Ultrasonics 54 (2014) 1395–1400
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Ultrasound biomicroscopy measurement of skin thickness change induced by cosmetic treatment with ultrasound stimulation Tak-Man Mak, Yan-Ping Huang, Li-Ke Wang, Yong-Ping Zheng ⇑ Interdisciplinary Division of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
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Article history: Received 5 July 2013 Received in revised form 27 January 2014 Accepted 13 February 2014 Available online 24 February 2014 Keywords: Skin thickness Ultrasound biomicroscopy Ultrasound stimulation Lotion Hydration
a b s t r a c t Moisturizing creams and lotions are commonly used in daily life for beauty and treatment of different skin conditions such as dryness and wrinkling, and ultrasound stimulation has been used to enhance the delivery of ingredients into skin. However, there is a lack of convenient methods to study the effect of ultrasound stimulation on lotion absorption by skin in vivo. Ultrasound biomicroscopy was adopted as a viable tool in this study to investigate the effectiveness of ultrasound stimulation on the enhancement of lotion delivery into skin. The forearm skin of 10 male and 10 female young subjects was tested at three different sites, including two lotion treatment sites with (Ultrasound Equipment – UE ON) and without (UE OFF) ultrasound stimulation and a control site without any lotion treatment. 1 MHz ultrasound with a duty cycle of 1.7%, a spatial peak temporal peak pressure of 195 kPa and an average power of 0.43 W was used for the stimulation. The skin thickness before, immediately after (0 min), and 15 and 30 min after the treatment was measured by an ultrasound biomicroscopic system (55 MHz). It was found that the skin thickness significantly increased immediately after the lotion treatment for both UE ON (from 1.379 ± 0.187 mm to 1.466 ± 0.182 mm, p < 0.001) and UE OFF (from 1.396 ± 0.193 mm to 1.430 ± 0.194 mm, p < 0.001) groups. Further comparison between the two groups revealed that the skin thickness increase of UE ON group was significantly larger than that of UE OFF group (6.5 ± 2.4% vs. 2.5 ± 1.3%, p < 0.001). Furthermore, it was disclosed that the enhancement of lotion delivery by ultrasound stimulation was more effective for the female subjects than the male subjects (7.6 ± 2.3% vs. 5.4 ± 2.0% immediately after treatment, p = 0.017). In conclusion, this study demonstrated that ultrasound biomicroscopy was a feasible method for studying the effectiveness of lotion treatment in vivo, and ultrasound stimulation was effective to enhance the rate of lotion absorption into skin. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, the use of ultrasound in beauty industry has been very common and demanding all over the world, especially in well developed cities such as Hong Kong. One common application is to use ultrasound to enhance the delivery of cosmetic lotion, a method called ‘‘ultrasound massage’’. Manufacturers of ultrasound massagers often claim that the use of these devices enhances the absorption of lotion ingredients into skin. However, the effectiveness of ultrasound stimulation on enhancing the lotion delivery has been scarcely reported in the literature.
⇑ Corresponding author. Address: Interdisciplinary Division of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China. Tel.: +852 27667664; fax: +852 23342429. E-mail addresses: [email protected] (T.-M. Mak), hti.huang@ connect.polyu.hk (Y.-P. Huang), [email protected] (L.-K. Wang), ypzheng@ ieee.org (Y.-P. Zheng). http://dx.doi.org/10.1016/j.ultras.2014.02.015 0041-624X/Ó 2014 Elsevier B.V. All rights reserved.
Low weight molecules (20 MHz) ultrasound images, which can be used for the measurement of skin thickness. The measurement of skin thickness has been successfully applied in studies of skin-related pathologies such as patch test and hormonal treatment [8–11]. It is a potential method to study the effectiveness of lotion delivery with the stimulation of ultrasound. This study aims to test the hypothesis that ultrasound stimulation can enhance lotion delivery. Therapeutic ultrasound (1 MHz) was used as a stimulation method to potentially enhance the delivery of cosmetic lotion to skin, and the skin thickness before and after the treatment was quantitatively assessed by a high frequency (55 MHz) ultrasound biomicroscopy on 20 subjects (10 male and 10 female). The change of skin thickness with and without ultrasound stimulation as well as the gender dependence of such change was reported. Details of the current study are presented as follows.
2. Materials and methods 2.1. Equipment and materials An ultrasound biomicroscopic system (Vevo 770, Visualsonics Inc., Toronto, Canada) with a transducer of 55 MHz in central frequency was used for the skin imaging. The axial resolution of this transducer was approximately 30 lm, the lateral resolution was approximately 70 lm and 2-D imaging was achieved by a mechanical scanning of the transducer in a linear way. To the best of the authors’ knowledge, this equipment is one of the best known ultrasound systems available in the commercial market for high frequency ultrasound imaging of skin in vivo. The spatial resolution provided by the 55 MHz probe is suitable for studying the skin; therefore, it was adopted in the current study. A 1-MHz ultrasound stimulation device (ST-302S Multi-Function Sonic Massager, AIKO Beauty Products Ltd., Hong Kong) (Fig. 1) was used for the ultrasound stimulation in this study. The head of the device with an embedded single-element ultrasound transducer had a diameter of 48.5 mm (Fig. 1). Through measurement in water using a needle hydrophone (HNP-0400, HN-series hydrophone, ONDA Corp., Sunnyvale, CA, USA), it was found that the ultrasound transducer of the stimulation device worked in a long tone-burst mode with a repetition period of 46 ms and a pulse duration of 768 ls, i.e., a duty cycle of 1.7%. The spatial peak temporal peak pressure was 195 kPa measured at a plane 5 mm away from the probe surface, which resembled the situation of treatment on skin. The total power measured over the acoustic beam was 0.43 W for the stimulation. The cosmetic product used in this study was a very common lotion widely available in the market (Baby Simply Soft Lotion, Johnson & Johnson Hong Kong Ltd., Hong Kong). This lotion is an oil-inwater emulsion which contains mostly water and also some other minor components such as glycerin and petrolatum.
Fig. 1. A picture of the ultrasound stimulation device. The use of various parts of the device as seen in the front view was briefly introduced.
Committee and all the subjects were asked to sign an informed consent form before assessment. 2.3. Assessment protocol and experimental test The subjects were instructed not to use any cosmetics on forearms one day before the experiment. Before test, they were asked to wash both forearms with liquid soap, and were instructed to stay in the test room for at least 30 min. The room temperature (20 ± 2 °C) was controlled by air conditioners while the humidity (40 ± 2%) was controlled by a dehumidifier placed in the test room during the whole experiment. The forearms of the subjects were divided into two regions including the proximal and distal parts (Fig. 2). The proximal parts of both forearms were used as control sites where no lotion or
2.2. Subjects Ten male and ten female subjects (age: 21 ± 1 years) were recruited for test on their forearms in the study. Subjects with skin irritation, food irritation, scar, sunburn or other skin problems were excluded from the study. In addition, those who had the habit of daily use of cosmetics on forearms were also excluded. Human ethical approval was obtained from the University Ethics
Fig. 2. The two arm locations used for lotion treatment with the ultrasound stimulation device. The stainless steel head was applied to massage the skin circularly and slowly for five minutes during lotion treatment.
