FormalPara Key Summary Points

Why carry out this study?

Despite the abundance of preexisting studies on various treatment modalities and therapeutic agents, complete resolution and long-term control of melasma remain elusive.

Radiofrequency has been shown to produce dermal changes in melasma lesions, whereas polynucleotides have been investigated for antimelanogenesis effects.

This study aimed to compare the efficacy of a pulsed, bipolar, microneedling radiofrequency device combined with polynucleotides versus radiofrequency alone for the treatment of melasma.

What was learned from the study?

Improvements in objective and subjective outcome measures were observed in 26 patients after 3 treatments at 2-week intervals. Recurrence of melasma was observed in three patients at 2, 3, and 4 months after the last treatment.

Pulsed, bipolar, microneedling radiofrequency with polynucleotides was not superior to microneedling radiofrequency alone in the treatment of melasma.

Microneedling radiofrequency may be considered as a safe adjunctive treatment option for melasma.

Introduction

Melasma is a common acquired pigmentation disorder characterized by tan to dark-brown macules and patches on the central, malar, or mandibular areas of the face. It affects predominantly, but not exclusively, middle-aged women with darker Fitzpatrick skin phototypes. Ultraviolet (UV) radiation, female sex hormones, inflammation, and genetic predisposition are primary drivers of this disease. Its histopathological characteristics include increased melanin in keratinocytes and macrophages, increased mast cells, dilated vessels in the dermis, solar elastosis, and a disrupted basement membrane [1]. Such findings have recently led to the proposal that melasma is not only a disease of melanocytes but also a photoaging skin disorder, with treatment modalities such as laser and light systems, and energy-based devices aiming to address this aspect being proposed as possible options [2, 3].

Melasma has proven challenging to treat due to its chronicity, a tendency to relapse, and inherent recalcitrance. Topical depigmenting agents and the judicious use of broad-spectrum sunscreens with visible light coverage remain the first line and mainstay of treatment. The second line consists of supplementation with chemical peels. Lasers and light-based modalities are considered third-line treatment options, best suited for refractory cases. Among these, intense pulsed light, Q-switched lasers, picosecond lasers, and fractionated resurfacing lasers, both ablative and nonablative, have been studied extensively [4]. However, they have produced variable results, with recurrence, postinflammatory hyperpigmentation (PIH), and rebound hyperpigmentation limiting their widespread application.

Radiofrequency (RF) devices initially became popular due to their efficacy in skin tightening and rejuvenation, with a high safety profile in darker skin phototypes and minimal posttreatment recovery time [5]. With delivery through microneedles, electrical currents reach deeper target tissues at a predictable penetration depth [6, 7]. Advances in technology enabled the delivery of pulsed-type RF, which produces thermal and nonthermal effects through gated RF oscillations, in contrast to continuous-type RF that produces thermal ablative effects by continuous delivery of high-frequency energy over set conduction times [6, 8]. Its thermal and nonthermal effects, together with deeper tissue penetration, have led to the use of pulsed-type microneedling RF in the treatment of refractory melasma [9, 10]. Proposed theories behind the observed improvement of melasma were enhanced permeability to topical treatments, and dermal effects ranging from changes in lesion vasculature and melanin washout [9], to neocollagenesis and repair of damaged basement membranes [10]. Studies that followed attributed RF’s efficacy to the observed loosening of melanocyte–keratinocyte attachments, promoting transdermal melanin elimination [11], and thermo-modulated shrinkage of CD31-positive blood vessels with decreased VEGF-A mRNA expression [8]. Radiofrequency reversed cellular and gene mediators of dermal senescence [12], repaired basement membranes, and reduced melanogenesis markers of the microphthalmia-associated transcription factor (MITF) pathway [13]. As melasma’s pathogenesis involves an interplay of various factors, the definitive role of RF in its treatment remains to be validated.

