pro g r a m. R eferences 1. Nohynek GJ, Lademann J, Ribaud C, et al. Grey goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol 2007; 37: Popov AP, Priezzhev AV, Lademann J, et al. TiO 2 nanoparticles as an effective UV-B radiation skin-protective compound in sunscreens. Journal of Physics D: Applied Physics 2005; 38: Borm PJ, Robbins D, Haubold S, et al. The potential risks of nanomaterials: a review carried out for ECETOC. Part Fibre Toxicol 2006; 3:11 4. Nel A, Xia T, Madler L, et al. Toxic potential of materials at the nanolevel. Science 2006; 311: Oberdorster G, Maynard A, Donaldson K, et al. Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Part Fibre Toxicol 2005; 2:8 6. Hidaka H, Kobayashi H, Koike T, et al. DNA Damage Photoinduced by Cosmetic Pigments and Sunscreen Agents under Solar Exposure and Artifical UV Illumination. J Oleo Sci 2006; 55: Dunford R, Salinaro A, Cai L, et al. Chemical oxidation and DNA damage catalysed by inorganic sunscreen ingredients. FEBS Lett 1997; 418:87 8. Uchino T, Tokunaga H, Ando M, et al. Quantitative determination of OH radical generation and its cytotoxicity induced by TiO (2) -UVA treatment. Toxicol In Vitro 2002; 16: Sayes CM, Wahi R, Kurian PA, et al. Correlating nanoscale titaniam structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicol Sci. 2006; 92: Wang JJ, Sanderson BJ, Wang H. Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells. Mutat Res 2007; 628: Wakefield G, Lipscomb S, Holland E, et al. The effects of manganese doping on UVA absorption and free radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing organic sunscreen components. Photochem Photobiol Sci 2004; 3: Lee WA, Pernodet N, Li B, et al. Multicomponent polymer coating to block photocatalytic activity of Ti O 2 nanoparticles. Chem Commun 2007; Baroli B, Ennas MG, Loffredo F, et al. Penetration of Metallic Nanoparticles in Human Full-Thickness Skin. J Invest Dermatol 2007; 127: Cross SE, Innes B, Roberts MS, et al. Human Skin Penetration of Sunscreen Nanoparticles: In-vitro Assessment of a Novel Micronized Zinc Oxide Formulation. Skin Pharmacol Physiol 2007; 20: Gamer AO, Leibold E, Van-Ravenzwaay B. The in vitro absorption of microfine zinc oxide and titanium dioxide through porcine skin. Toxicol In Vitro 2006; 20: Lademann J, Weigmann H, Rickmeyer C, et al. Penetration of titanium dioxide microparticles in a sunscreen formulation into the horny layer and the follicular orifice. Skin Pharmacology and Applied Skin Physiology 1999; 12: Schlumpf M, Cotton B, Conscience M, et al. In vitro and in vivo estrogenicity of UV screens. Environ Health Perspect 2001; 109:239 22

17 Studio prospettico sull evoluzione delle ferite chirurgiche trattate con gel al silicone Vincenzo De Giorgi Serena Sestini Barbara Alfaioli Marta Grazzini Agata Janowska Andrea Saggini Torello Lotti SU M M A R Y Study on the effectiveness of a silicone gel in treating surgical wounds The management of scars originating either from surg e ry or trauma is of notable significance in preventing the formation of evident scarring. What matters nowadays is not only addressing the functional alterations caused by these scars, but also the minor esthetic alterations that can cause serious psychological problems for patients. The s u rgeon is thus becoming more and more involved, beyond the surgical operation itself, in managing esthetic results and can deploy a range of therapeutic pro c e d u res to minimize the formation of prominent scars. This is also true in view of the incre a s i n g demand for purely aesthetic surg e ry ranging from the removal of common seborrh e i c keratoses, to blepharoplasty or major breast re c o n s t r u c t i o n. The aim of our study was thus the evaluation of the effectiveness of a silicone gel (Zeraderm ultra silicone gel ) in treating surgical wounds compared with a contro l g roup of the same phenotype and same scar site for which no product was advised. We evaluated the minimum aesthetic d a m a g e following surg e ry, in particular. T