T.-M. Mak et al. / Ultrasonics 54 (2014) 1395–1400
ultrasound treatment was applied. The distal parts of both forearms were treated with 5 g lotion, which was weighted by an electronic balance. Then the stainless steel head of the ultrasound enhancer was gently placed on the distal part of one forearm (grouped as Ultrasound Equipment – ‘‘UE ON’’) to massage the skin for five minutes (Fig. 2). For each participant, only one forearm would be treated with ultrasound, and the other one would be massaged by moving the probe without ultrasound turned on (grouped as ‘‘UE OFF’’). Half male and half female subjects were randomly assigned to have the ultrasound massage on their left forearms and the other half subjects had the massage on their right forearms. After five minutes, the excessive lotion remained on the skin was gently removed using tissue paper to assure a fair comparison between groups. The skin thickness was measured by the 55 MHz ultrasound biomicroscopic system (1) before any treatment (initial thickness), (2) immediately after treatment (thickness at 0 min), (3) 15 min after treatment (thickness at 15 min), and (4) 30 min after treatment (thickness at 30 min). For the control sites, the initial thickness and the thickness after 30 min were measured. Before scanning, a gel pad (Ultra Phonic Focus Conforming Gel Pad, Pharmaceutical Innovation, Inc., Newark, NJ, USA) was placed between the 55 MHz ultrasound probe and skin (Fig. 3). The reason for not using ultrasound coupling gel was to avoid the potential effect of water in the coupling gel on the skin thickness during the ultrasound measurement. For skin thickness assessment, the ultrasound probe was held steadily, in gentle contact with the gel pad, and perpendicular to the skin surface for optimal ultrasound imaging. For each site and each time point, 10 B-mode images of the skin were obtained along the transverse and longitudinal directions of the arm. The skin layer can be differentiated from the subcutaneous fat and the gel pad in the images obtained by the ultrasound biomicroscopy, as it shows hyper-echogenicity compared to the underneath tissue. For the thickness measurement, the upper and lower boundaries of the skin in the ultrasound image were manually identified and then a vertical line perpendicular to the boundaries was drawn to represent the skin thickness (Fig. 4). The above procedure was repeated for five times for each image, and the five measurement results were averaged. Furthermore, the results of the 10 images obtained from each site were averaged and the mean value was used for statistical analysis. To test the reproducibility of the skin thickness measurement in ultrasound images, which involved manual identification of boundaries, an intra-operator test–retest analysis was conducted.
Fig. 3. Skin thickness measurement using ultrasound biomicroscopy. An ultrasound probe was used for the imaging of skin. A gel pad was placed between the ultrasound probe and skin for acoustic coupling.
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Measurements were taken by the same investigator twice at the same site for each subject on two different days. Each time 10 images were collected so a total of 20 images were obtained for each subject for the reproducibility analysis. All the 20 subjects received the reproducibility test. The reliability study was not totally blinded and only the intra-rater reliability was assessed but the effect of non-blindness was thought to be minimal for the purpose of this study. 2.4. Statistical analysis Based on the current study, an intra-class correlation coefficient (ICC, Model 3) [12] was adopted to test the reproducibility of the skin thickness measurement. Repeated measure one-way ANOVA was used to analyze the skin thickness change before and after the treatments at the testing sites. Paired t-test was used to test the change of skin thickness at the control sites between the initial status and 30 min after the initial test. If a significant change was found along time after treatment, paired t-test would be applied to test the difference of the skin thickness in percentage with respect to its initial status at the testing sites with and without ultrasound stimulation, to reveal the effect of ultrasound stimulation on skin thickness. Independent t-test was used to test the difference of initial value and change of skin thickness between genders. A statistical level of p < 0.05 was used to judge the existence of a significant difference. All the statistical analyses were conducted using SPSS (Version 15, IBM Inc., Chicago, IL, USA). 3. Results ICC was 0.985 for the reproducibility test of skin thickness measurement, indicating that the measurement was highly reproducible. The results for all the subjects and also different genders were summarized in Table 1. The results showed that there was no significant change of the skin thickness at the control sites between the initial status and post 30 min for both UE ON (1.310 ± 0.151 mm vs. 1.308 ± 0.150 mm, p = 0.36) and UE OFF groups (1.299 ± 0.152 mm vs. 1.300 ± 0.147 mm, p = 0.33), suggesting that without the lotion treatment, the forearm skin should have no thickness change in the test environment. However, a significant change of the skin thickness after the lotion treatment was found for both the UE ON (p < 0.001) and UE OFF (p < 0.001) groups, but with different change patterns along post-treatment time for the two groups. Fig. 5 shows the different patterns of the skin thickness change for the UE ON and UE OFF groups. For the UE OFF group, the skin thickness increased significantly from 1.396 ± 0.193 mm to 1.430 ± 0.194 mm immediately after lotion treatment (p < 0.001), and then it continuously increased to 1.445 ± 0.194 mm from 0 min to 15 min (p = 0.048) and to 1.453 ± 0.197 mm from 15 min to 30 min after the treatment (p = 0.101). However, for the UE ON group, an overshoot phenomenon was observed. The thickness increased significantly from 1.379 ± 0.187 mm to 1.466 ± 0.182 mm (p < 0.001) immediately after the ultrasound treatment, then decreased slightly to 1.455 ± 0.177 mm from 0 min to 15 min (p = 0.089) and remained nearly unchanged to 1.450 ± 0.177 mm from 15 min until 30 min (p = 0.441). However, the skin thickness at 15 min and 30 min after treatment was still significantly larger than the initial thickness for both UE ON (both p < 0.001) and UE OFF (both p < 0.001), which might reflect the effectiveness of lotion treatment regardless of the ultrasound stimulation. Comparison between the UE ON and UE OFF groups revealed that the percentage increase of the skin thickness for the UE ON group was significantly larger than that of group UE OFF at 0 min (6.5 ± 2.4% vs. 2.5 ± 1.3%, p < 0.001), 15 min (5.7 ± 2.5% vs. 3.6 ± 2.3%, p < 0.001) and 30 min (5.4 ± 3.0%
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Fig. 4. Measurement of skin thickness based on ultrasound images. (a) Raw image of the forearm skin and (b) upper and lower boundaries were identified in the skin image as reference for skin thickness measurement.
