Time Analysis of Recidivism in Acquired Cholesteatoma Using the Kaplan-Meier Method.

OBJECTIVE: To analyze the time trends of recidivism of acquired cholesteatoma using the Kaplan-Meier method. STUDY DESIGN: We conducted a retrospective, observational study of 256 patients having their first cholesteatoma surgery. The cumulative recidivism-free rate was calculated using Kaplan-Meier survival analysis related to the follow-up period, pathophysiology, the extent of the disease, and recidivism pathologies. RESULTS: Pars flacida cholesteatoma with tympanic cavity progression had a high likelihood of recurrence disease. Pars tensa cholesteatoma led to more recurrence of the disease than the residual disease. In both pars flacida and pars tensa cholesteatoma, the incidence of disease recurrence increased even 3 years after surgery. On the contrary, the incidence of residual disease peaked within 3 years after surgery, and thereafter, the incidence of residual disease tended to be small. In particular, pars flacida cholesteatoma extending into the mastoid cavity or tympanic cavity tended to recur up to 5 years postoperatively. CONCLUSIONS: We calculated the cumulative recidivism-free rates of 256 patients with cholesteatoma using Kaplan-Meier survival analysis. These results can lead to better estimates of the length of the follow-up period. LEVEL OF EVIDENCE: Level IV evidence from case-control studies.

Transplantation of a human induced pluripotent stem cell-derived airway epithelial cell sheet into the middle ear of rats.

INTRODUCTION: Early postoperative regeneration of the middle ear mucosa is essential for the prevention of postoperative refractory otitis media and recurrent cholesteatoma. As a means for intractable otitis media management, we focused on human induced pluripotent stem cell (hiPSC)-derived airway epithelial cells (AECs), which have been used in upper airway mucosal regeneration and transplantation therapy. In this study, we transplanted hiPSC-derived AECs into the middle ear of immunodeficient rats. METHODS: Following the preparation of AEC sheets from hiPSCs, the bilateral middle ear mucosa of X-linked severe combined immunodeficient rats was scraped, and the AEC sheets were transplanted in the ears unilaterally. RESULTS: Human nuclear antigen (HNA)-positive ciliated cells were observed on the transplanted side of the middle ear cavity surface in three of six rats in the 1-week postoperative group and in three of eight rats in the 2-week postoperative group. No HNA-positive cells were found on the control side. The percentage of HNA-positive ciliated cells in the transplanted areas increased in the 2-week postoperative group compared with the 1-week group, suggesting survival of hiPSC-derived AECs. Additionally, HNA-positive ciliated cells were mainly located at sites where the original ciliated cells were localized. Immunohistochemical analysis showed that the transplanted AECs contained cytokeratin 5- and mucin 5AC-positive cells, indicating that both basal cells and goblet cells had regenerated within the middle ear cavity. CONCLUSIONS: The results of this study are an important first step in the establishment of a novel transplantation therapy for chronic otitis media.

Preclinical assessment of transplantable human nasal mucosal epithelial cell sheets.

INTRODUCTION: We previously reported a new cell transplantation therapy for patients with intractable otitis media using autologous nasal mucosal epithelial cell sheets, manufactured using temperature-responsive cell culture inserts. The current study aimed to verify whether the transplantable cell sheets could be manufactured for application in clinical trials, according to standard operational procedures (SOP), in a cell processing facility (CPF). METHODS: Human nasal mucosal epithelial cells from four volunteer donors were aseptically cultured and transplantable cell sheets successfully manufactured, with reproducibility, using temperature-responsive cell culture inserts in the CPF. During the manufacture of cell sheets, the CPF environment was confirmed to be aseptic by sterilization tests. Purity of the cell sheets was confirmed by histological analysis and flow cytometry. Both safety and quality of the human nasal mucosal epithelial cell sheets were validated. RESULTS: The cultured and manipulated human nasal mucosal epithelial cells showed no evidence of malignant transformation in vitro. The study confirmed the safety and suitability of the manufactured human nasal mucosal epithelial cell sheets for use in clinical trials. CONCLUSIONS: The results led to the establishment of a coherent system in which transplantation could be achieved smoothly.

Management of Intractable Petrous Bone Cholesteatoma With a Combined Translabyrinthine-Transsphenoidal Approach.

