Silk fibroin-hydroxyapatite scaffolds promote the proliferation of adipose-derived mesenchymal stem cells by activating the ERK signal.

Adipose-derived mesenchymal stem cell (Ad-MSC) with capacities of releasing trophic factors and chondrogenic differentiation was a promising candidate for tracheal reconstruction. Silk fibroin (SF)- hydroxyapatite (HA) scaffolds were fabricated by the freeze-drying method. And Ad-MSCs were co-cultured on the scaffolds for 14 days in vitro. The role of the SF-HA scaffold in regulating the adhesion, growth, and proliferation of Ad-MSCs, and its potential mechanisms were investigated. The identity of Ad-MSCs was confirmed by cell morphology, surface markers, and differentiation characteristics. Cell proliferation, viability, and morphology were observed via CCK-8, live/dead assay, and scanning electron microscopy (SEM). Gene mRNA and protein levels were examined using quantitative real-time polymerase chain reaction and western blotting, respectively. SF-HA scaffolds showed excellent properties of promoting Ad-MSCs adhesion, growth, and proliferation for at least 14 days. In the CCK-8 assay, the relative OD value of Ad-MSCs cultured on SF-HA scaffolds increased (p < 0.001). Furthermore, live/dead staining showed that the fluorescent coverage increased with time (p < 0.05). SEM also showed that 3 days after inoculation, the coverage of Ad-MSCs on the SF-HA scaffolds was 78.15%, increased to 92.91% on day 7, and reached a peak of 94.38% on day 14. Extracellular signal-regulated kinase (ERK) mRNA and phosphorylated ERK (pERK) protein expression increased at day 3 (p < 0.05), followed by a significant decline at day 7 (p < 0.05). And ERK mRNA expression was positively correlated with Ad-MSCs proliferation (p < 0.05). In summary, the SF-HA scaffold co-cultured with Ad-MSCs is a promising biomaterial for tracheal repair by activating the ERK signal pathway.

High frequency hearing loss may act as a screening index evaluating otolith function in vertigo patients with normal semi-circular canal function.

Objective: To investigate whether otolith dysfunction is related to hearing impairment in vertigo patients with normal semicircular canal function, and to clarify the types of hearing impairment that may be related to otolith organ damage. Methods: The demographic data, pure tone threshold audiometry (PTA) results (air-conduction), data of bithermal and video-head impulse test (vHIT), and vestibular evoked myogenic potential (VEMP) results (reaction threshold, P1-N1 amplitude) of patients with vertigo in outpatient clinic from April 2017 to January 2020 were collected. The clinical records of 51 vertigo patients with normal semicircular canal function were included in this study. Low-frequency, speech-frequency, high-frequency, full-frequency PTA were defined as the average of PTA in different frequency bands, respectively (low: 0.125, 0.25, 0.5 kHz; speech: 0.5, 1, 2 kHz, high: 4, 8 kHz, full 0.125-8 kHz). The correlations between hearing impairment in different frequency bands and otolith function impairment were analyzed. Results: The mean thresholds of 51 patients (102 ears) in low-PTA, speech-PTA, high-PTA, full-PTA were 20.95 ± 6.01, 21.92 ± 6.90, 40.12 ± 17.47, 26.97 ± 8.53 dB nHL, respectively. Among 102 ears, 87 ears (85.3%) could elicit c-VEMP waveforms and 65 ears (63.7%) had o-VEMP waveforms. The mean threshold and P1-N1 amplitude of c-VEMP were 83.10 ± 6.96 dB nHL and 176.79 ± 103.10 uV, while those of o-VEMP were 87.92 ± 5.99 dB nHL and 21.45 ± 32.22 uV. The mean threshold in high-PTA was significantly linearly correlated with c-VEMP threshold (P = 0.01) and P1-N1 amplitude (P = 0.028). There were not significant linear correlations between the mean threshold in each frequency band of PTA and o-VEMP threshold (low-PTA: P = 0.266, speech-PTA: P = 0.33, high-PTA: P = 0.311) or P1-N1 amplitude (low-PTA: P = 0.414, speech-PTA: P = 0.069, high-PTA: P = 0.08). Conclusions: There is a positive linear correlation between saccule dysfunction and high-frequency hearing impairment in vertigo patients with normal semi-circular canal function. High frequency hearing loss can be expected in patients who have saccular damage. It suggests that high frequency hearing loss in PTA may act as a screening index that otolith organ function should be comprehensively evaluated.

