Transcriptome analysis reveals the impact of NETs activation on airway epithelial cell EMT and inflammation in bronchiolitis obliterans.

Bronchiolitis obliterans (BO) is a chronic airway disease that was often indicated by the pathological presentation of narrowed and irreversible airways. However, the molecular mechanisms of BO pathogenesis remain unknown. Although neutrophil extracellular traps (NETs) can contribute to inflammatory disorders, their involvement in BO is unclear. This study aims to identify potential signaling pathways in BO by exploring the correlations between NETs and BO. GSE52761 and GSE137169 datasets were downloaded from gene expression omnibus (GEO) database. A series of bioinformatics analyses such as differential expression analysis, gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA) were performed on GSE52761 and GSE137169 datasets to identify BO potential signaling pathways. Two different types of BO mouse models were constructed to verify NETs involvements in BO. Additional experiments and bioinformatics analysis using human small airway epithelial cells (SAECs) were also performed to further elucidate differential genes enrichment with their respective signaling pathways in BO. Our study identified 115 differentially expressed genes (DEGs) that were found up-regulated in BO. Pathway enrichment analysis revealed that these genes were primarily involved in inflammatory signaling processes. Besides, we found that neutrophil extracellular traps (NETs) were formed and activated during BO. Our western blot analysis on lung tissue from BO mice further confirmed NETs activation in BO, where neutrophil elastase (NE) and myeloperoxidase (MPO) expression were found significantly elevated. Transcriptomic and bioinformatics analysis of NETs treated-SAECs also revealed that NETs-DEGs were primarily associated through inflammatory and epithelial-to-mesenchymal transition (EMT) -related pathways. Our study provides novel clues towards the understanding of BO pathogenesis, in which NETs contribute to BO pathogenesis through the activation of inflammatory and EMT associated pathways. The completion of our study will provide the basis for potential novel therapeutic targets in BO treatment.

Cartilage progenitor cells combined with PHBV in cartilage tissue engineering.

BACKGROUND: Bone marrow-derived stem cells (BMSCs) and chondrocytes have been reported to present "dedifferentiation" and "phenotypic loss" during the chondrogenic differentiation process in cartilage tissue engineering, and cartilage progenitor cells (CPCs) are novel seeding cells for cartilage tissue engineering. In our previous study, cartilage progenitor cells from different subtypes of cartilage tissue were isolated and identified in vitro, but the study on in vivo chondrogenic characteristics of cartilage progenitor cells remained rarely. In the current study, we explored the feasibility of combining cartilage progenitor cells with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics of cartilage progenitor cells with those of bone marrow-derived stem cells and chondrocytes. METHODS: These three cells combined with PHBV were cultured in vitro for 1 week without chondrogenic induction and then transplanted subcutaneously into nude mice for 6 weeks. The cell-PHBV constructs were evaluated by gross observation, histological staining, glycosaminoglycan content measurement, biomechanical analysis and RT-PCR. RESULTS: The chondrocyte-PHBV constructs and CPC-PHBV constructs became an ivory-whitish cartilage-like tissue, while the BMSC-PHBV constructs became vascularized 6 weeks after the subcutaneous implantation. Histological examination showed that many typical cartilage structures were present in the chondrocyte group, some typical cartilage structures were observed in the CPC group, while no typical cartilage structures were observed in the BMSC group. CONCLUSIONS: Cartilage progenitor cells may undergo chondrogenesis without chondrogenic induction and are better at chondrogenesis than BMSCs but worse than chondrocytes in the application of cartilage tissue engineering.

Effective Adsorption of Diesel Oil by Crab-Shell-Derived Biochar Nanomaterials.