Table 1 The skin thickness measured at the initial state without any treatment, 0 min, 15 min and 30 min after treatment. The skin thickness at the control sites were measured at the initial state and after 30 min. The results for the male and female subjects were also given for comparison. Test site (mm)
Control site (mm)
Initial
At 0 min
At 15 min
At 30 min
Initial
At 30 min
UE ON SD UE OFF SD
1.379 0.187 1.396 0.193
1.466 0.182 1.430 0.194
1.455 0.177 1.445 0.194
1.450 0.177 1.453 0.197
1.310 0.151 1.299 0.152
1.308 0.150 1.300 0.147
UE ON SD UE OFF SD
1.521 0.144 1.545 0.139
1.602 0.138 1.579 0.133
1.586 0.137 1.591 0.136
1.580 0.135 1.597 0.140
1.423 0.125 1.412 0.114
1.422 0.116 1.411 0.112
UE ON SD UE OFF SD
1.236 0.090 1.247 0.102
1.330 0.099 1.282 0.112
1.324 0.098 1.300 0.118
1.319 0.102 1.309 0.128
1.196 0.065 1.185 0.084
1.194 0.071 1.188 0.075
All
Male
Female
vs. 4.1 ± 2.9%, p = 0.022) after lotion treatment (Fig. 5b), suggesting the effectiveness of ultrasound stimulation for the improvement of skin hydration. When comparing male and female subjects, it was found that the forearm skin of the male subjects (initial thickness 1.533 ± 0.138 mm) was significantly thicker than that of the female subjects (initial thickness 1.242 ± 0.094 mm) (p < 0.001, Fig. 6). Immediately after lotion treatment, the percentage change of the skin thickness of the female (7.6 ± 2.3%) was significantly larger than that of male (5.4 ± 2.0%) for the UE ON group (p = 0.017) while no significant difference between genders was found for the UE OFF group (2.7 ± 1.2% vs. 2.3 ± 1.4%, p = 0.224). Similar results were also found between genders at 15 and 30 min post treatment for both the UE ON group (7.1 ± 2.7% vs. 4.3 ± 1.4%, p = 0.004 and 6.7 ± 3.4% vs. 4.0 ± 1.7%, p = 0.018, respectively) and UE OFF group (4.1 ± 2.9% vs. 3.0 ± 1.3%, p = 0.145 and 4.8 ± 3.8% vs. 3.4 ± 1.3%, p = 0.144, respectively) in terms of the percentage change of the skin thickness. 4. Discussion In this study, we investigated the effect of lotion treatment on skin thickness and also the effectiveness of ultrasound stimulation for the enhancement of lotion delivery into skin. A unique characteristic of our study is that ultrasound was used for both the stimulation and the monitoring purpose, although the range of frequency was quite different (1 MHz vs. 55 MHz) for the two applications. According to the results, lotion treatment was effective to increase the skin thickness, no matter with or without ultrasound stimulation; however, the ultrasound stimulation indeed enhanced the delivery effect as shown by a larger skin thickness
increase (6.5 ± 2.4% vs. 2.5 ± 1.3%, immediately after treatment). Furthermore, an overshoot phenomenon was observed for the change of the skin thickness with the UE ON group while this was not observed in the UE OFF group. The ultrasound stimulation seemed to be more effective for the female subjects, as indicated by a larger increase of skin thickness in the female subjects compared to the male subjects (7.6 ± 2.3% vs. 5.4 ± 2.0%, immediately after treatment). The increase of skin thickness detected in the current study was hypothesized to be caused by the increase of hydration and thus swelling of the skin after absorption of lotion. Chemicals that might increase the skin hydration after the application of lotion included the glycerin and petrolatum that were part of the lotion ingredients [13]. Skin thickness is most significantly contributed by a solid part including the collagen fibers and cellular substances, and a liquid part including the interstitial fluid content. It could be suggested that lotion treatment would not cause obvious change to the amount of solid substances in such a short duration and thus the variation of the skin thickness might most probably come from the change of water content of the skin. In a previous study, using a canine model, Diana et al. showed that the skin thickness significantly increased by 13–21% after the intravenous injection which was used to increase the level of skin hydration [14]. However, an increase of less than 10% was found in the current study. The disagreement in the change of skin thickness might be due to the different skin species (canine vs. human) tested and the different methods (intravenous injection vs. lotion treatment with ultrasound stimulation) adopted for changing the skin hydration. The use of ultrasound stimulation was found to have an enhanced effect on the increase of skin thickness immediately after
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Fig. 5. The change of skin thickness in (a) absolute and (b) percentage value with respect to its initial status along time before and after lotion treatment, with and without ultrasound stimulation. Different patterns of skin thickness change were revealed for the UE ON and UE OFF groups. An overshoot phenomenon for UE ON group could be observed in the figure. The curve for the UE OFF group was shifted a little to the right of the x-axis for a better comparison of the results.
treatment as observed in the UE ON group compared to the UE OFF group (6.5% vs. 2.5%, Fig. 5b). In a previous study, Park et al. used 1 MHz ultrasound with an intensity of peak pressure 400 kPa and a duty cycle of 10% to treat the skin in vitro for 60 min. They showed that more glycerol could be absorbed in the skin after ultrasound treatment compared to the control group where glycerol solution was used without ultrasound stimulation [15]. They also found that the use of contrast agents in the solution could further improve the effect of glycerol absorption, supporting that cavitation might be an important mechanism for the enhancement of skin permeability. Xu and Zhu also demonstrated the feasibility of ultrasound stimulation for the increased absorption of glycerol for the purpose of optical clearing [16]. Possible reasons for the effect of ultrasound stimulation might be some generally known phenomena of ultrasound studied in drug delivery, including cavitation, heating and convection. However, the specific mechanisms of action were not investigated in our study which warrants further studies using specific techniques such as confocal laser microscopy, surface temperature or tissue conductivity measurement. The skin thickness increase induced by ultrasound enhanced lotion delivery could be maintained for at least 30 min after the treatment. After application of the lotion, the skin thickness of the UE OFF group continuously increased, but that of the UE ON group increased first and then decreased slightly, exhibiting an overshoot phenomenon. This phenomenon might be due to that
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the moisturizing components in the lotion had been almost completely delivered into the skin layer and immediately acted as active humectants for increasing the hydration of skin. After the ultrasound stimulation was turned off, no extra moisturizing ingredients could be absorbed thus the hydration remained or even decreased slightly. In comparison, for the UE OFF group, the diffusion of moisturizing ingredients into the skin might be slower. It might take time before they could fully be absorbed, acted as active humectants and formed an occlusive layer for the increase of skin hydration. Therefore the skin thickness continuously increased during the observation period. Further investigation is needed to explain the explicit reason why different patterns were observed for the two treatment groups. It should be noted that the effect of ultrasound stimulation was not caused by the pure movement of the ultrasound massager probe on the skin as we also used sham massage through the massager probe movement without turning on ultrasound stimulation in the UE OFF group. In this study, we found the forearm skin of the male subjects was significantly thicker than that of the female subjects (1.533 ± 0.138 mm vs. 1.242 ± 0.094 mm, Fig. 6), which was in accordance with a previous study of skin thickness based on ultrasound imaging [17]. Through a histological study, Lee and Hwang reported that the forearm skin thickness of male was significantly thicker than that of female in Koreans [18]. The difference was assumed to be caused by the different amount of sex steroid in different genders [19,20]. After the ultrasound stimulation, it was found that the increase of skin thickness of female was significantly larger than that of the male subjects (7.6% vs. 5.4%, immediately after treatment). However, for the UE OFF group, no significant difference in the skin thickness change was observed between female and male (2.7% vs. 2.3%, immediately after treatment). This may suggest that the skin of the female was more reactive and sensitive to the ultrasound stimulation which enhanced the rate of moisturizer diffusion through stratum corneum. Again, the difference of sensitivity to ultrasound stimulation might come from the difference of skin structure such as the epidermis, especially stratum corneum, but the exact reason needs to be further investigated. There were some limitations for the current study. Firstly, this study only focused on the skin thickness change in response to ultrasound stimulation and lotion treatment within 30 min because we aimed to observe only a short term effect of the lotion delivery with and without ultrasound stimulation. How long the
Fig. 6. The change of skin thickness with time for the male and female subjects. It was found that the forearm skin of the male subjects was significantly thicker than that of the female subjects. The curves for the UE OFF group were shifted a little to the right of the x-axis for a better comparison of the results.
