Dynamic compression improves chondrogenesis in the tissue engineered model of cartilage.
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ABSTRACT:
PROVIDER: PRJNA1177463 | ENA |
REPOSITORIES: ENA
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Project description:Hyaline cartilage is a dense avascular tissue with low regenerative potential, present at the ends of the diarthrodial joints and in the cartilage growth plate. Skeletal diseases often result from extracellular changes in this tissue; however, studies of these are hindered by the tissue complexity, the difficulty in obtaining human material, and the cost of generating animal models. Recent developments in tissue engineering are opening possibilities to develop mechanoresponsive zonally stratified models of cartilage in vitro. In this study, we optimised a 3D model of cartilage using chondroprogenitor cells cultured for 21 days in 2% agarose hydrogel constructs with daily dynamic compression. Our hydrogel constructs developed pericellular matrices with nanostiffness comparable to native murine tissue and showed increased production of extracellular matrix components and expression of chondrogenic and differentiation markers. Daily dynamic compression resulted in progressive increase in mechanoresponsive gene expression and promoted a juvenile cartilage phenotype, decreasing expression of dedifferentiation and cartilage degradation markers. Our study highlights the potential of hydrogel-enhanced chondrogenesis and proposes an adaptable and scalable in vitro model to study mechanoresponses, intracellular signals and pericellular matrix involvement in cartilage development and disease.
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Project description:Murine primary rib-cage chondrocytes (mRCs) were isolated from E15.5 WT and HS-deficient Ext1gt/gt mice and expanded for 6-7 days. Tissue-engineered cartilage was generated by a 14-day chondrogenic culture of mRCs at 5×10^5 cells per 25 µl agarose carrier. Engineered cartilage was exposed to cyclic unconfined compression for 3 hours: 10-minute cycles of dynamic unconfined compression (25 % dynamic compression at 1 Hz superimposed to 10 % static off-set) were intermitted by 10-minute breaks (10 % static off-set).
2021-03-14 | E-MTAB-10258 | biostudies-arrayexpress
Project description:The circadian clock in murine articular cartilage is a critical temporal regulatory mechanism for tissue homeostasis and osteoarthritis. However, translation of these findings into humans has been hampered by the difficulty in obtaining circadian time series human cartilage tissues. As such, a suitable model is needed to understand the initiation and regulation of circadian rhythms in human cartilage. We used a chondrogenic differentiation protocol on human embryonic stem cells (hESCs) as a proxy for early human chondrocyte development. Chondrogenesis was validated using histology and expression of pluripotency and differentiation markers. The molecular circadian clock was tracked in real time by lentiviral transduction of human clock gene luciferase reporters. Differentiation-coupled gene expression was assessed by RNAseq and differential expression analysis.
2022-08-16 | E-MTAB-11758 | biostudies-arrayexpress
Project description:Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression and cellular processes; however, their roles in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified a novel lncRNA, Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND), as a regulator of mesenchymal stem cell (MSC) chondrogenesis, and we investigated its basic molecular mechanism and its potential application towards regenerative medicine. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SRY-Box 9 (SOX9), but not Transforming Growth Factor Beta 3 (TGF-β3). Utilizing the established model of pellet formation for MSC chondrogenesis, we showed that the silencing of GRASLNDresulted in lower accumulation of cartilage-like extracellular matrix, while GRASLND overexpression, either via transgene ectopic expression or by endogenous activation via CRISPR, significantly enhanced cartilage matrix production. GRASLND acts to inhibit IFN-γ by binding to eukaryotic initiation factor-2 kinase PKR. We further demonstrated that GRASLND exhibited a protective effect in engineered cartilage against interferon type II, and that GRASLND exerted this effect similarly in engineered cartilage across different sources of progenitors. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.