Preparation of a novel regenerated silk fibroin-based hydrogel for extrusion bioprinting.

Three-dimensional (3D) bioprinting technology, allowing rapid prototyping and personalized customization, has received much attention in recent years, while regenerated silk fibroin (RSF) has also been widely investigated for its excellent biocompatibility, processibility, and comprehensive mechanical properties. However, due to the difficulty in curing RSF aqueous solution and the tendency of conformational transition of RSF chains under shearing, it is rather complicated to fabricate RSF-based materials with high mechanical strength through extrusion bioprinting. To solve this problem, a printable hydrogel with thixotropy was prepared from regenerated silk fibroin with high-molecular-weight (HMWRSF) combined with a small amount of hydroxypropyl methylcellulose (HPMC) in urea containing aqueous solution. It was found that the introduction of urea could not only vary the solid content of the hydrogel to benefit the mechanical properties of the 3D-bioprinted pre-cured hydrogels or 3D-bioprinted sponges, but also expand the "printable window" of this system. Indeed, the printability and rheological properties could be modulated by varying the solid content, the heating time, the urea/HMWRSF weight ratio, etc. Moreover, the microstructure of nanospheres stacked in these lyophilized 3D-bioprinted sponges was interesting to observe, which indicated the existence of microhydrogels and both "the reversible network" and "the irreversible network" in this HMWRSF-based pre-cured hydrogel. Like other HMWRSF materials fabricated in other ways, these 3D-bioprinted HMWRSF-based sponges exhibited good cytocompatibility for dental pulp mesenchymal stem cells. This work may inspire the design of functional HMWRSF-based materials by regulating the relationship between structure and properties.

Transoral Endoscopic Localization of the Parapharyngeal Internal Carotid Artery.

Objectives To define transoral endoscopic surgical landmarks for the parapharyngeal segment of the internal carotid artery (ppICA) using cadaveric dissection. Methods Ten fresh cadaveric heads were dissected to demonstrate the parapharyngeal space anatomy and course of the ppICA as seen in a transoral approach. Anatomical measurements of the distance between the ppICA and bony landmarks were recorded and analyzed. Results The stylohyoid ligament, styloglossus, and stylopharyngeus could be considered to be the safe anterior boundary of the ppICA in the transoral approach; among them, the styloid ligament was the most rigid tissue. Dissection between the stylopharyngeus muscle and superior pharyngeal constrictor muscle provides direct access to the ppICA. At the level of the skull base, the distance from the root of the styloid process to the lateral margin of the external aperture of the carotid canal on the left side and on the right side was 8.57 ± 1.97 and 8.80 ± 1.21 mm, respectively. At the level of the maxillary tuberosity, the distance from the ppICA to the maxillary tuberosity on the left side and on the right side was 31.48 ± 2.24 and 31.01 ± 2.88 mm, respectively. Conclusion The endoscopic-assisted transoral approach can facilitate exposure of the ppICA. The root of the styloid process, styloid ligament, and maxillary tuberosity are critical landmarks in the identification of the ppICA in the transoral approach.

Porous Three-Dimensional Silk Fibroin Scaffolds for Tracheal Epithelial Regeneration in Vitro and in Vivo.

The regeneration of functional epithelial lining is critical for artificial grafts to repair tracheal defects. Although silk fibroin (SF) scaffolds have been widely studied for biomedical application (e.g., artificial skin), its potential for tracheal substitute and epithelial regeneration is still unknown. In this study, we fabricated porous three-dimensional (3D) silk fibroin scaffolds and cocultured them with primary human tracheobronchial epithelial cells (HBECs) for 21 days in vitro. Examined by scanning electronic microscopy (SEM) and calcein-AM staining with inverted phase contrast microscopy, the SF scaffolds showed excellent properties of promoting cell growth and proliferation for at least 21 days with good viability. In vivo, the porous 3D SF scaffolds (n = 18) were applied to repair a rabbit anterior tracheal defect. In the control group (n = 18), rabbit autologous pedicled trachea wall without epithelium, an ideal tracheal substitute, was implanted in situ. Observing by endoscopy and computed tomography (CT) scan, the repaired airway segment showed no wall collapse, granuloma formation, or stenosis during an 8-week interval in both groups. SEM and histological examination confirmed the airway epithelial growth on the surface of porous SF scaffolds. Both the epithelium repair speed and the epithelial cell differentiation degree in the SF scaffold group were comparable to those in the control group. Neither severe inflammation nor excessive fibrosis occurred in both groups. In summary, the porous 3D SF scaffold is a promising biomaterial for tracheal repair by successfully supporting tracheal wall contour and promoting tracheal epithelial regeneration.

