Isolation of Epidermal Progenitor Cells from Rat Tympanic Membrane.

The eardrum is an important structural component for hearing, but it is delicate and subject to traumatic injury and disease. Healing mechanisms are activated after injury but sometimes healing fails and chronic perforations develop, requiring surgical intervention. To model the wound healing responses we established a simple method for isolating keratinocytes and progenitors from individual eardrums. The central region of the eardrum contains epidermal proliferative centers that produce keratinocytes which migrate to cover the eardrum surface. We dissected out the central region and explanted it to the plastic membrane of a culture well insert. Epidermal cells grew from the explant onto the surface of the insert membrane. The cells could be serially harvested and passaged for continuous culture and characterization. Magnetic immunoseparation methods were used to enrich for epithelial cells with stem cell-like characteristics. Proliferation and migration in vitro was demonstrated, and the cells were shown suitable for tissue engineering applications.

Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis.

Angiogenesis, a morphogenic event endothelial cells (ECs) undergo in response to 3-D environmental triggers, is critical to the survival and ultimate functional capacity of engineered tissue constructs. Here we present a new collagen mimetic peptide (CMP) architecture consisting of multiple anionic charges at the peptide's N-terminus designed to attract growth factors by charge-charge interactions and bind to collagen by CMP-collagen interaction. The anionic CMPs exhibited specific binding affinity to type I collagen substrates while attracting vascular endothelial growth factors (VEGFs), which led to enhanced morphological features of ECs, indicative of tubulogenesis. The results show that these new CMPs could be used to direct proliferation and differentiation of cells in collagen scaffolds by localization and sustained delivery of growth factors and other morphogens.

Spatio-temporal modification of collagen scaffolds mediated by triple helical propensity.

Functionalized collagen that incorporates exogenous compounds may offer new and improved biomaterials applications, especially in drug-delivery, multifunctional implants, and tissue engineering. To that end, we developed a specific and reversible collagen modification technique utilizing associative chain interactions between synthetic collagen mimetic peptide (CMP) [(ProHypGly) chi; Hyp = hydroxyproline] and type I collagen. Here we show temperature-dependent collagen binding and subsequent release of a series of CMPs with varying chain lengths indicating a triple helical propensity driven binding mechanism. The binding took place when melted, single-strand CMPs were allowed to fold while in contact with reconstituted type I collagens. The binding affinity is highly specific to collagen as labeled CMP bound to nanometer scale periodic positions on type I collagen fibers and could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, bound CMPs discharged from the collagen at a sustained rate that correlated with CMP's triple helical propensity, suggesting that sustainability is mediated by dynamic collagen-CMP interactions. We also report on the spatially defined modification of collagen film with linear and multi-arm poly(ethylene glycol)-CMP conjugates; at 37 degrees C, these PEG-CMP conjugates exhibited temporary cell repelling activity lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic applications and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices.

A Rare Cause of a Fluctuating Cystic Lesion in the External Auditory Canal.

Temporomandibular joint (TMJ) ganglionic cyst is an uncommon entity and only a few have been reported in the literature. TMJ ganglion within the external auditory canal presenting clinically as a fluctuating cystic lesion has never been reported. Here, we present a unique case of such a lesion together with otoscopic and radiological images as well as provide a descriptive review of TMJ ganglionic cysts.

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.

Squamous cell carcinoma arising from a thyroglossal duct cyst: A case report and review of the literature.

OBJECTIVES: Squamous cell carcinoma in a thyroglossal duct cyst is exceedingly rare with only 26 reported cases in the literature so far, which only account for 6% of the patients. METHODS: We report a unique case of squamous cell carcinoma arising from a thyroglossal duct cyst in a 49-year-old male who was primarily diagnosed as a thyroglossal duct cyst with inflammation. The patient underwent Sistrunk procedure with wide local excision and radiation therapy as well as chemotherapy post-operatively and had no evidence of recurrence or metastasis for 24 months. In addition, we reviewed the relevant literatures. RESULTS: Whether squamous cell carcinoma actually arises from thyroglossal duct cyst is still controversial; however, carcinoma originating from metaplasia of columnar and squamous epithelium in thyroglossal duct cyst has been well accepted. The gold-standard diagnostic method is fine needle aspiration biopsy with ultrasound guidance. Sistrunk procedure alone or with wide excision is likely to be beneficial. Neck dissection is necessary in patients with positive cervical lymphadenopathy. Radiation therapy and chemotherapy have not yet been clearly defined. CONCLUSION: Squamous cell carcinoma arising from thyroglossal duct cyst is really a rare disease, whose origin, treatments and prognosis still remain uncertain. These are solely based on case reports, case series and expert opinions. Hence, more investigations about squamous cell carcinoma will be conducted in the near future.