2020-04-06 | GSE129985 | GEO
Project description:Purpose: To identify how axitinib stabilized the rabbit's engineered cartilage through the niche Methods: Rabbit's engineered cartilage was encapsulated in the PCL/collagen nanofibrous membrane containing no axitinib (0%-Axitinib group) or 3% axitinib (3%-Axitinib group) and implanted into nude mice. After 12 weeks of implantation, samples from the 0%-Axitinib group and 3%-Axitinib group were harvested, with the murine tissues entangled. Transcriptional profiles were generated by deep sequencing, in triplicate, using an Illumina HiSeqTM2000 machine, and filtered using Fastp software. The trimmed reads were mapped to the mouse genome (mm10, genome.UCSC.edu) using the STAR software to identify the transcriptional information in the niche of engineered cartilage. Then, the DESeq2 “counts” function of the R package was used to normalize gene expression levels based on the number of total reads of each sample. Differentially expressed genes between the 0%-Axitinib group and 3%-Axitinib group were identified as meeting the criteria of fold change ≥ 1.5 and P-value＜0.05. Lastly, we used Metascape (http://metascape.org) for gene ontology analysis. Results: Using an optimized data analysis workflow, we mapped about 20 to 30 million sequence reads per sample to the mouse genome (build mm10) and identified 20318 transcripts in the 0%-Axitinib and 3%-Axitinib groups. 837 genes were differentially expressed by more than 1.5-fold, p-value <0.05, with 313 genes downregulated and 524 genes upregulated in the 3%-Axitinib group. Gene ontology (GO) analysis revealed that the genes downregulated in the 3%-Axitinib group were mainly enriched in endothelial development, angiogenesis, and immune response. Genes upregulated in the 3%-Axitinib group were mainly enriched in response to hypoxia, Hif-1 signaling pathway, cartilage differentiation and development, and muscle structure development. Altered expression of 15 genes was further illustrated by heatmap, demonstrating the inhibited angiogenesis and improved chondrogenesis. Especially, Timp1, as a response to hypoxia, was remarkably upregulated, which also in turn inhibited the activity of matrix metalloproteinases (MMPs) and played an important role in preventing ossification. Conclusion: In this study, we proposed the first detailed transcriptional analysis of the effects of the sustainable and localized release of axitinib on the subcutaneous niche of engineered cartilage. The sustainable and localized release of axitinib created a relatively avascular, hypoxia, and low immune response niche in the 3%-Axitinib group. In this niche, Timp1 was remarkably upregulated, which inhibited the activity of MMPs and prevented endochondral ossification.
2021-08-16 | GSE160825 | GEO
Project description:Analysis of gene expression differences between human neonatal articular cartilage and human mesenchymal stem cells (hMSCs)-derived cartilage, to identify novel molecular signature for tissue engineered articular cartilage Abstract: Cellular differentiation comprises a progressive, multistep program that drives cells to fabricate a tissue with specific and site distinctive structural and functional properties. Cartilage constitutes one of the potential differentiation lineages that mesenchymal stem cells (MSCs) can follow under the guidance of specific bioactive agents. Single agents such as transforming growth factor beta (TGF-b) and bone morphogenetic protein 2 in unchanging culture conditions have been historically used to induce in vitro chondrogenic differentiation of MSCs. Despite the expression of traditional chon- drogenic biomarkers such as type II collagen and aggrecan, the resulting tissue represents a transient cartilage rather than an in vivo articular cartilage (AC), differing significantly in structure, chemical composition, cellular phenotypes, and mechanical properties. Moreover, there have been no comprehensive, multicomponent parameters to define high- quality and functional engineered hyaline AC. To address these issues, we have taken an innovative approach based on the molecular interrogation of human neonatal articular cartilage (hNAC), dissected from the knees of 1-month-old cadaveric specimens. Subsequently, we compared hNAC-specific transcriptional regulatory elements and differentially expressed genes with adult human bone marrow (hBM) MSC-derived three-dimensional cartilage structures formed in vitro. Using microarray analysis, the transcriptome of hNAC was found to be globally distinct from the transient, cartilage-like tissue formed by hBM-MSCs in vitro. Specifically, over 500 genes that are highly expressed in hNAC were not expressed at any time point during in vitro human MSC chondrogenesis. The analysis also showed that the differences were less variant during the initial stages (first 7 days) of the in vitro chondrogenic differentiation program. These observations suggest that the endochondral fate of hBM-MSC-derived cartilage may be rerouted at earlier stages of the TGF-b-stimulated chondrogenic differentiation program. Based on these analyses, several key molecular dif- ferences (transcription factors and coded cartilage-related proteins) were identified in hNAC that will be useful as molecular inductors and identifiers of the in vivo AC phenotype. Our findings provide a new gold standard of a molecularly defined AC phenotype that will serve as a platform to generate novel approaches for AC tissue engineering.
