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Cell

Biology
International

Cell Biology International 28 (2004) 255265

www.elsevier.com/locate/cellbi

Effects of ascorbic acid and ascorbic acid 2-phosphate, a


long-acting vitamin C derivative, on the proliferation and
differentiation of human osteoblast-like cells
Shinji Takamizawa a,b, Yojiro Maehata a, Katsuyuki Imai c, Haruki Senoo c,
Sadao Sato b,d, Ryu-Ichiro Hata a,d*
a
Department of Biochemistry and Molecular Biology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan
Department of Craniofacial Growth and Development Dentistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan
c
Department of Cell Biology and Histology, Akita University School of Medicine, Hondo, Akita 010-8543, Japan
d
Research Center of Advanced Technology for Craniomandibular Function, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan
b

Received 3 July 2003; revised 30 October 2003; accepted 10 January 2004

Abstract
In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C
derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells
with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations
in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its
concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis
and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis,
indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a
three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.
! 2004 Elsevier Ltd. All rights reserved.
Keywords: Ascorbic acid 2-phosphate; Ascorbic acid; Long-acting vitamin C; Human osteoblasts; Osteoblast differentiation

1. Introduction
Multifarious activities of -ascorbic acid (vitamin C,
AsA) have been demonstrated in many biological systems and in various scientific fields (Burns et al., 1987;
Hata, 1996). One of the essential functions of AsA may
be its role as a cofactor for the hydroxylation of proline
and lysine residues in collagen, which is the most abundant protein in the body (Kielty et al., 1993; Prockop
and Kivirikko, 1995). It is also an essential supplement
for the differentiation of various kinds of cells in culture
(Franceschi, 1992). One difficulty with the handling of
AsA is its instability in solution, especially under the
* Corresponding author. Tel.: +81-46-822-8840;
fax: +81-46-822-8839
E-mail address: ryuhata@kdcnet.ac.jp (R. Hata).
Abbreviations: Asc 2-P, ascorbic acid 2-phosphate; AsA, ascorbic
acid.
1065-6995/$ - see front matter ! 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2004.01.010

normal culture conditions of neutral pH and 37 (C.


Previously, we reported that a phosphate derivative
of AsA, ascorbic acid 2-phosphate (Asc 2-P), had a
cofactor activity for collagen biosynthesis by human
fibroblasts in culture, as did AsA. Asc 2-P was also
found to be very stable under culture conditions (Hata
and Senoo, 1989). Asc 2-P has since been used as a
supplement, instead of AsA, in a variety of cultures,
including that of osteoblastic cells (Fermor et al., 1998;
Gallagher et al., 1996; Gundle and Beresford, 1995;
references in Hata, 1996; Hitomi et al., 1992; Mizutani
et al., 2001; Senoo et al., 1989; Torii et al., 1994).
Both AsA and Asc 2-P have been shown to stimulate
differentiation of various kinds of cells, but there is little
information about any direct comparison of their effects.
Over a decade ago, we reported the stimulation of
growth and differentiation of human dermal fibroblasts
by both Asc 2-P and AsA (Hata et al., 1988; Hata and

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Senoo, 1989). Both of them are frequently used in


cultures of osteoblastic cells derived from various
kinds of animal species, but there are no comparative
reports about the effects of both factors on the growth
and differentiation of these cells. Here, we report
our results on such a comparison using MG-63 cells,
which are a widely used cell line of human osteoblastlike cells.

