Hyperbaric oxygen promotes bone regeneration by activating the mechanosensitive Piezo1 pathway in osteogenic progenitors.

Background: Hyperbaric oxygen (HBO) therapy is widely used to treat bone defects, but the correlation of high oxygen concentration and pressure to osteogenesis is unclear. Methods: Bilateral monocortical tibial defect surgeries were performed on 12-week-old Prrx1-Cre; Rosa26-tdTomato and Prrx1-Cre; Piezo1fl/+ mice. Daily HBO treatment was applied on post-surgery day (PSD) 1-9; and daily mechanical loading on tibia was from PSD 5 to 8. The mice were euthanized on PSD 10, and bone defect repair in their tibias was evaluated using µCT, biomechanical testing, and immunofluorescence deep-tissue imaging. The degree of angiogenesis-osteogenesis coupling was determined through spatial correlation analysis. Bone marrow stromal cells from knockout mice were cultured in vitro, and their osteogenic capacities of the cells were assessed. The activation of genes in the Piezo1-YAP pathway was evaluated using RNA sequencing and quantitative real-time polymerase chain reaction. Results: Lineage tracing showed HBO therapy considerably altered the number of Prrx1+ cells and their progeny in a healing bone defect. Using conditional knockdown mice, we found that HBO stimulation activates the Piezo1-YAP axis in Prrx1+ cells and promotes osteogenesis-angiogenesis coupling during bone repair. The beneficial effect of HBO was similar to that of anabolic mechanical stimulation, which also acts through the Piezo1-YAP axis. Subsequent transcriptome sequencing results revealed that similar mechanosensitive pathways are activated by HBO therapy in a bone defect. Conclusion: HBO therapy promotes bone tissue regeneration through the mechanosensitive Piezo1-YAP pathway in a population of Prrx1+ osteogenic progenitors. Our results contribute to the understanding of the mechanism by which HBO therapy treats bone defects. The Translational Potential of this Article: Hyperbaric oxygen therapy is widely used in clinical settings. Our results show that osteogenesis was induced by the activation of the Piezo1-YAP pathway in osteoprogenitors after HBO stimulation, and the underlying mechanism was elucidated. These results may help improve current HBO methods and lead to the formulation of alternative treatments that achieve the same functional outcomes.

Cellular Scale Curvature in Bioceramic Scaffolds Enhanced Bone Regeneration by Regulating Skeletal Stem Cells and Vascularization.

Critical-sized segmental bone defects cannot heal spontaneously, leading to disability and significant increase in mortality. However, current treatments utilizing bone grafts face a variety of challenges from donor availability to poor osseointegration. Drugs such as growth factors increase cancer risk and are very costly. Here, a porous bioceramic scaffold that promotes bone regeneration via solely mechanobiological design is reported. Two types of scaffolds with high versus low pore curvatures are created using high-precision 3D printing technology to fabricate pore curvatures radius in the 100s of micrometers. While both are able to support bone formation, the high-curvature pores induce higher ectopic bone formation and increased vessel invasion. Scaffolds with high-curvature pores also promote faster regeneration of critical-sized segmental bone defects by activating mechanosensitive pathways. High-curvature pore recruits skeletal stem cells and type H vessels from both the periosteum and the marrow during the early phase of repair. High-curvature pores have increased survival of transplanted GFP-labeled skeletal stem cells (SSCs) and recruit more host SSCs. Taken together, the bioceramic scaffolds with defined micrometer-scale pore curvatures demonstrate a mechanobiological approach for orthopedic scaffold design.

Evidence of Microplastics in Bronchoalveolar Lavage Fluid among Never-Smokers: A Prospective Case Series.

Microplastics (MPs) are abundant in air, but evidence of their deposition in the respiratory tract is limited. We conducted a prospective case series to investigate the deposition of microplastics in bronchoalveolar lavage fluid (BALF) and determine the internal dose of MPs via inhalation. Eighteen never-smokers aged 32-74 years who underwent fiberoptic bronchoscopy with BALF were recruited from Zhuhai, China. Control samples were obtained by performing the same procedure using isotonic saline instead of BALF. Laser direct infrared spectroscopy combined with scanning electron microscopy detected the presence and characteristics of MPs and quantitatively analyzed the microplastic in BALF and control samples. Concentrations of total and specific MPs in BALF and control samples were compared using the Wilcox test. Thirteen types of MPs were observed in 18 BALF samples. Polyethylene (PE, 86.1%) was the most abundant in BALF, followed by poly(ethylene terephthalate) (PET, 7.5%) and polypropylene (PP, 1.9%). Compared with the control samples, BALF had significantly higher concentrations of PE (median [IQR] of BALF: 0.38 [8.05] N/g), PET (0.26 [0.54] N/g), polyurethane (0.16 [0.24] N/g), PP (0.16 [0.11] N/g), and total MPs (0.91 [6.58] N/g). The presence of MPs in BALF provides novel evidence that MPs penetrate deep into the respiratory tract.

