Induction of hair growth in hair follicle cells and organ cultures upon treatment with 30 kHz frequency inaudible sound via cell proliferation and antiapoptotic effects

Abstract

Androgenic alopecia is a hair loss disease mediated by dihydrotestosterone (DHT) and is currently treated using minoxidil, finasteride, or low-level laser therapy. However, these treatments have side-effects, indicating the need for an alternative treatment. In the present study, it was demonstrated that inaudible sound at 30 kHz significantly induced proliferative and anti-apoptotic effects in human dermal papilla cells (hDPCs) and outer root sheath keratinocytes. Cell viability assay, ELISA, reverse transcription quantitative PCR and TUNEL assays were performed to evaluate the effect of inaudible sound. Inaudible sound was also demonstrated to significantly inhibit the hair loss signals induced by DHT treatment in hDPCs. Furthermore, inaudible sound significantly induced hair follicle (HF) elongation and hair matrix keratinocyte proliferation in human HF organ culture. Overall, the results suggested that inaudible sound may be effective in treating hair loss and could be used to develop a new hair loss treatment approach.

Keywords: antiapoptotic; hair follicles; hair growth; human dermal papilla cells; inaudible sound; outer root sheath; proliferation.

Figure 1

Inaudible sound stimulates proliferation and expression of hair growth-related factors in human dermal papilla cells. (A) Electrical sine waves were generated using a custom made function generator and amplifier set. (B) Cells were treated with inaudible sound (30 kHz) for 4 h and the Cell Counting Kit-8 assay was performed on day 2. (C) VEGF mRNA expression levels were assessed using reverse transcription-quantitative PCR. The mRNA expression levels of VEGF were normalized against the mRNA expression levels of RPLP0, a house-keeping gene. (D) VEGF concentration was determined using ELISA. Data are presented as the mean ± SD of three independent experiments. Statistically significant differences were determined using a one-way ANOVA followed by Tukey's honestly significant difference test or a paired Student's t-test. ^**P<0.01 and ^***P<0.001. VEGF, vascular endothelial growth factor; RPLP0, ribosomal protein lateral stalk subunit P0; NT, non-treated control.

Figure 2

Inaudible sound abrogates DHT-induced catagen-related secretion factors in human dermal papilla cells. Cells were treated with inaudible sound at 30 kHz for 4 h and DHT for 48 h, or with both 30 kHz inaudible sound and DHT. (A) DKK-1 and (B) TGF-β1 concentrations were determined using ELISA on day 2. Data are presented as the mean ± SD of three independent experiments. Statistically significant differences were determined using a one-way ANOVA followed by the Tukey's honestly significant difference test. ^**P<0.01, ^***P<0.001, ^#P<0.05 and ^##P<0.01. DHT, dihydrotestosterone; DKK-1, Dickkopf-1; NT, non-treated control.

Figure 3

Inaudible sound inhibits DKK-1 and TGF-β1-mediated apoptosis and regulates the expression of apoptosis-related genes in outer root sheath keratinocytes. Cells were treated with a combination of apoptosis stimulator (DKK-1 and TGF-β1) or inaudible sound at 30 kHz or with a combination of both. The (A) Cell Counting Kit-8 and (B) TUNEL assays were subsequently performed on day 2. (C) Bax and Bcl-2 mRNA expression levels were analyzed using reverse transcription-quantitative PCR. mRNA expression levels were normalized against the mRNA expression levels of RPLP0, a housekeeping gene. Data are presented as the mean ± SD of three independent experiments. Statistically significant differences were determined using a one-way ANOVA followed by the Tukey's honestly significant difference test. ^**P<0.01, ^***P<0.001, ^#P<0.05, ^##P<0.01 and ^###P<0.001. DKK-1, Dickkopf-1; RPLP0, ribosomal protein lateral stalk subunit P0; NT, non-treated control.

Figure 4

Inaudible sound promotes HF elongation and hair matrix keratinocyte proliferation in human HF organ culture. In total, 180 human scalp HFs with intact dermal papillae were obtained and treated for 5 days with inaudible sound at 30 kHz or with MNX. (A) HF length was analyzed under a stereomicroscope on day 2 and 5. (B) Relative length of each hair shaft. MNX (50 µM) served as the positive control for stimulating HF growth. Red dotted line, hair starting point. (C) Immunofluorescence staining of Ki-67 (green fluorescence) was performed after treating human HFs with inaudible sound at 30 kHz for 4 h every day, for 3 days. Nuclei were counterstained with DAPI (blue fluorescence). (D) For quantitative analysis, the number of Ki-67⁺ cells were counted and normalized to the number of DAPI-stained cells. Data are presented as the mean ± SD of three independent experiments. Statistically significant differences were determined using a one-way ANOVA followed by Tukey's honestly significant difference test or a paired Student's t-test. ^*P<0.05 and ^**P<0.01. HF, hair follicle; MNX, minoxidil; NT, non-treated control.

Figure 5

Potential mechanism for the stimulation of hair growth by inaudible sound. A schematic summarizing the proposed mechanism of action of inaudible sound. The present study demonstrated that inaudible sound potentially induced cell proliferation and the release of VEGF, a hair growth-related growth factor and abrogated DHT-induced catagen-related secretion factors, such as DKK-1 and TGF-β1 in human dermal papilla cells. Furthermore, inaudible sound was demonstrated to prevent apoptosis induced in HFs by catagen-related secretion factors and induced the proliferation of HF keratinocytes. VEGF, vascular endothelial growth factor; DHT, dihydrotestosterone; DKK-1, Dickkopf-1; HF, hair follicle.