Injectable Antioxidant and Oxygen-Releasing Lignin Composites to Promote Wound Healing

Abstract

The application of engineered biomaterials for wound healing has been pursued since the beginning of tissue engineering. Here, we attempt to apply functionalized lignin to confer antioxidation to the extracellular microenvironments of wounds and to deliver oxygen from the dissociation of calcium peroxide for enhanced vascularization and healing responses without eliciting inflammatory responses. Elemental analysis showed 17 times higher quantity of calcium in the oxygen-releasing nanoparticles. Lignin composites including the oxygen-generating nanoparticles released around 700 ppm oxygen per day at least for 7 days. By modulating the concentration of the methacrylated gelatin, we were able to maintain the injectability of lignin composite precursors and the stiffness of lignin composites suitable for wound healing after photo-cross-linking. In situ formation of lignin composites with the oxygen-releasing nanoparticles enhanced the rate of tissue granulation, the formation of blood vessels, and the infiltration of α-smooth muscle actin⁺ fibroblasts into the wounds over 7 days. At 28 days after surgery, the lignin composite with oxygen-generating nanoparticles remodeled the collagen architecture, resembling the basket-weave pattern of unwounded collagen with minimal scar formation. Thus, our study shows the potential of functionalized lignin for wound-healing applications requiring balanced antioxidation and controlled release of oxygen for enhanced tissue granulation, vascularization, and maturation of collagen.

Keywords: calcium peroxide; lignosulfonate; reactive oxygen species; vascularization; wound healing.

Figure 1

Elemental analysis of NPs of SLS-PLGA with or without CaO₂ by microPIXE. The concentration (arbitrary units, a.u.) of Ca, Cl, P, S, and K in the NPs normalized to the NPs of TLS. TLS also had trace amounts of Mn and Fe. The bars represent counting uncertainties (1σ) in a single measurement analyzed by the GeoPIXE software; mean ± SEM.

Figure 2

Quantification of O₂ from lignin composites with NPs of SLS-PLGA/CaO₂. The area under the curve (AUC) is calculated, and the differential between CPO and CPOc lignin composites over 1440 min is reported. One-way ANOVA with Tukey’s post hoc test; no significant difference to each other; mean ± SD, n = 4.

Figure 3

Swelling ratios and degradation of CPO and CPOc lignin composites. (a) Swelling ratios normalized to TLS lignin composites. No statistical difference between CPO and CPOc lignin composites at the same concentration and between two different concentrations at each composite type. Student’s t test shows no statistically significant difference; mean ± SD, n = 3. Degradation of lignin composites, TLS (b), CPO (c) and CPOc (d), in the solution of 0.5 U/mL collagenase (enzyme) or serum-free medium (SF medium). In panels b–d, black and brown symbols represent the concentrations of NPs (SLS-PLGA with or without CaO₂) at 4 and 40 mg/mL, respectively; mean ± SD, n = 3.

Figure 4

Oscillating rheometry of lignin composites. (a) Viscosity of each precursor before photo-cross-linking. (b) Axial stresses are plotted against compression varying from 0 to 20%. (c) Frequency sweeping of lignin composites. Solid and open symbols represent G′ (storage modulus) and G″ (loss modulus), respectively. (d) Loss tangent (δ) of lignin composites from 0.1 to 10 rad/s. Mean ± SD, n = 3 for all samples.

Figure 5

Morphometric analysis of wounds treated with lignin composites at 7 days post wounding. Wounds in WT C57BL/6 N mice were treated with lignin composites (a) to measure the epithelial gap (b) and granulation tissue area (c). In panel a, hematoxylin (blue, nuclei) and eosin (red, ECM and cytoplasm) stained wound sections from different treatments are shown. The left panels show the cross section of the wounds from edge to edge, and the right panels show the corresponding higher magnification of boxed areas (inset) of the granulating wound bed with biomaterial interface. Scale bar, 100 μm. (b) Quantification of the epithelial gap (distance between the blue margins) and granulation tissue area is shown. One-way ANOVA with the Kruskal–Wallis test followed by Dunn’s multiple comparison test was performed. *p < 0.05 and **p < 0.01; 4 ≤ n ≤ 7. Bar plots indicate mean ± SD, with individual values from each mouse wound indicated. Details of compositions of UNTX, TLS, CPOc, and CPO are in Table 1.

Figure 6

Assessment of neovascularization in the wounds treated with lignin composites at 7 days post wounding. Wounds in WT C57BL/6 N mice were treated with lignin composites (a), and the extent of neovascularization was assessed with immunostaining (CD31, brown) and hematoxylin counterstaining (blue, nuclei). CD31⁺ cells (b) and vessel formation (c) per HPF were quantified. The infiltration of αSMA⁺ cells in the wounds is visualized (d) and quantified per HPF (e). Scale bars, 50 μm in panel a and 3 mm in panel d, respectively. One-way ANOVA with Tukey’s post hoc tests, **p < 0.01 and *p < 0.05; 3 ≤ n ≤ 6. Bar plots indicate mean ± SD with individual values per each mouse wound indicated. Details of compositions of UNTX, TLS, CPOc, and CPO are in Table 1.

Figure 7

Assessment of inflammatory responses in the wounds treated with lignin composites at 7 days post wounding. Wounds in WT C57BL/6 N mice were treated with lignin composites (a), and the extent of inflammatory responses was assessed with immunohistochemical staining and hematoxylin counterstaining (blue, nuclei). CD45⁺ leukocytes (b); Ly6G⁺ monocytes, granulocytes, and neutrophils (c); F4/80⁺ pan macrophages (d); and CD206 M2 macrophages (e) per HPF are quantified. In panel a, black scale bars: 50 μm and gray scale bars: 125 μm, respectively; 3 ≤ n ≤ 6. Bar plots indicate mean ± SD with individual values per mouse wound indicated. Details of compositions of UNTX, TLS, CPOc, and CPO are in Table 1.

Figure 8

Scar assessment in the wounds treated with lignin composites at 28 days after surgery. Wound sections from WT C57BL/6N mice treated with lignin composites were stained with trichrome (blue, collagen), and the collagen content was assessed. Representative trichrome images of the wounds at low (top row) and high magnification (bottom row) of the area enclosed in yellow boxes are shown (a). Collagen content is quantified per HPF using color thresholding in ImageJ (b). Photographs of wounds at 28 days after surgery. For scar assessment, photographs were taken from all four treatment groups before harvest (c). Scale bar, 50 μm; 3 ≤ n ≤ 6. Bar plots indicate mean ± SD with values per mouse wound indicated. Details of compositions of UNTX, TLS, CPOc, and CPO are in Table 1.