A simple semi-quantitative approach studying the in vivo degradation of regenerated silk fibroin scaffolds with different pore sizes.

The biocompatibility and in vivo degradation rate of biomaterials represent critical control points in the long-term success of scaffolds for tissue restoration. In this study, new three-dimensional (3D) regenerated silk fibroin scaffolds (RSFs) were prepared by the freezing-defrosting procedure, and then were implanted beneath the dorsal skin of rats. This study aims to develop a kinetic semi-quantitative approach to assess in vivo degradation rate and biocompatibility of this kind of RSFs with different pore sizes for the first time, and to evaluate the relationship between the biodegradation and tissue responses by measuring the thickness of residual scaffolds, fibrous capsules and infiltrated tissues through integrated techniques of histology, optical imaging and image analysis. Our results showed that scaffolds with both pore sizes (74.35±10.84µm and 139.23±44.93µm, respectively) were well tolerated by host animals and pore size was found to be the rate limiting factor to the biodegradation in the subcutaneous implantation model. In addition, the biodegradation of RSFs was inflammation-mediated to a certain degree and fibroblasts may play a critical role in this process. Overall, such semi-quantitative approach was demonstrated to be a simple and effective method to assess the in vivo degradation rate, and the prepared RSFs were presented to have promising potential in tissue engineering applications.

Efficacy of silk fibroin-nano silver against Staphylococcus aureus biofilms in a rabbit model of sinusitis.

BACKGROUND: Staphylococcus aureus biofilms contribute significantly to the recalcitrant nature of chronic rhinosinusitis. In previous studies, it has been shown that silk fibroin-nano silver solution can eliminate S. aureus biofilms in vitro, which suggests a potential role of this novel agent in the treatment of biofilm-associated diseases, such as sinusitis. OBJECTIVE: The aim of this study was to investigate the efficacy of silk fibroin-nano silver solution as a topical anti-biofilm agent in a rabbit model of sinusitis. METHODS: Biofilm-associated sinusitis models were established in 24 New Zealand White rabbits by gelatin sponge placement and S. aureus inoculation through a hole drilled into the anterolateral wall of the right maxillary sinus. After 4 weeks, indwelling catheters were placed into the maxillary sinus. Different concentrations of silk fibroin-nano silver solution or normal saline were irrigated slowly into the maxillary sinus via the indwelling catheters. After 7 days of irrigation, the rabbits were sacrificed. The sinus mucosa was harvested and examined for biofilm biomass as well as morphological integrity of the epithelium by scanning electron microscopy. RESULTS: Silk fibroin-nano silver solution was found to be most effective in reducing the biomass of the S. aureus biofilms at a concentration of 384 mg/L, followed by the concentration of 153.6 mg/L, when compared with saline. After treatment with 384 mg/L silk fibroin-nano silver solution, the biofilms were completely eliminated and the injured epithelium was almost restored with regenerated cilia on the surface. CONCLUSION: Silk fibroin-nano silver solution was found to be an effective topical agent against S. aureus biofilms in the rabbit model of sinusitis, and its effect was concentration-dependent.

Normal saline solution nasal-pharyngeal irrigation improves chronic cough associated with allergic rhinitis.

