Anne Paumier

Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.

Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures.

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self‐repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)‐2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long‐term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real‐time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP‐2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP‐2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP‐2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT. J. Cell. Biochem. 111: 1642–1651, 2010.

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

BackgroundArticular 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.ResultsChondrocytes 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.ConclusionThe 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.

Investigating conversion of mechanical force into biochemical signaling in three-dimensional chondrocyte cultures

The culture of chondrocytes embedded within agarose hydrogels maintains chondrocytic phenotype over extended periods and allows analysis of the chondrocyte response to mechanical forces. The mechanisms involved in the transduction of a mechanical stimulus to a physiological process are not completely deciphered. We present protocols to prepare and characterize constructs of murine chondrocytes and agarose (1 week pre-culture period), to analyze the effect of compression on mRNA level by RT-PCR (2–3 d), gene transcription by gene reporter assay (3 d) and phosphorylation state of signaling molecules by western blotting (3–4 d). The protocols can be carried out with a limited number of mouse embryos or newborns and this point is particularly important regarding genetically modified mice.

Bone morphogenetic protein-2 stimulates chondrogenic expression in human nasal chondrocytes expanded in vitro.

Articular cartilage contains an extracellular matrix with characteristic macromolecules such as type II collagen. Because this tissue is avascular and mature chondrocytes do not proliferate, cartilage lesions have a limited capacity for healing after trauma. Autologous chondrocyte implantation (ACI) is widely used for the treatment of patients with focal damage to articular cartilage. However, this method faces a major issue: dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to determine if the step of cell amplification required for ACI could benefit from the use of bone morphogenetic protein (BMP)-2, a potent regulator of chondrogenic expression. Chondrocytes were isolated from human nasal cartilage, a hyaline cartilage like articular cartilage and were serially cultured in monolayers. After one, two or three passages, BMP-2 was used to evaluate the chondrogenic potential of the dedifferentiated chondrocytes, at the gene and protein level. We found that BMP-2 can reactivate the program of chondrogenic expression in dedifferentiated chondrocytes. To gain insight into the molecular mechanisms involved in the responsiveness of chondrocytes to BMP-2, we examined the phosphorylation of Smad proteins and the interaction of the Sry-type high-mobility-group box (Sox) transcription factors with the cartilage-specific enhancer of the type II procollagen gene. Our results show that BMP-2 acts by stimulating Smad phosphorylation and by enhancing DNA-binding of the Sox transcription factors to the specific enhancer of the type II procollagen gene. Thus, this study reveals the potential use of BMP-2 as a stimulatory agent in conventional ACI strategies.

Alteration of cartilage mechanical properties in absence of β1 integrins revealed by rheometry and FRAP analyses

CONTEXT Mechanical properties are essential for biological functions of the hyaline cartilage such as energy dissipation and diffusion of solutes. Mechanical properties are primarily dependent on the hierarchical organization of the two major extracellular matrix (ECM) macromolecular components of the cartilage: the fibrillar collagen network and the glycosaminoglycan (GAG)-substituted proteoglycan, mainly aggrecan, aggregates. Interaction of chondrocytes, the only cell type in the tissue, with the ECM through adhesion receptors is involved in establishing mechanical stability via bidirectional transduction of both mechanical forces and chemical signals. In this study, we aimed to determine the role of the transmembrane β1 integrin adhesion receptors in cartilage biomechanical properties by the use of genetic modification in mice. METHODS Costal cartilages of wild type and mutant mice lacking β1 integrins in chondrocytes were investigated. Cartilage compressive properties and solute diffusion were characterized by rheometric analysis and Fluorescence Recovery After Photobleaching (FRAP), respectively. Cartilage tissue sections were analyzed by histology, immunohistochemistry and transmission electron microscopy (TEM). RESULTS At the histological level, the mutant costal cartilage was characterized by chondrocyte rounding and loss of tissue polarity. Immunohistochemistry and safranin orange staining demonstrated apparently normal aggrecan and GAG levels, respectively. Antibody staining for collagen II and TEM showed comparable expression and organization of the collagen fibrils between mutant and control cartilages. Despite the lack of gross histological and ultrastructural abnormalities, rheological measurements revealed that the peak elastic modulus in compression of mutant cartilage was 1.6-fold higher than the peak elastic modulus of wild-type sample. Interestingly, the diffusion coefficient within the mutant cartilage tissue was found to be 1.2-fold lower in the extracellular space and 14-fold lower in the pericellular (PCM) space compared to control. CONCLUSION The results demonstrate that the absence of β1 integrins on the surface of chondrocytes increases the stiffness and modifies the diffusion properties of costal cartilage. Our data imply that β1 integrins-mediated chondrocyte-matrix interactions directly affect cartilage biomechanics probably by modifying physical properties of individual cells. This study thus highlights the crucial role of β1 integrins in the cartilage function.

Réponse des chondrocytes humains à la bone morphogenetic protein-2 après leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage

AIM OF THE STUDY Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic. MATERIALS AND METHODS Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored. RESULTS Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages. CONCLUSION The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.

Predictors of failed intrauterine balloon tamponade for persistent postpartum hemorrhage after vaginal delivery

Objective To identify the predictors of intrauterine balloon tamponade (IUBT) failure for persistent postpartum hemorrhage (PPH) after vaginal delivery. Design Retrospective case-series in five maternity units in a perinatal network. Setting All women who underwent IUBT for persistent PPH after vaginal delivery from January 2011 to December 2015 in these hospitals. Methods All maternity apply the same management policy for PPH. IUBT, using a Bakri balloon, was used as a second line therapy for persistent PPH after failure of bimanual uterine massage and uterotonics to stop bleeding after vaginal delivery. Women who required another second line therapy (embolization or surgical procedures) to stop bleeding after IUBT were defined as cases, and women whom IUBT stopped bleeding were defined as control group. We determined independent predictors for failed IUBT using multiple regression and adjusting for demographics with adjusted odds ratios (aORs) and 95% confidence intervals (95% CI). Results During the study period, there were 91,880 deliveries in the five hospitals and IUBT was used in 108 women to control bleeding. The success rate was 74.1% (80/108). In 28 women, invasive procedures were required (19 embolization and 9 surgical procedures with 5 peripartum hysterectomies). Women with failed IUBT were more often obese (25.9% vs. 8.1%; p = 0.03), duration of labor was shorter (363.9 min vs. 549.7min; p = 0.04), and major PPH (≥1,500 mL) before IUBT was more frequent (64% vs. 40%; p = 0.04). Obesity was a predictive factor of failed IUBT (aOR 4.40, 95% CI 1.06–18.31). Major PPH before IUBT seemed to be another predictor of failure (aOR 1.001, 95% CI 1.000–1.002), but our result did not reach statistical significativity. Conclusion Intrauterine balloon tamponade is an effective second line therapy for persistent primary PPH after vaginal delivery. Pre-pregnancy obesity is a risk factor of IUBT failure.