Mesotherapy for skin rejuvenation: assessment of the subepidermal low-echogenic band by ultrasound evaluation with cross-sectional B-mode scanning
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1 Dermatologic Therapy, Vol. 21, 2008, S1 S5 Printed in the United States All rights reserved Blackwell Publishing Inc 2008 Wiley Periodicals, Inc. DERMATOLOGIC THERAPY ISSN Mesotherapy for skin rejuvenation: assessment of the subepidermal low-echogenic band by ultrasound evaluation with cross-sectional B-mode scanning FRANCESCO LACARRUBBA, AURORA TEDESCHI, BEATRICE NARDONE & GIUSEPPE MICALI Dermatology Clinic, University of Catania, Catania, Italy ABSTRACT: Skin-targeted ultrasound is a noninvasive technique that has been extensively used to evaluate age-related dermal changes, and the presence of a subepidermal low-echogenic band (SLEB) has been related to chronic UVR exposure in several studies. Since SLEB echogenicity is photoage-related, the aim of this study was to evaluate, through ultrasound imaging, the effects on skin photoaging of mesotherapy, a treatment approach currently used in cosmetic dermatology for skin rejuvenation. Twenty women (mean age: 46.7 range years) with physical signs of moderate photoaging on the dorsum of the hands were enrolled and treated with multiple microinjections of hyaluronic acid (HA) salts of biotechnological origin (1.000 Kd) every week for 4 weeks. In all subjects, ultrasound evaluation was performed at each visit and 1 week after the last treatment to evaluate SLEB echogenicity changes during treatment. At the end of study, a statistically significant (p < 0.001) increase of SLEB echogenicity (with a mean increase of pixel numbers equal to 31.3%) was observed in 15 of 19 subjects who completed the study. Our preliminary study suggests that mesotherapy with HA may be an effective treatment for skin photoaging, as confirmed by ultrasound. Follow-up investigations on larger series of patients are necessary to further evaluate the safety, effectiveness, and duration of effect of this possible therapeutic approach to skin photoaging. KEYWORDS: mesotherapy, biorejuvenation, hyaluronic acid, ultrasound Introduction Address correspondence and reprint requests to: Giuseppe Micali, MD, Sezione di Dermatologia e Venereologia, Dipartimento di Specialità Medico-Chirurgiche, Università di Catania, Via Santa Sofia 78, Catania, Italy, Catania, Italy, or cldermct@nti.it. Disclosures/conflicts of interest: none declared. Skin aging includes intrinsic aging, mainly influenced by genetic, environmental, and hormonal factors, and photoaging, defined as the addition of chronic ultraviolet (UV)-induced damage to intrinsic aging, which accounts for most ageassociated skin changes (1 3). Alterations of connective tissue components, namely reduction of fibroblasts, collagen, and elastic fibers, are responsible for many of the morphologic and mechanical changes that result in skin wrinkling, sagging, loss of elasticity, and dryness (4,5). Some authors have recently proposed the term dermatoporosis to include all the aspects of aged skin, in which an important role is played by decreased hyaluronate content and expression of the major cell surface hyaluronate receptor, CD44 (3,6). Skin-targeted ultrasound (7,8) is a noninvasive technique that has been extensively used to evaluate age-related dermal changes. In 1989, some authors (9) described a low-echogenic band-like structure, termed as subepidermal low-echogenic S1
2 Lacarrubba et al. Table 1. Treatment schedule and ultrasound evaluation Week 0 (baseline) Week 1 Week 2 Week 3 Week 4 Treatment schedule X X X X Ultrasound evaluation X X X X X band (SLEB), located immediately below the epidermal entrance echo of photoaged skin. Following this observation, SLEB has further described as related to chronic UVR exposure in several studies (10 13). In addition, it has been demonstrated that SLEB echogenicity inversely correlates to chronic UV radiation exposure (10 13). Since SLEB echogenicity is photoage-related (12), the aim of this study was to evaluate through ultrasound imaging the effects on skin photoaging of mesotherapy (also called biorejuvenation, biorevitalization, or mesolift), a treatment approach currently used in cosmetic dermatology for skin rejuvenation (14 18). Materials and methods In this open-label pilot study, 20 subjects (mean age: 46.7 range years) were enrolled after giving written informed consent. Inclusion criteria were: female gender, age ranging from 40 to 65 years, and presence of physical signs of moderate photoaging on the dorsum of the hands associated with evidence of SLEB at ultrasound. Exclusion criteria were: superficial chemical peels performed within 4 weeks, history of allergic and/or irritant contact hand dermatitis, systemic disease, psychiatric illness, and pregnancy. Each patient was treated with multiple microinjections of hyaluronic acid (HA) salts (sodium chloride, chloride sodium phosphate) of biotechnological origin (1.000 Kd) (Viscoderm ); 