Decellularized tracheal scaffold as a promising 3D scaffold for tissue engineering applications.

Tissue engineering is a science that uses the combination of scaffolds, cells, and active biomolecules to make tissue in order to restore or maintain its function and improve the damaged tissue or even an organ in the laboratory. The purpose of this research was to study the characteristics and biocompatibility of decellularized sheep tracheal scaffolds and also to investigate the differentiation of Adipose-derived stem cells (AD-MSCs) into tracheal cells. After the decellularization of sheep tracheas through the detergent-enzyme method, histological evaluations, measurement of biochemical factors, measurement of DNA amount, and photographing the ultrastructure of the samples by scanning electron microscopy (SEM), they were also evaluated mechanically. Further, In order to check the viability and adhesion of stem cells to the decellularized scaffolds, adipose mesenchymal stem cells were cultured on the scaffolds, and the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay was performed. The expression analysis of the intended genes for the differentiation of mesenchymal stem cells into tracheal cells was evaluated by the real-time PCR method. These results show that the prepared scaffolds are an ideal model for engineering applications, have high biocompatibility, and that the tracheal scaffold provides a suitable environment for the differentiation of ADMSCs. This review provides a basis for future research on tracheal decellularization scaffolds, serves as a suitable model for organ regeneration, and paves the way for their use in clinical medicine.

Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

High-precision targeting workflow for volume electron microscopy.

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

The tracheal system of scutigeromorph centipedes and the evolution of respiratory systems of myriapods.

The tracheal system of scutigeromorph centipedes (Chilopoda) is special, as it consists of dorsally arranged unpaired spiracles. In this study, we investigate the tracheal systems of five different scutigeromorph species. They are strikingly similar to each other but depict unique characters compared to the tracheal systems of pleurostigmophoran centipedes, which has engendered an ongoing debate over a single versus independent origin of tracheal systems in Chilopoda. Up to now, only the respiratory system of Scutigera coleoptrata was investigated intensively using LM-, TEM-, and SEM-techniques. We supplement this with data for species from all three families of Scutigeromorpha. These reveal interspecific differences in atrial width and the shape and branching pattern of the tracheal tubules. Further, we investigated the tracheal system of Scutigera coleoptrata with three additional techniques: light sheet microscopy, microCT and synchrotron radiation based microCT analysis. This set of techniques allows a comparison between fresh versus fixed and dried material. The question of a unique vs. multiple origin of tracheal systems in centipedes and in Myriapoda as a whole is discussed with regard to their structural similarities and differences and the presence of hemocyanin as an oxygen carrier. We used morphological and molecular data and the fossil record to evaluate the alternative hypotheses.

Herb pair of Ephedrae Herba-Armeniacae Semen Amarum alleviates airway injury in asthmatic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedrae Herba (EH, Ephedra sinica Stapf.) and Armeniacae Semen Amarum (ASA, Prunus armeniaca L. var. ansu Maxim.) have been used to treat asthma, cold, fever, and cough in China for thousands of years. AIM OF THE STUDY: In this study, we aimed to investigate the optimal ratio of EH and ASA compatibility (EAC) to reduce airway injury in asthmatic rats and its possible mechanism. METHODS: Rats were sensitized with a mixture of acetylcholine chloride and histamine bisphosphate 1 h before sensitization by intragastric administration of EAC or dexamethasone or saline for 7 days. Subsequently, the ultrastructure of rat airway epithelial tissue changes, apoptosis of the airway epithelial cells, and the expression of mRNA and protein of EGRF and Bcl-2 were detected. RESULTS: Transmission electron microscope: EAC (groups C and E) had the most prominent effect on repairing airway epithelial cells' ultrastructural changes in asthmatic rats. TUNEL: dexamethasone and EAC (groups B、C、E and F) inhibited the apoptosis of airway epithelial cells in asthmatic rats (P < 0.05). In situ hybridization: EAC (group E) inhibited the overexpression of EGFR and Bcl-2 mRNA (P < 0.05).Western Blotting: EAC (groups A、B、C、E and F) inhibited the upregulation of airway epithelial EGFR and Bcl-2 protein expression (P < 0.01). CONCLUSIONS: Our findings indicate that EAC can inhibit abnormal changes in airway epithelial structure and apoptosis of airway epithelial cells, thereby alleviating airway injury. In this study, the best combination of EH and ASA to alleviate airway epithelial injury in asthmatic rats was group E (EH: ASA = 8: 4.5).

CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components.