2. Materials and methods


2.1. Cell culture
MG-63 human osteoblast-like cells were obtained
from American Type Culture Collection (Rockville,
MD, USA). The cells were divided into ampoules and
stored in liquid nitrogen. Cells from one ampoule were
grown and used for a series of experiments. The cells
responded to active vitamin D3 by expressing alkaline
phosphatase (ALP) activity, an early osteoblast differentiation marker, and osteocalcin, a marker of terminally differentiated osteoblasts (Franceschi and Young,
1990; Hata et al., 2002; Lian et al., 1999; Maehata et al.,
2002). The cells were plated at 2"105 per 100 mm
culture dishes and cultured in DMEM-10, which is
Dulbeccos modified Eagles medium (DMEM) (Nissui
Pharmaceutical, Tokyo, Japan) supplemented with an
antibiotic (gentamycin sulfate, 50 mg/l), an anti-fungal
reagent (Fungizone, 0.25 mg/l), buffered with N-2hydroxyethylpiperazine-N#-2-ethansulfonic acid (3 g/l,
DMEM-0), and containing 10% heat-inactivated fetal
bovine serum (FBS; Trance Scientific, Melbourne,
Australia). Cells were passaged once a week after dispersion with 0.1% trypsin (Wako Pure Chemical Industries,
Ltd, Osaka, Japan) in Dulbeccos Mg2+-/Ca2+-free phosphate buffered saline (DPBS [#]; TaKaRa, Tokyo,
Japan) for 15 min at 37 (C. Cells were counted using a
Coulter Z1 Counter (Coulter Electronics Ltd, England)
after dispersion of the cells with 0.1% trypsin solution in
DPBS (#).
2.2. Determination of growth rate
MG-63 cells were plated at 1"105 per 35 mm dish
(FALCON 3046, Becton Dickinson and Co., NJ, USA)
and cultured in DMEM-0, supplemented or not with the
sodium salt of -ascorbic acid (AsA, 0.251 mM; Wako
Pure Chemical Industries, Ltd, Osaka, Japan) or
-ascorbic acid 2-phosphate magnesium salt (Asc 2-P,
0.251 mM; Wako Pure Chemical Industries, Ltd) in the
absence or presence of 1% or 10% FBS. Phase-contrast
photomicrographs were taken with a DIAPHOT-TMD
(Nikon, Tokyo, Japan). DNA content was determined
by a modification of the fluorometric method described
previously (Hata et al., 1984).

2.3. Determination of alkaline phosphatase activity


After selected periods of culture, the medium was
removed, and the cell layer was rinsed twice with DPBS
(#) and then lysed with cell lysis buffer consisting of 10
mM TrisHCl, pH 7.5, containing 0.4% Nonidet P-40
(Iwaki Kagaku Co., Tokyo, Japan). Phenylmethylsulfonyl fluoride (Wako Pure Chemical Industries, Ltd)
was added to a final concentration of 4 mM (Franceschi
and Young, 1990). One half of the lysate was used
for determination of the DNA content as described
above and the remaining half for measurement of
ALP activity, using phosphophenol as the substrate
(Alkaline Phospha K, Wako Pure Chemical Industries,
Ltd).
2.4. Reverse transcriptasepolymerase chain reaction
(RTPCR)
Total RNA was isolated from the cells with TRIzol
(Invitrogen, Tokyo). Complementary DNAs were synthesized using the Superscript First Strand Synthesis
System (Invitrogen, Tokyo), following the manufacturers protocol. Aliquots (5%) of the total cDNA were
amplified in each PCR in a 20 l reaction mixture that
contained 5 pmol of 5# and 3# primers, 1"PCR buffer,
2.0 mM MgCl2, 0.2 mM of each deoxytrinucleotide, and
0.5 U of Ex Taq polymerase (TaKaRa, Tokyo). Each
cDNA sample was run in duplicate for every PCR.
Amplification was performed using a Mastercycler
gradient (Eppendorf-Netheler-Hinz GmbH, Hamburg).
The first denaturation step at 94 (C for 2 min was
followed by 15, 20 and 25 cycles of PCR for !-actin,
each consisting of denaturation at 94 (C for 30 s,
annealing at 60 (C for 2 min and extension at 72 (C for
2 min; and by 20, 25 and 30 cycles for bone/liver/kidney
ALP, each consisting of denaturation at 94 (C for 30 s,
annealing at 57 (C for 2 min, and extension at 72 (C for
2 min. In all cases, a final extension at 72 (C for 10 min
was performed before storing the samples. Specific
primers used were: for human !-actin, AGCCATGTA
CGTTGCTA (sense), and AGTCCGCCTAGAAGCA
(antisense), for amplification of 800 base pairs (bps); and
for ALP, ACGTGGCTAAGAATGTCATC (sense),
and CTGGTAGGCGATGTCCTTA (antisense) for
amplification of 475 bps (Rickard et al., 1996). PCR
products were separated by 2% agarose (NuSieve
3:1 agarose, BioWhittaker Molecular Applications,
Rockland, ME, USA) at least twice.
The separated DNA fragments were visualized by
ethidium bromide staining and quantified with a
FluorImager 595 (Amersham Phamacia Biotech, Tokyo)
using excitation at 514 nm, emission at 610 nm and
Image Quant for calculations. Twenty cycle-amplified
!-actin and 25 cycle-amplified ALP PCR products were
within the linear range of the amplification curves and
thus used for semi-quantitative analysis. Differences in

