Swallowing rehabilitation of patients undergoing T-tube implantation for the treatment of laryngotracheal stenosis.

OBJECTIVE: Patients with laryngotracheal stenosis (LTS) often have dysphagia after laryngotracheal reconstruction with T-tube insertion, which affects the quality of life. The purpose of this study is to observe the effect of swallowing rehabilitation therapy on the improvement of quality of life in patients of otolaryngology-head and neck surgery with dysphagia undergoing T-tube implantation treatment through longitudinal study. METHODS: Thirty-eight patients with LTS who experienced dysphagia after laryngotracheal reconstruction and T-tube implantation were recruited. All patients received swallowing rehabilitation therapy. The assessment of swallowing function was performed using the 10-item Eating Assessment Tool (EAT-10), the 30 mL water swallow test (WST), and flexible endoscopic evaluation of swallow (FEES). RESULTS: After swallowing rehabilitation therapy, timing of swallowing, grade of dysphagia, performance on FEES and 30 mL WST, and EAT-10 score all improved. Thirty-eight patients successfully transitioned to oral feeding and were able to remove their nasogastric tubes without experiencing any complications, including aspiration pneumonia. CONCLUSION: For patients with LTS who experienced dysphagia after laryngotracheal reconstruction and T-tube implantation, swallowing rehabilitation therapy could improve swallowing function of the patients, so as to reduce the potential harm caused by the pain and complications of surgery experienced by patients.

Silver(I)-catalyzed highly para-selective phosphonation of 2-aryloxazolines.

A silver-catalyzed phosphonation of 2-aryloxazolines has been accomplished. This protocol provides highly regioselective access to para-phosphonation products with good functional group tolerance and moderate to good yields via cross-dehydrogenation coupling. Mechanistic studies have shown that para-phosphonation products are obtained via a radical pathway. Furthermore, the directing oxazoline group in the para-phosphonation products is removable and can be converted to benzoic esters.

Visible-Light-Mediated Bimetal-Catalyzed meta-Alkylation of Arenes.

A mild approach to the visible-light-mediated bimetal-catalyzed meta-alkylation of arenes has been accomplished. The regioselective meta-alkylation is realized by a bimetallic ruthenium-palladium system. Ruthenium acts as a catalyst for the directing effect and as a photosensitizer, while the cocatalyst palladium behaves as a catalyst for the generation of fluoroalkyl radicals. This reaction not only is suitable for two-component meta-fluoroalkylation of arenes but can also be extended to three-component reactions to achieve bifunctionalization of olefins.

Visible-Light-Promoted C4-Selective Phosphorylation of Pyridine and Quinoline Derivatives.

The visible-light-promoted C4-selective phosphorylation of unprefunctionalized pyridine and quinoline derivatives has been accomplished. This Minisci-type protocol provides highly regioselective access to C4-phosphorylation products with good functional group tolerance and moderate to good yields via cross-dehydrogenation coupling under mild conditions. Mechanistic studies have shown that the C4-phosphorylation products are obtained via a radical pathway.

Palladium-catalyzed decarboxylative ortho-amidation of O-methyl ketoximes with oxamic acids.

The first palladium-catalyzed ortho-amidation of ketoximes has been developed with readily available, easy to handle and environment-friendly N,N-disubstituted oxamic acids as the amidation sources. When N-monosubstituted oxamic acids are used as the substrates, the formed ortho-amidated ketoximes undergo further intramolecular cyclization to provide 3-methyleneisoindolinones.

BINOL derivatives with aggression-induced emission.

As fluorescent probes, the small Stokes shift and ACQ effect limit the application of BINOL derivatives. Herein, a new series of BINOL derivatives were synthesized which could be turned from ACQ to AIE fluorophores by changing the electron withdrawing group. Among these compounds, BIN-COP exhibits an obvious AIE property with low cytotoxicity. The bioimaging performance indicated that the designed fluorophores could be successfully used for bioimaging.

[Study on decellularized laryngeal scaffold in dogs].