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treatment effect could be maintained needs further investigation. The situation might possibly be that after some time the skin thickness starts to reduce until returns to its initial value. For the UE ON group, ultrasound stimulation induced an overshoot phenomenon and the decrease of skin thickness had already been observed immediately after the ultrasound stimulation. For the UE OFF group, the thickness might reach a peak at some time after lotion application and decrease thereafter. A further longitudinal study can be planned to see the effect of lotion treatment and ultrasound stimulation over a period of several weeks or several months, so that the long term effectiveness of ultrasound massage can be assessed. Secondly, with respect to the ultrasound stimulation, this study only used it for five minutes, which was the default duration of a treatment session set by the manufacturer. According to the literature, time of ultrasound exposure for stimulation varied a lot and many studies used the duration of a few minutes for stimulation [21]; it is interesting to study the effect of a longer time of treatment session on the skin thickness change. The effects of other factors such as the ultrasound frequency and intensity can also be studied based on high frequency ultrasound imaging of skin. The findings could provide scientific reference for the function of lotion treatment associated with ultrasound stimulation on the skin change and help optimize protocols recommended for the use of ultrasound massage. Finally, it should be noted that in the current study, the results were obtained from a small number of young subjects with two main limitations. The first limitation caused by the small sample size is the error of estimation for the variance. The second but more important limitation associated with the small sample size is the low statistical power for the t-test, which increases the possibility of making a Type II error. Therefore, future research with a large sample size among more diverse age groups is desirable to confirm the results obtained in this study. 5. Conclusions Our study showed that lotion treatment was effective to increase the skin thickness, possibly due to an increase of water content in skin. The ultrasound stimulation could further enhance the rate of delivery, resulting in a significantly larger skin thickness increase immediately after lotion treatment (6.5% vs. 2.5%). It was interesting to see that female subjects experienced more significant skin thickness increase (7.6% vs. 5.4% immediately after ultrasound treatment) upon the application of ultrasound stimulation in comparison with the male subjects. In summary, this study demonstrated that 55 MHz ultrasound imaging was a useful tool to study the lotion delivery into skin enhanced by the ultrasound stimulation in vivo. Acknowledgements The project was partially supported by The Research Grant Council of Hong Kong (PolyU5354/08E), The Hong Kong Innovation
and Technology Fund (GHP/047/09) and The Hong Kong Polytechnic University (G-YL74). We would like to thank Mr. David Ng of AIKO Beauty Products Ltd., Hong Kong for providing the ultrasound massagers. We would also like to thank all the subjects for their participation in the experiment. References [1] R.C. Wester, H.I. Maibach, Individual and regional variation with in vitro percutaneous absorption, in: L.B. Robert, I.M. Howard (Eds.), In vitro Percutaneous Absorption: Principles, Fundamentals, and Applications, CRC Press, Boca Raton, 1991, pp. 26–30. [2] J.D. Bos, M. Meinardi, The 500 Dalton rule for the skin penetration of chemical compounds and drugs, Exp. Dermatol. 9 (2000) 165–169. [3] S.C. McNeill, R.O. Potts, M.L. Francoeur, Local enhanced topical delivery (LETD) of drugs - does it truly exist, Pharm. Res. 9 (1992) 1422–1427. [4] A.J. Singer, C.S. Homan, A.L. Church, S.A. McClain, Low-frequency sonophoresis: pathologic and thermal effects in dogs, Acad. Emerg. Med. 5 (1998) 35–40. [5] T. Fujimura, K. Tsukahara, S. Moriwaki, T. Kitahara, T. Sano, Y. Takema, Treatment of human skin with an extract of Fucus vesiculosus changes its thickness and mechanical properties, J. Cosmet. Sci. 53 (2002) 1–9. [6] D.S. Orth, Y. Appa, Glycerine: a natural ingredient for moisturizing skin, in: M. Lodén, H.I. Maibach (Eds.), Dry Skin and Moisturizers: Chemistry and Function, CRC Press, Boca Raton, London, 2000, pp. 213–228. [7] J.W. Fluhr, A. Bornkessel, E. Berardesca, Glycerol – Just a moisturizer? Biological and biophysical effects, in: M. Lodén, H.I. Maibach (Eds.), Dry Skin and Moisturizers: Chemistry and Function, CRC Press, Boca Raton, London, 2005, pp. 227–244. [8] L. Chen, M. Dyson, J. Rymer, P.A. Bolton, S.R. Young, The use of high-frequency diagnostic ultrasound to investigate the effect of hormone replacement therapy on skin thickness, Skin Res. Technol. 7 (2001) 95–97. [9] C. Eisenbeiss, J. Welzel, W. Schmeller, The influence of female sexhormones on skin thickness: evaluation using 20 MHz sonography, Br. J. Dermatol. 139 (1998) 462–467. [10] J. Serup, B. Staberg, P. Klemp, Quantification of cutaneous edema in patch test reactions by measurement of skin thickness with high frequency pulsed ultrasound, Contact Dermatitis 10 (1984) 88–93. [11] D.H. Turnbull, B.G. Starkoski, K.A. Harasiewicz, J.L. Semple, L. From, A.K. Gupta, D.N. Sauder, F.S. Foster, 40–100 MHz B-scan ultrasound backscatter microscope for skin imaging, Ultrasound Med. Biol. 21 (1995) 79–88. [12] G. Rankin, M. Stokes, Reliability of assessment tools in rehabilitation: an illustration of appropriate statistical analyses, Clin. Rehabil. 12 (1998) 187– 199. [13] M. Loden, The clinical benefit of moisturizers, J. Eur. Acad. Dermatol. Venereol. 19 (2005) 672–688. [14] A. Diana, C. Guglielmini, F. Fracassi, M. Pietra, E. Balletti, M. Cipone, Use of high-frequency ultrasonography for evaluation of skin thickness in relation to hydration status and fluid distribution at various cutaneous sites in dogs, Am. J. Vet. Res. 69 (2008) 1148–1152. [15] D. Park, J. Yoon, J. Park, B. Jung, H. Park, J. Seo, Transdermal drug delivery aided by an ultrasound contrast agent: an in vitro experimental study, Open Biomed. Eng. J. 4 (2010) 56–62. [16] X.Q. Xu, Q.H. Zhu, Feasibility of sonophoretic delivery for effective skin optical clearing, IEEE Trans. Biomed. Eng. 55 (2008) 1432–1437. [17] S. Seidenari, A. Pagnoni, A. Dinardo, A. Giannetti, Echographic evaluation with image analysis of normal skin – variation according to age and sex, Skin Pharmacol. 7 (1994) 201–209. [18] Y. Lee, K. Hwang, Skin thickness of Korean adults, Surg. Radiol. Anat. 24 (2002) 183–189. [19] H. Dao, R.A. Kazin, Gender differences in skin: a review of the literature, Gend. Med. 4 (2007) 308–328. [20] P.U. Giacomoni, T. Mammone, M. Teri, Gender-linked differences in human skin, J. Dermatol. Sci. 55 (2009) 144–149. [21] B.E. Polat, D. Blankschtein, R. Langer, Low-frequency sonophoresis: application to the transdermal delivery of macromolecules and hydrophilic drugs, Expert Opin. Drug Deliv. 7 (2010) 1415–1432.