BACKGROUND: Upper airway inflammation is one of the most commonly identified causes of chronic cough, although the underlying mechanism is not clear. This study compared normal saline solution nasal-pharyngeal irrigation (NSNPI) and fluticasone propionate nasal spray (FPNS) treatment for chronic cough associated with allergic rhinitis (AR). METHODS: Patients with suspected AR to house-dust mite were enrolled, and the symptom of cough was assessed by a cough symptom score and the Leicester Cough Questionnaire, and cough response to capsaicin was evaluated. AR was assessed by using the visual analog scale (VAS) and the Mini Juniper Rhinoconjunctivitis Quality of Life Questionnaire (MiniRQLQ). Mediators, including histamine, leukotriene C4, and prostaglandin D2, and the major basic protein from nasal lavage fluid (NLF) were examined. The patients were treated with NSNPI (the NSNPI group) or FPNS (the FPNS group) for 30 days, after which they were reassessed. RESULTS: Forty-five of 50 patients completed this study. The scores of the cough symptom and the Leicester Cough Questionnaire, and the capsaicin cough threshold all improved statistically after NSNPI but did not change after FPNS. There were statistically significant changes in the evaluations of the MiniRQLQ and the mediators, including histamine and leukotriene C4, in the NLF in the NSNPI group. However, significant changes were found in the assessments of VAS, MiniRQLQ, and all above mediators including histamine, leukotriene C4, and prostaglandin D2, and the major basic protein in the NLF of the FPNS group. Furthermore, the assessments of VAS and all the mediators were reduced more in the FPNS group compared with those in the NSNPI group. CONCLUSION: The patients with suspected AR to house-dust mite reported a better relief of the cough symptom after 30 days of treatment with NSNPI compared with that after nasal corticosteroid.

[Diagnostic value of portable monitor device in patients with potential obstructive sleep apnea hypopnea syndrome].

OBJECTIVE: To evaluate the diagnostic value of portable monitor device (PMD) in potential obstructive sleep apnea hypopnea syndrome (OSAHS) patients. METHODS: All patients met the inclusion criteria were asked to finish the questionniar and underwent anthropometric measurements, and then completed polysomnography (PSG) test and PMD test simultaneously. The correlation between AHI-PMD and AHI-PSG, between MinSaO2-PMD and MinSaO2-PSG were analyzed by Spearman analysis. T test was used to compare the correlation coefficient between the two groups; ROC analysis was used to evaluate the sensitivity and specificity of PMD in diagnosis of OSAHS, and got the Cut-off value between moderate and severe OSAHS and mild OSAHS. RESULTS: Through PSG test, of all the 111 cases, including 4 simple snoring cases, accounting for 3.6%, OSAHS patients with 107 cases, accounting for 96.4% which including 11 patients (9.9%) with mild, 17 patients (15.3%) with moderate, 79 patients (71.2%) with severe. The correlation of AHI-PMD and AHI-PSG between moderate and severe OSAHS patients was stronger than simple snoring and mild OSAHS patients. The coefficient test between the two groups was statistically significant (P=0.026). The correlation of MinSaO2-PMD and MinSaO2-PSG was statistically significant (P<0.001), the correlation of MinSaO2-PMD and MinSaO2-PSG between moderate and severe OSAHS group and snoring and mild OSAHS group was not statistically significant (P=0.270). A statistically significant correlation between AHI-PMD and AHI-PSG was found (P<0.001). PMD had a sensitivity and specificity of 96.9% and 86.7%, respectively (AUC=0.990, 95%CI 0.970-1.000). The cut-off value between moderate and severe OSAHS and mild OSAHS was AHI-PMD≥12 times/h. CONCLUSION: PMD had a satisfactory sensitivity and specificity for diagnosing and judging the severity of moderate and severe OSAHS.

The Intervention of CRAC Channels Alleviates Inflammatory Responses in Nasal Polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with Th2-dominant inflammation. However, effective treatments for CRSwNP have not yet been found. This study aimed to investigate the expression of Orai1 in nasal polyps (NP) and the influence on them of the intervention of Ca2+ release-activated Ca2+ (CRAC) channels. MATERIALS AND METHODS: Nasal samples were obtained from normal subjects or subjects with CRSwNP. We studied the distribution of Orai1 protein in NP and normal mucosa (normal group) using immunohistochemistry. These tissues in cultures were then maintained in the absence (control group) or presence of 2-aminoethoxydiphenyl borate (2-APB) for 24 h. Orai1 was examined by means of enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (RT-PCR). The Ca2+ mean fluorescence intensity (MFI) was evaluated by flow cytometry, and the inflammatory mediators, including interleukin (IL)-4, IL-5, IL-13, Dermatophagoides pteronyssinus-specific IgE, leukotriene C4 and eosinophil cation protein in cultures, were analyzed with ELISA and real-time RT-PCR. RESULTS: The expression of Orai1 was localized to the cytoplasmic membrane of inflammatory cells, and upregulated in NP compared to that in the normal group. However, Orai1 protein was decreased in polyp tissues after the 2-APB treatment. The levels of Ca2+ MFI and above inflammatory mediators were also elevated in NP, and reduced after the 2-APB administration compared to those in the control group. CONCLUSIONS: Orai1 and CRAC channels may play a crucial role in NP formation and development, and the 2-APB intervention of Orai1 protein may alleviate inflammatory responses in NP.

