2. Innovative regenerative treatment for the tympanic membrane perforation New York University, May 5 2011, New York, USA Shin-ichi Kanemaru, M.D., Ph.D. 1) Hiroo Umeda, M.D. 2), Yoshiharu Kitani, M.D. 2), Satoshi Ohno, M.D. 2), Tsuyoshi Kojima, M.D. 2), Tatsuo Nakamura, M.D., Ph.D. 3), Shigeru Hirano, M.D., Ph.D. 2), Juichi Ito, M.D., Ph.D. 2) 1) Department of Otolaryngology–Head and Neck Surgery, Medical Research Institute, Kitano Hospital, Osaka, Japan 2) Department of Otolaryngology–Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 3)Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Medical Research Institute Kitano Hospital

3. Disadvantages of TM perforation? Background Hearing loss, Decline of speech articulation Easy and recurrent infection Tinnitus, aural fullness and etc. Restrictions of daily life activities

4. Cancellation effect

5. Collision of sounds in the cochlea Rapid attenuation of energy

6. Greatest disadvantage of TM perforation Large TMP often causes over 50dBHL Hearing aid amplifies the "cancellation effect" conversation ： 40-60dB 50dBHL Hearing Aid

7. ？ What are the present treatments of TM perforation ？ Necessity of skin incision and harvest of auto-tissue Necessity of hospitalization Failure and sequelae of operation Mental/physical burden and costsOperation

8. Cells Scaffold Regulatory factors in situ tissue engineering Tissue engineering Approach for Regeneration of TM Gelatin sponge Gelatin sponge B-FGF B-FGF Good regenerative conditions Seal by fibrin glue Seal by fibrin glue Regeneration of TM

9. Method and Procedures Gelatin sponge b-FGF TM perforation Fibrin Glue After 3 weeks Disruption of the perforation edge Disruption of the perforation edge

10. Patients who are susceptible to this treatment Dry TM and tympanic cavity without active inflammation during the previous 3 years Proper aeration and no regions of soft tissue density in the mastoid and tympanic cavities based on Temporal bone CTs Intact ossicular chains No cholesteatoma and no invasion of epithelia into tympanic cavity

11. Patients Patients/ears: n=140/158 (M/F:59/81), Age: 10-91 Causes of b-FGF group Control group TM perforation n=148 n=10 Otitis media 90 5 Postoperatively 14 2 Old trauma 20 1 Residual perforation after operation/ventilation tube 24 2 insertion

13. Subtotal perforation Disruption of the perforation edge Gelatin Sponge with b-FGF Case 1. 65y.o. male OMC for 30years

14. After 3 weeks After 4 months Hearing Level Before: 61dB After: 33dB

15. 0.125 0.250 0.5 1 2 4 8 kHz 3 months after Before treatment dB 0 10 20 30 40 50 60 70 80 90 100 Conversation range

16. Case 2. 39-y.o. female After 1 month Total perforation after TM tube insertion After 3 months Hearing Level Before: 50dB After: 10dB

17. 3 months after Before treatment dB 0 10 20 30 40 50 60 70 80 90 100 0.125 0.250 0.5 1 2 4 8 kHz

18. Overall Results of b-FGF group Grade I ： PS 2/3 NA: Average hearing level of 0.5, 1 and 2 kHz LA: Average hearing level of 0.125, 0.25 and 0.5 kHz *TO: Temporary otorrhea **RTM: Retraction of tympanic membrane ***Chole: Cholesteatoma Classification by Grade I Grade II Grade III perforation size (n=37) (n=64) (n=47) Number of times 1-3 1-4 1-4 for treatment （ Ave. ） (1.31) (1.31) (1.95) Closure rates 94.6 ％ 85.9 ％ 83.0% (35/37) (55/64) (39/47) ImprovementNA:14.1dB 20.6dB 24.5dB of the ave. HLLA :28.7dB 31.1dB 35.3dB Adverse *TO: n=3 n=10 n=12 events ** RTM: n=2 n=5 n=5 ***Chole: n=0 n=2 n=2