Polynucleotides (PN) are highly purified natural DNA molecules comprising deoxyribonucleotide polymers of 50–200 base pairs [14]. Polynucleotides possess antiinflammatory, antiischemic, proangiogenic, and stimulatory effects on various cells [15]. Their capacity to accelerate tissue repair and regeneration led to aesthetic applications in rejuvenation of the face and body [16]. Their antimelanogenesis and skin-whitening properties were investigated by Noh et al. [17]. In the in vitro arm of the study, human melanocytes were incubated with polydeoxyribonucleotide. Decreased melanin synthesis and intracellular tyrosinase levels were observed. In the clinical study arm, lightening of lesions was observed in six patients (three with melasma, two with mottled pigmentation, and one with pigmented contact dermatitis) after receiving intradermal injections of polydeoxyribonucleotide every 4 weeks for a total of three treatment sessions. The study demonstrated that polydeoxyribonucleotides suppressed melanogenesis via reduction of MITF signaling and its downstream targets [17]. Similar findings were obtained in an in vitro study by Kim et al. [18], further demonstrating PN’s antimelanogenesis property. However, more clinical studies are needed to confirm PN’s role in the treatment of melasma.

Given that the pathogenesis of melasma is complex and involves an interplay of photoinduced changes such as solar elastosis, increased vascularity, and cellular signaling pathways leading to increased melanogenesis, the avenue of treatment modalities that could potentially address these factors remains to be explored. Thus, the present study aimed to investigate the efficacy and safety of pulsed microneedle RF in combination with PN for the treatment of melasma.

Methods

Subjects and Ethics

This pilot study was conducted at the Siriraj Skin Laser Center, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Thirty volunteer subjects were initially enrolled. They were between 18 and 60 years old, had Fitzpatrick skin types III to V, and had been diagnosed with bilateral facial melasma by a board‐certified dermatologist. A Wood’s lamp examination was performed to determine the type of melasma of each volunteer. Patients were not enrolled if they were pregnant, had an active infection or inflammatory dermatoses on the face, had a history of keloid or hypertrophic scar formation, or were taking oral anticoagulant or antiplatelet medications. Individuals were also excluded if they used topical treatments for melasma in the past month or received laser or light-based treatments for the face during the past 6 months.

Before this research began, its protocol was approved by the Ethics Committee of the Siriraj Institutional Review Board, Faculty of Medicine Siriraj Hospital, Mahidol University (reference number 409/2563). The study adhered to the guidelines of the Declaration of Helsinki of 1964 and its later amendments. Written informed consent was obtained from all study subjects. In addition, this work was registered with the Thai Clinical Trials Registry (identification number TCTR20210804002).

Before initiating RF treatment, block randomization was used to assign one side of the face of each participant to RF plus PN treatment. The contralateral side became the control and was treated with RF plus normal saline solution (NSS).

Materials

The RF device was an invasive pulsed-type bipolar RF with noninsulated needles (SYLFIRM; Viol, Kyunggi, Korea). Pulsed-type RF was delivered at a frequency of 2 MHz, with a 10 × 10 mm disposable tip comprising 25 noninsulated, penetrating microneedles in a uniform 5 × 5 array. The microneedles were made of surgical stainless steel, each 0.3 mm thick and 3.5 mm long. The parameters for each treatment session were as follows: first pass (whole face): level II, 1.5-mm penetration depth; second pass (over melasma lesions only): level II, 1.5 mm penetration depth; third pass (whole face): level II, 0.8 mm penetration depth. Each pass was done with 10–30% overlap.

A transparent, colorless, and odorless liquid containing 0.3% PN as the primary component (REVS-NCFS 140 HPn; Reanzen, Gyeonggi-do, Korea) was used as the topical product for the designated treatment hemiface. At each treatment session, the designated hemiface received 3–5 drops (approximately 0.2 ml) of the PN.