OBJECTIVE: To explore the survivorship and character of decellularized laryngeal scaffold in pectoralis major muscle flap in canine. METHODS: Eighteen donor larynx in experimental group were decellularized by perfusing sodium dodecyl sulphate. Three of them were used to detect the character of histology. The other fifteen ones were embedded in right pectoralis major muscle flap of acceptor canine. Donor larynx in control group were not perfused. Other experimental procedure was the same as experimental group. The specimens were harvested at two weeks, one month and two months after operation, respectively. Macroscopic view, histological examination and trypan blue staining were performed in the experimental group and control group. RESULTS: The size of the specimens decreased remarkably into disappearance in control group, there was statistical significance between the experimental group and the control group (which used least significant difference t test P < 0.05). There was only little neutrophils and lymphocytes infiltrating around the laryngeal scaffold at 2 weeks in the experimental group. One month after operation, loose connective tissue begin to form around the laryngeal scaffold. After two months of transplantation, the connective tissue became thicker and the number of blood vessels increased than before. There was a large number of lymphocytes and neutrophil infiltration around the laryngeal specimens in the control group at 2nd week. The perichondrium in the control group was damaged at one month post operation. The cartilage cells could not be detected two months after surgery. The survival rate of cartilage cell between experimental group (86.8% ± 3.2%) and the control group (88.6% ± 3.1%) did not show statistical significance before implantation (χ(2) = 0.19, P > 0.05). The survival rate of cartilage cell decreased insignificantly in experimental group while the survival rate declined obviously in the control group at two weeks and one month after operation, the difference had statistical significance (χ(2) were respectively 5.52 and 20.55, P were respectively < 0.05 and < 0.01), the survival rate of cartilage cell in experimental group was (65.8% ± 2.6%) at two months after operation, while the cartilage cell all disappeared in control group. CONCLUSIONS: Perfused decellularation technique can construct a low immunogenicity laryngeal cartilage scaffold which can survive in the chest muscle package and establish a good blood supplement. The decellularized laryngeal scaffold could be used as a biological scaffold for whole laryngeal reconstruction.

[Effects of dexamethasone on IL-13-induced mCLCA3 and Muc5ac expressions in nasal mucosa of rats].

AIM: To study the effects of dexamethasone on the IL-13-induced mCLCA3 and Muc5ac expressions in nasal mucosa of rats, and the role in mucus secretion of allergic rhinitis. METHODS: Thirty SD rats were randomly divided into control group, IL-13 group and dexamethasone group. Expressions of mCLCA3 mRNA and Muc5ac protein in nasal mucosa were detected by RT-PCR and immunohistochemistry, respectively. RESULTS: No mCLCA3 mRNA expression in the nasal mucosa was detected in the control group, while it was 0.319±0.121 in the IL-13 group (P<0.05 vs control group) and 0.144±0.105 in the dexamethasone group (P<0.05 vs IL-13 group). The expression of Muc5ac protein increased in the IL-13 group (1.389±0.499) as compared with the control group (0.300±0.145, P<0.05). After treatment with dexamethasone, the expression of Muc5ac protein was notably lower (0.901±0.390) than that in IL-13 group (P<0.05). CONCLUSION: IL-13 up-regulates the expressions of mCLCA3 mRNA and Muc5ac protein in nasal mucosa, which may play a pivotal role in the mucus overproduction of nasal mucosa. Dexamethasone substantially down-regulates the expressions of mCLCA3 mRNA and Muc5ac protein, which may have an inhibiting effect on mucus overproduction of nasal mucosa.

MicroRNA-203 leads to G1 phase cell cycle arrest in laryngeal carcinoma cells by directly targeting survivin.

Previous studies have shown that miR-203 acts as a tumor-suppressive microRNA in various cancers, but its roles in laryngeal carcinoma are still contradicted. Here, we found that miR-203 inhibited the growth of laryngeal cancer cells and survivin was a direct target of miR-203. Moreover, silencing of survivin recapitulated the effect of miR-203 on cell cycle progression, whereas overexpression of survivin reversed this effect. Additionally, qRT-PCR showed the reciprocal relationship between miR-203 and survivin in laryngeal cancer tissues. These findings indicate that miR-203 inhibits the proliferation of laryngeal carcinoma cells by directly targeting survivin, suggesting its application in anti-cancer therapeutics.

