Intracranial supramaximal facial nerve stimulation: a murine model.

PURPOSE: Supramaximal facial nerve stimulation is an applied current sufficient to evoke a maximal electromyographic response of facial musculature. It is used during cerebellopontine angle surgery for prognostication of postoperative nerve function. We utilized a rat model to examine safe parameters for intracranial electrical stimulation. MATERIALS AND METHODS: Intracranial facial nerve stimulation with electromyographic monitoring of 14 rats was performed. Supramaximal current level was determined and 50 additional pulses of supramaximal (4 rats), 3 times supramaximal (4), 10 times supramaximal (3), or zero (3) current were applied. To monitor progression of facial nerve injury, video recordings of vibrissae movements and eye closure were captured at 1, 3 and 28 days after surgery; animals were sacrificed on day 28, when nerve morphometry was performed. RESULTS: One rat in the supramaximal stimulation group (of 4), and one rat in the 10 times supramaximal stimulation group (of 3) demonstrated persistent impairment of facial nerve function as evidenced by decreased amplitude of vibrissae sweeping and eye closure impairment. The remainder of rats in all experimental groups demonstrated symmetric and normal facial nerve function at all time points. CONCLUSIONS: A novel animal model for supramaximal stimulation of the rat intracranial facial nerve is described. A small proportion of animals demonstrated functional evidence of nerve injury postoperatively. Function was preserved in some animals after stimulation with current order of magnitude higher than supramaximal levels. Further study with this model is necessary to definitively isolate the effects of surgical trauma from those of supramaximal electrical stimulation.

MRI Study of Jellyfish Collagen, Hyaluronic Acid, and Cadaveric Dermis for Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Test a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice. STUDY DESIGN: Animal model. METHODS: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross-linked jellyfish collagen (MX-JC) and adipose derived stem cells (ADSCs), Group 2, MX-JC alone, Group 3, cross-linked hyaluronic acid (X-HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology. RESULTS: After 100 µL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 µL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 µL, Group 3: 27.4 ± 12 and 10.6 ± 8 µL, and Group 4: 37.5 ± 11 and 9.85 ± 1 µL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4. CONCLUSIONS: MX-JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX-JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX-JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2452-E2460, 2021.

Chondrogenic Predifferentiation Inhibits Vascular Endothelial Growth Factor Angiogenic Effect in Pericranium-Derived Spheroids.

Craniofacial reconstruction of critical bone defects typically requires a bone graft. As graft availability may be restricted by disease or comorbidities, tissue engineering approaches are actively sought. The pericranium could provide new bone graft material. During development and repair, bone transitions through a chondrogenic phase. However, with tissue engineering, pluripotent cells can differentiate directly into bone cells. Does ability to recapitulate bone formation in vitro affect osteogenesis and vascularization of pericranium grafts? To answer this, we obtained tissue from nine patients with preplanned craniotomy surgery and studied three-dimensional osteogenesis and angiogenesis of pericranium-derived spheroids. First, we established growth and differentiation conditions on Matrigel. For each spheroid sample, we investigated (i) continuous osteogenic differentiation (COD) and (ii) osteogenic differentiation preceded by chondrogenesis (CD → OD). The effect of vascular endothelial growth factor (VEGF) was compared to VEGF supplemented with fibroblast growth factor, interleukin (IL)-1, IL-6, platelet-derived growth factor, and tumor necrosis factor-α, a growth factor mix (GFM) with possible synergistic effects. In this limited sample, we observed no age- or sex-related differences in cell expansion. Similarly, no statistically significant differences in osteogenic or angiogenic scores between COD or CD → OD spheroids were noted with regular media. In COD, however, VEGF statistically significantly increased angiogenesis compared to control media (p = 0.007). Also, in COD, both VEGF and VEGF + GFM increased osteogenesis (p = 0.047 and p = 0.038, respectively). By contrast, in CD → OD, neither VEGF nor VEGF + GFM yielded statistically significant angiogenesis or osteogenesis scores compared to control media. To understand these results, we characterized spheroid protein expression by nanoliquid chromatography coupled to tandem mass spectrometry. Nine angiogenic proteins were either uniquely expressed or upregulated in COD compared to CD → OD: (i) endothelial markers JUP, PTGIS, PTGS2, and TYMP, (ii) tissue remodeling factors CHI3L1 and MMP14, and (iii) metabolic pathways modulators ANGPTL4, ITGA5, and WNT5A. ANGPTL4, ITGA5, PTGIS, PTGS2, and WNT5A define a conserved angiogenic network and were >2-fold increased in VEGF compared to VEGF + GFM. Finally, we examined bone formation on printable poly-(propylene-fumarate) (PPF) scaffolds for individualized grafting. Under COD + VEGF conditions, PPF scaffolds loaded with pericranium-derived cells displayed hallmarks of spongiform-like bone formation. Thus, the human pericranium may be a potential repository for bone-generating cells with applications in craniofacial bone repair using tissue printing.

