Abstract
To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial ‘design of experiments’ approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.
Highlights
IntroductionThe field of tissue engineering (TE) is evolving towards the development of complex, three-dimensional (3D) constructs (i.e. cells/tissue combined with scaffold) to mediate the repair of severe defects
The field of tissue engineering (TE) is evolving towards the development of complex, three-dimensional (3D) constructs to mediate the repair of severe defects
Staining time did not significantly influence the normalized contrast, indicating that both contrast agents had infiltrated the neo-tissue within 30 minutes, even for the largest neo-tissue volume (Fig 5B and 5C)
Summary
The field of tissue engineering (TE) is evolving towards the development of complex, three-dimensional (3D) constructs (i.e. cells/tissue combined with scaffold) to mediate the repair of severe defects. The standard technique for evaluating tissue formation is histological sectioning It has a high discriminative power, both on tissue and cellular level. In standard settings it only allows assessment of tissue distribution in 2D, with loss of information due to a restricted sectioning orientation and with limited depth resolution [1,2,3]. Other standard techniques to assess the quality of a construct are Live/ Dead, DNA content (providing cell number estimation), histology and weight measurements. Live/Dead staining gives additional important information concerning cell state, it has limitations for internal, 3D visualization of tissue formation in a construct. Both DNA content and weight measurements are bulk measurement techniques not providing spatial information. There is a need for more advanced, quantitative 3D imaging techniques [4]
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