BACKGROUND AIMS: Suspension mononuclear cells (MNCs) can be differentiated into osteoblasts with the induction of ascorbic acid and β-glycerophosphate. The aim of this study was to determine the ability of suspension MNCs to differentiate into osteoblasts using ascorbic acid only. METHODS: Suspension MNCs were obtained by a combination of gradient centrifugation and culture selection. Suspension MNCs were subjected to differentiation assay by culturing them inside proliferation medium supplemented with 10 μg/mL, 30 μg/mL, 50 μg/mL, 60 μg/mL, 90 μg/mL and 500 μg/mL of ascorbic acid. Proliferation medium supplemented with 50 μg/mL ascorbic acid and 10 mmol/L β-glycerophosphate was used as a positive control for osteoblast induction, and proliferation medium without ascorbic acid was used as a negative control. Differentiation analysis was performed using alkaline phosphatase (ALP) assay, von Kossa staining and expression of osteoblast-related genes. RESULTS: With all concentrations of ascorbic acid used, there was a significant increase (P < 0.05) in ALP-specific activity and mineralized nodule formation throughout the differentiation course compared with negative control. Ascorbic acid was also able to activate the expression of osteopontin (SPP1), osteonectin (SPARC) and runt-related transcription factor 2 (RUNX2) messenger RNA in positive control and ascorbic acid-induced MNCs (30 μg/mL and 90 μg/mL) but not in negative control. CONCLUSIONS: Ascorbic acid alone was sufficient to induce osteoblast differentiation from suspension MNCs; 30-90 μg/mL of ascorbic acid was found to be the optimal concentration. Ascorbic acid can be used as a nutritional supplement for cellular therapy of bone-related disease.
BACKGROUND AIMS: Suspension mononuclear cells (MNCs) can be differentiated into osteoblasts with the induction of ascorbic acid and β-glycerophosphate. The aim of this study was to determine the ability of suspension MNCs to differentiate into osteoblasts using ascorbic acid only. METHODS: Suspension MNCs were obtained by a combination of gradient centrifugation and culture selection. Suspension MNCs were subjected to differentiation assay by culturing them inside proliferation medium supplemented with 10 μg/mL, 30 μg/mL, 50 μg/mL, 60 μg/mL, 90 μg/mL and 500 μg/mL of ascorbic acid. Proliferation medium supplemented with 50 μg/mL ascorbic acid and 10 mmol/L β-glycerophosphate was used as a positive control for osteoblast induction, and proliferation medium without ascorbic acid was used as a negative control. Differentiation analysis was performed using alkaline phosphatase (ALP) assay, von Kossa staining and expression of osteoblast-related genes. RESULTS: With all concentrations of ascorbic acid used, there was a significant increase (P < 0.05) in ALP-specific activity and mineralized nodule formation throughout the differentiation course compared with negative control. Ascorbic acid was also able to activate the expression of osteopontin (SPP1), osteonectin (SPARC) and runt-related transcription factor 2 (RUNX2) messenger RNA in positive control and ascorbic acid-induced MNCs (30 μg/mL and 90 μg/mL) but not in negative control. CONCLUSIONS:Ascorbic acid alone was sufficient to induce osteoblast differentiation from suspension MNCs; 30-90 μg/mL of ascorbic acid was found to be the optimal concentration. Ascorbic acid can be used as a nutritional supplement for cellular therapy of bone-related disease.