Synthetic pre-vascularized porous scaffold as an engineered environment for the in vitro osteosarcoma model

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Menshikh, Ksenia (Center for Translational Research on Autoimmune and Allergic Disease, Università del Piemonte Orientale )


One of the biggest challenges in the field of tumour engineering is providing cells with a proper environment mimicking physiological conditions of cancer from the biomechanical point of view. Hard tissue cancers, such as osteosarcoma, are no exception, especially taking into account the complexity of the bone microenvironment. The golden standard is the use of human-derived tissue-engineered scaffolds, but this meets other difficulties connected to regulations, low availability and reproducibility. This work aims to find an approach for providing osteosarcoma cell spheroids with the physiological environment independently of human tissue availability.
To form stable cell aggregates, GFP-transfected human osteosarcoma cells (U2OS) were cultured in a multiwell agarose-coated plate. The dynamics of cell metabolic activity in spheroids and their morphology was monitored by resazurin reduction assay, as well as visually by light and fluorescent microscopy. Alginate and bioactive glass were the components of choice for producing the matrices due to their proven reputation in the biomaterials field. Polymerized alginate samples enriched with bioactive glass particles were frozen, freeze-dried, cut and analysed by scanning electron microscopy (SEM). Human endothelial cells (EA.hy926) were suspended in the collagen and seeded on the porous alginate-based matrices. The cell-loaded scaffolds were then cultured with the addition of vascular endothelial growth factor (VEGF) to trigger tubule-like network formation. After the network development was confirmed by fluorescent microscopy and histological assays, previously obtained osteosarcoma spheroids were placed within each scaffold and monitored during the cultivation period to assess metabolic activity and sprouting.
Throughout the set-up of the method and considering the data from the analyses above, the optimal parameters of preparation of the matrices were established. Endothelial cells seeded on the porous matrices showed sufficient viability rates and the ability to form a tubule-like network throughout the chosen periods. Also, osteosarcoma cell spheroids demonstrated high metabolic activity and tumour-like behaviour in terms of invasion the surrounding matrix when placed within the pre-vascularized scaffolds.
The viability and morphology of endothelial cells and, consequently, osteosarcoma spheroids, allowed us to conclude that the obtained alginate-based pre-vascularized porous scaffolds are suitable as an environment for 3D in vitro modelling of osteosarcoma. The architecture of the scaffolds resembles trabecular bone tissue, while the inclusion of bioactive glass particles allows avoiding biochemical activation of osteogenic conditions. Moreover, the proposed system is tuneable, which offers the possibility of introducing various cell lines to simulate osteoblast-osteoclast crosstalk, as well as interaction with immune cells, and setting up dynamic conditions. The described approach can be admitted as promising in terms of developing antitumor drug test systems and studying peculiarities of primary bone cancer.


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