Plastic Surgery Research Council

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Utilization of MRI for Localization of Neocartilage on Human Chondrocyte-seeded Ear Scaffolds
Qing Yu, PhD1, Robin DiFeo Childs, MS1, Frank Reinauer, BS2, Stephanie Grom, BS2, Tobias Wolfram, PhD2, William Landis, PhD3, Noritaka Isogai, MD, PhD4, John Shelestak, BS5, Robert Clements, PhD5, Dylan Childs, MD1, Ananth Murthy, MD1
1Akron Children's Hospital, Akron, OH, 2KLS-Martin, Mühlheim, Germany, 3University of California, San Francisco, San Francisco, CA, 4Kindai University, Osaka, Japan, 5Kent State University, Kent, OH

PURPOSE: An alternative approach such as tissue engineering a biodegradable ear to substitute for current surgical procedures for ear reconstruction is a promising advance in regenerative medicine. The aim of this study was to utilize magnetic resonance imaging (MRI), histology and histomorphometry for identification and localization of neocartilage in implanted tissue-engineered human auricular chondrocyte-seeded ear scaffolds.
METHODS: Two human ear-shaped polycaprolactone (KLS Martin, Germany)/Neoveil nano (D15, Gunze Ltd., Japan) scaffolds with different framework designs and porosity were separately labeled as scaffold A (N = 1) and B (N = 1) and each was seeded with 600 million human auricular chondrocytes obtained surgically. The seeded scaffolds were implanted subcutaneously in the dorsum of athymic rats for 20 weeks. Harvested specimens were fixed in 10% neutral buffered formalin for 7 days prior to MRI scanning. The samples were immersed in fluorinert (FC-40; 3M, Saint Paul, MN) and placed in a custom-built chamber to provide enhanced contrast during MR scanning. Ear scaffolds were imaged with a Bruker ICON 1 Tesla small animal MRI using standard acquisition protocols (T1, T2 relaxation times). MR images were analyzed and recombined using ImageJ followed by registration with histological specimens for comparison. The presence of proteoglycan and elastin as markers for regenerated auricular cartilage in each implanted ear scaffold was evaluated by Safranin-O and Verhoeff staining.
RESULTS: After 20 weeks of implantation, neocartilage was observed in both scaffolds regardless of the differences in the scaffold framework and porosity. The MRI and histology results obtained from scaffold A were compared to like data from scaffold B (Figures A and B, respectively). Correlated cross-sectional MR imaging (T1 relaxation, Figures A-b, B-b), Safranin-O (Figures A-a, B-a), and Verhoeff staining (not illustrated) showed minimal cartilage formation on scaffold A and elastic cartilage regeneration in scaffold B. Rendered and projected images were also obtained to localize neocartilage on both scaffolds three-dimensionally.
CONCLUSION: A methodology for utilization of MRI to identify and localize neocartilage on human chondrocyte-seeded 3D-printed ear scaffolds has been developed. The present study confirmed the primary role of MRI in assessment of cartilage development as well as localization of neocartilage on implanted human auricular chondrocyte-seeded scaffolds. The study also revealed correlation between MR and histology results and demonstrated the capability of MRI techniques in tracing non-invasively the progress of the formation of auricular cartilage in vivo.


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