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.

Amelioration of Cigarette Smoke-Induced Mucus Hypersecretion and Viscosity by Dendrobium officinale Polysaccharides In Vitro and In Vivo.

Chronic obstructive pulmonary disease (COPD), characterized by oxidative stress and inflammation, is one of the leading causes of death worldwide, in which cigarette smoke (CS) is the major risk factor. Dendrobium officinale polysaccharides (DOPs) are the main active ingredients extracted from Dendrobium officinale, which have been reported to have antioxidant and anti-inflammatory activity as well as inhibition of mucin gene expression. This study is aimed at investigating the effect of DOPs on CS-induced mucus hypersecretion and viscosity in vitro and in vivo. For in vitro study, primary normal human bronchial epithelial cells (HBECs) differentiated at the air-liquid interface (ALI) culture for 28 days were stimulated with cigarette smoke medium (CSM) in the absence or presence of various concentrations of DOPs or N-acetylcysteine (NAC) for 24 hours. For in vivo study, male Sprague-Dawley rats were randomized to sham air (SA) as control group or CS group for 56 days. At day 29, rats were subdivided and given water as control, DOPs, or NAC as positive control as a mucolytic drug via oral gavage for the remaining duration. Samples collected from apical washing, cell lysates, bronchoalveolar lavage (BAL), and lung tissues were evaluated for mucin gene expression, mucus secretion, and viscosity. DOPs ameliorated the CS-induced mucus hypersecretion and viscosity as shown by the downregulation of MUC5AC mRNA, MUC5AC secretary protein, and mucus viscosity via inhibition of mucus secretory granules in both in vitro and in vivo models. DOPs produced its effective effects on the CS-induced mucus hypersecretion and viscosity via the inhibition of the mucus secretory granules. These findings could be a starting point for considering the potential role of DOPs in the management of the smoking-mediated COPD. However, further research is needed.

Morphological characterization of brush cells in the rat trachea.

Brush cells have recently been classified as solitary chemosensory cells. However, tracheal brush cells have not been morphologically and immunohistochemically characterized yet. In the present study, the morphological and immunohistochemical characteristics of tracheal brush cells were analyzed using immunohistochemistry and scanning, and transmission electron microscopies. Brush cells in the tracheal epithelium were barrel-like or columnar in shape and were immunoreactive for villin. Scanning and transmission electron microscopies revealed densely arranged thick microvilli on the apical surface of tracheal brush cells and tubular membranous elements and/or vesicular formations in the supranuclear region. A morphometrical analysis of tracheal whole-mount preparations showed that the density of brush cells was greater in the cranial third and the mucosa on the annular ligament. Double immunofluorescence revealed that the morphology of villin-immunoreactive brush cells was distinct from other non-ciliated cells in the tracheal epithelium, i.e., MUC5AC-immunoreactive mucous cells, SNAP25-immunoreactive neuroendocrine cells, and GNAT3-immunoreactive solitary chemosensory cells. On the other hand, tracheal brush cells were immunoreactive for the marker proteins for intestinal brush cells, CK18, DCLK1, and Cox1; however, these antibodies also recognized cells other than brush cells. Furthermore, immunoreactivity for PKD2L1, a cation channel subunit, was detected in brush cells. The present results demonstrated that tracheal brush cells are independent cell types. These brush cells may be activated by acid and the secretion of prostaglandins. In conclusion, the present study revealed that tracheal brush cells are independent cell types based on the morphological and immunohistochemical characteristics.

MiR-196-5p involvement in selenium deficiency-induced immune damage via targeting of NFκBIA in the chicken trachea.

