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Just Accepted by Journal of Cosmetic and Laser Therapy

Comparative histometric analysis of the effects of high intensity focused ultrasound (HIFU) and radiofrequency (RF) on skinDong Hye Suh, Jeong Hwee Choi, Sang Jun Lee, Ki-Heon Jeong, Kye Yong Song, Min Kyung Shin

Doi: 10.3109/14764172.2015.1022189

Abstract

Introduction High-intensity focused ultrasound (HIFU) and radiofre-quency (RF) are used for non-invasive skin tightening. Neocollagen-esis and neoelastogenesis have been reported to have a mechanism of controlled thermal injury.Objective To compare neocollagenesis and neoelastogenesis in each layer of the dermis after each session of HIFU and monopolar RF.Methods We analyzed the area fraction of collagen and elastic fib-ers using the Masson’s trichrome and Victoria blue special stains, respectively, before and after 2 months of treatments. Histometric analyses were performed in each layer of the dermis, including the papillary dermis, and upper, mid, and deep reticular dermis.Results Monopolar RF led to neocollagenesis in the papillary der-mis, and upper, mid, and deep reticular dermis, and neoelastogen-esis in the papillary dermis, and upper and mid reticular dermis. HIFU led to neocollagenesis in the mid and deep reticular dermis and neoelastogenesis in the deep reticular dermis. Among these treatment methods, HIFU showed the highest level of neocollagen-esis and neoelastogenesis in the deep reticular dermis.Conclusions HIFU affects deep tissues and impacts focal regions. Monopolar RF also affects deep tissues, but impacts diffuse regions. We believe this data provide further insight into effective skin tightening.

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Comparative histometric analysis of the effects of high intensity

focused ultrasound (HIFU) and radiofrequency (RF) on skin

Dong Hye Suh,*1 Jeong Hwee Choi,*2 Sang Jun Lee,1 Ki-Heon Jeong,2 Kye Yong Song,3

Min Kyung Shin2

1Department of Dermatology, Arumdaun Nara Dermatologic Clinic, Seoul, Korea, 2Department of Dermatology, College of Medicine, Kyung Hee University, Seoul, Korea, 3Department of Pathology, Chung-Ang University, Seoul, Korea

*Contributed equally to this work

Correspondence: Min Kyung Shin, M.D., Ph. D., Department of Dermatology, College of Medicine, Kyung Hee University, #1 Hoeki-Dong, Dongdaemun-Ku, Seoul, 130-702, Korea. Tel: 82-2-958-8300. Fax: 82-2-969-6538. E-mail: [email protected]

Abstract

Introduction High-intensity focused ultrasound (HIFU) and radiofrequency (RF) are used for non-invasive skin tightening. Neocollagenesis and neoelastogenesis have been reported to have a mechanism of controlled thermal injury.

Objective To compare neocollagenesis and neoelastogenesis in each layer of the dermis after each session of HIFU and monopolar RF.

Methods We analyzed the area fraction of collagen and elastic fibers using the Masson’s trichrome and Victoria blue special stains, respectively, before and after 2 months of treatments. Histometric analyses were performed in each layer of the dermis, including the papillary dermis, and upper, mid, and deep reticular dermis.

Results Monopolar RF led to neocollagenesis in the papillary dermis, and upper, mid, and deep reticular dermis, and neoelastogenesis in the papillary dermis, and upper and mid reticular dermis. HIFU led to neocollagenesis in the mid and deep reticular dermis and neoelastogenesis in the deep reticular dermis. Among these treatment methods, HIFU showed the highest level of neocollagenesis and neoelastogenesis in the deep reticular dermis.

Conclusions HIFU affects deep tissues and impacts focal regions. Monopolar RF also affects deep tissues, but impacts diffuse regions. We believe this data provide further insight into effective skin tightening.