Absorbable Hemostatic Aggregates.

Topical absorbable hemostats are routinely utilized in surgical procedures to assist in controlling intraoperative bleeding. SURGICEL Original Absorbable Hemostat, one of the most frequently used adjunctive hemostats, is composed of oxidized regenerated cellulose (ORC). We report here that a novel powdered form of ORC, composed of aggregates of ORC fine fibers, provides additional valuable hemostatic performance characteristics and retains the biochemical and bactericidal profile of the parent ORC fabric. The ORC aggregates are more effective in promoting coagulation than their constituent ORC fine fibers because of more favorable surface energetics and surface area. Aggregates with similar particle size distributions that have higher sphericity values exhibit better coagulation efficacy. Finally, ORC aggregates more effectively promote clot formation than starch-based hemostatic particles. The results of this investigation indicate that the efficacy of this novel powdered hemostat is based on its chemical composition, morphology, and particle surface energetics.

An in vivo comparison of the efficacy of hemostatic powders, using two porcine bleeding models.

BACKGROUND: Usage of topical hemostatic agents in surgery is increasing, including use during minimally invasive procedures, and even for surgeries that have a low risk of bleeding complications. A novel product, Surgicel® Powder - Absorbable Hemostatic Powder (SP), made from oxidized regenerated cellulose (ORC) fabric, has been developed for adjunctive use in surgical procedures to assist in control of oozing bleeding over broad areas and where access could be difficult with a fabric ORC product. This study compares the new SP to other commercially available hemostatic powder products in two in vivo models. METHODS: Hemostatic efficacy of SP was compared to two polysaccharide-based hemostats in a porcine liver punch biopsy model and to three polysaccharide-based hemostats and one non-regenerated oxidized cellulose hemostat in a porcine liver abrasion model. Primary outcomes measured were hemostatic efficacy, defined as hemostasis within 10 minutes of application, and time-to-hemostasis (TTH). RESULTS: In the punch biopsy model, SP displayed significantly higher effective hemostasis rates than one of the polysaccharide hemostats (p=0.047) and faster TTH than both (p<0.001). In the liver abrasion model, SP had significantly higher effective hemostasis rates (p≤0.002) and faster TTH (p<0.001) than the other four hemostatic agents. The amount of powder applied within the ranges used did not appear to affect hemostatic efficacy. CONCLUSION: In both the liver punch biopsy model of mild to moderate bleeding and the liver abrasion model of mild but diffuse oozing, SP provided more effective hemostasis and faster TTH than other marketed hemostatic powders. The results from this in vivo study suggest that Surgicel Powder may be useful in clinical applications where control of oozing capillary, mild venous, and small arterial hemorrhage is required including bleeding in difficult-to-access locations.

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.

Rat model of chronic tympanic membrane perforation: Ventilation tube with mitomycin C and dexamethasone.