S. Takamizawa et al. / Cell Biology International 28 (2004) 255265

257

Fig. 1. Schematic representation of metabolism of -ascorbic acid 2-Phosphate (Asc 2-P) and -ascorbic acid (AsA). Bold arrows indicate rapid
reactions.

the quantitative determinations between ALP PCR


product bands were calculated after normalizing the
values to the corresponding 20 cycle-amplified !-actin
PCR product band. Measurements were repeated at
least three times.
2.5. Metabolic labeling and collagen assay
The cells were incubated in 1 ml of fresh DMEM-10
containing [3H]proline (370 kbq/ml, -[2,3-3H]proline,
37 MBq/ml in ethanol:water [2:98] from PerkinElmer,
Boston, USA) and 0.25 mM Asc 2-P for the last 24 h in
the presence or absence of azetidine 2-carboxylic acid
(Az-C, 0.5 and 1.0 mM; Sigma-Aldrich Japan, Tokyo)
or ethyl-dihydroxybenzoate (EDHBA, 0.2 and 0.4 mM;
Sigma-Aldrich). The combined cell layer and medium
was processed for the determination of collagen and
noncollagenous protein synthesis using nonspecific
protease-free collagenases, as described previously (Hata
et al., 1980). The relative rate of collagen synthesis was
calculated assuming that collagen has an amino acid
content 5.4 times higher than that of other proteins
(Peterkofsky and Diegelmann, 1971).
2.6. Electron microscopy
The cells were fixed, processed and observed as
described previously (Hata and Senoo, 1989), except
that ultra thin sections were cut with an ultramicrotome
2088-V (LKB, Bromma, Sweden) and examined under
a transmission electron microscope (JEOL-1200EX,
Tokyo) at an acceleration voltage of 80 kV.

2.7. Statistical analysis


Data were expressed as mean$S.D. Significance of
differences was determined using Students t-test.
3. Results
3.1. Effect of AsA and Asc 2-P on the growth of MG-63
cells
Asc 2-P is stable under culture conditions and
liberates AsA by the action of ALP on the plasma
membrane of various kinds of cells. AsA thus produced
is then incorporated into the cells (Fig. 1).
In order to evaluate the effects of AsA and Asc 2-P on
the growth of cells, we first plated MG-63 cells in
DMEM-10 and cultured them for 24 h. During this
period, the cells adhered well to the culture dish surface.
Then the culture medium was changed to DMEM
containing various concentrations of FBS, AsA or Asc
2-P. The cells grew for 3 days in DMEM containing 1%
FBS (Fig. 2B) or 10% FBS (Fig. 2C), becoming three
times and eight times more numerous, respectively,
compared to day 0, as determined by the increase in
DNA content (Fig. 3A). The cell number did not
increase during this period in the absence of serum
(Fig. 2A and Fig. 3A).
When the medium was supplemented with AsA, no
significant growth stimulation was observed (Fig. 2E, F,
H, and I). Instead, disintegrated cells were observed in
the presence of AsA under serum-free culture conditions
(Fig. 2D, G, and J) or in the presence of 1.0 mM AsA

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Fig. 2. Effect of various concentrations of serum, AsA and Asc 2-P on the morphology of MG-63 cells. The cells were plated (1"105/35 mm dish)
and cultured in DMEM-10 for 24 h. Then the culture medium was changed to fresh DMEM containing various concentrations of FBS (0, 1, 10%)
in the absence (control, A, B, C) or presence of AsA (+AsA: 0.25 [D, E, F], 0.5 [G, H, I], or 1.0 mM [J, K, L]), or Asc 2-P (+Asc 2-P; 0.25 [d, e,
f], 0.5 [g, h, I], or 1.0 mM [j, k, l]). Phase-contrast photomicrographs were taken 3 days after the addition of AsA or Asc 2-P. Magnification "200.