Identification of RNA Modification-Associated Alternative Splicing Signature as an Independent Factor in Head and Neck Squamous Cell Carcinoma.

Objective: Head and neck squamous cell carcinoma (HNSCC) is a highly heterotopic malignant tumor. Alternative splicing (AS) and RNA modification have been reported to be involved in tumorigenesis. Therefore, we constructed RNA modification-associated AS (RMA-AS) signature model to predict the prognosis of HNSCC. Methods: AS events and RNA-modified gene expression information were downloaded from TCGA-HNSCC database. Univariate Cox regression analysis was employed for analyzing prognosis-related AS events. RMA-AS events were obtained by constructing a coexpression network between RNA modification-associated genes and AS events using WGCNA package. The prognostic signatures were analyzed by LASSO, univariate Cox, and multivariate Cox regression. Kaplan-Meier survival analysis, proportional hazard model, and ROC curve were performed to verify the prognostic value. "ESTIMATE" R package, ssGSEA algorithm, and CIBERSORT method were used to detect immune microenvironment in HNSCC. Cytoscape was utilized to build a regulatory network of splicing factor-regulated RMA-AS. Results: There were 16,574 prognostic AS events and 4 differentially expressed RNA modification-associated genes in HNSCC. Based on RMA-AS events, we obtained a risk model consisting of 14 AS events, named RMA-AS_Score. The samples were divided into RMA-AS_Score high- and RMA-AS_Score low-risk groups, according to the risk score. The RMA-AS_Score high group was related to poor prognosis. Moreover, the RMA-AS_Score signature was an independent prognostic predictor and was related to tumor grade. Meanwhile, the AUC value of RMA-AS_Score was 0.652, which is better than other clinical characteristics. Besides, a nomogram prediction model of quantitative prognosis has also been developed, which has robust effectiveness in predicting prognosis. In addition, the prognostic signature was observably related to immune microenvironment and immune checkpoint. Finally, 14 splicing factors were identified and constructed into a network of splicing factor-regulated RMA-AS. Conclusion: We identified the RMA-AS signature of HNSCC. This signature could be treated as an independent prognostic predictor.

Single-cell spatiotemporal analysis reveals cell fates and functions of transplanted mesenchymal stromal cells during bone repair.

Mesenchymal stromal cells (MSCs) transplantation could enhance bone repair. However, the cell fate of transplanted MSCs, in terms of their local distribution and spatial associations with other types of cells were poorly understood. Here, we developed a single-cell 3D spatial correlation (sc3DSC) method to track transplanted MSCs based on deep tissue microscopy of fluorescent nanoparticles (fNPs) and immunofluorescence of key proteins. Locally delivered fNP-labeled MSCs enhanced tibial defect repair, increased the number of stem cells and vascular maturity in mice. fNP-MSCs persisted in the defect throughout repair. But only a small portion of transplanted cells underwent osteogenic differentiation (OSX+); a significant portion has maintained their expression of mesenchymal stem cell and skeletal stem cell markers (SCA-1 and PRRX1). Our results contribute to the optimization of MSC-based therapies. The sc3DSC method may be useful in studying cell-based therapies for the regeneration of other tissue types or disease models.

Tropheryma whipplei detection by metagenomic next-generation sequencing in bronchoalveolar lavage fluid: A cross-sectional study.

