Thymosin beta-4 – A potential tool in healing middle ear lesions in adult mammals

Abstract

Acute tympanic membrane perforations primarily occur due to injury or infection in humans. In acute cases, nearly 80–94 % of the perforations heal spontaneously. In chronic cases, non-surgical treatment becomes significantly limited, and the perforation can be restored only by myringoplasty. In addition to classical grafts such as the fascia or cartilage, promising results have been reported with various biological materials including silk or acellular collagen. However, despite of all the efforts, healing remains insufficient. Consequentially, a need for substances which actively promote tympanic cell migration and proliferation is deemed essential.

In our study, we utilized Thymosin beta-4 (TB4), a 43aa peptide possessing many regenerative properties in various organ systems. Our aim was to reveal the impact of externally administered TB4 regarding impairments of the middle ear, particularly the tympanic membrane. We harvested tympanic membranes from adult mice and treated these with TB4 or PBS on both collagen gel matrixes and in the form of floating, ex vivo explants. Cell migration and proliferation was measured, while immunocytochemical analyses were performed to determine cell type and the nature of the targeted molecules.

We discovered the peptide affects the behavior of epidermal and epithelial cells of the tympanic membrane in vitro. Moreover, as our initial results imply, it is not the differentiated, yet most likely the local epidermal progenitor cells which are the primary targets of the molecule.

Our present results unveil a new, thus far undiscovered field regarding clinical utilization for TB4 in the future.

1. Introduction

Impaired hearing may influence quality of life, as it often leads to exclusion from communication resulting in social isolation, frustration, distress or even unemployment. More than 1.5 billion individuals experience some degree of hearing loss throughout their lifetime, and the vast majority of the affected population originate in low- and middle-income countries [2]. In addition, hearing impairment takes a significant toll on the economy. The WHO estimates unaddressed impaired hearing poses an annual global cost of 750 billion USD [1]. Hearing impairment may be caused by numerous factors. Beyond genetic alterations and malformations, infection, perinatal complications, age related sensorineural degeneration, injuries and environmental exposition can equally lead to hearing problems. Hearing loss can be broadly separated into two categories: conductive and sensorineural [3]. Sensorineural hearing loss (SNHL) occurs during inner ear or auditory pathway pathologies such as viral or bacterial infections, vascular occlusion, Menierés disease, damage by ototoxic drugs or various tumors [4]. Impairments of the outer and middle ear affect the sound conduction and amplification leading to conductive type of hearing loss (CHL) [5,6]. CHL can be caused by external or middle ear malformations, external auditory canal occlusion, otosclerosis, otitis media or tympanic membrane perforation [4,7] (Fig. 1.a).

Fig. 1.

a. Schematic of the human ear. Tympanic membrane (TM) is located between the external auditory canal (EAC) and the tympanic cavity (TC). M: Malleus, b. Endoscopic and a schematic image of the human tympanic membrane. PT: Pars tensa, PF: Pars flaccida, *: Malleolar folds, U: Umbo, M: Malleus. c. Hematoxylineosin staining of the mouse tympanic membrane. d. Schematic of the histological structure of the tympanic membrane.

The tympanic membrane (TM) is a cone-shaped organ tightly connected to the handle of the malleus and located between the external auditory canal (EAC) and the tympanic cavity (TC) (Fig. 1.a). Its inferior part, the pars tensa (PT), is fixed to the tympanic bone through the fibrocartilaginous annulus, whereas the malleus is attached to the bone with the malleolar ligaments forming the anterior and posterior malleolar folds on the surface of the tympanic membrane. The malleolar folds separate the superior part, the pars flaccida (PF), from the pars tensa (Fig. 1.b, c). Histologically, the TM consists of three layers. The outer surface contains the stratified squamous keratinized epithelium, which is continuous with the epithelium of the EAC. The inner surface is covered by simple cuboidal epithelium and the middle layer is made of fibroblastic connective tissue, containing the vessels and nerves which supply the membrane. While the pars tensa consists of organized radial and circular fibers, the pars flaccida has loosely arranged elastic collagen (Fig. 1.c, d).

