Novel Histological Evidence of Collagen and Elastin Regeneration in Fractional RF-Treated Acne Scars

Introduction: Fractional radiofrequency (RF) technology has been shown to be a gold standard therapy for acne scars. We have previously published clinical and histological results from the treatment of 8 patients with active acne and acne related scarring using a fractional RF device. Evidence of safety and efficacy was demonstrated, including long term follow-up clinical results from 4 patients, up to two years post last fractional treatment. In the current article we present histology evidence of acne scar skin renewal 3.5 years post treatment. Methods: Skin biopsies from acne scars in a concealed facial area were taken before, after four treatments and at 3.5 years after the last fractional treatment. Biopsies were examined with standard hematoxylin and eosin (H&E) stain, as well as Verhoeff’s Van Gieson (VVG) and Shikata stains for elastic fibers. Results: Histological findings demonstrate regeneration of elastic fibers and reduction of the dermal fibrosis. Scar depth of Patient #2 was reduced by 80% in the corresponding follow-up period. Conclusion: The long-term histological results further support the previously published findings that fractional RF is a safe and effective treatment for acne related scars.


Introduction
Permanent scarring from acne is an unfortunate prevalent complication of acne vulgaris which may be associated with considerable psychological distress. Dermatologists are frequently presented with the challenge of evaluating and pro-J. Cosmetics, Dermatological Sciences and Applications viding treatment recommendations to patients with acne scars [1].
Many different treatments, including chemical peels, surgical excision, punch grafting, dermabrasion, and tissue augmentation with a variety of filler substances, have been used to ameliorate atrophic scars with varying degrees of success [2] [3]. Recontouring of atrophic facial scars with CO 2 and Er:YAG lasers has become popular over the last decade [4] [5]; however, due to the extended post-laser recovery period and the potential risks, especially to candidates with dark skin type, patients have been less willing to undergo ablative laser skin resurfacing [6].
Fractional resurfacing technologies using either CO 2 fractional laser or fractional RF (FRF) energy delivered through multi-electrode pins, have demonstrated visible clinical benefits on atrophic acne scars and minor side effects comparing to non-fractional technologies [7].
In previously published articles we demonstrated our experience with an FRF device in the treatment of eight patients with active acne and acne-related scarring [8]. Long term clinical follow-up results of four out of eight patients who participated in the study were demonstrated, up to two years post last fractional treatment [9]. In the current publication we present long term histological evidence of acne scar skin renewal 3.5 years following completion of FRF treatment.

Methods
During the original study, all patients were informed about the risks, benefits and alternative treatments and all provided an informed consent based on the 1975 Declaration of Helsinki. Patients having acne scars, with or without active acne lesions, were treated with an array of 24 RF conducting pins.
Each pin is 2500 μm long (Fractora, InMode Ltd., Israel). As previously published, this study was originally carried out in the private clinic of Dr Judith Hellman in NY, NY. The treatment procedure was specified in the previously published articles [8] [9].
According to the typical treatment protocol, each patient underwent four facial treatments, usually four weeks apart. The first patient (PT1)-a male, 21 years old, with Fitzpatrick skin type II was treated with regular Fractora tip and the second patient (PT2)-a female, 19 years old, with Fitzpatrick skin type IV was treated with Fractora tip having pins with insulating coating along 2000μm that provided additional epidermal protection for dark skin.
Representative skin biopsies from two patients were taken before, after the fourth treatment and approximately 3.5 years after last treatment. The biopsies were taken from the same scarred areas of the face of each participant, to keep the results consistent, and the new biopsy was taken adjacent without overlapping to avoid the effect of the previous biopsy. Histological sections were fixed and examined with standard Hematoxylin and Eosin (H&E) stain as well as with Verhoeff's elastic stain-(VEG) and Shikata stains for elastic fibers. In the interim period between the fourth treatment and the follow-up period, the patients did not undergo any treatment or use any topicals as the acne stopped breaking out and scars do not benefit from topicals.

