Isolation and culture of human umbilical cord mesenchymal stem cells

The study was approved by the Ethics Committee of Shantou University Medical College (SUMC, Shantou, China). Umbilical cords were obtained from healthy pregnant women (excluding those with HIV, hepatitis, and syphilis) at term (38–40 weeks) through cesarean section in the Obstetrics and Gynaecology Department of the Second Affiliated Hospital of SUMC, after obtaining written informed consent. HuMSCs were prepared as previously described [26]. The umbilical cord was cut into 2–3 cm pieces with a sterile scalpel and cleaned with sterile phosphate-buffered saline (PBS). After removing the umbilical arteries, veins, and umbilical cord adventitia from the umbilical cord, Wharton’s jelly was obtained. Wharton's Jelly tissues were cut into 2 mm2 pieces and attached to the bottom of 100 mm cell culture dishes (JET BIOFIL, China). The culture dishes were subsequently inverted at 5% CO2 and 37 ℃ for 15–30 min until tissue blocks adhered to the bottom of the dishes. Dulbecco’s High Glucose Modified Eagle’s Medium (DMEM, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 2% fetal bovine serum (Gibco, USA), 1% penicillin–streptomycin (Beyotime, Shanghai, China), transferrin (Sigma-Aldrich Co., USA), basic fibroblast growth factor (bFGF, Zhuhai Essex Pharmaceutical Co., LTD., China), insulin (Sigma-Aldrich, USA), and selenium acid (Aldrich, Sigma-Aldrich, Co., USA), as previously described [30], was added, and the tissue blocks were incubated in a humidified CO2 incubator at 37 °C. The medium was replaced after HuMSCs grew out of the tissues after 5–7 days.

Conditioned medium from human umbilical cord mesenchymal stem cells seeded in gelatin sponge

The conditioned medium of HuMSCs used was the 3rd-5th passage. GS-HuMSCs-CM was prepared as previously described [29]. Briefly, gelatin sponges (Xiang’en, Jiangxi Medical Technology Development Co., LTD., China) were cut into 20 mm × 20 mm × 10 mm pieces in a sterile environment. Subsequently, gelatin sponges were coated with PLL (Beyotime, China). The third-generation HuMSCs were seeded in PLL-coated gelatin sponges (GS-HuMSCs). Each gelatin sponge piece was seeded with 1.5 mL HuMSCs suspension (3 × 106 cells /mL) and incubated in a 6-well plate at 5% CO2, 37 ℃ for 30 min. GS-HuMSCs were cultured in a 6 mL culture medium with 1 μL/mL ascorbic acid at 37 °C with 5% CO2 for 48 h. Conditioned medium was harvested from three groups: a group of HuMSCs at 100% confluency, a group of monolayer culture HuMSCs (Day 3 after confluency), and a group of GS-HuMSCs-CM (Day 3 after seeded in gelatin sponge). One day before the conditioned medium collection, the culture medium was changed to be serum-free. The CM was collected and centrifuged at 3000 rpm for 10 min to remove floating cells and cell debris, and then allotted and preserved at −80 °C prior to ELISA analysis. The cells were harvested and counted after CM collection.

Evaluation of growth factor levels (ELISA assay)

The CM of HuMSCs at 100% confluence, HuMSCs-CM (Day 3), and GS-HuMSCs-CM (Day 3) were collected, and the levels of VEGF, IGF-1, KGF, and HGF in CM were quantified by ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. All data were compiled from a minimum of three replicate experiments, with each performed in duplicate, and growth factor levels were calculated from four-parameter logistic curves. Finally, the growth factor concentration was adjusted to be ng (or pg) /ml/million cells.

Animals and ethical approval

6–7 week-old wild-type male C57BL/6J mice, weighing 20–22 g, were purchased from the Animal Center of Shantou University Medical College (Shantou, China). All protocols were approved by the Ethics Committee of the SUMC, and all animal experiments were approved by the Institutional Animal Care and Use Committee of the Shantou University Medical College and conducted according to the guidelines of the National Health and Medical Research Council (China).

Animal experiments

The mice were maintained in a 12 h light–dark cycle under a room temperature of 26 ± 2 °C. The C57BL/6J male mice were kept for one week before the experiments. The mice were anesthetized by inhaling isoflurane. The dorsal hair (2 cm × 4 cm) was shaved with an electric clipper and completely removed using hair remover cream (Reckitt Benckiser France, CEDEX, France). The mice were randomly assigned to three groups (n = 9 each): the control group (normal saline), HuMSCs group, and GS-HuMSCs-CM group. Mice in the control group were administered normal saline, mice in the HuMSCs group were administered 1 × 107 fresh HuMSCs suspended in normal saline, and mice in the GS-HuMSCs-CM group were administered GS-HuMSCs-CM, 16 points for intradermal injection, and 100 uL/point, once per three days for three times. Intradermal injections were administered with a 31-gauge needle at a depth of approximately 1 mm. (Fig. 1).

Fig. 1

Schematic diagram of the preparation of HuMSCs, GS-HuMSCs-CM, experimental model and treatment process. Saline (control), HuMSCs, GS-HuMSCs-CM, respectively, were injected subcutaneously in the dorsal skin of each mouse once per 3 days for three times. HuMSCs Human umbilical cord mesenchymal stem cells, GS gelatin sponge, CM conditioned medium

Hair growth observation

Skin darkening and hair growth were monitored. The hair growth score was evaluated as described by Vegesna et al. [31]. Briefly, a score of 0 indicated that the dorsal skin is still pink compared with the start time of the hair growth induction, and a score of 10 indicated full hair regrowth in the entire depilated area. The morphological changes of each group injected with normal saline (control), HuMSCs, or GS-HuMSCs-CM at day 0, day 7, day 10, day 14, and day 21 were captured by a digital camera (EOS 6D, Canon, Japan). The hairs of three areas (1 cm2) on the back of mice on day 14 and day 21 were obtained and weighed on a Sartorius BSA224S-CW 1/10,000 analytical balance (Sartorius, Beijing, China). On day 14 and day 21, thirty hairs were randomly selected from the same area of each mouse, the longest and the shortest hairs were excluded, and ten hairs were retained for length and thickness measurement using a micrometer (SangNond, Shenzhen, China) with a 0.01 mm at 10 × magnification accuracy under an inverted microscope (Olympus CKX31).

Hematoxylin and eosin (H&E) staining

On day 7, day 14, and day 21 after treatment, three mice were randomly selected from each treatment group and sacrificed. Skin samples were harvested, fixed, embedded in paraffin, and sectioned at 4 µm. Sections were rehydrated, dewaxed, and stained with hematoxylin–eosin (H&E) according to respective protocols. The images were captured and viewed using a microscope at 40 × and 400 × magnification. The hair-growth conditions and the hair follicle’s growth phase were observed under the microscope. The hair follicles in six randomly selected fields of view (200 ×) were counted under a microscope, and the average was calculated.

Statistical analysis

Each experiment was repeated at least three times. The data were statistically analyzed using one-way ANOVA and the least-squares difference test for the comparison among groups using SPSS ver. 25.0 (SPSS Inc., Chicago, IL, USA) for Windows statistical package. Significance was defined as P < 0.05. All data were reported as a mean ± standard error of the mean (SEM) of at least 3 separate experiments.