Antibacterial-Agent-Immobilized Gelatin Hydrogel as a 3D Scaffold for Natural and Bioengineered Tissues

Abstract

Hydrogels and their medical applications in tissue engineering have been widely studied due to their three-dimensional network structure, biocompatibility, and cell adhesion. However, the development of an artificial bile duct to replace the recipient's tissue is still desired. Some challenges remain in the tissue engineering field, such as infection due to residual artifacts. In other words, at present, there are no established technologies for bile duct reconstruction as strength and biocompatibility problems. Therefore, this study investigated hydrogel as an artificial bile duct base material that can replace tissue without any risk of infectious diseases. First, an antibacterial agent (ABA), Finibax (an ABA used for the clinical treatment of biliary tract infection), was immobilized in gelatin using a crosslinking agent, and the antibacterial properties of the gel and its sustainability were tested. Furthermore, the immobilized amount and the improvement of the proliferation of the human umbilical vein endothelial cells (HUVECs) were cultured as the ABA-Gelatin hydrogel was introduced to prepare a 3D scaffold. Finally, we performed hematoxylin and eosin (H&E) staining after subcutaneous implantation in the rat. Overall, the ABA-Gelatin hydrogel was found to be viable for use in hydrogel applications for tissue engineering due to its good bactericidal ability, cell adhesion, and proliferation, as well as having no cytotoxicity to cells.

Keywords: hydrogel, antibacterial agent, gelatin, 3D scaffold, biomaterials for tissue engineering, cytotoxicity, cell culture.

Figure 1

Descriptions of the images of the experiment: (a) The dropped position of the antibacterial agent (ABA) solutions in a dish; (b) the ABA concentrations: 2–500 µg/mL on the point; and (e) the spectral wavelength of the antibacterial agents, namely Finibax, Flomoxef, and Biapenem. Photographs of the action of the ABAs in a dish of transformed Escherichia coli: (c) control or ABA-free; (d) Finibax; (f) Flomoxef; and (g) Biapenem after 1 day of the inoculation. The bar represents 30 mm.

Figure 2

(a) Photograph of the ABA-Gelatin gel- the bar represents 5 mm; (b) the molecular structure of Finibax as an ABA; (c) the release percent of ABA in the gels; (d) the immobilized ABA amount in the ABA-free, ABA-mixed, and ABA-immobilized gelatins; and (e) the immobilized ABA amount at different concentrations of the crosslinking agent.

Figure 3

(a) Calibration curve of the number of E. coli (CFU/mL) and the turbidity value; (b) temporal change of the turbidity at different concentrations of the ABA; (c) calibration curve of the turbidity and the concentration of the ABA after three hours; and (d) evaluation of the activity of the ABA in free, mixed, and immobilized cases.

Figure 4

Photographs of the antibacterial evaluation on E. coli: (a) ABA-free gelatin gel and (b) ABA-immobilized gelatin gel. The bars represent 1 cm. Measurements of bacterial count on the gel surface: (c) PBS and (d) LB medium. Agar medium coated with PBS immersed in gel: (e) Gelatin without an antibacterial agent and (f) gelatin mixed with an antibacterial agent.

Figure 5

ABA-free gelatin gel and ABA-immobilized gelatin gel: (a) Turbidity increase rate with respect to the number of liquid changes and (b) evaluation of the number of viable cells in the human umbilical vein endothelial cells (HUVEC) culture on the gels.

Figure 6

Cell photographs of the human umbilical vein endothelial cell (HUVEC) culture on gelatin gels: 1 day after seeding with (a) an ABA-free gelatin gel and (b) an ABA-immobilized gelatin gel; 4 days after seeding with (c) an ABA-free gelatin gel and (d) an ABA-immobilized gelatin gel. The bars represent 200 µm.

Figure 7

ABA-free gelatin gel: (a) 1 week after transplantation; (b) after the extraction of the gel and (c) hematoxylin and eosin (H&E) staining of the gel after subcutaneous implantation for 1 week in the rat; ABA-immobilized gelatin gel: (d) 1 week after transplantation; (e) after the extraction of the gel and (f) H&E staining of the gel after subcutaneous implantation for 1 week in the rat. Bars represent 1 cm and the arrows indicate the gel sample.