Efficacy of topical vitamin D combined with microneedling in the treatment of vitiligo: A comparative study

Vitiligo is a depigmenting disorder caused by the destruction of melanocytes by various mechanisms which affect melanocyte function and survival. Different therapeutic approaches for vitiligo include nonsurgical and surgical methods but effective therapy is still challenging. There are few studies that suggest the role of vitamin D analogs in the repigmentation process with encouraging results. To our knowledge, this is the first study to evaluate the effect of topical vitamin D (cholecalciferol) combined with microneedling in the treatment of depigmented patches of vitiligo.


| INTRODUC TI ON
Vitiligo is an asymptomatic depigmenting disease that causes progressive damage of functional melanocytes which may be related to other autoimmune disorders. 1 It has a psychological effect on the quality of life and stigmatization, especially in dark-skinned patients.
The course of the disease is variable and unpredictable. 2 Although the etiopathogenesis of vitiligo is still unclear, there are many theories accused of its etiology, which include autoimmune mechanisms, genetics, psychological, and environmental factors. 3 There are many treatment modalities to regain pigmentation; however, there is yet no acceptable treatment for this challenging disease. 4,5 Microneedling can be used in the treatment of vitiligo as transdermal drug delivery. 6 It also produces micro-injuries in the epidermal layer, which stimulates wound healing resulting in the production of various growth factors that enhances melanocyte migration and helps repigmentation. 4 Vitamin D is postulated to maintain self-tolerance and stimulate the immune system against infections. 7 The advance in the understanding of 1,25 dihydroxy vitamin D3 and its immunomodulatory character endorses a broader use of this hormone in treating autoimmune disorders and even in the prevention of allograft rejection. 8 Topical vitamin D3 analogs (D3A) can be used with other therapies as an adjuvant to its immunomodulatory effects suppressing Tcells, positive effect on melanocyte differentiation, and stimulating melanogenesis. 7 In this study, we are evaluating the effect of microneedling alone versus microneedling combined with Topical Vitamin D (cholecalciferol) in stable vitiligo. This is the first study to date that evaluate the effect of topical cholecalciferol with microneedling to enhance repigmentation.

| PATIENTS AND ME THODS
A same-patient comparative prospective study was carried out on 25 patients complaining of stable vitiligo. Cases were recruited from the outpatient clinic from the interval of October 2019 until May 2021. Informed written consent was taken from each participant.
Approval from the Research Ethics Committee of the faculty of medicine of our university was also achieved.

| Patients
Patients complaining of stable vitiligo with at least two or more vitiligenous patches. Patients should have no increase in the extent of the lesion regarding number and size for a minimum 6-month duration.
We excluded patients with acral vitiligo, patients under the age of 18 years old, and pregnant or nursing females. Patients with coagulation disorders, active infections, and patients with viral hepatitis B or C, and HIV were also excluded. Patients with other dermatological and systemic diseases, patients with needle phobia, or previous treatment in the previous 3 months were also excluded from this research.

| Treatment protocol
Full history was taken including the history of systemic illnesses or dermatological disorders for all patients. History of previous treatment of vitiligo in the previous 3 months.

| Technique
The procedure was explained to every patient; then, microneedling was done for 2 vitiliginous patches. The treated site was cleaned with 70% alcohol and then anesthetized with topical lidocaine cream under occlusion for about 30 min. Microneedling was performed with dermapen (Dr. Pen model ultima A1-W) with needle length (0.5-1 mm) according to the treated site in all directions until multiple bleeding points appeared.
The first patch was treated with microneedling alone, and in the other patch, drops of topical cholecalciferol (Devarol-S 200 000 I.U solution) 0.5-2 ml according to the size of the vitiligo patch were applied during microneedling till a pinpoint bleeding point was seen, and then, the remaining vitamin D3 was applied after microneedling.
The procedure was done every 2 weeks with a maximum number of 12 sessions (6 months). Follow-up was done monthly for 3 months after the end of treatment sessions to detect any adverse effects or recurrence.

| Assessments
Photographs were taken at the starting point, before each session, and 3 months after the last session. The repigmentation responses were assessed according to the investigator's global assessment (IGA) by the researchers and two blinded dermatologists as follows: No change, poor improvement if the repigmentation is <25%, good response if 26%-50% pigmentation occurs, very good if 51%-75% pigmentation, and excellent response if 76%-100% improvement. 9 The skin texture, color, and pattern of pigmentation were examined every session.

| Patient's satisfaction
After the follow-up period, patients were asked about their degree of satisfaction and the ease of the session according to the following: 0 = not satisfied at all, 1 = poor satisfaction, 2 = moderate satisfied, and 3 = totally satisfied.
Patients were assessed clinically and asked to report any complications: pain, erythema, ulceration, ecchymosis, infection, burning sensation, hyperpigmentation, or any allergic reactions.