h e re f o re, not only we considered the formation or absence of the classic keloid or h y p e r t rophic scar, but above all whether the early application of the product on the wound led to, compared to the control group, the formation of more acceptable and minimum a e s t h e t i c damage even within the parameters of so-called p h y s i o l o g i- c a l scars. KEY WORDS: Silicone gel, Surgical wounds Dipartimento di Scienze Dermatologiche, Università di Firenze I ntroduzione La gestione di ferite, sia chirurgiche, sia traumatiche, assume un importanza notevole al fine di evitare la formazione di cicatrici particolarmente evidenti. Al giorno d oggi infatti sono diventate estremamente importanti, non soltanto le alterazioni funzionali che tali cicatrici possono provocare, ma anche piccole alterazioni estetiche, che portano in alcuni pazienti gravi problematiche psicologiche. È sempre più compito e dovere del chirurgo occuparsi, al di là del vero e proprio intervento, anche dei risultati estetici di tale intervento e mettere in atto tutte le procedure terapeutiche al fine di minimizzare la formazione di cicatrici evidenti. Tutto ciò anche vista la continua e crescente richiesta di interventi a fini puramente estetici, dall asportazione di una banale cheratosi seborroica, ad una blefaroplastica e ad una importante ricostruzione mammaria. Da un analisi della letteratura ci accorgiamo che, mentre è unanimemente descritta e riconosciuta la cicatrice cheloidea ( cicatrice che si estende oltre il tessuto danneggiato e ricopre i tessuti normali ), non vi è consenso su quando la cicatrice sia da considerarsi normale e fisiologica e quando invece debba essere considerata ipertrofica. Questa confusione genera spesso negli stessi chirurghi una gestione inappropriata delle ferite, che per la maggior parte dei casi 25

18 V. De Giorgi, S. Sestini, B.Alfaioli, M. Grazzini, A. Janowska, A. Saggini, T. Lotti si estrinseca in un non trattamento della ferita, una volta avvenuta la rimozione dei punti. È infatti esperienza comune sentire il detto l a cicatrice la fa il paziente. Tale enunciato, che ha in parte anche delle giuste basi scientifiche, poteva essere appropriato qualche decennio fa, ma attualmente non ha più giustificazione. Infatti il trattamento e la prevenzione delle cicatrici si caratterizza oggigiorno per l ampia varietà di terapie e tecniche utilizzate. Molti di questi trattamenti si sono affermati a seguito di una ampia diffusione nel corso degli ultimi anni, m e n t re solo una minoranza risulta eff e t t i v a m e n- te supportata da studi prospettici che abbiano incluso dei gruppi di controllo adeguati. Le valutazioni sull efficacia sono state ulteriormente limitate dalla difficoltà di quantificare le modificazioni obiettive delle lesioni cicatriziali e dal fatto che le cicatrici tendono naturalmente a migliorare nel tempo. 1 L utilizzo del silicone in varie forme ha rappresentato un opzione per il trattamento delle cicatrici a partire dall inizio degli anni 80. Esistono in letteratura più di dieci studi randomizzati e controllati, che dimostrano come l utilizzo del silicone costituisca una scelta terapeutica sicura ed efficace per le cicatrici cheloidee. 1-8 Inizialmente erano utilizzate piastre al silicone, che potevano risultare scomode per i pazienti, anche a seconda delle sedi della ferita, e quindi mal tollerate. 4 L impiego, invece, di prodotti in gel a base di silicone risulta estremamente comodo, raggiungendo una buona compliance da parte del paziente. 