Tropheryma whipplei is the bacterium associated with Whipple's disease (WD), a chronic systemic infectious disease primarily involving the gastrointestinal tract. T. whipplei can also be detected in different body site of healthy individuals, including saliva and feces. Traditionally, Tropheryma whipplei has a higher prevalence in bronchoalveolar lavage fluid (BALF) of immunocompromised individuals. Few studies have explored the significance of the detection of T. whipplei in BALF. Herein, we retrospectively reviewed 1725 BALF samples which detected for metagenomic next-generation sequencing (mNGS) from March 2019 to April 2022 in Zhuhai, China. Seventy BALs (70/1725, 4.0%) from 70 patients were positive for T. whipplei. Forty-four patients were male with an average age of 50 years. The main symptoms included cough (23/70), expectoration (13/70), weight loss (9/70), and/or dyspnea (8/70), but gastrointestinal symptoms were rare. Chronic liver diseases were the most common comorbidity (n=15, 21.4%), followed by diabetes mellitus (n=13, 18.6%). Only nine patients (12.9%) were immunocompromised. Twenty-four patients (34.3%) were finally diagnosed with reactivation tuberculosis and 15 patients (21.4%) were diagnosed with lung tumors, including 13 primary lung adenocarcinoma and two lung metastases. Fifteen patients (21.4%) had pneumonia. Among the 20 samples, T. whipplei was the sole agent, and Mycobacterium tuberculosis complex was the most common detected other pathogens. Among the non-tuberculosis patients, 31 (31/46, 67.4%) had ground glass nodules or solid nodules on chest CT. Our study indicates that T. whipplei should be considered as a potential contributing factor in some lung diseases. For non-immunocompromised patients, the detection of T. whipplei also needs attention. The mNGS technology improves the detection and attention of rare pathogens. In the future, the infection, colonization, and prognosis of T. whipplei in lung still need to be studied.

Metallic Scaffold with Micron-Scale Geometrical Cues Promotes Osteogenesis and Angiogenesis via the ROCK/Myosin/YAP Pathway.

The advent of precision manufacturing has enabled the creation of pores in metallic scaffolds with feature size in the range of single microns. In orthopedic implants, pore geometries at the micron scale could regulate bone formation by stimulating osteogenic differentiation and the coupling of osteogenesis and angiogenesis. However, the biological response to pore geometry at the cellular level is not clear. As cells are sensitive to curvature of the pore boundary, this study aimed to investigate osteogenesis in high- vs low-curvature environments by utilizing computer numerical control laser cutting to generate triangular and circular precision manufactured micropores (PMpores). We fabricated PMpores on 100 µm-thick stainless-steel discs. Triangular PMpores had a 30° vertex angle and a 300 µm base, and circular PMpores had a 300 µm diameter. We found triangular PMpores significantly enhanced the elastic modulus, proliferation, migration, and osteogenic differentiation of MC3T3-E1 preosteoblasts through Yes-associated protein (YAP) nuclear translocation. Inhibition of Rho-associated kinase (ROCK) and Myosin II abolished YAP translocation in all pore types and controls. Inhibition of YAP transcriptional activity reduced the proliferation, pore closure, collagen secretion, alkaline phosphatase (ALP), and Alizarin Red staining in MC3T3-E1 cultures. In C166 vascular endothelial cells, PMpores increased the VEGFA mRNA expression even without an angiogenic differentiation medium and induced tubule formation and maintenance. In terms of osteogenesis-angiogenesis coupling, a conditioned medium from MC3T3-E1 cells in PMpores promoted the expression of angiogenic genes in C166 cells. A coculture with MC3T3-E1 induced tubule formation and maintenance in C166 cells and tubule alignment along the edges of pores. Together, curvature cues in micropores are important stimuli to regulate osteogenic differentiation and osteogenesis-angiogenesis coupling. This study uncovered key mechanotransduction signaling components activated by curvature differences in a metallic scaffold and contributed to the understanding of the interaction between orthopedic implants and cells.

Mechanical loading attenuated negative effects of nucleotide analogue reverse-transcriptase inhibitor TDF on bone repair via Wnt/ß-catenin pathway.

The nucleotide analog reverse-transcriptase inhibitor, tenofovir disoproxil fumarate (TDF), is widely used to treat hepatitis B virus (HBV) and human immunodeficiency virus infection (HIV). However, long-term TDF usage is associated with an increased incidence of bone loss, osteoporosis, fractures, and other adverse reactions. We investigated the effect of chronic TDF use on bone homeostasis and defect repair in mice. In vitro, TDF inhibited osteogenic differentiation and mineralization in MC3T3-E1 cells. In vivo, 8-week-old C57BL/6 female mice were treated with TDF for 38 days to simulate chronic medication. Four-point bending test and µCT showed reduced bone biomechanical properties and microarchitecture in long bones. To investigate the effects of TDF on bone defect repair, we utilized a bilateral tibial monocortical defect model. µCT showed that TDF reduced new bone mineral tissue and bone mineral density (BMD) in the defect. To verify whether mechanical stimulation may be a useful treatment to counteract the negative bone effects of TDF, controlled dynamic mechanical loading was applied to the whole tibia during the matrix deposition phase on post-surgery days (PSDs) 5 to 8. Second harmonic generation (SHG) of collagen fibers and µCT showed that the reduction of new bone volume and bone mineral density caused by TDF was reversed by mechanical loading in the defect. Immunofluorescent deep tissue imaging showed that chronic TDF treatment reduced the number of osteogenic cells and the volume of new vessels. In addition, chronic TDF treatment inhibited the expressions of periostin and ß-catenin, but increased the expression of sclerostin. Both negative effects were reversed by mechanical loading. Our study provides strong evidence that chronic use of TDF exerts direct and inhibitory impacts on bone repair, but appropriate mechanical loading could reverse these adverse effects.