OBJECTIVE: To evaluate whether a combined translabyrinthine-transsphenoidal approach can be used to achieve adequate surgical resection of an extensive petrous bone cholesteatoma and create a debris drainage route for the residual cholesteatoma that is maintained long-term. PATIENT: A 71-year-old man with residual petrous temporal bone cholesteatoma that had spread extensively to the internal carotid artery and posterior cranial fossa. INTERVENTION: Surgical resection of the cholesteatoma via a translabyrinthine approach and creation of a debris drainage route into the nasopharynx via a transsphenoidal approach. MAIN OUTCOME MEASURES: Control of unresectable petrous temporal bone cholesteatoma and occurrence of cholesteatoma- or surgery-related complications. RESULTS: Although complete removal of the cholesteatoma was attempted via a translabyrinthine approach, this was not possible because the epithelium of the cholesteatoma was strongly attached to the internal carotid artery and posterior cranial fossa. A debris drainage route leading to the nasopharynx was created by drilling the clivus on the side of the lesion via a transsphenoidal approach. The patient has had no complications since surgery. The drainage route remains open, and the cholesteatoma has been controlled for 57 months. CONCLUSIONS: A markedly advanced petrous temporal bone cholesteatoma can be managed safety and reliably by combining a translabyrinthine approach with a transsphenoidal approach. Creation of a debris drainage route into the nasopharynx can prevent isolation of the cholesteatoma and mastoid cavity problems after surgery. This one-stage surgery may be a suitable method for keeping cholesteatoma under control in patients with unresectable petrous bone cholesteatoma.

Development of a nasal mucosa-removal model for evaluating cell therapy.

INTRODUCTION: Endoscopic sinus surgery is an effective surgical procedure for treating chronic sinusitis; however, extensive exposure of the bone in the nasal cavity can result in permanent disability postoperatively. Particularly, closure of the sinus drainage pathway due to bone hyperplasia associated with bone exposure can trigger the recurrence of sinusitis. It is essential to regenerate the nasal mucosa after surgery to avoid bone hyperplasia. Regenerative medicine, including cell therapy, could be one of the leading options for nasal mucosa regeneration. To date, there is a lack of effective models for evaluating treatments for prevention of bone hyperplasia that occurs after sinus surgery. The purpose of this study was to develop a model of nasal mucosal removal to evaluate cellular therapies. METHODS: The model was created in rabbits, a species with a wide nasal structure, and was generated by approaching the maxillary sinus from the nasal bone side and solely removing the maxillary sinus mucosa without destroying the structures in the nasal cavity. Adipose-derived mesenchymal stromal cell sheets prepared in temperature-responsive cell culture dishes were examined for the effect of transplantation in the animal model. Intranasal evaluation was assessed by micro-computed tomography and tissue staining. RESULTS: Significant bone hyperplasia in the maxillary sinus occurred on the side of mucosal removal, and no bone hyperplasia occurred in the control sham side in the same rabbits on postoperative day 28. Bone hyperplasia was observed over a short time period, with the presence of bone hyperplasia in the maxillary sinus on day 14 and calcification of the bone on day 28. The adipose-derived mesenchymal stromal cell (ADSC) sheet was transplantable in a nasal mucosa-removal model. No significant differences in bone hyperplasia were found between the transplantation side and the sham side in terms of the effect of transplantation of the ADSC sheet; however, bone hyperplasia tended to be suppressed on the transplantation side. CONCLUSIONS: This animal model is simple, highly reproducible, and does not require special equipment or drugs. In addition, this model can be used for various therapeutic interventions, including cell therapy. The presence or absence of the nasal mucosa affects bone remodeling, which highlights the importance of regeneration of the nasal mucosa. In the nasal mucosal regeneration therapy, the ADSC sheet had an inhibitory effect on bone hyperplasia. The nasal mucosa-removal model allows observation of conditions associated with nasal mucosa removal and evaluation of the effectiveness of cell therapy.

Imaging Analysis for Cholesteatoma Extension to the Perilymphatic Space in Labyrinth Fistulae.

OBJECTIVES/HYPOTHESIS: Disturbed perilymph behind a labyrinth fistula can lead to hearing deterioration; thus, delicate manipulation is required during surgery for cholesteatomatous fistulae with matrix extension to the perilymphatic space (EPS). However, it remains challenging to identify the EPS preoperatively. This study aimed to evaluate the diagnostic value of computed tomography (CT) for preoperative prediction of the EPS of cholesteatomatous fistulae. STUDY DESIGN: Retrospective study. METHODS: We included serial high-resolution CT images showing a cholesteatomatous bone defect in the lateral semicircular canal (LSC) requiring mastoidectomy. CT and intraoperative findings were analyzed retrospectively. Using axial CT planes, we evaluated the length and angle between the margins of bone defects. Receiver operating characteristic (ROC) curves were constructed to determine the cutoff points. RESULTS: We extracted data from 30 bone defects, of which six (20.0%) showed EPS intraoperatively. Bone defects with EPS (n = 6) had significantly greater length and angle values than those without EPS (n = 24) (P < .001 for both, Wilcoxon rank sum test). For length and angle, the area under the curve was 0.944 (95% confidence interval [CI]: 0.858-1.000) and 0.951 (95% CI: 0.875-1.000), respectively, according to the ROC analysis, and the optimal cutoff values were 3.65 mm and 71.6°, respectively, with 100% sensitivity and 91.67% specificity for both. CONCLUSIONS: Results demonstrated that a length >3.65 mm and an angle >71.6° for LSC bone defects on axial CT images are reliable diagnostic markers of EPS. Preoperative high-resolution CT analysis can provide surgeons with a more conscientious preparation for handling deeper labyrinth fistulae. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E1301-E1307, 2021.