TM perforation arises primarily due to injury or inflammation, a result of acute otitis media (AOM) or granular myringitis. Although 80–94% of all acute perforations spontaneously heal within three months, the remaining cases become inveterate resulting in a chronic suppurative otitis media (CSOM) (Fig. 2.a) [8]. In addition to CHL due to perforation and consequential insufficient amplification of the TM (Fig. 2.a white arrow), CSOM limits the patient’s quality of life, especially when participating in aquatic activities or bathing. Moreover, severe complications such as deafness, dizziness, meningitis or brain abscess may also occur. Clinically, CSOM primarily requires surgical intervention [9]. Currently, there are several technologies and materials available, which may be utilized for the reconstruction of the lesion. Autologous grafts such as the temporal fascia, perichondrium, cartilage or lobulus fat are commonly employed tissue types and result in a closure rate of 71–97% [10]. Additional materials like paper, silk, chitosan, collagen, polydimethyl siloxane or even acellular dermis as allograft effectively serve in mending reconstruction [11] (Fig. 2.b). Recently, the utilization of various bioactive molecules, such as basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) emerged as promising means to stimulate and heal TM perforations in various species including humans [12–15]. Despite the wide variety of methods and materials, unsuccessful surgeries and re-perforations may occur during everyday practice. Moreover, in addition to a significant complication risk among the elderly, the high number of surgical procedures implies a painful burden on the economy, which drives industry towards developing new technologies to accelerate critically needed healing procedures.

Fig. 2.

a, Endoscopic view of a left tympanic membrane with a chronic perforation in the superior-posterior quadrant (white arrow). The incudostapedial joint and the round window niche (black arrowhead) is visible through the perforation (white arrowhead). b, Schematic of the reconstruction of the perforation featuring an underlaid technique. The tympanic membrane is elevated and a temporal fascia graft is positioned on the medial surface of the tympanic membrane. c, Schematic representing cellular dynamics in the TM. A distinct population of stem cells are located at the malleolar fold (blue triangles) and committed progenitor cells have been described along the malleolar manubrium and in the annular region (purple dots) [16]. Out of these progenitor population, the keratinocytes of the TM derive and migrate towards the annulus and the outer ear canal (stars). Arrows represent the direction of cell migration.

There are numerous theories in published literature describing the potential cellular mechanism of spontaneous TM closure following perforation. Some of the related submissions declare the fibrous and mucosal layers or the keratin itself may serve as potential driving forces of regeneration [17–19]. Most recent studies however indicate the existing keratinocytes may be the primary role players in leading the healing process [20,21]. Moreover, special areas of the tympanic membrane have already been identified in which stem cells and/or already committed progenitor cells are located and being activated following perforation. Accordingly, progenitor cells were detected in the malleolar folds whereas committed progenitor cells were located near the handle of the malleus and in the annular regions (Fig. 2.c) [16,20,22].

Thymosin beta-4 (TB4) is a small, secreted molecule first described in 1966 by A. Goldstein and A. White [23]. Since it’s novel discovery, TB4 is a subject for many ongoing trials and was proven effective in healing dermal, ocular and cardiac impairments [24]. One of its first revealed intracellular functions is the capability to form a 1:1 complex with G-actin, resulting, among others, in altered cell migration [25]. TB4 also affects the DNA mismatch repair pivotal enzyme, hMLH1, and it was proven to be effective during corneal wound healing through actin, laminin-332 and other proteases in human ophthalmology trials [26,27]. Members of our research team equally confirmed, external administration of the molecule promotes myocardial and endothelial cell migration when cultured on three dimensional collagen matrixes and in the adult heart [28]. Moreover, stimulation of keratinocyte migration by TB4 both in vitro and in vivo has been confirmed [29].

To our surprise, despite of TB4′ s beneficial impact regarding tissue regeneration in various organs, the potential otological applications regarding TB4 have not yet been thoroughly studied. To fulfill these discrepancies, in this present study we first investigated the impact of TB4 on the cells of the middle ear. We utilized mouse tympanic membrane explants in both in vitro and ex vivo conditions and observed the alterations implemented on cell migration and cell proliferation respectively. Moreover, we intended to determine the impact of TB4 on the tympanic membrane’s own progenitor cells via immunohistochemical marker analyses.

2. Materials and methods

2.1. Animals and surgical procedures

In our experiments, we utilized C57BL/6 male and female mice 2–6 months of age. All animal procedures were carried out in strict accordance with the animal handling and welfare guidelines of the University of Pecs and the National Scientific Ethical Committee on Animal Experimentation of Hungary (Permission ID: BA02/2000-10/2021.). Mice were safely housed at the Szentagothai Research Center’s Animal Core Facility under standardized and continuously monitored conditions. During surgical procedures, adult mice were euthanatized by cervical dislocation, and the temporal bones encasing the TM were isolated. Next, the tympanic bulla was inferiorly opened using a 27G needle. The tendon of tensor tympani muscle was cut, and the tympanic membrane together with the malleus and incus was separated from the stapes and the inner ear.

