The Influence of Novel, Biocompatible, and Bioresorbable Poly(3-hydroxyoctanoate) Dressings on Wound Healing in Mice

Abstract

The human body's natural protective barrier, the skin, is exposed daily to minor or major mechanical trauma, which can compromise its integrity. Therefore, the search for new dressing materials that can offer new functionalisation is fully justified. In this work, the development of two new types of dressings based on poly(3-hydroxyoctanoate) (P(3HO)) is presented. One of the groups was supplemented with conjugates of an anti-inflammatory substance (diclofenac) that was covalently linked to oligomers of hydroxycarboxylic acids (Oli-dicP(3HO)). The novel dressings were prepared using the solvent casting/particulate leaching technique. To our knowledge, this is the first paper in which P(3HO)-based dressings were used in mice wound treatment. The results of our research confirm that dressings based on P(3HO) are safe, do not induce an inflammatory response, reduce the expression of pro-inflammatory cytokines, provide adequate wound moisture, support angiogenesis, and, thanks to their hydrophobic characteristics, provide an ideal protective barrier. Newly designed dressings containing Oli-dicP(3HO) can promote tissue regeneration by partially reducing the inflammation at the injury site. To conclude, the presented materials might be potential candidates as excellent dressings for wound treatment.

Keywords: CD68* macrophages; P(3HO); angiogenesis; bioresorption; dressing materials; poly(3-hydroxyoctanoate); polyhydroxyalkanoates; porous patches; wound healing.

Figure A1

GPC profile of oli-dicP(3HO).

Figure A2

Calibration curve of GPC prepared using polystyrene (PS) standards.

Figure 1

1H NMR of oli-dic-P(3HO) analyses, where (1) denotes the CH2 groups from DIC attached to oligomers and (1′) denotes groups from free DIC. The pictures also show labels of the assigned hydrogen atoms and the structural formulas of the compounds of the reaction mixture.

Figure 2

SEM image (A–C) of P(3HO) foams; (D–F) of P(3HO)/oli-dicP(3HO). View (A,D) ×220; (B,E) ×1000; (C,F) ×5000. In the figure, red arrows mark the pores, and blue arrows mark the grains of unleached salt.

Figure 3

(A–C) The reconstructed cross-section of P(3HO) patch, obtained through the µ-CT method. Yellow arrows indicate porogen grains present in the prepared material.

Figure 4

The Young’s modulus (A), tensile strength (B), and elongation at break (C) of prepared foams. The results are statistically significant, where: * p < 0.05, *** p < 0.001.

Figure 5

The cytotoxic assessment of P(3HO) enriched with different concentrations of diclofenac-modified oligomers (oli-dicP(3HO)) on MEF3T3 cells. Glass substrate was used as negative control. The results are statistically significant, where * p < 0.05, ** p < 0.001.

Figure 6

Microscopic images showing cells’ arrangement along the edges of the pores (orange dashed lines and arrows—placed inside the pore). The actin cytoskeleton is visualised in red (A–C) and cell nuclei (A,B) in blue. Panels A and B present images captured under 40× objective, panel C was imaged using a smaller (10×) magnification to exhibit a group of cells localised on the substrate.

Figure 7

Three-dimensional image reconstruction in orthogonal projection shows cells’ tendency to overgrow pores and penetrate the 3D structure of the material. Some cells form protrusions and stretch out at the edges of the pore (orange arrow). The shape of the substrate is marked with a dashed line, which shows that the cells penetrate all irregularities and pores in the material. Actin filaments are presented in red, and the cell nucleus is in blue.

Figure 8

The images of the stained actin skeleton (red) and nuclei (blue) show the tendency of fibroblasts to form elongated protrusions that line the edge of the pore (marked with orange rectangles).

Figure 9

Cluster-like cell structures imaged in fluorescent mode (A,C) and 3D image reconstructions showing the spatial distribution of cell bodies (B,D). Cell attachment points at the pore edges are marked with orange arrows.

Figure 10

Schematic representation of the experimental procedure. In the beginning, rectangular (10 × 5 mm) wounds were created (photo at the top). The wound was left untreated (control group, C on the left side) or treated with unmodified (P(3HO)) or modified (P(3HO)/oli-dicP(3HO)) dressing (middle and right side, respectively). Created with BioRender.com.

Figure 11

Comparison of average dressing thickness before operation and after 14 days of wound healing. Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval). Box plot showing the median (horizontal line); X (mean value). The box covers values from the first to the third quartile and the whiskers cover the total range. Significant digits (*) for p-value **** 0.0001.

Figure 12

Confocal microscope photos show the presence of CD11b-positive cells (A–C) and mice skin γδ T cells (D–F) in the injured area. Scale bars—100 µm.

Figure 13

Relative gene expression of pro-inflammatory (IL1, IL6) (A,B) and anti-inflammatory cytokines (TGF-β) (C), as well as fibrocytes (ITGAM) (D), from the damaged tissue and the surrounding area on days 7 (I) and 14 (II). Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval), significant digits (*) for p-value * 0.0332, ** 0.0021, *** 0.0002, **** 0.0001. The plot shows the median (horizontal line), where X represents the mean value, the box covers values from the first to the third quartile, and whiskers represent the total range.

Figure 14

Average absorbance values of pro-inflammatory (IL1, IL6) (A,B) and anti-inflammatory cytokines (IL10) (C) in serum were obtained from mice’s peripheral blood on days 7 (I) and 14 (II). Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval), significant digits (*) for p-value * 0.0332. The box plot shows the median (horizontal line), where X represents the mean value, the box covers values from the first to the third quartile, and the whiskers represent the total range.

Figure 15

Confocal microscope photos show new blood vessels (ACTA2, red marked with white arrows) present in the healed area in control (A), P(3HO) (B) and P(3HO)/oli-dicP(3HO) (C) group and numerous CD68-positive cells (green). Scale bars—100 µm.

Figure 16

Confocal microscope images showing cells growing inside dressings—cell nuclei (Hochest (blue))—marked with white arrows (B,C), as well as fibronectin deposition (red) present in the healed tissue and P(3HO)/oli-dicP(3HO) dressing (C). Control group on picture (A). Scale bars—50 µm.

Figure 17

Relative gene expression of Krt14 from the damaged tissue and the surrounding area on days 7 (I) and 14 (II). Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval), Significant digits (*) for p-value * 0.0332, ** 0.0021, **** 0.0001. Box plot showing the median (horizontal line), where X shows the mean value, the box covers values from the first to the third quartile, and the whiskers represents total range.