Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

Enhanced chondrogenesis of bone marrow-derived stem cells by using a combinatory cell therapy strategy with BMP-2/TGF-ß1, hypoxia, and COL1A1/HtrA1 siRNAs.

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-ß1. The best chondrogenic induction, with the least hypertrophic induction, was obtained with the combination of BMP-2 and TGF-ß1 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-ß1 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.

Shell extracts of the edible mussel and oyster induce an enhancement of the catabolic pathway of human skin fibroblasts, in vitro.

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. In this study, we investigated the effects of matrix macromolecular components extracted from the shells of two edible molluscs of economic interest, i.e., the blue mussel Mytilus edulis and the Pacific oyster Crassostrea gigas. The potential biological activities of these organic molecules were analysed on human dermal fibroblasts in primary culture. Our results demonstrate that shell extracts of the two studied molluscs modulate the metabolic activities of the cells. In addition, the extracts caused a decrease of type I collagen and a concomitant increase of active MMP-1, both at the mRNA and the protein levels. Therefore, our results suggest that shell extracts from M. edulis and C. gigas contain molecules that promote the catabolic pathway of human dermal fibroblasts. This work emphasises the potential use of these shell matrices in the context of anti-fibrotic strategies, particularly against scleroderma. More generally, it stresses the usefulness to valorise bivalve shells that are coproducts of shellfish farming activity.

Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering.

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-ß1 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.

Hypoxia inducible factor 1 alpha down-regulates type i collagen through Sp3 transcription factor in human chondrocytes.

Cartilage engineering is one challenging issue in regenerative medicine. Low oxygen tension or hypoxia inducible factor-1 (HIF-1α) gene therapy are promising strategies in the field of cartilage repair. Previously, we showed that hypoxia and its mediator HIF-1 regulate matrix genes expression (collagens and aggrecan). Here, we investigated the molecular mechanism involved in the regulation of type I collagen (COL1A1) by HIF-1 in human articular chondrocytes. We show that HIF-1α reduces COL1A1 transcription, through a distal promoter (-2300 to -1816 bp upstream transcription initiation site), containing two GC boxes that bind Sp transcription factors (Sp1/Sp3). Sp1 acts as a positive regulator but is not induced by HIF-1. COL1A1 inhibition caused by HIF-1 implies only Sp3, which accumulates and competes Sp1 binding on COL1A1 promoter. Additionally, Sp3 ectopic expression inhibits COL1A1, while Sp3 knockdown counteracts the downregulation of COL1A1 induced by HIF-1. In conclusion, we established a new regulatory model of COL1A1 regulation by HIF-1, and bring out its relationship with Sp3 transcription factor. In a fundamental level, these findings give insights in the mechanisms controlling COL1A1 gene expression. This may be helpful to improve strategies to impair type I collagen expression during chondrocyte differentiation for cartilage engineering. © 2016 IUBMB Life, 68(9):756-763, 2016.

Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the -112/-61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications.

Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction.

BACKGROUND: Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes. SCOPE OF REVIEW: This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions. MAJOR CONCLUSIONS: Current research involves the use of chondrocytes or progenitor stem cells, associated with "smart" biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process. GENERAL SIGNIFICANCE: This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.