Cytotoxicity Evaluation of Hydrogel Sheet Dressings Fabricated by Gamma Irradiation: Extract and Semi-Direct Contact Tests

Abstract

Cytotoxicity presents one of the required criteria in the biological evaluation of medical devices. In this study, the semi-direct contact test was used to evaluate the potential cytotoxicity of hydrogel wound dressings compared to the conventional extract test. Three types of hydrogel sheets were fabricated from poly(vinyl alcohol) (PVA) by gamma irradiation: Bare sheets, silver (Ag)-coated sheets, and Aloe vera (AV)-coated sheets. In the extract test, L929 cells were cultured in the extract derived from the elution of hydrogel samples. For the semi-direct contact test, the cells were cultured in situ with the hydrogel samples placed inside transwell inserts above the cell monolayers. At the endpoint of both tests, MTT assay was performed, and the cell viability was determined from the absorbance of formazan. Only the bare and AV-coated hydrogel sheets showed cell viability above the 70 % threshold that ensured the non-cytotoxicity by both tests. For Ag-coated sheets, less than 70 % cell viability occurred when Ag coating was ³ 0.2 mg/cm2. Interestingly, the formazan-depleted area underneath the Ag-coated sample could be clearly observed by the semi-direct contact test. The release of Ag in the form of nanoparticles was confirmed by UV-Vis absorption at 420 nm. In conclusion, the semi-direct contact test can serve as a reliable alternative to the conventional extract test in evaluating the potential cytotoxicity of hydrogel wound dressings.
 HIGHLIGHTS
 
 A semi-direct contact test with transwell inserts was investigated as an alternative approach to the conventional extract test in evaluating the cytotoxicity of hydrogel wound dressings
 Leachable substances from the hydrogel dressings permeated through the porous membrane of transwell inserts to the reach the cell monolayer underneath
 Cell viability results based on MTT assay obtained from semi-direct contact test were in agreement with the extract test
 Only the semi-direct contact test provided the visual assessment of localized toxicity caused by the release of silver nanoparticles from hydrogel sheets onto the cell monolayer
 
 GRAPHICAL ABSTRACT

Antibacterial Hydrogel Sheet Dressings Composed of Poly(vinyl alcohol) and Silver Nanoparticles by Electron Beam Irradiation

“…In terms of the sensitivity of the different bacteria to the AgNPs, our findings also agreed with a previous report that the MIC of AgNPs against S. aureus was higher than the level required for E. coli [29]. Based on our previous study, the release of AgNPs from the hydrogels was qualitatively confirmed by UV-Vis spectrophotometry [30]. In the present study, the amount of released AgNPs at varied AgNO 3 loadings was quantitatively measured in the form of ionized Ag + by ICP-MS (Figure 6), which decreased proportionally from treatments A-E. For hydrogels A, B, and C incorporated with AgNPs at 1, 0.5, and 0.25 mg/cm 2 , respectively, the amount of released AgNPs increased sharply by approximately 26-43% between 2 and 24 h. After, the level of the released AgNPs remained relatively unchanged for up to 72 h. Interestingly, for treatments D and E, with a much lower initial AgNO 3 loading, the released AgNPs continued to rise linearly with the incubation time from 2 up to 72 h. Regarding the AgNP levels of 59 ± 5 ppm for hydrogel B and 32 ± 2 ppm for hydrogel C measured by ICP at 24 h, it can be derived from our experiment that at least ~30 and 60 ppm of AgNPs is required for effective antibacterial action against S. aureus and E. coli, respectively.…”

“…In the present study, the amount of released AgNPs at varied AgNO 3 loadings was quantitatively measured in the form of ionized Ag + by ICP-MS (Figure 6), which decreased proportionally from treatments A-E. For hydrogels A, B, and C incorporated with AgNPs at 1, 0.5, and 0.25 mg/cm 2 , respectively, the amount of released AgNPs increased sharply by approximately 26-43% between 2 and 24 h. After, the level of the released AgNPs remained relatively unchanged for up to 72 h. Interestingly, for treatments D and E, with a much lower initial AgNO 3 loading, the released AgNPs continued to rise linearly with the incubation time from 2 up to 72 h. Regarding the AgNP levels of 59 ± 5 ppm for hydrogel B and 32 ± 2 ppm for hydrogel C measured by ICP at 24 h, it can be derived from our experiment that at least ~30 and 60 ppm of AgNPs is required for effective antibacterial action against S. aureus and E. coli, respectively. Based on our previous study, the release of AgNPs from the hydrogels was qualitatively confirmed by UV-Vis spectrophotometry [30]. In the present study, the amount of released AgNPs at varied AgNO3 loadings was quantitatively measured in the form of ionized Ag + by ICP-MS (Figure 6), which decreased proportionally from treatments A-E. For hydrogels A, B, and C incorporated with AgNPs at 1, 0.5, and 0.25 mg/cm 2 , respectively, the amount of released AgNPs increased sharply by approximately 26-43% between 2 and 24 h. After, the level of the released AgNPs remained relatively unchanged for up to 72 h. Interestingly, for treatments D and E, with a much lower initial AgNO3 loading, the released AgNPs continued to rise linearly with the incubation time from 2 up to 72 h. Regarding the AgNP levels of 59 ± 5 ppm for hydrogel B and 32 ± 2 ppm for hydrogel C measured by ICP at 24 h, it can be derived from our experiment that at least ~30 and 60 ppm of AgNPs is required for effective antibacterial action against S. aureus and E. coli, respectively.…”

Hydrogel Based Skin Contacting Medical Devices and Cytotoxicity: An Overview of Challenges and Recommendations from a Regulatory Perspective

“…In this method, the tested materials are placed in direct touch with the cell culture in order to identify any potential toxicity even from weak toxic materials (17) . The direct contact method is highly sensitive and able to detect weak cytotoxicity.…”