The Dorsal Skinfold Chamber as a New Tympanic Membrane Wound Healing Model: Intravital Insights into the Pathophysiology of Epithelialized Wounds.

BACKGROUND: Tympanic membrane perforations (TMPs) are a common complication of trauma and infection. Persisting perforations result from the unique location of the tympanic membrane. The wound is surrounded by air of the middle ear and the external auditory canal. The inadequate wound bed, growth factor, and blood supply lead to circular epithelialization of the perforation's edge and premature interruption of defect closure. Orthotopic animal models use mechanical or chemical tympanic membrane laceration to identify bioactive wound dressings and overcome premature epithelialization. However, all orthotopic models essentially lack repetitive visualization of the biomaterial-wound interface. Therefore, recent progress in 3D printing of customized wound dressings has not yet been transferred to the unique wound setup of the TMP. Here, we present a novel application for the mice dorsal skinfold chamber (DSC) with an epithelialized full-thickness defect as TMP model. METHODS: A circular 2-mm defect was cut into the extended dorsal skinfold using a biopsy punch. The skinfold was either perforated through both skin layers without prior preparation or perforated on 1 side, following resection of the opposing skin layer. In both groups, the wound was sealed with a coverslip or left unclosed (n = 4). All animals were examined for epithelialization of the edge (histology), size of the perforation (planimetry), neovascularization (repetitive intravital fluorescence microscopy), and inflammation (immunohistology). RESULTS: The edge of the perforation was overgrown by the cornified squamous epithelium in all pre-parations. Reduction in the perforation's size was enhanced by application of a coverslip. Microsurgical preparation before biopsy punch perforation and sealing with a coverslip enabled repetitive high-quality intravital fluorescence microscopy. However, spontaneous reduction of the perforation occurred frequently. Therefore, the direct biopsy punch perforation without microsurgical preparation was favorable: spontaneous reduction did not occur throughout 21 days. Moreover, the visualization of the neovascularization was sufficient in intravital microscopy. CONCLUSIONS: The DSC full-thickness defect is a valuable supplement to orthotopic TMP models. Repetitive intravital microscopy of the epithelialized edge enables investigation of the underlying pathophysiology during the transition from the inflammation to the proliferation phase of wound healing. Using established analysis procedures, the present model provides an effective platform for the screening of bioactive materials and transferring progress in tissue engineering to the special conditions of tympanic membrane wound healing.

Multifaceted tannin crosslinked bioinspired dECM decorated nanofibers modulating cell-scaffold biointerface for tympanic membrane perforation bioengineering.

Tympanic membrane (TM) perforation leads to persistent otitis media, conductive deafness, and affects life quality. Ointment medication may not be sufficient to treat TM perforation (TMP) due to the lack of an underlying tissue matrix and thus requiring a scaffold-based application. The engineering of scaffold biointerface close to the matrix via tissue-specific decellularized extracellular matrix (dECM) is crucial in instructing cell behaviour and regulating cell-material interaction in the bioengineering domain. Herein, polycaprolactone (PCL) and TM-dECM (from Sprague-Dawley rats) were combined in a different ratio in nanofibrous form using an electrospinning process and crosslinked via tannic acid. The histological and biochemical assays demonstrated that chemical and enzymatic decellularization steps removed cellular/immunogenic contents while retaining collagen and glycosaminoglycan. The morphological, physicochemical, thermomechanical, contact angle, and surface chemical studies demonstrated that the tannin crosslinked PCL/dECM nanofibers fine-tune biophysical and biochemical properties. The multifaceted crosslinked nanofibers hold the tunable distribution of dECM moieties, assembled into a spool-shaped membrane, and could easily insert into perforated sites. The dECM decorated fibers provide a preferable biomimetic matrix for L929 fibroblast adhesion, proliferation, matrix adsorption, and f-actin saturation, which could be crucial for bioengineering. Overall, dECM patterning, surface hydrophilicity, interconnected microporosities, and multifaceted nanofibrous biosystem modulate cell-scaffold performance and could open opportunities to reconstruct TMP in a biomimetic fashion.

