Pengcheng Cui

Structural integrity, ECM components and immunogenicity of decellularized laryngeal scaffold with preserved cartilage

Decellularization techniques have been widely used as an alternative strategy for organ reconstruction. However, the compliance of tracheal or laryngeal tissues can be increased during the decellularization process, which might cause postoperative stenosis due to elimination of chondrocytes. The purpose of our study was to construct a decellularized, whole, laryngeal scaffold with preserved chondrocytes using perfusion techniques and to evaluate the immunogenicity of the decellularized scaffold in vitro and in vivo in a rat model. The cellular components and immunogenicity of the scaffold were decellularized after 14 h of perfusion with detergent and 48 h of perfusion with phosphate buffered saline. However, the cartilage was well preserved via histological analysis and a chondrocyte viability test. Compared to the fresh larynx, the decellularized larynx did not show the presence of major histocompatibility complex antigens via immunohistochemical analysis in vitro and no significant immune rejection occurred 12 weeks post-implantation. In conclusion, decellularization via perfusion can achieve a decellularized, whole-laryngeal scaffold with the cell components removed and the cartilage and extra-cellular matrix well preserved. T cell-mediated immune rejection was significantly reduced in decellularized laryngeal cartilaginous scaffolds in vivo.

Tissue-engineered larynx using perfusion-decellularized technique and mesenchymal stem cells in a rabbit model

Abstract Conclusion: Reseeding mesenchymal stem cells (MSCs) into the decellularized laryngeal muscle matrix for construction of a tissue-engineered larynx is feasible. This in vivo maturation in the omentum could be the first step before in situ implantation of the construct. This construct could facilitate a tissue-engineered method for laryngeal reconstruction. Objectives: The extracellular matrix (ECM) and MSCs have been widely used for organ reconstruction. Our study aimed to prepare a soft tissue decellularized laryngeal scaffold with intact laryngeal cartilages utilizing a perfusion-decellularized technique, reseeding cells on it, and then construct a recellularized larynx. Methods: Perfusion-decellularized larynges were obtained from 20 rabbits by perfusion of the common carotid arteries with detergents. Twelve perfused larynges were observed by macroscopic visualization, histological examination, scanning electron microscopy (SEM), and cartilage viability. The remaining eight perfusion constructs were reseeded with induced MSCs aspirated from eight receptor rabbits. Composites were transferred into greater omentums of receptor rabbits after adherence for 1 day in vitro. Rabbit larynges were harvested after 4 weeks and 8 weeks, respectively. Macroscopic visualization, histological examination, and immunohistochemistry were performed. Results: Larynges perfused by sodium dodecyl sulfate became transparent after 2 h of perfusion. Histology and SEM indicated that the perfusion method showed a better decullularized effect. Almost no intact cells or nuclei were found, while more pores and collagen fibers were retained in the decellularized matrix. The chondrocyte vitality assay indicated that chondrocyte vitality was high. Vascularization was clearly seen by 4 weeks and relatively integrated cartilage frameworks remained by 8 weeks. Histological and immunohistochemical examinations clearly showed muscle bundles and vessels.

SIS with tissue-cultured allogenic cartilages patch tracheoplasty in a rabbit model for tracheal defect.

Conclusions: In the rabbit model, small intestinal submucosa (SIS) compounded with tissue-cultured allogenic cartilages appeared to be an efficacious method for the patch repair of partial circumferential tracheal defects instead of autologous grafts. SIS appears to be a safe and promising means of facilitating neovascularization and tissue regeneration. The long-term use of SIS and tissue-cultured allogenic cartilages warrants further investigation. Background: Tracheal defect reparation remains a challenging surgical problem that can require reconstruction using autologous grafts or artificial stents. This study was performed to evaluate the efficacy of SIS, a biocompatible, acellular matrix, compounded with different tissue-cultured allogenic cartilages, in the repair of a critical-size tracheal defect. Materials and methods: A full-thickness defect (4×8 mm) was created in tracheal rings four to six in adult rabbits. A piece of 8-ply SIS sandwiched in thyroid cartilage, auricular cartilage, or without cartilage, respectively (designated experiment 1, 2, or 3, respectively), was sutured to the edges of the defect with interrupted 4-0 polypropylene sutures. In control animals, the defect was closed with lamina praetrachealis. All animals were followed until signs of dyspnea became apparent or for 4 or 12 weeks. After follow-up and euthanasia, the trachea was harvested and prepared for histologic evaluation using conventional techniques. Results: All animals tolerated the procedure well but two animals in group 1 (n=5), three in group 2 (n=5), and one in group 3 (n=5) had stridor after operation and expired within <1 month with different degrees of obstruction. The other animals in these groups and the control animals (n =3) all survived >1 month. Histologically, neovascularization of the patch was noted with moderate inflammation. The surface of the SIS patch was covered with a lining of ciliated epithelial cells. The tissue-cultured allogenic cartilages degraded to some extent.

