Adult mammals are generally believed to have limited ability to regenerate complex tissues and instead, repair wounds by forming scars. In humans and across mammalian species, the tympanic membrane (TM) rapidly repairs perforations without intervention. Using mouse models, we demonstrate that the TM repairs itself through a process that bears many hallmarks of epimorphic regeneration rather than typical wound healing. Following injury, the TM forms a wound epidermis characterized by EGFR ligand expression and signaling. After the expansion of the wound epidermis that emerges from known stem cell regions of the TM, a multi-lineage blastema-like cellular mass is recruited. After two weeks, the tissue architecture of the TM is largely restored, but with disorganized collagen. In the months that follow, the organized and patterned collagen framework of the TM is restored resulting in scar-free repair. Finally, we demonstrate that deletion of Egfr in the epidermis results in failure to expand the wound epidermis, recruit the blastema-like cells, and regenerate normal TM structure. This work establishes the TM as a model of mammalian complex tissue regeneration.

Drug treatments for middle ear diseases are currently delivered systemically, or locally after opening the impermeable tympanic membrane (TM). We previously used bacteriophage display to discover novel peptides that are actively transported across the intact TM, with a variety of transport rates. Peptide structures were analyzed for evidence regarding the mechanism for this unexpected transport, which was then tested by the application of chemical inhibitors. Primary sequences indicated that trans-TM peptides share one of two amino acid motifs. Secondary structures revealed that linear configurations associate with higher transport rates than coiled structures. Tertiary analysis indicated that the shared sequence motifs are prominently displayed at the free ends of rapidly transported peptide phage. The shared motifs were evaluated for similarity to known motifs. The highest probability matches were for protein motifs involved in transmembrane transport and exosomes. Overall, structural findings suggest that the shared motifs represent binding sequences. They also implicate transcytosis, a polarized cell transport mechanism consisting of endocytosis, transcellular transport, and exocytosis. Inhibitor studies indicated that macropinocytosis, retrograde transport through Golgi and exocytosis participate in transport across the TM, consistent with transcytosis. This process can be harnessed to noninvasively deliver therapeutics to the middle ear.

Localized and non-invasive delivery of therapeutics across barriers in the body is challenging. Examples include the flux of drugs across the tympanic membrane (TM) for the treatment of middle ear infections, and across the round window to treat inner ear disease. With the emergence of macromolecular therapies, the question arises as to whether such delivery can be achieved with macromolecules. Here, we have used polyethylene glycols (PEGs) in solutions to investigate macromolecular permeation across the TM in the chinchilla ex vivo. As the molecular weight of PEG increased, flux across the TM decreased, with an exponential relationship between the apparent diffusion coefficient and the molecular weight of the polymers. PEG flux was further decreased if it was released from a poloxamer 407 hydrogel, and lessened with increasing hydrogel concentration. Our results provide a framework for understanding the permeation of macromolecules noninvasively across barriers.

Review of the English language literature finds little documentation of the relation of otology or otolaryngology outcomes to a surgeon's age, years in practice, or numbers of cases previously performed. Because of one surgeon's adoption of a new tympanoplasty technique for uncomplicated tympanic membrane perforations, our institution was situated to report an example of a surgical learning curve with its outcome. Experience versus outcome is worth establishing objectively because these relationships reflect on training and certification.

We previously identified peptides that are actively transported across the intact tympanic membrane (TM) of rats with infected middle ears. To assess the possibility that this transport would also occur across the human TM, we first developed and validated an assay to evaluate transport in vitro using fragments of the TM. Using this assay, we demonstrated the ability of phage bearing a TM-transiting peptide to cross freshly dissected TM fragments from infected rats or from uninfected rats, guinea pigs and rabbits. We then evaluated transport across fragments of the human TM that were discarded during otologic surgery. Human trans-TM transport was similar to that seen in the animal species. Finally, we found that free peptide, unconnected to phage, was transported across the TM at a rate comparable to that seen for peptide-bearing phage. These studies provide evidence supporting the concept of peptide-mediated drug delivery across the intact TM and into the middle ears of patients.

Neurological conditions such as traumatic brain injury (TBI) and hydrocephalus may lead to intracranial pressure (ICP) elevation. Current diagnosis methods rely on direct pressure measurement, while CT, MRI and other expensive imaging may be used. However, these invasive or expensive testing methods are often delayed because symptoms of elevated ICP are non-specific. Invasive methods, such as intraventricular catheter, subdural screw, epidural sensor, lumbar puncture, are associated with an increased risk of infection and hemorrhage. On the other hand, noninvasive, low-cost, accurate methods of ICP monitoring can help avoid risks and reduce costs while expediting diagnosis and treatment. The current study proposes and evaluates a novel method for noninvasive ICP monitoring using tympanic membrane pulsation (TMp). These signals are believed to be transmitted from ICP to the auditory system through the cochlear aqueduct. Fifteen healthy subjects were recruited and TMp signals were acquired noninvasively while the subjects performed maneuvers that are known to change ICP. A custom made system utilizing a stethoscope headset and a pressure transducer was used to perform these measurements. Maneuvers included head-up-tilt, head-down-tilt and hyperventilation. When elevated ICP was induced, significant TMp waveform morphological changes were observed in each subject (p < 0.01). These changes include certain waveform slopes and high frequency wave features. The observed changes were reversed by the maneuvers that decreased ICP (p < .01). The study results suggest that TMp waveform measurement and analysis may offer an inexpensive, noninvasive, accurate tool for detection and monitoring of ICP elevations. Further studies are warranted to validate this technique in patients with pathologically elevated ICP.

The tympanic membrane (TM) is critical for hearing and requires continuous clearing of cellular debris, but little is known about homeostatic mechanisms in the TM epidermis. Using single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we show that homeostatic TM epidermis is distinct from other epidermal sites and has discrete proliferative zones with a three-dimensional hierarchy of multiple keratinocyte populations. TM stem cells reside in a discrete location of the superior TM and generate long-lived clones and committed progenitors (CPs). CP clones exhibit lateral migration, and their proliferative capacity is supported by Pdgfra+ fibroblasts, generating migratory but non-proliferative progeny. Single-cell sequencing of the human TM revealed similar cell types and transcriptional programming. Thus, during homeostasis, TM keratinocytes transit through a proliferative CP state and exhibit directional lateral migration. This work forms a foundation for understanding TM disorders and modeling keratinocyte biology.

To establish whether covering the tympanic membrane perforation after war blast injury with silicon foil can enhance the ear drum healing rate and to determine the appropriate timing of silicon patching.

Injury or inflammation of the middle ear often results in the persistent tympanic membrane (TM) perforations, leading to conductive hearing loss (HL). However, in some cases the magnitude of HL exceeds that attributable by the TM perforation alone. The aim of the study is to better understand the effects of location and size of TM perforations on the sound transmission properties of the middle ear.

Otitis media (OM) is the most common infectious disease of children under six, causing more antibiotic prescriptions and surgical procedures than any other pediatric condition. By screening a bacteriophage (phage) library genetically engineered to express random peptides on their surfaces, we discovered unique peptides that actively transport phage particles across the intact tympanic membrane (TM) and into the middle ear (ME). Herein our goals were to characterize the physiochemical peptide features that may underlie trans-TM phage transport; assess morphological and functional effects of phage peptides on the ME and inner ear (IE); and determine whether peptide-bearing phage transmigrate from the ME into the IE. Incubation of five peptide-bearing phage on the TM for over 4hrs resulted in demonstrably superior transport of one peptide, in level and in exponential increase over time. This suggests a preferred peptide motif for TM active transport. Functional and structural comparisons revealed unique features of this peptide: These include a central lysine residue, isoelectric point of 0.0 at physiological pH and a hydrophobic C-terminus. When the optimal peptide was applied to the TM independent of phage, similar transport was observed, indicating that integration into phage is not required. When 109 particles of the four different trans-TM phage were applied directly into the ME, no morphological effects were detected in the ME or IE when compared to saline or wild-type (WT) phage controls. Comparable, reversible hearing loss was observed for saline controls, WT phage and trans-TM peptide phage, suggesting a mild conductive hearing loss due to ME fluid. Perilymph titers after ME incubation established that few copies of trans-TM peptide phage crossed into the IE. The results suggest that, within the parameters tested, trans-TM peptides are safe and could be used as potential agents for noninvasive delivery of drugs, particles and gene therapy vectors to the ME.

The most common etiologies of tympanic membrane perforation are infections and trauma.

This study aimed to investigate the effectiveness of topical allantoin application on wound healing in a rat chronic tympanic membrane perforation (TMP) model.