10 subjects received the HA salts at a concentration of 16 mg/ml, and 10 subjects received 20 mg/ml, to the dorsum of both hands, every week for 4 weeks. Each treatment consisted of about 30 injections for each hand, with a maximum total injected volume of 1 ml. Patients were instructed to apply maximum protection sunscreen cream (SPF 50) on the back of their hands every day for the entire study duration. In all subjects, ultrasound evaluation was performed at each visit and 1 week after the last treatment, on an area corresponding to the second metacarpal web space of the left hand, by the same investigator under constant environmental conditions, to evaluate SLEB echogenicity FIG. 1. Ultrasound image of photoaged skin: presence of a subepidermal low-echogenic band (SLEB) located below the epidermal entrance echo. changes during treatment. Treatment schedule and ultrasound evaluation are summarized in Table 1. Cross-sectional B-mode scans were obtained with a 22-MHz ultrasound system (EasyScan Echo, Business Enterprise, Trapani, Italy). For each examined field, the amplitudes of echoes of single image elements (pixels) of the SLEB were ascribed to a numerical scale (0 255), and mean gray values were quantified with ImageJ public domain software (available at Finally, statistical analyses of data obtained before and after treatment were performed using the parametric t-test for paired samples. p-values of 0.05 were considered to be statistically significant. Results At baseline, prior to HA injections, ultrasound showed the presence of SLEB in all subjects (FIG. 1). At the same visit, after HA injections, ultrasound showed the absence of the normal structure of the skin due to the presence of a microwheal resulting from microinjections. Therefore, the ultrasound evaluation was done 1 week later just before the following HA injection. Nineteen subjects completed the study and their data were evaluated. An evident increase of SLEB S2
3 Mesotherapy and ultrasound Table 2. Results: subepidermal low-echogenic band (SLEB) echogenicity of each examined field at baseline, after 4 weeks of treatment and percentage variation SLEB echogenicity at baseline (pixels) SLEB echogenicity after 4 weeks (pixels) Variation (%) Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz Pz FIG. 2. Pt. 2: ultrasound of the dorsum of the hand. Subepidermal low-echogenic band (SLEB) evidence at baseline (a). Increased SLEB echogenicity after 4 weeks of treatment (b). echogenicity was observed in 15 of 19 subjects, with a mean increase of pixel numbers equal to 31.3% (p < 0.001) (Table 2, FIGS. 2 3). No difference was detected between the two different HA concentrations. FIG. 4 shows SLEB echogenicity changes during treatment. Discussion During the last decade, an area of increasing research has been the development of new methods to treat and/or prevent the signs of skin photoaging. In this regard, a quantitative, precise, and noninvasive evaluation of skin photoaging would be desirable for in vivo studies. Mesotherapy is mildly invasive, does not involve tissue augmentation, and consists of injection, through dermal multipunctures, of bioactive substances, such as HA in a fluid formulation (16 18). A recent expert committee conference supported treatment with native HA as a useful therapeutic option for skin photoaging (17). Mesotherapy with HA in photoaged skin aids in the restoration of S3
4 Lacarrubba et al. FIG. 3. Pt. 12: ultrasound of the dorsum of the hand. Subepidermal low-echogenic band (SLEB) evidence at baseline (a). Increased SLEB echogenicity after 4 weeks of treatment (b). FIG. 4. Results: subepidermal low-echogenic band echogenicity changes of each examined field during treatment. more complete metabolic function, restores cutaneous water retention and hydration, and reestablishes skin tone and elasticity: all serving to diminish visible signs of photoaging. HA promotes skin rejuvenation by recreating a favorable environment to facilitate fibroblast activation and to facilitate exchange and interaction between cells and within the extracellular matrix (5,14,16 18). Use of high-frequency ultrasonography has recently served to visualize and quantify age-related dermal changes. It has been shown that some tissue characteristics, in particular dermal collagen type, content, and orientation have an effect on dermal echogenicity. These factors determine the number of collagen matrix interfaces and hence the extent of sound scatter. As dermal echoes are generated at the boundaries between collagen fibers and the surrounding water-rich ground substance, changes in both the fiber and the fluid component are likely to contribute to echogenicity. In particular, elastosis and basophilic degradation of collagen are the main causes of the decrease in echogenicity and SLEB formation, as confirmed when comparing ultrasound images with histolopathologic features (10,12). S4