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Ultrasonics journal homepage: www.elsevier.com/locate/ultras
Ultrasound biomicroscopy measurement of skin thickness change induced by cosmetic treatment with ultrasound stimulation Tak-Man Mak, Yan-Ping Huang, Li-Ke Wang, Yong-Ping Zheng ⇑ Interdisciplinary Division of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
a r t i c l e
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Article history: Received 5 July 2013 Received in revised form 27 January 2014 Accepted 13 February 2014 Available online 24 February 2014 Keywords: Skin thickness Ultrasound biomicroscopy Ultrasound stimulation Lotion Hydration
a b s t r a c t Moisturizing creams and lotions are commonly used in daily life for beauty and treatment of different skin conditions such as dryness and wrinkling, and ultrasound stimulation has been used to enhance the delivery of ingredients into skin. However, there is a lack of convenient methods to study the effect of ultrasound stimulation on lotion absorption by skin in vivo. Ultrasound biomicroscopy was adopted as a viable tool in this study to investigate the effectiveness of ultrasound stimulation on the enhancement of lotion delivery into skin. The forearm skin of 10 male and 10 female young subjects was tested at three different sites, including two lotion treatment sites with (Ultrasound Equipment – UE ON) and without (UE OFF) ultrasound stimulation and a control site without any lotion treatment. 1 MHz ultrasound with a duty cycle of 1.7%, a spatial peak temporal peak pressure of 195 kPa and an average power of 0.43 W was used for the stimulation. The skin thickness before, immediately after (0 min), and 15 and 30 min after the treatment was measured by an ultrasound biomicroscopic system (55 MHz). It was found that the skin thickness significantly increased immediately after the lotion treatment for both UE ON (from 1.379 ± 0.187 mm to 1.466 ± 0.182 mm, p < 0.001) and UE OFF (from 1.396 ± 0.193 mm to 1.430 ± 0.194 mm, p < 0.001) groups. Further comparison between the two groups revealed that the skin thickness increase of UE ON group was significantly larger than that of UE OFF group (6.5 ± 2.4% vs. 2.5 ± 1.3%, p < 0.001). Furthermore, it was disclosed that the enhancement of lotion delivery by ultrasound stimulation was more effective for the female subjects than the male subjects (7.6 ± 2.3% vs. 5.4 ± 2.0% immediately after treatment, p = 0.017). In conclusion, this study demonstrated that ultrasound biomicroscopy was a feasible method for studying the effectiveness of lotion treatment in vivo, and ultrasound stimulation was effective to enhance the rate of lotion absorption into skin. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, the use of ultrasound in beauty industry has been very common and demanding all over the world, especially in well developed cities such as Hong Kong. One common application is to use ultrasound to enhance the delivery of cosmetic lotion, a method called ‘‘ultrasound massage’’. Manufacturers of ultrasound massagers often claim that the use of these devices enhances the absorption of lotion ingredients into skin. However, the effectiveness of ultrasound stimulation on enhancing the lotion delivery has been scarcely reported in the literature.
⇑ Corresponding author. Address: Interdisciplinary Division of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China. Tel.: +852 27667664; fax: +852 23342429. E-mail addresses: [email protected] (T.-M. Mak), hti.huang@ connect.polyu.hk (Y.-P. Huang), [email protected] (L.-K. Wang), ypzheng@ ieee.org (Y.-P. Zheng). http://dx.doi.org/10.1016/j.ultras.2014.02.015 0041-624X/Ó 2014 Elsevier B.V. All rights reserved.
Low weight molecules (20 MHz) ultrasound images, which can be used for the measurement of skin thickness. The measurement of skin thickness has been successfully applied in studies of skin-related pathologies such as patch test and hormonal treatment [8–11]. It is a potential method to study the effectiveness of lotion delivery with the stimulation of ultrasound. This study aims to test the hypothesis that ultrasound stimulation can enhance lotion delivery. Therapeutic ultrasound (1 MHz) was used as a stimulation method to potentially enhance the delivery of cosmetic lotion to skin, and the skin thickness before and after the treatment was quantitatively assessed by a high frequency (55 MHz) ultrasound biomicroscopy on 20 subjects (10 male and 10 female). The change of skin thickness with and without ultrasound stimulation as well as the gender dependence of such change was reported. Details of the current study are presented as follows.
2. Materials and methods 2.1. Equipment and materials An ultrasound biomicroscopic system (Vevo 770, Visualsonics Inc., Toronto, Canada) with a transducer of 55 MHz in central frequency was used for the skin imaging. The axial resolution of this transducer was approximately 30 lm, the lateral resolution was approximately 70 lm and 2-D imaging was achieved by a mechanical scanning of the transducer in a linear way. To the best of the authors’ knowledge, this equipment is one of the best known ultrasound systems available in the commercial market for high frequency ultrasound imaging of skin in vivo. The spatial resolution provided by the 55 MHz probe is suitable for studying the skin; therefore, it was adopted in the current study. A 1-MHz ultrasound stimulation device (ST-302S Multi-Function Sonic Massager, AIKO Beauty Products Ltd., Hong Kong) (Fig. 1) was used for the ultrasound stimulation in this study. The head of the device with an embedded single-element ultrasound transducer had a diameter of 48.5 mm (Fig. 1). Through measurement in water using a needle hydrophone (HNP-0400, HN-series hydrophone, ONDA Corp., Sunnyvale, CA, USA), it was found that the ultrasound transducer of the stimulation device worked in a long tone-burst mode with a repetition period of 46 ms and a pulse duration of 768 ls, i.e., a duty cycle of 1.7%. The spatial peak temporal peak pressure was 195 kPa measured at a plane 5 mm away from the probe surface, which resembled the situation of treatment on skin. The total power measured over the acoustic beam was 0.43 W for the stimulation. The cosmetic product used in this study was a very common lotion widely available in the market (Baby Simply Soft Lotion, Johnson & Johnson Hong Kong Ltd., Hong Kong). This lotion is an oil-inwater emulsion which contains mostly water and also some other minor components such as glycerin and petrolatum.
Fig. 1. A picture of the ultrasound stimulation device. The use of various parts of the device as seen in the front view was briefly introduced.
Committee and all the subjects were asked to sign an informed consent form before assessment. 2.3. Assessment protocol and experimental test The subjects were instructed not to use any cosmetics on forearms one day before the experiment. Before test, they were asked to wash both forearms with liquid soap, and were instructed to stay in the test room for at least 30 min. The room temperature (20 ± 2 °C) was controlled by air conditioners while the humidity (40 ± 2%) was controlled by a dehumidifier placed in the test room during the whole experiment. The forearms of the subjects were divided into two regions including the proximal and distal parts (Fig. 2). The proximal parts of both forearms were used as control sites where no lotion or
2.2. Subjects Ten male and ten female subjects (age: 21 ± 1 years) were recruited for test on their forearms in the study. Subjects with skin irritation, food irritation, scar, sunburn or other skin problems were excluded from the study. In addition, those who had the habit of daily use of cosmetics on forearms were also excluded. Human ethical approval was obtained from the University Ethics
Fig. 2. The two arm locations used for lotion treatment with the ultrasound stimulation device. The stainless steel head was applied to massage the skin circularly and slowly for five minutes during lotion treatment.
