Research Article

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In situ time-series monitoring of collagen fibers
produced by standing-cultured osteoblasts using
a second-harmonic-generation microscope
EIJI HASE,1 OKI MATSUBARA,1 TAKEO MINAMIKAWA,2 KATSUYA SATO,2 AND TAKESHI YASUI2,*
1

Graduate School of Advanced Technology and Science, Tokushima University, 2-1 Minami-Josanjima, Tokushima 770-8506, Japan
Institute of Technology and Science, Tokushima University, 2-1 Minami-Josanjima, Tokushima 770-8506, Japan*Corresponding author: yasui.takeshi@tokushima‑u.ac.jp

2

Received 24 December 2015; revised 18 March 2016; accepted 18 March 2016; posted 18 March 2016 (Doc. ID 256429); published 15 April 2016

In bone tissue engineering and regeneration, there is a considerable need for an unstained method of monitoringcollagen fibers produced by osteoblasts. This is because collagen fibers play an important role as a bone matrix andcontinuous monitoring of their temporal dynamics is important in clarifying the organization process towardforming 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 needfor staining. Use of the 19 fs near-infrared pulsed light enables us to visualize the temporal dynamics in a thinlayer of collagen fibers produced by a single layer of osteoblasts in high-contrast SHG images. While the collagenfibers were produced and stored inside the osteoblasts at an early stage of culturing, the network structure ofcollagen 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. © 2016 Optical Society of America
OCIS codes: (180.4315) Nonlinear microscopy; (170.3880) Medical and biological imaging; (190.4160) Multiharmonic generation;(170.6930) Tissue.
http://dx.doi.org/10.1364/AO.55.003261

1. INTRODUCTION
Bone is the tissue that functions as a pivotal of the human bodyduring motion. Therefore, bone pain, deformities, and fractures
reduce quality of life significantly. However, since the metabolism of bone tissue is quite slow compared with other tissues, it isdifficult for the elderly to heal the damaged bone or increase asufficient amount of bone during a short period. Therefore, there
is a considerable need for bone tissue engineering and regeneration to locally replace damaged bone in the elderly [1,2].
Bone formation and regeneration in tissue engineering proceed via the following three steps. First, osteoblasts are differentiated and induced from induced pluripotent stem cells or
mesenchymal stem cells. Second, osteoblasts produce collagenfibers while increasing in number. Third, mineral is deposited
between the collagen fibers. Laboratory techniques for achieving the first step have already been established [3,4], and bone
tissue engineering is now in the stage of the second and third
steps. In these two steps, it is important to monitor the density
and structure of collagen fibers because collagen fibers play animportant role in constructing bone tissue as an extracellular1559-128X/16/123261-07 Journal © 2016 Optical Society of America

matrix and the structural maturity of them, namely, collagenmaturity, is related to bone mineralization.
Staining procedures have been widely used to selectively
visualize collagen fibers in histology [5]. However, such procedures cannot be used for continuously monitoring the temporal
dynamics of collagen fibers in situ during the culturing period
due to the invasiveness of the staining procedure and the damage it causes to cells. If the distribution of collagen fibers could
be visualized without the need for staining, in situ time-series
monitoring of collagen dynamics during the culturing period
would become possible. This would be useful for studying
how collagen fibers behave during the second and third steps,
namely, the organization process toward forming bone tissue.
Second-harmonic-generation (SHG) microscopy is a promising tool for in situ observation of collagen fibers in tissues
without the need for staining [6]. This microscopy has highselectivity and good image contrast to collagen molecules aswell as high spatial resolution, optical three-dimensional (3D)sectioning, minimal invasiveness, and moderate penetration.
Therefore, it has been widely used for visualization of collagen3262

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fibers in native tissues, such as skin [7], tendon [8], cornea [9],bone [10], and cartilage [11]. Furthermore, there are a few
reports in applying SHG microscopy for the tissue engineering
using fibroblast [12,13]. However, there have been no attempts
to use SHG microscopy in the field of the bone tissue engineering using osteoblasts.
In the work described here, we constructed an SHG microscope using 110 fs and 19 fs near-infrared pulsed light, andcompared SHG images of collagen fibers produced by cultured
osteoblasts. Then, we visualized the temporal evolution ofcollagen fibers produced by standing-cultured osteoblasts during a culturing period of 4 weeks in situ. Finally, we perform
image analysis of the SHG image based on the two-dimensional
Fourier transform (2D-FT) to extract a quantitative parameterfor the maturity degree of collagen fibers in the standingcultured osteoblast samples.
2. MATERIALS AND METHODS
A. Samples

