3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

Abstract

A medium perfusion system is expected to be beneficial for three-dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast-like MC 3T3-E1 cells were 3D-cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell-seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering.

Three-Dimensional Characterization of Tissue-Engineered Constructs by Contrast-Enhanced Nanofocus Computed Tomography

“…Images obtained by standard experimental techniques are mainly limited to surface restricted TE construct characterization as they are line-of-sight techniques and only provide limited depth information. 50,52 Not taking into account the latter limitation may result in the misinterpretation of statically cultured TE constructs as being full of ECM, while in reality only an outer layer of ECM exists, as confirmed by CE-nano-CT (Fig. 4).…”

“…Fluid flow is known to enhance matrix synthesis within TE constructs during perfusion culture. 19,40,50 Furthermore, differences in fibrillar collagen organization of the ECM, which are flow dependent, 51 could also explain the ECM differences that were observed by PTA staining as the ECM morphology could influence the binding mechanism of PTA to the ECM proteins. In Figure 5, it may also be observed that the difference in collagen content, as determined by Picrosirius Red staining, for the different flow rates is less pronounced than the one observed with the PTA staining.…”

Hydrodynamic 3D Culture for Bone Tissue Engineering

“…Images of scaffolds observed at 10x objective of a fluorescence stereomicroscope are shown afcording to the regions indicated by the broad views on the left. (Du et al, 2009) In contrast, the scaffolds cultured by the oscillatory flow had a relatively uniform distribution of livnig cells throughout the scaffolds. The inhomogeneous living cells distribution of the unidirectional perfusion culture was further verified by higher magnificaion, as shown in Figure 14.…”

“…A: The unidirectional perfusion system; (b) the oscillatory perfusion system. (Du et al, 2009) A gas-permeable bag was set in the circuit loop as a gas exchanger. The unifirectional media flow was driven bya syringe ponp.…”

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

“…Although static seeding is the most commonly used method, low seeding efficiencies and non-uniform cell distributions are often G Bouet et al In vitro 3D bone culture model reported (Alvarez-Barreto and Sikavitsas, 2007;Burg et al, 2000;Holy et al, 2000). Therefore, based on the literature (Du et al, 2009;Godbey et al, 2004;Janssen et al, 2006a), a dynamic seeding technique was used in this study. Combined with the architecture of the scaffolds, this resulted in a uniform cell colonisation and distribution within the scaffold interior.…”