Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress
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The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia.
MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved.
The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha (HIF-1α), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation.
Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.
KeywordsHypoxia Osteoblast Laser p38 PKD
This study was supported by National Research Foundation of Korea (NRF-2018R1A2B2006546) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI17C0708).
JHH carried out the molecular genetic studies and prepared the manuscript. JHC participated in the design of the study and performed the statistical analysis. IRK carried out the molecular studies. BSP conceived of the study, and participated in its design and coordination and helped to draft the manuscript. YDK designed the study, drafted the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
There are no animal experiments carried out for this article.
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