Evaluation of efficacy and safety of vitiligo treatment with micro‐needling combined with N‐Acetylcysteine and micro‐needling alone: A double‐blinded randomized controlled clinical trial

Vitiligo is a skin pigmentation disorder caused by the selective degradation of melanocytes. This study investigates the therapeutic effects of microneedling with and without N‐acetylcysteine (NAC) in patients with persistent and limited vitiligo.

The onset of oxidative stress results from decreased enzymatic activity in glutathione peroxidase and reduced glutathione production.5][16][17][18][19][20][21][22] Microneedling, a novel therapeutic modality in dermatology, activates the wound healing cascade by inducing physical trauma through needle penetration into the epidermis.It enhances the absorption of topical therapies beyond the thick stratum corneum.In recent years, microneedling has been extensively applied as a simple, costeffective, and tolerable treatment with both cosmetic and therapeutic benefits.The efficacy of microneedling in combination with other vitiligo treatments remains unknown and warrants further study.[25][26]

| Objectives
It's noteworthy that, to the best of our knowledge, few therapeutic trials have investigated the effect of glutathione and NAC on vitiligo.This double-blind intervention assesses the satisfaction, effectiveness, side effects, and tolerability of meso-microneedling treatment with 5% NAC ampoules and topical 4.7% NAC in vitiligo treatment as a novel approach compared to microneedling alone, as discussed in previous studies.

| Participants
Inclusion criteria included stable, symmetrical vitiligo lesions, aged 15 to 75 years, the absence of new lesions or lesion progression within the past year, and no prior treatments administered in the last 2 months.Exclusion criteria comprised allergies to N-acetylcysteine (NAC), current pregnancy or breastfeeding, intentions to conceive within the next 6 months, a history of hypertrophic or keloid scars, and the presence of pathological lesions, wounds, or infections at the microneedling application site.
The study adhered to ethical guidelines and commenced after obtaining ethics approval.

| Treatment protocol
Duration and Sessions: The treatment protocol spanned a duration of 2.5 months, involving six meticulously planned sessions with precisely spaced two-week intervals.Subsequent to the completion of the sessions, a comprehensive one-month follow-up was conducted, resulting in a total study duration of 3.5 months.Meso-Microneedling Procedure: Meso-microneedling, a key component of the intervention, was implemented using the NESOYA microneedling device.The application was randomized, targeting either the right or left side of the patient's face or body.The microneedles penetrated the epidermis to a depth of 1 mm, microneedling was continued until the presence of erythema or pinpoint bleeding.
Intervention in the Case Group Lesions: In the case group, following microneedling with the application of 2 mL of a 5% Nacetylcysteine (NAC) ampoule.The subsequent needling process extended from the lesion's periphery towards its central region, ensuring comprehensive coverage.the higher improvement rate with the application of NAC, the difference was not significant.

K E Y W O R D S mesotherapy, microneedling, NAC, N-acetylcysteine, vitiligo
Post-microneedling, a sterile gauze bandage was expertly applied, enhancing wound protection and minimizing potential complications.
Patients received explicit instructions to refrain from washing the treated area for a designated period of approximately 6 h, promoting optimal absorption of the applied therapeutic agents.
During treatment intervals, a formulated solution comprising 4.7% NAC in an Eucerin base were directed to apply specifically to the marked side of the case group vitiligo patch.This approach aimed to sustain the therapeutic impact of the intervention between sessions.
Microneedling in the Control Group: Conversely, in the placebo group, microneedling sessions were administered to lesions on the opposing side of the body.Distilled water was applied as the control substance during the microneedling procedure in this group.
This rigorous design allowed for a direct comparison between the effects of NAC intervention and the placebo, enabling a comprehensive evaluation of the efficacy of the treatment.

| Randomization
Simple randomization was employed.If a person had hypopigmented skin lesions on both sides, one side underwent microneedling with NAC, while the other side underwent microneedling without NAC.
In cases where all lesions were on the same side, lesions were randomly microneedled with or without NAC.

| Statistical methods
Data analysis followed the intention-to-treat principle, using SPSS statistical software version 22.0.Qualitative data were expressed as percentages, while quantitative data were presented as Mean ± SD.
Non-parametric tests, like the Mann-Whitney U test, were used for comparing means, with a significance level of 0.05 applied.All data were collected and analyzed anonymously, adhering to the Declaration of Helsinki.The study received ethical committee approval and was assigned the ethics code: IRCT20200826048531N1. (Ethics committee code: IR.IUMS.FMD.REC.1399.309).

