75% ethanol disinfection preserves biocompatibility and mechanical properties of swim bladder for cardiovascular applications

Abstract

Objectives: To evaluate the effects of 75% ethanol on the disinfection efficacy, biocompatibility, extracellular matrix (ECM) integrity, and mechanical properties of swim bladders from Hypophthalmichthys nobilis, a promising biomaterial for cardiovascular applications.

Methods: Swim bladders were immersed in 75% ethanol for 30, 60, or 120 minutes, followed by phosphate-buffered saline (PBS) rinsing. Disinfection efficacy was assessed using bacterial cultures; cytotoxicity and blood compatibility were evaluated using CCK-8 assays and hemolysis tests. The decellularization efficiency, ECM integrity, and mechanical properties were analyzed through histological staining, DNA quantification, and uniaxial tensile test.

Results: 75% ethanol effectively disinfected the swim bladder with minimal cytotoxicity and good hemocompatibility. Histological staining demonstrated preservation of collagen fibers, elastin, and glycosaminoglycans (GAGs), indicating minimal impact on the ECM structure. The uniaxial tensile test revealed that ethanol immersion caused tissue dehydration and increased stiffness, but these effects were reversible after PBS rinsing, with mechanical properties returning to baseline. DNA content and tissue thickness normalized following PBS washing, suggesting that ethanol did not impair decellularization.

Conclusions: 75% ethanol is an effective disinfectant for swim bladder biomaterials, preserving biocompatibility, ECM structure, and mechanical properties after rinsing. These findings support the use of swim bladder-derived scaffolds in cardiovascular tissue engineering applications.

Keywords: Swim bladder; ethanol; extracellular matrix; hemocompatibility; mechanical properties.

Figure 1

Flow chart of the study. CCK-8, cell counting kit-8; ECM, extracellular matrix; H&E stain, hematoxylin and eosin stain.

Figure 2

The results of bacterial culture. In the fresh group, bacterial colonies appeared on both Colombia Blood Agar and TSA plates within the first day of incubation. No bacterial colony was observed in any groups treated with 75% ethanol in 72 hours of incubation. TSA, Tryptic Soy Agar.

Figure 3

The results of hemolytic test in vitro and cytotoxicity analysis. A, B. Hemolytic test and hemolysis rate; C. CCK-8 for cytotoxicity analysis.

Figure 4

Changes in H&E staining (20×), DNA quantification, and tissue weight. H&E staining: (A) Fresh, (B) A30, (C) A60, (D) A120, (E) AP30, (F) AP60, (G) AP120, (H) Quantitative DNA testing, (I) Weight changes. Bar=50 μm. *; P<0.05, **; P<0.01 compared with the fresh group.

Figure 5

Uniaxial tensile test of the groups. A. Thickness; B. Elastic modulus; C. Elongation at break; D. Ultimate tensile stress; E. Stress-Strain curve. *; P<0.05, **; P<0.01.

Figure 6

EVG stain (20×), Modified Saffron-O and fast Green stain (20×), Sirius red stain (20×), and Alcian blue stain of the groups (20×). Bar=50 μm. EVG, Elastica van Gieson.