The possible mechanism of Hippophae fructus oil applied in tympanic membrane repair identified based on network pharmacology and molecular docking.

OBJECTIVE: This study aimed to explore the mechanisms of Hippophae fructus oil (HFO) in the treatment of tympanic membrane (TM) perforation through network pharmacology-based identification. METHODS: The compounds and related targets of HFO were extracted from the TCMSP database, and disease information was obtained from the OMIM, GeneCards, PharmGkb, TTD, and DrugBank databases. A Venn diagram was generated to show the common targets of HFO and TM, and GO and KEGG analyses were performed to explore the potential biological processes and signaling pathways. The PPI network and core gene subnetwork were constructed using the STRING database and Cytoscape software. A molecular docking analysis was also conducted to simulate the combination of compounds and gene proteins. RESULTS: A total of 33 compounds and their related targets were obtained from the TCMSP database. After screening the 393 TM-related targets, 21 compounds and 22 gene proteins were selected to establish the network diagram. GO and KEGG enrichment analyses revealed that HFO may promote TM healing by influencing cellular oxidative stress and related signaling pathways. A critical subnetwork was obtained by analyzing the PPI network with nine core genes: CASP3, MMP2, IL1B, TP53, EGFR, CXCL8, ESR1, PTGS2, and IL6. In addition, a molecular docking analysis revealed that quercetin strongly binds the core proteins. CONCLUSION: According to the analysis, HFO can be utilized to repair perforations by influencing cellular oxidative stress. Quercetin is one of the active compounds that potentially plays an important role in TM regeneration by influencing 17 gene proteins.

Bioprinted Collagen-Based Cell-Laden Scaffold With Growth Factors for Tympanic Membrane Regeneration in Chronic Perforation Model.

With the recent development of bioprinting technology, various attempts have been made to replace bioprinting technologies and regenerative medicine are more directed towards transplantation/reconstructive surgeries only with the implantation of scaffolds. The purpose of this study is to determine whether the growth factors, human umbilical cord serum (hUCS) and bFGF (basic fibroblast growth factor), have a synergistic effect on eardrum regeneration, when used with a cell-printed scaffold in a chronic tympanic membrane perforation (TMP) model. In this study, in vitro cellular activities for bioprinted cell-laden collagen scaffolds using human adipose stem cells (hASCs) and supplemented with 10 [Formula: see text]/mL hUCS and 10 ng/mL bFGF were performed. The mixture of the growth factors in the cell-laden structures effectively affects various in vitro cellular responses including the proliferation of hASCs and the migration of keratinocytes due to the synergistic effect of the growth factors and hASCs. For the in vivo evaluation, a rat TMP model was used, and the TMP regeneration was assessed by otoscopic examination, hearing threshold measurement, and histologic examination. Although the cell-laden structure containing hUCS was more enhancing effect compared to the structure with bFGF, more synergistic effect in the structure using hUCS/bFGF was observed. Based on the results, we believe that the cell-laden structure incorporating hUCS and bFGF can induce significant regeneration of chronic tympanic membrane perforation.

Expression of collagen type III in healing tympanic membrane.

OBJECTIVES: Type III collagen plays significant role in skin wound healing, forming provisional matrix guiding the inflammatory cells and fibroblasts into the wound site. Our preliminary study performed on rat's tympanic membrane (TM) using Rat Wound Healing RT2 Profiler PCR Array revealed up-regulated expression of collagen type III α1 chain mRNA also during healing of TM. This study was undertaken to confirm and evaluate collagen type III protein expression and distribution during TM healing process. METHODS: Sixty rats were used, of which 10 served as controls and the others had their TM perforated. The experimental animals were divided into five subgroups on the basis of time points (03, 06, 09, 14, 20 day after injury). Videootoscopy and histology were employed to assess morphology of the healing process. The expression of collagen type III was evaluated using Western blot analysis and its tissue localization was determined by the immunohistochemical method. RESULTS: The expression of collagen type III remained on the same level as in control TM up to day 06. On day 09 abrupt (p = 0.01) increase of the collagen type III expression was observed and it maintained on the same level to the end of observation period. In perforated TM collagen type III was detected in the healing area along the perforation border and around dilated blood vessels. On day 14 and 20 collagen type III was found in the connective tissue filling up the TM previous defect. CONCLUSIONS: Taking into consideration our recent and previous data, as well as results obtained by other authors, is seems possible that the increase of collagen type III expression in the late stage of TM healing contributes to proper scar formation.