Treatment Protocol

Before each treatment session, 2.5% lidocaine and 2.5% prilocaine cream (Liprikaine; T. Man Pharma Company Ltd., Bangkhunthian, Thailand) was applied under occlusion for 1 h. The participants were then asked to wash and dry their faces thoroughly, and the treatment areas were then cleaned with alcohol immediately before receiving RF treatment. Each patient received three passes of the RF treatment as previously described, with the treatment endpoint of mild erythema. Upon completion of the RF treatment, a research associate not involved with evaluation applied the PN and NSS to the designated hemifaces of each patient. The treating physicians as well as the patients were unaware of which hemiface received treatment with PN and which served as control. Immediately after the procedure, both treatment and control sides were occluded with a transparent dressing (Tegaderm; 3M Healthcare, St. Paul, MN, USA) for 1 h. Posttreatment instructions were to avoid the sun and apply a broad-spectrum sunscreen daily. The study participants were not allowed to use topical bleaching agents or any form of treatment for melasma. Treatment sessions were performed at 2-week intervals for a total of three sessions.

Treatment Evaluations

Subjective Assessment

Standard digital photographs were obtained at baseline and following the final treatment session (at 2 weeks and 1, 2, 3, and 6 months after). All digital photographs were taken with a facial photo fixture using a Canon PowerShot G9 standoff camera (OMNIA Imaging System; Canfield Scientific, Inc., Fairfield, NJ, USA).

Two blinded physicians independently determined the modified melasma area severity index (mMASI) score of each hemiface at baseline and each follow-up. For a hemiface, calculating the mMASI score is based on the percentage of the area involved (A) and darkness (D) of the lesion, applied to the formula: frontal 0.15 × (D × A) + malar 0.30 × (D × A) + chin 0.05 × (D × A) [19]. Area of involvement was rated from 0 to 6, where 0 indicates absent; 1, < 10%; 2, 10–29%; 3, 30–49%; 4, 50–69%; 5, 70–89%; and 6, 90–100%. Darkness was rated from 0 to 4, where 0 indicates absent; 1, slight; 2, mild; 3, marked; 4, severe [20]. Treatment side effects were recorded at each treatment session and follow-up visit. The study participants were also asked to evaluate the degree of improvement of their melasma lesions as slightly better (< 25%), fair (26%–50%), good (51%–75%), and excellent (> 75%). Pain levels during treatment were rated using a visual analog scale ranging from 0 (no pain) to 10 (severe pain).

Objective Assessment

The Melanin Index (MI) and Erythema Index (EI) were measured bilaterally using a Mexameter (Courage–Khazaka, Köln, Germany) at the same assessment time points as above. A translucent sheet was used to mark and map the designated areas on the face for location consistency. Skin roughness was captured using macrophotography using the Antera 3D system (Miravex, Dublin, Ireland).

Statistical Analyses

Mean values were obtained for the MI, EI, skin roughness, and mMASI scores together with their corresponding standard deviation. Due to the split-face study design, the comparison of MI, EI, skin roughness, and mMASI scores obtained over six time points was assessed using a generalized estimating equation (GEE) with exchangeable correlation structure. Treatment, time, and treatment × time interaction were entered as independent variables in the full model. Interaction was found nonsignificant and was thus excluded from the final model. Data were analyzed using PASW Statistics for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). Significance was set at a value of p ≤ 0.05. Descriptive statistics were used for the improvement evaluations of the participants.

Results

Of the 30 participants initially enrolled, 26 (86.67%) were included in the analyses. Four patients (13.3%) were excluded from the study: three developed melasma recurrence, and one could not complete follow-up. All study participants were women, with a mean age of 43.2 ± 7.0 years. Mixed melasma predominated (61.5%; n = 16), and the mean duration of the disease was 8.9 ± 6.5 years. Fifteen patients received PN on the right hemiface, and 15 received PN on the left hemiface. Demographic characteristics and number of patients randomized to treatment and control sides are summarized in Table 1.