Time-course mass spectrometry data of adipose mesenchymal stem cells acquiring chondrogenic phenotype.

OBJECTIVES: Rabbit adipose mesenchymal stem cells were used for the purpose of studying acquisition of the chondrogenic phenotype over time at 1, 14 and 28 days after in vitro incubation with differentiation media, using nano-liquid chromatography electrospray ionization tandem mass spectrometry analysis. This was part of a preliminary study of the behavior of differentiated adipose stem cells for use in a rabbit model of laryngoplasty. DATA DESCRIPTION: The data comprise .MGF, .RAW, .MZID, and .XLSX, lists of peaks, peptides and proteins identified by nano-flow liquid chromatography electrospray ionization tandem mass spectrometry analysis upon incubation with non-differentiating (ND) or chondrogenic differentiating (CHD) media (ProteomeXchange ID PXD010236). XLSX files contain the following information: day 1 CT (control, N = 3499 proteins), day 14 ND (N = 3106 proteins), day 28 ND (N = 3116 proteins), day 14 CHD (N = 2901 proteins), and day 28 CHD (N = 2876 proteins). Proteins are characterized with respect to their - 10lgP value, percent coverage, number of total as well as unique peptides after trypsin digestion, derivatization method (carbamidomethylation, oxidation, or combined carbamidomethylation + oxidation), average mass, and include a full description.

Treatment of eosinophilic otitis media with pegylated interferon-α 2a and 2b.

OBJECTIVE/HYPOTHESIS: Eosinophilic otitis media (EOM) is a variant of chronic otitis media that is characterized by the development of thick mucoid middle ear effusion, adult onset bronchial asthma, sinonasal polyposis, and aspirin sensitivity. EOM is typically refractory to corticosteroid therapy and surgical intervention. Pegylated interferon (PEG-IFN) has effectively treated hypereosinophilic syndrome in clinical trials; however, the efficacy of this medication for EOM treatment remains undefined. STUDY DESIGN: Retrospective, case series, tertiary academic center. METHODS: A retrospective chart review was performed on EOM patients from 2008-2014. A total of 32 patients met the clinical criteria for EOM according to established diagnostic guidelines. Outcomes of all patients with severe, refractory EOM who initiated PEG-IFN therapy are reported. RESULTS: Eight patients were treated with pegylated interferon-α 2a or 2b for refractory EOM. Half of the patients had significant side effects with interferon treatment. Three of these were able to continue at a reduced dosage without side effect reoccurrence, and one patient stopped the medication permanently. Four of eight (50%) patients had a complete clinical response with total resolution of otorrhea and normalization of middle ear mucosa, and were able to discontinue corticosteroid treatment. Two patients attempted to stop PEG-IFN therapy after prolonged symptom remission and had recurrent otorrhea. Both patients had symptom resolution after PEG-IFN reinitiation. CONCLUSIONS: These data demonstrate that pegylated interferon-α 2a and 2b therapy may benefit patients with severe, refractory EOM. Further larger studies with long-term follow-up are required to validate these early but promising results. LEVEL OF EVIDENCE: 4. Laryngoscope, 127:1208-1216, 2017.

Assessment of Scaffolding Properties for Chondrogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells in Nasal Reconstruction.

