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. 2023 Jul 28;14(8):403.
doi: 10.3390/jfb14080403.

In Vitro and In Vivo Evaluation of Epidermal Growth Factor (EGF) Loaded Alginate-Hyaluronic Acid (AlgHA) Microbeads System for Wound Healing

Maqsood Ali  1 , Si Hyun Kwak  2 , Je Yeon Byeon  2 , Hwan Jun Choi  2
Affiliations

In Vitro and In Vivo Evaluation of Epidermal Growth Factor (EGF) Loaded Alginate-Hyaluronic Acid (AlgHA) Microbeads System for Wound Healing

Maqsood Ali et al. J Funct Biomater. .

Abstract

The management of skin injuries is one of the most common concerns in medical facilities. Different types of biomaterials with effective wound-healing characteristics have been studied previously. In this study, we used alginate (Alg) and hyaluronic acid (HA) composite (80:20) beads for the sustained release of epidermal growth factor (EGF) delivery. Heparin crosslinked AlgHA beads showed significant loading and entrapment of EGF. Encapsulated beads demonstrated biocompatibility with rat L929 cells and significant migration at the concentration of AlgHAEGF100 and AlgHAEGF150 within 24 h. Both groups significantly improved the expression of Fetal Liver Kinase 1 (FLK-1) along with the Intercellular Adhesion Molecule-1 (ICAM-1) protein in rat bone Mesenchymal stem cells (rbMSCs). In vivo assessment exhibited significant epithelialization and wound closure gaps within 2 weeks. Immunohistochemistry shows markedly significant levels of ICAM-1, FLK-1, and fibronectin (FN) in the AlgHAEGF100 and AlgHAEGF150 groups. Hence, we conclude that the EGF-loaded alginate-hyaluronic acid (AlgHA) bead system can be used to promote wound healing.

Keywords: alginate; epidermal growth factor; heparin; hyaluronic acid.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) Visual photographs of AlgHAHep beads, (b) the percentages of bead size distribution, (c) illustration of possible AlgHAHepEGF chemical composition, and (d) EDS spectrum indicating the composition of the bead system.
Figure 2
Figure 2
Characterization of prepared AlgHAHepEGF beads, (a) degradation of the bead system (n-3), (b) pH change of PBS with the AlgHAHepEGF100, and (c) the FTIR analysis of the bead system showing the peaks related to alginate, hyaluronic acid, and amide bonds.
Figure 3
Figure 3
(a) EGF loading on beads (AlgHA and AlgHAHep), (b) EGF entrapment efficiency (%) of the beads (AlgHA and AlgHAHep), and (c) heparin and EGF release from the beads; AlgHA/Hep shows the cumulative release of heparin from the beads and AlgHA100 (EGF loaded with no heparin) and AlgHAHepEGF100 (heparin crosslinked EGF) show the cumulative release of EGF (n = 3, *** p < 0.001, 200 mg/group).
Figure 4
Figure 4
Biocompatibility testing of L929 with the AlgHA scaffold groups by MTT (a) after 1, 3, and 5 days of treatment and (b) nucleus fluorescence microscopic analysis of the L929 cells for cell proliferation by Hoechst staining (Scale bar 500 µm).
Figure 5
Figure 5
Representation of cell migration (a) control group along with the EGF group’s effect on L929 after 24 h and (b) representative images of L929 cell migration (n = 3, ns: non-significant, ** p < 0.005, *** p < 0.001).
Figure 6
Figure 6
Representative Western blots graph and images. (a) Flk-1 protein expression obtained by three independent experiments, (b) ICAM-1 expression, and (c) representative blot images of Flk-1, ICAM-1, and beta-actin expressions in the control and different AlgHAHepEGF groups (n = 3, ns: non-significant, * p < 0.005, ** p < 0.001, **** p < 0.0001).
Figure 7
Figure 7
(a) Wound-healing behavior of the control and AlgHA groups is shown with relative comparison and statistics of 1 week and 2 weeks after wound closure. (b) Representative photos of the wound sites in the three groups at 1 and 2 weeks (n = 3, ns: non-significant, *** p < 0.001).
Figure 8
Figure 8
Histological findings showing that AlgHAHepEGF150 promotes wound healing. (a) 1 week and 2 weeks after inducing the wound, defect sites show contraction in 2-week images (scale bar 1 mm), (b) magnified images of the defect and AlgHA groups showing epithelial thickness and formation of the epidermal layer across the wound area (scale bar 100 µm), magnified images of the defect and treatment groups showing vascularization, (c) MT staining of all the groups at 1 and 2 weeks; images show collagen formation and granulation tissue formation in all the groups (scale bar 1 mm), (d) epithelial score between the two ends of defect and treatment groups, (e) epidermal layer formation graph of all the 2 weeks groups, and (f) wound closure gap of the two edges of the defect in AlgHAHepEGF100 and AlgHAHepEGF150 groups. NE (neo-epithelization) n = 3, unpaired t-test analysis, ** p < 0.01, *** p < 0.001) (Red squares shows wound sites while arrows pointing new epidermal layer in the wound area).
Figure 9
Figure 9
Immunohistochemistry for ICAM-1, Flk-1, and fibronectin in rat skin. (a) Microscopic images of ICAM-1, Flk-1, and FN for defect, AlgHAHepEGF100, and AlgHAHepEGF150 groups and (b) % integral optical density (% IOD) of ICAM-1, Flk-1, and FN (n = 3, ** p < 0.005, *** p < 0.001, **** p < 0.0001) (Blue arrows identifying stained areas in the image).
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