2.1.

Cell Culture

MC3T3-E1 osteoblast-like cells (Riken BRC, Tsukuba, Japan) were precultured in alpha-minimum essential medium ($\alpha \text{-}\mathrm{MEM}$) (Nacalai tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS) (Nacalai tesque, Kyoto, Japan) and were maintained at 37°C and 5% ${\mathrm{CO}}_2$ in a humidified incubator until subconfluent. Cells were then seeded onto a polydimethylsiloxane (PDMS) rubber chamber (STB-CH-04, STREX, Osaka, Japan) coated with fibronectin (Wako, Osaka, Japan) at a density of $5.0\times {10}^4\text{cells}/{\mathrm{cm}}^2$ and cultured in the culture medium that contains $\alpha \text{-}\mathrm{MEM}$ supplemented with 10% FBS, 1% penicillin-streptomycin (Nacalai tesque, Kyoto, Japan). MC3T3-E1 is an osteoblast precursor cell line. We added an osteoblast-inducer reagent to the culture medium (composition of 1% ascorbic acid, 0.2% hydrocortisone, and 2% $\beta$-Glycerophosphate) (TaKaRa, Shiga, Japan) to differentiate cells into osteoblast-like cell and to maintain the differentiated state of cell during experiment. Cells were statically incubated for 1 day before stretch application to ensure sufficient cell adhesion. The culture medium was exchanged semiweekly.

2.2.

Application of Mechanical Stimuli

To apply stretch stimulus to the cells, we developed a custom cell-stretching device as shown in Fig. 1. The device consists of a stepping motor and a ball screw. The rotational motion of the stepping motor is transmitted into linear reciprocating motion by the ball screw. The PDMS chamber with the cells was mounted on the stretching device. The entire device was put into the ${\mathrm{CO}}_2$ incubator during the experimental period. Cell culture periods were set to 1, 2, and 3 weeks. During these periods, stretch stimulus was applied to the cells for 5 min/day or 3 h/day with 5% strain magnitude and 0.5 Hz loading frequency. The cells cultured statically (no stretch application) in the PDMS chamber were defined as the control group. The number of samples in each stretch condition group is three.

Fig. 1

(a) Schematic of the cell stretch device, (b) mechanism to convert rotational motion into linear motion, and (c) photograph of the cell stretch device.

2.3.

Collagen Staining and Observation

To compare the image obtained using conventional collagen staining method and the image obtained by our SHG microscopy setup, we performed another experiment. Cells were fixed at 1 and 2 weeks culture periods with and without stretching stimulation (1 h/day, 5% strain magnitude, and 0.5-Hz frequency) using 4% paraformaldehyde, and stained with the collagen stain kit (TaKaRa, Shiga, Japan), which contains sirius red dye. Stained collagen was observed using the phase contrast inverted microscope (CKX-41, Olympus, Tokyo, Japan) with $\times 4$ dry objective lens and recorded with the digital camera (DP71, Olympus, Tokyo, Japan). Obtained RGB color images were converted into cyan, magenta, yellow, and black (CMYK) four color model. We assessed the depth of sirius red staining color using the key plate value of CMYK image.

2.4.

Experimental Setup for Second-Harmonic-Generation Microscopy

We used SHG microscopy to evaluate the amount and maturity of collagen fibers synthesized by the osteoblasts as shown in Fig. 2. 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 ($\mathrm{NA}=0.90$, $\mathrm{WD}=2\mathrm{mm}$). The laser spot was scanned in a two-dimensional plane on the sample surface using galvano mirrors with relay lenses. The SHG signal was collected by the photo multiplier tube (PMT) (Photon counting head, H8259, Hamamatsu photonics K. K., Shizuoka, Japan), which was mounted on backward to the sample. In all experiments, PMT detector gain was set constant. The scanning area was $260\mu \mathrm{m}\times 260\mu \mathrm{m}$ with a 256-pixel resolution in each direction. Scanning rate was also set constant for all experiments. The scanning area was expanded to include 16 total images acquired sequentially with an electromechanically controlled stage, resulting in a total imaging area of $1040\mu \mathrm{m}\times 1040\mu \mathrm{m}$ comprising 1,048,576 total pixels ($256\times 256\times 16$).