Keywords: High intensity focused ultrasound; Monopolar radiofrequency; Neocollagenesis; Neoelastogenesis

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Introduction Many different laser and other light-based systems have been developed and

evaluated for their capability to reverse photodamage and age-associated

rhytides, a process referred to as photorejuvenation (1, 2). Although ablative

lasers remain the gold standard for photodamaged skin rejuvenation, their use

is associated with side effects as well as a prolonged and unpleasant

posttreatment downtime (3). Thus, in recent years, non-invasive and non-

ablative procedures have attracted attention with the hope of achieving skin

tightening effects close to those of ablative lasers while avoiding long

recovery times and potential complications (4-7). Nonablative rejuvenation

(NAR) devices have been designed to induce thermal injury within the

dermis without epidermal damage. NAR devices in use include intense

pulsed light, radiofrequency (RF), neodymium-doped yttrium aluminum

garnet (Nd:YAG), and pulsed dye lasers (8-10). However, laser energy can

be diffracted, absorbed, or scattered, resulting in suboptimal energy

penetration (11, 12). So several technologies that utilize energis other than

light and laser have been developed, such as RF and focused ultrasound (13).

Monopolar RF is the one of the first and most-studied non-invasive skin

tightening treatments demonstrated to be effective in numerous trials (14-18).

Monopolar RF therapy delivers uniform heat at a controlled depth in dermal

layers, causing direct collagen contraction and immediate skin tightening (2,

19). Because RF energy is produced by an electric current rather than by a

light source, there are no diminutions by tissue scattering or absorption by

epidermal melanin. As such significant thermal energies can be generated

safely within the deeper tissue layers (20). Subsequent remodeling and

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reorientation of collagen bundles and the formation of new collagen is

achieved during the months after treatment (14).

One of the recent advances in non-invasive skin tightening is the use of high

intensity focused ultrasound (HIFU). This can produce small, micro-thermal

lesions at precise depths in the mid to deep reticular dermis up to the fibro-

muscular layer, potentially up to 6 – 7.8 mm deep, causing thermally-induced

contraction of collagen and tissue coagulation with subsequent collagenesis,

while sparing the epidermis (21-23). This deep tissue heating ability of HIFU

is comparable with those of RF, laser, and infrared light sources, which are

up to 2 – 4 mm deep within the dermis (4-6).

There have been no studies comparing neocollagenesis and

neoelastogenesis in the layers of the dermis by histometric analysis. The

purpose of this study is to evaluate and compare the tightening effects of

these two NAR devices by using histometric analysis in each layer of the

dermis in Asian skin.

Materials and methods Study population

A total of 33 patients were enrolled in this study. Patient age ranged from 27

to 76 years (mean, 49.5 years) with a gender breakdown of 31 females and 2

males. Exclusion criteria were prior cosmetic facial surgery or placement of

tissue fillers, scarring in the treatment region, and allergy to topical

anesthetics.

We adhered to the recommendations of the current version of the

Declaration of Helsinki. Informed consent was obtained from all patients

before treatment.

Monopolar RF and HIFU

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Monopolar RF and HIFU were used in this study. For monopolar RF, a TC

tip and Frame tip were used, respectively.

Monopolar RF with a TC tip (Thermage NXT® Inc., Hayward, CA) was

used at energy levels between 2.0 and 3.5 (20 - 38 J/cm2), using a “big fast”

3-cm2 tip for 3 - 5 passes and a total of 300 shots (pulses). Monopolar RF

with a frame tip (Thermage CPT® Inc., Hayward, CA) was used at energy

levels set between 2.0 and 3.5 (20 - 38 J/cm2), using a “total” tip for 8 - 10

passes and a total of 600 shots (pulses).

HIFU (Ulthera® LLC, Mesa, AZ) was applied using a handpiece with a

frequency of 4.4 MHz and a focal depth of 4.5 mm. Pulses were arranged in a

linear array with each pulse spaced 1.5 mm apart and the entire linear array

was up to 25 mm long.

Tissue samples

We collected paraffin-embedded tissue samples from patients who were

treated with monopolar RF or HIFU before and after two months of each

session of monopolar RF and HIFU. A total 33 tissue samples were collected

before and after the treatment. There were 11 tissue samples treated with

monopolar RF (TC tip), 11 tissue samples treated with monopolar RF (frame

tip), and 11 tissue samples treated with HIFU. A 2-mm punch biopsy was

used to sample the lateral side of the cheek. The tissue samples were all

stained with hematoxylin and eosin, and Masson’s trichrome and Victoria

blue stains were used for collagen and elastic fibers, respectively.

