In bone tissue engineering and regeneration, there is a considerable need for an unstained method of monitoring collagen fibers produced by osteoblasts. This is because collagen fibers play an important role as a bone matrix and continuous monitoring of their temporal dynamics is important in clarifying the organization process toward forming bone tissue. In the work described here, using a second-harmonic-generation (SHG) microscope, we performed in situ time-series monitoring of collagen fibers produced by cultured osteoblasts without the need for staining. Use of the 19 fs near-infrared pulsed light enables us to visualize the temporal dynamics in a thin layer of collagen fibers produced by a single layer of osteoblasts in high-contrast SHG images. While the collagen fibers were produced and stored inside the osteoblasts at an early stage of culturing, the network structure of collagen fibers was formed and locally condensed at a late stage. Furthermore, we extracted a quantitative parameter of collagen maturity degree in the cultured sample by use of image analysis based on a two-dimensional Fourier transform of the SHG image. The proposed method will be useful for in situ quality and quantity control of collagen fibers in bone tissue engineering and regeneration.

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.

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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-harmonicgeneration (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/cm 2 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.