and 1% FBS (Fig. 2K). In fact, dose-dependent inhibition of cell growth was indicated by the decrease in
DNA content, when the medium was supplemented with
AsA under serum-free culture conditions (Fig. 3B).
Similar apparent growth inhibition was observed by
supplementation of the culture medium with 1.0 mM
AsA and 1% FBS (Fig. 2K, Fig. 3B). On the other hand,
no apparent growth inhibition was observed when Asc
2-P was the supplement, whether FBS was present or not
(Fig. 2d to i). Instead, significant growth stimulation was
observed by supplementation with 1.0 mM Asc 2-P,
irrespective of the FBS concentration (Fig. 3C). Growth
stimulative effects of Asc 2-P were also observed at
lower concentrations, when FBS was present in the
culture medium (Fig. 3C).
3.2. Effects of AsA and Asc 2-P on ALP activity and
mRNA levels of MG-63 cells
AsA at 0.25 mM, the concentration normally used for
culture, significantly stimulated ALP activity in MG-63

cells. Asc 2-P also increased the enzyme activity, giving


a value much higher than that obtained with AsA
(Fig. 4B). Cell growth was also stimulated in cultures
containing Asc 2-P, but not in those supplemented with
AsA (Fig. 4 A). The effect of these factors on the level of
ALP mRNA was examined by RTPCR. After gel
electrophoresis and staining, we detected a band that
moved a little bit faster than the 500 bps marker band;
and it corresponded with the size (475 bps) of the
RTPCR product expected for human bone/liver/kidney
type ALP. We confirmed the DNA sequences of RT
PCR products after cloning (data not shown). The
bands of ALP-specific PCR products were denser when
mRNAs were obtained from cultures having AsA or
Asc 2-P, than in those from the control. The differences
were observed regardless of the cycle number of PCR
(Fig. 5A). Semi-quantitative determination of the
density of the amplified bands indicated a significant
increase in ALP PCR products when the cells were
cultured for 7 days with 0.25 mM AsA or Asc 2-P
(Fig. 5B).

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Fig. 3. Effect of various concentrations of serum, AsA and Asc 2-P on the growth of MG-63 cells. The cells were plated (1"105/35 mm dish) and
cultured in DMEM-10 for 24 h. Then the medium was exchanged for fresh medium containing FBS at 0%, 1%, and 10% without (A, control) or with
AsA (B, +AsA: 0.25, 0.5, 1.0 mM) or Asc 2-P (C, +Asc 2-P: 0.25, 0.5, 1.0 mM). Values are the means of quadruplicate assays$S.D. Significantly
different from respective controls: *P<0.05, **P<0.01.

3.3. Collagen is essential for stimulation of cell growth


and ALP activity by Asc 2-P
When 0.25 mM Asc 2-P was present in the cultures,
collagen synthesis, as well as total protein synthesis,
increased (data not shown). The relative rate of collagen

synthesis to total protein synthesis also increased from


1.4% to 2.3% in the presence of Asc 2-P (Fig. 6A); and
the Asc 2-P-induced increase in cell growth, as seen from
DNA content and ALP activity (Figs. 3 and 4), was
confirmed (Fig. 6B, C, and D). Various concentrations
of inhibitors of collagen synthesis, such as Az-C and

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Fig. 4. Effects of AsA and Asc 2-P on the growth and ALP activity of MG-63 cells. The cells were plated (1"105/35 mm dish) and cultured in
DMEM-10 in the absence (Control) or presence of AsA (0.25 mM) or Asc 2-P (0.25 mM) for 6 days. The medium was changed 3 days after seeding.
A: DNA content per 35 mm dish. B: ALP activity shown as femtomoles of phenol released per minute per g DNA. Values are the means of
quadruplicate assays $S.D. Significant differences are indicated by brackets: *P<0.05, **P<0.01.