Histometric analysis

Histologic photographs were analyzed with Image J software

(http://rsb.info.nih.gov/ij). We measured the thickness of the dermis in all

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tissue samples by using slides stained with hematoxylin and eosin. We also

analyzed slides stained with Masson’s trichrome and Victoria blue by

calculating the area fractions of collagen and elastic fibers, respectively,

before and after the treatment at the following areas: the papillary dermis,

upper reticular dermis, mid reticular dermis, and deep reticular dermis.

Statistical Analysis

For statistical analysis, a paired T test was performed using Excel 2007

software (v.12.0; Microsoft Co., USA) to compare and identify any

significant differences between before and after the treatment. A value of p <

0.05 was regarded as statistically significant.

Results

Monopolar RF caused a significant increase in both neocollagenesis and

neoelastogenesis in the upper reticular dermis. HIFU led to a significant

increase in both of the above parameters, but in the deep reticular dermis

(Fig. 1, 2 and Table 1 - 4).

Monopolar RF (TC tip) caused a significant increase in both

neocollagenesis and neoelastogenesis in the upper reticular dermis.

Specifically, we observed neocollagenesis in the upper, mid, and deep

reticular dermis and neoelastogenesis in the papillary dermis and upper

reticular dermis (Fig. 1, 2 and Table 1).

Monopolar RF (frame tip) led to a significant increase in both

neocollagenesis and neoelastogenesis in the papillary dermis, and upper and

mid reticular dermis. Specifically, we observed neocollagenesis in the

papillary dermis, and upper, mid, and deep reticular dermis.

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Neoelastogenesis was observed in the papillary dermis, and upper and mid

reticular dermis (Fig. 1, 2 and Table 2).

HIFU caused a significant increase in both neocollagenesis and

neoelastogenesis in the deep reticular dermis. Specifically, we observed

neocollagenesis in the mid and deep reticular dermis and neoelastogenesis in

the deep reticular dermis (Fig. 1, 2 and Table 3).

We conducted comparisons of each device in terms of area fraction changes

(%) (Table 4); areas of highest change were analyzed among all devices.

Monopolar RF (TC tip) led to the highest degree of neocollagenesis in the

upper reticular dermis, while monopolar RF (frame tip) demonstrated the

highest degree of neocollagenesis in the papillary dermis and mid reticular

dermis and neoelastogenesis in the papillary dermis, and upper and mid

reticular dermis. HIFU caused the highest degree of neocollagenesis in the

deep reticular dermis and neoelastogenesis in the deep reticular dermis.

Discussion

Treatment for aged skin has included surgery, such as rhytidectomy,

blepharoplasty, and brow lift. However, minimally invasive procedures have

gained popularity because of various benefits, including less postoperative

downtime (13). Ablative and nonablative laser devices have conventionally

been used to improve skin laxity, but these modalities have primarily focused

on treating the superficial layers of the skin because of limitations in

penetration depth (11-13, 23, 24). Several technologies have been developed

that utilize sources of energy other than light and laser to overcome aging,

such as RF and focused ultrasound (13).

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The RF device is different from cosmetic lasers in that it produces an

electric current rather than light. The energy produced is not liable to be

diminished by tissue diffraction or absorption by epidermal melanin.

Therefore, the thermal effect of RF is independent of skin color, which is an

advantage for Asian patients, and significant thermal energies can be

generated safely within the deeper tissue layers (20). However, poor

technique leading to cryo-injury or over-heating with excessive fluences may

potentially lead to cutaneous compromise with blistering, crusting and the

subsequent development of postinflammatory hyperpigmentation, especially

in skin of color (25). These adverse effects are thought to be due to the

reflection of infrared light energy from prominent bony areas of the face,

poor contact cooling, or bulk tissue heating from overlapping pulses (14, 26,

27). Heat generated by RF results in collagen fibril denaturation with

immediate contraction (2). Over time, as part of a thermally mediated healing

response, fibroblasts are stimulated to enhance new collagen deposition and

remodeling, resulting in further collagen tightening and an overall increase in

collagen content (12). Also, as RF energy usually follows the path of least

resistance, fibrous septa of the subcutaneous fat lobules are preferentially

heated and account for the deeper thermal effects of RF devices (20, 28).

