Abstract
In vitro 3D tissue-engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co-culture of TE skeletal muscle and bone are investigated. High-glucose Dulbecco's modified Eagle medium (HG-DMEM) supplemented with 20% fetal bovine serum followed by HG-DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co-cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen-based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co-culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.
Highlights
In vitro bio-toxicity testing of developmental pharmaceuticals, biomaterials and medical devices is performed on in vitro monolayer cell culture models (ISO10993)
Alkaline Phosphatase (ALP) concentration was not significantly different between cultures co-culture levels tended to be lower (Figure 6C). These results indicate that coculture bone cell populations exhibit a greater osteogenic potential than control cultures, and demonstrates that C2C12s positively interact with TE85 cultures in 3D
Whilst co-culture systems described within the literature report the influence of one cell type on the other, these papers have yet to describe the conditions which allow for the successful culture and differentiation of both cell types in a single medium system through experimental comparisons [52,53,54,55]
Summary
In vitro bio-toxicity testing of developmental pharmaceuticals, biomaterials and medical devices is performed on in vitro monolayer cell culture models (ISO10993). Monolayer models are capable of identifying cytotoxic effects through morphological and biochemical assays, or by assessing changes in gene expression [1]. These results do not effectively translate across to in vivo tissue systems [1,2]. This is due to a general failure to accurately recapitulate the complex nature of native tissue structures and the biochemical pathways that accompany such architecture, justifying the use of animal models [3,4,5,6]. There is a growing need for more complex in vitro models, which provide more representative structures and physiology than conventional cell cultures, without the complexities of animal research
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