IMPORTANCE: Nasal reconstruction in patients who are missing a significant amount of structural nasal support remains a difficult challenge. One challenge is the deficiency of cartilage left within the nose as a consequence of rhinectomy or a midline destructive disease. Historically, the standard donor source for large quantities of native cartilage has been costal cartilage. OBJECTIVE: To enable the development of protocols for new mesenchymal stem cell technologies as alternative procedures with reduced donor site morbidity, risk of infection and extrusion. DESIGN, SETTING, AND MATERIALS: We examined 6 popular scaffold materials in current practice in terms of their biodegradability in tissue culture, effect on adipose-derived mesenchymal stem cell growth, and chondrogenic fate commitment. Various biomaterials of matching size, porosity, and fiber alignment were synthesized by electrospinning and overlaid with rabbit adipose-derived mesenchymal cells in media supplemented or not with chondrogenic factors. Experiments were performed in vitro using as end points biomarkers for cell growth and chondrogenic differentiation. Polydioxanone (PDO), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), PHBV-polycaprolactone, poly(L-lactide-co-caprolactone), poly(lactic-co-glycolic acid), and polystyrene scaffolds of 60% to 70% porosity and random fiber alignment were coated with poly(L)-lysine/laminin to promote cell adhesion and incubated for 28 days with 2.5 to 3.5 × 105 rabbit adipose mesenchymal cells. MAIN OUTCOMES AND MEASURES: Cell growth was measured by fluorometric DNA quantitation and chondrogenic differentiation of stem cells by spectrophotometric sulfated glycosaminoglycan (sGAG) assay. Microscopic visualization of cell growth and matrix deposition on formalin-fixed, paraffin-embedded tissue sections was performed, respectively, with nuclear fast red and Alcian blue. RESULTS: Of 6 scaffold materials tested using rabbit apidose mesenchymal cells, uncoated scaffolds promoted limited cell adhesion but coating with poly(L)-lysine/laminin enabled efficient cell saturation of scaffold surfaces, albeit with limited involvement of scaffold interiors. Similar growth rates were observed under these conditions, based on DNA content analysis. However, PDO and PHBV/PCL scaffolds supported chondrogenic fate commitment better than other materials, based on soluble sGAG analysis and microscopic observation of chondrogenic matrix deposition. The mean (SD) sGAG scaffold values expressed as fold increase over control were PDO, 2.26 (0.88), PHBV/PCL, 2.09 (0.83), PLCL, 1.36 (0.39), PLGA, 1.34 (0.77), PHBV, 1.07 (0.31), and PS, 0.38 (0.14). CONCLUSIONS AND RELEVANCE: These results establish materials, reagents, and protocols for tissue engineering for nasal reconstruction using single-layer, chondrogenically differentiated, adipose-derived mesenchymal stem cells. Stackable, scaffold-supported, multisheet bioengineered tissue may be generated using these protocols. LEVEL OF EVIDENCE: NA.

Human Adipose-Derived Mesenchymal Stem Cells for Osseous Rehabilitation of Induced Osteoradionecrosis: A Rodent Model.

Objective Human adipose-derived mesenchymal stem cells (ADSCs) were used to rehabilitate bone damaged by osteoradionecrosis (ORN) in an established animal model. Study Design Prospective animal study. Setting Academic department laboratory. Subjects and Methods After institutional review board and Institutional Animal Care and Use Committee approval, 24 athymic nude rats were divided into 5 groups: 4 groups irradiated (20 Gy) by brachytherapy catheter placed at the left hemimandible and 1 mock irradiation control (n = 4). For all groups, ORN was initiated by extraction of the central molar 1 week later. After 28 days, animals (n = 5/group) received injection at the extraction site with saline (SAL), ADSCs, platelet-rich plasma and collagen (PRP/COL), or ADSCs + PRP/COL. Rats were sacrificed 28 days later and their mandibles harvested for histopathology analysis (osteoblasts, osteoclasts, and fibrosis) and bone volume measurement using 3-dimensional micro-computed tomography. Results All but 1 rat survived the experiment period (23/24). Radiographic and histological analysis revealed 60% bone loss in the SAL group compared with the nonirradiated control. Injection of ADSCs increased jaw region bone volume by up to 36% ( P < .01). All experimental groups (ADSC, PRP/COL, and ADSC + PRP/COL) showed dramatically decreased osteoclast counts ( P < .001) while injection of PRP/COL with or without ADSCs increased osteoblasts. Increased fibrosis was observed after ADSC injection ( P < .05). Conclusion The application of human ADSCs to an induced mandibular osteoradionecrosis model in athymic rats results in increased deposition or preservation of bone, demonstrated both histologically and radiographically. This offers an encouraging possible treatment option for translational research in this difficult disease.

B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma.

