In situ time-series monitoring of collagen fibers produced by standing-cultured osteoblasts using a second-harmonic-generation microscope

Abstract

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.

Quantification of collagen fiber properties in alcoholic liver fibrosis using polarization-resolved second harmonic generation microscopy

“…In contrast, stage F4 was dominated by complete and stable fibrosis, resulting in less variation in the data. This speculation was also supported by the finding that total SHG intensity was significantly higher in the stage F4 samples than in the others, as it has been suggested that SHG luminosity is higher in thicker, mature collagen fibers 28 , 29 . Thus in liver fibrosis that repeatedly progresses and regresses, the PR-SHG might be useful in precisely identifying the pathological state of the patients, and in the future, it is expected to be combined with morphological classifications such as the new Inuyama classification and METAVIR score to enable more detailed classification of liver fibrosis.…”

Bone quality assessment of osteogenic cell cultures by Raman microscopy

“…These differences could in part be explained by the fact that bone mineral formed in two‐dimensional osteogenic cell cultures do not fully capture the hierarchical or anisotropic collagen organization found in native bone . A recent in situ second‐harmonic‐generation study has also shown that collagen fibers produced by preosteoblast MC3T3‐E1 cell cultures are in general less structured and nonspecifically orientated due to immature structure . In addition, the collagen amide III band at ~1,270 cm −1 is known to be strongly polarization‐dependent compared with the ~1,240 cm −1 band and therefore would be more sensitive to the orientation of collagen fibrils, especially in native bone.…”

Quantitative in situ time-series evaluation of osteoblastic collagen synthesis under cyclic strain using second-harmonic-generation microscopy

“…In a previous study, we succeeded in visualizing the collagen fibers synthesized by osteoblasts using SHG microscopy without any fixation. 11 In this study, we succeeded in imaging collagen fibers synthesized by osteoblasts under cyclic stretch stimulation over time. Results showed the amount of collagen fibers produced by the stretched groups was significantly higher compared with the statically cultured group.…”

“…The optical setup of the SHG microscope was essentially the same as was previously reported. 11 A mode-locked Ti:Sapphire laser light (pulse duration at the focal point of 19 fs, center wavelength of 787 nm, repetition frequency of 81.8 MHz, laser power of 20 mW measured at the optical path which guides laser to the microscope) is focused on the sample by an objective lens for water immersion (NA ¼ 0.90, WD ¼ 2 mm). The laser spot was scanned in a two-dimensional plane on the sample surface using galvano mirrors with relay lenses.…”

“…Previously, we demonstrated in situ time-series SHG imaging of collagen fibers synthesized by osteoblasts. 11 However, this experiment was performed under a standing culture condition without a mechanical stimulus.…”