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.
We used Fourier-transform second-harmonic generation (FT-SHG) microscopy to analyze the orientation of collagen fibers in healing rabbit tendons recovered from an artificial transection. The histological difference between normal and healing tendons can be observed from normal SHG images, whereas the percentage of anisotropic (preferred orientation) regions obtained from the orientation analysis was well correlated (R2 = 0.63) with Young’s modulus obtained from tensile testing of the same sample. Since Young’s modulus reflects the degree of mechanical healing, our results indicate that FT-SHG microscopy have a unique potential as a non-destructive and non-invasive modality for a simultaneous assessment of the histological and mechanical healing degree in injured tendons.
The aim of this study is to provide an in situ method to non-invasively monitor osteoblastic collagen synthesis under mechanical stimulation. We applied second-harmonic-generation (SHG) microscopy to monitor the collagen fibers produced by osteoblast-like cells. To evaluate the influence of mechanical stimulation on collagen synthesis and maturation, we compared SHG images of osteoblast-produced collagen fibers with and without a cyclic stretch stimulus. Image analysis of the average SHG intensity indicated that the amount of osteoblastic collagen synthesis was significantly enhanced by the cyclic stretch. Furthermore, the maturity of the collagen fibers was not affected in the early stage of bone formation by the mechanical stimulus.
Tendon rupture is a trauma that is difficult to fully recover from. Therefore, non-destructive and non-invasive evaluation method for the tendon healing is strongly required. In this study, we performed the orientation analysis of collagen fiber in healing tendon by two-dimensional Fourier transform (2D-FT) of SHG image. The extracted 2D-FT power spectra imply the correlation with the degree of the tendon healing. These results indicate that SHG microscopy has a unique potential as a non-destructive and non-invasive indicator of tendon healing.
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.
Tendon rupture is a trauma difficult to recover the condition before injury. In previous researches, tensile test and staining method have been widely used to elucidate the mechanism of the repair process from the viewpoints of the mechanical property and the histological findings. However, since both methods are destructive and invasive, it is difficult to obtain both of them for the same sample. If both the mechanical property and the histological findings can be obtained from the same sample, one may obtain new findings regarding mechanisms of tendon repairing process.
In this paper, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, deep penetration, and without additional staining. Since SHG light intensity sensitively reflects the structural maturity of collagen molecule and its aggregates, it will be a good indicator for the repairing degree of the ruptured tendon. From comparison of SHG images between the 4-weeks-repaired tendon and the sound tendon in the animal model, we confirmed that SHG light intensity of the repaired tendon was significantly lower than that of the sound tendon, indicating that the collagen structure in the repaired tendon is still immature. Furthermore, we performed both SHG imaging and the tensile test for the same sample, and confirmed a correlation between them. This result shows a potential of SHG light for an indicator of the histological and mechanical recovery of the ruptured tendon.
Second-harmonic-generation (SHG) microscopy is a new tool for observing the collagen fiber in tissue in vivo. Conventional SHG microscopy equipped with 100-fs pulse laser is insufficient to visualize low-order-structured, immature collagen with high contrast (for example, collagen fiber produced by cultured cell) because of low nonlinear susceptibility χ(2). To enhance the image contrast while avoiding the sample damage, one should increase a peak power of the laser light while maintaining the same average power as the 100-fs laser. In this paper, we constructed sensitive SHG microscopy equipped with a 10-fs Ti:Sapphire laser and succeeded to visualize collagen fibers produced by the cultured osteoblasts in vivo.
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