Although the Vibrant Soundbridge is one of the most frequently used active middle ear implants, data regarding how middle ear ventilation disorders may affect the transmission behavior of its floating mass transducer are still insufficient. Studies involving coupling the floating mass transducer to the stapes head are particularly lacking. This temporal bone study evaluated the influence of simulated middle ear ventilation disorders on the middle ear transfer function in the reconstructed middle ear. The middle ear transfer function was measured using Laser Doppler Vibrometry after vibroplasty onto the stapes head, with or without tympanic membrane reconstruction. Middle ear ventilation disorders were simulated through changes in static pressure via the external ear channel with a maximum pressure of +3 kPa. Slice thickness of tympanic membrane reconstruction material was measured using micro-CT. When the reconstructed ossicular chain and the reconstructed tympanic membrane were mechanically excited by the floating mass transducer under conditions of ambient static pressure, the transmission behavior was found to be independent of the type of tissue used. Increase in static pressure up to +3 kPa caused maximum low frequency transmission loss of 15 dB when elastic grafts were used and 5 dB when stiff tissue was inserted. At high frequencies, measured loss of up to 5 dB was relatively independent of the tissue stiffness. Increase in static pressure led to displacement of the tissues towards the vestibulum and caused stiffening, especially of the annular ligament. Stiffening-induced transmission losses were mainly found at low frequencies and could not be compensated by the floating mass transducer in this range. Above 1300 Hz, the continuous force spectrum of the actuator sufficiently protected against loss of amplitude. To minimize postoperative transmission loss due to persisting ventilation disorders, choosing a very stiff tympanic membrane reconstruction material seems to be appropriate.

Chronic infections are often connected to biofilm formation. In presence of implants, this can lead to loss of the implant. Systemic or local application of drugs is relatively ineffective in case of biofilm formation. One technique to provide antibacterial properties on demand is the antibacterial photodynamic therapy (aPDT). Using this technique, these properties can be "switched on" by light illumination. In the middle ear with the semitransparent tympanic membrane, it might be possible in future to activate the antibacterial effect without opening the membrane. Therefore, we investigated the optical absorbance spectra of the tympanic membrane. Optical absorbance spectra were measured in ex vivo preparations from neonatal and adult rats with the membrane still being attached to the surrounding bony ring and four human samples. After performing area scans, the spot with the lowest absorbance being surrounded by a ring like structure with higher absorbance was chosen as region of interest for scanning wavelengths between 300 and 900 nm. Absorbance is generally higher at lower wavelengths with a local absorbance maximum at 420 nm and a weak second maximum with two neighbouring peaks at 540 / 580 nm and is significantly higher in adult rats compared to neonatal rats where about 10% of light was transmitted. The human samples show similar characteristics with a little higher absorbance. For activation of aPDT through the tympanic membrane, larger wavelengths are more promising. Whether the amount of light transmitted through the membrane would be sufficient to induce aPDT remains to be tested in further experiments.

The airpuff startle stimulus consists of two modalities, tactile and acoustic. Tympanic membrane rupture (TMR) effectively deafens a rat, thus preventing it from perceiving the acoustic component of the airpuff and permitting study of the tactile component in isolation. Previous studies have shown that the tactile modality is sufficient to drive the cardiovascular response to the airpuff, but cannot elicit the full behavioral startle response. In the present study Fos protein was used as a marker of neuronal activation to identify brain regions activated by the airpuff in both intact and TMR rats. Results show an attenuation of Fos expression following TMR in the dorsal and ventral cochlear nuclei, ventral nucleus of the lateral lemniscus and medial geniculate nucleus. In contrast, Fos expression following TMR was unchanged in the locus coeruleus, the laterodorsal tegmental nucleus, the supramammilary nucleus, and the ventromedial hypothalamic nucleus. Analysis of behavioral data confirmed that the startle response to the airpuff was diminished following TMR. These data are the first of which we know to employ an immediate early gene approach to discriminate between brain regions activated by the tactile and acoustic startle stimulus modalities. The results are discussed in terms of the classical acoustic startle circuit, and the central autonomic pathways activated by the tactile component of the airpuff.

