No abstract

Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

Umbilical cord blood (UCB) is a promising alternative source of mesenchymal stem cells (MSCs), because UCB-MSCs are abundant and harvesting them is a painless non-invasive procedure. Potential clinical applications of UCB-MSCs have been identified, but their ability for chondrogenic differentiation has not yet been fully evaluated. The aim of our work was to characterize and determine the chondrogenic differentiation potential of human UCB-MSCs (hUCB-MSCs) for cartilage tissue engineering using an approach combining 3D culture in type I/III collagen sponges and chondrogenic factors. Our results showed that UCB-MSCs have a high proliferative capacity. These cells differentiated easily into an osteoblast lineage but not into an adipocyte lineage. Furthermore, BMP-2 and TGF-β1 potentiated chondrogenic differentiation, as revealed by a strong increase in mature chondrocyte-specific mRNA (COL2A1, COL2B, ACAN) and protein (type II collagen) markers. Although growth factors increased the transcription of hypertrophic chondrocyte markers such as COL10A1 and MMP13, the cells present in the neo-tissue maintained their phenotype and did not progress to terminal differentiation and mineralization of the extracellular matrix after subcutaneous implantation in nude mice. Our study demonstrates that our culture model has efficient chondrogenic differentiation, and that hUCB-MSCs can be a reliable source for cartilage tissue engineering.

Umbilical cord blood (UCB) is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs) to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2) and transforming growth factor-β (TGF-β1)) and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O2) or hypoxia (<5% O2) for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-β1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13) expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering.

Mesenchymal stem cells (MSCs) hold promise for cartilage engineering. Here, we aimed to determine the best culture conditions to induce chondrogenesis of MSCs isolated from bone marrow (BM) of aged osteoarthritis (OA) patients. We showed that these BM-MSCs proliferate slowly, are not uniformly positive for stem cell markers, and maintain their multilineage potential throughout multiple passages. The chondrogenic lineage of BM-MSCs was induced in collagen scaffolds, under normoxia or hypoxia, by BMP-2 and/or TGF-β1. The best chondrogenic induction, with the least hypertrophic induction, was obtained with the combination of BMP-2 and TGF-β1 under hypoxia. Differentiated BM-MSCs were then transfected with siRNAs targeting two markers overexpressed in OA chondrocytes, type I collagen and/or HtrA1 protease. siRNAs significantly decreased mRNA and protein levels of type I collagen and HtrA1, resulting in a more typical chondrocyte phenotype, but with frequent calcification of the subcutaneously implanted constructs in a nude mouse model. Our 3D culture model with BMP-2/TGF-β1 and COL1A1/HtrA1 siRNAs was not effective in producing a cartilage-like matrix in vivo. Further optimization is needed to stabilize the chondrocyte phenotype of differentiated BM-MSCs. Nevertheless, this study offers the opportunity to develop a combinatory cellular therapy strategy for cartilage tissue engineering.

A significant association between a polymorphism in the D repeat of the gene encoding asporin and osteoarthritis, the most frequent of articular diseases, has been recently reported. The goal of the present study was to investigate the expression of this new class I small leucine-rich proteoglycan (SLRP) in human articular chondrocytes. First, we studied the modulation of asporin (ASPN) expression by cytokines by Western blot and reverse transcription-polymerase chain reaction. Interleukin-1β and tumor necrosis factor-α downregulated ASPN, whereas transforming growth factor-β1 (when incubated in a serum-free medium) upregulated it. Similarly to proinflammatory cytokines, chondrocyte dedifferentiation induced by a successive passages of cells was accompanied by a decreased asporin expression, whereas their redifferentiation by three-dimensional culture restored its expression. Finally, we found an important role of the transcription factor Sp1 in the regulation of ASPN expression. Sp1 ectopic expression increased ASPN mRNA level and promoter activity. In addition, using gene reporter assay and electrophoretic mobility shift assay, we showed that Sp1 mediated its effect through a region located between -473 and -140 bp upstream of the transcription start site in ASPN gene. In conclusion, this report is the first study on the regulation of asporin expression by different cytokines in human articular chondrocytes. Our data indicate that the expression of this gene is finely regulated in cartilage and suggest a major role of Sp1.

Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-β) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-β strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model.