Dietary selenium (Se) deficiency can induce multifarious immune injury in tissues, accompanied by inflammation and a decreased expression of selenoproteins. The results of previous studies indicated that these issues are associated with Se-mediated microRNAs involved in immune regulation, although the specific mechanisms associated with these interactions have not been reported in the trachea of chickens. To explore the effects of Se deficiency in the trachea of chickens and the role of miR-196-5p, we established correlational models of tracheal injury in chickens. One hundred broilers were divided into four groups, including a control group (C group), a Se deficient group (L group), a lipopolysaccharide (LPS)-induced control group (C + LPS group) and a LPS-induced Se deficient group (L + LPS group). Light microscopy observations indicated that the infiltration of inflammatory cells was the major histopathological change caused by Se deficiency. Furthermore, ultrastructural observation of the tracheal epithelium and ciliary showed typical inflammatory signs owing to Se deficiency. We determined the targeting relationship between miR-196-5p and NFκBIA by bioinformatics analysis. In the case of Se deficiency, the changes were detected as follows: 19 selenoproteins showed different degrees of decrease (p < 0.05). Significant inhibition of both antimicrobial peptides and immunoglobulin production were observed (p < 0.05). IκB-α (NFκBIA) expression degraded with the increasing miR-196-5p (p < 0.05), and the NF-κB pathway was activated. Thereafter, we can see a significant increase in the mRNA levels of inflammatory cytokines-related genes (tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E (PTGE), interleukin (IL)-1ß, IL-6) and protein expression of NF-κB/iNOS pathway-related genes (NF-κB, iNOS, TNF-α, COX-2) (p < 0.05). The release of IL-2, interferon (IFN)-γ inhibited (p < 0.05) and the secretion of IL-4, IL-6 increased, suggesting the imbalance of Th1/Th2 (Th, helper T cell) cytokines. Compared to the control, the mRNA and protein expression levels of the anti-inflammatory system components with antioxidant activity (PPAR-γ/HO-1) were in an inhibitory state (p < 0.05). Antioxidases (SOD, CAT, GSH-Px) activities were suppressed. The activities of the peroxide markers (MDA, H2O2) were enhanced (p < 0.05). In addition, Se deficiency had a positive effect on the pathological changes of inflammation and the exceptional immunity in LPS-treated groups (p < 0.05). The results confirmed the relationship between miR-196-5p and NFκBIA in chickens, revealing that Se deficiency causes respiratory mucosal immune dysfunction via the miR-196-5p-NFκBIA axis, oxidative stress and inflammation. Moreover, Se deficiency exacerbates the inflammatory damage stimulated by LPS. Our work provides a theoretical basis for the prevention of tracheal injury owing to Se deficiency and can be used as a reference for comparative medicine. Furthermore, the targeted regulation of miR-196-5p and NFκBIA may contribute to the protection of the tracheal mucosa in chickens.

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism. METHODS: In this case series, patients with a positive antemortem or post-mortem SARS-CoV-2 result were considered eligible for enrolment. Post-mortem examinations were done on 14 people who died with COVID-19 at the King County Medical Examiner's Office (Seattle, WA, USA) and Snohomish County Medical Examiner's Office (Everett, WA, USA) in negative-pressure isolation suites during February and March, 2020. Clinical and laboratory data were reviewed. Tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative RT-PCR. FINDINGS: The median age of our cohort was 73·5 years (range 42-84; IQR 67·5-77·25). All patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. The major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. Coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. Lymphocytic myocarditis was observed in one patient with viral RNA detected in the tissue. INTERPRETATION: The primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. Microthrombi, where observed, were scarce and endotheliitis was not identified. Although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of SARS-CoV-2 infection requires further examination. FUNDING: None.

Human lung microvascular endothelial cells as potential alternatives to human umbilical vein endothelial cells in bio-3D-printed trachea-like structures.

BACKGROUND: We have been trying to produce scaffold-free structures for airway regeneration using a bio-3D-printer with spheroids, to avoid scaffold-associated risks such as infection. Previous studies have shown that human umbilical vein endothelial cells (HUVECs) play an important role in such structures, but HUVECs cannot be isolated from adult humans. The aim of this study was to identify alternatives to HUVECs for use in scaffold-free structures. METHODS: Three types of structure were compared, made of chondrocytes and mesenchymal stem cells with HUVECs, human lung microvascular endothelial cells (HMVEC-Ls), and induced pluripotent stem cell (iPSC)-derived endothelial cells. RESULTS: No significant difference in tensile strength was observed between the three groups. Histologically, some small capillary-like tube formations comprising CD31-positive cells were observed in all groups. The number and diameters of such formations were significantly lower in the iPSC-derived endothelial cell group than in other groups. Glycosaminoglycan content was significantly lower in the iPSC-derived endothelial cell group than in the HUVEC group, while no significant difference was observed between the HUVEC and HMVEC-L groups. CONCLUSIONS: HMVEC-Ls can replace HUVECs as a cell source for scaffold-free trachea-like structures. However, some limitations were associated with iPSC-derived endothelial cells.

Comparative Morphological Features of Syrinx in Male Domestic Fowl Gallus gallus domesticus and Male Domestic Pigeon Columba livia domestica: A Histochemical, Ultrastructural, Scanning Electron Microscopic and Morphometrical Study.