| Participant flow diagram
As illustrated in Diagram 1, of the 29 evaluated patients, 22 met the inclusion criteria.However, two patients, one with two lesions and the other with three lesions (six lesions in total, 2 from the intervention group and 4 from the control group), were excluded during the treatment process.It is noteworthy that some patients had more than two lesions, and some lesions were not the same on both sides for all patients.Thus, out of 52 lesions, 46 successfully completed the treatment process.Of these, 10 lesions were on the face and trunk, and 36 were in the acral area.Twenty-four lesions were randomly selected and treated with meso-microneedling with NAC (intervention group), and 22 lesions were treated with microneedling with distilled water (control group).

| Descriptive analysis
Patients' mean age was 36.4 years (range: 18-58).Ten patients were female (50%), and 10 patients were male (50%).The mean duration of the disease was 12.4 years (see Figure 1).The mean duration of vitiligo stability in patients was 3.9 years (see Figure 2).The mean extent of lesions decreased from 100% at baseline to 89.8% and 82.3% in the intervention group and 92.5% and 89.5% in the control group 2 weeks after the third treatment session and 1 month after the treatment cessation, respectively.
The mean extent of lesions in acral lesions reached 94.4% and 90.6%, and 79% and 68.5%in non-acral lesions, respectively, 2 weeks after the third treatment session and 1 month after the end of the treatment.
Repigmentation grade scores reached 0.2 and 0.5 in the intervention group, 0.1 and 0.2 in the control group, 0.1 and 0.2 in acral areas, and 0.3 and 1 in non-acral areas 2 weeks after the third session and 1 month after the end of the treatment.
The mean tensity of lesions (stage) decreased from 3.5 at baseline to 3 and 2.5 in the intervention group, from 3.4 to 3 and 2.9 in the control group, from 3.5 to 3.2 and 2.9 in the acral areas, and from 3.3 to 2.2 and 1.8 in non-acral areas 2 weeks after the third session and 1 month after the end of the treatment, respectively.
The initial repigmentation grade score for all lesions was considered zero; from 46 lesions, 41 lesions still had a zero repigmentation grade score 2 weeks after the third session, four lesions had a Repigmentation grade scores in the intervention group showed more improvement than those in the control group, but this difference was not statistically significant (Figures 3 and 4).
The samples of treatment results in the intervention and control groups are shown in Figures 5 and 6.

| Analytical analysis
The results are presented in Tables 1-5.Two weeks after the third session, the extent of lesions (%) was significantly different in the intervention group (p < 0.01), but the repigmentation grade score was not (p = 0.1).However, both were significantly different 1 month after the treatment.
Tensity of lesions (stage) significantly changed 2 weeks after the third session and 1 month after treatment in the intervention group (p < 0.01).
The extent of lesions (%) was significantly different (p < 0.01), but the repigmentation grade score was not 2 weeks after the third session and 1 month after the treatment in the control group.
According to the results, despite the higher rate of improvement in the intervention group, the differences between the intervention and control groups were not significant, neither statistically nor clinically.
The extent of lesions (%) was significantly different (p < 0.01), but the repigmentation grade score was not (p = 0.08) 2 weeks after the third session in acral lesions.However, both were significantly different 1 month after treatment.Tensity of lesions (stage) was significantly According to Table 5, significant improvement was evident in the treatment process of non-acral lesions compared to acral lesions.
After a one-month follow-up period following the completion of treatment, there were no instances of vitiligo lesion recurrence observed among the patients in either treatment group.

| DISCUSS ION
This study aimed to evaluate the efficacy, side effects, tolerability, and satisfaction of microneedling treatment with and without topical NAC in the management of persistent vitiligo.2 weeks after the third session and 1 month after the end of treatment, a comprehensive assessment was conducted.
The healing process of patients' lesions during microneedling treatment, with or without medication, demonstrated significant improvement.Improvement in the extent of lesions (%), repigmentation grade score, and tensity of lesions (stage) in acral lesions was excellent.Since acral lesions are resistant to treatment and no effective treatment has been reported for them yet, our finding is a new achievement.Also in the present study, significant improvement was seen in the healing process of non-acral lesions compared to acral areas.
Previous studies have not investigated the effect of microneedling with NAC mesotherapy broadly.Few studies have examined F I G U R E 3 Mean extent of lesions (%) 2 weeks after the 3rd session and 1 month after the 6th session in the intervention and control groups.

