CoCl2 -simulated hypoxia potentiates the osteogenic differentiation of fibroblasts derived from tympanosclerosis by upregulating the expression of BMP-2.

Tympanosclerosis (TS) is a result of long-standing middle ear inflammation characterized by fibroblasts ossification. Fibrosis is the last revertible stage in the progress of middle ear inflammation to TS. It was hypothesized that chronic hypoxia could be modulating fibrosis, which in turn additionally further aggravated hypoxia via decreasing oxygen diffusion. However, the effects of hypoxia on osteoinductive activity of fibroblasts have not been explored. Herein, we purposed to explore the role of hypoxia in osteogenic differentiation of fibroblasts derived from TS. The expression of bone morphogenetic protein-2 (BMP-2), hypoxia-inducible factor-1α (HIF-1α), and Vimentin in the human surgical specimens of tympansclerosis was investigated by immunofluorescent staining. Furthermore, cultured fibroblasts were stratified into the following study groups: control, 25, 50, and 100 µM cobaltous chloride (CoCl2 ) group. BMP-2, as well as HIF-1α levels of expression were detected via western blotting and immunofluorescence analysis. We found that the expression of BMP-2 and HIF-1α was significantly upregulated in TS tissues and these fibroblasts, which was vimentin positive surrounding sclerotic plaques, were also expressing HIF-1α positive. The results also demonstrated that CoCl2 treatment increased nuclear HIF-1α protein level in the fibroblast. Furthermore, treatment with CoCl2 significantly increased BMP-2 expression and remarkably elevated alkaline phosphatse activity and the mineralized nodules area. These data illustrate that hypoxia may play an osteogenic role in TS fibroblasts via the elevated expression of a possible osteogenic factor, BMP-2.

The Possible Role of Sclerostin in the Pathogenesis of Tympanosclerosis.

OBJECTIVE: The aim of this study was to investigate sclerostin (SOST) expression in a rat model of experimental tympanosclerosis (TS) and its possible role in the formation of TS. MATERIALS AND METHODS: Thirty-four SD rats were randomly divided into 2 groups: experimental group (n = 17) and normal group (n = 17). The left tympanic cavities in the experimental group were inoculated with methicillin-resistant Staphylococcus aureus. The changes of tympanic membranes were examined and recorded under otoendoscope. Haematoxylin-eosin staining was adopted to detect the morphological changes in the tympanic membrane and middle ear mucosa. Immunohistochemistry and Western blot analysis were used to observe the expression of SOST, Wnt3a, ß-catenin, and P-ERK1/2. RESULTS: In the experimental group, sclerotic lesions were observed in 54.5% ears in the end of 6 weeks. Morphological changes such as mucosa incrassation, inflammatory cells infiltration, fibrous tissue proliferation, and interstitial tissue incrassation prominently appeared in the tympanic membrane and middle ear mucosa. SOST protein was mainly distributed in the cytoplasm of epithelial cells and gland cells, the expression of which increased significantly in the calcified experimental ears. In addition, expression levels of Wnt3a, ß-catenin, and P-ERK1/2 increased significantly in the calcified group too. CONCLUSION: The upregulated expression level of SOST may be involved in the formation of TS, first, through the pro-phosphorylation of ERK1/2 in the inflammatory stage, and then through the enhancement of Wnt3a in the osteogenic stage.

Gene expression profile of collagen types, osteopontin in the tympanic membrane of patients with tympanosclerosis.

BACKGROUND: Tympanosclerosis is a pathological process involving the middle ear. The hallmark of this disease is the formation of calcium deposits. In the submucosal layer, as well as in the right layer of the tympanic membrane, the calcium deposits result in a significant increase in the activity of fibroblasts and deposition of collagen fibers. OBJECTIVES: The aim of our study was to examine the expression level of genes encoding collagen type I, II, III and IV (COL1A1, COL2A1, COL3A1, COL4A1) and osteopontin (SPP1) in the tympanic membrane of patients with tympanosclerosis. MATERIAL AND METHODS: The total RNA was isolated from middle ear tissues with tympanosclerosis, received from 25 patients and from 19 normal tympanic membranes. The gene expression level was determined by real-time RT-PCR. The gene expression levels were correlated with clinical Tos classification of tympanosclerosis. RESULTS: We observed that in the tympanic membrane of patients with tympanosclerosis, the expression of type I collagen is decreased, while the expression of type II and IV collagen and osteopontin is increased. Moreover, mRNA levels of the investigated genes strongly correlated with the clinical stages of tympanosclerosis. CONCLUSIONS: The strong correlations between the expression of type I, II, IV collagen and osteopontin and the clinical stage of tympanosclerosis indicate the involvement of these proteins in excessive fibrosis and pathological remodeling of the tympanic membrane. In the future, a treatment aiming to modulate these gene expressions and/or regulation of the degradation of their protein products could be used as a new medical approach for patients with tympanosclerosis.