Many studies have been carried out to investigate the morphological structure of the syrinx in many bird species. However, the cellular organization of the syrinx in the fowls and pigeons is still unclear. The current study revealed that in fowl and pigeon, the syrinx is formed of three main parts including tympanum (cranial) part, intermediate syringeal part, and bronchosyringeal (caudal) part, in addition to pessulus and tympaniform membranes. A great variation in the structural characteristics of syrinx of fowl and pigeon was recorded. In fowl, the tympaniform membranes showed a characteristic distribution of elastic and collagen fibers which increase the elasticity of tympaniform membranes. Moreover, the bony pessulus helps the medial tympaniform membranes to be stiffer, vibrate more strongly so that louder sound will be generated. In pigeon, the lateral tympaniform membrane is of greater thickness so that the oscillation of this membrane is reduced and the amplitude is lower. Moreover, the pessulus is smaller in size and is formed mainly of connective tissue core (devoid of cartilaginous or bony plates), resulting in the failure of stretching and vibrating of the medial tympaniform membranes, that leads to the generation of deeper sound. Electron microscopic examination of the syringes of fowls and pigeons revealed numerous immune cells including dendritic cells, plasma cells, mast cells, and lymphocytes distributed within syringeal mucosa and invading the syringeal epithelium. Telocytes were first recorded in the syrinx of fowls and pigeons in this study. They presented two long telopodes that made up frequent close contacts with other neighboring telocytes, immune cells, and blood capillaries.

Electron cryotomography of intact motile cilia defines the basal body to axoneme transition.

Cells use motile cilia to generate force in the extracellular space. The structure of a cilium can be classified into three subdomains: the intracellular basal body (BB) that templates cilium formation, the extracellular axoneme that generates force, and the transition zone (TZ) that bridges them. While the BB is composed of triplet microtubules (TMTs), the axoneme is composed of doublet microtubules (DMTs), meaning the cilium must convert between different microtubule geometries. Here, we performed electron cryotomography to define this conversion, and our reconstructions reveal identifying structural features of the BB, TZ, and axoneme. Each region is distinct in terms of microtubule number and geometry, microtubule inner proteins, and microtubule linkers. TMT to DMT conversion occurs within the BB, and microtubule geometry changes to axonemal by the end of the TZ, followed by the addition of axoneme-specific components essential for cilium motility. Our results provide the highest-resolution images of the motile cilium to date and reveal how BBs template axonemes.

Fast cyclical-decellularized trachea as a natural 3D scaffold for organ engineering.

Commonly reported decellularization protocols for trachea may take up from several weeks to months in order to remove the cellular materials. Two years ago, we significantly reduced the time of decellularization trachea process using trypsin. Despite the positive outcome, the protocol was useful to produce 5 cm graft length, an unsuitable length graft for most patients with tracheal disorders. In this work we improved the decellularization procedure for longer sections up to 10 cm without considerable extension in the necessary time process (2 weeks). Herein, for the first time, we completely describe and characterize the process for pig tracheal bioactive scaffolds. Histological and molecular biology analysis demonstrated effective removal of cellular components and nuclear material, which was also confirmed by the Immunohistochemical (IHC) analysis of the major histocompatibility complexes (MHCs) and DNA stain by 4'-6-diamidino-2-phenylindole (DAPI). The images and data obtained from scanning electron microscopy (SEM) and thermal analysis showed conservation of the hierarchical structures of the tracheal extracellular matrix (ECM), the biomechanical tests showed that decellularization approach did not lead to a significant alteration on the mechanical properties. In this paper, we demonstrate that the proposed cyclical-decellularization protocol allowed us to obtain a non-immunological 10 cm natural tracheal scaffold according to the in vivo immunological assessment. Furthermore, the recellularization of the matrix was successfully achieved by demonstrating first-stage cellular differentiation from stem cells to chondrocytes expressed by the SOX9 transcription factor; this organ-engineered tracheal matrix has the potential to act as a suitable template for organ regeneration.

Establishment of rapid risk assessment model for cigarette smoke extract exposure in chronic obstructive pulmonary disease.

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 µg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.

De-epithelialization of porcine tracheal allografts as an approach for tracheal tissue engineering.

Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.

Calaxin is required for cilia-driven determination of vertebrate laterality.

