Prostacyclin suppresses twist expression in the presence of indomethacin in bone marrow-derived mesenchymal stromal cells

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/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.

Figure 1

Immunocytochemical staining of CD34, CD45, CD105, Col1, Runx2, Twist, OC and ALP in BMSC of donor A without administration of iloprost or indomethacin at Days 10 and 28. The hematopoietic markers CD34 and CD45 could not be detected in almost any assay (−), whereas CD105 was always present (+++). Graduation according to the proportion of stained cells as described in the running text. Optical magnification: 400×.

Figure 2

LDH-Assay and calculated cell count of Donors A and B without supplementation of iloprost or indomethacin at different points in time.

Figure 3

Specific ALP activity of Donor A (A), B (B) and C (C) at Days 10 and 28 with and without additional administration of indomethacin and/or iloprost. Standard deviations and statistical significances are displayed: * for p<0.05 and ** for p<0.01 [paired two-sample t test for Days 10 and 28, respectively, compared to the control group (0)]. Concentrations of 10⁻⁵ M (A) and 10⁻⁶ M (B) indomethacin alone were not investigated.