Frédéric Mallein-Gerin

Chondrocytes or adult stem cells for cartilage repair: the indisputable role of growth factors.

Articular cartilage is easily injured but difficult to repair and cell therapies are proposed as tools to regenerate the defects in the tissue. Both differentiated chondrocytes and adult mesenchymal stem cells (MSCs) are regarded as cells potentially able to restore a functional cartilage. However, it is a complex process from the cell level to the tissue end product, during which growth factors play important roles from cell proliferation, extracellular matrix synthesis, maintenance of the phenotype to induction of MSCs towards chondrogenesis. Members of the TGF-β superfamily, are especially important in fulfilling these roles. Depending on the cell type chosen to restore cartilage, the effect of growth factors will vary. In this review, the roles of these factors in the maintenance of the chondrocyte phenotype are discussed and compared with those of factors involved in the repair of cartilage defects, using chondrocytes or adult mesenchymal stem cells.

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.

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.

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24xa0days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.

BMP-2 and TGF-β1 differentially control expression of type II procollagen and α10 and α11 integrins in mouse chondrocytes

Bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and protein level. In this study, the expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression.

Identification of four alternatively spliced transcripts of the Ucma/GRP gene, encoding a new Gla-containing protein.

The Ucma protein (Upper zone of growth plate and cartilage matrix associated protein) has recently been described as a novel secretory protein mainly expressed in cartilage and also as a novel vitamin-K-dependent protein named GRP (Gla-rich protein). This protein has the highest Gla content of any protein known to date. In this article, we identify four alternatively spliced variants of Ucma/GRP gene transcripts in mouse chondrocytes. We show that the expression of all four isoforms is associated with the early stages of chondrogenesis. The Ucma/GRP gene encodes four proteins named Ucma/GRP-F1, -F2, -F3, and -F4, which differ by exon 2, exon 4, or both. Among them, only Ucma/GRP-F1 and -F3 were secreted into the culture medium of transfected chondrocytes, while Ucma/GRP-F2 and -F4 accumulated in the cells. Using HeLa cells or freshly isolated embryonic mouse chondrocytes transfected with enhanced green fluorescent protein fusions, microscopy analysis revealed that Ucma/GRP-F1 and -F3 were localized in the Golgi complex, whereas Ucma/GRP-F2 and -F4 formed aggregates. This aggregation was microtubule-dependent since disruption of microtubules with nocodazole reduced Ucma/GRP-F2 and -F4 aggregation in a reversible manner. Using biochemical fractionation and Western blot analysis, Ucma/GRP-F1 and -F3 isoforms were detected in the soluble fraction while Ucma/GRP-F2 and -F4 were found in an insoluble-enriched fraction. We conclude that the co-expression of soluble and insoluble isoforms also Gla-rich and Gla-deleted isoforms may be finely tuned. Imbalance in isoform expression may therefore be involved in skeletal pathology.

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

CONTEXTnMechanical 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.nnnMETHODSnCostal 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).nnnRESULTSnAt 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.nnnCONCLUSIONnThe 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.

Stable subclones of the chondrogenic murine cell line MC615 mimic distinct stages of chondrocyte differentiation

Fourteen stable subclones derived from the murine chondrogenic cell line MC615 were established and characterised regarding their differentiation stages and responsivity to BMP2. Based on their gene expression profiles which revealed remarkable variances in Col2a1 and Col10a1 expression, subclones could be grouped into at least three distinct categories. Three representative subclones (4C3, 4C6 and 4H4) were further characterised with respect to gene expression pattern and differentiation capacity. These subclones resembled (i) weakly differentiated chondrogenic precursors, strongly responding to BMP2 stimulation (4C3), (ii) collagen II expressing chondrocytes which could be induced to undergo maturation (4C6) and (iii) mature chondrocytes expressing Col10a1 and other markers of hypertrophy (4H4). Interestingly, BMP2 administration caused Smad protein phosphorylation and stimulated Col10a1 expression in all clones, but induced Col2a1 expression only in precursor‐like cells. Most remarkably, these clones maintained a stable gene expression profile at least until the 30th passage of subconfluent culture, but revealed reproducible changes in gene expression and differentiation pattern in long term high density cultures. Thus, the newly established MC615 subclones may serve as a potent new tool for investigations on the regulation of chondrocyte differentiation and function. J. Cell. Biochem. 108: 589–599, 2009.