Supplementary Material for: Mapping the Regenerative Pattern in the Human Tympanic Membrane

Introduction The reason why chronic tympanic membrane (TM) perforations fail to heel is an enigma. A better insight into the premises and cellular events in this process is fundamental. Previous large studies on human TMs are lacking. The main objective of the present study was to map potential stem cells in the human TM and to address the regenerative capacity in the TM keratinocytes. Further, the study aimed to assess the regenerative premises for three regions of the TM: the malleus, the annulus, and the unattached portion of the pars tensa (UPT), including different anatomical levels (inferior, intermediate, and superior) within each region. Methods Ten healthy human TMs were harvested during lateral skull base surgery. Using IHC, the TMs were stained for α6 and β1 integrin, CK19, p63, and Ki-67. Semi-quantification was made with two single-blinded observers scoring the staining of the slides. Inter-rater reliability was evaluated with weighted Cohen’s kappa and group differences between the mean grading were further calculated. Results Antibodies for α6 and β1 integrin, CK19, p63, and Ki-67 could be detected along the malleus, annulus and UPT of human TMs displaying a diversity in the regenerative capacity of the human TM. The staining of Ki-67 and p63 was more prevalent in the annulus and malleus compared to in the UPT. α6 staining was more prevalent in the annulus while CK19 staining was more prevalent in the malleus. Finally, several antibodies displayed a higher staining intensity in the inferior anatomical levels of the TM. Conclusion The present study shows the presence of putative stem cells in all regions of the human TM, with a stronger regenerative capacity close to the annulus and malleus compared to the UPT. These findings may set a baseline for the localization and diversity of putative stem cells in the human TM.