Fig. 2

(a) SHG microscopy experimental setup. The light source is an ML–Ti:S laser and its pulse width is 18.9 fs at the focal spot, and laser power is 20 mW. The laser light is scanned two-dimensionally by Galvano mirrors ($256\times 256\text{pixels}$), and the scanning area is expanded by sequential image acquisition using a mechanical control stage. The total image size is $1040\mu \mathrm{m}\times 1040\mu \mathrm{m}$ and the total number of pixels is 1,048,576 ($256\times 256\times 16$). (b) Mechanism of occurring SHG light. The SHG light occurs only from noncentrosymmetric materials (e.g., collagen fibers). The wavelength of the incident light is converted to half wavelength by entering the collagen fiber.

2.5.

Statistics

We used Student’s $t$-test to evaluate the effect of mechanical stimulation on osteoblastic collagen synthesis. We assumed a statistical significance threshold of 0.05 for all experimental data.

3.1.

Comparison of Conventional Collagen Staining Image and Second-Harmonic-Generation Microscopy Image

Figure 3 shows the phase contrast images of Sirius red staining collagen synthesized by MC3T3-E1 cells in culturing periods of 1 and 2 weeks with and without stretching stimulation. Figure 4 shows SHG microscopy images of cells that were fixed with 4% paraformaldehyde. SHG imaging was performed before sirius red staining. The observed area and magnification did not match between the phase contrast image and the SHG image. These images obtained using two kinds of different methods show qualitatively good agreement of distribution of osteoblastic synthesized collagen. Figure 5 shows the histogram of SHG signal intensity in the obtained SHG images. As shown in the graph, according to the culturing time periods and application of stretch stimulation, the peak and foot of right side (meaning high SHG signal intensity) in the histogram curve shift toward the right. Figure 6 shows the correlation diagram between the mean value of SHG signal intensity and the mean key plate value of CMYK image calculated from the sirius red-stained collagen image. There is a good correlation between the SHG microscopy image and conventional sirius red staining image. From these results, 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.

Fig. 3

Osteoblasts synthesized collagen stained by sirius red dye. The area of stained collagen and the color depth of sirius red dye are increased according to the culturing time period and the application of stretching mechanical stimuli.

Fig. 4

SHG image of fixed cell and collagen. SHG imaging was conducted before sirius red staining. The observed region was not same with that of resius red stained image presented in the Fig. 3. The area and the intensities of SHG signal are increased according to the culturing time period and the application of mechanical stimuli. These SHG images show qualitatively good agreement with the sirius red stained image. (a), (b) and (c) Representative magnified images of collagen. Cropped region is indicated by the red rectangle in the entire SHG image. (a) There are distributed mist-like SHG signals, but fibrous structure cannot be observed. This SHG image might detect nonfibrillar collagen synthesized by osteoblastic cells. (b) SHG signals with fragmented thin fibrous structure can be observed. We speculate that is an immature fibrillar collagen. (c) There are thick and dense fibrous structure. We considered that this SHG image detects matured collagen fibers. (d) Representative SHG image that is used to define the threshold SHG signal intensity range. The cropped region shown by the red rectangle in (d) is used to calculate average SHG intensity and standard deviation.

Fig. 5

Histograms of SHG signal intensity of SHG images obtained from fixed cell samples. According to the culturing time periods and application of stretch stimulation, the peak and foot of right side in the histogram curve shift toward the right.

Fig. 6

Correlation diagram between the mean value of SHG signal intensity in fixed cell samples and the mean key plate value of CMYK image calculated from the sirius red stained collagen image. There is a good correlation between the SHG microscopy image and conventional sirius red staining image.

3.2.

Definition of Matured Fibrillar Collagen Using Second-Harmonic-Generation Signal Intensity

In Figs. 4(a)–4(c) shows the representative images that reflect the maturation process of osteoblastic-synthesized collagen in vitro obtained by the SHG microscopy. These images are cropped from the entire SHG image, respectively. In the SHG image obtained at 1 week culture period with stretch stimulation [indicated as Fig. 4(a)], there are distributed mist-like SHG signals, but fibrous structure cannot be observed. This SHG image might detect nonfibrillar collagen synthesized by osteoblastic cells. On the other hand, in the SHG image at 2 weeks without stretch stimulation (control sample) [Fig. 4(b)], SHG signals with fragmented thin fibrous structure can be observed. We speculate that this is an immature fibrillar collagen. As shown in Fig. 4(c), the SHG image obtained at the 2 weeks culture period with stretch stimulation, there are thick and dense fibrous structures. We considered that this SHG image detects matured collagen fibers.