OBJECTIVE: Chronic tympanic membrane perforation (TMP) in a clinical setting may attract surgical intervention. With the advent of modern biomaterials, new options are available for myringoplasty but safety and efficacy need evaluation in a chronic TMP animal model. The aim of this study was to evaluate the efficacy of ventilation tube (VT) insertion in conjunction with topical application of mitomycin C/dexamethasone (M/D) for the creation of chronic TMP in rats. METHODS: Thirty male Sprague-Dawley rats underwent myringotomy of the right tympanic membrane (TM) and were divided into three experimental groups: spontaneous healing (myringotomy control), VT insertion for 2 weeks and VT insertion for 2 weeks in conjunction with topical application of M/D (VT-M/D). All TMs were regularly assessed by otoscopy for 10 weeks and then animals were sacrificed for histological evaluation. RESULTS: In the VT-M/D group, seven out of ten (70%) perforations were patent at 10 weeks (mean patency, 57.9 days; P<0.01). The VT group had two out of ten (20%) perforations patent at 10 weeks (mean patency, 26.5 days; P<0.01), while all TMPs from the myringotomy control group were closed by day 9 (mean patency, 7.2 days). Histologically, the TMPs patent at week 10 had a stratified squamous epithelialized rim, keratinocyte layer thickening around the perforation edge as well as increased collagen deposition and macrophage infiltration. CONCLUSION: Chronic TMP in a rat model was successfully created by VT insertion and the efficacy was increased in combination with topical application of M/D.

Generation of kidney cancer-specific antitumor immune responses using peripheral blood monocytes transduced with a recombinant adenovirus encoding carbonic anhydrase 9.

PURPOSE: Carbonic anhydrase 9 (CA9) is the most promising molecular marker described for renal cell carcinoma (RCC) to date. We investigated whether transduction of monocytes from peripheral blood with adenovirus encoding the CA9 gene (AdV-CA9) could stimulate a T-cell mediated immune response against cancer cells expressing CA9. The ability to consistently generate a T-cell response is an important step toward the development of a CA9-specific RCC vaccine. EXPERIMENTAL DESIGN: AdV-CA9 was generated using the AdEasy system. AdV-CA9-transduced peripheral blood mononuclear cell (PBMC)-derived monocytes were used to raise CTLs from autologous peripheral blood lymphocytes (PBLs). The ability of CTLs to lyse targets expressing CA9 was assessed by (51)Cr-release. RESULTS: Monocytes were efficiently transduced with AdV-CA9. In five of six experiments, AdV-CA9-transduced monocytes were able to induce a population of CTLs from bulk PBLs. CTLs were capable of lysing autologous, but not allogeneic monocytes expressing CA9. Furthermore, CTLs were able to lyse autologous RCC tumor cells expressing CA9. The ability of CTLs to lyse relevant targets was blocked by anti-CD3, anti-CD8, and anti-MHC class I antibodies demonstrating a MHC class I restricted response. CONCLUSIONS: These results suggest that PBMC-derived monocytes transduced with AdV-CA9 can generate RCC-specific MHC class I restricted CTLs capable of lysing CA9-expressing cancer cells. Transduction of PBMC-derived monocytes with adenovirus provides a simple and effective alternative to the use of dendritic cells for the induction of antigen-specific CTL.

Searching for a rat model of chronic tympanic membrane perforation: Healing delayed by mitomycin C/dexamethasone but not paper implantation or iterative myringotomy.

OBJECTIVES: Surgical intervention such as myringoplasty or tympanoplasty is an option in the current clinical management of chronic tympanic membrane perforation (TMP). Animal models of chronic TMP are needed for pre-clinical testing of new materials and to improve existing techniques. We evaluated several reported animal model techniques from the literature for the creation of chronic TMPs. The aim of this study was to evaluate production of chronic TMPs in a rat model using topical mitomycin C/dexamethasone, paper insertion into middle ear cavity (MEC) or re-myringotomy. METHODS: Forty male Sprague-Dawley rats underwent myringotomy of the right tympanic membrane (TM) and were randomly divided into 3 experimental groups: application of topical mitomycin C/dexamethasone, paper insertion into middle ear cavity, or re-myringotomy. Control perforations were allowed to close spontaneously. TMs were assessed regularly with otoscopy for 8 weeks. At the end of 8 weeks, animals were sacrificed for histology. RESULTS: The closure of TMPs was significantly delayed by mitomycin C/dexamethasone (mean patency, 18.9 days; P≤0.01) compared with the control (mean patency, 7 days), but was not significantly delayed in the paper insertion group (mean patency, 9.4 days; P=0.74). Repeated myringotomy of closed perforations (mean number of myringotomies, 8.9 per ear) stimulated acceleration of closure rather than delay. Histologically, the mitomycin C/dexamethasone group had almost normal TM morphology, while the paper insertion group revealed inflammatory and granulomatous responses. The re-myringotomy group had a thickened TM fibrous layer with collagen deposition. CONCLUSIONS: Mitomycin C/dexamethasone delayed TMP closure in rats but the effect was not sufficiently long-lasting to be defined as a chronic TMP. Neither paper insertion into middle ear cavity nor re-myringotomy created chronic TMP in rats.