[Biomechanical study on decellularized laryngeal scaffold in dogs].

OBJECTIVE: To evaluate the biomechanical characteristics of the decellularized laryngeal scaffold. METHODS: Ten Chinese adult dogs were randomly divided into two groups: perfusion group (n = 5) and control group (n = 5). The acellular larynx scaffold was obtained from dogs through cranial thyroid arteries perfusion with detergents. Comparative examinations were performed by the macroscopic view, histological view (hematoxylin and eosin stain, Alcian blue stain and Masson stain), scanning electron microscope (SEM) and biomechanical properties between perfusion group and control group. RESULTS: Macroscopic view showed that the decellularized laryngeal scaffold appeared pale asphyxia. HE stain indicated that there were little acellular traces of muscle and mucosa. Alcian blue stain, Masson stain and scanning electron microscope (SEM) suggested that there were no obvious changes about glycosaminoglycan and collagen. The compressive modulus of thyroid cartilage was (1.06 ± 0.07) MPa (x(-) ± s) in experimental groups and (1.15 ± 0.11) MPa in control group, showing no significant difference (t = 1.424, P > 0.05), neither in compressive modulus of annular cartilage (1.68 ± 0.11) MPa in experimental groups and (1.67 ± 0.09) MPa in control group (t = 0.185, P > 0.05). The tensile strength of thyroid cartilage between experimental (5.74 ± 0.88) MPa and control groups (6.18 ± 1.33) MPa did not have the statistical significance (t = 0.627, P > 0.05). CONCLUSION: These results indicate that perfusion method can construct a perfect biomechanical acellular larynx scaffold which could be a better selection for laryngeal reconstruction with tissue engineering method.

Reconstruction of tracheal wall defect with a mesh patch of nickel-titanium shape-memory alloy.

OBJECTIVES: We explored the feasibility of reconstructing tracheal wall defects with a mesh patch fashioned from a nickel-titanium shape-memory alloy. METHODS: A tracheal wall defect was first constructed surgically by resecting the anterior half of the tracheal wall between the second and sixth tracheal rings. The defect was reconstructed in 8 experimental animals by replacing the resected tracheal mucosa and tracheal cartilage with a pedicle skin flap, which was then enclosed in the mesh patch. In 4 control animals, only a pedicle skin flap with strap muscles was used in the reconstruction procedure. The performance of the animals was observed after surgery. At the end of the experiments, the reconstructed segment was harvested for anatomic evaluation. RESULTS: In the experimental group, 1 animal died 5 days after the operation. Endoscopic and anatomic examination of the 7 animals that survived the observation period showed that the reconstructed trachea was stable, with sufficient airway space for breathing. All 4 control animals died after the operation. After observing successful completion of this operation in animals, we successfully used this method to repair a tracheal wall defect in a human victim of a traffic accident. CONCLUSIONS: Tracheal defects can be successfully reconstructed by use of a mesh patch of nickel-titanium shape-memory alloy as an extraluminal stent--a method that avoids complications associated with intraluminal stents.

[Immunological study on decellularized whole laryngeal scaffold].

OBJECTIVE: To evaluate the immunogenicity of the decellularized laryngeal scaffold. METHODS: Twelve perfused, decellularized laryngeal scaffolds were obtained from rabbits through common carotid artery perfusion with detergents. The twelve decellularized laryngeal scaffolds and the twelve fresh larynxes were then implanted in para-laryngeal muscles of rabbits and harvested after two weeks, four weeks, twelve weeks and twenty-four weeks, respectively. Macroscopic view, histological examination and lymphocyte infiltration test were performed. RESULTS: The decellularized larynxes were implanted and preserved the laryngeal extracellular matrix and laryngeal architecture. The decellularized larynx did not show obvious immunological rejection after implanted into the para-laryngeal muscles of the recipient rabbits. The volume of implanted larynx became smaller but retained cartilage scaffold. The larynxes in the control group presented the serious immunological rejection and the majority tissues of the larynxes were disintegrated and substituted by the fibrous connective tissues after four weeks. The peripheral tissues were damaged and necrotic at different degrees. The quantity of the lymphocyte infiltration in the control group was higher than that in the experiment group and the result had the statistical significance (P < 0.01). CONCLUSIONS: Perfused, decellularized technique can construct a low immune laryngeal cartilage scaffold which could be a satisfactory material for laryngeal repair.

