Steven Dayan, Shridhar Ventrapragada, Anil R. Shah
Low frequency ultrasound is used in many arenas of medicine to allow for deeper penetration of topical agents. However, there have only been limited studies examining ultrasound and its effects on skin rejuvenation. This study examined an ultrasound delivery system with and without the use of a mild exfoliating agent on the epidermal and dermal response in both a porcine model and a prospective human trial. Six 3×3 cm areas of a Yucatan minipig were treated with either ultrasound, an exfoliant or a combination of the two and examined for histologic differences in a controlled, prospective manner. A subsequent prospective human trial examined ultrasound with or without a mild exfoliant compared to pre and post treatment results. Physician-graded scores demonstrated statistical improvement in the combined treatment modality of salicylic acid and ultrasound in uneven pigmentation, fine wrinkles, skin laxity, lack of fullness, and overall improvement(p<.01). The treatment group of ultrasound alone improved in the category of fine wrinkles (p<.05). Patient subjective scores improved significantly (p<.01) with ultrasound alone and in combination with 2.5% salicylic acid in fine wrinkles, uneven pigmentation, skin laxity, brown spot size, brown spot color, pore size, and overall improvement (p<.05). The histopathologic pig model did not demonstrate changes with regard to dermal collagen content, elastin content or epidermal thickness following ultrasound treatment This study demonstrated that ultrasound in combination with salicylic acid appears to offer modest improvement in skin rejuvenation.
Initially introduced into the German medical community in the 1930’s, ultrasound has been used in North American medicine since the 1940’s. Ultrasound technology has a wide range of applications in medicine ranging from vascular and radiological studies to physical therapy and wound healing.1,2 Recently, there has been renewed attention in the ability of ultrasound to enhance permeation of pharmaceuticals through the skin’s barrier.3-13 However, there have only been limited studies examining the effects of ultrasound on skin rejuvenation.
Clinical researchers have been working towards alternative methods of effectively rejuvenating the skin’s appearance without the requisite extended recovery period. Alpha and beta hydroxy agents have a storied history and are widely used for reducing signs of facial aging.14,15 Both agents are used in a variety of over-the-counter products at low doses and provide minimal skin rejuvenatory effects with prolonged courses. It appears that in order to achieve a significant improvement in the appearance of the skin the topical agents need to penetrate the tough skin barrier (the stratum corneum).16 However, this requires a potent concentration, which may not only involve a significant recovery period, but also the potential of a chemical burn.15
More recently attention has been focused on lasers and intense pulsed light sources used in a non-ablative manner to stimulate collagen reformation without adversely affecting the epidermis.17-20 Demand for these procedures is high, as patients with tempered expectations are appreciating the subtle improvements in their skin’s appearance with minimal downtime. Laser technology, while effective, is expensive and highly regulated. Also, many of the reported lasers with success are not easily movable from one ambulatory setting to another. Efforts are being instituted to find alternative methods of rejuvenating facial skin.
Ultrasound, a ubiquitously used technology, has a long history of safe, successful use in many other realms of health care. The purpose of this study was to determine if low frequency ultrasound energy delivered with and without the addition of low dose topical agents could result in an increase in dermal collagen and elastin content in the animal model and improve the skin’s appearance in a clinical model.
Materials and Methods:
Study procedures were submitted, reviewed, and performed in accordance with institutional guidelines regarding animal experimentation. One hairless, Yucatan minipig was used for the study.
Six three x three centimeter (cm) treatment areas plus one control three x three cm area were marked bilaterally along the flank of the pig. The treatment areas were positioned two cm off midline starting at the midportion of the scapula and subsequently proceeding inferiorly. A two cm space was maintained between the next corresponding treatment areas. Sites were treated once a day for four weeks. Biopsies were taken two and four weeks after the completion of the study. Samples were then sent for histological processing and evaluation.
Experimental areas were treated with either the SkinMaster delivery system, which uses a piezoelectric spatula oscillating at 25 kHz (Symedex, Bloomington, MN), fifteen percent salicylic acid, thirty five percent glycolic acid or a combination of the above delivery system and one of the above exfoliants. (Table 1)
All sample sites were degreased with acetone prior to each procedure. Four by four (Johnson and Johnson) gauze pads were cut to fit each treatment area and saturated with their appropriate solution, either fifteen percent salicylic acid or thirty five percent glycolic acid and applied to their corresponding area. Topical agents were applied for three minutes. After the application time had expired, the solutions were neutralized with saline soaked gauze pads. Treatment areas were moisturized daily after the corresponding treatment using 0.5 cc to each sample site of a hyaluronic acid squalene moisturizing solution. Areas treated with ultrasound alone had application of only the ultrasound and the daily moisturizing solutions. Areas treated with both ultrasound and either the salicylic or glycolic acid were first subjected to three minutes ultrasound, neutralized with saline, then after a lapse of three minutes the corresponding solution was applied for three minutes.
