Brief communicationZebrafish: A possible tool to evaluate bioactive ions
Graphical abstract
Introduction
The zebrafish (Danio rerio), a freshwater vertebrate, has been used as a model system since the early 1970s. The reasons for its popularity are many: easy maintenance at a low cost and large number of larvae produced per mating, which is ideal for high-throughput experiments; optical transparency of the embryos and rapid embryonic development, which enables observation of organ function within a few days [1], [2], [3]. The zebrafish skeleton shows a high similarity to human bone, with matrix proteins and signaling pathways such as Wnt and BMP (bone morphogenic protein) [4]. Just like the human bone, theirs are also vascularised and remodeled by osteoblasts, osteoclasts and osteocytes [1], [5]. In 2005 Fleming et al. suggested a high-throughput in vivo model to assay compounds that affected bone density using the transparent zebrafish larvae. The results showed that vitamin D, glucocorticoid, bisphosphonates and PTH treatments responded on the zebrafish skeleton in the same way as in humans and rodents [6], [7]. Considering the advantages of the zebrafish model it is surprising how little this organism has been used in the development of biomaterials. There are certainly several areas were this model system could be useful, such as when evaluating the effects of dissolution products on bone formation. However, since zebrafish have other mechanisms for calcium homoeostasis, calcium ions are not good candidates for this type of studies [5].
There are several aspects in which the zebrafish are superior to isolated cell cultures: Firstly the fish contains the whole bone remodeling system i.e. interactions of osteoblasts, osteoclasts and osteocytes, thus being close to a mammalian in vivo situation. Secondly mineralisation assays in cell cultures are often tedious and time consuming with time spans of 20–30 days while fish larvae develop mineralised tissue within 3–4 days.
The aim of this work was to assess the feasibility of using a zebrafish larvae for evaluating bioactive ions' effect on bone formation. Silicate ion was chosen for this purpose since the ion is recognised as being important for bone formation [8], by stimulating osteoblast mineralisation [9], [10], [11] and inhibiting osteoclast differentiation [12]. In biomaterials intended for bone regeneration this ion has been used in various formulations: Bioglass [13], silicon substituted calcium phosphates [14], [15] and silica-polymer composites [16], [17]. In most cases the silicate ions are supposed to leak out from the material and thereby stimulate the surrounding bone tissue [18], [19]. Most often osteoblastic cells are used to evaluate the bone stimulating property of these ions, by measuring alkaline phosphatase (ALP) activity and bone matrix mineralisation [20], [21]. In this study the effect of silicate ion on zebrafish skeleton was evaluated and compared to the results obtained from a commonly used osteoblastic cell line, MC3T3-E1. A murine calvaria preosteoblast cell line that produce a mineralised matrix very similar to bone [22]. It has been suggested that this cell line is suitable for biomaterial evaluation [23]. To the authors' knowledge this type of study has not previously been done.
Section snippets
Cell culture
The murine osteoblastic cell line MC3T3-E1 subclone 14 was obtained from American Type Culture Collection (Rockville, USA). The cells were cultured in a complete medium: α-MEM (Hyclone, USA) supplemented with 10% foetal bovine serum (FBS; Hyclone, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Lonza, USA). The cell culture was maintained at 37 °C, 5% CO2 in a humidified atmosphere. Cells were trypsinised (Lonza, USA) and seeded with a density of 10,000 cells/cm2 in three 96 well plates and
Cell proliferation and ALP activity
None of the sodium metasilicate concentrations used in this study were toxic to the cells, as seen from the proliferation data (Fig. 1a). Higher proliferation was observed for cells treated with the highest silicate concentration. By day 7 the proliferation levelled out for all groups.
No ALP activity could be detected at day 3; however, the activity had increased considerably at day 7 and continued increasing until day 10 (Fig. 1b). For cells treated with 625 μM silicate a notably higher ALP
Discussion
The zebrafish model combines the scalability of an in vitro system with the relevance of a vertebrate in vivo model. Zebrafish bone is highly similar to human bone and has a rapid development, within a few days, thus, making the model system ideal for evaluating compounds that affect both bone formation and loss. Previous studies have shown that zebrafish skeleton respond to bone anabolic substances in a similar way as in humans and rodents [6]. These results raise the question if they also
Conclusion
Osteoblastic cells, MC3T3-E1, subjected to silicate ion treatment showed an increase in ALP activity and mineral formation compared to non-treated. The same trend; increase in mineralised area in response to higher silicate concentrations, was observed for zebrafish larvae 9 days post fertilisation. For earlier time points no difference in mineralised area could be detected in the larvae. Since the fish larvae encompass the whole bone remodeling system it is advantageous over isolated in vitro
Acknowledgement
This work was supported by the Science for Life Laboratory Zebrafish Technology Platform in Uppsala. Funding from the Swedish Research Council (GA 621-2011-3399) is acknowledged.
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