F I G U R E 4
Mean of tensity of lesions (stage) at the initial session, 2 weeks after the 3rd session and one month after the 6th session in the intervention and control groups.the effect of microneedling alone, in combination with other topical medications such as tacrolimus, latanoprost, and 5-fluorouracil, or in combination with phototherapy.
A study in 2020 by Samar Saleh, which used microneedling with 70% TCA for fixed vitiligo lesions, showed a significant improvement in the repigmentation of fixed vitiligo lesions. 27However, they treated patients in one group, and it is not possible to determine how much repigmentation is related to microneedling.In the present study, we had intervention and control groups; therefore, the results can be evaluated more precisely.Furthermore, Kumar et al. 28 in 2019 used microneedling and 5-fluorouracil cream for the treatment of fixed and non-fixed lesions of vitiligo and reported a significant improvement without any side effects, which is in line with our study.
In the study by Khashaba et al. 29 in 2018, vitiligo lesions were classified into three treatment groups: NBUVB phototherapy, microneedling with topical triamcinolone, and a combination of phototherapy and microneedling.No significant difference was observed among the treatment groups, although the best result was obtained from the third group.
Another study by Amira et al. 30 in 2019 compared the effects of microneedling combined with phototherapy.They reported a significantly improved response in the group that received microneedling before phototherapy compared to phototherapy alone.
In a study by Lima et al., 31 who used microneedling and 0.1% tacrolimus for four sessions with intervals of 15 days to treat resistant vitiligo lesions, facial lesions showed better response to treatment, which is similar to our study.However, they did not report repigmentation in acral lesions.Although in our study, a significant response was observed in refractory acral lesions.
Another study by Howyda et al. 32 on permanent vitiligo lesions, divided patients into three groups: microneedling with tacrolimus, microneedling alone, and tacrolimus twice daily for 6 months.The treatment was 12 sessions every 2 weeks.The rate of improvement in the combination therapy group (66.6%) was significantly F I G U R E 5 Sample of treatment results in intervention group at the initial session (A), 2 weeks after the 3rd session (B) and 1 month after the 6th session (C).
F I G U R E 6 Sample of treatment results in control group at the initial session (A), 2 weeks after the 3rd session (B) and 1 month after the 6th session (C).higher than that in the other two groups (33.3%) and also the rate of improvement in the combination therapy group was significantly higher in the extremities.
The mechanism underlying microneedling revolves around the deliberate creation of controlled micro-injuries, a process known to stimulate collagen synthesis and potentially enhance drug delivery. 33This aligns seamlessly with research conducted by Mekawy, K. M. M. et al. 33 and Ghandehari, R. et al., 34 both delving into the application of microneedling for the delivery of tranexamic acid in the treatment of melasma.While these studies differ in their specific focuses, they converge on highlighting the safety and effectiveness of microneedling in delivering therapeutic agents.
Limited studies on pigmentary diseases, including the research by Debasmita, B.et al., 35 have compared the efficacy of Q-switched Nd:YAG laser and microneedling, both combined with topical tranexamic acid (TA), in the treatment of melasma.The results indicated comparable efficacy of both modalities, aligning with our study's focus on evaluating microneedling for vitiligo. 35rthermore, investigations such as Mekawy, K. M. M. et al. and Ghandehari, R. et al. 33,34 compared micro-needling and fractional CO2 laser for drug delivery of tranexamic acid in the treatment of melasma, demonstrating the equivalence of these modalities.While our study focuses on vitiligo, these findings provide valuable insights into the broader landscape of microneedling and laser treatments.
Considering the mechanism of melanocyte stimulation, studies conducted by Bailey, A. J. M. et al., and Mamdouh Kamal Dawaud, S. et al. 36,37 delve into the efficacy of microneedling and lasers in delivering tranexamic acid for the treatment of hyperpigmentation.While not directly related to vitiligo, these studies significantly contribute to our understanding of the mechanisms underlying the effects of microneedling and laser treatments on pigmentation.
In the continuum of existing evidence, our study carves a unique niche by specifically investigating the impact of microneedling with NAC mesotherapy in the treatment of permanent vitiligo lesions.In contrast to previous studies that explored microneedling alone or in combination with various topical medications, our controlled intervention, coupled with a comparison with a control group, adds precision to the evaluation of outcomes.

