Tokushima University, Graduate School of Technology, Industrial and Social Sciences, Tokushima City,, Japan.
Japan Synchrotron Radiation Research Institute, Research and Utilization Division, Sayo, Hyogo, Japan.
J Biomed Opt. 2019 Jan;24(3):1-8. doi: 10.1117/1.JBO.24.3.031019.
The aim of this study is to evaluate the osteoblastic collagen synthesis under mechanical stimulation using second-harmonic-generation (SHG) microscopy. We apply SHG microscopy to monitor the collagen fibers synthesized by osteoblast-like cells (MC3T3-E1) without the need for fixation and staining. To quantitatively evaluate the influence of mechanical stimulation on osteoblastic collagen synthesis, we compare SHG images of osteoblast-synthesized collagen fibers with and without a cyclic stretch stimulus applied using a lab-made stretching device. We acquire SHG images every 7 days for 3 weeks at different stimulus conditions (5 min/day and 3 h/day with a strain magnitude of 5% and a frequency of 0.5 Hz). Image analysis of the average SHG intensity indicates that the amount of osteoblastic collagen synthesis is significantly enhanced by the cyclic stretch compared with the nonstretched condition, while there is no significant difference between the two mechanical stimulation conditions. Furthermore, the maturity of the collagen fibers in the early stage of bone formation is not affected by the mechanical stimulation. The results can be used in bone regenerative medicine to apply feedback control of collagen synthesis by artificial stimulation.
本研究旨在利用二次谐波产生（SHG）显微镜评估机械刺激下的成骨细胞胶原合成。我们应用 SHG 显微镜来监测成骨样细胞（MC3T3-E1）合成的胶原纤维，而无需固定和染色。为了定量评估机械刺激对成骨细胞胶原合成的影响，我们比较了使用实验室自制拉伸装置施加循环拉伸刺激前后成骨细胞合成的胶原纤维的 SHG 图像。我们在不同的刺激条件下（每天 5 分钟和每天 3 小时，应变幅度为 5%，频率为 0.5 Hz）每 7 天获取一次 SHG 图像，持续 3 周。平均 SHG 强度的图像分析表明，与未拉伸状态相比，循环拉伸显著增强了成骨细胞胶原的合成，而两种机械刺激条件之间没有显著差异。此外，在骨形成的早期阶段，胶原纤维的成熟不受机械刺激的影响。这些结果可用于骨再生医学，通过人工刺激对胶原合成进行反馈控制。
