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Sodium Tungstate for Promoting Mesenchymal Stem Cell Chondrogenesis.

ABSTRACT: Articular cartilage has a limited ability to heal. Mesenchymal stem cells (MSCs) derived from the bone marrow have shown promise as a cell type for cartilage regeneration strategies. In this study, sodium tungstate (Na₂WO₄), which is an insulin mimetic, was evaluated for the first time as an inductive factor to enhance human MSC chondrogenesis. MSCs were seeded onto three-dimensional electrospun scaffolds in growth medium (GM), complete chondrogenic induction medium (CCM) containing insulin, and CCM without insulin. Na₂WO₄ was added to the media leading to final concentrations of 0, 0.01, 0.1, and 1?mM. Chondrogenic differentiation was assessed by biochemical analyses, immunostaining, and gene expression. Cytotoxicity using human peripheral blood mononuclear cells (PBMCS) was also investigated. The chondrogenic differentiation of MSCs was enhanced in the presence of low concentrations of Na₂WO₄ compared to control, without Na₂WO₄. In the induction medium containing insulin, cells in 0.01?mM Na₂WO₄ produced significantly higher sulfated glycosaminoglycans, collagen type II, and chondrogenic gene expression than all other groups at day 28. Cells in 0.1?mM Na₂WO₄ had significantly higher collagen II production and significantly higher sox-9 and aggrecan gene expression compared to control at day 28. Cells in GM and induction medium without insulin containing low concentrations of Na₂WO₄ also expressed chondrogenic markers. Na₂WO₄ did not stimulate PBMC proliferation or apoptosis. The results demonstrate that Na₂WO₄ enhances chondrogenic differentiation of MSCs, does not have a toxic effect, and may be useful for MSC-based approaches for cartilage repair.

SUBMITTER: Khader A

PROVIDER: S-EPMC5165671 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Sodium Tungstate for Promoting Mesenchymal Stem Cell Chondrogenesis.

Khader Ateka A Sherman Lauren S LS Rameshwar Pranela P Arinzeh Treena L TL

Stem cells and development 20161017 24

Articular cartilage has a limited ability to heal. Mesenchymal stem cells (MSCs) derived from the bone marrow have shown promise as a cell type for cartilage regeneration strategies. In this study, sodium tungstate (Na<sub>2</sub>WO<sub>4</sub>), which is an insulin mimetic, was evaluated for the first time as an inductive factor to enhance human MSC chondrogenesis. MSCs were seeded onto three-dimensional electrospun scaffolds in growth medium (GM), complete chondrogenic induction medium (CCM) c ...[more]

PMID: 27615276

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Project description:BACKGROUND:Although mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis. METHODS:Aggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3?weeks in presence and absence of transforming growth factor-beta 1 (TGF-?1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence. RESULTS:Reducing ACN from 500?k to 70?k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to ?-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a ?-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue. CONCLUSIONS:This study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.

Project description:Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact the chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models, including three-dimensional (3D) microribbon (?RB) scaffolds, two-dimensional hydrogel (2D-HG) substrates, 3D hydrogels (3D-HGs), and pellet. For each culture model involving biomaterials, four polymers were compared, including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in ?RB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D-HG or 3D-HG models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial compositions can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting that N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSC-based cartilage regeneration in different culture models. Impact Statement Here we assessed the effects of Rho-associated protein kinase (ROCK) inhibition on mesenchymal stem cell (MSC) chondrogenesis in different culture models, including three-dimensional (3D) microribbon scaffolds, two-dimensional hydrogel substrates, 3D hydrogels, and pellet culture. Our results demonstrate that effects of ROCK inhibition on MSC chondrogenesis differ substantially depending on culture models. Furthermore, MSC chondrogenesis cannot be predicted using ROCK signaling alone. The results from this study fill in a gap of knowledge in the correlation between ROCK signaling and MSC chondrogenesis, which may be used to enhance MSC-based cartilage regeneration in different culture models.

Dataset Information

Sodium Tungstate for Promoting Mesenchymal Stem Cell Chondrogenesis.

Publications

Sodium Tungstate for Promoting Mesenchymal Stem Cell Chondrogenesis.

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