| Autor: |
Liew LJ; 1 Ear Sciences Centre, School of Medicine, University of Western Australia , Perth, Australia .; 2 Ear Science Institute Australia , Perth, Australia ., Chen LQ; 2 Ear Science Institute Australia , Perth, Australia .; 3 School of Veterinary and Life Sciences, Murdoch University , Perth, Australia ., Wang AY; 1 Ear Sciences Centre, School of Medicine, University of Western Australia , Perth, Australia .; 2 Ear Science Institute Australia , Perth, Australia .; 4 Department of Otolaryngology, Head and Neck, Skull Base Surgery, Sir Charles Gairdner Hospital , Perth, Australia ., von Unge M; 5 Akershus University Hospital and University of Oslo , Oslo, Norway .; 6 Centre for Clinical Research Västerås, University of Uppsala , Uppsala, Sweden ., Atlas MD; 1 Ear Sciences Centre, School of Medicine, University of Western Australia , Perth, Australia .; 2 Ear Science Institute Australia , Perth, Australia ., Dilley RJ; 1 Ear Sciences Centre, School of Medicine, University of Western Australia , Perth, Australia .; 2 Ear Science Institute Australia , Perth, Australia .; 7 The Centre for Cell Therapy and Regenerative Medicine, School of Medicine, University of Western Australia , Perth, Australia . |
| Abstrakt: |
Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane (TM) and localized specifically to the umbo and annulus regions. While they have been proposed to play a role in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we compared different explant techniques using normal and perforated rat TM tissues and investigated their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal stem cell markers integrin β1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most effective technique. Explants made from perforated TM produced cells before those from unperforated TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells that were capable of culture expansion by proliferation and cell migration when subject to a wound assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved suitable for tissue engineering of the TM. |
