Imaging Cells in Polymer Scaffolds by X-Ray Microcomputed Tomography
Shauna M. Dorsey, Sheng Lin-Gibson, Carl Simon Jr.
We have investigated the ability of X-ray microcomputed tomography (CT) to make quantitative, three-dimensional (3D) measurements of cell adhesion and proliferation in polymeric tissue engineering scaffolds. The most common method for examining cells in scaffolds is microscopy . Sectioning followed by histology can image the scaffold interior but is destructive, tedious and only semi-quantitative . Fluorescence microscopy can be quantitative when high-throughput approaches are applied  and confocal fluorescence microscopy can yield 3D images . However, neither can "see through" opaque materials to image the interior of a scaffold. Other common methods for measuring cell presence include the colorimetric and fluorometric soluble assays for enzymes (dehydrogenase) , protein (BCA)  or DNA (Picogreen) . These soluble assays are quantitative but do not yield information on cell distribution. In contrast, CT generates 3D images, can penetrate deep into the scaffold interior, is non-destructive and is inherently quantitative [7-10]. For these reasons, we have investigated the sensitivity of using CT to image and measure cell adhesion and proliferation in polymeric tissue engineering scaffolds. In order to test the ability of CT to assess cell adhesion and proliferation in polymer scaffolds, cells were seeded onto polymer scaffolds at six different concentrations (0, 5000, 10000, 25000, 100000 and 400000 cells per scaffold) and measured at different time points (1 d, 7 d and 14 d). Poly(-caprolactone) (PCL) was chosen as the material for scaffold fabrication because it is biocompatible and has been cleared by FDA for use in biomedical implants. A salt-leaching approach was chosen because it is a common and effective method for scaffold fabrication. The MC3T3-E1 osteoblast cell line was used because it is a well-characterized murine osteoblast model which has been widely applied for regenerative orthopaedic applications. Cell adhesion and proliferation on the scaffolds was assessed by three techniques: fluorescence microscopy, a soluble assay for DNA (Picogreen) and CT. Results from the three approaches were compared so that the usefulness of CT for detecting cells in tissue engineering scaffolds could be evaluated.