Skin samples were fixed in ten percent formalin solution. Following a forty-eight hour fixation period, tissue specimens were dehydrated and embedded in paraffin. Seven-micrometer paraffin sections were obtained and stained by the following methods: 1) hematoxylin-eosin method, 2) Gomori’s trichrome, 3) Verhoeff elastin stain. (Figure 1)
An oculometer eyepiece calibrated with a slide micrometer was used to measure epidermal thickness. To avoid sampling bias, the thickness of the epidermis was measure from the non-keratinized cellular epidermal layer up to the deepest portion of the epidermal ridge. The keratinized noncellular layers of the epidermis were not measured because of fragmentation of this layer during micrometry.
A point-counting method described elsewhere was used to assess a change in the number of dermal structures between the control sample and the treated skin.17
Patients were recruited from the senior author’s private practice. Inclusion criteria included age greater than eighteen, mild to severe wrinkle formation and willingness to comply with the requirements of the study. Exclusionary criteria included botulinum toxin treatment within the last 90 days and recent history of chemical peel, microdermabrasion or facial cosmetic surgery. Those with carotid artery disease, skin cancer, vitiligo, pregnancy, lactation, psiorasis, herpes simplex infection, propensity to form keloids, previous electrolysis, and use of isotretinoin anisatil were also excluded. Home skin care regimens were evaluated and standardized to eliminate potential confounding factors. Patients were given samples of gentle moisturizers and sunscreens (Maroque; MDAesthetics, Northbrook, Ill).
Following informed consent, patients were treated with either Ultrasound alone for ten minutes (Sound Skin, Chicago, Illinois) 2.2Hz at a setting of 2.0 or a combination of Salicylic Acid (2.5%) Maroque, MD Aesthetics, Northbrook Illinois) applied to the skin followed immediately by 10 minutes of ultrasound to the each side of the face. The ultrasound was applied from the nasolabial fold medially, preauricular crease laterally, mandibular border inferiorly, and to the orbital rim superiorly. Topical anesthesia was not necessary. Treatments were repeated biweekly for a total of 8 treatments.
Standardized full-face, oblique, and lateral view photographs were taken before beginning treatment and after the last treatment using a digital photograph camera (Fuji S-100, Valhalla, New York). Before treatment sessions, all makeup was removed. Digital photographs were taken prior to, after each treatment and four weeks following the eighth treatment.
Each participant was required to perform a subjective self-evaluation before each treatment and after completion of the study. Patients rated the following variables on a 9-point scale, with 9 being the most severe: fine wrinkles, coarse wrinkles, visual dryness, skin roughness, uneven pigmentation, skin laxity, and overall improvement.
Unlabeled pre and the final post photographs for each patient were imported into power point and blindly evaluated by three physician observers. The physician graders were masked to the preoperative and postoperative status of the photographs. Post-treatment photographs were taken immediately before the last treatment session. Using a 9-point grading system, the graders reviewed the following variables: fine wrinkles, coarse wrinkles, skin laxity, pigmentation, and overall rating. Preoperative and post-treatment scores in each category were compared. A 1-tailed, paired comparative statistical analysis of the 2 groups was conducted using the t test.
Figure 1 shows the percentage collagen fibers in the dermis. Neither ultrasound, nor exfoliants applied alone caused an increase in the amount of collagen. Collagen arrangement was unchanged, and there was no subsequent differences seen in the thickening or density of collagen.
Figure 2 shows the percentage of elastin fibers in the dermis. There is an unappreciable difference between the two and four week samples. The elastin fibers seem to be identical in shape, character, and density.
No appreciable difference was noted in epidermal thickness from treatment sites when compared to non-treated control sites. The dermal width was also noted to be unchanged. In addition, the hematoxylin-eosin stained slides did not demonstrate an inflammatory response or structural damage to the integrity of the epidermis or dermis.
48 patients completed eight treatments, had post-treatment photographs, and were entered into the study. All of the participants were female ranging in age from 26-63.
