Tissue Eng Regen Med.  2018 Dec;15(6):793-801. 10.1007/s13770-018-0167-1.

Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress

• Heo JH
• Choi JH
• Kim IR
• Park BS
• Kim YD

• KMID: 2441270
• DOI: http://doi.org/10.1007/s13770-018-0167-1

Abstract

BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSION
Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxiccultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Keyword

Hypoxia; Osteoblast; Laser; p38; PKD

MeSH Terms

Alkaline Phosphatase
Anoxia
Blotting, Western
Cell Culture Techniques
Collagen Type I
Humans
In Vitro Techniques
Low-Level Light Therapy
Miners*
Osteoblasts*
Oxygen
Phosphorylation
Phosphotransferases
Transcription Factors
Alkaline Phosphatase
Collagen Type I
Oxygen
Phosphotransferases
Transcription Factors