Abstract
Burn injuries, the most common and destructive forms of wounds, are generally accompanied with life-threatening infections, inflammation, reduced angiogenesis, inadequate extracellular matrix production, and lack of growth factor stimulation. In the current study, a new antimicrobial carbopol-based hydrogel formulated with boron and pluronic block copolymers was evaluated for its healing activity using in vitro cell culture techniques and an experimental burn model. Cell viability, gene expression, and wound healing assays showed that gel formulation increased wound healing potential. In vitro tube-like structure formation and histopathological examinations revealed that gel not only increased wound closure by fibroblastic cell activity, but also induced vascularization process. Moreover, gel formulation exerted remarkable antimicrobial effects against bacteria, yeast, and fungi. Migration, angiogenesis, and contraction-related protein expressions including collagen, α-smooth muscle actin, transforming growth factor-β1, vimentin, and vascular endothelial growth factor were considerably enhanced in gel-treated groups. Macrophage-specific antigen showed an oscillating expression at the burn wounds, indicating the role of initial macrophage migration to the wound site and reduced inflammation phase. This is the first study indicating that boron containing hydrogel is able to heal burn wounds effectively. The formulation promoted burn wound healing via complex mechanisms including stimulation of cell migration, growth factor expression, inflammatory response, and vascularization.
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Acknowledgments
This study was supported by Yeditepe University and by grants from the Scientific and Technological Research Council of Turkey (TUBITAK, project code: 112 M495). The current formulation has a patent application to the Turkish patent institute with the application number of 2013/04209.
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The authors have no conflict of interest.
Ethical Approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
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Supplementary Figure 1
a Schematic diagram of the whole experimental plan. Cell viability analysis of NIH 3T3 cells treated with different concentrations of b NaB (1–500 μg/ml), c F68 (5–20 μg/ml), and d F127 (5-20 μg/ml). e NaB and gel formulation stimulated NIH 3T3 cell migration after 36 h incubation period (objective ×4). f Tube formation of HUVEC cells treated with NaB, F68, and F127 (alone or in combination) after 24 h (objective ×4). Black arrows indicate defined concentrations for NaB (15 μg/ml), F68 (10 μg/ml), and F127 (10 μg/ml).Scale bar, 400 μm. Data represent the mean values ± SD per sample from three separate experiments performed in triplicate. Comparisons were performed by ANOVA (Tukey’s post hoc). * P < 0.05: comparison with control group. Control: growth medium-treated NIH 3T3 cells (for b, c, d, and e sections) or HUVECs (for f section). F68: 10 μg/ml pluronic F68; F127: 10 μg/ml pluronic F127; F68-F127: combination of 10 μg/ml pluronic F68 and F127; NaB: 15 μg/ml sodium pentaborate pentahydrate; Gel: combination of 15 μg/ml NaB, 10 μg/ml pluronic F68 and F127 containing growth medium (GIF 37 kb)
Supplementary Figure 2
Immunocytochemical analysis of NIH 3T3 cells treated with different reagents for 24 h. Gel formulation resulted in remarkable increase in Col I and VIM expression levels. On the other hand, α-SMA was weakly expressed in NaB group. DAPI used for nucleus staining. Scale bar, 50 μm. Control: growth medium-treated NIH 3T3 cells. F68: 10 μg/ml pluronic F68; F127: 10 μg/ml pluronic F127; F68-F127: combination of 10 μg/ml pluronic F68 and F127; NaB: 15 μg/ml sodium pentaborate pentahydrate; Gel: combination of 15 μg/ml NaB, 10 μg/ml pluronic F68 and F127 containing growth medium. α-SMA: alpha-smooth muscle actin, Col I: collagen type I, and VIM: vimentin (GIF 60 kb)
Supplementary Figure 3
a Macroscopic images of burn wounds at days 3, 7, and 14. b Macroscopic evaluation of deep second-degree burn wound. c Hematoxylin and eosin staining of second-degree burn wound (GIF 48 kb)
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Demirci, S., Doğan, A., Karakuş, E. et al. Boron and Poloxamer (F68 and F127) Containing Hydrogel Formulation for Burn Wound Healing. Biol Trace Elem Res 168, 169–180 (2015). https://doi.org/10.1007/s12011-015-0338-z
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DOI: https://doi.org/10.1007/s12011-015-0338-z