Rates of revision and device failure in cochlear implant surgery: a 30-year experience.

OBJECTIVES/HYPOTHESIS: To characterize revision cochlear implant surgery and quantify rates of revision and device failure. STUDY DESIGN: Retrospective review of 235 cases of revision cochlear implant surgery performed at the Sydney Cochlear Implant Center over a period of 30 years, between January 1982 and June 2011. METHODS: Patient demographics and characteristics of revision surgery were retrospectively extracted from a centralized database. Analyses of overall and cumulative rates were performed. RESULTS: During the study period, 2,827 primary cochlear implantations were performed in 2,311 patients, with 201 primary implants in 191 patients of this cohort (109 children and 82 adults) undergoing 235 revision surgeries. The most common indication for revision surgery was device failure (57.8%), followed by migration/extrusion (23.4%), infection/wound complication (17.0%), and poor outcome/secondary pathology (6.4%). The majority of revision surgeries were reimplantations. Overall revision and device failure rates were 8.3% and 4.8%, respectively. The cumulative revision rate for primary implants at all ages increased linearly by 1% per year. The cumulative revision rate was significantly higher in children, and decreased with more recently performed implantations and with newer generations of implants. CONCLUSIONS: The cumulative revision rate for primary implants suggests an ongoing linear relationship between the time of postprimary implantation and the need for revision surgery. We have formed an evidence base that characterizes the nature and frequency of revision surgery in a high-volume setting, allowing clinicians to effectively counsel prospective patients and clinics to understand the burden of revision surgery and device failure.

Relapse of Non-Hodgkin's Lymphoma Involving the Trachea: Acute Subglottic Obstruction.

Relapsing lymphoma involving the trachea causing tracheal obstruction is exceedingly uncommon. Despite its rarity, it should be considered in the differential diagnosis when a patient with known lymphoma presents with signs of airway obstruction such as stridor. We report an unusual case of relapsing non-Hodgkin's lymphoma with tracheal involvement in a 57-year-old female and review the relevant literature. It is highly unusual for relapsing lymphoma to involve the trachea causing tracheal obstruction. Despite its rarity, it can present with life-threatening airway obstruction which may be rapidly progressive requiring immediate surgical intervention such as tracheostomy.

Animal models of chronic tympanic membrane perforation: in response to plasminogen initiates and potentiates the healing of acute and chronic tympanic membrane perforations in mice.

Tympanic membrane perforations (TMP) are relatively common but are typically not treated in their acute stage, as most will heal spontaneously in 7-10 days. Those cases which fail to heal within 3 months are called chronic TMP which attract surgical intervention (e.g. myringoplasty), typically with a temporalis fascia autograft. New materials for the repair of chronic TMP are being developed to address deficiencies in the performance of autografts by undergoing evaluation in animal models prior to clinical study. However, there is currently a lack of ideal chronic TMP animal models available, hindering the development of new treatments. Various techniques and animal species have been investigated for the creation of chronic TMP with varied success. In the present commentary, we bring to the attention of readers the recent report by Shen et al. in Journal of Translational Medicine. The study reported the creation of a chronic TMP animal model in plasminogen gene deficient mice. However, the short observation time (9, 19 days), lack of success rate and the scarcity of solid evidence (e.g. otoscopic & histologic images) to confirm the chronicity of TMP warrant a more thorough discussion.

Animal models of chronic tympanic membrane perforation: a 'time-out' to review evidence and standardize design.