ROCK inhibitor combined with Ca2+ controls the myosin II activation and optimizes human nasal epithelial cell sheets.

The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca2+ concentration. However, it was not known whether a combination would influence the behavior of cultured epithelial cells through changes in the phosphorylation of non-muscle myosin light chain II (MLC). Here we show that the combination of ROCK inhibition with Ca2+ elevation regulated the phosphorylation of MLC and improved both cell expansion and cell-cell adhesion during the culture of human nasal mucosal epithelial cell sheets. During explant culture, Ca2+ enhanced the adhesion of nasal mucosal tissue, while ROCK inhibition downregulated MLC phosphorylation and promoted cell proliferation. During cell sheet culture, an elevation of extracellular Ca2+ promoted MLC phosphorylation and formation of cell-cell junctions, allowing the harvesting of cell sheets without collapse. Moreover, an in vitro grafting assay revealed that ROCK inhibition increased the expansion of cell sheets three-fold (an effect maintained when Ca2+ was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca2+ could facilitate the fabrication of many types of cell graft.

The impact of middle ear aeration on surgical outcome after intact canal wall tympanoplasty for cholesteatoma.

OBJECTIVE: The aeration status of the middle ear is presumed to be one of the factors affecting the outcome in acquired cholesteatoma. The present study investigated the impact of postoperative middle ear aeration on hearing and recurrence after intact canal wall tympanoplasty. METHODS: We conducted a retrospective chart review of 127 pars flaccida cholesteatoma patients who underwent primary intact canal wall tympanoplasty at a tertiary academic medical center. We collected data on clinical characteristics, including extent of cholesteatoma, surgical procedure, pre- and postoperative middle ear aeration, hearing level, and recurrence. The aeration was measured before and at one year after operation on two axial computed tomography (CT) planes: at the lateral semicircular canal (mastoid cavity) and at the oval window (tympanic cavity). Based on the postoperative air-bone gap (ABG), patients were categorized into two groups: the successful hearing (ABG ≤15 dB) group and the unsuccessful hearing (ABG >15 dB) group. We used aeration ratio to assess the relationship between postoperative aeration improvement and hearing outcome or recurrence. Multivariate logistic regression analysis was used to identify the factors associated with hearing outcome. RESULTS: At one year after operation, aeration ratio in both mastoid and tympanic cavities was significantly improved than the preoperative status (p < 0.001, p < 0.001, respectively, Wilcoxon signed-rank test). The positive correlation was found between postoperative aeration ratios of mastoid cavity and tympanic cavity (r = 0.348, p < 0.001, Spearman's rank-correlation coefficient). In mastoid and tympanic cavities, the postoperative aeration ratio in successful hearing group (n = 57) was significantly higher than that in the unsuccessful hearing group (n = 45) at one year after operation (p < 0.001, p = 0.028, respectively, Mann-Whitney U test). Multivariate logistic regression analysis demonstrated that postoperative aeration ratio in mastoid cavity and preoperative ABG were significant independent prognostic factors for successful hearing (odds ratio [95% confidence interval]: 2.630 [0.985 - 7.024], p = 0.045; 0.891[0.840 - 0.944], p < 0.001, respectively). However, postoperative aeration ratios did not significantly differ between with (n = 14) and without recurrence (n = 113) groups in both cavities. CONCLUSION: Our results suggest that better postoperative aeration in mastoid cavity is independently associated with satisfactory hearing outcome after intact canal wall tympanoplasty in pars flaccida cholesteatoma. However, no significant differences were observed between middle ear aeration at one year after operation and recurrence.

Transplantation of autologous oral mucosal epithelial cell sheets inhibits the development of acquired external auditory canal atresia in a rabbit model.