This study, for the first time, rendered crab shell activated biochar modified by potassium hydroxide (KOH) impregnation (CSAB), revealing a new potential application in the removal of diesel oil from oily wastewater. The structural characteristics of crab shell biochar (CSB) and CSAB were investigated by SEM, and the crystal structure and optical properties of as-prepared samples were analyzed using XRD and FTIR. Results showed that CSAB had stratified surface structure morphology, abundant functional groups, and that its high specific surface area could reach up to 2441 m²/g, which was about eight times larger than that of untreated CSB (307 m²/g). An adsorption isotherm study indicated that the actual adsorption process both of CSAB and CSB were found to fit better with the Freundlich equation. Moreover, chemical interaction controlled the adsorption kinetics efficiency while the adsorption equilibrium capacity was 93.9 mg/g. Due to its highly developed pore structure, unique surface characteristics, and effective adsorption performance, this low-cost activated carbon had the potential to serve as an efficient adsorbent for water pollution purification.

Exosomes derived from mature chondrocytes facilitate subcutaneous stable ectopic chondrogenesis of cartilage progenitor cells.

BACKGROUND: Developing cartilage constructed with the appropriate matrix composition and persistent chondrogenesis remains an enduring challenge in cartilage defects. Cartilage progenitor cell (CPC)-based tissue engineering has attracted recent attention because of its strong chondrogenic differentiation capacity. However, due to the lack of a suitable chondrogenic niche, the clinical application of CPC-regenerated cartilage in the subcutaneous environment remains a challenge. In this study, exosomes derived from chondrocytes (CC-Exos) were used to provide the CPC constructs with a cartilage signal in subcutaneous environments for efficient ectopic cartilage regeneration. METHODS: Rabbit CPC-alginate constructs were prepared and implanted subcutaneously in nude mice. CC-Exos were injected into the constructs at the same dose (30 µg exosomes per 100 µL injection) after surgery and thereafter weekly for a period of 12 weeks. Exosomes derived from bone mesenchymal stem cells (BMSC-Exos) were used as the positive control. The mice in the negative control were administered with the same volume of PBS. At 4 and 12 weeks after implantation, the potential of CC-Exos and BMSC-Exos to promote chondrogenesis and stability of cartilage tissue in a subcutaneous environment were analyzed by histology, immunostaining, and protein analysis. The influences of BMSC-Exos and CC-Exos on chondrogenesis and angiogenic characteristics in vitro were assessed via coculturing with CPCs and human umbilical vein endothelial cells. RESULTS: The CC-Exos injection increased collagen deposition and minimized vascular ingrowth in engineered constructs, which efficiently and reproducibly developed into cartilage. The generated cartilage was phenotypically stable with minimal hypertrophy and vessel ingrowth up to 12 weeks, while the cartilage formed with BMSC-Exos was characterized by hypertrophic differentiation accompanied by vascular ingrowth. In vitro experiments indicated that CC-Exos stimulated CPCs proliferation and increased expression of chondrogenesis markers while inhibiting angiogenesis. CONCLUSIONS: These findings suggest that the novel CC-Exos provides the preferable niche in directing stable ectopic chondrogenesis of CPCs. The use of CC-Exos may represent an off-the-shelf and cell-free therapeutic approach for promoting cartilage regeneration in the subcutaneous environment.

[ISOLATION, CULTURE AND IDENTIFICATION OF CARTILAGE DERIVED STEM CELLS FROM THREE SUBTYPES OF CARTILAGES].