In Vitro Treatment with 2-APB Inhibits the Inflammation in Nasal Polyps.

OBJECTIVE: Glucocorticoids are considered the main treatment option for chronic rhinosinusitis with nasal polyps (CRSwNP), but their effect rate ranges from 60.9% to 80%. Novel therapeutic means should be studied. The purpose of this study was to investigate the expression of Orai1 in nasal polyps (NPs) and the influence of intervention of Orai1 on NPs after in vitro treatment of 2-aminoethoxydiphenyl borate (2-APB). STUDY DESIGN: Prospective cross-sectional study. SETTING: University hospital. SUBJECTS AND METHODS: Nasal biopsy samples were obtained from normal subjects or subjects with CRSwNP. We studied the localization of Orai1 protein in NPs by using immunohistochemistry. Then these tissues in cultures were maintained in the absence or presence of dexamethasone (DEX) or 2-APB. Orai1 was examined by Western blot, enzyme-linked immunosorbent assay (ELISA), and real-time reverse transcription-polymerase chain reaction (RT-PCR). Inflammatory mediators including interleukin (IL)-1ß, IL-5, eosinophil cation protein (ECP), leukotriene (LT)C4, interferon (IFN)-γ, and dermatophagoides pteronyssinus (DP)-specific immunoglobulin E (sIgE) as well as mucins (MUCs) including MUC5B and MUC7 in cultures were analyzed with ELISA and real-time RT-PCR. RESULTS: The expression of Orai1 was localized to cytoplasmic membrane of inflammatory cells and submucosal glandular cells and was upregulated in NPs compared with normal nasal mucosa. Orai1 was decreased in NPs after in vitro treatment of 2-APB but not after DEX intervention. The levels of inflammatory mediators and mucins were reduced more after 2-APB treatment when compared with those after DEX treatment. CONCLUSION: Orai1 may play crucial roles in NP formation, and the intervention of Orai1 may inhibit NP development.

A simple animal model of Staphylococcus aureus biofilm in sinusitis.

BACKGROUND: Previous research suggested that the biofilm of Staphylococcus aureus contributes greatly to the recalcitrant nature of chronic rhinosinusitis (CRS). However, the lack of a simple and stable animal model limited further study in this field. The aim of this study was to create a convenient animal model of S. aureus biofilms in the maxillary sinus of rabbit. METHODS: New Zealand white rabbits were used as model animals and incised vertically along the median line of the nasal dorsum to expose the anterolateral wall of maxillary sinus, on which a 1.5-mm-diameter hole was drilled to enter the sinus cavity. Through the hole, a piece of gelatin sponge was inserted and then inoculated bacterial suspension into the maxillary sinus. One to 8 weeks after the surgery, the sinus mucosa were harvested and examined with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. RESULTS: All rabbits tolerated the surgical procedures and had developed sinusitis by the time they were killed. SEM revealed that biofilms were presented in 100% of rabbits who had bacteria infected for ≥2 weeks, during which the ciliated epithelial cells were encapsulated and gradually destroyed. H&E staining revealed morphological changes of the epithelial cells and infiltration of inflammatory cells in the subepithelial layer, which showed a strong correlation with the results of SEM. CONCLUSION: This biofilm model of sinusitis avoids excessive damage to the nasal cavity and sinuses of the rabbits. It may be a desirable animal model for studying the pathogenesis and eradication strategies of bacterial biofilms in sinusitis.

[Pilomatricoma: a retrospective study of 399 cases].