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ultrasound treatment was applied. The distal parts of both forearms were treated with 5 g lotion, which was weighted by an electronic balance. Then the stainless steel head of the ultrasound enhancer was gently placed on the distal part of one forearm (grouped as Ultrasound Equipment – ‘‘UE ON’’) to massage the skin for five minutes (Fig. 2). For each participant, only one forearm would be treated with ultrasound, and the other one would be massaged by moving the probe without ultrasound turned on (grouped as ‘‘UE OFF’’). Half male and half female subjects were randomly assigned to have the ultrasound massage on their left forearms and the other half subjects had the massage on their right forearms. After five minutes, the excessive lotion remained on the skin was gently removed using tissue paper to assure a fair comparison between groups. The skin thickness was measured by the 55 MHz ultrasound biomicroscopic system (1) before any treatment (initial thickness), (2) immediately after treatment (thickness at 0 min), (3) 15 min after treatment (thickness at 15 min), and (4) 30 min after treatment (thickness at 30 min). For the control sites, the initial thickness and the thickness after 30 min were measured. Before scanning, a gel pad (Ultra Phonic Focus Conforming Gel Pad, Pharmaceutical Innovation, Inc., Newark, NJ, USA) was placed between the 55 MHz ultrasound probe and skin (Fig. 3). The reason for not using ultrasound coupling gel was to avoid the potential effect of water in the coupling gel on the skin thickness during the ultrasound measurement. For skin thickness assessment, the ultrasound probe was held steadily, in gentle contact with the gel pad, and perpendicular to the skin surface for optimal ultrasound imaging. For each site and each time point, 10 B-mode images of the skin were obtained along the transverse and longitudinal directions of the arm. The skin layer can be differentiated from the subcutaneous fat and the gel pad in the images obtained by the ultrasound biomicroscopy, as it shows hyper-echogenicity compared to the underneath tissue. For the thickness measurement, the upper and lower boundaries of the skin in the ultrasound image were manually identified and then a vertical line perpendicular to the boundaries was drawn to represent the skin thickness (Fig. 4). The above procedure was repeated for five times for each image, and the five measurement results were averaged. Furthermore, the results of the 10 images obtained from each site were averaged and the mean value was used for statistical analysis. To test the reproducibility of the skin thickness measurement in ultrasound images, which involved manual identification of boundaries, an intra-operator test–retest analysis was conducted.
Fig. 3. Skin thickness measurement using ultrasound biomicroscopy. An ultrasound probe was used for the imaging of skin. A gel pad was placed between the ultrasound probe and skin for acoustic coupling.
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Measurements were taken by the same investigator twice at the same site for each subject on two different days. Each time 10 images were collected so a total of 20 images were obtained for each subject for the reproducibility analysis. All the 20 subjects received the reproducibility test. The reliability study was not totally blinded and only the intra-rater reliability was assessed but the effect of non-blindness was thought to be minimal for the purpose of this study. 2.4. Statistical analysis Based on the current study, an intra-class correlation coefficient (ICC, Model 3) [12] was adopted to test the reproducibility of the skin thickness measurement. Repeated measure one-way ANOVA was used to analyze the skin thickness change before and after the treatments at the testing sites. Paired t-test was used to test the change of skin thickness at the control sites between the initial status and 30 min after the initial test. If a significant change was found along time after treatment, paired t-test would be applied to test the difference of the skin thickness in percentage with respect to its initial status at the testing sites with and without ultrasound stimulation, to reveal the effect of ultrasound stimulation on skin thickness. Independent t-test was used to test the difference of initial value and change of skin thickness between genders. A statistical level of p < 0.05 was used to judge the existence of a significant difference. All the statistical analyses were conducted using SPSS (Version 15, IBM Inc., Chicago, IL, USA). 3. Results ICC was 0.985 for the reproducibility test of skin thickness measurement, indicating that the measurement was highly reproducible. The results for all the subjects and also different genders were summarized in Table 1. The results showed that there was no significant change of the skin thickness at the control sites between the initial status and post 30 min for both UE ON (1.310 ± 0.151 mm vs. 1.308 ± 0.150 mm, p = 0.36) and UE OFF groups (1.299 ± 0.152 mm vs. 1.300 ± 0.147 mm, p = 0.33), suggesting that without the lotion treatment, the forearm skin should have no thickness change in the test environment. However, a significant change of the skin thickness after the lotion treatment was found for both the UE ON (p < 0.001) and UE OFF (p < 0.001) groups, but with different change patterns along post-treatment time for the two groups. Fig. 5 shows the different patterns of the skin thickness change for the UE ON and UE OFF groups. For the UE OFF group, the skin thickness increased significantly from 1.396 ± 0.193 mm to 1.430 ± 0.194 mm immediately after lotion treatment (p < 0.001), and then it continuously increased to 1.445 ± 0.194 mm from 0 min to 15 min (p = 0.048) and to 1.453 ± 0.197 mm from 15 min to 30 min after the treatment (p = 0.101). However, for the UE ON group, an overshoot phenomenon was observed. The thickness increased significantly from 1.379 ± 0.187 mm to 1.466 ± 0.182 mm (p < 0.001) immediately after the ultrasound treatment, then decreased slightly to 1.455 ± 0.177 mm from 0 min to 15 min (p = 0.089) and remained nearly unchanged to 1.450 ± 0.177 mm from 15 min until 30 min (p = 0.441). However, the skin thickness at 15 min and 30 min after treatment was still significantly larger than the initial thickness for both UE ON (both p < 0.001) and UE OFF (both p < 0.001), which might reflect the effectiveness of lotion treatment regardless of the ultrasound stimulation. Comparison between the UE ON and UE OFF groups revealed that the percentage increase of the skin thickness for the UE ON group was significantly larger than that of group UE OFF at 0 min (6.5 ± 2.4% vs. 2.5 ± 1.3%, p < 0.001), 15 min (5.7 ± 2.5% vs. 3.6 ± 2.3%, p < 0.001) and 30 min (5.4 ± 3.0%
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Fig. 4. Measurement of skin thickness based on ultrasound images. (a) Raw image of the forearm skin and (b) upper and lower boundaries were identified in the skin image as reference for skin thickness measurement.
