J. Biomedical Science and Engineering, 2010, 3, 751-757 JBiSE
doi:10.4236/jbise.2010.38100 Published Online August 2010 (http://www.SciRP.org/journal/jbise/).
Published Online August 2010 in SciRes. http:// www. scirp. org/journal/jbise
Dextran coating on and among fibers of polymer sponge
scaffold for osteogenesis by bone marrow cells in vivo
Masataka Yoshikawa1,2, Norimasa Tsuji1, Hideyuki Kakigi1, Takayoshi Yabuuchi1,
Yasunori Shimomura1, Hiroyuki Hayashi1, Hajime Ohgushi2
1Department of Endodontics, Osaka Dental University, Osaka, Japan;
2Tissue Engineering Research Group, Health Research Institute, National Institute of Advanced, Industrial Science and Technology,
Amagasaki, Japan.
Email: yosikawa@cc.osaka-dent.ac.jp
Received 25 May 2010; revised 8 June 2010; accepted 20 June 2010.
Although hydroxyapatite is commonly used as a
scaffold for bone regeneration, sponges may be suit-
able because of the adaptability to the defect. To use
as a scaffold, the fiber of sponge would be coated with
any adhesive to storage stem cells in the sponges. Fiber
in the structure of commercially available sponges was
coated by immersion in dextran solution and air
dried. After seeding of rat bone marrow cells (rBMCs),
the sponges were implanted subcutis of rats for esti-
mate osteogenesis in vivo. The level of osteocalcin was
25.28 ± 5.71 ng/scaffold and that of Ca was 129.20 ±
19.69 μg/scaffold. These values were significantly high-
er than those in sponges without dextran coating (p <
0.01). It was thought that rBMCs could be stored on
the shelf by dextran deposition in the fiber of the
sponge. In vivo examination, dextran induced osteo-
genesis by rBMCs in many spaces in the inner struc-
ture of the sponge.
Keywords: Dextran; Cell Adhesion; Scaffold; Bone
Marrow Cells; Osteogenesis
A part or total tooth regeneration has been studied by
many researchers [1-5]. In a basic study for tooth regen-
eration, it was shown that stem cells were detected in the
tooth pulp [6]. And it was shown in the report that the
stem cells differentiated to osteoblasts, chondrocytes,
adipocytes or neurocytes. Development of the removed
tooth germ to mature tooth was also attempted in vitro
[7]. However, tooth regeneration by tissue engineering is
particularly difficult because the constitution and con-
figuration of a tooth is complex. Odontoblasts, amelo-
blasts and cement blasts will be required for tooth re-
generation. Methods to isolate these cells from tissue or
technique for differentiation and induce these blast cells
from stem cells are not still established. This may be one
of the reasons that tooth regeneration is not realized.
Restoration of partial defect of the tooth or reproduction
of the whole tooth that had been missed for any reason
should be a definitive goal of the regenerative therapy in
the dentistry. Also, for regeneration of tooth, isolation
from the tissue of odontoblasts, ameloblasts and cemen-
toblasts, and induction of differentiation from stem cells
to these blast cells would be required. However, these
methods have not yet been established.
It is well-known that bone consists of an apatite
structure like dentine. Furthermore, osteocalcin which
should be synthesized by osteoblasts is present in den-
tine as calcium-binding protein [8,9]. Therefore, for the
restoration of teeth, a method of substituting bone which
resembles dentine in properties should be established.
For the regeneration of tooth or bone with a three-di-
mensional structure, a scaffold for proliferation and dif-
ferentiation of bone marrow stem cells and osteogenesis
is required. Sufficient resistance to load in the living
body is required for regenerated hard tissue.
The morphology of the scaffold should be easily
modifiable in order to apply the technique to any con-
figuration of defects. The intensity of the sponge as a
scaffold is extremely low, but modification of the shape
is easy. Polyvinyl alcohol (PVA) sponge with bone mar-
row cells was used for bone regeneration in the defective
part of the bone [10]. In this study, commercially avail-
able polyvinyl formal (PVF) sponge was selected as the
scaffold. PVF sponge consists of PVA cross-linked con-
struction by formalin. Stem cells must attach to the
structure of the scaffold to induce osteogenesis by the
cells in a porous scaffold [11,12]. However, the sponge
is inappropriate for keeping cells because of the fibrous
construction. One of the methods for seeding many cells
to attach in the pores of scaffold is coating with a
752 M. Yoshikawa et al. / J. Biomedical Science and Engineering 3 (2010) 751-757
Copyright © 2010 SciRes. JBiSE
chemical substance that promotes cell adhesion on the
scaffold [13]. Dextran, which has superior biocompati-
bility [14], is a natural polymer with linkage of a large
number of D-glucose, and is a kind of polysaccharide
existing in extracellular matrix. Dextran promotes adhe-
sion between protein and cells [15].