Expression of collagens type I and V in healing rat's tympanic membrane.

OBJECTIVES: Our preliminary study performed on perforated rat's tympanic membrane (TM) using Rat Wound Healing RT2 Profiler PCR Array showed significantly increased levels of mRNA for collagens type I and V. Enhanced expression of those genes does not assure that their protein products are indeed present, and in what quantity. Therefore, this study was undertaken to analyze the collagen type I and V content in the healing TM. METHODS: Sixty rats were used, of which 10 served as controls and the others had their TM perforated. The experimental animals were divided into five subgroups on the basis of time points (03, 06, 09, 14, 20 day after injury). Videootoscopy and histology were employed to assess the morphology of the healing process. The expression of collagen type I and V was evaluated using Western blot analysis. Tissue localization of collagens was determined by the immunofluorescence method. RESULTS: The collagen type I expression was three times higher on the third day after injury and remained on that level for whole period of observation, up to day 20. The increase of the collagen type V expression was gradual, reaching the highest level on day 14 following injury. In comparison to the control TM statistically significant increase in the level of expression was observed starting from day 09 to the end of observation period. In healing TM immunofluorescent labeling of collagen type I and V was seen on the surface of remnants of previous lamina propria and in the loose proliferating fibrous tissue. On day 20 immunofluorescence was present mainly on the surface of thin connective tissue layers forming the scar in the place of previous perforation. CONCLUSION: Although the collagens type I and V are present only in subepithelial layer in the normal rat's TM they play significant role in TM healing process.

Tympanic Membrane Derived Stem Cell-Like Cultures for Tissue Regeneration.

Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane (TM) and localized specifically to the umbo and annulus regions. While they have been proposed to play a role in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we compared different explant techniques using normal and perforated rat TM tissues and investigated their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal stem cell markers integrin ß1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most effective technique. Explants made from perforated TM produced cells before those from unperforated TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells that were capable of culture expansion by proliferation and cell migration when subject to a wound assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved suitable for tissue engineering of the TM.

Repair of Tympanic Membrane Perforations with Customized Bioprinted Ear Grafts Using Chinchilla Models.

The goal of this work is to develop an innovative method that combines bioprinting and endoscopic imaging to repair tympanic membrane perforations (TMPs). TMPs are a serious health issue because they can lead to both conductive hearing loss and repeated otitis media. TMPs occur in 3-5% of cases after ear tube placement, as well as in cases of acute otitis media (the second most common infection in pediatrics), chronic otitis media with or without cholesteatoma, or as a result of barotrauma to the ear. About 55,000 tympanoplasties, the surgery performed to reconstruct TMPs, are performed every year, and the commonly used cartilage grafting technique has a success rate between 43% and 100%. This wide variability in successful tympanoplasty indicates that the current approach relies heavily on the skill of the surgeon to carve the shield graft into the shape of the TMP, which can be extremely difficult because of the perforation's irregular shape. To this end, we hypothesized that patient specific acellular grafts can be bioprinted to repair TMPs. In vitro data demonstrated that our approach resulted in excellent wound healing responses (e.g., cell invasion and proliferations) using our bioprinted gelatin methacrylate constructs. Based on these results, we then bioprinted customized acellular grafts to treat TMP based on endoscopic imaging of the perforation and demonstrated improved TMP healing in a chinchilla study. These ear graft techniques could transform clinical practice by eliminating the need for hand-carved grafts. To our knowledge, this is the first proof of concept of using bioprinting and endoscopic imaging to fabricate customized grafts to treat tissue perforations. This technology could be transferred to other medical pathologies and be used to rapidly scan internal organs such as intestines for microperforations, brain covering (Dura mater) for determination of sites of potential cerebrospinal fluid leaks, and vascular systems to determine arterial wall damage before aneurysm rupture in strokes.