EDHBA, were added together with Asc 2-P, in order to


see the relationship between collagen synthesis, cell
growth and ALP activity. The presence of collagen
synthesis inhibitors suppressed collagen synthesis in the
cells in a dose dependent manner (Fig. 6A). In addition,
cell growth (DNA content) and ALP activity were
repressed by the presence of either inhibitor. These
results indicate that stimulation of collagen synthesis
is essential for stimulation of both growth and ALP
activity in the cells by Asc 2-P.
3.4. Formation of ECM rich tissue-like substance by
MG-63 cells
Electron microscopy revealed multi-layered cells, and
well-developed rough endoplasmic reticulum (rER) and
Golgi apparatus (Fig. 7B, D) in cultures in the presence
of Asc 2-P (+Asc 2-P, Fig. 7B and 7D), whereas in its

absence, multi-layering and these organelles were less


pronounced (Control, Fig. 7A, C). Cells surrounded
by striated fibers typical of fibrous collagen were
prominent in Asc 2-P supplemented cultures (Fig. 7B,
insert), but not evident in the control cultures (Fig. 7A,
insert). These data indicate Asc 2-P-stimulated matrix
maturation and formation of a tissue-like substance by
MG-63 cells.
4. Discussion
It has been reported that AsA is essential for
the differentiation of osteoblastic cells in culture
(Franceschi, 1999; Franceschi and Young, 1990; Owen
et al., 1990). In their pioneering work using MG-63 cells
to study the regulation of osteoblast differentiation,
Franceschi and Young (1990) reported that AsA

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261

Fig. 5. RTPCR analysis of ALP mRNA levels of MG-63 cells cultured in the absence or presence of 0.25 mM AsA or Asc 2-P for 7 days. The cells
were plated (1"105/35 mm dish) and cultured in DMEM-10 in the absence (Control) or presence of AsA (0.25 mM) or Asc 2-P (0.25 mM) for
7 days. Total RNAs were prepared and used for RTPCR as described in the text. A: Aliquots of RTPCR product were separated by 2% agarose
gel electrophoresis and visualized by staining with ethidium bromide. B: Relative amounts of ALP mRNA expression were determined using a Fluor
Imager. Values are means$S.D. of triplicate determinations using two different preparations of total RNAs. Significant differences are indicated by
brackets: *P<0.05, **P<0.01.

repressed the cell growth and stimulated the differentiation of these human osteoblast-like cells. We are
interested in this work, because these cells respond to
active vitamin D3 by expressing ALP, an early osteoblast differentiation marker, and osteocalcin, a marker
of terminally differentiated osteoblasts (Franceschi and
Young, 1990; Hata et al., 2002; Lian et al., 1999;
Maehata et al., 2002). When we used Asc 2-P, a longacting ascorbic acid derivative, as a supplement in the
culture medium of MG-63 cells, it stimulated the nascent

growth of the cells, irrespective of the concentration


used (Figs. 2 and 3).
On the other hand, the growth of the cells was either
repressed or unaffected, depending on the concentration
of AsA and FBS (Figs. 2 and 3). The effect also varied
depending on the freshness of the medium, DMEM-10
and cell density (data not shown). These variations in
the effect on cell growth have also been seen in other
culture systems (Clement et al., 2001; Makino et al.,
1999). Such variations may be due to opposing effects of

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Fig. 6. Effects of Asc 2-P and inhibitors of collagen synthesis on collagen synthesis, cell proliferation and ALP activity of MG-63 cells. The cells were
inoculated (1"105/35 mm dish) and cultured in DMEM-10 in the absence (Control, open bars) or presence of 0.25 mM Asc 2-P. Azetizine
2-carboxylic acid (Az-C, 0.5 or 1.0 mM) or ethyl-3,4-dihydroxybenzoate (EDHBA, 0.2 or 0.4 mM), inhibitors of collagen synthesis were added to
some of the Asc 2-P-containing cultures, and the cells were cultured for a further 7 days. A: Relative rate of collagen synthesis was determined by
a method using purified bacterial collagenases. B: DNA content was determined by fluorometric assay. C: ALP activity indicated as picomoles of
phenol released per minute per dish. D: ALP activity expressed as femtomoles of phenol released per minute per g DNA. Significantly different
from control values: *P<0.05, **P<0.01 and significantly different from Asc 2-P-supplemented cultures, aP<0.05, bP<0.01.

