CRISPR/Cas12a powered air-displacement enhanced evanescent wave fluorescence fiber-embedded microfluidic biochip for nucleic acid amplification-free detection of Escherichia coli O157:H7.

Simple yet ultrasensitive and contamination-free quantification of environmental pathogenic bacteria is in high demand. In this study, we present a portable clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) powered Air-displacement enhanced Evanescent wave fluorescence Fiber-embedded microfluidic Biochip (AEFB) for the high-frequency and nucleic acid amplification-free ultrasensitive detection of Escherichia coli O157:H7. The performance of AEFB was dramatically enhanced upon employing a simple air-solution displacement process. Theoretical assays demonstrated that air-solution displacement significantly enhances evanescent wave field intensity on the fiber biosensor surface and increases the V-number in tapered fiber biosensors. Consequently, light-matter interaction is strengthened, and fluorescence coupling and collection efficiency are improved, considerably enhancing sensitivity. By integrating the CRISPR biosensing mechanism, AEFB facilitated rapid, accurate, nucleic acid amplification-free detection of E.coli O157:H7 with polymerase chain reaction (PCR)-level sensitivity (176 cfu/mL). To validate its practicality, AEFB was used to detect E.coli O157:H7 in surface water and wastewater. Comparison with RT-PCR showed a strong linear relationship (R2 = 0.9871), indicating the excellent accuracy and reliability of this technology in real applications. AEFB is highly versatile and can be easily extended to detect other pathogenic bacteria, which will significantly promote the high-frequency assessment and early-warning of bacterial contamination in aquatic environments.

Augmented drug resistance of osteosarcoma cells within decalcified bone matrix scaffold: The role of glutamine metabolism.

Due to the lack of a precise in vitro model that can mimic the nature microenvironment in osteosarcoma, the understanding of its resistance to chemical drugs remains limited. Here, we report a novel three-dimensional model of osteosarcoma constructed by seeding tumor cells (MG-63 and MNNG/HOS Cl no. 5) within demineralized bone matrix scaffolds. Demineralized bone matrix scaffolds retain the original components of the natural bone matrix (hydroxyapatite and collagen type I), and possess good biocompatibility allowing osteosarcoma cells to proliferate and aggregate into clusters within the pores. Growing within the scaffold conferred elevated resistance to doxorubicin on MG-63 and MNNG/HOS Cl no. 5 cell lines as compared to two-dimensional cultures. Transcriptomic analysis showed an increased enrichment for drug resistance genes along with enhanced glutamine metabolism in osteosarcoma cells in demineralized bone matrix scaffolds. Inhibition of glutamine metabolism resulted in a decrease in drug resistance of osteosarcoma, which could be restored by α-ketoglutarate supplementation. Overall, our study suggests that microenvironmental cues in demineralized bone matrix scaffolds can enhance osteosarcoma drug responses and that targeting glutamine metabolism may be a strategy for treating osteosarcoma drug resistance.

Sophisticated Magneto-Mechanical Actuation Promotes In Situ Stem Cell Assembly and Chondrogenesis for Treating Osteoarthritis.

Abnormal mechanical loading often leads to the progressive degradation of cartilage and causes osteoarthritis (OA). Although multiple mechanoresponsive strategies based on biomaterials have been designed to restore healthy cartilage microenvironments, methods to remotely control the on-demand mechanical forces for cartilage repair pose significant challenges. Here, a magneto-mechanically controlled mesenchymal stem cell (MSC) platform, based on the integration of intercellular mechanical communication and intracellular mechanosignaling processes, is developed for OA treatment. MSCs loaded with antioxidative melanin@Fe3O4 magnetic nanoparticles (Magcells) rapidly assemble into highly ordered cell clusters with enhanced cell-cell communication under a time-varying magnetic field, which enables long-term retention and differentiation of Magcells in the articular cavity. Subsequently, via mimicking the gait cycle, chondrogenesis can be further enhanced by the dynamic activation of mechanical signaling processes in Magcells. This sophisticated magneto-mechanical actuation strategy provides a paradigm for developing mechano-therapeutics to repair cartilage in OA treatment.

Inhibition of fibroblast activation protein ameliorates cartilage matrix degradation and osteoarthritis progression.