Table 1 Patient demographics and sum of hemifaces that received PN (n = 26)
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Figures 1, 2, 3, and 4 show means with standard deviation of the MI, EI, skin roughness, and mMASI scores for the PN (treatment) and NSS (control) sides from baseline and the five follow-up time points after the final treatment session, showing a decreasing trend. Comparison of the above outcome measures for the treatment and control sides over six time points was done using a generalized estimating equation, resulting in the values presented in Table 2. Comparing treatment with control, there was no significant difference in the results obtained for MI, EI, skin roughness, and mMASI score based on the obtained p-values. The MI, EI, skin roughness, and mMASI scores were decreased in both sides compared with baseline. With regards to time point, MI for both treatment and control groups showed a significant decrease starting from 1 month after (p = 0.010) through to 6 months after the last treatment session (p = < 0.001) compared with baseline. Erythema index for both groups showed a significant decrease at 2 months after the last treatment session (p = 0.009). Skin roughness and modified MASI scores obtained for both PN and NSS sides were significantly decreased as early as 2 weeks until 6 months after the last treatment session.

Fig. 1
figure 1

Mean Melanin Index (MI) values for treatment (PN) and control (NSS) sides from baseline and across all assessment points

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Fig. 2
figure 2

Mean Erythema Index (EI) values for treatment (PN) and control (NSS) sides from baseline and across all assessment points

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Fig. 3
figure 3

Mean skin roughness scores determine using Antera 3D for treatment (PN) and control (NSS) sides from baseline and across all assessment points

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Fig. 4
figure 4

Mean Modified Melasma Area and Severity Index scores for treatment (PN) and control (NSS) sides from baseline and across all assessment points

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Figure 5 illustrates the patient evaluations of improvement in their melasma lesions. Subjects in both treatment and control groups rated their improvement as “good to excellent” (51% to ≥ 75%) as early as 2 weeks after the last treatment session, with a gradual and steady increase in the number of patients reporting a higher rating of improvement at 1, 2, and 3 months after the last treatment. This trend continued until the sixth month, and was similar for both treatment and control sides. Figure 6 shows representative clinical photographs of two study participants, with comparisons between assessment points.

Fig. 5
figure 5

Patient’s subjective evaluation of melasma lesion improvement from baseline across all assessment time points, rated as follows: slightly better (< 25%), fair (26–50%), good (51–75%), and excellent (> 75%)

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Fig. 6
figure 6

Representative clinical photos of lesions on the malar areas in two study participants. A steady decrease in darkness, area, and homogeneity of patches is noted on both the PN (right) and NSS (left) sides from baseline until 6 months after the last treatment session. For each patient, a similar degree of improvement is seen on the control and treatment sides

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Table 2 Comparison of melanin index, erythema index, mMASI score, and skin roughness over six time points using generalized estimating equation. b, regression coefficient
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All study participants tolerated treatment well, with a reported mean pain score of 2.0 ± 1.8 (minimum, 0; maximum, 6). Only mild, transient erythema was observed after treatment. As mentioned above, recurrence of melasma occurred in three patients (10% recurrence rate) at 2, 3, and 4 months after the last treatment session, respectively. These patients opted to be treated with topical medications and no longer followed up throughout the course of the study.

Discussion

Chronic UV exposure is central to the dermal changes observed in melasma [21]. Metalloproteinases are stimulated by UV, and together with tryptase, type IV collagen is degraded leading to basement membrane disruption [22]. Melanocytes and melanin descend into the dermis, and crosstalk between epidermal melanocytes and dermal senescent fibroblasts [23] leads to the stimulation of melanogenesis [12, 24]. Solar elastosis and granzyme B from mast cells lead to increased vascularity in melasma lesions, a vicious cycle then ensues wherein endothelin-1 produced by endothelial cells further promotes melanogenesis [25]. Given its multifaceted pathogenesis, treatment protocols aimed at addressing the various structural aspects of melasma have been proposed [26].