Thirty two of the patients received salicylic acid plus ultrasound, sixteen received ultrasound alone. All of the patients tolerated the study well and found the ultrasound therapy soothing. No adverse events were noted
In most categories, patients found subjective benefit with and without an exfoliant. The evaluations were significantly improved in the coarse wrinkles, visual dryness, skin pigmentation, skin laxity, brown spot size, fine wrinkles, brown spot color, pore size, and overall improvement with ultrasound. (Table 2) Only roughness did not improve statistically after sonographic therapy. When ultrasound was combined with salicylic acid (2.5%), patient evaluations improved in all parameters except for roughness and visual dryness. (Table 3 )
An objective difference was seen in the masked physician arm in most categories when a mild exfoliant was added. The categories with significant improvements included skin laxity, fine wrinkles, uneven pigmentation, and overall improvement. (Table 4 ) Only coarse wrinkles did not show significant improvement. In the patients receiving ultrasound only as a skin rejuvenation therapy, objective improvement was only identified in fine wrinkle, all other categories were without significant change.(Table 5 )
Ultrasound’s benefits are delivered via thermal and non-thermal mechanisms. Ultrasound, defined as sound waves with a greater frequency than that which is heard by the human ear are transmitted from a piezoelectric transducer to the skin through a coupling media.1 Oscillated waves delivered to the soft tissues are absorbed or scattered. Areas of high collagen content absorb much of the energy and convert the mechanical energy to heat. Bone and joint capsular structures, high in collagen content, preferentially absorb the waves; therefore causing generation of heat in tendons and periosteum.
Therapeutic ultrasound frequency ranges from 1-3 MHz. At 1 MHz, ultrasound waves may extend down beyond 2 cm reaching the musculoskeletal soft tissues. At the higher frequency, 3 MHz range, ultrasound energy is subject to much more friction and is attenuated by dermal and subcutaneous collagen therefore penetrating less than 2 cm.1 Ultrasound generated heat has been shown to increase collagen tissue extensibility, alter blood flow, and increase enzymatic activity.1 Many of the benefits of ultrasound energy may also be attributed to its non-thermal mechanisms.
Ultrasound non-thermal mechanisms include acoustic streaming, sonophoresis, and cavitation. Rapid oscillation of tissue and fluid as a result of ultrasound energy promotes movement of fluid waves against cells. This may result in alterations of the cellular membrane permeability and ion concentrations between the inner and outer cell wall, which may stimulate an intracellular cascade resulting in increased fibroblastic activity and collagen formation.1,21
Sonophoresis is the notion that ultrasound can facilitate the passage of topical products through the skin’s hydrophobic barrier. Evidence has mounted that the method by which agents are assisted through the skin is not convective but rather through the phenomenon of cavitation.7
Cavitation, the most novel of the ultrasound mechanisms, involves the generation of gaseous bubbles in a medium and is due to the nucleation of small gaseous cavities during the negative pressure cycles of ultrasound, followed by the growth of these bubbles throughout subsequent pressure cycles.7-22 Mitragotri et. al. theorized and found supporting evidence that ultrasound caused cavitation in the keratinocytes lipid bilayer resulting in structural disorder and permeability. This was found to be the most important cause for ultrasonic enhancement of transdermal transport.7 On average, these defects measure 20 microns and allow for pathways in which pharmaceuticals can pass.12 Ultimately, resulting in deeper penetration of these agents and possibly increased absorption. Extensive and detailed research has established low frequency ultrasound in the range of 20 kHz to be the ideal wavelength for enhancing skin permeability. Salicylic acid, in particular had a 300-fold increase in permeability following low frequency ultrasound treatment.8 Additionally this permeability lasted for up to 12 hours after the treatment was completed.
