Maintaining Inducibility of Dermal Follicle Cells on Silk Fibroin/Sodium Alginate Scaffold for Enhanced Hair Follicle Regeneration

Abstract

The extracellular matrix (ECM) is important for maintaining cell phenotype and promoting cell proliferation and differentiation. In order to better solve the problem of skin appendage regeneration, a combination of mechanical/enzymatic digestion methods was used to self-extract dermal papilla cells (DPCs), which were seeded on silk fibroin/sodium alginate scaffolds as seed cells to evaluate the possibility of skin regeneration/regeneration of accessory organs. Scanning electron microscopy (SEM) graphs showed that the interconnected pores inside the scaffold had a pore diameter in the range of 153-311 μm and a porosity of 41-82%. Immunofluorescence (IF) staining and cell morphological staining proved that the extracted cells were DPCs. The results of a Cell Counting Kit-8 (CCK-8) and Calcein-AM/PI live-dead cell staining showed that the DPCs grew well in the composite scaffold extract. Normal cell morphology and characteristics of aggregation growth were maintained during the 3-day culture, which showed that the silk fibroin/sodium alginate (SF/SA) composite scaffold had good cell-compatibility. Hematoxylin-eosin (H&E) staining of tissue sections further proved that the cells adhered closely and aggregated to the pore wall of the scaffold, and retained the ability to induce differentiation of hair follicles. All these results indicate that, compared with a pure scaffold, the composite scaffold promotes the adhesion and growth of DPCs. We transplanted the SF/SA scaffolds into the back wounds of SD rats, and evaluated the damage model constructed in vivo. The results showed that the scaffold inoculated with DPCs could accelerate the repair of the skin and promote the regeneration of the hair follicle structure.

Keywords: cell-compatibility; composite scaffold; dermal papilla cells; extracellular matrix; hair follicle structure; self-extract.

Figure 1

Gross observation and SEM images of different scaffolds. (a) FCC, (b) AC, and (c) FA. Scale bars = 200 μm. The freezing temperature of each sample was −20 °C.

Figure 2

IF staining of dermal papilla cells (DPC) specific proteins and cell morphology. (a) Strong positive expression of alpha-SMA (red), (b) negative expression of CK-19 (only nuclei expression), (c) nuclei/cell microfilaments staining after 3-day of cell culture, including cell microfilaments (green), nuclei (blue), and their combination, (d) a partial view of (c). Scale bars, (a,b,d) = 100 μm, (c) = 200 μm.

Figure 3

The proliferation and growth of DPCs in the extracted media of different scaffolds. The fluorescent staining images of live-dead cells after 3-day of culturing in: (a) blank, (b) FCC, (c) AC, (d) FA extract, respectively. Live cells showed green, and dead cells showed red. (e) The cell proliferation after 24, 48, and 72-h culture in the extracted media of different scaffolds tested using CCK-8. * indicates statistically significant differences. (n = 4, per group, p < 0.05). Scale bars = 200 μm.

Figure 4

Morphology of DPCs in the extracted media of different scaffolds. Cell microfilaments staining images of DPCs cultured in extract for 2 days. (a) Blank group, (b) FCC, (c) AC, and (d) FA. Scale bars = 100 μm.

Figure 5

Adhesion of DPCs to the scaffolds. Three days after the DPCs were grown on the scaffolds, the nuclei were stained with DAPI (blue). The arrows point to the cells. (a) FCC, (b) AC, (c) FA. Scale bars = 100 μm.

Figure 6

H&E staining showed the distribution of DPCs seeded on the scaffolds for 3-day in vitro culture. (a) FCC, (b) AC, and (c) FA. (d–f) Corresponding to the partial enlarged diagrams of a–c. The arrows point mostly to the area where the cells aggregated. Scale bars, (a–c) = 50 μm, (d–f) = 20 μm.

Figure 7

H&E staining image of normal skin tissue of SD rats. Scale bar = 200 μm.

Figure 8

H&E staining of sections of wounds treated by different materials on different days, respectively. (a) Control group without any treatment, (b) B/M scaffold not inoculated with DPCs, (c) M/C scaffold inoculated with DPCs. Scale bar = 200 μm.