2.2. Collagen gel migration assay

Collagen gel matrixes were prepared as previously described [28]. Shortly, one ml of 1 mg/ml type I Rat tail collagen in acetic acid (Roche, Basel, Switzerland) was first mixed with 100 μl of 10x M199 (Thermo Fischer Scientific; Waltham, ME, USA) and next adjusted with 100 μl of 2.2% NaCO₃ until pH 7.4 was achieved. A total of 280 μl of collagen per well was placed into four well plates (VWR; Radnor, PA, USA) and incubated for 15 min at room temperature (RT) under sterile conditions. 300 μl DMEM (Thermo Fischer Scientific) growing media containing 10% fetal bovine serum (FBS) (ATCC, Manassas, VA, USA) and 1% Pen/Strep antibiotics (Thermo Fischer Scientific) was applied at 37 °C in a CO₂ incubator to hydrate the gels. Following 1hr of incubation, the medium was exchanged until further utilization. Prior to the addition of the tympanic membrane explants onto the gels, the growing medium was carefully removed. Next, dissected TM was placed with its outer side facing down onto the collagen matrix (referred to as Day 0). Cell migration was visually inspected to confirm adherence. Following confirmation (3–4 days), 300 μl of DMEM enriched with 10% FBS and 1% P/S was added, and the explants were treated with 3 μl of 100 ng/ul TB4 in PBS (Thermo Fischer Scientific) or with 3 μl of PBS only as a control, respectively. The opposite of the treated TM per each animal was utilized as untreated control. Explants were kept under standard cell culture conditions at 37 °C and monitored/photo documented daily. The culturing media with proper treatment was exchanged every two days over a span of 12 days. Finally, TM membrane explants were terminated via 4% paraformaldehyde for 5 min at RT and washed and stored in PBS at 4 °C until further utilization.

2.3. Ex vivo floating explant preparation

For preparation of floating explants, TMs were isolated en bloc with the tympanic bone and soft tissue. Next, TMs were placed into 500 μl of DMEM and treated with 5μl of 100 ng/μl TB4 or PBS alone as control in 5% CO₂ at 37 °C. The culturing medium was refreshed every two days, incubated for two weeks, terminated via 4% paraformaldehyde for five minutes and stored in PBS at 4 °C until further analyses.

2.4. Collagen gel cell migrational distance measurements

The scale of cell migration was measured and defined between the edge of the TM and the migratory cells in pixel units utilizing ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA) (Fig. 3.a, b). Migratory cell identification was performed by visual observation of the cell morphology. Namely, cobblestone-shaped cells were defined as epidermal, while spindle shaped elongated cells were characterized as mesenchymal cell types under the microscope. Migrational distance of the various cell types was analyzed separately.

Fig. 3.

a, b, Representative photomicrographs depict the rate of epithelial cell migration of TM cells on collagen gel matrixes. (Black arrows indicate how measurements were performed). c, High magnification image of the boxed area of (a) represents the front of migrating cobblestone shaped epithelial cells (black arrowheads) and spindle-like mesenchymal cells (black arrows) d, Distance of epithelial cell migration of TM epithelial cells on collagen gel matrixes. Significant alteration between treated and untreated groups was first detectable at day nine of the explantation. (n = 4/each) Means and standard deviation bars with 95% confidence limits are shown. *p < 0.05.

2.5. Cell proliferation assays

Cell proliferation assays were performed on both collagen gel matrixes and ex vivo floating explant systems utilizing Click-iT Alexa Fluor 488 imaging kit (Thermo Fischer Scientific), respectively. The modified thymidine analogue EdU was supplemented in the culture media at 10 μM concentration during treatment of both collagen and floating TM explants. The experiment was terminated after 12 (collagen matrix) and 14 (floating explant) days. TM samples were fixed using 4% paraformaldehyde, and EdU incorporation was detected according to the manufacturer’s recommended protocol. EdU positive cells were counted and analyzed by investigating three equal size representative areas of the migrating TM cells on collagen matrixes and the area of the pars tensa on “floating explants” utilizing high power images, respectively.