In this report, we demonstrate that B7-H1, a B7 family molecule implicated in tumor immune evasion, is constitutively expressed on 66% of freshly isolated squamous cell carcinomas of the head and neck (SCCHN). To define the potential impact of tumor-associated B7-H1 on immunotherapy, the B7-H1-negative mouse SCC line, SCCVII, was transfected to express B7-H1. Although all of the animals succumbed to B7-H1/SCCVII tumors even after adoptive T-cell immunotherapy, the infusion of B7-H1 blocking monoclonal antibody with activated T cells cured 60% of animals. These data support B7-H1 blockade as a new approach to enhance the efficacy of T-cell immunotherapy.

An Athymic Rat Model for Mandibular Osteoradionecrosis Allowing for Direct Translation of Regenerative Treatments.

OBJECTIVE: We aim to create a model of mandibular osteoradionecrosis in athymic rats. Athymic rats provide an immunosuppressed environment whereby human stem cells and biomaterials can be used to investigate regenerative solutions for osteoradionecrosis, bridging the gap between in vivo testing and clinical application. STUDY DESIGN: Prospective animal study. SETTING: Academic otolaryngology department laboratory. SUBJECTS AND METHODS: After Institutional Animal Care and Use Committee approval, 10 athymic nude rats were divided into 2 groups. The experimental group (n = 6) underwent irradiation (20 Gy), while the control group (n = 4) underwent sham irradiation catheter placement only. All 10 rats underwent extraction of the second mandibular molar 7 days later. The rats were sacrificed 28 days after dental extraction, and their mandibles were harvested. The mandibles were examined with histologic analysis and bone volume analysis based on 3-dimensional micro-computed tomography. RESULTS: All 10 rats survived the experiment period. Radiographic and histologic analysis revealed decreased bone formation in the experimental group compared with the control group. Jaw region volume ratio was 0.83 for the experimental group versus 0.97 in the control group (P = .003). The region-of-interest volume ratio was 0.75 in the experimental group and 0.97 in the control group (P = .005). Histologically, there were increased osteoclasts (P = .02) and decreased osteoblasts (P = .001) as well as increased fibrosis in the experimental group versus the control group. CONCLUSION: Mandibular osteoradionecrosis can be effectively and reproducibly produced in an athymic rat model. This will allow further research to study regenerative medicine in an athymic rat model.

Inhibition of MEK pathway in vestibular schwannoma cell culture.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to evaluate the Ras GTPase (Ras) to extracellular signal-regulated kinase (ERK) pathway in vestibular schwannoma (VS) cell cultures and patient excised schwannoma tumors. Mitogen-activated protein kinase kinase (MEK) inhibitor CI-1040 (PD184352) was utilized to evaluate the effect of specific MEK inhibition on benign schwannoma cell culture proliferation and apoptosis. STUDY DESIGN: Prospective evaluation of human schwannoma cell lines and tumors. METHODS: Western blotting was completed with phospho-antibodies for proteins in the Ras-ERK pathway. Increasing concentrations of CI-1040 were utilized in schwannoma cell cultures to evaluate cell proliferation and apoptosis. Proliferation was measured with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation assay, and apoptosis was monitored with flow cytometry of annexin V/propidium iodide-stained cells. RESULTS: The most consistent Ras-ERK pathway alterations were found in phospho-MEK and ERK. Phospho-MEK was not overexpressed in the schwannoma cell lines, but six out of 10 VS showed significant increases compared to benign Schwann cell controls. Similarly, nine of 10 VS tumors showed increased phospho-ERK expression. CI-1040 showed significantly reduced schwannoma cell proliferation at the 50 and 100 µM (IC(50) 20 µM and 30 µM) concentrations when compared to carrier only controls in two out three schwannoma cell lines. The remaining schwannoma cell line was relatively refractory to the antiproliferative effects of CI-1040 at doses up to 100 µM (IC(50) 58 µM). Cumulative data of four separate schwannoma cell lines demonstrated that apoptosis was increased in treated schwannoma cells at CI-1040 concentrations of 50 and 100 µM at 72 hours. CONCLUSIONS: There is overexpression of phosphorylated (activated) proteins in the Ras-ERK pathway in schwannoma cultures and tumors as compared to benign human Schwann cell culture controls. MEK inhibitor, CI-1040, created significantly decreased schwannoma cell proliferation and increased apoptosis in cell culture. These data justify the use of MEK inhibitors in animal treatment studies of VS.

B7-H1 expression in vestibular schwannomas.