This is thought to be important in subsequent remodeling of subcutaneous

tissue and tightening of the skin (11, 29).

HIFU delivers geometrically micro-focused ultrasound energy at precise

and consistent depths on a prescribed tissue plane, which can be from the

dermis down to the subdermal connective tissue of the superficial

musculoaponeurotic system (SMAS), depending on the selected transducer

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used (30, 31). The device has also been shown to be safe, with transient

erythema and edema being the most common side effects (21, 32). Also the

absorption of ultrasound energy is independent of the melanin content of skin

(33, 34). Therefore, in contrast to light-based devices, the thermal effect of

HIFU is independent of skin color and chromophores, and thus is

advantageous for Asian skin, similar to the RF system. Ultrasound waves

induce vibration in tissue, thus creating friction between molecules, which

eventually generates heat. Overall, selective coagulative change is effected

within the focal region of the beam, but other tissue proximal and distal to

the focal region of the ultrasound field is preserved (21, 22, 30-32, 35). Also,

deep energy delivery to the level of the SMAS in a fractionated pattern is

thought to be most effective in inducing skin tightening (36).

The penetration depths of monopolar RF and HIFU were to subcutaneous

tissue and SMAS, respectively (37). Thermal imaging has revealed that RF

delivery is more diffuse, tends to affect the dermis, and travels along

connective tissue septae into the subdermis (37). In contrast to monopolar

RF, HIFU is sharply focused (22).

In this study, we observed that HIFU showed increased dermal collagen in

the mid and deep reticular dermis and rearrangement of elastic fibers in the

deep reticular dermis. Furthermore, when comparing each device, HIFU

showed the highest neocollagenesis and neoelastogenesis in the deep

reticular dermis (Table 4). This result indicates that HIFU has an effect on

deep tissues and affects focal regions, which is consistent with previous

reports (22, 23, 32). On the other hand, monopolar RF caused

neocollagenesis in the papillary dermis, and upper, mid, and deep reticular

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dermis and neoelastogenesis in the papillary dermis, and upper and mid

reticular dermis. This result indicates that monopolar RF also has effects on

deep tissues, but tends to affect diffuse regions and is less effective than

HIFU in the deep reticular dermis, as stated in previous reports (11, 12, 28,

29, 37).

In monopolar RF, we used 2 different tips: TC tip and frame tip. The TC tip

caused a significant increase in both neocollagenesis and neoelastogenesis in

the upper reticular dermis. However, the frame tip had the same effect even

in the deeper regions and up to the mid reticular dermis. Furthermore, the

frame tip a greater degree of change (%) than the TC tip in all layers, except

in terms of neocollagenesis in the upper reticular dermis. This may be due to

the greater number of shots performed with the frame tip (frame tip, 600

shots; TC tip, 300 shots). However, it is clear that the frame tip has an effect

on deeper regions than the TC tip.

Our limitation is the short interval of follow up, 2 months, which may be

insufficient to show neocollagenesis and neoelastogenesis. However, we

previously proved this period to be sufficient to show effectiveness (23).

This study is the first to compare the effects of monopolar RF and HIFU on

Asian skin and reports histological data in each layer of the dermis. We

believe this data provide basic informations that will contribute to effective

skin tightening. Combining these two devices, simultaneously or

sequentially, may exert synergistic tightening effects by compensating for the

different effects in each skin layer.

Funding sources: None

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Declaration of interest: The authors report no declaration of interest. The

authors alone are responsible for the content and writing of the paper.

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Ultrasound (IUS). Lasers Surg Med. 2008; 40:67-75.

31. Laubach HJ, Makin IR, Barthe PG, Slayton MH, Manstein D. Intense

focused ultrasound: evaluation of a new treatment modality for precise

microcoagulation within the skin. Dermatol Surg. 2008; 34:727-34.

32. Alam M, White LE, Martin N, Witherspoon J, Yoo S, West DP.

Ultrasound tightening of facial and neck skin: a rater-blinded

prospective cohort study. J Am Acad Dermatol. 2010; 62:262-9.

33. Goss SA, Johnston RL, Dunn F. Comprehensive compilation of

empirical ultrasonic properties of mammalian tissues. J Acoust Soc Am.

