Micro-Injury Induces Hair Regeneration and Vitiligo Repigmentation Through Wnt/ β-Catenin Pathway

Background: Extrinsic injury can evoke intrinsic stimulation subquently initiate physiological repair process. Several kinds of injury have been studied to promote hair growth and skin pigmentation. In this study, we ask if proper injury could be employed to create local stimuli subsquently to induce hair regeneration and vitiligo repigmentation. Methods: We rstly manufactured a novel designed device to precisely control all micro-injury parameters. Then the most appropriate micro-injury extent was evaluated without over-damage to skin. The effects of micro-injury on hair regeneration and vitiligo repigmentation were examined by macroscopical observation, histological staining, gene and protein expression analysis. Results: We discover that proper micro-injury effectively induces hair regeneration by activating the hair follicle stem cell proliferation and migration downwards to hair matrix, nally shifting the hair follicle stage from telogen into anagen. On vitiligo model mice, micro-injury also induces the hair follicle melanocyte stem cell migrate upwards to interfollicular epidermis, activate and give rise to melanocytes to repopulate vitiligo lesion. Mechanism analysis indicates that the canonical Wnt/b-catenin pathway plays a key role in the micro-injury induced regeneration process. Conclusions: The present study demonstrates that micro-injury has great potential in inducing hair regeneration and vitiligo repigmentation, laid a foundation to develop micro-injury based treatment method in alopecia and vitiligo.


Background
Hair follicle harbors heterogenous stem cell populations contributing to hair cycle, hair color, epithelial renewal and skin color under physiological condition and/or stress condition. Extrinsic environmental stimuli, such as hair plucking [1] and mechanical stretch [2] can induce hair growth. Morever, large full thickness skin excision can induce hair negeosis in the central area of wound, a phenomenon called wound-induced HF neogenesis (WIHN) in mice [3] . These elegant studies inspire us that proper physical method could be employed to promote hair growth.
Physical stimuli, such as PUVA [4] , UVB (NB-UVB) [5] , CO 2 fractional laser [6] , microneeding [7] were also employed in clinic to treat skin depigmentation disorders including vitiligo. In addition, clinical observation shows that vitiligo recovery usually experinces a process called perifollicular repigmentaion [8] . Recent study using lineage tracing mice exactly shows that hair follicle melanocyte stem cell (HF-McSCs) give rise to epidermal melanocyte after injury or UVB treatment [9] . All these studies suggest that hair follicle serve as a cell reservoir for fuctional interfollicular epidermal melanocytes upon injuries.
Wnt signal plays a vital role both in hair regeneration and vitiligo repigmentation. The canonical Wnt signaling pathway hallmarked by the event of cytosolic β-catenin stabilization and its subsequent translocation to the cell nucleus, where it interacts with LEF/TCF transcription factors to activate downstream gene transcription [10] , including the key melanogenesis genes Mitf, TRP1, TRP2 (also known as DCT) and tyrosinase (TYR) [11,12] .
However, what extent of proper injury can induce local reaction and whether the injury stimuli can induce hair growth and/or skin pigmentation need more exploration. In this study, by a ne customer-made instrument we discover that proper injury stimulation effect both on hair regeneration and vitiligo repigmentation through injury induced Wnt/β-catenin pathway.

Methods And Materials
Customer-made micro-injury instrument To standardize the micro-injury, we designed and made a ne instrument to control the depth, density and temperuture of the injury (Fig. S1A). The density was achieved by a disposable multi-needle head ( Fig.   S1B), in the present study we set at 0, 10, 20, 30, 40, 50, 60 needles/cm 2 , which create the corresponding micro-injuries/cm 2 . The depth was controlled by a fastener on the head, in the present study was set at 2 mm, which approximately reach dermis. The temperuture is controlled by an electromagnetic heating unit to make the needles at 500℃ for easily acupucturing into skin. All parameters were set by the instrument instead of controlling by manual operation. Animals C57BL/6 mice were obtained and feed at Laboratory Animal Center of the Third Military Medical University, Chongqing, China. Dct-LacZ transgenic mice were generated by Prof. Ian Jackson [13] , then were backcrossed with C57BL/6J. and were kindly provided by Prof. Haiying Guo [14] . All animal procedures were performed under the ethical guidelines of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University.

Vitiligo mouse model
Adult C57BL/6 mice tail were employed for vitiligo model because melanocytes resist in tail epidermis. We established vitiligo model as previously [15] . Brie y, 50 µg TRP2-180 (Anaspec, Fremont, CA, USA), 5 µg LPS (Invivogen, San Diego, CA, USA) and 5 µg ODN 1826 (invivogen, San Diego, CA, USA) were mixed for immune-induced depigmentation. Seven week-old male C57BL/6 mice were immunized subcutaneously at the hind footpad once a week for 2 weeks, followed by immunizing intradermally in the tail dermis once a week for 2 weeks. Control mice were immunized with PBS. Four to 5 weeks after the last immunization, immunized mice should develop a depigmented skin lesion around the tail injection site.

Micro-injury, XAV939 injection and sample collection
For hair regeneration, 7 week-old male C57BL/6 mice whose hair cycle enter in telogen were employed for micro-injury. After anaesthesia and hair cut, mice were made micro-injuries distributedly on their dorsal skin twice a week at different dose or different time. Self-control (sham injury) was made on neighber area of each dorsal skin. Hair growth was monitored by taking pictures before each times of injury. Skin samples from dose group were harvested for histological examination at the end of 3 weeks. Skin samples from time group were harvested 2 days after the last injury.
For vitiligo repigmentation, established vitiligo mice were employed for micro-injury once a week. We chose 30 micro-injuries/cm 2 because this dose could make greatest injury stimulaiton without obvious skin damage. Tail skin color was monitored by taking pictures before each times of injury. Skin samples were harvested for histological examination, gene and protein analysis at indicated timepoint.

