Effect of basic fibroblast growth factor with collagen/gelatin fixture in a rabbit model of nasal septum perforation.

Introduction: The treatment of nasal septum perforation solely by surgical intervention presents significant challenges. This study evaluated the effect of basic fibroblast growth factor (bFGF) in combination with collagen/gelatin on wound healing of nasal septum perforation in a rabbit animal model. Methods: A nasal septum perforation rabbit model was created. bFGF was added to a collagen/gelatin fixture and placed adjacent to the perforation, which is a complete defect. The rabbits were divided into three groups: the sham group that underwent the surgical procedure only, bFGF (-) group that received collagen/gelatin fixture without bFGF, and bFGF(+) group that received collagen/gelatin fixture with bFGF. The dimensions of the perforations were measured after 4 weeks, and the septum was subjected to histological examination. Results: All perforations remained open in the sham group (closure rate: 20.4%-83.1%). The closure rates of the bFGF(-) and bFGF(+) groups were 49.4%-68.8% and 72.7%-100%, respectively. No significant difference was noted in the closure rates between the sham and bFGF(-) groups; however, significant differences were observed between the sham and bFGF(+) groups, and the bFGF(-) and bFGF(+) groups (p < 0.05), indicating that bFGF promoted perforation closure. Conclusions: The study demonstrated that bFGF with collagen/gelatin carrier promoted wound healing in a rabbit model of nasal septum perforation.

Analysis of the potential of human cultured nasal epithelial cell sheets to differentiate into airway epithelium.

Understanding the expected efficacy and safety of a new regenerative therapy requires analysis of the fate of the transplanted cell graft. We have shown that transplantation of autologous cultured nasal epithelial cell sheets onto the middle ear mucosa can improve middle ear aeration and hearing. However, it remains unknown whether cultured nasal epithelial cell sheets have the potential to gain mucociliary function in the environment of the middle ear because sampling cell sheets after transplantation is challenging. The present study re-cultured cultured nasal epithelial cell sheets in different culture media and evaluated whether the sheets have the potential to differentiate into airway epithelium. Before re-cultivation, cultured nasal epithelial cell sheets fabricated in keratinocyte culture medium (KCM) contained no FOXJ1-positive and acetyl-α-tubulin-positive multiciliated cells or MUC5AC-positive mucus cells. Interestingly, multiciliated cells and mucus cells were observed when the cultured nasal epithelial cell sheets were re-cultured in conditions that promote differentiation of airway epithelium. However, multiciliated cells, mucus cells and CK1-positive keratinized cells were not observed when cultured nasal epithelial cell sheets were re-cultured in conditions that promote epithelial keratinization. These findings support the suggestion that cultured nasal epithelial cell sheets have the ability to differentiate and gain mucociliary function in response to an appropriate environment (possibly including the environment found in the middle ear) but are unable to develop into an epithelial type that differs from its origins.

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.

Retraction notice to "Preclinical assessment of transplantable human nasal mucosal epithelial cell sheets" [Regenerative Therapy 18 (2021) 59-65].

[This retracts the article DOI: 10.1016/j.reth.2021.03.006.].

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.

Radiological and Audiological Prediction for Ossicular Fixation in Chronic Otitis Media and Tympanic Membrane Perforation.

OBJECTIVES: Recurrent middle-ear infection can lead to ossicular fixation, adversely affecting post-tympanoplasty hearing outcomes. Preoperative prediction of ossicular fixation remains challenging. We aimed to investigate potential predictors of ossicular fixation in patients with chronic otitis media. STUDY DESIGN: Retrospective. SETTING: Tertiary academic medical center. PATIENTS: Patients with noncholesteatomatous chronic otitis media and tympanic membrane perforation, without ossicular discontinuities. INTERVENTIONS: Diagnostic. MAIN OUTCOME MEASURES: The fixation of each ossicle was assessed during tympanoplasty. The impact of preoperative otoscopic findings, computed tomography (CT) features, and hearing levels on the prediction of ossicular fixation was evaluated using uni- and multivariable logistic regression analyses. RESULTS: One hundred thirty-five patients were included. Soft-tissue density between the malleus head and the anterior wall (odds ratio, 3.789 [95% confidence interval, 1.177-12.196]; p = 0.0255) and poor development of mastoid cells (16.826 [2.015-134.520]; p = 0.0078) were independent predictors of malleus fixation. In addition, ≥50% tympanic membrane perforation (5.412 [1.908-15.353]; p = 0.0015), poor development of mastoid cells (3.386 [1.039-11.034]; p = 0.0431), and a ≥40-dB preoperative air-bone gap (ABG) at 500 Hz (4.970 [1.732-14.261]; p = 0.0029) were independent predictors of incus fixation. Soft-tissue density surrounding the stapes (18.833 [1.856-191.104]; p = 0.0119) and a ≥40-dB preoperative ABG at 500 Hz (13.452 [1.640-∞]; p = 0.0138) were correlated with stapes fixation. CONCLUSIONS: The accurate prediction of ossicular fixation in patients with chronic otitis media based on CT features and the ABG may facilitate decision-making regarding the need for ossiculoplasty, possibly avoiding unnecessary manipulation or overlooking of fixation.

