Tympanic membrane regeneration : clinical and experimental human studies

<p dir="ltr">The keratinized squamous epithelium of the tympanic membrane (TM) possesses a great inborn capacity for cell migration and proliferation. This regenerative capacity is reflected in the fact that most tympanic membrane perforations (TMP) heal spontaneously within a few weeks. However, when early complete healing does not occur, the perforation may become chronic, leading to persistent discharge and hearing loss. The reasons why some TMPs fail to heal remain unclear. Therefore, a deeper understanding of the early stages of TM regeneration is essential for developing strategies to prevent chronic TMPs (cTMP).</p><p dir="ltr">The overall aim of this thesis is to gain understanding of the healing process in the human TM and why some cTMPs do not heal.</p><p dir="ltr">In Paper I we studied the presence and specific locations of epidermal progenitor cells in the keratinocyte layer in the healthy human TM. Ten healthy human TMs were stained with a panel of antibodies specific against epidermal stem cell (SC) and proliferation markers (Ki-67, CK19, p63, and the integrins a6 and B1). The results showed presence of progenitor cells along the whole keratinocyte layer in the TM and with a significantly higher presence close to the malleus and annulus region.</p><p dir="ltr">In Paper II, by inducing a local trauma to the TM in vivo, we explored the early regenerative events in the human TM by mapping the presence and changes of epidermal progenitor cells. Twenty hours after traumatization there was significant upregulation of CK19 and a6 integrin in the unattached pars tensa (UPT) close to the trauma and with comparable levels of those in the previously shown regenerative regions in Paper I. The proliferation marker Ki-67 was stronger seen in the UPT far away from the location of the traumatization. CK19, Ki-67, and p63 were still strongly seen close to the malleus and annulus confirming the regenerative regions seen in Paper I. This first study of the acute healing response in the keratinocyte layer of human TMs following in vivo traumatization provide important insights through the use of an experimental model.</p><p dir="ltr">In Paper III we evaluated the investigational medicinal product Plasminogen (human) 10 in patients with cTMP in a phase 1 clinical study, aiming to set a base for a new cost-effective and safe treatment method for cTMPs. By subcutaneous injections of plasminogen (PLG) close to the TM in patients with cTMP, the safety, feasibility and effect of the drug was measured. The results showed no serious adverse events (AE). The AEs reported were overall mild and of transient character. Patients could tolerate the treatment of three PLG injection days in one week. No differences were seen in the active treatment group compared to placebo regarding healing outcome. The results suggest future trials on PLG, including dose escalating studies.</p><p dir="ltr">In Paper IV risk factors for an unhealed TMP after surgical intervention with myringoplasty was identified. Swedish quality register data was analyzed for the years 2014-2019, including all patients undergoing a myringoplasty with a registered follow-up visit. The results indicated that age of the patient, surgical aim, history of prior ear surgery, and postoperative infection constituted risk factors for unsuccessful healing following myringoplasty. Interestingly, cases that required simultaneous ossiculoplasty showed a higher rate of successful healing. The study presents one of the largest cohorts on myringoplasty outcome and adds new insights on why cTMPs do not heal despite facilitating healing support.</p><p dir="ltr">In conclusion, this thesis contributes to an enhanced understanding of the natural regenerative processes of the TM and determines that there are putative SCs in the human TM. Further, the methodologies and findings presented herein provide a foundation for future research to improve treatment and management of cTMPs.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Sepehri E,</b> Engmér Berglin C, Liu Y, Eriksson PO, von Unge M, Arebro J. Mapping the Regenerative Pattern in the Human Tympanic Membrane. Audiology and Neurotology. 2025 Aug 16:1-24. doi: 10.1159/000547944.<br><a href="https://doi.org/10.1159/000547944">https://doi.org/10.1159/000547944<br></a><br></p><p dir="ltr">II. <b>Sepehri E,</b> Engmér Berglin C, Liu Y, Eriksson PO, von Unge M, Arebro J. The Early Regenerative Events in the Human Tympanic Membrane after In Vivo Traumatization. [Submitted]</p><p dir="ltr">III. <b>Sepehri E,</b> Tideholm B, Hellström S, Engmér Berglin C. Plasminogen - Safe for Treatment of Chronic Tympanic Membrane Perforation: a Phase 1 Randomized, Placebo-Controlled Study. Acta Otolaryngol. 2024 Jul-Aug;144(7-8):439-445. <a href="https://doi.org/10.1080/00016489.2024.2396488" rel="noreferrer" target="_blank">https://doi.org/10.1080/00016489.2024.2396488</a></p><p dir="ltr">IV. <b>Sepehri E,</b> Arebro J, Westman E, Eriksson PO. Unsuccessful Healing of Tympanic Membrane Perforations Following Myringoplasty - a Nationwide Study. [Submitted]</p>