Abstract: BACKGROUND: In recent years, there have been many novel tympanic membrane repair materials, including patches and 3D-printed scaffolds. However, the tympanic membrane repaired by these materials is different from the natural tympanic membrane in terms of thickness and internal structure.
OBJECTIVE: To explore the efficacy of bone marrow mesenchymal stem cells-loaded high-porosity polycaprolactone/collagen nanofiber membrane scaffolds in repairing chronic tympanic membrane perforation.
METHODS: Polycaprolactone, polycaprolactone-collagen, and high-porosity polycaprolactone-collagen nanofiber membranes were prepared by electrospinning technology, and the surface morphology, porosity and cell compatibility of the scaffolds were characterized. The tympanic membrane perforation model of 50 male SD rats was established by puncturing the posterior lower part of both eardrums with a sterile 23-measure needle combined with mitomycin C and hydrocortisone. After 12 weeks of modeling, the rats were divided into five groups by the random number table method. The blank control group did not receive any treatment. In the other four groups, polycaprolactone nanofiber membrane (polycaprolactone group), polycaprolactone-collagen nanofiber membrane (polycaprolactone-collagen group), high-porosity polycaprolactone-collagen nanofiber membrane (high-porosity polycaprolactone-collagen group) and high-porosity polycaprolactone-collagen nanofiber membrane containing bone marrow mesenchymal stem cells (high-porosity polycaprolactone-collagen group) were implanted at the perforation of the tympanic membrane, respectively. Each group consisted of 10 animals. The healing of the tympanic membrane was examined by otoendoscopy after 1, 2, 3 and 4 weeks of stent implantation. Hematoxylin-eosin staining, Masson staining, and Ki-67 immunohistochemical staining were performed on the tympanic membrane after 4 weeks of implantation. 
RESULTS AND CONCLUSION: (1) Scaffold characterization: Scanning electron microscopy showed that compared with other nanofiber membranes, the high-porosity polycaprolactone-collagen nanofiber membranes had more orderly nanofiber structure, larger surface pore size, and higher porosity (P < 0.001). Live/dead staining showed that bone marrow mesenchymal stem cells adhered well on the three scaffolds, and the number of living cells on the high-porosity polycaprolactone-collagen nanofiber membrane was more than that on the other two scaffolds. Almarin staining showed that the proliferation rate of bone marrow mesenchymal stem cells on the high-porosity polycaprolactone-collagen nanofiber membrane was higher than that of the other two fiber membranes. (2) Animal experiments: Except for the blank control group, the tympanic membrane of the other four groups healed gradually with the extension of the time of fibrous membrane implantation, among which the healing speed of the cell-loaded high-porosity polycaprolactone-collagen group was the fastest. Hematoxylin-eosin staining, Masson staining, and Ki-67 immunohistochemical staining showed that the tympanic membrane of rats in the cell-carrying high-porosity polycaprolactone-collagen group was moderate in thickness and a three-layer structure with uniform collagen fiber layers, similar to the normal tympanic membrane, and the repair quality of tympanic membrane was better than that of other fiber membrane groups. (3) The results showed that the high-porosity polycaprolactone-collagen nanofiber membrane containing bone marrow mesenchymal stem cells could not only rapidly repair the perforation of the tympanic membrane, but also the newly healed tympanic membrane was similar to normal tympanic membrane in structure and thickness.

Key words: ">electrospinning technology, chronic tympanic membrane perforation, polycaprolactone-collagen scaffold, mesenchymal stem cell, nanofiber membrane