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Micromass Cultures (micromass + culture)
Selected AbstractsTransgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cellsTHE JOURNAL OF GENE MEDICINE, Issue 1 2006Jung Park Abstract Background Adult primary mesenchymal cells of different origin which can be obtained with minor donor site morbidity are considered for articular cartilage repair. This study aims at a comparison of their chondrogenic potential. Methods Mesenchymal cells were isolated from perichondrium/periosteum, bone marrow or fat of adult rats and found to be positive for the stem-cell-related antigens Sca-1, c-Kit, CD10, CD13 and CD90 by reverse transcription polymerase chain reaction (RT-PCR). Chondrogenic differentiation was induced by applying recombinant bone morphogenetic protein-2 (BMP-2) or adenoviral vectors carrying BMP-2 cDNA, followed by micromass culture. The stimulated cells were characterized by RT-PCR, cell proliferation and apoptosis assays. Expression of aggrecan, collagen type I, II, IX and X and alkaline phosphatase genes was analyzed by RT-PCR, immunofluorescence and immunohistochemistry in comparison with unstimulated control cells. Adenovirally stimulated cells were transplanted into mechanically generated partial-thickness cartilage lesions in the patellar groove of the rat femur. Quality and integration of the repair tissues were assessed by histochemical and immunohistochemical methods. Results Stimulation with BMP-2 or AdBMP-2 led to an up-regulation of cartilage-specific gene expression in all three cell populations studied, most rapidly and prominently in the perichondrial/periosteal cells, which showed a 3200-fold increase of type II collagen mRNA and reached the highest absolute levels of type II and IX collagen transcripts after stimulation. Similar results were obtained for the bone marrow stromal cells (BMSC), while the respective transcript levels in fat stromal cells declined after an initial more than 30-fold elevation. Following transplantation in vivo, AdBMP-2-infected perichondrial/periosteal cells produced a proteoglycan-rich, type II collagen-positive matrix with only faint staining for type I collagen. The repair tissue originating from AdBMP-2-infected BMSC showed less intense type II collagen staining, but a relatively proteoglycan-rich matrix, weakly positive for type I collagen. Transgene-activated fat stromal cells formed rather fibrous tissue mainly composed of type I collagen. Unstimulated cells of the three different populations gave only rise to fibrous tissue. Conclusions Perichondrium/periosteum-derived cells and BMSC seem superior to cells isolated from fat with respect to forming hyaline cartilaginous tissue. A chondrogenic stimulus, e.g. by transfer of BMP-2 cDNA, appears to be required for initiation and support of chondrogenic differentiation. Copyright © 2005 John Wiley & Sons, Ltd. [source] Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro,DEVELOPMENTAL DYNAMICS, Issue 2 2002Anthony M. Delise Abstract During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell,cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1,2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation. © 2002 Wiley-Liss, Inc. [source] Vascular regression is required for mesenchymal condensation and chondrogenesis in the developing limbDEVELOPMENTAL DYNAMICS, Issue 3 2001Melinda Yin Abstract Vascular regression occurs during limb mesenchymal cell condensation and chondrogenesis, but it is unclear whether it is required for these processes or is a secondary phenomenon without major regulatory roles. To address this issue, beads presoaked with the potent angiogenic factor vascular endothelial growth factor (VEGF) were implanted in the vicinity of the prospective digit 2 in early chick embryo wing buds and the effects on angiogenesis and digit development were determined over time. We found that VEGF treatment caused a marked local increase in blood vessel number and density. Strikingly, this was accompanied by inhibition of digit 2 development as revealed by lack of expression of chondrogenic transcription factor Sox9 and absence of Alcian blue staining. Vascular distribution and skeletal development in adjacent areas remained largely unaffected. Inhibition of digit formation and excess vascularization were both reversible upon further embryonic growth and dissipation of VEGF activity. When supernumerary digits were induced at the anterior limb margin by retinoic acid treatment, their development was also preceded by vascular regression; interestingly, cotreatment with VEGF inhibited supernumerary digit development as well. Direct exposure of limb mesenchymal cells in micromass cultures to VEGF caused no obvious effects on condensation and chondrogenesis, indicating that VEGF effects are not due to direct action on skeletal cells. Our results are the first to provide evidence that vascular regression is required for mesenchymal condensation and chondrogenesis. A model of how patterning mechanisms and vascular regression may intersect and orchestrate limb skeletogenesis is proposed. © 2001 Wiley-Liss, Inc. [source] Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factorsARTHRITIS & RHEUMATISM, Issue 8 2010Rosa Maria Borzí Objective To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and ,-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. Results Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, ,-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13,remodeled or ,degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusion MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors. [source] | ||||||||||