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. 2021 Feb 26;22(5):2358.
doi: 10.3390/ijms22052358.

Differential Responses to Bioink-Induced Oxidative Stress in Endothelial Cells and Fibroblasts

Hatice Genç  1 , Jonas Hazur  2 , Emine Karakaya  2 , Barbara Dietel  3 , Faina Bider  2 , Jürgen Groll  4 , Christoph Alexiou  1 , Aldo R Boccaccini  2 , Rainer Detsch  2 , Iwona Cicha  1
Affiliations

Differential Responses to Bioink-Induced Oxidative Stress in Endothelial Cells and Fibroblasts

Hatice Genç et al. Int J Mol Sci. .

Abstract

A hydrogel system based on oxidized alginate covalently crosslinked with gelatin (ADA-GEL) has been utilized for different biofabrication approaches to design constructs, in which cell growth, proliferation and migration have been observed. However, cell-bioink interactions are not completely understood and the potential effects of free aldehyde groups on the living cells have not been investigated. In this study, alginate, ADA and ADA-GEL were characterized via FTIR and NMR, and their effect on cell viability was investigated. In the tested cell lines, there was a concentration-dependent effect of oxidation degree on cell viability, with the strongest cytotoxicity observed after 72 h of culture. Subsequently, primary human cells, namely fibroblasts and endothelial cells (ECs) were grown in ADA and ADA-GEL hydrogels to investigate the molecular effects of oxidized material. In ADA, an extremely strong ROS generation resulting in a rapid depletion of cellular thiols was observed in ECs, leading to rapid necrotic cell death. In contrast, less pronounced cytotoxic effects of ADA were noted on human fibroblasts. Human fibroblasts had higher cellular thiol content than primary ECs and entered apoptosis under strong oxidative stress. The presence of gelatin in the hydrogel improved the primary cell survival, likely by reducing the oxidative stress via binding to the CHO groups. Consequently, ADA-GEL was better tolerated than ADA alone. Fibroblasts were able to survive the oxidative stress in ADA-GEL and re-entered the proliferative phase. To the best of our knowledge, this is the first report that shows in detail the relationship between oxidative stress-induced intracellular processes and alginate di-aldehyde-based bioinks.

Keywords: alginate di-aldehyde; cell death; cell viability; gelatin; glutathione; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ATR-FTIR spectra of pristine alginate di-aldehyde (ADA) and alginate covalently crosslinked with gelatin (ADA-GEL). The relevant peaks are discussed in the text.
Figure 2
Figure 2
Schematic depiction of (a) ADA and gelatin separately and (b) ADA-GEL structure formed via Schiff base bond formation. (c) Solid state 13C NMR spectra of ADA-GEL, pristine ADA and alginate (from top to bottom).
Figure 3
Figure 3
Cell viability of NIH/3T3- (left) and EA.hy926-cells (right) embedded in alginate and ADA hydrogels (%DO: 13–26%) after incubation for 24 h and 72 h. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
Time-dependent comparison of primary endothelial cells (ECs) and fibroblasts grown in ADA. (a) Cell viability (DiI-positive cells); (b) apoptotic populations in total death cluster (DiI-negative, PI-negative staining) and necrotic populations in total death cluster (DiI-negative, PI-positive staining). Control: cells with DCFH-DA grown on plastic. * p < 0.05 indicates significant differences between the groups in cell viability (a) and number of apoptotic cells (b).
Figure 5
Figure 5
Time-dependent comparison of primary ECs and fibroblasts grown in ADA-GEL. (a) Cell viability (DiI-positive cells); (b) apoptotic populations in total death cluster (DiI-negative, PI-negative staining) and necrotic populations in total death cluster (DiI-negative, PI-positive staining). Control: cells with DCFH-DA grown on plastic. * p < 0.05 indicates significant differences between the groups in cell viability (a) and number of necrotic cells (b).
Figure 6
Figure 6
Comparison of intracellular reactive oxygen species (ROS) levels and cellular thiol levels in primary ECs and fibroblasts grown in a time dependent manner in ADA. (a) Mean value of DCF emission detected by flow cytometer; (b) intracellular thiol level of cells under the same conditions. Results obtained in the same cell population with MBB staining and mean value of emission are presented. Cell viability at respective time points is indicated above the columns. Control: cells with DCFH-DA grown on cell culture plastic.
Figure 7
Figure 7
Comparison of intracellular ROS levels and cellular thiol levels in primary ECs and fibroblasts grown in a time dependent manner in ADA-GEL. (a) Mean value of DCF emission detected by flow cytometer; (b) intracellular thiol content of cells under the same conditions. Results obtained in the same cell population with MBB staining and mean value of emission are presented. Cell viability at respective time points is indicated over the columns. Control: cells with DCFH-DA grown on cell culture plastic.
Figure 8
Figure 8
Immunofluorescence staining of proliferating cells (green channel) after 72 h of incubation. Representative images show Ki-67 expression in primary ECs and human fibroblast grown in ADA and ADA-GEL. Nuclei: Hoechst (Blue), Ki-67: Alexa Fluor 488 (green). The white arrows indicate scarce fibroblasts in ADA positive for Ki-67.
Figure 9
Figure 9
FTIR spectra of hydrogel samples from cell culture experiments after 6 h of incubation in comparison to pristine ADA and ADA-GEL. Cells were removed from the hydrogels prior to measurements, and “+ HUVEC” indicates samples that were obtained from cell culture with primary ECs. The characteristic peaks are discussed in the text.
Figure 10
Figure 10
Comparison of cell viability between primary ECs and fibroblasts grown in ADA versus ADA-GEL. * p < 0.05, ** p < 0.001.
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