Bioapplications of Bacterial Cellulose Polymers Conjugated with Resveratrol for Epithelial Defect Regeneration

Abstract

Excellent wound dressing is essential for effective wound repair and regeneration. However, natural polymeric skin substitutes often lack mechanical strength and hydrophilicity. One way to overcome this limitation is to use biodegradable polymers with high mechanical strength and low skin-irritation induction in wet environments. Bacterial cellulose (BC) is an attractive polymer for medical applications; unlike synthetic polymers, it is biodegradable and renewable and has a strong affinity for materials containing hydroxyl groups. Therefore, we conjugated it with resveratrol (RSV), which has a 4'-hydroxyl group and exhibits good biocompatibility and no cytotoxicity. We synthesized BC scaffolds with immobilized RSV and characterized the resulting BC/RSV scaffold with scanning electron microscopy and Fourier-transform infrared spectroscopy. We found that RSV was released from the BC in vitro after ~10 min, and immunofluorescence staining showed that BC was highly biocompatible and regenerated epithelia. Additionally, Masson's trichrome staining showed that the scaffolds preserved the normal collagen-bundling pattern and induced re-epithelialization in defective rat epidermis. These results indicated that RSV-conjugated BC created a biocompatible environment for stem cell attachment and growth and promoted epithelial regeneration during wound healing.

Keywords: bacterial cellulose; biodegradable polymer biomaterials; epidermal reconstruction; tissue engineering scaffolds; wound healing.

Figure 1

FT-IR spectrum of the resveratrol (RSV) particle. (A) Bacterial cellulose (BC)/RSV scaffold (purple line), (B) enlargement of (A), (C) type I collagen (COL)/RSV scaffold, and (D) enlargement of (C). Black squares are enlarged areas, and red squares indicate the region of the RSV spectrum. (E) FT-IR identification of the RSV regions in the scaffolds.

Figure 2

SEM analysis. (A) BC scaffold, (B) BC/RSV scaffold, (C) COL scaffold, and (D) COL/RSV scaffold. Red arrows indicate RSV particles. Scale bars = 5 μm (A,B) and 15 μm (C,D).

Figure 3

SEM images of human adipose stem cells line (hASCs) grown on biomaterials for 7 d. (A) BC scaffold, (B) BC/RSV scaffold, (C) COL scaffold, and (D) COL/RSV scaffold. Red arrows indicate hASCs. Scale bar = 20 μm.

Figure 4

Monitoring RSV release from biomaterials in artificial saliva solution at 37 °C. (A) BC/RSV scaffold and (B) COL/RSV scaffold.

Figure 5

Immunofluorescence staining of stem cells in the presence of hASCs. (A) Octamer-binding transcription factor 4 (OCT-4) and (B) bone morphogenic protein (BMP)4. Trypan blue staining of hASCs grown on biomaterials for 7 d. (C) BC, (D) BC/RSV, (E) COL, and (F) COL/RSV scaffolds. Scale bar = 50 μm.

Figure 6

Representative images showing BC and BC/RSV scaffold biocompatibility with hASCs. (A) BC and (B) BC/RSV scaffolds. Images are of formalin-fixed, frozen, and immunostained stem cells for OCT4, nestin, and the keratinocyte-differentiation marker involucrin. β-actin was used as an internal staining control. White arrows indicate positive antibody expression in the scaffold. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 500 μm.

Figure 7

Representative images showing COL and COL/RSV biocompatibility with hASCs. (A) COL and (B) COL/RSV scaffolds immunostained with the stem cell biomarkers OCT4, nestin, and the keratinocyte-differentiation marker involucrin. β-actin was used as an internal staining control. White arrows indicate positive antibody expression in the scaffold. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 500 μm.

Figure 8

Effect of scaffolds on dermal wound healing. (A) Skin defects induced on the back of a rat and filled with scaffolds; (B) wound contraction observed in each scaffold group on days 3, 7, and 14 after injury. [(L),(M)] Semi-quantitatively measure the wound area of the epidermal defect in each material group.

Figure 9

Histological assessment of scaffold implants in skin defects after 7 and 14 d. (A) Masson’s trichrome staining demonstrated that COL bundles formed after 7 d in the wound treated with RSV-loaded BC and COL scaffolds; (B) after 14 d, remodeling progress was improved in the presence of the BC/RSV scaffolds relative to the COL/RSV scaffolds. Relatively greater COL accumulation and deposition were observed in the COL scaffolds. Light micrographs of Masson’s trichrome staining show that the BC/RSV scaffolds preserved normal COL-bundling patterns and orientation.

Figure 10

Keratinocyte differentiation on BC- and BC/RSV-scaffold implants in skin defects after 7 and 14 d. Implant areas were detected by immunofluorescence staining for involucrin (green) and CK-14 (red) in wound tissue for different groups at 7 d after implantation. The BC/RSV group showed relatively higher levels of involucrin- and CK-14-positive keratinocytes. The re-epithelialization marker fibronectin (green) was present in the BC-scaffold group at 14 d after implantation. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 100 μm.

Figure 11

Keratinocyte differentiation on COL- and COL/RSV-scaffold implants in skin defects after 7 and 14 d. The COL/RSV group showed relatively higher levels of involucrin- (green) and CK-14- (red) positive keratinocytes. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 100 μm. The COL and the COL/RSV scaffolds displayed fibronectin (green) expression, indicating ECM formation and re-epithelialization during wound healing.