Acquired external auditory canal atresia is characterized by fibrous tissue formation in the ear canal, hearing loss and chronic otorrhea. Although the disease can be treated surgically, the recurrence rate is high. This study explored whether autologous oral mucosal epithelial cell sheets could be used as a novel therapy for ear canal atresia. We succeeded in generating a rabbit model of acquired external auditory canal atresia by dissecting the skin of the ear canal. Endoscopic and histological findings in this model indicated that atresia developed over a 4-week period and was not inhibited by the placement of polyglycolic acid sheets immediately after skin dissection. By contrast, transplantation of autologous oral mucosal epithelial cell sheets, which had been fabricated by culture on temperature-responsive inserts without a feeder layer, prevented the development of atresia during the 4-week period after skin dissection. Transplantation of autologous epithelial cell sheets after surgical treatment of acquired external auditory canal atresia could be a promising new method to reduce the risk of disease recurrence. STATEMENT OF SIGNIFICANCE: Acquired external auditory canal atresia is characterized by fibrous tissue formation in the ear canal, which leads to hearing loss and chronic otorrhea. Although surgical treatments are available, the recurrence rate is high. In this study, we successfully generated a rabbit model of acquired external auditory canal atresia by dissecting the skin of the ear canal. Furthermore, we utilized this new animal model to investigate whether the transplantation of autologous oral mucosal epithelial cell sheets could be used as a novel therapy for ear canal atresia. Our results raise the possibility that the transplantation of autologous epithelial cell sheets after surgical treatment of ear canal atresia could be a promising new method to reduce the risk of disease recurrence.

Analysis of human nasal mucosal cell sheets fabricated using transported tissue and blood specimens.

Previously, we succeeded in transplanting autologous nasal mucosal cell sheets in the middle ears of 5 patients, who underwent cholesteatoma resection, which prevents recurrence of cholesteatoma in clinical settings. Current good manufacturing practice (GMP) standards for human cell cultivation requires the establishment of cell processing centers (CPC) which act as germ-free facilities. However, due to practical difficulties involved in establishing and maintaining such facilities at each individual hospital, a functional transport system is felt to be needed for the continuation of effective regenerative therapy. In the current study, nasal mucosal tissue and autologous blood obtained from 3 human volunteers were transported for over 3 h. Disinfected nasal tissues were cultured using keratinocyte culture medium, which included autologous serum prepared from blood. After 24 d, cultured nasal mucosal cells were transported for over 3 h and subsequently assessed for cell number, viability and purity. Moreover, CK4, CK8, and CK18 were analyzed the suitability of these nasal mucosal cell sheets for middle ear regenerative therapy. Overall, we confirmed that nasal mucosal cell sheets can be fabricated using transported nasal mucosal tissue and blood. This study would be contribute to establish a new regenerative therapy for clinical application, accompanied with transportation between companies and hospitals.

Clinical Characteristics of Pars Tensa Cholesteatoma: A Comparative Study of Area-Based Classification Systems Proposed by the Japanese Otological Society and the European Academy of Otology - Neuro-Otology.

OBJECTIVES: To assess the clinical characteristics of extent patterns in pars tensa cholesteatoma. MATERIALS AND METHODS: This was a retrospective chart review. Forty-four patients with pars tensa cholesteatoma who underwent primary surgery at a tertiary academic medical center were included. The main outcomes measured were sex, age, clinical background, and stage classification of pars tensa cholesteatoma (including the extent of cholesteatoma and involvement of the sinus tympani) according to two staging classifications: criteria advocated by the Japanese Otological Society (JOS) and those advocated by the European Academy of Otology and Neuro-Otology (EAONO)/JOS joint consensus statements. RESULTS: The mean patient age ± standard deviation was 38.4±19.6 years. The patients comprised 19 men and 25 women. According to the JOS classification, 18 ears (40.9%) were classified as stage I, 22 (50.0%) as stage II, and 4 (9.1%) as stage III. According to the EAONO/JOS joint consensus statements, 14 ears (31.8%) were classified as stage I, 26 (59.1%) as stage II, and 4 (9.1%) as stage III. Fourteen ears (31.8%) demonstrated involvement of the sinus tympani. Four ears (9.1%) that were originally categorized as stage I cholesteatoma by the JOS criteria showed sinus tympani invasion and were subsequently categorized as stage II according to the EAONO/JOS criteria. CONCLUSION: We determined the clinical characteristics of pars tensa cholesteatoma based on the novel and well-defined classification criteria. Further studies including long-term outcomes are necessary to demonstrate the clinical relevance of the discrepancy between the two criteria with respect to involvement of the sinus tympani.

Explant culture of oral mucosal epithelial cells for fabricating transplantable epithelial cell sheet.