5 Mesotherapy and ultrasound In our study, a statistically significant increase of SLEB echogenicity was observed in 15 of 19 subjects. The ultrasound modifications we obtained are likely related to an increased density of dermal collagen fibers by fibroblast activation resulting from treatment. Interestingly, we noted that in four patients there were minimal or no changes in echogenicity, suggesting that not all patients respond uniformly to HA mesotherapy and some may do not respond at all. HA is a molecule that is involved in the skin aging process (19). It is also contained in many cosmetic products for the treatment of skin aging (4,17). Some authors suggested that with age, a progressive decrease in extracellular matrix and its major component HA (which has an important role in the stabilization of the intercellular structures via formation of a viscoelastic network together with collagen and elastic fibers) occurs, resulting in the loss of mechanical properties of skin (3). The relation between HA and its cell surface receptor CD44 has recently been evaluated by both in vitro and in vivo studies (3,6,19,20). UV irradiation significantly decreased both the content of HA and the expression of CD44 in the epidermis of hairless mice (20). Furthermore, CD44 and HA levels were found to be decreased in aging skin when compared with the skin of young individuals (19). In this regard, a defect in the interaction of HA and CD44 should be considered as a new target for the development of novel therapeutic strategies in those dermatologic conditions characterized by skin atrophy and extracellular matrix modifications (3). A recent study suggested that topical HA fragments application may provide attractive therapeutic options in the treatment of human skin atrophy by a CD44-dependent mechanism (20). Our preliminary study suggests that mesotherapy with HA may be an effective treatment for skin photoaging, as confirmed by ultrasound. The molecular weight of the product used in this study is around 1 million Daltons, very similar to the molecular weight of endogenous HA and the product is biocompatible. Follow-up investigations on larger series of patients are necessary to further evaluate the safety, effectiveness, and duration of effect of this possible therapeutic approach to skin photoaging. References 1. Yaar M, Gilchrest BA. Photoageing: mechanism, prevention and therapy. Br J Dermatol 2007: 15: Verdier-Sévrain S, Bonté F, Gilchrest B. Biology of estrogens in skin: implications for skin aging. Exp Dermatol 2006: 15: Lübeck RP, Berneburg M, Trelles M, et al. How best to halt and/or revert UV-induced skin ageing: strategies, facts and fiction. Exp Dermatol 2008: 17: Ramos-e, -Silva M, da Silva Carneiro SC. Elderly skin and its rejuvenation. Products and procedures for the aging skin. J Cosmet Dermatol 2007: 6: Kerscher M, Bayrhammer J, Reuther T. Rejuvenating influence of a stabilized hyaluronic acid-based gel of nonanimal origin on facial skin aging. Dermatol Surg 2008: 34: Kaya G, Saurat JH. Dermatoporosis: a chronic cutaneous insufficiency/fragility syndrome. Clinicopathological features, mechanisms, prevention and potential treatments. Dermatology 2007: 215: Jemec GBE, Gniadecka M, Ulrich J. Ultrasound in dermatology. Eur J Dermatol 2000: 10: Rallan D, Harland CC. Ultrasound in dermatology basic principles and applications. Clin Exp Dermatol 2003: 28: de Rigal J, Escoffer C, Querleux B, et al. Assessment of aging of the human skin by in vivo ultrasonic imaging. J Invest Dermatol 1989: 93: Gniadecka M, Jemec GBE. Quantitative evaluation of chronological ageing and photoageing in vivo: studies on skin echogenicity and thickness. Br J Dermatol 1998: 139: Gniadecka M. Effects of ageing on dermal echogenicity. Skin Res Tech 2001: 7: Sandby-Moller J, Wulf HC. Ultrasonographic subepidermal low-echogenic band, dependence of age and body site. Skin Res Tech 2004: 10: Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (I): blood flow, thickness, and ultrasound echogenicity. Skin Res Tech 2005: 11: Andre P. Hyaluronic acid and its use as a rejuvenation agent in cosmetic dermatology. Semin Cutan Med Surg 2004: 23: Verpaele A, Strand A. Restylane SubQ, a non-animal stabilized hyaluronic acid gel for soft tissue augmentation of the mid- and lower face. Aesthet Surg J 2006: 26: Lacarrubba F, Nardone B, Tedeschi A, Nordstrom R, Micali G. Ultrasound evaluation of mesotherapy for skin rejuvenation. In: Tosti A, De Padova MP, eds. Atlas of mesotherapy in skin rejuvenation. London, UK: Informa Healthcare Ltd, Wiest L, Kerscher M. Native hyaluronic acid in dermatology results of an expert meeting. J Dtsch Dermatol Ges 2008: 6: Iorizzo M, De Padova MP, Tosti A. Biorejuvenation: theory and practice. Clin Dermatol 2008: 26: Calikoglu E, Sorg O, Tran C, et al. UVA and UVB decrease the expression of CD44 and hyaluronate in mouse epidermis, which is counteracted by topical retinoids. Photochem Photobiol 2006: 82: Kaya G, Tran C, Sorg O, et al. Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism. PLoS Med 2006: 3: e493. S5
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