[A preliminary study on the preparation of perfusion-decellularized laryngeal scaffold and the feasibility of laryngeal muscle reconstruction].

OBJECTIVE: To prepare a decellularized whole laryngeal scaffold by utilizing a perfusion-decellularized technique, reseed cells on it, and construct decellularized laryngeal muscles. METHODS: Perfusion decellularized larynxes were obtained by common carotid arterious perfusion with detergents. Then they were performed by macroscopic view, histological examination, scanning electron microscopy (SEM) and cartilage viability. Decellularized laryngeal scaffold were then reseeded with inducted mesenchymal stem cells (MSCs). Composites were transferred into greater omentums of rabbits after one day's adherence and harvested after eight weeks. Macroscopic view, histological examination and immunohistochemistry were performed. RESULTS: Perfusion larynxes became transparent after two hours. Histology and SEM indicated that perfusion method showed better decullularized effect. More vintages and collagen fibers but no intact cell or nuclei were retained in the decellularized matrix. Porosity measured by Image pro plus 6.0 was 80.4% +/- 3.2% (x +/- s). Chondrocyte vitality assay indicated chondrocyte vitality rate in the perfusion group was 86.9% +/- 1.5%. After eight weeks, vascularization formed and integrated cartilage frameworks still remained. Histological examination could clearly show the presence of muscle bundles and vessels. Immunohistochemical examination indicated that sarcomeric-alpha actin expressed positively in corresponding areas. CONCLUSIONS: It is feasible to reseed MSCs into the decellularized laryngeal muscle matrix for constructing tissue-engineered laryngeal muscles. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.

[Experimental study on tracheal reconstruction using porous titanium rings and free skin flap].

OBJECTIVE: To investigate the feasibility and efficacy of the cervical tracheal reconstruction using porous titanium rings and free skin flap. METHODS: Twelve adult mongrel dogs were divided randomly into group I and group lI. A segment of cervical trachea (25 mm, 4 rings, about 2/3 circumference) was resected and a rectangular free skin flap was harvested from abdomen. The flap was sutured to the defect part and supported with two porous titanium rings (group I) or without (group II ). X ray and fiberscopic examinations were performed at the end of the first and the sixth months postoperatively. After six months the dogs were sacrificed and the grafts were examined macroscopically and microscopically. RESULTS: In group I, one dog was sacrificed for wound infection and skin flap necrosis with deflexion of titanium rings in the fifth day postoperatively. The other 5 of 6 survived until the end of six months. X-ray examination showed titanium rings were fastened well without displacement or deformity. Through fiberscopy, the trachea luminal patency was maintained well without stricture, shrinkage or necrosis. Histologic examination showed most of the inner surface of the flap was covered with ciliated columnar epithelium. In group II, 3 of 6 dogs died of suffocation within 24 hours postoperatively. The remaining 3 dogs survived from 7 to 16 days with dyspnea and fiberscopic examination showed narrowed trachea lumens. CONCLUSIONS: Porous titanium rings could recreate the framework for cervical tracheal reconstruction using free skin flap and would be one of the options for tracheal reconstruction.

[Preliminary study of primarily cultured C57 articular cartilage transfected with plasmid IDO-EGFP by lipofectamine].

