J Rheum Dis.  2024 Apr;31(2):79-85. 10.4078/jrd.2023.0043.

Effect of recombinant human bone morphogenetic protein-2 and osteoprotegerin-Fc in MC3T3-E1 cells

• Kim SH
• Choi HJ
• Lee SM
• Yoon DS
• Son CN

• KMID: 2554323
• DOI: http://doi.org/10.4078/jrd.2023.0043

Abstract

Objective
We compared the osteoblastogenesis by serially administrating recombinant human bone morphogenetic protein-2 (rhBMP-2) and osteoprotegerin-immunoglobulin Fc segment complex (OPG-Fc).
Methods
The MC3T3-E1 preosteoblast cell line was differentiated for 1, 3, and 7 days with a treatment of OPG-Fc in 10~200 ng/mL concentration and the cell viability was evaluated by Cell Counting Kit-8 analysis. The level of differentiation from MC3T3-E1 cells to osteoblasts was determined by alkaline phosphatase activity. The level of runt domain-containing transcription factor 2 (Runx2) and osteopontin (OPN) manifestation, involved in osteoblast differentiation, was examined by real-time polymerase chain reaction and western blotting.
Results
During MC3T3-E1 cell differentiation, the differentiation level was high with 1-day treatment using 100 ng/mL OPGFc. The treatment with 50 ng/mL rhBMP-2 for 7 days, followed by 1-day treatment with 100 ng/mL OPG-Fc produced the highest differentiation level, which was approximately 5.3 times that of the control group (p<0.05). The expression of Runx2 mRNA significantly increased, reaching 2.5 times the level of the control group under the condition of 7-day treatment with rhBMP-2 and 1-day treatment with OPG-Fc (p<0.001). The expression of Runx2 protein significantly increased to approximately 5.7 times that of the control group under the condition of 7-day treatment with rhBMP-2, followed by 1-day treatment with OPG-Fc (p<0.01). The expression of OPN protein showed no change from that of the control group under various conditions of rhBMP-2 and OPGFc combinations.
Conclusion
These results imply that the treating preosteoblasts with rhBMP-2 first and then with OPG-Fc increased osteoblast differentiation efficacy.

Keyword

Recombinant human bone morphogenetic protein-2; Osteoprotegerin-Fc; Osteoblast; Differentiation

Figure

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  Figure 1 Viability and alkaline phosphatase (ALP) activity in osteoblasts (MC3T3-E1) following treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2)/osteoprotegerin (OPG-Fc). A: control, B: 50 ng/mL rhBMP-2 for 8 days, C: 100 ng/mL OPG-Fc for 8 days, D: 50 ng/mL rhBMP-2 and 100 ng/mL OPG-Fc for 8 days, E: 50 ng/mL rhBMP-2 for 7 days and 100 ng/mL OPG-Fc for 1 day, F: 100 ng/mL OPG-Fc for 1 day and 50 ng/mL rhBMP-2 for 7 days. Error bars represent mean±standard deviation. *p<0.05 compared with the control group.

• 

  Figure 2 The levels of mRNA in osteoblasts (MC3T3-E1) following treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2)/osteoprotegerin (OPG-Fc). A: control, B: 50 ng/mL rhBMP-2 for 8 days, C: 100 ng/mL OPG-Fc for 8 days, D: 50 ng/mL rhBMP-2 and 100 ng/mL OPG-Fc for 8 days, E: 50 ng/mL rhBMP-2 for 7 days and 100 ng/mL OPG-Fc for 1 day, F: 100 ng/mL OPG-Fc for 1 day and 50 ng/mL rhBMP-2 for 7 days. Error bars represent mean±standard deviation. Runx2: runt domain-containing transcription factor 2, OPN: osteopontin. **p<0.01 and ***p<0.001, compared with the control group. #p<0.05 and ##p<0.01, compared with the B group.

• 

  Figure 3 Protein levels in osteoblasts (MC3T3-E1) following treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2)/osteoprotegerin (OPG-Fc). A: control, B: 50 ng/mL rhBMP-2 for 8 days, C: 100 ng/mL OPG-Fc for 8 days, D: 50 ng/mL rhBMP-2 and 100 ng/mL OPG-Fc for 8 days, E: 50 ng/mL rhBMP-2 for 7 days and 100 ng/mL OPG-Fc for 1 day, F: 100 ng/mL OPG-Fc for 1 day and 50 ng/mL rhBMP-2 for 7 days. Error bars represent mean±standard deviation. Runx2: runt domain-containing transcription factor 2, OPN: osteopontin. *p<0.05 compared with the control group. #p<0.05 compared with the B group.

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