This experiment investigated the effect of dextran to
induce stem cell differentiation in dextran-coated PVF
sponge. To confirm the effects of dextran and the avail-
ability the PVF sponge as a scaffold, coating of a PVF
sponge with dextran was accomplished by immersion in
dextran solution and in vivo examinations were per-
2.1. Experimental Animals
In this study, 6- and 7-week-old male Fischer 344 rats
(CLEA Japan Inc., Tokyo, Japan) were used. The Animal
Welfare Committee of Osaka Dental University ap-
proved the experimental procedures regarding use and
care of animals in this study. This study was performed
under the Guidelines for Animal Experimentation at
Osaka Dental University.
2.2. Rat Bone Marrow Cell (rBMC) Isolation
rBMCs were obtained from the bone shaft of femora of
six 6-week-old male Fischer 344 rats after euthanasia by
intraperitoneal overdose of sodium pentobarbital (Dai-
nippon-Sumitomo Pharmaceutical Co., Ltd., Osaka, Ja-
pan) essentially according to the methods described in
previous reports [16,17] with minimal modification.
Both ends of the femur were cut off at the epiphysis and
bone marrow was flushed out with 10 ml of minimum
essential medium (MEM: Nakalai Tesque Inc., Kyoto,
Japan) expelled from a syringe through a 21-gauge nee-
2.3. Primary Culture for Preparing rBMCs
rBMCs in 10 ml of MEM were removed in a cell culture
flask (T75: BD Biosciences, MA, USA). For primary
culture, MEM supplemented with 15% fetal bovine se-
rum (FBS: JRH Biosciences, KS, USA) and antibiotics
(100 units/ml penicillin, 100 g/ml streptomycin, and
0.25 g/ml amphotericin B; Sigma Chemical Co., MO,
USA) was prepared. Primary culture was performed for
1 week in T75 in 5% CO2 and 95% relative humidity at
37C in an incubator. The culture medium was renewed
After primary culture, rBMCs in T75 culture flask
were washed three times using phosphate buffer solution
without Ca2+ and Mg2+ (PBS (-): Nakalai Tesque Inc.)
and the cells were isolated from the bottom of T75 flask
with trypsin-EDTA (0.5 mg/ml trypsin and 0.53 mol
EDTA: Nakalai Tesque Inc.) solution. Harvested cells
were re-suspended in culture medium at 1 × 107 cells/ml
2.4. Dextran Coating of Polyvinyl Formal (PVF)
Sponge and rBMCs Seeding in the Sponges
PVF sponges made by formalization of polyvinyl al-
cohol were generously donated by Ione (Osaka, Japan).
PVF sponges with a cubic configuration (5 5 5 mm)
for use in this study were obtained by cutting from a
sheet. Pores were 130 m diameter on average. The
PVF sponges were sterilized in ethylene oxide gas
before use.
Dextran with 10 kDa of molecular weight was pur-
chased from Sigma-Aldich (MO, USA) and dissolved
at 2 g/dl concentration in ultra purified water. The liq-
uid was sterilized by filtration (0.22 m) (Millex®:
Milipore Japan, Tokyo, Japan). The sponges were im-
mersed in dextran solution for 24 hours. The sponges
used as a control were immersed in ultra purified water.
The sponges were air-dried under radiation with ultra-
violet light. Each of the PVF sponges with and without
immersion in dextran solution were respectively
seeded with 1106 rBMCs in a 0.1 ml cell suspension
and were incubated for 3 hours in 5% CO2 and 95%
relative humidity at 37C to promote cell adhesion in
the sponges.
The micro structures of PVF sponges with and with-
out immersion in dextran solution were observed by
scanning electron microscopy (SEM, JSM 5400: JEOL
DATUM Ltd., Tokyo, Japan). For SEM confirmation of
whether the dextran coating on sponge fiber would be
preserved during rBMCs seeding and subcutaneous im-
plantation, sponges that had been immersed in dextran
solution were stored in MEM for 4 weeks in 5% CO2
and 95% relative humidity at 37C.
2.5. Dorsal Subcutaneous Implantation of PVF
Sponge with rBMCs
For in vivo osteogenesis in PVF scaffolds, six of 7-
week-old male Fischer 344 rats were used. Under gen-
eral anaesthesia with intra-peritoneal injection of 0.04
mg/g body weight of sodium pentobarbital, the backs of
rats were shaved and disinfected with povidone iodine
(Isodine: Meiji Seika Kaisha Ltd., Tokyo, Japan). The
dorsal skin of the rat was incised close to the scapula on
both sides across the vertebra at right angles and subcu-
taneous pockets were made using a mucosal raspatory.