Functional Outcomes of Heparin-Binding Epidermal Growth Factor-Like Growth Factor for Regeneration of Chronic Tympanic Membrane Perforations in Mice.

We aim to demonstrate that regeneration of chronic tympanic perforations with heparin-binding epidermal growth factor-like growth factor (HB-EGF) delivered by an injectable hydrogel restored hearing to levels similar to that of nonperforated tympanic membranes. Chronic tympanic membrane perforation is currently managed as an outpatient surgery with tympanoplasty. Due to the costs of this procedure in the developed world and a lack of accessibility and resources in developing countries, there is a great need for a new treatment that does not require surgery. In this study, we show in a mouse model through measurement of auditory brainstem response and distortion product otoacoustic emissions that tympanic perforations lead to hearing loss and this can be predominantly recovered with HB-EGF treatment (5 µg/mL). Our animal model suggests a return to function between 2 and 6 months after treatment. Auditory brainstem response thresholds had returned to the control levels at 2 months, but the distortion product otoacoustic emissions returned between 2 and 6 months. We also show how the vibration characteristics of the regenerated tympanic membrane, as measured by laser Doppler vibrometry, can be similar to that of an unperforated tympanic membrane. Using the best available methods for preclinical evaluation in animal models, it is likely that HB-EGF-like growth factor treatment leads to regeneration of chronic tympanic membrane perforations and restoration of the tympanic membrane to normal function, suggesting a potential route for nonsurgical treatment.

Rat model of chronic tympanic membrane perforation: A longitudinal histological evaluation of underlying mechanisms.

OBJECTIVE: To evaluate histologically the progressive development and underlying mechanisms of chronic tympanic membrane perforation (TMP) in a rat model using a two-weeks ventilation tube (VT) treatment combined with topical application of mitomycin C/dexamethasone (VT-M/D), compared with normal tympanic membrane and acute TMPs. METHODS: Fifty male Sprague-Dawley rats were divided into three experimental groups: a normal control group (n = 5), an acute TMP group (n = 5) (i.e. 3 days post-myringotomy) and a VT-M/D group (n = 40). The TMs were regularly assessed by otoscopy. The normal control animals were sacrificed on day 0 and the acute TMP group was sacrificed 3 days post-myringotomy for histological and immunohistochemical evaluations. The VT-M/D group was sacrificed at various time points - 14 and 17 days, 3, 4, 6, 8 and 10 weeks. RESULTS: On longitudinal histological examination, compared with normal TM and acute TMP, the perforation edges at the later time points illustrated thickened stratified squamous epithelium rimming around the edges, significant increase in keratin and collagen deposition, increased macrophage infiltration as well as reduced cellular proliferation. Three phases of TMP healing process were identified - the acute healing phase (3-17 days), the transition phase (3-4 weeks) and the chronic phase (6-10 weeks). CONCLUSION: Based on the histological results of this study, the progressive development of chronic TMPs appeared to be associated with increased epidermal thickening, collagen and keratin deposition, macrophage infiltration and reduced cellular proliferation. After the 3-4 weeks of transition phase, the TMPs seemed to have transformed into a non-healing chronic TMP between 6 and 10 weeks.

Enhanced expression of hepatocyte growth factor in the healing of experimental acute tympanic membrane perforation.