OBJECTIVE: To study the clinical and pathological features of pilomatricoma. METHOD: The authors retrospectively investigated the clinical and pathological materials of 399 patients with pilomatricoma. RESULT: Single lesion occurred in most patients (99%) and 56.39% of them were younger than 30 years. The male-female ratio was 1:1.33. The lesions which sizes average 1.22 cm were commonly emerged in the head, neck, and upper extremity. CONCLUSION: Pilomatricoma is a slowly developed benign cutaneous tumour, but it can aggravate sometimes. It's manifestation is diversed and easily misdiagnosed. Early complete excision is recommended for hard or tenacious nodules on head, neck and upper extremity.

[Morphological observation of amifostine's protective effect on radiation induced early middle ear injury].

OBJECTIVE: To assess the radio-protective effect of amifostine on the middle ear in animal models by observing the morphological changes of middle ear mucosa. METHOD: Adult guinea pigs (n=38) were divided into the radiation group, the radiation-amifostine group and the control group. The first two groups were exposed to total 45 Gy of Gamma radiation which was administered to the right ear of each guinea pig with 3.0 Gy/fraction, 5 times per week, using a cobalt-60 machine. And the radiation+amifostine group was pretreated with the radio protector amifostine 100 mg/kg intra-peritoneally 30 min before each fraction of radiation. Sterile saline was administered intra-peritoneally in radiation group before ear irradiation. Two normal guinea pigs given no treatment were chosen as the control. The tympanic bullas were removed and the mucosae was processed for light microscope and scanning electron microscope examination on the 2nd and 30th days post irradiation to observe mucosa thickness, leukocyte and cilia. RESULT: Light microscope and scanning transmission electron microscope examination showed in radiation groups that the middle ear effusion occurred; the cilia and micro cilia fell off, fused or collapsed; their directions changed; the mucosa became thicker; and leukocytes were also found infiltrating into the mucosa. On the 30th day,the damage was more serious. As comparison, there was no manifest damage of the middle ear mucosa in radiation+amifostine group. The mucosa thickness and the leukocyte quantity of radiation group were obviously higher than that of radiation+amifostine group (P < 0.01). CONCLUSION: The middle ear mucosa was found to change over time after irradiation. Amifostine has a protective effect on radiation induced early middle ear injury.

The relationship between the protective effect of amifostine and decreased intercellular adhesion molecule 1 expression.

PURPOSE: The objective of our study was to characterize the relationship between the protective effect of amifostine and decreased intercellular adhesion molecule 1 (ICAM-1) expression in early-phase, radiation-induced otitis media and to illustrate the possible mechanism of early-phase radiation-induced otitis media. MATERIALS AND METHODS: A comparison study of middle ear tissue was performed by the expression of ICAM-1 and the electron microscope from total 38 guinea pigs. Group A, consisting of 2 pigs, was used as control, and these pigs were not irradiated. Groups B, C, D, and E, consisting of 9 pigs each, were irradiated. Sterile saline was administered intraperitoneally to the pigs in groups B and D before irradiation, and amifostine was administered intraperitoneally as an aqueous solution 30 minutes before irradiation to the pigs in groups C and E. The pigs in groups B and C were killed on the second day after irradiation, and the pigs in groups D and E were killed 30 days after irradiation. RESULTS: Intercellular adhesion molecule 1 was strongly expressed in the middle ear mucosa of the irradiated pigs after a 45-Gy dose of radiation was administered. Enhanced ICAM-1 expression was accompanied by pathomorphologic changes in the middle ear tissue. Scanning electron microscopy demonstrated the changes. Intercellular adhesion molecule 1 expression in the mucosa of the groups killed on the second day was stronger than that in the mucosa of the groups killed 30 days after irradiation. Amifostine protected the middle ear from radiation injury, and we found that the expression of ICAM-1 in the middle ear mucosa was down-regulated. However, slight expression of ICAM-1 remained 30 days after irradiation. CONCLUSIONS: Irradiation increased the expression of ICAM-1 in the middle ear mucosa. Amifostine protected the middle ear from early irradiation injury. There was a relationship between oxygen free radicals derived from irradiation and up-regulation of ICAM-1 expression. Continuous ICAM-1 expression might be related to stenosis of the eustachian tube.