MicroRNA-24 inhibits growth, induces apoptosis, and reverses radioresistance in laryngeal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein

BackgroundIncreasing evidence indicates that dysregulation of microRNAs is involved in tumor progression and development. The aim of this study was to investigate the expression of microRNA-24 (miR-24) and its function in laryngeal squamous cell carcinoma (LSCC).MethodsQuantitative RT-PCR (qRT-PCR) was used to detect miR-24 expression in LSCC cell lines and tissue samples. MTT, colony formation, and flow cytometry was performed to analyze the effects of miR-24 expression on growth, apoptosis, and radiosensitivity of LSCC cells. Dual-luciferase reporter assays were performed to examine regulation of putative miR-24 targets. Expression of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein, cleaved or total caspase-3, and cleaved or total PARP protein were detected by qRT-PCR and western blotting assays, respectively.ResultsmiR-24 expression levels in LSCC cell lines or tissue were significantly lower than in a normal human keratinocyte cell line or adjacent normal tissues. Functional analyses indicated that re-expression of miR-24 inhibits growth, reduces colony formation, and enhances apoptosis in LSCC cells. In addition, miR-24 upregulation increases LSCC sensitivity to irradiation by enhancing irradiation-induced apoptosis, and luciferase activity indicated that miR-24 binds to the 3′-untranslated region (3′-UTR) of XIAP mRNA. Upregulation of miR-24 inhibits XIAP protein expression in LSCC cells, and silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells. In addition, XIAP mRNA expression significantly increases in LSCC tissues and is inversely correlated with miR-24 expression.ConclusionsOur data suggest that miR-24 inhibits growth, increases apoptosis, and enhances radiosensitivity in LSCC cells by targeting XIAP. Therefore, miR-24 may be a potential molecular target for the treatment of human LSCC.

A model of canine tracheal stenosis induced by radiofrequency cauterization

OBJECTIVE The current methods of management of tracheal stenosis have disadvantages and are controversial, therefore ideal experimental animal models for the further studies are required. The aim of this study was to establish a new model of canine tracheal stenosis by radiofrequency cauterization. METHODS The tracheal cartilage ring was injured by radiofrequency at the level of the sixth tracheal ring in 12 mongrel dogs. A fibrolaryngoscope was used to guide the procedure. The animals were observed after operation and examined after euthanasia. Endoscopic and histological examinations were undertaken to evaluate the progress of stenosis. The degree of stenosis was calculated using the formula: degree of stenosis=(initial lumen area-final lumen area)/initial lumen area × 100%. RESULTS Tracheal stenosis had developed in all dogs by the 21st day post operation. Costal retraction was observed in all dogs after the 18th day post operation. At the end of the study, gross and endoscopic examinations showed that stenosis had been induced to a satisfactory degree and without any complications. The median of the degree of stenosis was 92%, with a range of 84-94%. Histological examination showed that cartilage was damaged and that granulation tissue and collagen fibres had formed. CONCLUSIONS The model of canine tracheal stenosis induced by radiofrequency cauterization is a relatively simple, reliable, and reproducible animal model. This model may be useful in the development of new methods of treatment for tracheal stenosis.

Both Central and Peripheral Auditory Systems Are Involved in Salicylate-Induced Tinnitus in Rats: A Behavioral Study

Objective This study was designed to establish a low dose salicylate-induced tinnitus rat model and to investigate whether central or peripheral auditory system is involved in tinnitus. Methods Lick suppression ratio (R), lick count and lick latency of conditioned rats in salicylate group (120 mg/kg, intraperitoneally) and saline group were first compared. Bilateral auditory nerves were ablated in unconditioned rats and lick count and lick latency were compared before and after ablation. The ablation was then performed in conditioned rats and lick count and lick latency were compared between salicylate group and saline group and between ablated and unablated salicylate groups. Results Both the R value and the lick count in salicylate group were significantly higher than those in saline group and lick latency in salicylate group was significantly shorter than that in saline group. No significant changes were observed in lick count and lick latency before and after ablation. After ablation, lick count and lick latency in salicylate group were significantly higher and shorter respectively than those in saline group, but they were significantly lower and longer respectively than those in unablated salicylate group. Conclusion A low dose of salicylate (120 mg/kg) can induce tinnitus in rats and both central and peripheral auditory systems participate in the generation of salicylate-induced tinnitus.

Management of Severe and Complex Hypopharyngeal and/or Laryngotracheal Stenoses by Various Open Surgical Procedures: A Retrospective Study of Seventeen Patients

Objective: To systematically study various surgical approaches for treating complex hypopharyngeal and/or laryngotracheal stenoses at a variety of sites and levels. Patients and Methods: We retrospectively analyzed the treatment of 17 patients with severe and complex hypopharyngeal and/or laryngotracheal stenosis at various sites and levels of severity. All of the 17 patients initially had a tracheostomy. Thirteen had failed the previous laser lysis and/or dilation treatment. Given the high severity and complexity of stenosis, all of these patients were treated by open surgical reconstruction techniques using repairing grafts (flaps), followed by stenting. Results: Thirteen of 17 patients had successful decannulation 1-8 months post-operation and had stable airway and adequate vocal and swallow function. Two patients with complex hypopharyngeal and esophageal stenosis had unsuccessful decannulation. Follow-up was lost in 1 patient with complex hypopharyngeal and esophageal stenosis and 1 patient with original hypopharyngeal stenosis and recurrent thoracotracheal stenosis. Conclusion: Despite the failure by the regular treatments using laser lysis and/or dilation therapy, severe and complex hypopharyngeal and/or laryngotracheal stenosis may be successfully treated by variable open surgical reconstruction techniques using different grafts (flaps) depending on the site and severity of the stenosis.

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.