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Three-dimensional characterization of tissue-engineered constructs by contrast-enhanced nanofocus computed tomography.

ABSTRACT: To successfully implement tissue-engineered (TE) constructs as part of a clinical therapy, it is necessary to develop quality control tools that will ensure accurate and consistent TE construct release specifications. Hence, advanced methods to monitor TE construct properties need to be further developed. In this study, we showed proof of concept for contrast-enhanced nanofocus computed tomography (CE-nano-CT) as a whole-construct imaging technique with a noninvasive potential that enables three-dimensional (3D) visualization and quantification of in vitro engineered extracellular matrix (ECM) in TE constructs. In particular, we performed a 3D qualitative and quantitative structural and spatial assessment of the in vitro engineered ECM, formed during static and perfusion bioreactor cell culture in 3D TE scaffolds, using two contrast agents, namely, Hexabrix® and phosphotungstic acid (PTA). To evaluate the potential of CE-nano-CT, a comparison was made to standardly used techniques such as Live/Dead viability/cytotoxicity, Picrosirius Red staining, and to net dry weight measurements of the TE constructs. When using Hexabrix as the contrast agent, the ECM volume fitted linearly with the net dry ECM weight independent from the flow rate used, thus suggesting that it stains most of the ECM. When using PTA as the contrast agent, comparing to net weight measurements showed that PTA only stains a part of the ECM. This was attributed to the binding specificity of this contrast agent. In addition, the PTA-stained CE-nano-CT data showed pronounced distinction between flow conditions when compared to Hexabrix, indicating culture-specific structural ECM differences. This novel type of information can contribute to optimize bioreactor culture conditions and potentially critical quality characteristics of TE constructs such as ECM quantity and homogeneity, facilitating the gradual transformation of TE constructs in well-characterized TE products.

SUBMITTER: Papantoniou I

PROVIDER: S-EPMC3936499 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Three-dimensional characterization of tissue-engineered constructs by contrast-enhanced nanofocus computed tomography.

Papantoniou Ioannis I Sonnaert Maarten M Geris Liesbet L Luyten Frank P FP Schrooten Jan J Kerckhofs Greet G

Tissue engineering. Part C, Methods 20131019 3

To successfully implement tissue-engineered (TE) constructs as part of a clinical therapy, it is necessary to develop quality control tools that will ensure accurate and consistent TE construct release specifications. Hence, advanced methods to monitor TE construct properties need to be further developed. In this study, we showed proof of concept for contrast-enhanced nanofocus computed tomography (CE-nano-CT) as a whole-construct imaging technique with a noninvasive potential that enables three ...[more]

PMID: 23800097

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Project description:BackgroundGross dissection is a widespread method for studying animal anatomy, despite being highly destructive and time-consuming. X-ray computed tomography (CT) has been shown to be a non-destructive alternative for studying anatomical structures. However, in the past it has been limited to only being able to visualise mineralised tissues. In recent years, morphologists have started to use traditional X-ray contrast agents to allow the visualisation of soft tissue elements in the CT context. The aim of this project is to assess the ability of contrast-enhanced micro-CT (μCT) to construct a three-dimensional (3D) model of the musculoskeletal system of the bird wing and to quantify muscle geometry and any systematic changes due to shrinkage. We expect that this reconstruction can be used as an anatomical guide to the sparrowhawk wing musculature and form the basis of further biomechanical analysis of flight.MethodsA 3% iodine-buffered formalin solution with a 25-day staining period was used to visualise the wing myology of the sparrowhawk (Accipiter nisus). μCT scans of the wing were taken over the staining period until full penetration of the forelimb musculature by iodine was reached. A 3D model was reconstructed by manually segmenting out the individual elements of the avian wing using 3D visualisation software.ResultsDifferent patterns of contrast were observed over the duration of the staining treatment with the best results occurring after 25 days of staining. Staining made it possible to visualise and identify different elements of the soft tissue of the wing. Finally, a 3D reconstruction of the musculoskeletal system of the sparrowhawk wing is presented and numerical data of muscle geometry is compared to values obtained by dissection.DiscussionContrast-enhanced μCT allows the visualisation and identification of the wing myology of birds, including the smaller muscles in the hand, and provides a non-destructive way for quantifying muscle volume with an accuracy of 96.2%. By combining contrast-enhanced μCT with 3D visualisation techniques, it is possible to study the individual muscles of the forelimb in their original position and 3D design, which can be the basis of further biomechanical analysis. Because the stain can be washed out post analysis, this technique provides a means of obtaining quantitative muscle data from museum specimens non-destructively.

Dataset Information

Three-dimensional characterization of tissue-engineered constructs by contrast-enhanced nanofocus computed tomography.

Publications

Three-dimensional characterization of tissue-engineered constructs by contrast-enhanced nanofocus computed tomography.

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