Abstract
.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.
Highlights
Bone cells, such as osteoblasts, osteoclasts, and osteocytes, play important roles in bone metabolism, such as bone formation and resorption.[1]
We concluded that our SHG microscopy setup can be used to assess the amount of osteoblastic collagen synthesis without fixation and staining, and to conduct in situ time-series evaluation
We conducted in situ time-lapse observations of collagen synthesized by osteoblasts using SHG microscopy without any fixation and staining to evaluate the influence of mechanical stimulation on osteoblastic collagen synthesis
Summary
Bone cells, such as osteoblasts, osteoclasts, and osteocytes, play important roles in bone metabolism, such as bone formation and resorption.[1]. As bone metabolism is quite slow, it is difficult for elderly to heal damaged bone or increase the sufficient amount of bone during a short period.[2] there is a considerable need for bone tissue engineering and regenerative medicine to locally replace damaged bone in older patients. Staining methods have been widely used to visualize collagen fibers in the tissue.[7,8] invasiveness due to fixation and staining makes it difficult to apply this method for in situ monitoring of collagen fiber production by osteoblasts over time. Second-harmonic-generation (SHG) microscopy has been used to visualize the collagen fibers in situ without the need for fixation and staining.[9,10] SHG light is generated from collagen molecules in biological tissues. SHG microscopy has high selectivity and good image contrast for collagen molecules as well as high spatial resolution, optical three-dimensional (3-D) sectioning, moderate penetration, and
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