| Statistical analysis
The collected data was revised, coded, and entered into the Statistical Package for Social Science (IBM SPSS) version 23. The quantitative data were presented as mean, standard deviations, and ranges when their distribution was found parametric, the comparison between groups with qualitative data was done by using chi-square test and Fisher's exact test instead of the chi-square only when the expected count in any cell was <5. The confidence interval was set to 95%, and the margin of error accepted was set to 5%. So, the p < 0.05 was considered significant.

| RE SULTS
This study included 25 patients who had stable vitiligo, 11 of the patients (44.0%) were males and 14 (56.0%) were females. The ages ranged from 18 to 52 years (mean ± SD: 29.8 ± 7.43 years). As regard the duration of vitiligo, it ranged from 1 to 8 years. Eleven patients (44%) had a positive family history of vitiligo while 14 (56.0%) had a negative family history.
The clinical type of vitiligo in the studied patients was as follows: 9 patients (76%) had non-segmental vitiligo, while 6 (24%) had segmental vitiligo.
In the patches treated with topical vitamin D after needling, 10 patches (40%) had an excellent response, 8 (32%) had a very good response, 4 (16%) showed a good response, and 3 patches (12%) showed a mild response. On the contrary, in the patches treated with microneedling only, an excellent response was detected in only 4 patches (16%), 6 and 8 patches (24% and 32%) had a very good response and good response, respectively, and 7 patches (28%) showed a mild response.
In the treated patches, there was no statistically significant difference between the two groups. Despite this, the response was slightly better in the patches treated with microneedling and vitamin D than in the patches treated with microneedling only ( Table 2).
There was no statistically significant difference as regard response to treatment between segmental vitiligo and non-segmental The patients' satisfaction showed no statistically significant difference between both groups. The pattern of pigmentation was different in the two groups, in which the dominant type in the patches treated with microneedling and vitamin D was the diffuse type of pigmentation, while in the patches treated with microneedling alone the pigmentation was perifollicular. There were statistically significant differences between the two groups (p = 0.013) ( Table 3).
For the patches treated with microneedling, an excellent response was reported in 25% of the patches on the trunk, 16.7% of the patches on the elbow, and 14.3% of the patches on the legs. On the contrary, in the patches treated with microneedling and vitamin D excellent response was seen, in 83.3% of the knee patches, 42.9% of the patches on the legs, and 25% of the patches on the trunk.
Regarding the side effects, there was no statistically significant difference between both groups considering pain and erythema.
The exfoliation was statistically significantly higher in the microneedling combined Vitamin D group where 11 patients (44.0%) had exfoliation whereas only 1 patient (4.0%) in microneedling group p value <0.001* (Table 4).

| DISCUSS ION
Microneedling is considered a minimally invasive technique that is used to treat different dermatological disorders. Microneedling is considered a safe and effective treatment method for vitiligo. The combination of microneedling with other treatment modalities showed increased efficacy. 1 Low vitamin D levels are found to be related to many autoimmune diseases, for example, systemic lupus and rheumatoid arthritis. 10 The mechanism by which vitamin D influences autoimmune disease is still unclear, but in vitro studies suggest the regulation of immune cells by vitamin D. Vitamin D is considered a vital hormone that is produced from the skin and has a role in skin pigmentation. 8 This is the first study to date that used topical cholecalciferol  There are few studies, that assessed the role of topical vitamin D in the treatment of vitiligo; however, this is the first study to evaluate the combined effect of topical vitamin D with microneedling.
Our hypothesis was that vitamin D has an enhancing effect on melanogenesis by stimulation of immature melanocytes precursors and its immunomodulatory effect. 8 Oh et al. 19 suggested that vitamin D3 enhances tyrosinase activ- This agrees with many studies with different treatment modalities that suggested that the results were better noticed on the face followed by the trunk and limbs. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] The pattern of pigmentation was statistically significantly different between the two groups; in the patches treated with microneedling and vitamin D, the pigmentation was diffuse, while in the patches treated with microneedling only the pigmentation was follicular.
The repigmentation pattern depends on available sources of melanocytes from the three sites: the hair follicle act as a reservoir for melanocytes, from the border of depigmented patches, and from survived melanocytes within depigmented patches. These melanocytes can initiate a perifollicular, marginal, and diffuse repigmentation pattern. 24 It has been reported that SV responds poorly to medical treatment and stabilizes after an initial rapid spread. 25 Accurate data about the response of treatment to SV is not available as it is considered resistant, and most studies do not distinguish this type of vitiligo. 26 In our study, the response to treatment of segmental versus non- and longer follow-up periods, are required to achieve the ideal concentration.

CO N FLI C T O F I NTE R E S T
There are no financial disclosure or conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.