9 Lo scopo dello studio è stato la valutazione dell efficacia di un gel al silicone (Zeraderm ultra gel ) nel trattamento delle ferite chirurgiche rispetto ad un gruppo di controllo con stesso fenotipo e stessa sede cicatriziale, a cui non è stato consigliato alcun prodotto. In particolare è stato valutato il minimo danno estetico residuato all intervento chirurgico. Non è stato quindi giudicato soltanto la formazione o non del classico cheloide o della cicatrice ipertrofica, ma soprattutto se l applicazione precoce del prodotto sulla ferita si estrinsechi, rispetto al gruppo di controllo, con la formazione di un migliore e minimo danno estetico anche nell ambito della cicatrice cosiddetta fisiologica. Inoltre è stata valutata la compliance del paziente rispetto allo stesso prodotto, valutando la presenza di dolore, prurito, senso di presenza della ferita in corso di cicatrizzazione. ateriali e metodi M Sono stati inclusi nello studio 110 pazienti (55 di sesso maschile, 55 di sesso femminile) (Tabella 1), sottoposti ad interventi di d e r m o c h i r u rgia ambulatoriale presso il Dipartimento di Scienze Dermatologiche dell Università di Firenze, nel periodo maggio-luglio I suddetti pazienti sono stati divisi in due gruppi: un gruppo di studio ed un gruppo di controllo. Tutti i pazienti sono stati sottoposti ad interventi di escissione chirurgica di lesioni cutanee melanocitarie (nevi melanocitici, melanomi), lesioni cutanee neoplastiche non melanocitarie (carcinoma basocellulare, carcinoma spinocell u l a re, angiocheratoma, tumori annessiali), lesioni cutanee benigne (cheratosi seborroiche, lipomi). Sono stati esclusi dallo studio i pazienti con escissioni chirurgiche di dermatofibromi, di cisti sebacee e di lesioni cutanee in flogosi. I pazienti arruolati presentavano il seguente fototipo secondo Fitzpatrick: 5% tipo I, 21% tipo II, 47% tipo III, 27% tipo IV. Tutti i casi sono stati operati dallo stesso chirurgo mediante lama fredda (VDG) e sono stati utilizzati gli stessi materiali per la sutura e per la N Età Mediana Range Lesioni Lesioni Lesioni Lesioni pazienti media età Arti sup. Arti inf. Tronco Volto (%) (%) (%) (%) Gruppo di studio % 20% 38% 17% (33 m, 32 f) Gruppo di controllo % 24% 34% 12% (22 m, 23 f) Tabella 1. Caratteristiche dei pazienti. 26

19 Studio prospettico sull evoluzione delle ferite chirurgiche trattate con gel al silicone a b c Figura 1. medicazione. A tutti i pazienti è stato detto di sospendere l attività sportiva per un periodo di 4 settimane. Al gruppo di studio (65 pazienti) è stato prescritto l uso di gel al silicone da applicare sulla ferita due volte al giorno per 60 giorni dopo la rimozione dei punti di sutura. Invece al gruppo di controllo (45 pazienti) non è stata prescritta alcuna terapia preventiva. Tutti i pazienti, sia del gruppo di studio, sia del gruppo di controllo, sono stati visitati a cadenza mensile per i primi tre mesi dall intervento e successivamente ogni 2 mesi per un follow-up complessivo di 8 mesi dalla data dell intervento, sempre dagli stessi dermatologi (VDG, SS) al fine di mantenere una valutazione riproducibile nei vari pazienti. Durante ogni visita di controllo è stata effettuata una documentazione iconografica ed è stata valutata l evoluzione della cicatrice nel tempo. In particolare è stato utilizzato, un videocapillaroscopio digitale per verificare la precoce comparsa sulla cicatrice di vascolarizzazione, testimoniata dalla presenza di teleangectasie. I n o l t re è stata valutata la presenza di allodinia e/o di p rurito evocato mediante l utilizzo di un fine pennello, sfruttando un test già utilizzato in neurologia per la valutazione delle pare s t e s i e. L induzione del dolore e/o prurito è stata scatenata attraverso il delicato movimento del pennello sulla cute, partendo a circa 5 cm dalla cicatrice (cute pericicatriziale) e venendo poi a spostarsi lentamente in direzione centripeta, verso il cent ro della cicatrice. Per ogni paziente il test è stato e ffettuato almeno 3 volte e la presenza di are e nelle quali il paziente sentiva dolore e/o prurito sono state marcate con una penna dermografica. Il paziente è stato istruito di informare l investig a t o re alla prima sensazione di dolore o di prurito provata. Le aree alle quali il pennello elicitava una risposta sono state marcate con una penna v e rde quando il paziente riferiva prurito e con una penna rossa quando provava dolore. A tutti i pazienti, ad ogni controllo, è stato chiesto di quantificare su una scala tarata da 0 a 10, il peggiore dolore e/o prurito provato nell ulti- 27

