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. 2019 Mar;23(2):142-52.
doi: 10.29252/.23.2.142. Epub 2018 Oct 31.

Exogenous Nitric Oxide Induced Early Mineralization in Rat Bone Marrow Mesenchymal Stem Cells via Activation of Alkaline Phosphatase

Mohammad Hussein Abnosi  1 , Sadieeh Pari  1
Affiliations

Exogenous Nitric Oxide Induced Early Mineralization in Rat Bone Marrow Mesenchymal Stem Cells via Activation of Alkaline Phosphatase

Mohammad Hussein Abnosi et al. Iran Biomed J. 2019 Mar.

Abstract

Background: Since the low concentration and short-time treatment with sodium nitroprusside (SNP), a nitric oxide (NO)–donor, cause no harm to rat bone marrow mesenchymal stem cells (MSCs), we studied the impact of SNP on MSCs differentiation.

Methods: MSCs were treated with 100 and 1000 µM of SNP for 1 hour in every 48 hours and after 5, 10, 15, and 21 days in osteogenic media. The viability and the level of mineralization were determined using MTT assay and alizarin red staining, respectively. Morphology of the cells was studied using fluorescent dye. Concentration of calcium and the activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were evaluated by commercial kits.

Results: SNP with the concentration of 1000 µM significantly reduced viability from day 5 to day 20, but 100 µM did not affect the viability until the day 15. The low concentration of SNP increased matrix deposition from day 10 and reached almost its maximum (4.40 ± 2.4) at the day 15. Also, increasing the activity of ALP (419 ± 2.2), due to low concentration of SNP, started at day 10 and continued till the day 20, while LDH (2026 ± 11) and AST (25.6 ± 0.4) elevations were observed from day 5 onwards. In case of ALT, we observed a significant decrease (36%) from day 5 till day 20.

Conclusion: Based on our findings, low concentrations of SNP might be useful in the promotion of bone repair.

Keywords: Alkaline phosphatase; Mesenchymal stem cells; Nitroprusside; Osteoblasts.

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Conflict of interest statement

None declared.

Figures

Fig. 1
Fig. 1
The cell viability of BMSCs after 5, 10, 15, and 21 days of treatment with 0, 100, and 1000 µM of SNP in osteogenic media, based on MTT assay. Values are means ± SD. The asterisks (*) and (**) represent the level of significant in each day compared to the control (p < 0.05 and p < 0.001, respectively; ANOVA, Tukey's test)
Fig. 2
Fig. 2
Fluorescent micrograph images of BMSCs stained with Hoechst, after 5, 10, 15, and 21days of incubation in osteogenic media treated with 0, 100 and 1000 µM of SNP. Nuclear condensation and deformation (arrows) of cells were observed after treatment with 1000 µM of SNP (magnification 200 ).
Fig. 3
Fig. 3
Fluorescent micrograph images of BMSCs stained with acridine orange, after 5, 10, 15, and 21 days of incubation in osteogenic media treated with 0, 100 and 1000 µM of SNP. Shrinkage and complete disappearance of cytoplasm in some cells (arrows) were observed after treatment with 1000 µM of SNP (magnification 200 )
Fig. 4
Fig. 4
Osteogenic confirmation. (A) Microscopic images (200 ) of the cell after alizarin red staining; (B) Camera photograph from plates after alizarin red staining
Fig. 5
Fig. 5
Mean activity of ALP (A), LDH (B), AST (C), and ALT (D) in the cells at osteogenic media after 5, 10, 15 and 21days of incubation and treatment with 0, 100, and 1000 µM of SNP. Values are means ± SD. The asterisks (*) and (**) represent the level of significant in each day compared to the control at p < 0.05 and p < 0.001, respectively (ANOVA, Tukey's test)
See this image and copyright information in PMC

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