Possible role of Dickkopf-1 protein in the pathogenesis of tympanosclerosis in a rat model.

OBJECTIVES: This study aimed to investigate the expression of DKK1 protein in an experimental model of tympanosclerosis and its possible role in the pathogenesis of this disorder. METHODS: Forty Sprague Dawley rats were included in the study: 20 in the control group (which received no treatment) and 20 in the experimental group (which received an incision to induce tympanosclerosis). Otomicroscopy was performed to observe the development of myringosclerosis. Haematoxylin and eosin staining was performed to observe the morphological changes. Western blot analysis and immunohistochemistry were performed to assess the expression of DKK1 protein. RESULTS: At day 15, sclerotic lesions were observed in 70 per cent of the tympanic membranes. Inflammatory infiltration and hyaline degeneration markedly appeared in the tympanic membranes and middle-ear mucosa. DKK1 protein was mainly distributed in the cytoplasm of epithelial cells, which were widely distributed in the tympanic membranes and middle-ear mucosa. The expression of DKK1 protein was significantly decreased in the calcified experimental ears. CONCLUSION: DKK1 protein is involved in the pathogenesis of tympanosclerosis by regulating the Wnt/ß-catenin signalling pathway.

PAI-1 inhibits development of chronic otitis media and tympanosclerosis in a mouse model of otitis media.

CONCLUSION: Bullae of type 1 plasminogen activator inhibitor (PAI-1) knockout (KO) mice showed low levels of inflammation against nontypable Haemophilus influenzae (NTHi) at the early stage of otitis media (OM). However, PAI-1 KO mice fail to terminate inflammation, which may significantly contribute to the development of tympanosclerosis in PAI-1 KO mice. OBJECTIVE: To investigate the role of PAI-1 in the pathogenesis of OM and subsequent tympanosclerosis. METHODS: OM was induced with NTHi in PAI-1 KO and background control C57BL/6 mice. mRNA expression of PAI-1, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA) was measured in the bullae of C57BL/6 mice. mRNA expression of interleukin (IL)-1ß, tumor necrosis factor (TNF) α, macrophage inflammatory protein (MIP-2), tPA, and uPA in PAI-1 KO and C57BL/6 mice was compared. Histological changes produced by OM were compared at 1, 3, and 7 days after NTHi inoculation. RESULTS: NTHi up-regulated the expression of PAI-1 and tPA in the bullae of C57BL/6 mice, but not uPA. mRNA expression of IL-1ß, TNFα, and MIP-2 was low in PAI-1 KO mice at early time points, but significantly higher at the later stage of OM. Similarly to the gene expression results, histological changes associated with OM were less at days 1 and 3 in PAI-1 KO mice. However, unlike the gradual resolution of OM pathologies in C57BL/6 mice, PAI-1 KO mice showed significant pathological changes of tympanosclerosis.

Altered response of fibroblasts from human tympanosclerotic membrane to interacting mast cells: implication for tissue remodeling.

Several lines of evidence suggest that a tympanosclerotic (TMS) lesion often develops secondary to acute and chronic otitis media. Histological findings indicate that fibroblasts and inflammatory cells, including mast cells, play a key role in the tympanosclerotic plaque formation. However, details on the functional characteristics of tympanosclerotic fibroblasts (Fs(TMS)) are scanty. Therefore the aim of our study was to examine the activity of human fibroblasts from tympanosclerotic lesions and to evaluate the influence of stimulated by crosslinking of IgE receptor mast cells (HMC-1(FcɛRI)) on fibroblast functional behavior. We observed that fibroblasts from normal tympanic membrane (Fs(TM)) released less TNF-α, TGF-ß1 and IL-6 compared to Fs(TMS). Fs(TMS) but not Fs(TM) upon interaction with HMC-1(FcɛRI) released increased quantities of TNF-α and TGF-ß1. Exposing the fibroblast to HMC-1(FcɛRI) cells resulted in an increased synthesis of proteins including collagen. We noted that the COL2A1 transcript level increased ∼5- and ∼12-fold in Fs(TM) and Fs(TMS) co-cultured with HMC-1(FcɛRI), respectively. Both Fs(TM) and Fs(TMS) upon maintenance in the primary culture released significant quantities of matrix metalloproteinase 9 (MMP-9). However, Fs(TMS) released ∼5-fold more MMP-9 activity compared to the Fs(TM) cultures. The mast cell-induced release of TNF-α, TGF-ß1 and MMP-9 sustained for a longer time in Fs(TMS) cultures compared to Fs(TM). Concluding, our data strongly indicate that increased fibroblast sensitivity to mast cell stimulation greatly contributes to the excessive fibrosis and pathological remodeling of the tympanic membrane. We postulate that the persistency of the Fs(TMS) activated state could be an important factor in the pathogenesis of tympanosclerosis.