AIM: To determine the transfection efficiency and transient expression of pIDO-EGFP gene in primarily cultured C57 articular cartilage of mice, and to establish a transfection method of the primarily cultured articular cartilage in mice. METHODS: Plasmid IDO-EGFP was amplified in Escherichia coli. The primarily cultured mouse chondrocytes which were initially obtained from articular cartilage were cultured in vitro and transfected with pIDO-EGFP by lipofectamine2000 reagent under optimized condition. Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscopy (LSCM), and transfection efficiency was determined by flow cytometry. RESULTS: There was obvious expression of EGFP at 24 h after transfection. The transfection efficiency of pIDO-EGFP into primarily cultured mouse chondrocytes reached 36.43% at 48 hours and the transfection did not affect the process of cell adherence. CONCLUSION: IDO gene has been successfully transfected into primarily cultured chondrocytes by means of lipofectamine2000 reagent and the chondrocytes can survive in vitro. Satisfactory efficiency of transient transfection can be reached under optimized condition, which will provide a basis for gene introduction and modification of tissue engineered cartilage.

[Anteroposterior cricoid split interposition grafting for severe glottic and subglottic stenosis].

OBJECTIVE: To study the effect of anterior and posterior cricoid splitting interposition grafting for severe glottic and subglottic stenosis. METHODS: This is a retrospective study, from 1991 to 2001 years, 25 patients (male 15, female 10, aged 9 to 46 years) with severe glottic and subglottic stenosis were operated with anterior and posterior cricoid splitting interposition grafting method at Tangdu Hospital. All of 25 patients were tracheostomy dependent before reconstruction. 19 patients had previously undergone 1 to 7 (average 2) surgical procedures. The surgical technique consisted of laryngotracheostomy, cricoid lamina midline vertical incision; rib cartilage graft (17 cases), muscular fasciae, perichondrium or split-thickness skin graft (15 cases), pedicle arytenoid cartilage graft (2 cases) and thyroid cartilage graft (1 case) interposition and silicon T-tube stenting for 3 to 6 months. RESULTS: Twenty-four patients (96%) were successfully decannulated and got an effective phonation. One patient failed decannulation. The follow-up period ranged from 1 to 10 years. All of the 24 patients had a stable airway and effective phonation. CONCLUSIONS: The anteroposterior cricoid split interposition graft technique was a safe and effective method for the treatment of severe glottic and subglottic stenosis. Careful split of the cricoid, avoiding injury of esophageal musculature, careful hemostasis, a tight suture graft and using stent were the keys of successful operation.

[Experimental research using human bone marrow mesenchymal stem cells as the seed cells for bone and cartilage tissue engineering].

AIM: To investigate the feasibility of human bone marrow mesenchymal stem cells(hMSCs) as the seed cells for bone and cartilage tissue engineering. METHODS: Purified hMSCs were cultured in-vitro and induced to differentiate into osteoblasts and chondrocytes. Cellular morphologies were observed under inverted and electron microscopes. The specific markers of the osteoblasts and chondrocytes were detected by histochemical staining, immunohistochemical staining, and RT-PCR. RESULTS: After the hMSCs were passaged for 15 generations, the choractenistic morphology and cell surface antigens of hMSCs remained unchanged. The level of alkaline phosphatase(ALP) in the culture supernatant of the osteoinduction groups was higher than those in the control groups (P<0.05). The morphology of the cells in the osteoinduction and chondroinduction groups changed from spindle-shaped cells into polygon-shaped cells. A large number of the dilated rough endoplasmic reticulua, Golgi apparatus and mitochondria could be seen under transmission electron microscope. Calcium deposition was detected on the surfaces of the hMSCs after osteoinduction. Collagen(COL)-like processes were detected under scanning electron microscope. The staining of the ALP, calcium nudis, COL-I and osteocalcin(OC) were positive, and expressions of the COL-I and OC mRNAs were detected after osteoinduction. The expression of COL-II was detected by immunohistochemical staining and RT-PCR and a lot of the metachromatic-staining matrix around the cells was observed with toluidine blue staining after chondroinduction. CONCLUSION: hMSCs from human bone marrow can be purified, expanded and differentiated into osteoblasts and chondrocytes in-vitro, providing an alternative source for bone and cartilage tissue engineering.