OBJECTIVE: To isolate and culture cartilage derived stem cells from different subtypes of cartilages, and to identify their characteristics. METHODS: Cartilage derived stem cells were isolated from different subtypes of cartilages (auricle cartilage, articular cartilage, and intervertebral cartilage) by using adhesive method of fibronectin. The expressions of positive surface markers (CD29 and CD90) and negative surface markers (CD34 and CD45) in cartilage derived stem cells were detected via flow cytometry. The single cell colony-forming efficiency of cartilage derived stem cells was determined by clonal formation unit test; the multipotent differentiation capacity was identified by chondrogensis, osteogenesis, and adipogenesis induction. RT-PCR was used to test the expression of osteogenic, chondrogenic, and adipogenic genes; and bone marrow mesenchymal stem cells (BMSCs) served as control. RESULTS: Three cell populations were successfully isolated from different subtypes of cartilages, which could express CD29 and CD 90 highly, but did not express CD34 and CD45. After 2 weeks of culture, single cartilage derived stem cell could form single cell colony. In addition, cartilage derived stem cells had high chondrogenesis, osteogenesis, and adipogenesis potentials. After osteogenic induction, the expressions of collagen type I and collagen type X in articular and intervertebral cartilage stem cells were significantly higher than those in BMSCs (P<0.05), while there was no significant difference between auricular cartilage stem cells and BMSCs (P>0.05). The expressions of Aggrecan and collagen type II in cartilage derived stem cells after chondrogenic induction were significantly higher than those in BMSCs (P<0.05). While the ability of adipogenic differentiation was lower than that in BMSCs, but no significant difference was found (P>0.05). CONCLUSION: Cartilage derived stem cells in different subtypes of cartilages possess typical characteristics of stem cells.

Chondrogenic differentiation of bone marrow­derived stem cells cultured in the supernatant of elastic cartilage cells.

Repair of cartilage defects remains a challenge for surgeons, owing to its poor self­repairing capacity. Cartilage tissue engineering, particularly marrow stem cell­based cartilage regeneration, provides a promising option for the regeneration of damaged cartilage. Although producing tissue­engineered cartilage from marrow stem cells appeared to be a feasible method, constructing certain sub­types of cartilage, including elastic cartilage, remains difficult. Therefore, the present study explored the feasibility of constructing elastic cartilage by culturing bone marrow­derived stem cells (BMSCs) in the supernatant of elastic cartilage cells to generate elastic cartilage. The elastic cartilage cells were obtained from the auricle cartilage of a newborn pig, and BMSCs were isolated from pig bone marrow aspirate. The supernatant of the chondrocytes was collected and then used to the culture BMSCs. At various time­points, the differentiation of BMSCs was evaluated by gross view, histological examination and quantitative polymerase chain reaction. BMSCs changed from spindle­shaped cells into polygonal cells with increasing culture time. The expression of collagen II and elastin was observed in the cells cultured in the supernatant of elastic chondrocytes, while no expression was observed in the control cells. Furthermore, the expression of collagen I and collagen X was downregulated in the cells cultured in the supernatant of elastic cartilage cells. The supernatant of elastic cartilage cells promoted the differentiation of BMSCs into elastic cartilage cells, which may be a promising method for constructing certain sub­types of tissue­engineered cartilage.

Isolation and identification of stem cells in different subtype of cartilage tissue.

BACKGROUND: Cartilage tissue engineering provided a promising therapy for the repair of cartilage defects, and seeding cells play a vital role in cartilage regeneration. Chondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs) were reported to be the ideal seeding cells, but 'dedifferentiation' and 'unstable phenotype' of tissue-engineered cartilage constructed by the two cell type hamper their clinical application. Recently, cartilage tissue was reported to possess a stem cell population, which may be a more superior cell source in cartilage tissue engineering. METHODS: In current study, we isolated a cell population from different subtype of cartilage tissue via a differential adhesion assay to fibronectin. RESULTS: Flow cytometry analysis demonstrates the cell lines expressed mesenchyme stem cell positive surface marker such as CD29 and CD90. Meanwhile, the cells are highly proliferative and multipotent. Reverse transcription-PCR detection showed the cell population expressed osteogenic and adipogenic differentiation under different induction conditions. More interesting, monolayer cells underwent chondrogenic differentiation in the presence of dexamethasone and insulin-like growth factor 1. In addition, the expression of chondrogenic genes in cartilage-derived stem cells (CSCs) was higher than those in BMSCs. CONCLUSION: CSC may become an ideal seeding cell in cartilage tissue engineering, owing to its stemness and chondrogenic characteristics.