Exploring Methacrylated Gellan Gum 3D Bioprinted Patches Loaded with Tannic Acid or L-Ascorbic Acid as Potential Platform for Wound Dressing Application

Abstract

To improve wound healing, advanced biofabrication techniques are proposed here to develop customized wound patches to release bioactive agents targeting cell function in a controlled manner. Three-dimensional (3D) bioprinted "smart" patches, based on methacrylated gellan gum (GGMA), loaded with tannic acid (TA) or L-ascorbic acid (AA) have been manufactured. To improve stability and degradation time, gellan gum (GG) was chemically modified by grafting methacrylic moieties on the polysaccharide backbone. GGMA patches were characterized through physicochemical, morphological and mechanical evaluation. Kinetics release and antioxidant potential of TA and AA as well as antimicrobial activity against common pathogens Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli in accordance with ISO 22196:2011 are reported. The cytocompatibility of the patches was demonstrated by direct and indirect tests on human dermal fibroblasts (HDF) as per ISO 10993. The positive effect of GGMA patches on cell migration was assessed through a wound healing assay. The results highlighted that the patches are cytocompatible, speed up wound healing and can swell upon contact with the hydration medium and release TA and AA in a controlled way. Overall, the TA- and AA-loaded GGMA patches demonstrated suitable mechanical features; no cytotoxicity; and antioxidant, antimicrobial and wound healing properties, showing satisfactory potential for wound dressing applications.

Keywords: 3D bioprinting; L-ascorbic acid; methacrylated gellan gum patches; tannic acid; wound healing.

Figure 1

ATR-FTIR spectra of gellan gum (GG) and methacrylated GG (GGMA) between 4000 and 650 cm⁻¹.

Figure 2

ATR-FTIR spectra of (A) tannic acid (TA), GGMA and GGMA/TA and (B) L-ascorbic acid (AA), GGMA and GGMA/AA between 4000 and 650 cm⁻¹.

Figure 3

Morphological, mechanical and physicochemical characterization. Scanning electron microscopy (SEM) images of (A) GGMA, (B) GGMA/TA, (C) GGMA/AA. Scale bars: 1 mm. High vacuum 10.00 kV, magnification 100×. (D) Storage modulus of bulk GGMA, GGMA/TA and GGMA/AA materials; (E) swelling behavior and (F) cumulative release of TA and AA from GGMA-based patches. ◊ with solid line indicates GGMA, □ with dashed line indicates GGMA/TA, Δ with dotted line indicates GGMA/AA. (G) 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities of GGMA, GGMA/TA and GGMA/AA. Data reported as mean value ± standard deviation (S.D.), n = 6.

Figure 4

Histograms show direct and indirect biocompatibility tests. (A,B) Histograms show the percentage of viable cells in direct biocompatibility tests at 24 and 48 h, respectively. (C,D) Histograms show the percentage of viable cells in indirect biocompatibility tests at 24 and 48 h, respectively. White column: HDFs, untreated cells; black column: HDFs with GGMA patch (A,B) or GGMA-patch-conditioned medium (C,D); grey column: HDFs with GGMA/AA patch (A and B) or GGMA/AA-patch-conditioned medium (C,D); dotted column: HDFs with GGMA/TA patch (A,B) or GGMA/TA-patch-conditioned medium (C,D). Data, means ± standard S.D., were normalized to the untreated group mean. Two-way ANOVA and one-way ANOVA followed by Bonferroni’s test were performed for direct and indirect tests, respectively. **** p < 0.0001.

Figure 5

(A) Representative optical microscope images showing the area covered by the cells at 0, 24 and 48 h after wounding in the four different conditions (magnification 10×). Black arrows indicate cell aggregates. (B) Time course curves of the wound healing assay: control, i.e., untreated cells, green line; GGMA-based patch-conditioned medium, red line; GGMA/AA-based patch-conditioned medium, blue line, GGMA/TA-based patch-conditioned medium, magenta line; (C) Histograms of the wound healing assay: control, i.e., untreated cells, first bars; GGMA-based patch-conditioned medium, second bars; GGMA/AA-based patch-conditioned medium, third bars, GGMA/TA-based patch-conditioned medium, fourth bars. Data are reported as means ± S.D., (two-way ANOVA followed by Bonferroni’s test). * p < 0.012; ** p < 0.0017.

Figure 6

Antimicrobial activity of films against tested bacteria. Statistical significance was tested within a pathogen. Letters on the bars of the figure indicate statistically significant differences (p < 0.05). Statistical analysis was performed using Past v. 4.15. ANOVA and Tukey’s post-hoc test were applied.

Figure 7

Representative scheme of producing 3D methacrylated gellan gum (GGMA), GGMA/tannic acid (TA) and GGMA/L-ascorbic acid (AA) patches, from 3D bioprinting to TA and AA loading.

Figure 8

(A) Timeline showing the steps/day of the biocompatibility (direct) test. (B) Timeline showing the steps/day of the indirect test. (C) Timeline showing the steps/day of the wound healing assay.