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Abstract Three-dimensional (3D) tissue engineering (TE) is a prospective treatment that can be used to restore or replace damaged musculoskeletal tissues such as articular cartilage. However, current challenges in TE include identifying materials that are biocompatible and have properties that closely match the mechanical properties and cellular environment of the target tissue, while allowing for 3D tomography of porous scaffolds as well as their cell growth and proliferation characterization. This is particularly challenging for opaque scaffolds. Here we use graphene foam (GF) as a 3D porous biocompatible substrate which is scalable, reproduceable, and a suitable environment for ATDC5 cell growth and chondrogenic differentiation. ATDC5 cells are cultured, maintained, and stained with a combination of fluorophores and gold nanoparticle to enable correlative microscopic characterization techniques, which elucidate the effect of GF properties on cell behavior in a three-dimensional environment. Most importantly, our staining protocols allows for direct imaging of cell growth and proliferation on opaque GF scaffolds using X-ray MicroCT, including imaging growth of cells within the hollow GF branches which is not possible with standard fluorescence and electron microscopy techniques. Abstract Figure
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Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice
Abstract BackgroundGiven the need for descriptive and increasingly mechanistic morphological analyses, contrast‐enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro‐level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA‐enhanced microCT. ResultsOur protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. ConclusionThese protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.
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- Award ID(s):
- 1731909
- PAR ID:
- 10458303
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Developmental Dynamics
- Volume:
- 249
- Issue:
- 4
- ISSN:
- 1058-8388
- Format(s):
- Medium: X Size: p. 573-585
- Size(s):
- p. 573-585
- Sponsoring Org:
- National Science Foundation
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