OBJECTIVES: The present study was performed to investigate the expression of hepatocyte (HGF), epidermal (EGF) and vascular endothelial (VEGF) growth factors in the course of healing of experimental tympanic membrane (TM) perforations in rats. The goal was to explain the role of these growth factors in the healing process of TM and to assess the possibility of their future application as healing promoters. METHODS: Seventy rats were used, of which 10 served as controls and the others had their TM perforated. The experimental animals were divided into six subgroups on the basis of time points (01, 03, 05, 07, 09, 15 day after injury). Videootoscopy and histology were employed to assess the morphology of the healing process. The expressions of HGF, EGF and VEGF were evaluated using Western blot analysis. Tissue localization of HGF was determined by the immunofluorescence method. RESULTS: HGF was hardly detectable in normal TM; however, a significant increase was noted in its expression starting from the third day after injury throughout the follow-up period, with the highest level on day 05. The analysis of HGF tissue localization with immunofluorescence revealed diffuse staining in the cytoplasm of proliferating epithelial cells. The expression of EGF was elevated on the first day after injury, not reaching statistical significance, and then returned to the level observed in the control TM. No significant differences were noted in the expression of VEGF. CONCLUSION: High expression of HGF during the healing process of acute TM perforations makes it a promising candidate for further studies oriented towards its possible use in augmentation of TM healing.

Growth factors expression in hyaluronic acid fat graft myringoplasty.

OBJECTIVES/HYPOTHESIS: To investigate the effect of hyaluronic acid (HA) associated to fat graft on growth factors expression during the healing process of tympanic membrane (TM) perforations in guinea pigs using the hyaluronic acid fat graft myringoplasty (HAFGM) technique. STUDY DESIGN: Prospective randomized animal study. METHODS: Thirty guinea pigs were divided equally into three groups: group I (control group), group II (fat graft myringoplasty technique), and group III (HAFGM technique). TMs were perforated on day 1 and then sampled on days 0, 3, 8, and 21 and tested for the expression of: epidermal growth factor (EGF), insulin-like growth factor (IGF), tumor necrosis factor α (TNF α), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). Five perforated TMs were taken at day 0 from group I to serve as a reference level. RESULTS: Group III showed an increased expression of all tested growth factors, except for KGF. EGF was highest in the early healing process; then IGF peaked at day 8 with statistically significant increase compared to groups I and II. TNF α in group III was significantly higher than group I throughout the study, with a peak level at day 21. VEGF was significantly higher in group III compared to group I at days 3 and 21. Neovascularization and scarless TM closure was obtained in group III, while spontaneous closure was associated with thin-layered and scarred TM in group I. CONCLUSIONS: HA association to fat graft in perforated TM increases the expression of the endogenous growth factors, suggesting that such an association is advantageous for healing. LEVEL OF EVIDENCE: N/A.

Collagen Type II is produced in healing pars tensa of perforated tympanic membranes: an experimental study in the rat.

HYPOTHESIS: Type II collagen is synthesized in the tympanic membrane during healing of a perforation. The time course and appearance of Type II collagen regrowth in the newly healed tympanic membrane is the subject of the present study. BACKGROUND: The arrangement of Type II collagen fibers in the tympanic membrane gives it a unique tensile strength important for sound conduction. Healing after tympanic membrane perforation can cause the tympanic membrane to lose its characteristic. At what phase Type II collagen in tympanic membrane is replaced during healing of a perforation has previously not been studied. METHODS: Rat tympanic membranes were perforated, and the animals were sacrificed 9 to 16 days after perforation. Tympanic membranes were stained with a Type II collagen antibody. RESULTS: At Day 9, a majority of the tympanic membranes had healed. Keratinizing epithelium and connective tissue had formed, but there was no Type II collagen. At Day 10, all tympanic membranes had healed, and staining for Type II collagen appeared. After Day 10, staining was more intense. Newly formed collagen did not show the parallel bundle arrangement seen in normal tympanic membranes but was more scattered in the tissue. CONCLUSION: Type II collagen was seen in tympanic membranes only after closure of the perforation. The fiber arrangement after healing was disturbed, which presumably has an impact on the function of the tympanic membrane. Understanding the formation of Type II collagen in the healing of tympanic membrane perforations could enable further research toward treating tympanic membrane perforations.

Tympanic membrane wound healing in rats assessed by transcriptome profiling.