Calaxin is a Ca2+-binding dynein-associated protein that regulates flagellar and ciliary movement. In ascidians, calaxin plays essential roles in chemotaxis of sperm. However, nothing has been known for the function of calaxin in vertebrates. Here we show that the mice with a null mutation in Efcab1, which encodes calaxin, display typical phenotypes of primary ciliary dyskinesia, including hydrocephalus, situs inversus, and abnormal motility of trachea cilia and sperm flagella. Strikingly, both males and females are viable and fertile, indicating that calaxin is not essential for fertilization in mice. The 9 + 2 axonemal structures of epithelial multicilia and sperm flagella are normal, but the formation of 9 + 0 nodal cilia is significantly disrupted. Knockout of calaxin in zebrafish also causes situs inversus due to the irregular ciliary beating of Kupffer's vesicle cilia, although the 9 + 2 axonemal structure appears to remain normal.

Fast degrading elastomer stented fascia remodels into tough and vascularized construct for tracheal regeneration.

Tracheal reconstruction remains a major surgical challenge, mainly owing to the scarce of resilient hollow grafts with identifiable vascular pedicle in humans. In this study, we developed a three-layer, elastomeric, trachea-like composite made of poly glycerol sebacate (PGS) and polycaprolactone (PCL), which presented appropriate resilient property, timely degradation and interconnected pores. C shape PCL rings fabricated with selective laser sintering (SLS) techniques are regularly positioned around porous PGS tubes and fixed by PCL electrospinning sheath. Such an elastomeric composite underwent host remodeling including rapid vascularization and tissue infiltration after fascia wrapping. With degrading of PGS, C rings well incorporated into growing fascia and lead to the formation of pedicled tracheal grafts, which attributes to the strong and resilient properties of generated hollow grafts thus enabled orthotopic transplantation in segmental tracheal defect. Progressive remodeling on such vascularized and mechanically stable grafts resulted in epithelium regeneration on luminal side as well as production of adequate amount of collagen and elastin, which warrantees the air passage during breathing. Future study employing large animal models more representative of human tracheal regeneration is warranted before clinical translation. Using fast degrading PGS combined with PCL rings represents a philosophical shift from the prevailing focus on tough grafts in airway reconstruction and may impact regenerative medicine in general.

Fine particulate matter (PM2.5) enhances airway hyperresponsiveness (AHR) by inducing necroptosis in BALB/c mice.

OBJECTIVE: To observe the effects of prolonged exposure to high concentrations of PM2.5 on the trachea and lungs of mice and to determine whether the damages to the trachea and lung are induced by necroptosis. METHODS: Six- to eight-week-old female Balb/C mice of PM group were restrained in an animal restraining device using a nose-only "PM2.5 online enrichment system" for 8 weeks, in Shijiazhuang, Hebei, China. Anti -Fas group was exposed to PM2.5 inhalation and anti-Fas treatment via intranasal instillation. The mice in the control group inhaled filtered clean air. PM2.5 sample was collected and analyzed. Airway Hyperresponsiveness (AHR) was tested. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for Hematoxylin and eosin (HE) staining, electron microscopy, cellular inflammation, cytokines, Tunel, Fas, RIPK3 and MLKL expression. RESULTS: Compared to the other two groups, PM group displayed significantly increased AHR, neutrophils in BALF, significant bronchitis and alveolar epithelial hyperplasia and inflammation and necroptosis which were indicated by increased TUNEL, Fas, RIPK3 and MLKL measure. CONCLUSION: Our findings suggest that PM2.5 can enhance AHR and these changes are induced by necroptosis-related inflammation.

Early studies of airway submucosal glands.

This historical article provides a comprehensive review of early research on the structure and function of airway submucosal glands. The literature before 1950 or so, is virtually unknown, but in addition to being of historical interest it contains much of relevance to current research. Airway glands were first mentioned in 1602. The first description of their general form, size, and distribution was in 1712. Gland morphology was determined in 1827 by injecting mercury into their openings. Wax was later used. Detailed comparative information for all regions of the tracheobronchial tree was provided by Frankenhauser in 1879 (Untersuchungen uber den bau der Tracheo-Bronchial-Schleimhaut). Histological studies began in 1870, and by the end of the 19th century, all the major histological features had been described. The first physiological studies on airway mucous secretion were published in 1892. Kokin, in 1896 (Archiv für die gesamte Physiologie des Menschen und der Tiere 63: 622-630), was the first to measure secretion from individual glands. It was not, however, until 1933 that gland secretion was quantified. This early literature raises important questions as to the role of the collecting duct epithelium in modifying primary secretions. It also provides perhaps the most accurate measure of basal gland secretion in vivo.