Fibroblast activation protein (Fap) is a serine protease that degrades denatured type I collagen, α2-antiplasmin and FGF21. Fap is highly expressed in bone marrow stromal cells and functions as an osteogenic suppressor and can be inhibited by the bone growth factor Osteolectin (Oln). Fap is also expressed in synovial fibroblasts and positively correlated with the severity of rheumatoid arthritis (RA). However, whether Fap plays a critical role in osteoarthritis (OA) remains poorly understood. Here, we found that Fap is significantly elevated in osteoarthritic synovium, while the genetic deletion or pharmacological inhibition of Fap significantly ameliorated posttraumatic OA in mice. Mechanistically, we found that Fap degrades denatured type II collagen (Col II) and Mmp13-cleaved native Col II. Intra-articular injection of rFap significantly accelerated Col II degradation and OA progression. In contrast, Oln is expressed in the superficial layer of articular cartilage and is significantly downregulated in OA. Genetic deletion of Oln significantly exacerbated OA progression, which was partially rescued by Fap deletion or inhibition. Intra-articular injection of rOln significantly ameliorated OA progression. Taken together, these findings identify Fap as a critical pathogenic factor in OA that could be targeted by both synthetic and endogenous inhibitors to ameliorate articular cartilage degradation.

Bibliometric analysis of research trends in stem cell therapy for knee osteoarthritis over the period 2001-2021.

Stem cell therapy is a promising treatment for knee osteoarthritis, but few bibliometric studies have been performed on the subject. Bibliometric analysis is helpful for identifying the most influential studies in a specific field and can evaluate the global research trends in stem cell therapy for knee osteoarthritis. The Web of Science Core Collection was searched for publications from 2001 to 2021. Publication performance was analyzed using several bibliometric parameters, including VOSviewer, to identify the research landscape of trends in topics, and CiteSpace was investigated to identify the keywords that have the strongest citation bursts. From 2001 to 2021, in total, 1,345 publications explored the research on stem cells in knee osteoarthritis. The United States contributed the largest number of publications and at the top list of international collaborations. Tokyo Medical and Dental University ranked first among institutions in the overall number of articles and citations. The journal of Osteoarthritis and Cartilage had the largest number of publications. Sekiya Ichiro was the most cited author, with 32 articles. The keywords with the most frequent occurrence were "osteoarthritis," "mesenchymal stem cells," and "cartilage," in descending order of frequency. "fibroblast growth factor" and "extracellular vesicle" were the first and last searched theme terms, respectively. The number of publications on stem cells for knee osteoarthritis stays growing. Cartilage repair and paracrine function are current research hotspots for the stem cell therapy mechanism. Stem cell therapy has gradually advanced from basic research to the clinical application stage.

Dual functions of microRNA-17 in maintaining cartilage homeostasis and protection against osteoarthritis.

Damaged hyaline cartilage has no capacity for self-healing, making osteoarthritis (OA) "difficult-to-treat". Cartilage destruction is central to OA patho-etiology and is mediated by matrix degrading enzymes. Here we report decreased expression of miR-17 in osteoarthritic chondrocytes and its deficiency contributes to OA progression. Supplementation of exogenous miR-17 or its endogenous induction by growth differentiation factor 5, effectively prevented OA by simultaneously targeting pathological catabolic factors including matrix metallopeptidase-3/13 (MMP3/13), aggrecanase-2 (ADAMTS5), and nitric oxide synthase-2 (NOS2). Single-cell RNA sequencing of hyaline cartilage revealed two distinct superficial chondrocyte populations (C1/C2). C1 expressed physiological catabolic factors including MMP2, and C2 carries synovial features, together with C3 in the middle zone. MiR-17 is highly expressed in both superficial and middle chondrocytes under physiological conditions, and maintains the physiological catabolic and anabolic balance potentially by restricting HIF-1α signaling. Together, this study identified dual functions of miR-17 in maintaining cartilage homeostasis and prevention of OA.

Synovial mesenchymal stem cell-derived extracellular vesicles containing microRN555A-26a-5p ameliorate cartilage damage of osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a degenerative disease characterized by cartilage damage. We aimed to improve the understanding of the protective mechanism of synovial mesenchymal stem cell (SMSC)-derived extracellular vesicles (EVs) in cartilage damage of OA. METHODS: SMSCs and SMSC-EVs were isolated from synovial biopsies of patients without OA and then identified. The pathological microenvironment of chondrocytes in OA was simulated by inducing SW1353 cells with interleukin (IL)-1ß, followed by SMSC-EV treatment to assess SW1353 cell proliferation, apoptosis and inflammation. Endocytosis of Dil-labeled EVs by SW1353 cells was observed. microRNA (miR)-26a-5p expression in EVs and EV-treated SW1353 cells was assessed. The effect of miR-26a-5p was evaluated after it was down-regulated in SMSCs, followed by extraction of EVs, which acted on SW1353 cells. The target relationship of miR-26a-5p and phosphatase and tensin homologue (PTEN) was predicted and confirmed. The role of PTEN in OA was evaluated after it was overexpressed. Functional assays were implemented in vivo to certify the role of SMSC-EVs in OA. RESULTS: SMSC-EVs enhanced IL-1ß-induced SW1353 cell proliferation, whereas they inhibited apoptosis and inflammation. EVs were endocytosed by SW1353 cells and delivered miR-26a-5p into SW1353 cells to overexpress miR-26a-5p. Down-regulation of miR-26a-5p in EVs attenuated the protection of EVs against IL-1ß-induced cell damage. miR-26a-5p targeted PTEN, for which overexpression spoiled the protection of EVs against IL-1ß-induced cell damage. SMSC-EVs carrying miR-26a-5p repaired cartilage damage of OA. CONCLUSIONS: SMSC-EVs carried miR-26a-5p into chondrocytes to up-regulate miR-26a-5p and inhibit PTEN, thereby inhibiting apoptosis and inflammation and ameliorating cartilage damage of OA.