OBJECTIVES/HYPOTHESIS: The aim of this study is to elucidate transcriptional changes that occur in response to tympanic membrane (TM) perforation in rats and to infer key genes and molecular events in the healing process. STUDY DESIGN: A prospective cohort study of 393 male Sprague-Dawley (Rattus norvegicus) rats. METHODS: Sprague-Dawley rats were randomly allocated into either control or perforation groups spanning a 7-day time period. Perforation groups consisted of 12-hour, 24-hour, 36-hour, 2-day, 3-day, 4-day, 5-day, six-day, and 7-day time points. The left TMs of all perforation groups were perforated and the RNA extracted at the specified time point postperforation. Subsequent analysis was performed using Agilent's 4 × 44 k whole rat genome arrays (40 in total) to assess wound-healing gene expression over a 7-day time period. RESULTS: Over a 7-day time course and at nine time points that encompassed the wounding and progression of healing, a total of 3,262 genes were differentially expressed. In this study the transcripts most upregulated occurred at 12 hours. These were Stefin A2 (344-fold), Stefin 2 (143-fold), and Natriuretic peptide precursor type B (222-fold). Those most downregulated also occurred at 12 hours. These were alcohol dehydrogenase 7 (13.1-fold) and gamma-butyrobetaine hydroxylase (10.4-fold). Results were validated by quantitative real-time polymerase chain reaction. CONCLUSIONS: The findings of this study provide a baseline against which to identify disease-related molecular signatures, biomarkers, and to develop new treatments for TM conditions based on molecular evidence.

Early age conductive hearing loss causes audiogenic seizure and hyperacusis behavior.

Recent clinical reports found a high incidence of recurrent otitis media in children suffering hyperacusis, a marked intolerance to an otherwise ordinary environmental sound. However, it is unclear whether the conductive hearing loss caused by otitis media in early age will affect sound tolerance later in life. Thus, we have tested the effects of tympanic membrane (TM) damage at an early age on sound perception development in rats. Two weeks after the TM perforation, more than 80% of the rats showed audiogenic seizure (AGS) when exposed to loud sound (120 dB SPL white noise, < 1 min). The susceptibility of AGS lasted at least sixteen weeks after the TM damage, even the hearing loss recovered. The TM damaged rats also showed significantly enhanced acoustic startle responses compared to the rats without TM damage. These results suggest that early age conductive hearing loss may cause an impaired sound tolerance during development. In addition, the AGS can be suppressed by the treatment of vigabatrin, acute injections (250 mg/kg) or oral intakes (60 mg/kg/day for 7 days), an antiepileptic drug that inhibits the catabolism of GABA. c-Fos staining showed a strong staining in the inferior colliculus (IC) in the TM damaged rats, not in the control rats, after exposed to loud sound, indicating a hyper-excitability in the IC during AGS. These results indicate that early age conductive hearing loss can impair sound tolerance by reducing GABA inhibition in the IC, which may be related to hyperacusis seen in children with otitis media.

Keratinocyte growth factor 1, fibroblast growth factor 2 and 10 in the healing tympanic membrane following perforation in rats.

The aim of this study was to provide a transcriptome profile of Keratinocyte Growth Factor (KGF)-1, Fibroblast Growth Factor (FGF) 2 and FGF10 (KGF2) in the healing rat tympanic membrane (TM) over 7 days and an immunohistochemical account over 14 days following perforation. KGF1, FGF2, and FGF10 play important roles in TM wound healing. The tympanic membranes of rats were perforated and sacrificed at time points over a 14-day period following perforation. The normalized signal intensities and immunohistochemical protein expression patterns at each time point for KGF1, FGF2, and FGF10 are presented. The primary role of both KGF1 and FGF2 appeared to be in the proliferation and migration of keratinocytes. Whereas the role of KGF1 appeared to be exclusively concerned with increased proliferation and migration at the perforation site, the continued expression of FGF2, beyond perforation closure, suggested it has an additional role to play. FGF10 (KGF2), whilst possessing the highest sequence homologous to KGF1, has a different role in TM wound healing. The effect of FGF10 on keratinocytes in wound healing appeared to emanate from the connective tissue layer.