19. % ****** *<0.001, **< 0.001, ***<0.001: Mann Whitney U test Comparison between the two groups

20. 0 40 50 60 70 80 90 100 dB % 100 90 80 70 60 Speech articulation Before treatment After treatment

21. Why can we easily achieve to TM regeneration? Gelatin sponge ＋ bFGF Gelatin sponge ＋ bFGF

22. Factors for making possible to regenerate TM Tissue stem cells/Progenitor cells Gelatin sponge as a scaffold b-FGF as a growth factor Creating optimal regenerative conditions

23. Cells Disruption of the perforation edge

24. Cells Auditory Epithelial Migration

25. Process of the TM regeneration 43 year old male, OMC for 28year Before after 9days after 1m 123

26. Cells There are tissue stem cells/progenitor cells that are origin of regenerative TM around the perforation edge. Disruption of the perforation edge

27. Scaffold Gelatin sponge Gelatin sponge is made of a protein extracted from collagen and has an open space structure. A sustained release substrate for b-FGF

28. Strong inducer for blood capillaries Fibroblast growth factor improvement in the local regenerative conditions Suitable for regeneration of the intermediate layer of TM Growth factor ： b-FGF B-FGF

29. Histology of TM Epithelial layer Intermediate Fibrous layer EAM side

30. Spontaneous regenerated part of TM

31. Regenerated TM by this treatment Before 2 ms after

32. Differences in growing speed Spontaneous regeneration Regenerated TM by this treatment I II III I II III I: epithelial layer, II: intermediate fibrous layer, III: mucosal layer Gelatin sponge with b-FGF

33. Seal by fibrin glue Ideal cell culture condition Creating optimal regenerative conditions Protection of dry and infection Fibrin glue

34. No skin incision and no harvest of autologous tissues Wide application for various kinds/sizes of the TM perforation including total perforations Only 10 minutes simple/easy treatments for outpatients Ideal hearing up and tinnitus reduction immediately after the treatment No restrictions of the patient’s daily life No severe sequelae and no disadvantages Cost-effective and alleviation of mental and physical burdens of the patients No skin incision and no harvest of autologous tissues Wide application for various kinds/sizes of the TM perforation including total perforations Only 10 minutes simple/easy treatments for outpatients Ideal hearing up and tinnitus reduction immediately after the treatment No restrictions of the patient’s daily life No severe sequelae and no disadvantages Cost-effective and alleviation of mental and physical burdens of the patients Remarkable advantages

35. Summary This study demonstrated that the combination of a gelatin sponge, b-FGF and fibrin glue was effective for regeneration of the TM perforation. This is the innovative regenerative therapy: easy, simple, cost-effective and minimum- invasive treatment for outpatients.

36. Our dream coming true! Medical Research Institute Kitano Hospital, Osaka, Japan

37. Hybrid Tympanoplasty

38. Tympanoplasty TM regeneration safety sequelae safety sequelae Hearing improvement Hearing improvement cost-effective adaptation Background

39. What is the Hybrid Tympanoplasty? After mastoidectomy and posterior tympanotomy, cleaning of the tympanic cavity through mastoid cavity No need to harvest of temporal fascia for reconstruction of TM No need to exfoliate soft tissue of EAM and TM To perform regeneration of the TM though external auditory meatus

40. III Mastoidectomy Posterior tympanotpmy III Regeneration of MACs IV Regeneration of TM Procedures of Hybrid Tympanoplasty

41. Merits of the Hybrid Tympanoplasty Day or short stay surgery. Minimum sequelae are associated with this procedure because of no Restrictions are not placed on the patient’s daily life. There are low risks of damage to chorda tympani nerve. It is possible to fully regenerate normal TM morphology and to improve hearing up to maximum level. Wide renge of applications.

42. Adaptation of Hybrid Tympanoplasty Chronic otitis media No adaptation for cholesteatoma, adhesive otitis media No adaptation for post operative cases Intact case of ossicular chains

43. 4 weeks after Hybrid Tympanoplasty Hearing level: 42.5dB/15.0dB (before/after)

44. Histology of TM