Immunohistochemical investigation of inducible nitric oxide synthase, osteopontin, and calcium-sensing receptor in a myringosclerosis/tympanosclerosis model.

HYPOTHESIS: To investigate roles of types of inflammation, inducible nitric oxide synthase (iNOS), osteopontin (OPN), and calcium sensing receptor (CaSR) in the tympanic membrane and middle ear in etiopathogenesis of myringosclerosis/tympanosclerosis (MT). BACKGROUND: Etiopathogenesis of myringosclerosis/tympanosclerosis is still unclear. Clinical and experimental observations demonstrate that hyperoxygenation might induce tympanosclerosis. METHODS: Seventy-five rats were divided into 3 groups: ventilation tube (VT) insertion, the Eustachian tube (ET) obliteration, and both procedures. Right ears were selected for mentioned interventions. Left ears served as controls. Then, histopathologic and immunohistochemical investigations were performed in tympanic bulla. MT and inflammation in tympanic membrane and middle ear space were investigated. Immunohistochemical investigation included staining with iNOS, OPN, and CaSR. RESULTS: Overall 42.7% of all rats developed MT. There was no significant difference in MT incidence among the groups (ET + VT group: 56%; ET group: 44%; VT group: 28%; p > 0.017). iNOS expression occurred in 30.6% of the intervention groups with insignificant differences (ET + VT group: 40%; ET group:36%; VT group:16%; p > 0.05). There was no significant difference in iNOS expression between tympanosclerotic (25%) and non tympanosclerotic ears (34.9%) (p = 0.359). OPN was expressed in 82.6% overall. It was the highest for ET group and ET + VT group (92% for each) followed by VT group (64%). There was a marginal significance in comparison of OPN staining between VT group and ET group and also between VT group and ET + VT group (p = 0.017). There was a significant difference in OPN expression between tympanosclerotic (100%) and nontympanosclerotic ears (69.8%) (p = 0.001). Neither control ears nor intervention groups showed CaSR expression. Comparisons of inflammation of the tympanic membrane and middle ear space between tympanosclerotic and non-tympanosclerotic ears yielded significant differences (p = 0.003, p = 0.002, respectively). Tympanosclerotic ears had a tendency to show chronic or mixed inflammation in contrast to non-tympanosclerotic ears (p < 0.017). Filled-middle ear space was seen in 25% of the intervention groups with no significant difference (p > 0.017). There was a significant difference in the incidence between tympanosclerotic (46.8%) and non-tympanosclerotic ears (7%) (p < 0.017). CONCLUSION: Based on these findings, iNOS may not be evident in stage of MT. OPN staining is strongly associated with the development of MT. CaSR has no role in formation of MT. The results proved roles of mixed or chronic inflammation and the presence of the filled-middle ear in development of MT.

Levels of reactive oxygen species in rat tympanic membranes after incisional versus radiofrequency myringotomy.

OBJECTIVE: A close relationship between reactive oxygen species (ROS) and myringosclerosis, which is a common complication of myringotomy, was recently reported. The objective of this study was to measure ROS levels directly in rat tympanic membranes using luminol-aided chemiluminescence (CL) in order to compare the levels of ROS after incisional and radiofrequency (RF) myringotomy. METHODS: Fifteen Sprague-Dawley rats were separated into three groups of five animals each. Bilateral myringotomies were made using an appropriate myringotomy lancet in Group 1 and RF in Group 2. Group 3 served as the control group with no myringotomy. Twenty-four hours after the procedure, all tympanic membranes were inspected with an otomicroscope and then excised for the measurement of ROS using luminol-aided CL. RESULTS: The mean ROS level in Group 1 was significantly higher than that in Groups 2 and 3 (p<0.05 for both). The difference in mean ROS level between Groups 2 and 3 was not significant (p>0.05). Otomicroscopy revealed increased vascularity and vessel dilation in all tympanic membranes that underwent myringotomy. Vascular dilation was observed in the annular region in the vessels that passed along the long arm of the malleus, in addition to the vessels feeding the anterior and posterior tympanomalleolar folds. CONCLUSIONS: Although the relationship between ROS and the development of myringosclerosis after myringotomy has been demonstrated, the present study is the first to compare incisional and RF myringotomy based on the measurement of ROS levels. Our results indicate that the increase in ROS due to myringotomy was greater following incisional myringotomy than RF myringotomy.