Based on these observation results, to evaluate the amount of collagen fibers synthesized by cultured osteoblasts under different loading conditions, we set the certain threshold range of SHG signal intensity to detect the matured collagen fibers. Figure 4(d) shows a representative SHG image of well-matured fibrous collagen fibers synthesized by osteoblastic cells in our study. We cropped one region in which matured collagen fibers exist. The histogram of SHG signal intensity in the cropped region is shown in Fig. 7. The mean value of SHG signal intensity from the matured collagen fibers was 90 with a standard deviation of 45. As this threshold range served as a criterion to determine whether the collagen fibers had matured, we used the values that the number of pixels whose SHG intensity was within the threshold range was divided by the total number of pixels in the SHG image to evaluate the amount of osteoblastic collagen synthesis.

Fig. 7

Histogram of SHG signal intensity calculated from the cropped SHG image, which represents the matured collagen fibers.

3.3.

In Situ Time-Series Evaluation of Collagen Synthesis Using Second-Harmonic-Generation Microscopy

We performed SHG imaging of collagen fibers synthesized by cultured osteoblasts under three different conditions (control, stretch for 5 min/day, and stretch for 3 h/day) as shown in Fig. 8. The representative magnified images are shown as Figs. 8(a)–8(c), respectively. Cropped regions are indicated by red rectangles in the entire SHG image. The collagen fibers were synthesized and secreted by osteoblasts with the lapse of time. As shown in Fig. 8(a), the nucleus appeared as a dark round area. More importantly, the brightness of the SHG images for each condition increased over time, indicating a temporal change of collagen distribution during the culturing period as shown in Figs. 8(b) and 8(c).

Fig. 8

SHG images of collagen synthesized by osteoblasts at each culturing time periods and each mechanical stimulus conditions. (a), (b), and (c) Magnified representative images of collagen fibers in each culturing time periods and mechanical stimulation conditions.

Figure 9 shows the number of pixels that had a measured SHG intensity within the threshold range for each week at each test condition. The graph shows the percentage of pixels within the threshold range calculated from the total number of pixels in each SHG images (1,048,576 pixels). The temporal change in the number of pixels in which SHG signal intensity was within the threshold range for each group was compared. Although the control group exhibited an increase in the number of pixels determined as a collagen fiber, the rate of increase was significantly smaller than those of under stretch conditions. In both stretch condition groups, the number of pixels within the threshold range increased significantly compared with the control group. This result implies that the amount of collagen fibers synthesized by osteoblasts was enhanced by the mechanical stimulation. For all three culture periods (1 to 3 weeks), there was a significant difference between the control group and both stretched groups. However, no significant difference was observed between the two stretch condition groups.

Fig. 9

Total number of pixels within the threshold range (an SHG intensity value of $95\pm 45$), which was considered to matured collagen fibers. These values were normalized by dividing the total number of pixels in the scanned area. Data represent the mean ± standard deviation. *Symbol indicates a significant difference ($p<0.05$, Student’s $t$-test).

To investigate the differences in collagen fiber maturity more precisely, we calculated the mean SHG intensity for those pixels determined as a collagen fiber. The maturity of collagen fiber includes fiber thickness, degree of cross linking and alignment of microfibrils in the collagen fiber. We hypothesized that more matured collagen fiber exhibits higher SHG signal intensity. Figure 10 shows the mean SHG intensity for the pixels determined as a collagen fiber as a function of time period in three groups. Although the number of pixels which SHG signal intensity was within the threshold range increased steadily during the culture periods (Fig. 9), the mean SHG intensity values for those pixels were almost constant during those three culture periods. This result suggests that the amount of osteoblastic collagen synthesis was steadily increased during the culture period and enhanced by the application of mechanical stimulation, whereas the maturity of the collagen fibers was not affected by the mechanical stimulation and culturing time period.

Fig. 10

Relationship between the mean values of the SHG intensity for the pixels within the threshold range. Data represent the mean ± standard deviation. There was no significant difference between all culturing time periods and all stimulation condition groups.