1978; 64:423-57.

34. Keshavarzi A, Vaezy S, Kaczkowski PJ, Keilman G, Martin R, Chi EY,

et al. Attenuation coefficient and sound speed in human myometrium

and uterine fibroid tumors. J Ultrasound Med. 2001; 20:473-80.

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35. Kennedy JE, Ter Haar GR, Cranston D. High intensity focused

ultrasound: surgery of the future? Br J Radiol. 2003; 76:590-9.

36. Har-Shai Y, Bodner SR, Egozy-Golan D, Lindenbaum ES, Ben-Izhak O,

Mitz V, et al. Mechanical properties and microstructure of the

superficial musculoaponeurotic system. Plast Reconstr Surg. 1996;

98:59-70; discussion 71-3.

37. Abraham MT, Vic Ross E. Current concepts in nonablative

radiofrequency rejuvenation of the lower face and neck. Facial Plast

Surg. 2005; 21:65-73.

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Table Legends Table 1. Average Area Fractions of Collagen, Elastin, and Dermal Thickness

Before and After Monopolar Radiofrequency (TC Tip) Treatment.

Mean (Standard Deviation)

Before treatment

2 months after treatment Change (%) p-value

Average area fractions of collagen (%)

Papillary dermis 80.87 (14.86) 83.11 (16.70) 2.78 0.399

Upper Reticular dermis 42.64 (8.83) 49.38 (10.61) 15.81 0.002

Mid Reticular dermis 54.06 (8.58) 57.76 (10.37) 6.83 0.007

Deep Reticular dermis 57.62 (11.22) 64.39 (12.96) 11.74 0.001

Average area fractions of elastin (%)





Dermal thickness (mm) 952.26 (92.98) 973.41 (91.57) 2.22 0.423

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Table 2. Average Area Fractions of Collagen, Elastin, and Dermal Thickness

Before and After Monopolar Radiofrequency (Frame Tip) Treatment.


Before treatment












Dermal thickness (mm) 1103.08 (198.87)

1174.71 (194.58) 6.49 0.042

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Table 3. Average Area Fractions of Collagen, Elastin, and Dermal Thickness

Before and After High Intensity Focused Ultrasound Treatment.


Before treatment












Dermal thickness (mm) 737.47 (119.00) 905.57 (164.28) 22.79 0.037

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Table 4. Change (%) of Area Fractions of Collagen, Elastin, and Dermal

Thickness Before and After Treatment with Each Device.

Monopolar RF (TC tip)

Monopolar RF (frame tip)

High Intensity Focused Ultrasound

Change (%) p-value Change

(%) p-value Change (%) p-value


Papillary dermis 2.78 0.399 14.92 0.028 1.28 0.779

Upper Reticular dermis 15.81 0.002 14.42 0.007 6.44 0.326

Mid Reticular dermis 6.83 0.007 17.87 0.022 11.32 0.045

Deep Reticular dermis 11.74 0.001 12.58 0.000 21.20 0.000


Papillary dermis 9.10 0.036 18.14 0.013 0.49 0.926

Upper Reticular dermis 10.25 0.002 14.12 0.041 2.79 0.602

Mid Reticular dermis 8.16 0.156 12.22 0.000 11.23 0.070

Deep Reticular dermis 5.14 0.118 11.56 0.060 13.48 0.000

Dermal thickness (mm) 2.22 0.423 6.49 0.042 22.79 0.037 RF: radiofrequency *p value: before and after each device

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Figure Legends

Figure 1. Histopathology of skin biopsy before (A, B, and C) and 2 months

after (D, E, and F) treatment with each device. (A, D) Monopolar RF with

TC tip; (B, E) Monopolar RF with frame tip; (C, F) HIFU. Masson’s

trichrome, original magnification x 40.

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Figure 2. Histopathology of skin biopsy before (A, B, and C) and 2 months

after (D, E, and F) treatment with each device. (A, D) Monopolar RF with

TC tip; (B, E) Monopolar RF with frame tip; (C, F) HIFU. Victoria blue,

original magnification x 40.

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comparative histometric analysis of the effects of high ......this provisional pdf corresponds to...

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