Emerging role of exosomal long non-coding RNAs in lung cancer.

BACKGROUND: Lung cancer is one of the most common malignancies worldwide. Also, it is the leading cause of cancer morbidity and mortality in men. Despite advances in lung cancer diagnosis and treatment, novel approaches are strongly needed to promote early diagnosis and effective treatment of lung cancer. Presently, accumulating data reveal that long noncoding RNAs (lncRNAs) are differentially enriched in exosomes and mediate multiple biological processes in lung cancer, suggesting the potential application of exosomal lncRNAs as diagnostic biomarkers and therapeutic targets. CONCLUSION: In this review, we described the emerging roles of lncRNAs specifically sorted into exosomes in lung cancer. We discussed the current knowledge of the exosomal lncRNA sorting mechanism and highlighted opportunities for exosome-derived lncRNAs as biomarkers in clinical practice. In particular, we systematically summarized the biological functions of exosomal lncRNAs in lung cancer.

Mechanical loading alleviated the inhibition of ß2-adrenergic receptor agonist terbutaline on bone regeneration.

The long-term use of adrenergic medication in treating various conditions, such as asthma, increases the chances of bone fracture. Dynamic mechanical loading at a specific time is a method for improving bone quality and promoting healing. Therefore, we hypothesized that precisely controlling the mechanical environment can contribute to the alleviation of the negative effects of chronic treatment with the common asthma drug terbutaline, which is a ß2-adrenergic receptor agonist that facilitates bone homeostasis and defect repair through its anabolic effect on osteogenic cells. Our in vitro results showed that terbutaline can directly inhibit osteogenesis by impairing osteogenic differentiation and mineralization. Chronic treatment in vivo was simulated by administering terbutaline to C57BL/6J mice for 4 weeks before bone defect surgery and mechanical loading. We utilized a stabilized tibial defect model, which allowed the application of anabolic mechanical loading. During homeostasis, chronic terbutaline treatment reduced the bone formation rate, the fracture toughness of long bones, and the concentrations of bone formation markers in the sera. During defect repair, terbutaline decreased the bone volume, type H vessel, and total blood vessel volume. Terbutaline treatment reduced the number of osteogenic cells. Periostin, which was secreted mainly by Prrx1+ osteoprogenitors and F4/80+ macrophages, was inhibited by treating the bone defect with terbutaline. Interestingly, controlled mechanical loading facilitated the recovery of bone volume and periostin expression and the number of osteogenic cells within the defect. In conclusion, mechanical loading can rescue negative effects on new bone accrual and repair induced by chronic terbutaline treatment.

Exosomal lncRNA SCIRT/miR-665 Transferring Promotes Lung Cancer Cell Metastasis through the Inhibition of HEYL.

Lung cancer remains the leading cause of cancer-related death worldwide. Recently, extracellular vesicles such as exosomes have attracted considerable interest both as a source for theranostic biomarkers and an essential participant in lung cancer progression. However, how specific exosomal cargos, such as noncoding RNAs, are selectively packaged into exosomes and promote lung cancer progression remains unclear. In this study, we identified miR-665 as the most elevated exosomal miRNA from both non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. We further demonstrated that lncRNA SCIRT was also increased in cancer cell exosomes and may facilitate the exosomal loading of miR-665 with the help of hnRNPA1. As a consequence, exosomal miR-665 promoted lung cancer cell invasion and migration by targeting Notch downstream transcription factor HEYL. In addition, we found that miR-665 and SCIRT were significantly upregulated in tumor tissue and plasma of patients with lung cancer, and both of them showed increased expression in metastatic disease samples. Our findings suggest that the exosomal transferring of miR-665 and SCIRT is a functional and mechanism-driven pathway that contributes to cancer progression and, thus, may provide novel diagnostic and therapeutic targets for lung cancer.