We used the mouse osteoblast cell line MC3T3-E1, which wasprovided by the RIKEN-BRC through the National Bio-Resource
Project of the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT), Japan. The cells were cultured with
α-MEM medium (Nacalai Tesque Co. Ltd.) to which was added
10% fetal bovine serum until the cultured cell concentration
reached 80% confluent. We used a chamber made of silicone
rubber (STREX Inc.) for cell culturing. The chamber was sterilized at 120°C for 20 min with an autoclave, and its surface wasthen coated with fibronectin solution (Wako Pure Chemical
Industries, Ltd.) for 6 h to enhance the cell adhesion on the culture surface. Then the chamber was rinsed twice with Dulbecco’s
phosphate buffered saline. After the cells were seeded onto thechamber, they were cultured in the medium with an osteoblastinducing reagent. This medium was an α-MEM medium towhich was added 1% ascorbic acid, 0.2% hydrocortisone, and
2% β-glycerophosphate. For sterilization, we also added 1% penicillin and streptomycin solutions (100 U/ml and 100 μg/ml, respectively). We performed a standing culture of the cells for one,
two, three, and four weeks. A single layer of osteoblasts adhered
onto the bottom of the chamber produced a thin layer of collagenfibers (typical thickness <10 μm) for the culturing periods.
To confirm the production of collagen fibers by the standingcultured osteoblasts, we applied the staining procedure using
Sirius Red with the samples [14]. Sirius Red stains both thecollagen fiber and the nuclear pink. The staining procedure
was applied to each of the samples cultured for different periods.
Figures 1(a)–1(e) show stained images of the standing-cultured
osteoblast samples at 0 day, 1 week, 2 weeks, 3 weeks, and 4
weeks, respectively, after starting the standing culture (image
size ! 1 mm by 1 mm). In the sample cultured for 0 day, only
osteoblasts should be stained with light pink because of no production of collagen fibers immediately after starting the standing
culture [see Fig. 1(a)]. In the sample cultured for 1 week, thepink color became deeper due to the increased number of osteoblasts and the production of collagen fibers [see Fig. 1(b)].
However, it is not clear how much collagen fibers were produced.
In the sample cultured for 2 weeks, a gradation of deep pink andlight pink was observed [see Fig. 1(c)]. Since the osteoblasts were

Fig. 1. Sirius-Red-stained images of collagen fibers produced in thestanding-cultured osteoblast samples at (a) 0 day, (b) 1 week, (c) 2
weeks, (d) 3 weeks, and (e) 4 weeks after starting the standing culture.
Image size is 1 mm by 1 mm. The collagen fibers and the osteoblasts
were stained pink in this staining procedure.

already confluent at 1 week after starting the standing culture,
the gradation is mainly due to increased density of collagenfiber rather than the increased number of osteoblasts. In otherwords, osteoblasts produced dense collagen in some regions. In
the sample cultured for 3 weeks, the gradation became clearer,
and the pink color in dense collagen regions became deeper [seeFig. 1(d)]. In the sample cultured for 4 weeks in Fig. 1(e), there
was no significant difference of the stained image compared withthe sample cultured for 3 weeks, implying that the productionof the collagen fibers was almost saturated. In this way, we confirmed the production of collagen fibers in the sample.
B. SHG Microscope

Figure 2 shows the experimental setup of the SHG microscope.
Here we used a 110 fs and 19 fs near-infrared pulsed light for
SHG imaging. The 110 fs pulsed light at a wavelength of
780 nm was obtained by a frequency-doubled mode-locked
erbium-doped fiber laser (FD-ML-ErF, Toptica, FFS. SYS-SHG;

Fig. 2. Experimental setup of the SHG microscope. ML-TiS, 10 fs,
787 nm mode-locked Ti:sapphire laser; NDC, negative dispersion
controller; FD-ML-ErF, 110 fs, 775 nm frequency-doubled modelocked erbium-doped fiber laser; GM, galvanometer mirror; PMT,
photon-counting photomultiplier.

