Correlative Imaging of 3D Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications

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.