Heparin binding-epidermal growth factor-like growth factor for the regeneration of chronic tympanic membrane perforations in mice

Abstract

We aim to explore the role of epidermal growth factor (EGF) ligand shedding in tympanic membrane wound healing and to investigate the translation of its modulation in tissue engineering of chronic tympanic membrane perforations. Chronic suppurative otitis media (CSOM) is an infected chronic tympanic membrane perforation. Up to 200 million suffer from its associated hearing loss and it is the most common cause of pediatric hearing loss in developing countries. There is a need for nonsurgical treatment due to a worldwide lack of resources. In this study, we show that EGF ligand shedding is essential for tympanic membrane healing as it's inhibition, with KB-R7785, leads to chronic perforation in 87.9% (n=58) compared with 0% (n=20) of controls. We then show that heparin binding-EGF-like growth factor (5 μg/mL), which acts to shed EGF ligands, can regenerate chronic perforations in mouse models with 92% (22 of 24) compared with 38% (10 of 26), also with eustachian tube occlusion with 94% (18 of 19) compared with 9% (2 of 23) and with CSOM 100% (16 of 16) compared with 41% (7 of 17). We also show the nonototoxicity of this treatment and its hydrogel delivery vehicle. This provides preliminary data for a clinical trial where it could be delivered by nonspecialist trained healthcare workers and fulfill the clinical need for a nonsurgical treatment for chronic tympanic membrane perforation and CSOM.

FIG. 1.

Developing KB-R7785 chronic tympanic membrane (TM) perforations. Figure demonstrates the lack of progression, through the proliferation and migration of keratinocytes of the TM wound following perforation and application of KB-R7785 with staining with Hematoxylin and Eosin (a, c) and Cytokeratin (b, d). Time points are day 2 (a), showing the perforation edge (arrow) and surrounding exudate, day 7 (b), showing keratinization inhibited (arrow), and day 44 (c), showing a perforation (arrow) and no keratinization, day 44 (d) showing a lack of keratinocyte proliferation at the handle of malleus attachment to the TM (arrow). Scale bar=10 μm (magnification 10×). Color images available online at www.liebertpub.com/tea

FIG. 2.

Changes in the middle ear with eustachian tube (ET) occlusion. Figure demonstrates the changes in the middle ear mucosa with occlusion of the ET. Cilia are present in the mucosa of the middle ear mucosa (arrows) with an inflammatory exudate (top left corner). These changes are seen throughout the middle ear mucosa in models created with ET occlusion. They are not seen in models without ET occlusion. Scale bar=10 μm (magnification 10×). Color images available online at www.liebertpub.com/tea

FIG. 3.

The perforation margin in the chronic suppurative otitis media (CSOM) model. Figure demonstrates the perforation edge showing a mucosal layer migrated laterally to be directly adjacent to the keratinocyte layer (arrow) with a thickened fibrous layer containing inflammatory cells. These findings are commonly observed in human chronic TM perforations. Scale bar=10 μm (magnification 40×). Color images available online at www.liebertpub.com/tea

FIG. 4.

Heparin binding–epidermal growth factor (HB-EGF) treatment of chronic TM perforations. Figure shows the otoscopic appearance of chronic TMs treated with HB-EGF (a) and those controls treated with polymer only (b) at 4 weeks following treatment. Figure also shows the otoscopic appearance of chronic TMs with ET occlusion treated with HB-EGF (c) and those controls treated with polymer only (d) at 6 weeks following treatment. Figure also shows the otoscopic appearance of CSOM treated with HB-EGF (e) and those controls treated with polymer only (f) at 4 weeks following treatment. A white X marks the area of residual perforation. Color images available online at www.liebertpub.com/tea

FIG. 5.

The healed TM following HB-EGF treatment compared with healed controls. Figure shows Cytokeratin (brown) staining to identify the keratinocytes and keratin layers in the TM. The HB-EGF-treated chronic perforation (a) shows normal TM layers, including a thick keratin layer compared with the control TM (b). The HB-EGF-treated TM (a) has a normal thickness connective tissue layer compared with the lack of keratinization and thick disorganized connective tissue layer of the control TM (b). Scale bar=10 μm (magnification 40×). Color images available online at www.liebertpub.com/tea

FIG. 6.

The auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) thresholds of HB-EGF-treated ears compared with control. Figure shows the (a) ABR and (b) DPOAE thresholds measured at 10 weeks in ears perforated and injected with the polymer hydrogel loaded with HB-EGF at 50 μg/mL (n=13), 10 times the effective dose, compared with control ears, which were injected with the polymer hydrogel only (n=13). There were no differences between groups demonstrating a lack of ototoxicity of HB-EGF in the mouse model.