20 V. De Giorgi, S. Sestini, B.Alfaioli, M. Grazzini, A. Janowska, A. Saggini, T. Lotti mo mese a livello cicatriziale/pericicatriziale. La severità della sensazione variava da nessun dolore/prurito alla fine della scala (severità = 0), al massimo dolore/prurito all altro estremo della scala (severità = 10). Ai pazienti è stato anche chiesto di riferire eventuali altre sensazioni o sintomi associati alla cicatrice ed eventuali altri trattamenti effettuati durante il periodo di osservazione, così da escludere dallo studio i pazienti che abbiano iniziato terapie influenzanti la cicatrizzazione (ad es. terapia cortico-steroidea), come anche i pazienti che non abbiano rispettato il protocollo di studio (2 applicazioni di prodotto al giorno per 60 giorni). R isultati Gruppo di Studio Tutti i 65 pazienti (33 maschi e 32 femmine) con un età media di 52 anni (range 26-81) che sono stati arruolati, hanno portato a termine lo studio. In 16 pazienti le lesioni sono localizzate a livello degli arti superiori (braccio, avambraccio e mano), in 13 pazienti a livello dell arto inferiore (coscia, gamba e piede), in 11 pazienti al volto (Figura 1) e nei rimanenti le lesioni sono localizzate a livello del tronco. In 18 pazienti (27%) abbiamo avuto la formazione di una cicatrice non fisiologica (Tabella 2). In particolare, in 10 pazienti (15%) abbiamo avuto una cicatrice diastasica, in 6 pazienti (9%) una cicatrice ipertrofica e in 2 pazienti (3%) una cicatrice atrofica. Non abbiamo invece registrato cicatrici cheloidee. I casi in cui abbiamo avuto una cicatrice diastasica erano localizzati agli arti inferiori in 6 casi e a livello del tronco nei rimanenti 4. Le cicatrici localizzate agli arti inferiori interessavano in 4 casi pazienti con un età superiore ai 65 anni, che all esame obiettivo presentavano i segni clinici di una lieve-moderata insufficienza venosa. Invece, le cicatrici ipertrofiche hanno interessato in 4 casi pazienti di età inferiore ai 48 anni, ed erano localizzate in 3 casi a livello sternale, in 2 casi in regione deltoidea e in 1 caso a livello addominale. Un paziente con cicatrice atrofica era diabetico. L aspetto clinico delle cicatrici mostrava in 10 pazienti (15%) un eritema perilesione ed in 8 pazienti (12%) la presenza di teleangectasie, monitorizzate mediante videocapillaro s c o p i o digitale. Tredici pazienti (20%) hanno riferito la presenza di dolore durante la fase di cicatrizzazione, la severità di tale sensazione era lieve-moderata, alla scala tarata variava da 3 a 5, alcuni pazienti parlavano di sensazione di fastidio, più che di un vero dolore. Invece, sei pazienti (9%) hanno riferito la presenza di prurito, di severità variabile da 3 a 6 alla scala tarata. Al test di stimolazione con un fine pennello, 12 pazienti (18%) hanno riferito la presenza di parestesie, in particolare prurito e dolore, il primo più frequentemente in sede pericicatriziale, mentre il secondo era prevalente al centro della cicatrice (Tabella 3). Da sottolineare che il lieve dolore era sempre riferito dai pazienti con cicatrice ipertrofica. Nessun paziente ha mostrato effetti collaterali all applicazione del gel al silicone. Il 30% dei pazienti si è lamentato del costo del prodotto. Gruppo di controllo Nei 45 pazienti (22 maschi e 23 femmine) del gruppo di controllo abbiamo rilevato una cicatrice alterata in 25 pazienti (55%) (Tabella 2). In particolare abbiamo registrato la formazione di cicatrici cheloidee in 5 pazienti (11%), cicatrici ipertrofiche in 10 pazienti (22%), cicatrici diastasiche in 8 pazienti (18%) e cicatrici atrofiche in 2 pazienti (4%). Le cicatrici cheloidee erano localizzate in 1 caso a livello di un arto superiore, in 2 casi a livello toracico e in 2 casi a livello deltoideo. Alterazioni Cicatrici Cicatrici Cicatrici Cicatrici cicatrice cheloidee ipertrofiche diastatiche atrofiche Gruppo di studio 27% 0% 9% 15% 3% Gruppo di controllo 55% 11% 22% 18% 4% Tabella 2. Caratteristiche cicatrici. 28