Hypoxia promotes differentiation of adipose-derived stem cells into endothelial cells through demethylation of ephrinB2.

BACKGROUND: Delivery of endothelial cells into the ischemic tissue is emerging as an alternative approach in revascularization of injured tissues by means of angiogenesis to restore organ function. Adipose-derived stem cells (ASCs) are a readily accessible source of the mesenchymal stem cell with rapid expansion and multidifferentiation potential. The view has emerged that endothelial cells (ECs) differentiated from ASCs is a step forward for adult vascular repair in regenerative medicine and construction of the blood vessel by tissue engineering approach. METHODS: In this study, differentiation of human ASCs (hASCs) into vascular EC lineage was induced by combined treatment of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-4 (BMP4) under hypoxia condition. The expression of CD31, VEGF-R2, and VE-cadherin was determined by immunofluorescent staining, real-time PCR, and western blot analysis. These differentiated cells acquired functional characteristics of mature ECs as determined by their tube formation ability, DiI-ac-LDL uptake, and nitric oxide secretion in vitro. The methylation status in the proximal promoter CpGs was determined by bisulfite sequencing. RESULTS: hASCs expressed endothelial cell markers including CD31, VEGF-R2, and VE-cadherin by combined treatment of VEGF and BMP4 under hypoxia condition. These differentiated cells exhibited the angiogenesis potential in vitro, and injection of these differentiated cells enhanced angiogenesis in the ischemic hindlimb of diabetic mice. Furthermore, it was found that hypoxia increased significantly EphrinB2 expression EC differentiation, which is greatly downregulated with EphrinB2 blockage. The methylation status in the proximal promoter CpG results showed that methylation of EphrinB2 promoter decreased in hASCs with exposure to hypoxia. CONCLUSION: Our data demonstrate that hASCs can be efficiently induced to differentiate into vascular EC lineages which are mediated by demethylation of ephrinB2 under hypoxia condition.

Intra-articular tracking of adipose-derived stem cells by chitosan-conjugated iron oxide nanoparticles in a rat osteoarthritis model.

Adipose-derived stem cells (ADSCs) hold great potential in cartilage tissue engineering due to their multipotency and ease of availability. MRI is an effective and noninvasive imaging approach to track cells and observe new tissue regeneration. It is essential to find a compatible and efficient imaging reagent without affecting the stemness of ADSCs. Herein, we developed chitosan-modified iron oxide nanoparticles (IO-CS) as the T 2 contrast reagent with good cell compatibility and high cellular uptake efficiency and used IO-CS for ADSC intra-articular imaging in a rat osteoarthritis (OA) model. TEM demonstrated the great morphology and size distribution of IO-CS nanoparticles with the size of 17 nm. Magnetization (29.4 emu per g) and MRI tests confirmed (R 2 of 184 mM-1 s-1) the feasibility of IO-CS nanoparticles as an MRI contrast reagent. In addition, the IO-CS nanoparticles showed good cellular compatibility and high labeling efficiency as compared to the commercial agent ferumoxytol. Moreover, incorporation of IO-CS nanoparticles did not alter the adipogenic, osteogenic and chondrogenic differentiation ability of ADSCs. Furthermore, the MRI transverse R 2 maps showed a persistence time of the IO-CS nanoparticles in ADSCs of 6 days in vitro. Then, we investigated the imaging capability of the IO-CS nanoparticle-labeled ADSCs in vivo with MRI for 5 weeks. The histological studies demonstrated the intra-articular biodistribution of the IO-CS nanoparticles, including in the cartilage superficial layer, synovial sublining layer, periosteum and bone marrow cavity. They provided systemic distribution information of the ADSCs in the OA rat model. In summary, we developed an accessible and effective T 2 imaging reagent with good biocompatibility and maintenance of the stemness of ADSCs. This showed the potential translational application of IO-CS nanoparticles as an MRI reagent in cartilage tissue engineering.