INTRODUCTION: Carrier-free autologous mucosal epithelial cell sheets have been clinically utilized as a cell therapy for various epithelial disorders. Fabrication of a transplantable oral mucosal epithelial cell sheet without mouse feeder layers requires a higher seeding density than that of a sheet with mouse feeder layer culture; therefore, a large amount of donor mucosal tissue is needed. However, cell grafts co-cultured with mouse feeder layers are classified by the US Food and Drug Administration (FDA) as xenogeneic products. The goal of this study was to evaluate the utility of oral mucosal epithelial cells expanded by primary explant culture for the fabrication of an adequate number of transplantable epithelial cell sheets without mouse feeder layers. METHODS: Small fragments derived from minced oral mucosal tissue were placed into culture dishes for primary explant culture in keratinocyte culture medium. After primary explant culture, the outgrown cells were treated with trypsin-EDTA and were seeded on a temperature-responsive cell culture insert. After subculture, the cultured cells were harvested as a confluent cell sheet from the culture vessel by temperature reduction. RESULTS: Carrier-free human oral mucosal epithelial cell sheets were fabricated in all human cases, and autologous transplantation of the harvested cell sheets showed rapid epithelial regeneration to cover epithelial defects in a rabbit model. The explant culture method, involving the use of small fragments for primary culture, was sufficient for preparing a large number of mucosal epithelial cells without mouse feeder layers. Moreover, oral mucosal epithelial cells derived from the primary explant culture after cryopreservation allowed for the fabrication of cell sheets. CONCLUSIONS: This method for fabricating transplantable oral mucosal epithelial cell sheets is an attractive technique for regenerative medicine. It offers a patient-friendly manufacturing method in which a small amount of biopsy material from the patient represents a sufficient epithelial cell source, and a manufacturing plan for preparing cell grafts can be easily tailored.

Practical analysis of pars flaccida cholesteatoma with classification and staging system proposed by Japan Otological Society: a comparative study.

BACKGROUND: The Japan Otology Society (JOS) proposed the classification and staging system for middle ear cholesteatoma. However, there was little analysis of the pathology of cholesteatoma using this staging system. AIMS/OBJECTIVES: To analyze the pathology of pars flaccida cholesteatoma using the staging system by JOS. MATERIAL AND METHODS: A total of 183 cases of fresh pars flaccida cholesteatoma treated between January 2009 and December 2015 were included. We used the staging system recommended by JOS (2015). The association of the following variables in each stage of pars flaccida cholesteatoma was examined: age, gender, preoperative hearing level, staging, statuses of mastoid cell growth and stapes, tympanic sinus invasion. RESULTS: Stage II disease showed the highest degree of progression, and peak incidence was observed in the third and fourth decades of life. Stage III significantly increased after the age of 40 years. The progression of the disease stage was significantly associated with deterioration of hearing level. Cholesteatoma invasion to tympanic sinus was recognized in 14.2% of cases. The state of the stapes is increasingly likely be to fracture as stage progresses. CONCLUSIONS: The JOS Staging System appropriately reflects the disease state, and it was found to be clinically meaningful in this study.

Middle ear mucosal regeneration by tissue-engineered cell sheet transplantation.

The recurrence of cholesteatoma after surgical treatment often occurs as a result of poor mucosal regeneration in the middle ear cavity and mastoid cavity and changes, such as granulation tissue formation, which impair gas exchange in the middle ear cavity. Conventional tympanoplasty often results in a lack of mucosal regeneration in the resected area of the mastoid cavity. In particular, mucosal regeneration in a poorly pneumatized mastoid cavity is extremely difficult. If the middle ear mucosa can be preserved or rapid postoperative regeneration of mucosa on the exposed bone surface can be achieved after middle ear surgery, the results of surgical treatment for otitis media, including cholesteatoma, can potentially be improved and the physiological function of the middle ear can be recovered. To overcome these limitations, we developed a novel treatment method combining tympanoplasty and autologous nasal mucosal epithelial cell sheet transplantation for postoperative regeneration of the middle ear mucosa. In clinical research, we endoscopically removed an approximately 10 × 10 mm2 piece of nasal mucosal tissue. Tissue-engineered autologous nasal mucosal epithelial cell sheets were fabricated by culturing the harvested cells in an aseptic environment in a good manufacturing practice-compliant cell processing facility. The cultivated cell sheets were transplanted, during tympanoplasty, onto the exposed bony surface of the attic of the tympanic and mastoid cavities where the mucosa had been lost. We performed this procedure on four patients with middle ear cholesteatoma and one patient with adhesive otitis media. All patients showed favorable postoperative course with no adverse events or complications and the patients' hearing ability post-transplantation remained good.