Table 1 The skin thickness measured at the initial state without any treatment, 0 min, 15 min and 30 min after treatment. The skin thickness at the control sites were measured at the initial state and after 30 min. The results for the male and female subjects were also given for comparison. Test site (mm)
Control site (mm)
Initial
At 0 min
At 15 min
At 30 min
Initial
At 30 min
UE ON SD UE OFF SD
1.379 0.187 1.396 0.193
1.466 0.182 1.430 0.194
1.455 0.177 1.445 0.194
1.450 0.177 1.453 0.197
1.310 0.151 1.299 0.152
1.308 0.150 1.300 0.147
UE ON SD UE OFF SD
1.521 0.144 1.545 0.139
1.602 0.138 1.579 0.133
1.586 0.137 1.591 0.136
1.580 0.135 1.597 0.140
1.423 0.125 1.412 0.114
1.422 0.116 1.411 0.112
UE ON SD UE OFF SD
1.236 0.090 1.247 0.102
1.330 0.099 1.282 0.112
1.324 0.098 1.300 0.118
1.319 0.102 1.309 0.128
1.196 0.065 1.185 0.084
1.194 0.071 1.188 0.075
All
Male
Female
vs. 4.1 ± 2.9%, p = 0.022) after lotion treatment (Fig. 5b), suggesting the effectiveness of ultrasound stimulation for the improvement of skin hydration. When comparing male and female subjects, it was found that the forearm skin of the male subjects (initial thickness 1.533 ± 0.138 mm) was significantly thicker than that of the female subjects (initial thickness 1.242 ± 0.094 mm) (p < 0.001, Fig. 6). Immediately after lotion treatment, the percentage change of the skin thickness of the female (7.6 ± 2.3%) was significantly larger than that of male (5.4 ± 2.0%) for the UE ON group (p = 0.017) while no significant difference between genders was found for the UE OFF group (2.7 ± 1.2% vs. 2.3 ± 1.4%, p = 0.224). Similar results were also found between genders at 15 and 30 min post treatment for both the UE ON group (7.1 ± 2.7% vs. 4.3 ± 1.4%, p = 0.004 and 6.7 ± 3.4% vs. 4.0 ± 1.7%, p = 0.018, respectively) and UE OFF group (4.1 ± 2.9% vs. 3.0 ± 1.3%, p = 0.145 and 4.8 ± 3.8% vs. 3.4 ± 1.3%, p = 0.144, respectively) in terms of the percentage change of the skin thickness. 4. Discussion In this study, we investigated the effect of lotion treatment on skin thickness and also the effectiveness of ultrasound stimulation for the enhancement of lotion delivery into skin. A unique characteristic of our study is that ultrasound was used for both the stimulation and the monitoring purpose, although the range of frequency was quite different (1 MHz vs. 55 MHz) for the two applications. According to the results, lotion treatment was effective to increase the skin thickness, no matter with or without ultrasound stimulation; however, the ultrasound stimulation indeed enhanced the delivery effect as shown by a larger skin thickness
increase (6.5 ± 2.4% vs. 2.5 ± 1.3%, immediately after treatment). Furthermore, an overshoot phenomenon was observed for the change of the skin thickness with the UE ON group while this was not observed in the UE OFF group. The ultrasound stimulation seemed to be more effective for the female subjects, as indicated by a larger increase of skin thickness in the female subjects compared to the male subjects (7.6 ± 2.3% vs. 5.4 ± 2.0%, immediately after treatment). The increase of skin thickness detected in the current study was hypothesized to be caused by the increase of hydration and thus swelling of the skin after absorption of lotion. Chemicals that might increase the skin hydration after the application of lotion included the glycerin and petrolatum that were part of the lotion ingredients [13]. Skin thickness is most significantly contributed by a solid part including the collagen fibers and cellular substances, and a liquid part including the interstitial fluid content. It could be suggested that lotion treatment would not cause obvious change to the amount of solid substances in such a short duration and thus the variation of the skin thickness might most probably come from the change of water content of the skin. In a previous study, using a canine model, Diana et al. showed that the skin thickness significantly increased by 13–21% after the intravenous injection which was used to increase the level of skin hydration [14]. However, an increase of less than 10% was found in the current study. The disagreement in the change of skin thickness might be due to the different skin species (canine vs. human) tested and the different methods (intravenous injection vs. lotion treatment with ultrasound stimulation) adopted for changing the skin hydration. The use of ultrasound stimulation was found to have an enhanced effect on the increase of skin thickness immediately after
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Fig. 5. The change of skin thickness in (a) absolute and (b) percentage value with respect to its initial status along time before and after lotion treatment, with and without ultrasound stimulation. Different patterns of skin thickness change were revealed for the UE ON and UE OFF groups. An overshoot phenomenon for UE ON group could be observed in the figure. The curve for the UE OFF group was shifted a little to the right of the x-axis for a better comparison of the results.
treatment as observed in the UE ON group compared to the UE OFF group (6.5% vs. 2.5%, Fig. 5b). In a previous study, Park et al. used 1 MHz ultrasound with an intensity of peak pressure 400 kPa and a duty cycle of 10% to treat the skin in vitro for 60 min. They showed that more glycerol could be absorbed in the skin after ultrasound treatment compared to the control group where glycerol solution was used without ultrasound stimulation [15]. They also found that the use of contrast agents in the solution could further improve the effect of glycerol absorption, supporting that cavitation might be an important mechanism for the enhancement of skin permeability. Xu and Zhu also demonstrated the feasibility of ultrasound stimulation for the increased absorption of glycerol for the purpose of optical clearing [16]. Possible reasons for the effect of ultrasound stimulation might be some generally known phenomena of ultrasound studied in drug delivery, including cavitation, heating and convection. However, the specific mechanisms of action were not investigated in our study which warrants further studies using specific techniques such as confocal laser microscopy, surface temperature or tissue conductivity measurement. The skin thickness increase induced by ultrasound enhanced lotion delivery could be maintained for at least 30 min after the treatment. After application of the lotion, the skin thickness of the UE OFF group continuously increased, but that of the UE ON group increased first and then decreased slightly, exhibiting an overshoot phenomenon. This phenomenon might be due to that
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the moisturizing components in the lotion had been almost completely delivered into the skin layer and immediately acted as active humectants for increasing the hydration of skin. After the ultrasound stimulation was turned off, no extra moisturizing ingredients could be absorbed thus the hydration remained or even decreased slightly. In comparison, for the UE OFF group, the diffusion of moisturizing ingredients into the skin might be slower. It might take time before they could fully be absorbed, acted as active humectants and formed an occlusive layer for the increase of skin hydration. Therefore the skin thickness continuously increased during the observation period. Further investigation is needed to explain the explicit reason why different patterns were observed for the two treatment groups. It should be noted that the effect of ultrasound stimulation was not caused by the pure movement of the ultrasound massager probe on the skin as we also used sham massage through the massager probe movement without turning on ultrasound stimulation in the UE OFF group. In this study, we found the forearm skin of the male subjects was significantly thicker than that of the female subjects (1.533 ± 0.138 mm vs. 1.242 ± 0.094 mm, Fig. 6), which was in accordance with a previous study of skin thickness based on ultrasound imaging [17]. Through a histological study, Lee and Hwang reported that the forearm skin thickness of male was significantly thicker than that of female in Koreans [18]. The difference was assumed to be caused by the different amount of sex steroid in different genders [19,20]. After the ultrasound stimulation, it was found that the increase of skin thickness of female was significantly larger than that of the male subjects (7.6% vs. 5.4%, immediately after treatment). However, for the UE OFF group, no significant difference in the skin thickness change was observed between female and male (2.7% vs. 2.3%, immediately after treatment). This may suggest that the skin of the female was more reactive and sensitive to the ultrasound stimulation which enhanced the rate of moisturizer diffusion through stratum corneum. Again, the difference of sensitivity to ultrasound stimulation might come from the difference of skin structure such as the epidermis, especially stratum corneum, but the exact reason needs to be further investigated. There were some limitations for the current study. Firstly, this study only focused on the skin thickness change in response to ultrasound stimulation and lotion treatment within 30 min because we aimed to observe only a short term effect of the lotion delivery with and without ultrasound stimulation. How long the
Fig. 6. The change of skin thickness with time for the male and female subjects. It was found that the forearm skin of the male subjects was significantly thicker than that of the female subjects. The curves for the UE OFF group were shifted a little to the right of the x-axis for a better comparison of the results.
