Recombinant humanized collagen combined with nicotinamide increases the expression level of rat basement membrane proteins and promotes hair growth

Abstract

Background: Hair follicle stem cells (HFSCs) play crucial roles in hair growth and are expected to be potential targets in regenerative medicine and tissue engineering.

Method: This study aims to investigate the positive effect on hair growth by the recombinant human collagen complex (RHC complex), composed of rhCOL III, rhCOL XVII, and rhCOL XXI, along with nicotinamide, both in vitro and in vivo, by HFSCs and rat models. The survival rate, function, and differentiation of HFSCs were investigated.

Results: The CCK-8 experiment showed that the RHC complex was non-toxic to HFSCs, and the cell survival rate exceeded 80% after 8 and 16 h of treatment. The ELISA method showed that the RHC complex significantly increased the intracellular vascular endothelial growth factor (VEGF) levels. In addition, the increase in the content of trichohyalin (a key structural protein of hair) indicates that the structure and function of hair follicles may be enhanced. The expression levels of β-integrin and p63 were significantly upregulated, which are crucial for cell adhesion, migration, and maintenance of HFSCs homeostasis. In the rat model, significant hair growth was observed after a 7-day treatment period. The period of vigorous hair growth in rats was selected for immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and hematoxylin-eosin (HE) staining analysis. The results showed that the RHC complex could promote the expression of Integrin, Laminin and Perlecan, which were conducive to maintaining the stability of the microenvironment of HFSCs. Additionally it facilitated the migration and differentiation of HFSCs, as evidenced by an increased number of hair follicles in HE-stained skin tissues. In conclusion, the RHC complex exhibited high HFSCs survival rates and enhanced the expression of HFSCs-associated factors and basement membrane proteins. These properties make the RHC complex a promising novel ingredient for promoting hair growth, preventing hair loss, and maintaining hair health.

Keywords: VEGF; hair follicle stem cells; hair loss; recombinant humanized collagen; trichohyalin.

FIGURE 1

Different concentrations of RHC complex were cultured with HFSCs for 8–16 h (A) HFSCs viability was assessed via CCK-8 assay (Cell Counting Kit-8) following 8-h treatment with/without nicotinamide and the RHC complex. (B) HFSCs viability was assessed via CCK-8 assay (Cell Counting Kit-8) following 16-h treatment with/without nicotinamide and the RHC complex. The results are expressed as mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the blank group).

FIGURE 2

Different concentrations of RHC complex were cultured with HFSCs for 8–16 h. The cell viability of HFSCs was measured after treatment with RHC complex using a cck-8 assay. The results are expressed as mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the blank group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with 1 μΜ minoxidil group).

FIGURE 3

The levels of different concentrations in HFSCs treated with RHC complex (0.01, 0.05, 0.1, 0.5, 2 mg/mL) for 8–16 h were measured using ELISA assay. (A) determination of VEGF level in HFSCs. (B) determination of p63 level in HFSCs. (C) determination of trichohyalin level in HFSCs. (D) determination of β-integrin level in HFSCs. The results are expressed as mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the blank group; ##p < 0.01, ###p < 0.001 compared with 1 μΜ minoxidil group).

FIGURE 4

The expression of collagen IV in HFSCs induced by different concentrations of RHC complex. (A) Fluorescence microscopy observation images. Each image represents three similar experiments. (Scale bar: 100 µm). (B) Quantitative analysis of collagen IV immunofluorescence images after 8h of administration. (C) Quantitative analysis of collagen IVimmunofluorescence images after 16 h of administration. The results are expressed as mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the blank group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with 1 μΜ minoxidil group).

FIGURE 5

The dorsal skin of rats was treated with different concentrations of the RHC complex (rhCOL III: rhCOL XVII: rhCOL XXI: niacinamide = 400:100:50:2), which was administered at 24h intervals. Changes in hair growth were observed daily and photographs were taken on days 7 and 14.

FIGURE 6

Expression of (A) Integrins, (B) Laminin, and (C) Perlecan proteins in the dorsal skin of rats in different concentrations of RHC complex-treated groups was examined by ELISA. RHC complex at 0 mg/mL was used as a blank group, and 5% minoxidil was used as a control group. The results are expressed as mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the blank group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the 5% minoxidil group).

FIGURE 7

IF images of Integrin, Laminin and Perlecan protein expression in dorsal rat skin treated with different concentrations of RHC complex. (A) Images of isolated skin tissue IF labelled with Intergin (green), Laminin (green), Perlcean (green) and DAPI (blue). (Scale bar: 0.5 mm). (B) Quantitative analysis of Integrin. (C) Quantitative analysis of Laminin. (D) Quantitative analysis of Perlecan. The results are expressed as mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the blank group; ##p < 0.01, ###p < 0.001 compared with the 5% minoxidil group).

FIGURE 8

Effects of different concentrations of RHC complex (rhCOL III: rhCOL XVII: rhCOL XXI: niacinamide = 400:100:50:2) on hair growth (morphology and number) compared with 5% minoxidil and blank (0 mg/mL). (A) Representative images of longitudinal and transverse sections of H&E-stained skin tissue on day 7. (Scale bar: 0.5 mm). Red arrows in the images mark the hair follicles. (B) Number of hair follicles in each group of rats. The results are expressed as mean ± SD of three independent experiments. (**p < 0.01, ***p < 0.001 compared with the blank group; ##p < 0.01, ###p < 0.001 compared with the 5% minoxidil group).

FIGURE 9

Physical quantification evaluation of hair in rats treated for 7 days, (A) Physical Ouantitative Assessment Growth. (B) Radar diagram for physical assessment ofhair growth.