2019-11-23 | GSE140861 | GEO
Project description:Cartilage tissue engineering seeks to replace degenerated or damaged cartilage following disease or injury. Mesenchymal stem/stromal cells (MSCs) provide an attractive cell source for cartilage repair; however, the underlying molecular pathways that drive chondrogenesis of these pools of adult stem cells remains poorly understood. Here, we generated a rich data set of high throughput RNA sequencing of human MSCs throughout chondrogenesis at six different time points. Our data is consisted of 18 libraries with three individual donors as biological replicates, each library possessing a sequencing depth of 100 million reads. We also performed flow cytometric, histological, and biochemical analyses in parallel to validate the quality of our in vitro engineered cartilage. Differential gene expression and gene ontology analyses identified dynamic changes in multiple biological pathways, namely downregulation in cell cycle and proliferation, and upregulation in extracellular matrix synthesis. Weighted gene correlation network analysis also identified an important chondrogenic gene subset, whose functional characterization promises to further harness the potential of MSCs for cartilage tissue engineering. Furthermore, we created a graphic user interface encyclopedia built with the goal of producing an open resource of transcriptomic regulation for additional data-mining and pathway analysis of the process of MSC chondrogenesis. The tool can be accessed at: http://msc-browser.guilaklab.com
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Project description:Deer antler-derived reserve mesenchyme cells (RMCs) are a promising source of cells for cartilage regeneration therapy due to their chondrogenic differentiation potential. However, the regulatory mechanism has not yet been elucidated. microRNAs (miRNAs) have been shown to regulate the differentiation of various mesenchymal stem cells (MSCs) and play an important role in the post-transcriptional regulation of chondrogenesis and hypertrophic differentiation. In this study, we demonstrated that the RMCs showed typical MSCs differentiation potentials. During chondrogenic differentiation, we obtained the expression profile of miRNAs, among which miR-145 was the most prominent candidate as key microRNA involved in the balance of chondral and endochondral differentiation. Knockdown of miR-145 promoted chondrogenesis and inhibits hypertrophy differentiation in RMCs. Mechanically, by online databases prediction combined with dual-luciferase reporter assay, SOX9 was suggested as a target of miR-145. Further validation experiments confirmed that knockdown of miR-145 contributed to the balance between endochondral versus chondral differentiation of RMCs by targeting SOX9. Additionally, RMCs transfected with the miR-145 knockdown mediated of lentiviral vector successfully promoted cartilage regeneration in vivo. In summary, our study suggested that the reciprocal negative feedback between SOX9 and miR-145 was essential for balancing between endochondral versus chondral differentiation of RMCs. Our study suggested that modification of RMCs using miRNAs transduction might be an effective treatment for cartilage defects.
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Project description:This study investigates the transcriptome response of meniscus fibrochondrocytes (MFCs) to the low oxygen and mechanical loading signals experienced in the knee joint using a model system. We hypothesized that short term exposure to the combined treatment would promote a matrix-forming phenotype supportive of inner meniscus tissue formation. Human MFCs on a collagen scaffold were stimulated to form fibrocartilage over 6weeks under normoxic (NRX, 20% O2) conditions with supplemented TGF -β3. Tissues experienced a delayed 24h hypoxia treatment (HYP, 3% O2) and then 5min of dynamic compression (DC) between 30 and 40% strain. Delayed HYP induced an anabolic and anti-catabolic expression profile for hyaline cartilage matrix markers, while DC induced an inflammatory matrix remodeling response along with upregulation of both SOX9 and COL1A1. There were 41 genes regulated by both HYP and DC. Overall, the combined treatment supported a unique gene expression profile favouring the hyaline cartilage aspect of inner meniscus matrix and matrix remodeling.
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Project description:Long-term dynamic compression enhanced the mechanical properties of MSC-seeded constructs only when loading was initiated after 21 days of chondrogenic differentiation. This study examined the molecular differences of chondrogenic MSCs compared to undifferentiated MSCs (TGF-beta vs no TGF-beta) and the effects of dynamic loading on MSC chondrogenesis (loading vs free-swelling).
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