HYPOTHESIS: B7-H1 is expressed in vestibular schwannomas. BACKGROUND: Little is known about how benign human vestibular schwannomas interact with antibody-mediated or cell-mediated immunity. We report on the aberrant expression of a novel T-cell coregulatory molecule, B7 homolog 1 (B7-H1), in vestibular schwannomas and discuss the implications of B7-H1 expression and tumor aggressiveness and a potential regulator of B7-H1 expression. METHODS: Immunohistochemical staining for B7-H1, CD8+, CD3+, and CD4+ lymphocytes were performed on 48 fresh-frozen vestibular schwannoma tissue specimens. A clinical review of patient presenting symptoms and tumor characteristics was performed. Real-time polymerase chain reaction was used to determine if there was differential expression of B7-H1 messenger RNA and microRNA-513, a known regulator of B7-H1, in several strongly positive and negative B7-H1 vestibular schwannomas. RESULTS: Nine (19%) of 48 tumors were negative, 23 (48%) tumors were 1+ mildly positive (<20% section area), and 16 (33%) stained 2+ strongly positive (>or=20% section area) for B7-H1. The average number of CD8 cells per high-power field was 2.1 for positive-staining tumors and 1.0 for negative tumors (p = 0.16). Failure of tumor control with stereotactic radiation (p = 0.029) was significantly greater in the strongly positive B7-H1 tumors. Real-time polymerase chain reaction did not show significant differential expression of microRNA-513 (p = 0.62) or B7-H1 messenger RNA (p = 0.35) between the tumors showing strong and negative immunohistochemical staining for B7-H1 protein. CONCLUSION: Vestibular schwannoma tumors express B7-H1, which has been associated with immune tolerance and adverse disease characteristics in several malignancies. Growing tumors that were surgically removed after failed stereotactic radiation therapy were significantly more likely to strongly express B7-H1 protein, which lends some credibility to the hypothesis that immuno-evasion may play some role in their continued growth. Although clinical trends were seen, greater statistical power is required to evaluate whether B7-H1 expression correlates with more aggressive tumor growth or poorer hearing class. B7-H1 seems to be expressed in equal amounts at the RNA level in all vestibular schwannoma tumors that suggests that differential protein expression is occurring at the posttranscriptional level. However, microRNA-513 does not regulate B7-H1 protein expression in these tumors.

Bioluminescent imaging of intracranial vestibular schwannoma xenografts in NOD/SCID mice.

HYPOTHESIS: Intracranial vestibular schwannoma xenografts can be successfully established and followed with bioluminescent imaging (BLI). BACKGROUND: Transgenic and xenograft mouse models of vestibular schwannomas have been previously reported in the literature. However, none of these models replicate the intracranial location of these tumors to reflect the human disease. Additionally, traditional imaging methods (magnetic resonance imaging, computed tomography) for following tumor engraftment and growth are expensive and time consuming. BLI has been successfully used to longitudinally follow tumor treatment responses in a noninvasive manner. BLI's lower cost and labor demands make this a more feasible approach for tumor monitoring in studies involving large numbers of mice. METHODS: Patient excised vestibular schwannomas were cultured and transduced with firefly luciferase expressing lentivirus. One million cells were stereotactically injected into the right caudate nucleus of 21 nonobese diabetic/severe combined immunodeficient mice. Schwannoma engraftment and growth was prospectively followed for 30 weeks after injection with BLI. After animal sacrifice, the presence of human tumor cells was confirmed with fluorescent in situ hybridization. RESULTS: Eight (38%) of 21 mice successfully engrafted the schwannoma cells. All of these mice were generated from 4 (67%) of the 6 patient excised tumors. These 8 mice could be differentiated from the nonengrafted mice at 21 weeks. The engrafted group emitted BLI of greater than 100,000 photons/s (range, 142,478-3,106,300 photons/s; average, 618,740 photons/s), whereas the nonengrafted group were all under 100,000 photons/s (range, 0-76,010 photons/s; average, 10,737 photons/s) (p < 0.001). Fluorescent in situ hybridization analysis confirmed the presence of viable human schwannoma cells in much greater numbers in those mice with stable or growing tumors compared with those whose tumors regressed. CONCLUSION: We have successfully established an intracranial schwannoma xenograft model that can be followed with noninvasive BLI. We hope to use this model for in vivo testing of schwannoma tumor therapies.