High speed mechanical oscillation at subthreshold ultrasound levels may also create vapor bubbles, which collapse when reaching areas of high pressure. This phenomenon when applied to the skin may provide for exfoliation of the upper levels of the epidermis. Zukowski, histologically studying the post auricular skin of six patients treated with high speed mechanical oscillations noted a 30-50% reduction in the stratum corneum of treated individuals following three passes with a piezoelectric spatula (Unpublished data). He concluded that his intervention provided for a particle free gentle exfoliation of stratum corneum. Others have also found piezoelectric generated low frequency sound waves as a method for exfoliating the horny layer of the epidermis. Additionally, when a glycolic acid was added after the ultrasound treatment significant improvement in skin elasticity and skin hydration was noted.16
Enhancing facial rejuvenation with topical agents such as glycolic acids and salicylic acids are surging in popularity. As a cosmetic agent their regulation is not bound by the same standards as pharmaceuticals and their efficacy at times can be in doubt.14 Defining parameters for these cosmetically enhancing topical agents are being elucidated. Evidence is surfacing that effectiveness is dependent on the ideal vehicle, concentration and product pH.14,15 However, the product’s potency may be dependant on its ability to penetrate the epidermis. Recognizing that the stratum corneum was being debrided and thinned in addition to a cavitation induced disruption of the lipid bilayer, we evaluated the effect on collagen and elastin content that the topical agents would have when applied in conjunction with low frequency ultrasound.
In our porcine model utilizing the same low frequency piezoelectric device as above, we did not notice a change in the epidermal depth of our treated samples when compared to controls four weeks following our intervention. Based on our findings there does not seem to be a significant change in the collagen and elastin content or epidermal thickness as a result of the ultrasound device we used. Though we did not notice any objective improvement, there does seem to be a subjective improvement. In particular, the skin treated with ultrasound alone appeared debrided of its horny layer, was smoother, without ulceration and more aesthetically pleasing. (Figure 5 and 6)
Our study examined two different ultrasound modalities, a pizoelectric spatula and an ultrasound wand. The pizoelectric spatula was used in the porcine model at low frequency and noted no objective histologic improvement. The ultrasound wand was used in the clinical study, also at low frequency, and noted objective visual change in the group receiving the combination of ultrasound and salicylic acid. Data from the histological arm suggest that dermal collagen is not increased. This suggest that objective improvements in facial skin noted in the clinical arm are not secondary to dermal change, rather improvements clinically reported could be due to simple exfoliation of stratum corneum translating to visible objective renewal, yet lacking permanent underlying structural changes.
Although the masked clinical arm could only distinguish benefit in those patients receiving salicylic acid in combination with ultrasound, all of the patients in this study found subjective significant improvement with ultrasound directed therapy. Discrepancies between patients’ subjective improvement and the physicians’ evaluation may in part be due to the exfoliating nature of salicylic acid and its ability to improve the pigmentary nature of the skin which is translated in photographic evaluation, while, any subtle three dimensional changes are often more difficult to graphically record. Additionally it is very likely that a positive placebo effect contributed to the patients perceived improvements.
Low frequency ultrasound was utilized in both the clinical and histopathologic arms of this study. Lower frequency ultrasound has been found to be effective for pharmaceutical delivery through the stratum corneum.14 Glycolic and salicylic acid’s effectiveness is directly dependant on its ability to penetrate the epidermis.1,22 This study does not support or refute the ability of ultrasound to increase skin permeability of topical resurfacing agents. It is also not clear whether use of a higher frequency ultrasound, which acts at a shallower depth, would have caused direct stimulation of the dermis and therefore increased collagenous content of the skin.
Other ultrasound devices manufactured with different configurations may have separate findings. Perhaps the greatest benefit derived from this treatment modality is a gentle exfoliation of the stratum corneum. As such, the effects resulting from ultrasound delivered via a piezoelectric spatula at 25 kHz both alone or in combination with gentle exfoliants are limited to the epidermis. Minimal and inconsequential effects on dermal structures can be expected.
A potential weakness of the design of this study is the lack of a low dose salicylic (2.5%) treatment arm. This dose of salicylic acid is much lower than that used for a peel or series of peels (typically 20-30%), and lower than most over the counter daily use salicylic acid products. In addition, the pH of the salicylic acid product used (Maroque) was high, similar to over the counter products, limiting its efficacy. The authors felt that the mild exfoliation created by salicylic acid used in similarly dosed over-the-counter products has been well studied and unlikely to provide any skin rejuvenation with eight limited treatments in such a low treatment dose and high pH by itself. However, an interesting comparison could be made between salicylic acid treatment group of increasing concentration with and without ultrasound to determine the degree of improvement in penetration provided by low frequency ultrasound.
Further work needs to be undertaken regarding optimal ultrasound exposure time, intensity level or topical agent. But our results can be used as a stepping-stone for future experiments.
Low frequency ultrasound is a safe and widely used application for a device often used by non-medical personnel under the guidance of a physician. When used alone at therapeutic energy levels its safety appears well established. Similar to microdermabrasion, it can provide a benefit to patients desiring a safe and effective method for exfoliating their stratum corneum. However, although not clearly demonstrated in our study, there exists the potential for an increased permeability of topically applied agents through a denuded barrier. This possibility warrants that greater attention be placed on the composition of agents applied to the skin during mechanical exfoliation and ultrasound application.
