Functional Outcomes of Heparin-Binding Epidermal Growth Factor-Like Growth Factor for Regeneration of Chronic Tympanic Membrane Perforations in Mice

Abstract

We aim to demonstrate that regeneration of chronic tympanic perforations with heparin-binding epidermal growth factor-like growth factor (HB-EGF) delivered by an injectable hydrogel restored hearing to levels similar to that of nonperforated tympanic membranes. Chronic tympanic membrane perforation is currently managed as an outpatient surgery with tympanoplasty. Due to the costs of this procedure in the developed world and a lack of accessibility and resources in developing countries, there is a great need for a new treatment that does not require surgery. In this study, we show in a mouse model through measurement of auditory brainstem response and distortion product otoacoustic emissions that tympanic perforations lead to hearing loss and this can be predominantly recovered with HB-EGF treatment (5 μg/mL). Our animal model suggests a return to function between 2 and 6 months after treatment. Auditory brainstem response thresholds had returned to the control levels at 2 months, but the distortion product otoacoustic emissions returned between 2 and 6 months. We also show how the vibration characteristics of the regenerated tympanic membrane, as measured by laser Doppler vibrometry, can be similar to that of an unperforated tympanic membrane. Using the best available methods for preclinical evaluation in animal models, it is likely that HB-EGF-like growth factor treatment leads to regeneration of chronic tympanic membrane perforations and restoration of the tympanic membrane to normal function, suggesting a potential route for nonsurgical treatment.

Keywords: HB-EGF; chronic perforation; tympanic membrane; tympanic regeneration.

FIG. 1.

Developing KB-R7785 chronic TM perforations. The figure shows the experimental study method for the mouse cohort. HB-EGF, heparin-binding epidermal growth factor-like growth factor; LDV, laser Doppler vibrometry; TM, tympanic membrane.

FIG. 2.

Hearing outcomes of HB-EGF treatment of chronic tympanic membrane perforations. The ABR and DPOAE thresholds in decibels (db) during the study. At baseline, both the treatment and control ears are not significantly different. As would be predicted, the treatment ears show a hearing loss after creation of a chronic perforation. This hearing loss returns in the treatment ear after 2 months as measured by the ABR and within 2–6 months as measured by the DPOAE. At the 6-month measurement, there is no significant difference between the treated ears and the control ears. ABR, auditory brainstem response; DPOAE, distortion product otoacoustic emission.

FIG. 3.

Dose escalation effect of HB-EGF. The figure shows a dose–response curve to determine the optimal dose, determined by number of perforations healed, to be used for these experiments; a preliminary cohort was treated as described above with escalating doses of HB-EGF. The numbers of chronic perforations at each dose are displayed above the curve at the time point.

FIG. 4.

LDV results of HB-EGF treatment of chronic tympanic membrane perforations. The figure shows the mean umbo velocity (normalized by pressure at the tympanic membrane) of the control and treatment groups, grouped into frequency bins corresponding to the frequencies used for the ABR and DPOAE measurements. Standard error bars are shown at each frequency. In both groups, the normalized velocity increases from 4.0 to 11.3 kHz before leveling off, although slight differences can be observed at higher frequencies. Overall, no significant difference was found between the two groups except at 31.9 and 46.1 kHz (p < 0.001, p < 0.001), with the treatment group having a smaller LDV score (with the treatment group scoring on average 0.73 and 0.53 for 31.9 and 46.1 kHz, respectively, compared with 1.29 and 1.08 for control).

FIG. 5.

Microscopic appearance of the tympanic membrane cohort. The figure shows the microscopic appearance of the TMs before measurement with the LDV. The samples are labeled according to mouse (M) and whether the sample is the control (C) side or the treatment (T) side. Sample M5C is shown as the LDV lining up the laser target onto the reflective bead to demonstrate the anatomical location of the measurements.

FIG. 6.

Sample tympanic membranes before and after debris removal. The figure shows examples (M6, M9) of the before and after cleaning images of the TMs (M6 before [a] and after [b], M9 before [c] and after [d]). The debris in both examples is partially adherent and with delicate cleaning, and it was thought that it could not be removed in its entirety without preserving the structure of the TM.