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10 November 2014 : Original article  

Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

Oliver KemperABC, Monika HertenB, Johannes FischerB, Marcel HaversathDEF, Sascha BeckE, Tim ClassenE, Sebastian WarwasC, Tjark TassemeierF, Stefan LandgraeberD, Sabine Lensing-HöhnB, Rüdiger KrauspeA, Marcus JägerA

DOI: 10.12659/MSM.890953

Med Sci Monit 2014; 20:2219-2227

Abstract

BACKGROUND: Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost.

MATERIAL AND METHODS: Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45–/14–). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10^–7 M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10^–6 M (Group 1, A), 10^–5 M (Group 2, B), 10^–4 M (Group 3, A/B), and together with 10^-7 M iloprost (Groups 4–6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34–, CD45–, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity).

RESULTS: Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently.

CONCLUSIONS: Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin, it completely suppressed Twist. Thus, in the treatment of avascular osteonecrosis or painful bone marrow edema, the undesirable effects of indomethacin might be counterbalanced by iloprost.

Keywords: Bone Marrow Cells - cytology, Antigens, CD - metabolism, Alkaline Phosphatase - metabolism, Cell Count, Cell Differentiation - drug effects, Cell Proliferation - drug effects, Collagen Type I - metabolism, Core Binding Factor Alpha 1 Subunit - metabolism, Epoprostenol - pharmacology, Iloprost - pharmacology, Indomethacin - pharmacology, L-Lactate Dehydrogenase - metabolism, Mesenchymal Stromal Cells - metabolism, Nuclear Proteins - metabolism, Osteocalcin - metabolism, Twist Transcription Factor - metabolism

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750