Cell sheet transplantation prevents inflammatory adhesions: A new treatment for adhesive otitis media.

INTRODUCTION: We developed a new treatment method that combines tympanoplasty with transplantation of autologous cultured nasal mucosal epithelial cell sheets to regenerate the mucosa of patients with adhesive otitis media, which has been difficult to treat effectively. We verified whether this procedure could be performed safely and measured its therapeutic efficacy. METHODS: Autologous nasal mucosal epithelial cell sheets were manufactured at a good manufacturing practice-compliant cell processing facility using autologous nasal mucosal tissue. We performed tympanoplasty and transplanted the cell sheets into the middle ear cavity in six patients with adhesive otitis media. RESULTS: The manufactured autologous cultured epithelial cell sheets met the predetermined quality standards and were successfully transplanted safely in all cases. Computed tomography findings after cell sheet transplantation showed that aeration in the tympanic cavity was maintained or restored in five of the six patients (83.3%). Four of the six (66.7%) patients had postoperative air-bone gap within 20 dB, which is considered a postoperative success in tympanoplasty for chronic middle ear disease. CONCLUSIONS: The results of this clinical study suggest that tympanoplasty with cell sheet transplantation can be used to treat adhesive otitis media by reliably preventing re-adhesion of the tympanic membrane.

Temporary Storage of the Human Nasal Tissue and Cell Sheet for Wound Repair.

Temporary storage of nasal tissues and nasal cell sheets, which entails transportation between hospitals and cell culture facilities, is an important issue in regenerative medicine. Herein, we investigated the preservation of chilled and frozen nasal tissues and expiry dates of ready-to-use nasal cell sheets. Although the cell number in preserved tissues was lower than that in fresh tissue, nasal cell sheets could be fabricated from tissues that had been refrigerated for 5 days and frozen-thawed over 5 days. Moreover, the nasal mucosal cell sheets were preserved in a non-hazardous buffer. The cell number, viability, and structure were not maintained in saline containing E-cadherin for 2 days; however, these were maintained in Hank's balanced salt solution for 2 days, but not for 5 days. To assess the proliferation capacity of cells in the stored cell sheets, we performed cell sheet grafting assays in vitro. Cell sheets stored in Hank's balanced salt solution for 2 days adhered to collagen gel and expanded normally. Our results show that nasal tissues can be stored temporarily in refrigerators or deep freezers, and Hank's balanced salt solution can be used for preservation of ready-to-use cell sheets for a few days.

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.

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.

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.

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.

Factors related to the satisfaction level of elderly hearing-impaired individuals with cochlear implants.

OBJECTIVE: This study investigated factors related to the satisfaction level of cochlear implants for the elderly. METHODS: A survey was conducted by sending an anonymous self-reported questionnaire to medical facilities specializing in cochlear implantation throughout Japan and members of cochlear implant self-help groups aged 65 years and older. The subjects were divided into two age-based groups (under 75 and 75 years and older) to analyze the usage of cochlear implants. Binary logistic regression was performed to analyze factors related to the satisfaction level of the recipients with hearing improvements provided by cochlear implants (p<0.05). RESULTS: Responses were received from 60 cochlear implant users. The mean age of the respondents was 74.9±6.87 (mean ± 1SD) years. The mean cochlear implant use was 12.4 ± 4.0 (mean ± 1SD) hours per day. Regarding satisfaction with the cochlear implants, 93.3% responded "somewhat satisfactory" or better, indicating at least moderate satisfaction. However, fewer respondents in the 75-years and older group reported feeling "satisfactory" or better (chi-square test, p<0.05). Concerning device operation and management, difficulties including volume adjustment, switching between program, and exchanging cables, were reported. Among the patient-reported indices of postoperative hearing improvements studied, their ability to hear and comprehend conversations with family members and information provided at reception desks were most associated with user satisfaction with cochlear implants. CONCLUSION: Many elderly patients were satisfied with their cochlear implants; however, respondents in the 75-years and older group had lower levels of satisfaction compared to those in respondents in the under-75-years group. Elderly patients had problems with more complex operations and management of their cochlear implants. Moreover, they were satisfied with their ability to comprehend familiar, everyday conversations. These factors related to satisfaction level may be useful in providing valuable rehabilitation for elderly patients with cochlear implants.

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.

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.

Lessons learned from conventional animals: Encouragement to use specific-pathogen-free animals.

Experimental rabbits provide evidence for translational research regarding the pathogenies or treatment of human diseases. We developed a novel method for regenerating the middle ear mucosa using autologous cultured nasal mucosal epithelial cell sheets, and evaluated the wound healing process in the middle ear mucosa of experimental rabbits. Nonetheless, vigilant microbiological monitoring of experimental animals is essential to effectively prevent a decline in their health conditions, which may affect the research results. We experimented with contamination of Pasteurella multocida in non-specific-pathogen-free (SPF) rabbits (without microbiological monitoring). Most non-SPF rabbits had otitis media, whereas SPF rabbits did not, which affected their results during the mucosal regeneration study. The contamination was resolved by changing the experimental design from using non-SPF rabbits to that using SPF rabbits. It is crucial to use the SPF animals for any surgical intervention studies.

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.

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.

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.