A gelatin/collagen/polycaprolactone scaffold for skin regeneration

Abstract

Background: A tissue-engineered skin substitute, based on gelatin ("G"), collagen ("C"), and poly(ε-caprolactone) (PCL; "P"), was developed.

Method: G/C/P biocomposites were fabricated by impregnation of lyophilized gelatin/collagen (GC) mats with PCL solutions, followed by solvent evaporation. Two different GC:PCL ratios (1:8 and 1:20) were used.

Results: Differential scanning calorimetry revealed that all G/C/P biocomposites had characteristic melting point of PCL at around 60 °C. Scanning electron microscopy showed that all biocomposites had similar fibrous structures. Good cytocompatibility was present in all G/C/P biocomposites when incubated with primary human epidermal keratinocytes (PHEK), human dermal fibroblasts (PHDF) and human adipose-derived stem cells (ASCs) in vitro. All G/C/P biocomposites exhibited similar cell growth and mechanical characteristics in comparison with C/P biocomposites. G/C/P biocomposites with a lower collagen content showed better cell proliferation than those with a higher collagen content in vitro. Due to reasonable mechanical strength and biocompatibility in vitro, G/C/P with a lower content of collagen and a higher content of PCL (GC_LP_H) was selected for animal wound healing studies. According to our data, a significant promotion in wound healing and skin regeneration could be observed in GC_LP_H seeded with adipose-derived stem cells by Gomori's trichrome staining.

Conclusion: This study may provide an effective and low-cost wound dressings to assist skin regeneration for clinical use.

Keywords: Adipose-derived stem cells; Collagen; Gelatin; Poly(ε-caprolactone)(PCL); Skin tissue engineering.

Figure 1. Cumulative protein release from G/C/P biocomposites in PBS at 37 °C.

Figure 2. SEM images of GC_LP_L, GC_LP_H, GC_HP_L, and GC_HP_H biocomposites.

Figure 3. Attachment and proliferation of (A) Primary human epidermal keratinocyte (PHEK) and (B) primary human dermal fibroblasts (PHDF) (C) human adipose-derived stem cells (ASCs) grown on G/C/P and C/P biocomposites.

The blank wells (tissue culture polystyrene, TCP) served as the control. ∗: indicates P < 0.05 between TCP and other groups.

Figure 4. Immunohistochemical assay for (A) PHEK grown on four G/C/P biocomposites at 1, 3 days and for (B) PHDF grown on four biocomposites at 1, 3 days, as the staining controls lacking primary antibody were present in the second set of columns.

CK-14 was a specific protein for PHEK, whereas alpha tubulin was a specific protein for PHDF. w/o, without. (scale bar: 250 µm).

Figure 5. Comparison of wound closure: (A) the appearance and (B) the area of wound in the full-thickness skin defect of nude mice covered with GC_LP_H scaffold and those seeded with ASCs until 21 days.

The untreated wounds were served as the control. ∗: indicates P < 0.05 between control and other groups.

Figure 6. Collagen deposition in the wound bed by Gomori’s trichrome staining for the histology of cultured skin model.

Collagen deposition in the wound bed by Gomori’s trichrome staining for the histology of cultured skin model in the groups of (A) open wound (B) GC_LP_H only (C) ASCs-seeded GC_LP_H (40×; scale bar: 30 µm) on the full-thickness skin defect of nude mice for 14 days. (D) to (F) were the magnified images of the box area (red rectangle) in (A) to (C) respectively (100×; scale bar: 5 µm).