Heo Jin-Ho, Choi Jeong-Hun, Kim In-Ryoung, Park Bong-Soo, Kim Yong-Deok
1Department of Oral and Maxillofacial Surgery, Pusan National University, 49 Busandaehak-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do 50612 Republic of Korea.
2Department of Oral Anatomy, Pusan National University, 49 Busandaehak-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do 50612 Korea.
Tissue Eng Regen Med. 2018 Nov 17;15(6):793-801. doi: 10.1007/s13770-018-0167-1. eCollection 2018 Dec.
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 .
本研究旨在评估低强度激光治疗（LLLT）与重组人骨形态发生蛋白-2（rhBMP-2）联合应用于低氧培养的MC3T3-E1成骨细胞的效果，并确定低氧条件下成骨细胞分化和矿化的潜在信号通路。
将MC3T3-E1细胞在1%氧张力下培养72小时。细胞培养物分为四组：常氧对照组、单独低强度激光（LLL）组、rhBMP-2联合LLLT组和低氧条件下的rhBMP-2组。在0、24和48小时进行激光照射。细胞用50 ng/mL的rhBMP-2处理。在第3、7和14天测量碱性磷酸酶活性以评估成骨细胞分化。在第7和14天用茜素红S染色确定细胞矿化情况。进行蛋白质免疫印迹分析以评估p38/蛋白激酶D（PKD）信号通路是否参与其中。
结果表明，LLLT和rhBMP-2协同增加碱性磷酸酶（ALP）活性和矿化。蛋白质免疫印迹分析显示，与单独的rhBMP-2组或LLL组相比，LLLT和rhBMP-2联合组中I型胶原蛋白、 runt相关转录因子2（RUNX2）和osterix（Osx）的表达增加，而缺氧诱导因子1-α（HIF-1α）的表达降低更多。此外，LLLT和rhBMP-刺激p38磷酸化，rhBMP-2和LLLT增加Prkd1磷酸化。
rhBMP-2和LLL联合治疗通过激活p38/PKD信号通路诱导低氧培养的MC3T3-E1成骨细胞的分化和矿化。
