Effects and mechanisms of polycaprolactone-cellulose acetate nanofiber scaffold loaded with rat epidermal stem cells on wound healing of full-thickness skin defects in rats

Objective To explore the effects and mechanisms of polycaprolactone-cellulose acetate (PCL-CA) nanofiber scaffold loaded with rat epidermal stem cells (ESCs) on wound healing of full-thickness skin defects in rats. Methods The experiment research method was applied. The primary ESCs were isolated from 1-3 d old Sprague-Dawley (SD) rats (undefined gender) by rapid adherent method and cultured by rapid adherent method. ESCs of the first passage were used for the subsequent experiments after the positive expressions of integrin β₁ and cytokeratin 19 (CK19) in primary cells were identified respectively by flow cytometey and immunofluorescence method. PCL-CA nanofiber scaffolds with polycaprolactone and cellulose acetate as components were prepared by electrospinning technique. The topological structure of the nanofiber scaffolds was determined and the diameter of 25 fibers was measured by scanning electron microscope. The constructed PCL-CA nanofiber scaffolds were used as the culture substrate for ESCs, which were cultured in keratinocytes (KCs) medium to construct ESCs-nanofiber scaffold complex (hereinafter referred to as ESCs scaffold). After 3 days of culture, the morphology of ESCs in the scaffold and their relationship was observed by scanning electron microscopy. The ESCs in ESCs scaffold were set as PCL-CA nanofiber scaffold group, and the ESCs cultured with KCs medium in culture dishes coated with type Ⅳ collagen were set as type Ⅳ collagen group. Western blotting was used to detect the protein expression level of CK19 in ESCs in the two groups after 3 days of culture (

=3). The protein expressions of CK19 and proliferating nuclear antigen (PCNA) in ESCs in the two groups were detected by immunofluorescence method after 7 days of culture. A circular full-thickness skin wound of about 2 cm in diameter was prepared on both left and right sides of the back of 15 male SD rats aged 6-8 weeks. The rats were then equally divided into blank control group without implantation, scaffold alone group implanted with PCL-CA nanofiber scaffold, and ESCs scaffold group implanted with ESCs scaffold which were constructed after 3 days of culture according to the random number table. The percentage of wound areas on post injury day (PID) 3, 7, 14, and 21 was calculated (

=5). The new skin tissue at the wound edge was collected on PID 21, the wound healing quality was evaluated by Masson staining, and the protein expression levels of Notch1, Jagged1, and Hes1, which are key proteins of Notch signaling pathway, were detected by Western blotting (

=3). Data were statistically analyzed with one-way analysis of variance, one-way analysis of variance, analysis of variance for repeated measurement, independent sample

test, and Bonferroni correction. Results The constructed PCL-CA nanofiber scaffolds had a porous, mesh-like, and multilayered three-dimensional structure, in which the surface of the fibers was smooth and non-porous, and the fiber diameter was (383±24) nm. The ESCs in ESCs scaffold showed intact cellular structures and were tightly attached to the scaffold after 3 days of culture. The cells were interconnected and fully extended on the surface of the scaffold to form a membrane. After 3 days of culture, the protein expression level of CK19 of ESCs in PCL-CA nanofiber scaffold group was significantly higher than that in type Ⅳ collagen group (

=24.56,

<0.01). After 7 days of culture, compared with those in type Ⅳ collagen group, there was no significant change in the proportion of PCNA positive cells of ESCs in PCL-CA nanofiber scaffold group, while the proportion of CK19 positive cells was higher. On PID 3, 7, 14, and 21, the percentages of wound areas of rats in ESCs scaffold group were (78.0±1.8)%, (40.9±2.0)%, (17.9±1.1)%, and (5.0±1.0)%, respectively, which were significantly lower than (84.2±1.9)%, (45.4±2.6)%, (21.8±1.7)%, and (10.1±1.1)% in blank control group (

=5.42, 3.09, 4.33, 7.58,

<0.05 or

<0.01) and (82.7±1.2)%, (44.8±2.0)%, (22.4±2.4)%, and (10.3±2.4)% in scaffold alone group (

=4.98, 3.11, 3.84, 4.57,

<0.05 or

<0.01), while the percentages of wound areas of rats between blank control group and scaffold alone group were similar (

=1.47, 0.39, 0.47, 0.22,

>0.05). On PID 21, the layer of new skin at the wound edge of rats in each group was intact; compared with that in blank control group or scaffold alone group, the new skin tissue at the wound edge of rats in ESCs scaffold group had more orderly collagen arrangement; the scaffolds in the new skin at the wound edge of rats were completely degraded in ESCs scaffold group and scaffold alone group. On PID 21, the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in scaffold alone group were similar to those in blank control group (

=1.70, 1.94, 0.18,

>0.05), while the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in ESCs scaffold group were significantly higher than those in scaffold alone group (

=13.31, 22.07, 20.71,

<0.01). Conclusions PCL-CA nanofiber scaffolds can inhibit the differentiation of ESCs of rats without affecting their proliferation

. ESCs scaffolds constructed through using PCL-CA nanofiber scaffolds as the carrier to culture ESCs of rats can significantly promote the wound healing of full-thickness skin defects in rats, and the mechanism may be related to the activation of Notch signaling pathway.