In situ quantitative evaluation of osteoblastic collagen synthesis under cyclic strain by using second-harmonic-generation microscope

Oki Matsubara; Eiji Hase; Takeo Minamikawa; Takeshi Yasui; Katsuya Sato

doi:10.1117/12.2214008

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14 March 2016 In situ quantitative evaluation of osteoblastic collagen synthesis under cyclic strain by using second-harmonic-generation microscope

Oki Matsubara, Eiji Hase, Takeo Minamikawa, Takeshi Yasui, Katsuya Sato

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Proceedings Volume 9712, Multiphoton Microscopy in the Biomedical Sciences XVI; 971222 (2016) https://doi.org/10.1117/12.2214008
Event: SPIE BiOS, 2016, San Francisco, California, United States

Abstract

Osteoblast-produced collagen matrix in bone is influenced by the mechanical stimulus from their surroundings. However, it has been still unclear how mechanical stimulus affects collagen production by osteoblasts. Therefore, it is strongly required to investigate the characteristics of osteoblastic bone regenerative tissue engineering. Recently, second-harmonic-generation (SHG) microscope has attracted attention for in situ visualization of collagen fiber because of less invasiveness, unstaining and no fixation, as well as high spatial resolution and 3D imaging. Using SHG microscopy, one can track the temporal dynamics of collagen fiber during the cultured period of the sample. We applied cyclic stretch strain to osteoblasts (MC3T3-E1) by using originally developed cell stretching device. The stimulation time was set to 5min or 3hours with same strain 5% and same frequency 0.5Hz. Cells were seeded onto the PDMS (polydimethylsiloxane) rubber chamber at a density of 50,000 cells/cm² and cultured in α-MEM with 10% FBS, 1% P/S, 1% Ascorbic acid, 0.2% hydrocortisone and 2% β-Glycerophosphate. SHG imaging was carried out every 7 days. As a result, we confirmed from SHG image that the collagen production was enhanced by the cyclic stretch strain, stretch stimulation time and stretch application term.

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Oki Matsubara, Eiji Hase, Takeo Minamikawa, Takeshi Yasui, and Katsuya Sato "In situ quantitative evaluation of osteoblastic collagen synthesis under cyclic strain by using second-harmonic-generation microscope", Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 971222 (14 March 2016); https://doi.org/10.1117/12.2214008

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