3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan

Abstract

This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

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“…These findings are supported by the results from previously published studies that showed increased expression of osteogenic factors and ALP in osteoblast-like MC3T3-E1 and MG63 cells in the 3D culture associated with increased gone mineral deposits [16]. Similar findings were previously reported using SaOS2, a human osteosarcoma cell line, where the expression levels of markers of early osteoblastic differentiation markers such as Runx2 and ALP were increased [17].…”

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“…3D cell culture is essential for studying the cell behaviors and interactions with the external environment . Cell transplantation after 3D culture can fill the tissue defects and induce the tissue regeneration [Figure (b)] . Laronda et al .…”

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“…Microglia may also proliferate in response to pro-inflammatory stimuli so an increase in viability may reflect an activation state. Different effects for similar treatments have previously been reported between 2D and 3D cultures, and while 2D culture models may over-estimate adverse effects 37,59,60 they are a well-established, and relatively costeffective model system, while 3D cultures permit the potential…”

“…Microglia may also proliferate in response to pro-inflammatory stimuli so an increase in viability may reflect an activation state. Different effects for similar treatments have previously been reported between 2D and 3D cultures, and while 2D culture models may over-estimate adverse effects 37,59,60 they are a well-established, and relatively costeffective model system, while 3D cultures permit the potential influence of extracellular matrix models to be investigated. Finally, as both the MTT (used in the 2D culture) calcein-AM fluorescence (used in the 3D culture) report on viability as a product of metabolic and enzymatic (esterase) activity, any treatments that alter this cellular activity will also alter the apparent cell viability, so interpretations and direct comparisons should be cautiously considered.…”