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spectral bandwidth!10 nm, repetition frequency!100 MHz).
The pulse duration at the focal point of an objective lens wasmeasured by a two-photon-fluorescence interferometric autocorrelator [15]. We confirmed that the pulse duration was almost
the same as that at the exit window of the laser cavity (110 fs). On
the other hand, the 19 fs pulsed light was achieved at the focal
point by precise dispersion control of an ultrashort mode-locked
Ti:sapphire laser light (ML-Ti:S, FEMTOLASERS Produktions
GmbH, Femtosource Scientific Pro; pulse duration ! 10 fs,
center wavelength ! 787 nm, spectral bandwidth ! 103 nm,
repetition frequency ! 81.8 MHz) with an external negative
dispersion controller (NDC, FEMTOLASERS Produktions
GmbH, MOSAIC PRO V), composed of a negatively chirped
mirror pair (group delay dispersion ! −900 to −6270 fs2 ) and apositively chirped wedge prism pair (group delay dispersion !
"50.8 to "356 fs2 ).
After selecting either 110 fs pulsed light or the 19 fs one
by a flipper mirror, the laser beam was focused onto the sample
with an objective lens (Olympus Corp., LUMPlanFI/IR;
magnification ! 60, NA ! 0.90, working distance ! 2 mm,
water immersion) and its focal point was two-dimensionallyscanned by a combination of the galvanometer mirrors (GMs)and the relay lenses (f ! 80 mm and 150 mm, magnification !
1.875). The forward-propagating SHG light was detected by aphoton-counting photomultiplier (PMT, Hamamatsu Photonics
K. K., H8259-01) after collecting SHG light with a condenser
lens and eliminating the laser light with optical filters.
Using the above GM optics, SHG images of a 260 μm by
260 μm region, composed of 256 pixels by 256 pixels, wereacquired at a rate of 0.1 image/s. The field of view in SHG
imaging is limited by a scanning angle of the galvanometer mirror and/or a magnification factor of the relay lenses. To further
enlarge the imaging region, we scanned the sample position horizontally or vertically at intervals of 260 μm using a steppingmotor-driven translation stage every time acquiring an SHG
image of 260 μm by 260 μm regions using the GMs. Finally,
we obtained a large SHG image with a size of 1.04 mm by
1.04 mm, corresponding to 1024 pixels by 1024 pixels, bystitching 16 SHG images together in a matrix of four rows andfour lines.
3. RESULTS
A. Comparison of SHG Images of CulturedOsteoblasts-Produced Collagen Fibers Obtained with
110 fs and 19 fs Pulsed Light

We visualized the spatial distribution of collagen fibers in a
cultured osteoblast sample (culturing period ! 3 weeks) by
SHG microscopy. Figures 3(a) and 3(b) show a comparison of
SHG images of collagen fibers obtained with 110 fs pulsed
light (average power ! 20 mW) and 19 fs pulsed light(average power ! 20 mW), respectively. The gray scales of the
SHG light intensity in these two images were corrected by considering the different repetition frequencies (100 MHz for
110 fs pulsed light and 81.8 MHz for 19 fs pulsed light),
allowing us to directly compare the intensity of SHG images.
The dimness in the edge of the SHG image is mainly due toinsufficient filling of water between the objective lens surface
and the bottom of the silicone-rubber chamber. In the SHG

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Fig. 3. SHG images of collagen fiber in the cultured osteoblast sample (culturing period ! 3 weeks) obtained using (a) 110 fs pulsed light(power ! 20 mW) and (b) 19 fs pulsed light (power ! 20 mW).
Image size is 1040 μm by 1040 μm, composed of 1024 pixels by1024 pixels. (c) Ratio image obtained by dividing two SHG images
obtained with 110 fs pulsed light and 19 fs pulsed light.