Statistical analysis
Data were presented as the mean ± S.D. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). A two-tailed Student's t-test was performed to calculate statistical signi cance for differences between two groups. In all cases, P ≤ 0.05 was considered statistically signi cant.

Micro-injury induces hair regeneration
For choosing an optimal injury density, 6 groups of mice were created with 10, 20, 30, 40, 50, 60 microinjuries/cm 2 on their dorsal skin twice a week for 3 weeks. Comapred to self-control (sham injury, upper circles), micro-injury (lower circles) can induce hair growth from 10-30 micro-injuries/cm 2 on a dosedependent manner. While 40-60 micro-injuries/cm 2 can not induce hair growth effectively, probably due to the over-damage to hair follicles (Fig. 1A). Histology results show that hair follicles entered anagen in 30 micro-injuries/cm 2 group while the hair follicles with sham-injury remain in telogen, histological analysis also indicates the over-damage of skin in 60 micro-injuries/cm 2 group, which could explain the delay of hair growth (Fig. 1B). Therefore we next chose the optimal 30 micro-injuries/cm 2 to study the times effect of micro-injury on hair regeneration. Macroscopical observation show that micro-injury promotes hair growth with micro-injury times increasing (Fig. 1C). H.E. staining show that micro-injuries can induce hair cycle transition from telogen to anagen in a time-dependent manner (Fig. 1D).
Immuno uorescence show that micro-injuries induce K15 + hair follicle stem cell proliferation (Fig. 1E). These results demonstrate that micro-injury induces hair regeneration by shifting the hair follicle stage from telogen into anagen.

Micro-injury induces vitiligo repigmentation
We rstly established a vitiligo model on mouse tail skin. TRP2-180 peptide immunization readily induced skin depigmentation ( Fig. 2A). Compared to the model group which depatched skin remain white after 2 months, micro-injury grandually induces repigmentation during 2 months, indicate the injury stimulation is effective on vitiligo treatment ( Fig. 2A). Fontana-Masson staining also verify that melanocytes regenerate increasely in vitiligo lesion (Fig. 2B). LacZ staining in model group locates the Dct-LacZ + hair follicle melanocyte stem cells (HF-McSCs) in hair bulge (Fig. 2C). While after micro-injury treatment, LacZ + HF-McSCs migrate into epidermis, indicating that HF-McSCs response for perifollicular repigmentation in vitiligo lesion (Fig. 2C). Gene expression analysis show that all melanogenesis gene expression level increase with micro-injury (Fig. 2D ). Western blotting also show that pigment-related proteins increase signi cantly (Fig. 2E).

Discussion
Studies have pointed out the importance of using injury-induced effect in promoting hair growth. The outstanding one of them is the study on hair plucking-induced hair regeneration [1] , which shows the hair plucking-created minor injury lead to a larger scale hair regeneration by activating the neighberhood hair follicle stem cells. The other representative one is the wound-induced hair neogenesis (WIHN) study [3] , showing us a large injury enviroment (> 1 cm 2 on mouse skin) simulates the embryonic development path thus forms new hair at the centre of the wound. Absolutely, these studies showed us a injury-based therapy potential on hair regrowth even regeneration. However, these studies are concept-veri ed and are currently di cult to apply in clinic treatment, for hair plucking is unacceptable for patients who are anxious about hair lossing and thirst for hair regrowth, as well as a large tramua is di cult to achieve in clinic practice. In this study, we used a customer-made heat micro-needle instrument to create standard minor injuries on skin and veri ed it's effective application in promting hair growth, establishing it's potential application in treating hair loss disorders.
As a classical type of depigmentation skin disorders, vitiligo treatment is still under developing. Usually clinicials treat the disorder from two steps, delay and hold the broadening of the white patch in progressive stage followed by re-establishing pigmentation during stable stage. Among several sources for the re-populate melanocytes, hair follicle represents a primary cell reservoir indicating by a familiar recovery process called hair follicle island [8] , which showing a hair-initiated pigmentation "island" in white patch "ocean". However, current rst-line method to treat vitiligo are time-consumered, thus needs to develop additional methods. Injury has showed potential in inducing skin pigmentation such as microneeding alone [7] or combined with phototherapy [16,17] , CO 2 fractional laser [6] . In this study, we showed an effective melanocytes regeneration induced by creating micro-injury on vitiligo leision, validating the conception that bulge McSCs migrate from the hair follicle to the epidermis after wounding and providing a scienti c basis for developing alternative method beyond current therapy methods.
Further studies, informed by the work reported here, are needed to complete our understanding of how the extrinsic environmental micro-injury evoke the intrinsic Wnt ligands subseqently leading to the initiation of the regeneration process, and what kind of suitable regimens for micro-injury treatment of AA and vitiligo in clinic treatments.

Conclusions
In conclusion, the present study designed and manufactured a precisely controlled instrument to create micro-injury on skin and tested their effect on hair regeneration and vitiligo repigmentation. Resluts demonstrate that proper micro-injury effectively mobilizated the HF-SC migrate downwards to hair matrix to contribute to hair growth, as well as the HF-McSCs migrate upwards to interfollicular epidermis to repopulate vitiligo melanocytes (Fig. 4C). Mechanism analysis show that Wnt/β-catenin pathway plays a key role in the micro-injury induced process. This study laid a foundation to develop micro-injury based treatment method in alopecia and vitiligo.

Consent for publication
All the authors give their consent for publication.