Overall, ultrasound therapy appears to be a well-tolerated adjunctive therapy which is most effective in combination with a mild peeling agent. No adverse effects or downtime were noted. Ultrasound is inexpensive, portable, and widely available technology which can be used for skin rejuvenation if proven effective. Although the results demonstrated modest improvement, they were statistically significant changes noted by photographic evaluation.
- Ziskin MC, Mcdiarmid T, Michlovitz SL:Therapeutic Ultrasound. In Thermal Agents in Rehabilitation, ed 2.F.A. Davis Co. Philadelphia, PA. 1990, p134.
- Byl NN, Hopf H: The use of Oxygen in Wound Healing. In Wound Healing Alternatives in Management, ed 2. F.A. Davis Co. Philiadelphia, PA. 1995. P. 394.
- Ahmet T, Ashley S, Mitragotre S:Investigation of the role of Cavitation in Low-frequency sonophoresis using acoustic spectrosocopy. J Pharmaceutical Sci; 2002:91:P444-453.
- Tezel A, Ashley S, Tuchscherer J, Mitragotri S. Frequency Dependence Of Sonophoresis. Pharm Res 2001:18:p.1694-1700.
- Mitragotri S, Farrell J, Tang H, Terahara T, Kost J, Langer R. Determination of threshold energy dose for ultrasound induced transdermal drug transport.J Contr rel 2000:63:p41-52.
- Fang JY, Fang CL, Sung KC, Chen HY. Effect of low frequency ultrasound on the in vitro percutaneous absorption of clobetasol 17-propionate. Int J Pharm 1999. 191:33-42.
- Mitragotri S, Edwards DA, Blankschtein D, Langer R. A mechanistic study of Ultrasonically enhanced transdermal drug delivery. J Pharm Sci 1995;84:p697-706.
- Mitragotri S, Blankschtein D, Langer R.Transdermal drug delivery using low frequency sonophoresis. Pharm Res 1996;13:p411-420.
- Singer A, Homan C, Church A, McClain SA. Low frequency sonophoresis:Acad Emerg Med 1998;5:35-40.
- Mitragotri S, Blanckschtein D, Langer R. An explanation for the variation of the sonophoretic transdermal transport enhancement from drug to drug. J Pharm Sci 1997;85:p1190-1192.
- Mitragotri S, Johnson ME, Blanckschtein D, Langer R. An analysis of the size of selectivity for solute partitioning, diffusion, and permeation across lipid bilayers. Biophys Jour 1999;77:p1268-1283.
- Wu J, Chappelow J, Yang J, Weimann L. Defects generated in human stratum corneum specimens by ultrasound. Uls Med Biol 1998;24:755-710.
- Mitragotri S. Effect of bilayer disruption on transdermal transport of low molecular weight hydrophobic solutes. Pharm Res 2001;18:1018-1023.
- Kurtzweil P. FDA Cons Mag 1998
- Draelos ZD.Topical skin care. In Atlas of Cosmetic Surgery W.B. Saunders Philadelphia PA.2002: p123-138.
- Morganti P, Randazzo SD, Fabrizi G. Enhancing the Glycolic acid efficacy by piezoelectric vibrations. J Appl Cosm 1997;15:147-159.
- Shim E, Barnette D, Hughes K, Greenway HT. Microdermabrasion: A clinical and histopathological study. Derm Surg 2001;27:524-530.
- Zelickson BD, Kilmer SL., Bernstein E., Chotzen VA., Dock J., Mehregan D., Coles C. Lasers Surg. Med. 1999.25:229-236.
- Kelly KM., Nelson JS., Lask GP., Geronemus RG., Bernstein LJ., Cryogen spray cooling in combination with Non ablative laser treatment of facial rhytides. Arch Dermatol. 1999;135:691-694.
- Goldberg DJ., Samady JA., Intense pulse light and Nd:YAG laser nonablative treatment of facial rhytids. Laser Surg Med. 2001;28:141-144.
- Mortimer AJ, Dyson M. The effect of therapeutic ultrasound on calcium uptake in fibroblasts. Ultrasound med biol 1988;14 (6):499-506
- Davies R, Ed; Cavitation in real liquids; American Elsvier:New York,1964.