image obtained with the 110 fs pulsed light, it is difficult todetermine the detailed distribution of collagen fibers due tothe too low SHG intensity [see Fig. 3(a)]. On the other hand,
the SHG image obtained with the 19 fs pulsed light clearly showsthat fine collagen fibers were distributed in the whole imaging
region, and their density increased locally [see Fig. 3(b)].
Next, to investigate the enhancement of SHG light intensity
quantitatively, we generated an image formed by calculating the
SHG light intensity ratio at each pixel of the two SHG images
in Figs. 3(a) and 3(b). Figure 3(c) shows the resulting ratio image. In 16 regions of interest (ROI) of this ratio image [see blackboxes in Fig. 3(c)], the ratios were distributed within 3.81 #0.11 (mean # standard deviation). Since the SHG light intensity has an inverse dependence on the pulse duration under theconstant pulse energy [16], an 8.7 times enhancement of the
SHG light intensity is expected from the difference in the pulse
durations (110 fs and 19 fs). However, the actual enhancementof the SHG image remained as 3.81. One reason for the lower
enhancement is in the residual chirping and pedestal components of the 19 fs pulse light. These hardly contribute to SHG
light generation due to lower peak power and lead to a decreasein SHG enhancement. Also, the achromatic aberration of theobjective lens may have decreased the signal enhancement of
the SHG image obtained with 19 fs pulsed light, because
the tight focusing of the broadband laser light is a critical factor
in SHG microscopy. Nevertheless, the obtained enhancement
factor of 3.81 enabled us to visualize the spatial distribution in athin layer of collagen fibers produced by a single layer of thecultured osteoblasts in situ without the need for staining. The
risk of photodamage in the cells induced by the ultrashortpulsed light is discussed later.

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B. In Situ Time-Series SHG Imaging of Collagen
Fibers Produced by Standing-Cultured Osteoblasts
During 4-Week Culturing Period

We performed in situ time-series SHG imaging of the collagenfibers produced in one and the same standing-cultured osteoblast sample. Figures 4(a)–4(e) show time-series SHG images of
the collagen fiber distribution in one and the same sample at 0day, 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively, after
starting the standing culture. Although SHG light might begenerated from microtubule in the cells in addition to collagen[17], the sample at 0 day, which is before producing the collagen fibers, indicated no SHG signals [see Fig. 4(a)]. Therefore,
we can conclude that microtubule did not contribute to the
SHG signal in this experiment. In the sample cultured for 1 to
4 weeks, it was clear that the collagen fibers were produced andtheir distribution changed with the elapsed time. In Fig. 4(b), thedistribution of the collagen fibers appeared as circular shapes,
implying that the collagen fibers were produced and stored in
the osteoblasts. In Fig. 4(c), a similar distribution was confirmed;
however, SHG light was more intense than that in Fig. 4(b),implying that the produced collagen fibers accumulated in theosteoblasts and their density increased. In Fig. 4(d), thin collagenFig. 4. In situ time-series SHG images of collagen fiber distribution
produced in one and the same sample of standing-cultured osteoblastsat (a) 0 day, (b) 1 week, (c) 2 weeks, (d) 3 weeks, and (e) 4 weeks
after starting the standing culture. Image size is 1040 μm by 1040 μm,
composed of 1024 pixels by 1024 pixels.

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fibers appeared as a network structure outside the osteoblasts inaddition to the circle-like collagen distribution inside them. In
Fig. 4(e), the collagen fibers became thicker, and the density ofcollagen fibers outside the osteoblasts increased locally. Fromthe comparison of these SHG images with the stained images
in Fig. 1, the rough behavior of the collagen fibers during thestanding culture was reasonably in agreement between them.
However, it should be noted that the SHG images visualized
more detailed changes of the collagen fiber distribution than thestained images.
C. Quantitative Analysis for the Maturity Degreeof Collagen Fibers