We present the results of our comparison of the efficacy and safety of noninsulated, bipolar, pulsed-type microneedle RF with PN versus RF alone to treat melasma. Objective and subjective assessments demonstrated consistent improvement at all follow-up time points compared with baseline. In particular, MI, skin roughness, and the mMASI scores showed significant improvements. There was also a concurrent improvement in the ratings given by participants for their melasma lesions. Furthermore, tolerance of the procedure was reported. More importantly, the degrees of improvement on the control and treatment sides were similar, indicating that efficacy is possible with RF alone. The recurrence however of melasma in three patients indicates that RF treatment may not suffice as monotherapy for melasma.

Our results are in line with those obtained by Park et al. [27], wherein pulsed-type microneedling RF was used to treat melasma in 24 Korean women. Patients received one full-face treatment once a week, every 2 weeks for a total of five treatment sessions. Similar to the current study, decreased MI and EI values were obtained, together with a similar degree of improvement versus baseline in physician and patient ratings. The patients also tolerated the treatment well. In contrast to the current study, melasma recurrence was not observed. A possible reason for this would be the improved efficacy of five microneedling RF treatment sessions compared with three sessions in the current study. Another possibility would be that the current study’s longer follow-up led to increased detection of melasma recurrence. The works of Yoon et al. [12] and Lee et al. [13] demonstrated the capacity of microneedling RF to decrease basal epidermal pigmentation, decrease expression of MITF and tyrosinase, reverse UV-induced premature senescence, and restore expression of collagen type IV in the basement membrane, leading to the clinically observed lightening of solar lentigo [12] and age-associated facial pigmentation [13]. The authors believe that the observed lightening of melasma lesions in the current study may be attributed to the aforementioned effects of microneedling RF.

Improved skin roughness in the current study was also observed in melasma studies utilizing microneedling RF as monotherapy [10] and in combination with Q-switched Nd:YAG (QSNY) laser toning [9, 13, 28]. Improved skin texture and tone was observed after 5–10 treatments at 2–3-week intervals with microneedling RF alone [10]. Combined with QSNY, improvements were achieved after 5–7 weekly treatments [9], and ten treatments done once a week [28] and every 2 weeks [13]. The current study differs in that improved skin roughness as measured by Antera 3D was obtained with three treatment sessions done every 2 weeks. Microneedling RF produces cocoon-shaped zones of subablative thermal injury in the dermis [29]. In turn, this triggers a wound healing response with dermal remodeling, neocollagenesis, and elastogenesis [30]. Clinically, this enhances skin texture, pore size, and brightness [31,32,33]. These effects may explain the improvement in skin roughness observed in this study.

We were not able to demonstrate added efficacy of PN to microneedling RF. The degree of improvement in MI, EI, and mMASI scores was similar for both control and treatment sides. The main difference between the study by Noh et al. [17] and the current study is the manner of PN delivery. Noh et al. administered PN via intradermal injections, thus possibly facilitating direct placement of PN at the target depth. The current study topically applied PN immediately after microneedling RF treatment, with the purpose of enhancing PN’s transdermal delivery. However, there is no certainty that PN did indeed reach the intended target depth upon application after RF microneedling. Polynucleotides have a molecular weight ranging from 50 to 1500 kilodaltons (kDa) [18], and although a microneedling therapy system was used to enhance its delivery in an animal study [34], its skin permeability characteristics have not been directly evaluated.

One of the main limitations of this study is its small sample size. Another would be the lack of concurrent histological and immunohistochemistry studies that would aid in confirming and further characterizing RF’s effects. Future studies with a bigger population, together with both histological and immunohistochemical confirmation, are thus warranted. With the observed recurrence of melasma in three of our patients, optimum treatment parameters and intervals remain to be determined. In addition, more studies on PN are needed to determine a mode of delivery that will be both efficacious and tolerable to the patient.

Conclusions

The combination of invasive, bipolar, pulsed-type microneedling RF with PN is not superior compared with microneedling RF alone in the treatment of melasma. Microneedling RF may be considered as safe and efficacious for the improvement of skin roughness, and as an adjunctive treatment option for melasma.