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Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells

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Abstract

The inductive effects of increased osmolarity on chondrogenesis are well approved. However, the effects of the osmolyte agent invoked to induce hyperosmolarity are largely neglected. Herein, we scrutinized how hyperosmotic conditions acquired by addition of different osmolytes would impact chondrogenesis. We briefly assessed whether such conditions would differentially affect hypertrophy and angiogenesis during MSC chondrogenesis. Chondrogenic and hypertrophic marker expression along with VEGF secretion during adipose-derived (AD)-MSC chondrogenesis under three osmolarity levels (350, 450, and 550 mOsm) using three different osmolytes (NaCl, sorbitol, and PEG) were assessed. MTT assay, qRT-PCR, immunocytochemistry, Alcian Blue staining, ELISA, and ALP assays proved osmolyte-type dependent effects of hyperosmolarity on chondrogenesis, hypertrophy, and angiogenesis. At same osmolarity level, PEG had least cytotoxic/cytostatic effect and most prohibitive effects on angiogenesis. As expected, all hyperosmolar conditions led to enhanced chondrogenesis with slightly varying degrees. PEG and sorbitol had higher chondro-promotive and hypertrophy-suppressive effects compared to NaCl, while NaCl had exacerbated hypertrophy. We observed that TonEBP was involved in osmoadaptation of all treatments in varying degrees. Of importance, we highlighted differential effects of hyperosmolarity obtained by different osmolytes on the efficacy of chondrogenesis and more remarkably on the induction/suppression of cartilage pathologic markers. Our study underlies the need for a more vigilant exploitation of physicobiochemical inducers in order to maximize chondrogenesis while restraining unwanted hypertrophy and angiogenesis.

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Acknowledgments

This work has been partly funded by the University of Tehran and partly by the Stem Cell Technology Research Center and Iranian Council for Stem Cell Science and Technology.

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Authors and Affiliations

  1. Department of Biotechnology, College of Science, University of Tehran, P.O.Box: 14155-6455, Tehran, Iran

    Sorour Ahmadyan & Mahboubeh Kabiri

  2. Department of Cellular and Molecular Biology, Kish International Campus, Kish Island, Iran

    Sorour Ahmadyan & Ali Farazmand

  3. Stem Cell Technology Research Center, Tehran, Iran

    Sorour Ahmadyan & Hana Hanaee-Ahvaz

  4. Faculty of Biology, College of Science, University of Tehran, Tehran, Iran

    Ali Farazmand

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  1. Sorour Ahmadyan
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  2. Mahboubeh Kabiri
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Correspondence to Mahboubeh Kabiri.

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Electronic supplementary material

Supplementary Figure 1

Characterization of stemness properties of the MSC isolated from adipose tissue. A) MSC were differentiated into adipocytes. Adipose vesicles can be observed as red droplets stained by Oil Red O. B) MSC were differentiated into Osteogenic lineage. Calcium crystals are stained in red using Alizarin Red staining. C) As expected the isolated cells are negative for hematopoietic stem cell markers, i.e., CD34 and CD45, while showing high expression of CD90 and CD73. Scale bar: 200 μm. (GIF 1.28 mb)

High resolution image (TIFF 4.32 mb)

Supplementary Figure 2

Quantification of Collagen II Staining intensity with Image Pro Plus software. A representative image analyzed with the software. B) The same image after color selection and mask application. The arrow shows sensitivity of color selection with unspecific staining being excluded. (GIF 337 kb)

High resolution image (TIFF 1.94 mb)

Supplementary Figure 3

A) Representative Col-X stained images analyzed with the Image Pro Plus software. B) The same image after color selection and applying mask. (GIF 195 kb)

High resolution image (TIFF 1.73 mb)

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Ahmadyan, S., Kabiri, M., Hanaee-Ahvaz, H. et al. Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells. Appl Biochem Biotechnol 185, 507–523 (2018). https://doi.org/10.1007/s12010-017-2647-5

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