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

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.

Cell specificity of magnetic cell seeding approach to hydrogel colonization.

Tissue-engineered scaffolds require an effective colonization with cells. Superparamagnetic iron oxide nanoparticles (SPIONs) can enhance cell adhesion to matrices by magnetic cell seeding. We investigated the possibility of improving cell attachment and growth on different alginate-based hydrogels using fibroblasts and endothelial cells (ECs) loaded with SPIONs. Hydrogels containing pure alginate (Alg), alginate dialdehyde crosslinked with gelatin (ADA-G) and Alg blended with G or silk fibroin (SF) were prepared. Endothelial cells and fibroblasts loaded with SPIONs were seeded and grown on hydrogels for up to 7 days, in the presence of magnetic field during the first 24 h. Cell morphology (fluorescent staining) and metabolic activity (WST-8 assay) of magnetically-seeded versus conventionally seeded cells were compared. Magnetic seeding of ECs improved their initial attachment and further growth on Alg/G hydrogel surfaces. However, we did not achieve an efficient and stable colonization of ADA-G films with ECs even with magnetic cell seeding. Fibroblast showed good initial colonization and growth on ADA-G and on Alg/SF. This effect was further significantly enhanced by magnetic cell seeding. On pure Alg, initial attachment and spreading of magnetically-seeded cells was dramatically improved compared to conventionally-seeded cells, but the effect was transient and diminished gradually with the cessation of magnetic force. Our results demonstrate that magnetic seeding improves the strength and uniformity of initial cell attachment to hydrogel surface in cell-specific manner, which may play a decisive role for the outcome in tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2948-2956, 2017.

Evaluation of hydrogel matrices for vessel bioplotting: Vascular cell growth and viability.

Developing matrices biocompatible with vascular cells is one of the most challenging tasks in tissue engineering. Here, we compared the growth of vascular cells on different hydrogels as potential materials for bioplotting of vascular tissue. Formulations containing alginate solution (Alg, 2%, w/v) blended with protein solutions (silk fibroin, gelatin, keratin, or elastin) at 1% w/v were prepared. Human umbilical vein endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were cultivated on hydrogels for 7 days. Cell number and morphology was visualised using fluorescent staining at day 3 and 7. Cell metabolic activity was analysed using WST assay. Compared to pure Alg, Alg/keratin, Alg/gelatin and Alg/silk fibroin provided superb surfaces for ECs, supporting their attachment, growth, spreading and metabolic activity. SMCs showed best colonization and growth on Alg/silk fibroin and Alg/keratin hydrogels, whereas on elastin-containing hydrogels, cell clustering was observed. Fibroblasts growth was enhanced on Alg/elastin, and strongly improved on silk fibroin- and keratin-containing hydrogels. In contrast to the previous studies with alginate dialdehyde-gelatin crosslinked gels, Alg/gelatin blend hydrogels provided a less favourable scaffold for fibroblasts. Taken together, the most promising results were obtained with silk fibroin- and keratin-containing hydrogels, which supported the growth of all types of vascular cells. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 577-585, 2016.