2.6. Immunolabeling and detection

Following fixation, collagen gel or whole mount TM explants were rinsed 2× in PBS. Permeabilization was performed for 15 mins with 0.5% TritonX-100 in PBS at RT with gentle shaking. Next, samples were rinsed three times with PBS for 5 min and blocked in 3% donkey serum in PBS for two hours. Explants were then incubated with Cytokeratin-19 (1:200; Santa Cruz Biotechnologies, Dallas, TX, USA) and Vimentin (1:200; Santa Cruz Biotechnologies, Dallas, TX, USA) primary antibodies in 1% donkey serum containing PBS at 4 °C overnight. Following three times rinsing and washing in PBS, anti-rabbit Cy3 (1:500) and anti-mouse-FITC (1:500) conjugated secondary antibodies (Jackson Immunoresearch Laboratories Inc., West Grove; PA, USA) were added and kept at 4 °C overnight with gentle shaking. Following incubation, explants were rinsed in PBS with 0.5% Tween-20 for 2 × 1 hours. Nuclei were counterstained with 1 μM DAPI in PBS. Finally, both, collagen and floating explants were carefully transferred to a microscopic slide and mounted. Images were captured utilizing Zeiss LSM 710 laser scanning confocal microscope (Zeiss, Jena, Germany) and Nikon Eclipse3 epifluorescent microscope (Nikon Corp., Tokyo, Japan), respectively. For image processing, Z-stack and tiles were interconnected using ImageJ software.

2.7. Statistics

Statistical significance regarding cell migration and EdU positive cell calculations of TB4 and PBS treated groups was determined via IBM SPSS Statistics software Ver 28.0 (IBM, Armonk, NY, USA). When p-value appeared < 0.05, statistical calculations were performed using a standard t-test of variables with 95% confidence intervals.

3. Results

3.1. Thymosin beta-4 alters the migration of tympanic membrane epithelial cells in vitro

Evidently, TB4 bestows a significant impact upon cardiac and corneal cell migration in vitro and in vivo [28]. To reveal whether TB4 may equally accelerate the movement of tympanic membrane cells, thus potentially support regeneration of TM perforations, we first checked the direct impact of TB4 on TM epithelial and mesenchymal cell behavior. We explanted whole tympanic membranes from adult C57/BL6 mice and placed them on collagen gel extracellular matrixes. Explants were treated externally via TB4 (1 ng/μl final concentration) or PBS as a control. Visually, when epithelial cells formed cobblestone structures, mesenchymal cells appeared as star or spindle-like formations on collagen. Analyzing and measuring the migrational distance of both cell types, we found TB4 influences epithelial cell movement (Fig. 3.) without influencing mesenchymal cell behavior (data not shown) in vitro. Daily photo documentation and measurement of the explants revealed the migration of TB4 treated explants became statistically significant on the ninth day following explantation (TB4, 1099.0 / 179.9 px; PBS, 709.2 / 86.6 px, p-value: 0.0054) and maintained being significant until termination of the experiment (Day 10: TB4, 1461.6+/−188.0 px; PBS, 916.3+/−135.1 px, p-value: 0.0029; Day 11: TB4, 1752.2+/−72.2 px; PBS, 1215.5+/−114.1 px, p-value: 0.0004; Day 12: TB4, 2068.2+/−140.4 px, PBS, 1559.4+/−130.8 px, p-value: 0.0020) (Fig. 3.) suggesting the peptide may serve in aiding the closure of TM perforations. Finally, there was no detectable difference regarding the migration of mesenchymal cells between treated and untreated groups under the given conditions (data not shown).

3.2. Thymosin beta-4 promotes TM cell proliferation in vitro and in ex vivo explant systems

In addition to tympanic membrane cell migration, next we investigated if TB4 may equally accelerate cell proliferation utilizing two distinct TM explant systems. To define the rates of cumulative cell proliferation, we supplemented the TM tissue samples with EdU for two weeks, respectively, and visualized EdU incorporation via fluorescent microscope as suggested by the manufacturer. Our results revealed TB4 significantly accelerates TM cell proliferation on collagen matrixes (Fig. 4.).

Fig. 4.

Effect of TB4 on TM cell proliferation. a, b, TM explant following two weeks of TB4 (a) and PBS (b) treatment showing increased number of cells accumulating EdU (green) on the malleolar, central and the annular regions of the explants. Notably, the level of cellular outgrowth is also increased. c, Bar graph illustrating the number of EdU positive proliferated cells in representative 50 × 50 pixel size annular regions following TB4 or PBS treatment. Means and standard deviation bars with 95% confidence limits are shown. a, annular region; c, central region; m, malleus.