Chitin nanofibrils (CNs) are an emerging bio-based nanomaterial. Due to nanometric size and high crystallinity, CNs lose the allergenic features of chitin and interestingly acquire anti-inflammatory activity. Here we investigate the possible advantageous use of CNs in tympanic membrane (TM) scaffolds, as they are usually implanted inside highly inflamed tissue environment due to underlying infectious pathologies. In this study, the applications of CNs in TM scaffolds were twofold. A nanocomposite was used, consisting of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer loaded with CN/polyethylene glycol (PEG) pre-composite at 50/50 (w/w %) weight ratio, and electrospun into fiber scaffolds, which were coated by CNs from crustacean or fungal sources via electrospray. The degradation behavior of the scaffolds was investigated during 4 months at 37 °C in an otitis-simulating fluid. In vitro tests were performed using cell types to mimic the eardrum, i.e., human mesenchymal stem cells (hMSCs) for connective, and human dermal keratinocytes (HaCaT cells) for epithelial tissues. HMSCs were able to colonize the scaffolds and produce collagen type I. The inflammatory response of HaCaT cells in contact with the CN-coated scaffolds was investigated, revealing a marked downregulation of the pro-inflammatory cytokines. CN-coated PEOT/PBT/(CN/PEG 50:50) scaffolds showed a significant indirect antimicrobial activity.

Stereotactic surgery is a widely used procedure in neuroscience research to study the brain's regulation of feeding behavior. In line with this notion, this study aims to assess how food consumption and feeding patterns are affected in response to the use of auditory bars that preserve or damage the tympanic membrane during stereotactic surgery. Our previous observations led us to hypothesize that the traumatic tympanic membrane rupture affects food intake and feeding patterns in rats undergoing stereotactic procedures. Thereby, female and male rats were cannulated in the third ventricle (3V) using both types of auditory bars. Post-surgical pain was assessed using the grimace scale. Food intake, meal patterns and weight gain or loss were analyzed for 5-7 consecutive days after surgery. Normal food intake, increased body weight and regular meal patterns were observed from postoperative day 2 when the stereotactic procedure was performed using auditory bars that maintain the integrity of the tympanic membrane. However, tympanic membrane rupture prevented the expected recovery of food intake and body weight. This effect was accompanied by an alteration in eating patterns, which was persistent over 7 days of recovery. Thus, tympanic membrane preservation during surgery is necessary to evaluate short-term feeding patterns. This study demonstrates auditory bars that do not damage the tympanic membrane should be used when performing stereotactic surgery for subsequent analysis of rat behavior.

Myringoplasty is a surgical procedure to reconstruct tympanic perforation. However, repair of anterior perforations is still challenging. To analyze the anatomical and hearing outcomes of myringoplasty with a new technique of chondroperichondrial graft via endoscopy, 23 adult patients were retrospectively analyzed. All patients had anterior perforations of tympanic membranes that were repaired with a composite strip-type cartilage-perichondrium graft through a total endoscopic transcanal approach. The anatomical graft success rate at postoperative 1 month was 86.96% (20/23) and reached 100% at the 6- and 12-month follow-up. Compared to the preoperative air conduction threshold (44.7 ± 13.56 dB) and air-bone gap (ABG) (22.35 ± 6.54 dB), the postoperative air conduction threshold and ABG decreased to 33.52 ± 10.88 dB and 12.52 ± 3.94 dB, respectively (P < .0001). Twenty-two (95.65%) patients had an ABG below 20 dB postoperatively. The mean ABG improvement in our cohort was 9.83 ± 5.00 dB. The functional graft success rate was 95.65% (22/23). The convenience, reliability, time, and labor savings accrued from the approach described here make it a good choice for repair of anterior perforation of tympanic membrane.

Similar to skin, epithelia in the tympanic membrane (TM) regenerate and move toward the opening of the external ear canal, a process called epithelial migration (EM). EM is important for maintaining healthy ears because this process removes cerumen and debris. Therefore, increasing the rate of EM or TM regeneration could be very important for healthy ear maintenance and function. Stem cells or their conditioned media have been used in medical therapy in humans to increase the rate and efficacy of EM. The purpose of this study was to evaluate the ability of canine stem cell conditioned media to accelerate EM in canine TMs. Canine adipose tissue derived-mesenchymal stem cell conditioned media (cAD-MSCCM), and several cytokines related to keratinocyte growth or migration within the media were quantified using ELISA. Ink drops were placed on the TMs of four normal beagles. Then, cAD-MSCCM was applied weekly, a total of three times to the TMs of one ear, and nothing was applied to the other eye. The results showed a higher TM EM rate in the treatment group than in the control group (p < 0.05). No adverse events were recorded. These results suggest that the weekly application of cAD-MSCCM accelerates the TM EM rate.

Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA(-/-)) or tPA (tPA(-/-)) after TM perforation. Delayed healing of TM perforations was observed in uPA(-/-) mice but not tPA(-/-) mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA(-/-) mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA(-/-) mice are most likely due to the reduced generation of plasmin.