AsA, i.e. its stimulatory effect on growth, and its inhibitory side effects under in vitro culture conditions, where
there is an excess of O2 (Makino et al., 1999; Oda et al.,
2001).
Ascorbyl radicals produced from AsA and H2O2 are
generated by self-reduction of oxidized AsA, due to an
excessive amount of it in the presence of excess O2. In
fact, 10 mM AsA and 0.25 mM H2O2 killed nearly 100%
of MG-63 cells (data not shown). Asc 2-P is resistant to
oxidation by O2 and releases free AsA by the action of
ALP in the cell membranes of most animal cells, including osteoblasts. There was no inhibitory effect on growth
by Asc 2-P up to 10 mM (Figs. 2 and 3, and data not
shown), suggesting that free AsA is formed from Asc 2-P
only at the cell surface and the AsA thus formed is
immediately incorporated by the cells. These data indicate that Asc 2-P is superior to AsA as a supplement
for in vitro cultures, so we used only the former in
subsequent experiments.

Asc 2-P also stimulated collagen synthesis and ALP


activity. In addition, specific inhibition of collagen
synthesis due to the presence of both Asc 2-P and the
proline analogue Az-C, which is incorporated instead
of proline into proto-procollagen polypeptides and
inhibits the maturation of collagen chains, attenuated
the stimulatory effects on growth and expression of
ALP activity (Fig. 6). This attenuation by Az-C may
not reflect an inhibitory effect on general protein
synthesis, because only Asc 2-P-stimulated ALP
activity, in addition to collagen synthesis, was inhibited
by it. Further evidence was obtained by the inhibition of collagen synthesis via a different molecular
mechanism.
The presence of Asc 2-P together with EDHBA, a
specific inhibitor of prolyl hydroxylase (Sasaki et al.,
1987) that is essential for the maturation of protoprocollagen chains, also showed a similar attenuative
effect on growth and ALP activity. These data indicate

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263

Fig. 7. Effect of Asc2-P on the ultrastructure of MG-63 cells in culture. The cells were cultured in the absence (A and C) or presence (B and D) of
0.25 mM Asc2-P for 6 weeks. For electron microscopy, the dishes were processed as described in the text. The electron photomicrographs are of
cultures sectioned perpendicular to the dish surface. G, Golgi apparatus; M, mitochondrion; rER, rough endoplasmic reticulum. Bars indicate 1 m
(A and B), 200 nm (inserts of A and B), and 500 nm (C and D), respectively.

that specific stimulation of collagen synthesis by Asc 2-P


may be essential for growth stimulation of the cells
and expression of ALP activity, the latter being a marker
for early osteoblast differentiation. Asc 2-P-induced
increases in the nascent growth rate, collagen synthesis

and ALP activity were also observed in normal osteoblastic cell cultures, even though their relationship was
not examined (Owen et al., 1990).
We have previously observed that Asc 2-P stimulates
the synthesis and maturation of collagen molecules

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(Hata and Senoo, 1989; Kurata and Hata, 1991; Kurata


et al., 1993). We also observed the formation of
3-dimensional tissue-like material by human dermal
fibroblasts, when cultured for long periods of time in the
presence of Asc 2-P (Hata and Senoo, 1989).
When MG-63 cells were cultured for 6 weeks in the
presence of Asc 2-P, the prominence of striated collagen
fibers was indicated by electron microscopic observation
of the cell layer, suggesting Asc 2-P-stimulated collagen
accumulation, as well as collagen synthesis. Thus, Asc
2-P supplementation of the culture medium facilitated
the formation of a 3-dimensional tissue-like material by
MG-63 cells.
The Asc 2-P-supplemented culture system described
here may be useful for analyzing the process of osteoblast differentiation, and for investigating the effects
of various hormones and other factors on the
differentiation and metabolism of osteoblastic cells.
Acknowledgements
We thank Drs Kazuhito Izukuri and Yasumasa Kato
for their helpful discussions. A part of this work was
supported by grants from the following: Ground
Research Announcement for Space Utilization promoted by the Japan Space Forum (R.H.); Bioventure
Research (S.S. and R.H.) and Scientific Research on
Priority Area (A; R.H.), from the Ministry of
Education, Culture, Sports, Science and Technology of
Japan and Scientific Research (B; R.H.) from the Japan
Society for Promotion of Science.
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