While three dimensional gel systems are excellent tools for investigating cell migration and endothelial-mesenchymal transformation in vitro [30], utilizing ex vivo systems to predict a candidate’s impact on a targeted organ bears major benefits [16]. Thus, we dissected adult tympanic membranes with the related tympanic bone and soft tissue and simply incubated them floating with or without TB4 in the culture medium (1 ng/μl final concentration. To analyze the cumulative rate of cell proliferation initiated by the peptide, we further supplemented our explants by adding EdU to the medium. Similar to the investigations in collagen, our experiments revealed a noticeably higher amount of EdU positive cells in the pars tensa, and a significantly increased number of proliferating cells in the annular region of the TM, when TB4 was present in the medium (Fig. 5).

Fig. 5.

a–d, Representative photomicrographs (a, c) and EdU accumulation in ex vivo floating TM explants two weeks following TB4 (b) and PBS (d) treatment presenting EdU signal accumulation in the central malleolar handle or umbo region and in the annular region (white arrows). e, TB4 treated TM contains significantly higher EdU positive cells in the annular regions (representative 50 × 50 pixel size) (white arrows of b, d). Means and standard deviation bars with 95% confidence limits are shown, (n = 3/each), *p = 0.005. m, malleolar handle; u, umbo; mf, malleolar fold.

3.3. Thymosin beta-4 alters the number of Vimentin and Cytokeratin-19 positive cells in ex vivo floating explants

Earlier investigations suggest, it is the TM’s own progenitors that are responsible for the membrane’s regeneration and repair following injury [16,31–34]. To investigate whether TB4 may alter the number of these cells, we performed whole mount immunohistochemistry via antibodies for Cytokeratin-19, a previously identified TM progenitor marker in rodents [16], and for Vimentin, a central role player of the epithelial-mesenchymal transition and participant of wound healing [35] utilizing ex vivo floating explants respectively (Fig. 6.).

Fig. 6.

a-h, Immunohistochemical investigations detecting Vimentin (green) and Cytokeratin-19 (red) positive progenitor cells in the annular region of ex vivo floating TM explants following TB4 (a–d) or PBS (e–h) treatments. Notably, in the TB4 treated group, the number of Vimentin and Cytokeratin-19 positive cells were significantly increased when compared to PBS treated controls (none), and the alteration was primarily detectable between the umbo region and the annular territories of the tympanic membrane. White arrowheads point at Cytokeratin-19 positive cells with small nuclei, while Vimentin positive cells (green arrowheads) are cells with larger nuclei (c, d). a: annulus.

Our results revealed TB4 increases the number of both cell types, and this strong positivity is primarily located at the inferior regions of the pars tensa, reaching to the middle annular region of the TM. In contrary to the TB4 treated membranes, almost no signal was detected in the control specimens (Fig. 6.).

4. Discussion

In contrary to most acute tympanic membrane perforations which spontaneously heal within 7–10 days [12], chronic lesions result in long-term conductive hearing loss significantly altering life qualities of the patient. Sadly, the irreversible condition requires surgical intervention such as myringoplasty or tympanoplasty, in which autologous tissues or allografts are classically utilized [10,36]. Although the overall surgical success rate is at nearly 90%, many factors, such as anterior localization of the perforation, sclerotic or atrophic tympanic membrane or former surgeries, bear a significant impact regarding efficacy [37]. Additionally, some of the elderly and polymorbid patients are simply not suitable for surgery.

To overcome the forementioned obstacles, many studies are focusing on revealing the cellular and molecular alterations of the healing procedure. We hypothesized, utilizing secreted molecules significant in natural wound healing may enhance the success rate of the surgical procedures, or even aid CSOM without invasive intervention. In contrast to classical wound healing processes, however, there are some special circumstances regarding tympanic membrane perforation. First and foremost, the tympanic membrane is suspended in air and therefore, no underlying scaffold is available to lead the healing. Regarding cellular activity, it was shown that keratinocytes of the outer epithelial layer are the most relevant cell type to seam perforations [22].