OBJECTIVE: To review the literature on techniques for creation of chronic tympanic membrane perforations (TMP) in animal models. Establishing such models in a laboratory setting will have value if they replicate many of the properties of the human clinical condition and can thus be used for investigation of novel grafting materials or other interventions. METHODS: A literature search of the PubMed database (1950-August 2014) was performed. The search included all English-language literature published attempts on chronic or delayed TMP in animal models. Studies of non English-language or acute TMP were excluded. RESULTS: Thirty-seven studies were identified. Various methods to create TMP in animals have been used including infolding technique, thermal injury, re-myringotomy, and topical agents including chemicals and growth factor receptor inhibitors. The most common type of animal utilized was chinchilla, followed by rat and guinea pig. Twenty three of the 37 studies reported success in achieving chronic TMP animal model while 14 studies solely delayed the healing of TMP. Numerous experimental limitations were identified including TMP patency duration of <8 weeks, lack of documentation of total number of animals attempted and absence of proof for chronicity with otoscopic and histologic evidence. CONCLUSION: The existing literature demonstrates the need for an ideal chronic TMP animal model to allow the development of new treatments and evaluate the risk of their clinical application. Various identified techniques seem promising, however, a need was identified for standardization of experimental design and evidence to address multiple limitations.

The vestibular schwannoma surgery learning curve mapped by the cumulative summation test for learning curve.

OBJECTIVE: To demonstrate and quantify the learning curve for microsurgical excision of vestibular schwannoma in a newly formed neurootologic team by using the cumulative summation test for learning curve (LC-CUSUM). To secondarily identify the factors influencing postoperative facial nerve outcome. STUDY DESIGN: Retrospective review. SETTING: Tertiary referral center. PATIENTS: Between 1999 and 2011, 153 consecutive cases of vestibular schwannoma excision. INTERVENTION: One-hundred and fifty-three patients underwent excision of vestibular schwannoma. MAIN OUTCOME MEASURES: Facial nerve outcomes were assessed using the House-Brackmann (HB) facial nerve grading system. Postoperative facial nerve outcomes at 12 months were analyzed using the LC-CUSUM method with HB Grades I to III being defined as successful outcomes. The factors that influence postoperative facial nerve outcome were analyzed. RESULTS: The constructed learning curve shows a gradual improvement in facial nerve outcomes. The learning curve crossed the derived LC-CUSUM barrier at the 56th procedure, indicating that sufficient evidence had accumulated to demonstrate that the surgeon had achieved optimal outcomes at this point. Tumor size (p = 0.008) and surgical approach (p = 0.005) were 2 additional significant factors influencing postoperative facial nerve outcome. CONCLUSION: The learning curve is evident in this series of microsurgical excisions of vestibular schwannoma. A newly formed team needs to perform at least 56 cases to gain sufficient experience to accomplish optimal results. Position along the learning curve, tumor size, and familiarity with a preferred surgical approach are the factors, which dominated facial nerve outcome. We recommend the use of LC-CUSUM test for learning curve analysis.

Predictors of outcome in patients with metastatic cutaneous head and neck squamous cell carcinoma involving cervical lymph nodes: Improved survival with the addition of adjuvant radiotherapy.

BACKGROUND: The purpose of this study was to compare the outcome of surgery against surgery plus radiotherapy in patients with metastatic cutaneous head and neck squamous cell carcinoma (HNSCC) to cervical nodes. METHODS: We conducted a 28-year retrospective analysis of 122 patients whom were treated for metastatic cutaneous HNSCC involving the cervical nodes (levels I-V). RESULTS: After surgery alone, 11 patients (55%) developed recurrence compared with 23 patients (23%) after surgery plus radiotherapy. On multivariate analysis, the following variables were significantly associated with disease-free survival (DFS): immunosuppression (p = .002), treatment modality (p < .001), extracapsular spread (p = .009), and pathological nodal stage (p = .04). Patients undergoing surgery plus radiotherapy had a significantly better 5-year DFS (74% vs 34%; p = .001) and 5-year overall survival (OS; 66% vs 27%; p = .003) compared with surgery alone. CONCLUSION: In patients with metastatic cutaneous HNSCC involving cervical nodes, survival was significantly improved with the addition of radiotherapy.