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treatment effect could be maintained needs further investigation. The situation might possibly be that after some time the skin thickness starts to reduce until returns to its initial value. For the UE ON group, ultrasound stimulation induced an overshoot phenomenon and the decrease of skin thickness had already been observed immediately after the ultrasound stimulation. For the UE OFF group, the thickness might reach a peak at some time after lotion application and decrease thereafter. A further longitudinal study can be planned to see the effect of lotion treatment and ultrasound stimulation over a period of several weeks or several months, so that the long term effectiveness of ultrasound massage can be assessed. Secondly, with respect to the ultrasound stimulation, this study only used it for five minutes, which was the default duration of a treatment session set by the manufacturer. According to the literature, time of ultrasound exposure for stimulation varied a lot and many studies used the duration of a few minutes for stimulation [21]; it is interesting to study the effect of a longer time of treatment session on the skin thickness change. The effects of other factors such as the ultrasound frequency and intensity can also be studied based on high frequency ultrasound imaging of skin. The findings could provide scientific reference for the function of lotion treatment associated with ultrasound stimulation on the skin change and help optimize protocols recommended for the use of ultrasound massage. Finally, it should be noted that in the current study, the results were obtained from a small number of young subjects with two main limitations. The first limitation caused by the small sample size is the error of estimation for the variance. The second but more important limitation associated with the small sample size is the low statistical power for the t-test, which increases the possibility of making a Type II error. Therefore, future research with a large sample size among more diverse age groups is desirable to confirm the results obtained in this study. 5. Conclusions Our study showed that lotion treatment was effective to increase the skin thickness, possibly due to an increase of water content in skin. The ultrasound stimulation could further enhance the rate of delivery, resulting in a significantly larger skin thickness increase immediately after lotion treatment (6.5% vs. 2.5%). It was interesting to see that female subjects experienced more significant skin thickness increase (7.6% vs. 5.4% immediately after ultrasound treatment) upon the application of ultrasound stimulation in comparison with the male subjects. In summary, this study demonstrated that 55 MHz ultrasound imaging was a useful tool to study the lotion delivery into skin enhanced by the ultrasound stimulation in vivo. Acknowledgements The project was partially supported by The Research Grant Council of Hong Kong (PolyU5354/08E), The Hong Kong Innovation
and Technology Fund (GHP/047/09) and The Hong Kong Polytechnic University (G-YL74). We would like to thank Mr. David Ng of AIKO Beauty Products Ltd., Hong Kong for providing the ultrasound massagers. We would also like to thank all the subjects for their participation in the experiment. References [1] R.C. Wester, H.I. Maibach, Individual and regional variation with in vitro percutaneous absorption, in: L.B. Robert, I.M. Howard (Eds.), In vitro Percutaneous Absorption: Principles, Fundamentals, and Applications, CRC Press, Boca Raton, 1991, pp. 26–30. [2] J.D. Bos, M. Meinardi, The 500 Dalton rule for the skin penetration of chemical compounds and drugs, Exp. Dermatol. 9 (2000) 165–169. [3] S.C. McNeill, R.O. Potts, M.L. Francoeur, Local enhanced topical delivery (LETD) of drugs - does it truly exist, Pharm. Res. 9 (1992) 1422–1427. [4] A.J. Singer, C.S. Homan, A.L. Church, S.A. McClain, Low-frequency sonophoresis: pathologic and thermal effects in dogs, Acad. Emerg. Med. 5 (1998) 35–40. [5] T. Fujimura, K. Tsukahara, S. Moriwaki, T. Kitahara, T. Sano, Y. Takema, Treatment of human skin with an extract of Fucus vesiculosus changes its thickness and mechanical properties, J. Cosmet. Sci. 53 (2002) 1–9. [6] D.S. Orth, Y. Appa, Glycerine: a natural ingredient for moisturizing skin, in: M. Lodén, H.I. Maibach (Eds.), Dry Skin and Moisturizers: Chemistry and Function, CRC Press, Boca Raton, London, 2000, pp. 213–228. [7] J.W. Fluhr, A. Bornkessel, E. Berardesca, Glycerol – Just a moisturizer? Biological and biophysical effects, in: M. Lodén, H.I. Maibach (Eds.), Dry Skin and Moisturizers: Chemistry and Function, CRC Press, Boca Raton, London, 2005, pp. 227–244. [8] L. Chen, M. Dyson, J. Rymer, P.A. Bolton, S.R. Young, The use of high-frequency diagnostic ultrasound to investigate the effect of hormone replacement therapy on skin thickness, Skin Res. Technol. 7 (2001) 95–97. [9] C. Eisenbeiss, J. Welzel, W. Schmeller, The influence of female sexhormones on skin thickness: evaluation using 20 MHz sonography, Br. J. Dermatol. 139 (1998) 462–467. [10] J. Serup, B. Staberg, P. Klemp, Quantification of cutaneous edema in patch test reactions by measurement of skin thickness with high frequency pulsed ultrasound, Contact Dermatitis 10 (1984) 88–93. [11] D.H. Turnbull, B.G. Starkoski, K.A. Harasiewicz, J.L. Semple, L. From, A.K. Gupta, D.N. Sauder, F.S. Foster, 40–100 MHz B-scan ultrasound backscatter microscope for skin imaging, Ultrasound Med. Biol. 21 (1995) 79–88. [12] G. Rankin, M. Stokes, Reliability of assessment tools in rehabilitation: an illustration of appropriate statistical analyses, Clin. Rehabil. 12 (1998) 187– 199. [13] M. Loden, The clinical benefit of moisturizers, J. Eur. Acad. Dermatol. Venereol. 19 (2005) 672–688. [14] A. Diana, C. Guglielmini, F. Fracassi, M. Pietra, E. Balletti, M. Cipone, Use of high-frequency ultrasonography for evaluation of skin thickness in relation to hydration status and fluid distribution at various cutaneous sites in dogs, Am. J. Vet. Res. 69 (2008) 1148–1152. [15] D. Park, J. Yoon, J. Park, B. Jung, H. Park, J. Seo, Transdermal drug delivery aided by an ultrasound contrast agent: an in vitro experimental study, Open Biomed. Eng. J. 4 (2010) 56–62. [16] X.Q. Xu, Q.H. Zhu, Feasibility of sonophoretic delivery for effective skin optical clearing, IEEE Trans. Biomed. Eng. 55 (2008) 1432–1437. [17] S. Seidenari, A. Pagnoni, A. Dinardo, A. Giannetti, Echographic evaluation with image analysis of normal skin – variation according to age and sex, Skin Pharmacol. 7 (1994) 201–209. [18] Y. Lee, K. Hwang, Skin thickness of Korean adults, Surg. Radiol. Anat. 24 (2002) 183–189. [19] H. Dao, R.A. Kazin, Gender differences in skin: a review of the literature, Gend. Med. 4 (2007) 308–328. [20] P.U. Giacomoni, T. Mammone, M. Teri, Gender-linked differences in human skin, J. Dermatol. Sci. 55 (2009) 144–149. [21] B.E. Polat, D. Blankschtein, R. Langer, Low-frequency sonophoresis: application to the transdermal delivery of macromolecules and hydrophilic drugs, Expert Opin. Drug Deliv. 7 (2010) 1415–1432.
