Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells |
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Authors: | Kosuke Sako Daisuke Sakai Yoshihiko Nakamura Jordy Schol Erika Matsushita Takayuki Warita Natsumi Horikita Masato Sato Masahiko Watanabe |
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Affiliation: | 1.Department of Orthopedic Surgery, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan; (M.S.); (M.W.);2.Center for Musculoskeletal Innovative Research and Advancement (C-MiRA), Tokai University Graduate School, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan;3.Research Center for Regenerative Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan; (Y.N.); (J.S.); (E.M.); (T.W.); (N.H.) |
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Abstract: | Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics. |
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Keywords: | intervertebral disc nucleus pulposus nucleus pulposus progenitor cell Tie2 receptor basic fibroblast growth factor chimera fibroblast growth factor ERK Akt whole tissue culture type II collagen |
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