Polydioxanone medical thread-embedding needles (Miracu) were purchased from Dongbang Acupuncture (Boryeong, Korea).
Seven-week-old male C57 black 6 (C57BL/6) mice were purchased from Samtaco Bio Korea, Ltd. (Osan, Korea) and were allowed to adapt to their new environment for 1 week. The mice were housed in certified standard laboratory cages and provided with food and water ad libitum prior to the experiment. A total of 15 mice were assigned to three groups (5 mice in each group): the normal saline- sprayed, minoxidil-sprayed, and thread-embedded groups. The animal protocol used in this study was reviewed and approved by Pusan National University’s Institutional Animal Care and Use Committee (PNU–IACUC) in accordance with established ethical procedures and scientific care (approval number: PNU-2014-0581).
As described in a previous study, the dorsal hair of 8-week-old C57BL/6 mice, whose hair follicles were in the telogen phase of the hair growth cycle, were depilated to induce homogeneous anagen induction . One day after removal of the dorsal hair, the mice in the negative and the positive control groups were treated with normal saline or 5% minoxidil, respectively, once a day for 16 days. Mice in the third group received TET 1 day after removal of the dorsal hair (Fig. 1). Fifteen threads (1 cm in length) were embedded in the dorsal skin of each mouse (1 cm2). The hair growth and thickness at the sites of the dorsal skin lesions in the C57BL/6 mice was measured by using dermoscopy (Sometech, Inc., Seoul, Korea).
The C57BL/6 mice were euthanized 16 days after the first treatment. Dorsal skin samples were fixed for 24 hours at room temperature in bouin’s solution and were then embedded in paraffin. Sections of tissue, 7-μm thick, were cut and mounted on glass slides, deparaffinizedin xylene, and processed for hematoxylin and eosin staining. Processed skin tissues were examined under light microscopy.
Bromodeoxyuridine (BrdU), which is used to label proliferating cells in tissue sections , was intraperitoneally injected at a dose of 50 μg/g of body weight (BW) twice per day for three consecutive days after hair removal, as described in a previous study . Dorsal skin tissues were collected on the 16 days after the first treatment and were subjected to immunostaining for BrdU.
The permeated tissue sections were incubated with a selected antibody according to the manufacturer’s instructions: mouse anti- BrdU antibody (1 : 200, Santa Cruz, CA, U.S.A.) for the detection of BrdU, proliferating cell nuclear antigen antibody (1 : 1000, Santa Cruz, CA, U.S.A.) for the detection of PCNA, fibroblast growth factor-7 (FGF-7) antibody (1 : 200, Santa Cruz, CA, U.S.A.) for the detection of FGF-7, or fibroblast growth factor-5 (FGF-5) antibody (1 : 200, Santa Cruz, CA, U.S.A.) for the detection of FGF-5. Bound antibodies were sequentially reacted with biotinylated goat anti-mouse immunoglobulin G (IgG) and avidin-biotinylated peroxidase complex (Vector Laboratories, Inc., Burlingame, CA, U.S.A.) for 30 minutes in a moisture chamber. Immunoreactivity was visualized by using diaminobenzidine.
Total ribonucleic acid (RNA) was isolated by using TRIzol reagent (Invitrogen) and was converted to complementary deoxyribonucleic acid (cDNA) by using AccuPower RT PreMix (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. Specific DNA sequences were amplified with AccuPower PCR PreMix (Bioneer, Daejeon, Korea). The oligonucleotide primer sequences were as follows: FGF-7, forward 5’-AGATCATGCTTCCACCTCGT-3’and reverse 5’-TGGGTCCCTTTCACTTTGCC-3’; FGF-5, forward 5’-ACCCGGATGGCAAAGTCAAT-3’ and reverse 5’-TGGTTTCACCGGTGGCTTTT-3’; GAPDH, forward 5’-GGAGCCAAAAGGGTCATCAT-3’ and reverse 5’-GTGATGGCATGGACTGTGGT-3’. Amplified products were analyzed in 1.0% agarose gel under ultraviolet light, and the imageswere captured using the GelDoc-It TS Imaging System (UVP, LLC, Upland, CA, U.S.A.).
Data were expressed as means ± standard deviations (SDs). Statistical differences between means were determined by using the one-way analysis of variance (ANOVA) for repeated measures. P-values less than 0.05 were considered significant.
The dorsal hairs of 8-week-old C57BL/6 mice in the telogen stage of the hair growth cycle were depilated to synchronize anagen induction. We assigned the mice into the following 3 groups (5 mice per group): group 1, normal saline- applied negative control; group 2, 5% minoxidil-treated positive control; group 3, medical thread-embedded experimental group. One day after depilation of the dorsal hair, the dorsal skin of the mice was topically sprayed with normal saline or 5% minoxidil or was embedded with medical thread. BrdU was administered twice a day for three consecutive days after hair removal. The dorsal skin was collected on the 16th day after treatment and was subjected to hematoxylin and eosin staining.