The characteristic changes in a series of SHG images in Fig. 4
include the accumulation of collagen fibers inside the osteoblasts and the subsequent formation of collagen fiber networks
outside the osteoblasts. We consider that such characteristicchanges are related to the maturity degree of the collagen fibers.
One promising method to extract the spatial characteristicsof those SHG images is use of the image analysis based on
the 2D-FT [18]. This image analysis is useful for the evaluationof matured native tissues having the specific orientation or theperiodic structure of collagen fibers. However, the collagenfibers produced by the cultured osteoblasts are less structured
and nonspecifically oriented due to immature structure [seeFigs. 4(d) and 4(e)]. To extend the SHG image analysis tothe sample with less structured and nonspecifically orientatedcollagen fibers, we have to use another approach of SHG image
analysis. To extract the maturity degree of collagen fibers from
SHG image quantitatively, we proposed another 2D-FT-based
image analysis and applied it for SHG images in Fig. 4.
We selected a ROI with size of 11 pixels by 11 pixels in theoriginal SHG image (pixel size ! 1024 × 1024), calculated the
2D-FT, and obtained the 2D-FT power spectrum. Afterthe curve fitting analysis of the 2D-FT power spectrum withthe 2D Gaussian function, we calculated a ratio of a long axis toa short axis (FT-L/S ratio) in the elliptical base of the function.
We repeated a similar procedure every time the ROI was shiftedhorizontally or vertically at 5 pixel intervals, and obtained the2D mapping of the FT-L/S ratio, namely, an FT-L/S image,with a size of 202 pixels by 202 pixels. The FT-L/S image contains information about the network structure of collagen fibers
rather than the density of them because the FT-L/S ratio is notdirectly related to the SHG light intensity. To take the densityof collagen fiber into consideration, we have to convolute theintensity information of the SHG image with the FT-L/S image. To this end, the size of the SHG image was reduced from1024 pixels by 1024 pixels to 202 pixels by 202 pixels by binning the data in each ROI, and the resized SHG image wasmultiplied by the FT-L/S image. The resulting image includes
information on both the network structure and the density ofcollagen fibers. We called this image a collagen maturity factor(CMF) image. After segmenting the CMF image (pixel size !
202 × 202) into 16 images (pixel size ! 50 × 50), we calculated the mean CMF value for each segmented image. Finally,
we performed the significance test of the mean CMF values
among the samples cultured for different periods.
Figure 5 shows the comparison of the original SHG image,
the FT-L/S image, the resized SHG image, and the CMF image

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4. DISCUSSION

Fig. 5. Comparison of the original SHG image (1024 pixels by1024 pixels), the FT-L/S image (202 pixels by 202 pixels), the resized
SHG image (202 pixels by 202 pixels), and the CMF image (202pixels by 202 pixels) with respect to the elapsed time of the standing
culture.

with respect to the elapsed time of the standing culture. We
consider that the CMF image reasonably reflected the temporal
evolution of the collagen fibers, or the maturity degree of collagen fibers, during the standing culture.
Figure 6 shows the relation between the culturing periods
and the mean CMF values for 16 segmented CMF images,
indicating the increase of the mean CMF value with respect
to the elapsed time. There were significant differencesamong the samples cultured for 0 day, 1 week, 2 weeks,and 3 weeks. On the other hand, no significant difference
between samples cultured for 3 weeks and 4 weeks may imply
nearly saturation of the collagen production during the standing culture.

Fig. 6. Relation between the culturing periods and the mean CMF
value for 16 segmented CMF images. The p value was calculated byStudent’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, notsignificant).

We succeeded in demonstrating in situ time-series SHG
imaging of the collagen fibers produced by standing-cultured
osteoblasts. Use of the 19 fs pulsed light enables sensitive visualization of the thin-layer collagen fibers produced by a single
layer of the osteoblasts; however, we could not visualize it
at high image contrast by use of the 110 fs pulsed light. Suchsensitive SHG imaging of osteoblast-produced collagen fibers
will open a new door for bone tissue engineering and regeneration. First, the specific selectivity to only collagen fibers, withhigh image contrast, allows simple and straightforward investigation of the collagen dynamics in cultured osteoblast samples. The high-contrast, background-free SHG images makethe image analysis more powerful for quantitative analysis.
It is in contrast to the stained procedure where not only thecollagen fibers but also the osteoblasts were stained with thesimilar pink color. Second, and more importantly, the stain-freevisualization of collagen fibers allows us to in situ monitor one
and the same sample in a time series. This feature is useful for
determining the response of collagen fibers to various external
stimuli as well as the standing culture. For example, it is known
that a mechanical stimulus [19] or chemical one [20] promotes
the production of collagen fibers by cultured osteoblasts.
Furthermore, to achieve the desired mechanical properties ofbone tissue, it may be possible to control the production, superorganization, and/or orientation of collagen fibers in bone
tissue by stimulus feedback based on the monitoring of SHG
images. This method will open the door for in situ feedback
control of the quality and quantity in collagen fibers in bone
tissue engineering.
Next, we consider the relation between the collagen maturity degree and the start of the bone mineralization because
osteoblasts create the nanocomposite structure of bone bysecreting a collagenous extracellular matrix on which apatite
crystals subsequently form [21]. Figures 7(a)–7(c) show thephase contrast image of the standing-cultured osteoblast samples at 7 days, 21 days, and 31 days after starting the culture,