Expression of fibroblast growth factor receptors 1-4 in human chronic tympanic membrane perforation.

OBJECTIVE: To locate fibroblast growth factor receptor (FGFRs) 1-4 in human chronic tympanic membrane (TM) perforation. METHODS: A sample of human chronic TM perforation was harvested during myringoplasty. The sample was immediately fixed in 4% paraformaldehyde and embedded in OCT compound. Immunohistochemistry was performed with FGFR 1-4 polyclonal antibodies. RESULTS: FGFRs 1-4 were strongly and weakly expressed in the epidermal and mucosal layer of the TM perforation, respectively. CONCLUSIONS: As it is impossible to perform quantitative analysis based on the fluorescence intensity of each immunoreactivity, the presence of FGFRs 1-4 in the human chronic TM perforation is shown. The expressions of FGFRs 1-4 indicated that the clinical use of bFGF agent is useful for myringoplasty.

Efecto del factor de crecimiento endotelio vascular (VEGF) sobre perforaciones timpánicas en ratas Long-Evans / Effect of vascular endothelial growth factor (VEGF) on tympanic perforations in Long-Evans rats

Introducción: La anglogenesis es el desarrollo de nuevos vasos sanguíneos desde una red vascular existente, contempla una secuencia de eventos complejos y es fundamental en el proceso reparativo. Existen múltiples factores estimulantes de la angiogénesls, entre ellos se encuentran factores de crecimiento como el VEGF (factor de crecimiento endotelio vascular). Debido a su rol reparativo se han utilizado factores proanglogénicos para reparar perforaciones timpánicas. Objetivo: Estudiar el efecto del VEGF sobre perforaciones timpánicas de ratas Long-Evans. Material y método: Se usan 15 ratas adultas, se realizan perforaciones timpánicas bilaterales, se instilan al azar las perforaciones con solución fisiológica y VEGF, se realiza visualización microscópica de los tímpanos a los días 9,15 y 21 posperforación. Las ratas son sacrificadas el día 21 y se realiza estudio histológico del grosor timpánico. Resultados: No se aprecia un efecto inductivo del VEGF sobre el cierre de las perforaciones timpánicas, se produce un aumento en el grosor timpánico de las ratas tratadas con VEGF.

Plasmin/plasminogen is essential for the healing of tympanic membrane perforations.

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

The collagen structure of the tympanic membrane: collagen types I, II, and III in the healthy tympanic membrane, during healing of a perforation, and during infection.

OBJECTIVE: To analyze the distribution of 3 collagen types in healthy tympanic membranes, during healing of a perforation, and during infection. DESIGN: Immunohistochemical study of collagen types I, II, and III in the tympanic membranes of healthy rats as well as during healing of a perforation and in the presence of infection with Streptococcus pneumoniae at various time points. SETTING: Laboratory research center at a university hospital. RESULTS: Type II collagen was a main constituent of the lamina propria of the pars tensa, whereas type I collagen was found mainly in the pars flaccida. Collagen types I and III were found at the insertion to the malleus handle and in the loose connective tissue surrounding the main collagen layer of the pars tensa. After myringotomy, collagen types I and III were found at the perforation border and around dilated blood vessels early in the healing phase. During infection, the collagen layer was thickened and stained strongly for type II collagen. Collagen types I and III were found in the edematous connective tissue around the main collagen layer and around dilated blood vessels. Three months after perforation or infection, all 3 collagens were present in the lamina propria of the tympanic membrane. Extensive amounts of all 3 collagen types were present in the scar tissue in the tympanic membranes of rats that had undergone myringotomy during the presence of acute otitis media. CONCLUSIONS: The lamina propria of the pars tensa is mainly made up of type II collagen, whereas type I collagen is found in the pars flaccida. Thus the fibrous structure of the pars tensa and flaccida is composed of diverse collagen types, which reflects the different physiological properties of these tissues. Collagen types I and III are present in the acute healing phase after myringotomy and infection, and the collagen content of the tympanic membrane is modified during the inflammatory and healing process.