Considering molecular support, a significant number of studies are focusing on investigating the effect of bioactive molecules on tympanic membrane cells and healing. Among them, bFGF and EGF were the most investigated in the last decades [12,13,38,39]. EGF is thought to stimulate proliferation and keratinization of epidermal cells and bFGF acts on both endo- and epithelial cells and fibroblasts. Unlike bFGF, EGF was shown to be present in the normal intact TM, the expression of both molecules however increases following tympanic membrane perforation with a peak of three days following injury [38]. These observations led to the conclusion, extrinsically applied EGF or bFGF can serve beneficial in healing tympanic membrane perforations. Scientific and clinical investigations proved it is EGF, which has the most significant impact on acute perforations, whereas bFGF supports primarily the healing of chronic perforations, especially when combined with biological scaffolds [13].

TB4, a small secreted actin sequestering peptide was recently introduced to play significant role regarding cell migration, progenitor activation, tissue repair and regeneration of various injured organs in vivo [28,40,41]. Excitingly, while its capability of enhancing keratinocyte migration and cell maturation was also reported [25,42], clinical application of TB4 on nonhealing corneal surface defects initiated dramatic improvement following topical therapy [43,44]. Considering the beneficial impacts of TB4 on wound healing in the heart and eye, we asked whether it is equally capable of aiding middle ear lesions. In our present study, we investigated the influence of extrinsic TB4 administration on tympanic membrane explants.

Enhancing migration of tympanic cells proved to be one of the key components regarding TM wound healing [9]. Our results proved, the presence of TB4 compelled epithelial cells to migrate significantly further on collagen matrix, than when treated with PBS (Fig. 3.). The difference became significant nine days following the harvesting of the TM and remained significant until the end of the follow-up (Fig. 3. c). Notably, migration of mesenchymal-like cells was equally investigated, however, no impact regarding TB4 was detected. Epithelial cells, especially migratory keratinocytes deriving from TM stem and committed progenitor cells were reported as important role players during tympanic membrane regeneration [9,16]. Our results regarding enhanced epithelial TM cell migration and the investigation of others focusing on corneal epithelia [45] both provide strong foundation to suspect the peptide may act similar in humans in vivo.

In vitro culturing of TMs on collagen gel extracellular matrixes, even if it resembles the general cellular dynamics of TM cells, as they migrate towards the annulus and outwards from the central progenitor pool, differs from the in vivo environment. Doubtlessly, developing and utilizing ex-vivo systems similar to natural conditions is critical and supportive regarding future clinical applications. In our experiments, we adapted [22] and developed an ex vivo floating explant model to further predict the impact of TB4 upon the membrane. In this system, without any additional extracellular matrixes, the TM cells are in their original locus, thus suitable for investigating the presence, differentiation, proliferation and behavior of primary or differentiated progenitor cell populations (Fig. 2.c). To our surprise, our results proved TB4 accelerates the overall migration and proliferation of TM cells in both systems. The experiments in the floating explants, however, further revealed, the areas in which TB4 enhances cell proliferation are primarily located at the pars tensa and annular regions of the TM (Fig. 5.). Moreover, by utilizing immunocytochemistry, we localized a vast number of Cytokeratin-19 and Vimentin positive cells in the annular region (Fig. 6.). In addition to playing a significant role during epithelial-mesenchymal transformation and wound healing [35,46], both proteins have been previously described as potential markers of tympanic membrane progenitors [16]. Consequently, the increase in Vimentin and Cytokeratin-19 positive cells with elevated cell proliferation by TB4 at the annular region strongly suggest, the peptide may target the endogenous progenitor pool of the injured TM to accelerate and support healing in vivo.

Naturally, there are many questions left unanswered prior to the molecule’s safe clinical utilization. Even if TB4 proved beneficial in healing various lesions of many organs [28,41,45,47–50], it is fundamentally relevant to investigate the protein’s impact in the presence of chronic and acute TM perforations utilizing both, in vitro and in vivo conditions. Moreover, since wound healing is a dynamic process, a detailed timeline analysis with precise topographical tracking of the proliferating and migratory cells are equally crucial to further understand action dynamics. Finally, revealing the molecular alterations initiated by TB4 to fully comprehend its mechanism is equally critical.

Regardless of the aforementioned shortcomings and in consideration that TB4 was investigated in many organ systems previously, to our knowledge, this is the first work published dissecting and demonstrating the effects of TB4 on tympanic membrane cells. Given the data presented in this paper, we strongly believe our results warrant validity in promoting and accelerating painless healing of chronic tympanic membrane lesions in the near future.

Data availability

Data will be made available on request.