Fig. 7. Phase contrast image of the standing-cultured osteoblast
samples at (a) 7 days, (b) 21 days, and (c) 31 days after starting theculture, respectively. The black parts indicate the apatite crystals andthe pink color is mainly due to the culture medium.

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respectively. The black parts in these images indicate the apatite
crystals and the pink color is mainly due to the culture medium.
In Fig. 7(a), the osteoblasts proliferated and became confluent,whereas the apatite crystals did not form yet. After that, the apatite crystals started to appear [see Fig. 7(b)] and increased theirarea with the lapse of time [see Fig. 7(c)]. These temporal behaviors of the apatite crystals were significantly in accord with thesaturation of collagen production, or the collagen maturity,
indicated by the mean CMF value in Fig. 6. Therefore, the mean
CMF value may be used as an indicator for the start of bonemineralization during the culture.
While use of the sub-20 fs pulsed light effectively enhanced
the image contrast in the SHG microscopy of the skin tissue[22] and the fibroblast-produced collagen fibers [13] in addition to the osteoblast-produced collagen fibers in this study, we
may have to consider its invasiveness to cells. When reducing
the pulse duration below 100 fs, the high peak energy increases
the risk of nonlinear photodamage and decreases the viability ofliving cells. For example, in the previous study for the Chinese
hamster ovarian cell, the risk of the photodamage in multiphoton microscopy increased inversely proportional to the pulse
duration [23]. Also, a similar result was obtained in bovine
adrenal chromaffin cells [24]. However, there have been noattempts to investigate the photodamage of the osteoblasts.
During the culturing period of 4 weeks in Fig. 4, we monitored
the morphological change of the osteoblasts using the phase
contrast microscopy together with SHG microscopy. However,
we did not observe the structural change of them (not shown).
Furthermore, SHG images in Fig. 4 indicate that the laserbeam-irradiated osteoblasts adequately produced collagenfibers during the culturing period in the same manner as theosteoblasts without irradiation of the laser beam (not shown).
However, these are no more than the preliminary findings
about the photodamage. The more careful analysis of the photodamage is required to conclude whether the osteoblasts arephotodamaged or not. Future work is to quantitatively investigate the influence of the laser irradiation on the osteoblast bymeasuring alkaline phosphatase activity, which is a marker ofbone production activity [25].
5. CONCLUSIONS
We constructed a SHG microscope for in situ time-series
monitoring of collagen fibers produced by standing-cultured
osteoblasts. Use of the 19 fs pulsed light enables us to visualize
a thin layer of collagen fibers produced by a single layer ofthe osteoblasts in high-contrast SHG images. Using thisSHG microscope, the temporal evolution of the collagen fiber
distribution in the standing-cultured osteoblast samples wascontinuously monitored during a culturing period of 4 weeks
in the form of high-contrast, time-series SHG images. From acomparison with the staining procedure, we confirmed theability to obtain knowledge about the detailed behavior of collagen fibers during the culturing period from the SHG images.
Furthermore, we succeeded in extracting a quantitative parameter of the collagen maturity degree in the cultured sample
by use of the image analysis based on 2D-FT of SHG images.
Although 2D SHG imaging was performed in this paper,
3D imaging capability in SHG microscopy will be useful forvolume evaluation of the osteoblast-produced collagen fibers
if the 3D culturing model of osteoblasts is prepared by useof collagen gel or scaffold. Such in situ visualization and thequantitative analysis of the osteoblast-produced collagen fibers
will be a powerful tool for the quality control of cultured and/or
regenerated tissues in bone tissue engineering.
Funding. Ministry of Education, Culture, Sports, Science,
and Technology (MEXT) (25560200); Tokushima University.
Acknowledgment. The authors are grateful to Dr. S.
Fukushima of Osaka University for fruitful discussions.
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