Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression

<p>Abstract</p> <p>Background</p> <p>Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice.</p> <p>Results</p> <p>Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell^®Compression Plus™ System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading.</p> <p>Conclusion</p> <p>The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.</p

The epigenetic players and the chromatin marks involved in the articular cartilage during osteoarthritis

Epigenetics defines the modifications of the genome that do not involve a change in the nucleotide sequence of DNA. These modifications constitute a mechanism of gene regulation poorly explored in the context of cartilage physiology. They are now intensively studied by the scientific community working on articular cartilage and its related pathology such as osteoarthritis. Indeed, epigenetic regulations can control the expression of crucial gene in the chondrocytes, the only resident cells of cartilage. Some epigenetic changes are considered as a possible cause of the abnormal gene expression and the subsequent alteration of the chondrocyte phenotype (hypertrophy, proliferation, senescence…) as observed in osteoarthritic cartilage. Osteoarthritis is a joint pathology, which results in impaired extracellular matrix homeostasis and leads ultimately to the progressive destruction of cartilage. To date, there is no pharmacological treatment and the exact causes have yet to be defined. Given that the epigenetic modifying enzymes can be controlled by pharmacological inhibitors, it is thus crucial to describe the epigenetic marks that enable the normal expression of extracellular matrix encoding genes, and those associated with the abnormal gene expression such as degradative enzyme or inflammatory cytokines encoding genes. In this review, only the DNA methylation and histone modifications will be detailed with regard to normal and osteoarthritic cartilage. Although frequently referred as epigenetic mechanisms, the regulatory mechanisms involving microRNAs will not be discussed. Altogether, this review will show how this nascent field influences our understanding of the pathogenesis of OA in terms of diagnosis and how controlling the epigenetic marks can help defining epigenetic therapies

A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton’s jelly samples

Transplantation of mesenchymal stem/stromal cells (MSCs) has emerged as an effective method to treat diseased or damaged organs and tissues, and hundreds of clinical trials using MSCs are currently under way to demonstrate the validity of such a therapeutic approach. However, most MSCs used for clinical trials are prepared in research laboratories with insufficient manufacturing quality control.In particular, laboratories lack standardized procedures for in vitro isolation of MSCs from tissue samples, resulting in heterogeneous populations of cells and variable experimental and clinical results. MSCs are now referred to as Human Cellular Tissue-based Products or Advanced Therapy Medicinal Products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and from the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate production of these cells. These guidelines, commonly called “Good Manufacturing Practices” (GMP), include recommendations about laboratory cell culture procedures to ensure optimal reproducibility, efficacy and safety of the final medicinal product. In particular, the Food and Drug Administration divides ex vivo cultured cells into “minimally” and “more than minimally” manipulated samples, in function of the use or not of procedures “that might alter the biological features of the cells”. Today, minimal manipulation conditions have not been defined for the collection and isolation of MSCs (Torre et al. 2015)(Ducret et al. 2015).Most if not all culture protocols that have been reported so far are unsatisfactory, because of the use of xeno- or allogeneic cell culture media, enzymatic treatment and long-term cell amplification that are known to alter the quality of MSCs. The aim of this study was to describe a standardized procedure for recovering MSCs with minimal handling from two promising sources, the dental pulp (DP) and the Wharton’s jelly (WJ) of the umbilical cord. The quality and homogeneity of the expanded cell populations were assessed by using flow cytometry with criteria that go beyond the International Society of Cellular Therapy (ISCT) guidelines for MSC characterization

Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A

Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification

International audienceMesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31 − cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31 − DP cell population contained 1.4% of CD56 + , 1.5% of CD146 + , 2.4% of CD271 + and 6.3% of MSCA-1 + cells but very few Stro-1 + cells (≤1%). CD56 + , CD146 + , CD271 + , and MSCA-1 + cell subpopulations expressed various levels of these markers. CD146 + MSCA-1 + , CD271 + MSCA-1 + , and CD146 + CD271 + cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56 + , CD271 + , MSCA-1 + , and Stro-1 + cells, and MSCA-1 by 15-30% of CD56 + , CD146 + , CD271 + , and Stro-1 + cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56 + cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146 + CD56 + , MSCA-1 + CD56 + , and CD146 + MSCA-1 + cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products

Improvement of the chondrocyte-specific phenotype upon equine bone marrow mesenchymal stem cell differentiation. Influence of TGF-ß1 or TGF-ß3, associated with BMP-2 and type I collagen siRNAs

International audienceArticular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Consequently, cartilage markers, such as type II collagen, are degraded whereas atypic molecules, such as type I collagen, are newly synthetized. Another essential phenomenon occurring in OA is the upregulation of HtrA1, a serine protease targeting upstream receptors of signalling pathways involved in the synthesis of articular cartilage markers. OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. The aim of this study is to improve the autologous chondrocyte implantation strategy by enhancing the chondrogenic differentiation of mesenchymal stem cells (MSCs) in order to increase the type II collagen/ type I collagen ratio

ZAK beta is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAK beta is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKO's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.Peer reviewe

Structure et formation des fibres de collagene : etude ultrastructurale apres congelation rapide suivie de cryosubstitution ou cryofracture

SIGLECNRS T 56282 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc