Development of New Regeneration Methods in Animal Models with Chronic Tympanic membrane Perforations Using Tissue Engineering Techniques

Tympanic Membrane (TM) perforation, in particular chronic otitis media, is one of the most frequent clinical problems in the otolaryngology field, which sometimes presents conductive healing loss. This study consists of three purposes and experiments; 1) we developed new animal models for chronic tympanic membrane perforation (CTMP). 2) We explored an approach for TM regeneration that latent progenitors or stem cells within epithelial layers may play an important role as a regulator of TM regeneration in both acute and chronic perforations. 3) And we also developed new regeneration methods to treat CTMPs. For the first purpose, nine methods were applied to 100 Sprague-Dawley rats; I) simple perforation, II) mitomycin C (MC) gelfoam for 10min, III) MC for 10min with saline gelfoam for 1 week, IV) MC for 10min and dexamethasone for 1 week, V) MC for 1 week (called as ‘Choung’s COM model II’), VI) dexamethasone for 1 week, VII) perforation by heated micropicks, VIII) heated perforations followed by dexamethasone for 1 week, and IX) dexamethasone application for 1 week after thermal-perforating with MC for 10min (called as ‘Choung’s COM model I’). For the second purpose, the neonatal rats (P3-5) were used for culture of TM cells. The cultured TM cells were immunostained with cytokeratin 19 (CK19), integrin β1 (INGβ1), P63 (epidermal stem cell markers), Ki67 (proliferation marker), and DAPI. Rat models for acute or chronic perforations in TMs were prepared. Chronic perforations were produced by a new method - ‘Choung’s COM model I’. The TMs were regularly obtained from rat models with acute or chronic perforations and immunostained with CK19, INGβ1, and DAPI. For the third purpose, we evaluated cell proliferation effects of insulin-like growth factor-binding protein (IGFBP), epidermal growth factor (EGF) and fibroblast growth factor (FGF) on in vitro cultures of TM cells using an MTT assay, and then constructed an EGF-releasing-chitosan patch scaffold (EGF-CPS) based on previous studies. We analyzed its toxicity and strength, and we studied it using scanning electron microscopy. We developed the IGFBP-chitosan patch scaffold (IGFBP-CPS) and EGF-CPS to healing in the CTMPs. In the first experiments, the perforation maintenance rates after 8 weeks were 0 % for method I, II, VI, VII, and VIII, 5.5% for III, 11.5% for IV, 65% for V, and 75% for IX. We then evaluated the reproducibility of the most effective methods using extra 70 rats. The average successful perforation rate was 71.6% (59 in 81 TMs) in method IX and 67.5% (27 in 40 TMs) in method V, which revealed constant reproducibility for CTMPs. From the second experiments, we found that the TMs expressed the highly positive staining of the epithelial stem cell markers. In the normal TMs, the TM stem cells were weakly expressed in all areas of TMs. On the other hand, the TM stem cells were highly expressed in perforated TMs, especially in the perforated regions near malleus handle and annulus, regardless of the acute and chronic perforations. In the third experiments, the EGF group, which was treated with EGF-CPS, showed healing in 56.5% of the animals (13/23), while the control group, which did not receive any treatment, revealed 20.8% healing (4/24) (P=0.04). Transmission electron microscopic studies of regenerated TMs in the EGF group showed much greater preservation of histological features, and TMs of the EGF group were thinner than spontaneously healed TMs. When we applied the IGFBP-CPS, 43.8% of the perforated TMs were regenerated completely in the animal models with CTMP. In conclusions, animal models with CTMPs can be efficiently created by thermal damage, MC, and dexamethasone. TMs contain progenitor or epidermal stem cells in the areas close to malleus handle or annulus, which may be key areas for TM regeneration. TM regeneration ability is not critically weakened even in chronic perforations. Novel EGF-CPS or IGFBP-CPS can be used as a nonsurgical intervention technique for treatment of CTMPs.

Author dc:creator

• 김, 승원

Contributors dc:contributor

• 정, 연훈
• 대학원 의학과
• 200824680

• tympanic membrane
• chronic otitis media
• regeneration
• stem cells
• scaffold
• insulin-like growth factor-binding protein
• epidermal growth factor
• 고막
• 만성중이염
• 줄기세포
• 재생
• 스캐폴드
• 인슐린유사성장인자 결합단백질
• 상피세포증식인자

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000013925
000000013925

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/8641