| Limitations
The limitations of this study included irregular patient visits for treatment sessions, posing challenges in maintaining a consistent treatment schedule.Additionally, the study faced the limitation of not being able to directly evaluate the accuracy of NAC topical ointment use by patients at home.Future studies may benefit from incorporating strategies such as patient diaries or electronic monitoring to address this limitation.

2 | ME THODS 2 . 1 |
Trial designThis clinical trial employed a double-blind, randomized, controlled design.Human participants were randomly selected from individuals exhibiting symmetrical vitiligo lesions on their trunk, limbs, or face.Recruitment occurred at Rasool Akram Medical Complex's dermatology clinic between September 2020 and September 2021.
At the study's initiation, demographic data, initial Modified Vitiligo Extent Tensity Index (VETI) scores, VisioFace® documents, and initial photographs were meticulously documented by the primary researcher.A checklist recorded potential microneedling and drug-related complications for each patient.Additionally, patients' responses to drug tolerance, compliance with regular usage, and therapeutic satisfaction were recorded.A second researcher, blinded to the study, evaluated patients based on VisioFace® images, specialized clinical observations, and the Modified VETI score.The Modified VETI Score attributed 100% involvement to each lesion for a more accurate evaluation of extent, with severity determined by lesion tensity, percentage of repigmentation, and repigmentation severity.In the first session, all lesions were assigned 100% involvement and zero repigmentation grade scores.Lesion tensity (stage), based on cutaneous and hair pigmentation in vitiligo lesions, was classified as follows: Stage zero: Natural pigmentation.Stage 1: Hypopigmentation (including lighter skin pigmentation and hair).

Stage 2 :
Complete depigmentation with black hair and perifollicular pigmentation.
Among the study participants, eight had a positive family history, and six mentioned a history of hypothyroidism.The mean satisfaction score with the treatment was 4.6 out of 10, with 12 patients giving a satisfaction score above five.

D I A G R A M 1
Details of number of patients and lesions during enrollment, allocation, follow-up and data analysis.repigmentation grade score of one, and one lesion had a repigmentation grade score of two.After 1 month of treatment, four lesions still had a zero repigmentation grade score, eight lesions had a repigmentation grade score of one, three lesions had a repigmentation grade score of two, and one lesion had a repigmentation grade score of three.

F I G U R E 1
Distribution of disease duration of patients.F I G U R E 2 Distribution of duration of vitiligo stability of patients.different 2 weeks after the third session and 1 month after the treatment in acral lesions (p < 0.01, both of them).

3
Comparison of extent of lesions (%), repigmentation grade score and tensity of lesions (stage) 2 weeks after the 3rd session and 1 month after the 6th session between the intervention and control groups.
The robustness of our findings is fortified by the randomized clinical trial design, which sets our study apart from others.Utilizing the density of lesions (stage) and repigmentation grade score as evaluation criteria aligns with established measures, particularly the Extent of lesions (%), repigmentation grade score and tensity of lesions 2 weeks after the 3rd session and 1 month after the 6th session in the intervention group and comparison with the baseline.
Collectively, our study, when viewed in conjunction with relevant literature, underscores the significance of considering diverse treatment modalities and their mechanisms of action within the vitiligo landscape.The findings not only consolidate the existing knowledge but also advocate for further exploration of microneedling with NAC mesotherapy as a promising avenue for managing persistent vitiligo lesions.

in Two weeks after the 3rd session One month after the 6th session Location p-Value Location p-Value Acral Non-acral Acral Non-acral
Extent of lesions (%), repigmentation grade score and tensity of lesions 2 weeks after the 3rd session and 1 month after the 6th session in acral lesions and comparison with the baseline.Comparison of extent of lesions (%), repigmentation grade score and tensity of lesions 2 weeks after the 3rd session and 1 month after the 6th session between acral and non-acral lesions.
TA B L E 5