3D Tissue-Engineered Construct Analysis via Conventional High-Resolution Microcomputed Tomography Without X-Ray Contrast

Abstract

As the field of tissue engineering develops, researchers are faced with a large number of degrees of freedom regarding the choice of material, architecture, seeding, and culturing. To evaluate the effectiveness of a tissue-engineered strategy, histology is typically done by physically slicing and staining a construct (crude, time-consuming, and unreliable). However, due to recent advances in high-resolution biomedical imaging, microcomputed tomography (μCT) has arisen as a quick and effective way to evaluate samples, while preserving their structure in the original state. However, a major barrier for using μCT to do histology has been its inability to differentiate between materials with similar X-ray attenuation. Various contrasting strategies (hardware and chemical staining agents) have been proposed to address this problem, but at a cost of additional complexity and limited access. Instead, here we suggest a strategy for how virtual 3D histology in silico can be conducted using conventional μCT, and we provide an illustrative example from bone tissue engineering. The key to our methodology is an implementation of scaffold surface architecture that is ordered in relation to cells and tissue, in concert with straightforward image-processing techniques, to minimize the reliance on contrasting for material segmentation. In the case study reported, μCT was used to image and segment porous poly(lactic acid) nonwoven fiber mesh scaffolds that were seeded dynamically with mesenchymal stem cells and cultured to produce soft tissue and mineralized tissue in a flow perfusion bioreactor using an osteogenic medium. The methodology presented herein paves a new way for tissue engineers to identify and distinguish components of cell/tissue/scaffold constructs to easily and effectively evaluate the tissue-engineering strategies that generate them.

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

“…For example, the information obtained by destructive scanning electron microscopy (SEM) remains 2D and rather elusive even when multiple sample slices are analyzed. 8,25,27 Thus, one has to be careful when using 2D validated techniques to draw 3D conclusions. Proper validation under 3D conditions and/or complementary use of additional real 3D information requires substantial additional research, but it is a much safer route to obtain reliable information of 3D cell and tissue behavior.…”

“…However, the value of X-ray CT-based information for TE will ultimately be a trade-off of factors such as physicochemical, morphological, and dimensional TE construct properties, contrast-enhanced agent, and equipment limitations ( Table 2). According to specific study objectives, a customized strategy will be required to extract the necessary information in a robust way, ranging from (1) accurate and detailed (100s of mm scale) identification and quantification of different TE construct components limited to small TE construct samples and using phase-contrast imaging, 25 to (2) using standard desktop micro-or nano-CT as a routine 3D imaging technique for whole TE construct analysis (mm to cm scale) and quantification of mineralization after in vivo implantation. 10,13 The latter approach, without the use of a contrast agent, has also been used for static or bioreactor in vitro cultures, 16,18,19 showing distinct contrast differences between the mineralized matrix and scaffold.…”

“…10,13 The latter approach, without the use of a contrast agent, has also been used for static or bioreactor in vitro cultures, 16,18,19 showing distinct contrast differences between the mineralized matrix and scaffold. Phase-contrast imaging [24][25][26] or micro-CT combined with osmium tetroxide as a contrast agent 28 has shown its potential to assess nonmineralized ECM in an in vitro engineered TE construct. However, the limited availability of synchrotron radiation or CT systems allowing phase-contrast imaging, the toxicity of osmium tetroxide, and the high cost associated with its disposal, all hamper their use as routine quality control for TE constructs.…”

“…22,23 Several studies have shown that when using phase-contrast imaging, in most cases only available by synchrotron radiation, the nonmineralized ECM formed in vitro in 3D TE constructs can be visualized. [24][25][26] However, routine access to systems allowing phase-contrast imaging is limited and there are restrictions on the sample specifications. On the other hand, by using the more routinely available desktop micro-CT in a standard absorption mode, without the use of a contrast agent, it has not been possible yet to visualize an in vitro produced nonmineralized ECM in 3D scaffolds.…”

1984 : on monitoring cell fate in three-dimensional polymeric scaffolds for tissue engineering applications

“…Bone, fibrous tissue and ceramic scaffolds present different coefficients of X-ray absorption, therefore their 3D structures can be separated and corresponding quantitative data such as bone volume, thickness, growth, destruction, remodeling and changes in bone density can be obtained [220,222]. However, contrast between distinct types of soft tissue with similar X-ray attenuation is limited [223].…”