Results
In a previously published article, histological sections of skin biopsies taken before treatment and after four fractional treatments from one patient were presented [8]. In the current publication, we compare baseline and results post four fractional treatments with histological sections from biopsies taken at 3.5 years after the last fractional treatment. Figure 1 and Figure 2 demonstrate biopsies taken prior to treatment from two subjects. Figure 1 shows that the dermal fibrosis or acne scarring extends deep past the isthmus level (past the insertion of the pili erector muscles). The bracket highlights the depth of the dermal scar. In this area there is a loss of adnexal structures, notably the hair follicle. There are retained sebaceous glands beneath the fibrosis but the follicle is lost. The initial depth of the scarring process is 1.5 mm for PT1. It should be noted that in case of PT2, the pretreatment biopsy did not reflect the deepest part of the scar, only a depth of 0.50 mm was shown (Figure 2(b)). This may be due to sampling variants that occurred in the sectioning process of the histological slices. Figure 2 shows biopsies performed prior to treatment stained with Shikata and VEG stains which are specific for elastic fibers. The Shikata preparation stains the elastic fibers black for PT1 and the VEG stain, a comparable elastic fibers silver stain, highlights elastic fibers in PT2 sample. Both histologies demonstrate dermal scar which replaces the normal elastic fibers and both stains clearly reveal areas with a loss of elastic fibers, replaced by fibrous tissue. In addition, the Shikata preparation of the biopsy demonstrates adjacent adnexal structures in the normal area with a drop out of adnexal structures in the scar tissue.
Figures 3-5 present PT1 outcome after four fractional treatments. Figure 3 demonstrates the reduction of scar depth to almost half for PT1 after four treatments. The maximal depth of scarring was measured as 0.8 mm vs. 1.5 mm before treatment.
In Figure 3 and Figure 4 regenerative near normalized wispy collagen fibers are present in the reticular dermis; the scar is reduced to the superficial dermis. In addition, elastic fibers that were devoid of the original scar tissue repopulated the treated area ( Figure 5). Figure 6 presents VEG stain of PT2 histology results after 4 treatments. In PT2, there appears to be sebaceous glands above the dermal scarring process as a result of adjacent reactive sebaceous hyperplasia. Below the sebaceous glands, there is clear dermal fibrosis with loss of elastic fibers which indicates definitive dermal fibrosis extending to depth measurement of 1.13 mm. Of note, in the superficial dermis there are regenerative dystrophic elastics fibers, indicating elastic fiber regeneration and repopulation.         There is loss of elastin in the circled area but regenerative elastic fibers in the superficial papillary dermis above the dermal scar and near the circled area are clearly observed (×10).

Discussion
Our previous articles reported clinical and histological experience with the FRF device (Fractora, InMode, Ltd., Israel) for the treatment of active acne lesions and acne scarring [8] [9]. Treatment with FRF led to visible improvement in the appearance of the active acne and of acne scars. The depth of the scars was substantially reduced after four treatments and scars were regressed higher into the superficial dermis, apparently pushed up by new near normal collagen fibers. In addition, there was a repopulation of new elastic fibers in the superficial dermis previously occupied by scar tissue [8].
Long term clinical evidence was presented, demonstrating visual improvement of active acne lesions, acne scarring, pores and general skin texture after up to two years post last fractional treatment [9].
Long term follow-up histological results are not frequently available due to patient's compliance. In a study comparing a 1450 nm diode laser and a 1320 nm Nd:YAG laser in the treatment of atrophic facial scars, evaluations of cutaneous biopsies obtained before treatment, immediately after the first treatment, and at 1, 3, 6, and 12 months after the third treatment were performed [10]. Histological evaluation demonstrated an increase in dermal collagen on both the 1320 nm Nd:YAG and 1450 nm diode laser treating split-face halves 6 months after the final laser procedure. Additional neocollagenesis was not observed at the 12-month follow-up visit [10]. In an earlier study, prolonged clinical and histological effects from CO 2 laser resurfacing of atrophic acne scars were investigated [4].   [9]. Regeneration of collagen fibers and of elastic fibers was clearly observed in the current biopsies. In the case of PT1, an 80% reduction in scar depth was noted when comparing the depth measurement of 1.5 mm in pretreatment histologies with the follow-up measurement of 0.3 mm after 3.5 years.
A similar depth reduction was noted in PT2 case, when comparing post four fractional treatments, the depth measurement of 1.13 mm to the follow-up measurement of 0.25 mm after 3.5 years. Comparison to baseline was not possible since accurate pretreatment histology was not available. This was due to sectioning angle variations, leading to a sampling error in which the biopsy was not representative of the deepest dermal scar. It is postulated though, that the pretreatment scar depth was even deeper than the post four treatments measurement. Even without such an assumption, however, there was a visible clinical improvement in the appearance of the acne scars, reflecting the 80% reduction in scar depth in the case of PT2.
The finding of the substantial improvement in scar depth, and the fact that new elastic fibers are regenerated, may be helpful in treating various indications caused by irreversible elastin damage such as scarring alopecia, solar elastosis and smokers' wrinkles. In the latter example, free radicals destroy the elastic fibers and activate elastase, thus regeneration of elastic fibers may have a profound efficacy for the treatment of such indications.
It should be noted that in order to scientifically support these initial histological findings, more cases should be followed similarly for a long period after treatment. Results from more subjects will substantiate the safety and efficacy of the FRF modality for treatment of acne scars.

Conclusion
Skin biopsies clearly demonstrate the efficacy of the Fractora treatment. Substantial reduction of acne scar depth and repopulation of the scar tissue by collagen and elastic fibers are visible 3.5 years after fractional treatments.