All rats were respectively implanted with four scaffolds
in individual subcutaneous pockets alongside the verte-
bra. Two scaffolds without dextran coating but with
rBMCs seeding were inserted in the left subcutaneous
M. Yoshikawa et al. / J. Biomedical Science and Engineering 3 (2010) 751-757 753
Copyright © 2010 SciRes. JBiSE
pocket of three rats and the other two with dextran
coated and rBMCs seeding were inserted in the right
side of the other three rats. The incised wounds were
sutured, and adhesive (Aron alpha: Toa-gosei Co., Ltd.,
Tokyo, Japan) was applied over the wounds. Implanted
scaffolds were removed from the dorsal subcutaneous
tissue of the rats 4 weeks postoperatively after euthana-
sia by intraperitoneal injection of excessive dose of so-
dium pentobarbital.
2.6. Histological Examination of Osteogenesis in
Implanted Scaffolds
Three of each removed scaffold with or without dextran
coating and with rBMCs seeding were fixed in 10%
buffered formalin solution (pH 7.2). Specimens were
decalcified in 10% formic acid solution for two days,
embedded in paraffin, and 6 m serial sections were
made. All sections were stained with hematoxylin-eosin
and examined under an optical microscope.
2.7. Immunochemical and Biochemical
Examination of Osteogenesis in
Implanted Scaffolds
The other remaining sponges removed from rat subcuta-
neous tissue were frozen in liquid nitrogen and crushed
respectively using a Mixer Mill (MM 301: Retsch Co.,
Ltd., Tokyo, Japan). The crushed samples were respec-
tively added to 1 ml of buffer solution (pH 7.4) consist-
ing of 10 mM Tris-HCl, 1 mM EDTA and the 100 mM
NaCl. The cells in the solution were sonicated for 30
seconds at 3C (BIORUPTOR UCW-201: Tosho Electric
Co., Ltd., Tokyo, Japan). The samples were centrifuged
for 1 minute at 16,000 g. The supernatants were used
for quantitative analysis of osteocalcin (Rat Osteocalcin
ELISA kit DS®; DS Pharma Biomedical Co., Ltd., Osaka,
Japan). Then, 1 ml of formic acid was individually
added to each precipitation and samples were decalci-
fied for 72 hours for analyze calcium quantity in each
sponge using Calcium-E test WAKO® (Wako Pure Che-
mical Co. Inc., Osaka, Japan).
Data were presented as mean standard deviations.
Statistical comparisons between the mean values of os-
teocalcin in implanted scaffolds were performed using
two-way unrepeated ANOVA followed by post hoc
analysis using Tukey-Kramer’s test. Differences of p <
0.01 were considered significant.
3.1. SEM Examination of PVF Sponges with and
without Immersion in Dextran Solution
The SEM image of the PVF sponge without immersion
in dextran solution showed reticular configuration as in
Figures 1(a) and (b). Sizes of the fibers ranged from
about 5 m in the fine portion to 150 m in the thick
portion. The large nodes formed a spacious shelf and
extended fibrous branches in every direction. The major
axes were 150 to 250 m. The fibers of sponges im-
mersed in dextran solution were larger in diameter than
those of sponges without immersion in dextran solution
(Figures 2(a) and (b)). Each fiber presented a thick plate
with a width of 150-300 m. The fibers of the sponge
seemed to be covered with viscous substance. The pos-
sibility that the dextran coating on the fiber was pre-
served under rBMCs seeding and during subcutaneous
tissue implantation was shown by SEM observation of
sponges that had been kept in MEM for 4 weeks after
immersion in dextran solution (Figure 3). However, the
surface of the substance on the fiber of sponge just after
immersion in dextran solution was smoother than the
surface of the sponge stored in MEM.
Figure 1. (a) SEM image of PVF sponge without immersion
in dextran solution Fine fibers connected with each other
were observed in the interior of the sponge. (Bar: 300 m);
(b) This illustrate shows image of higher magnification of
Figure 1(a). Fine fibers were clearly seen. (Bar: 100 m).
754 M. Yoshikawa et al. / J. Biomedical Science and Engineering 3 (2010) 751-757
Copyright © 2010 SciRes. JBiSE
Figure 2. (a) SEM image of PVF sponge following im-
mersion in the dextran solution. The fibers seemed to be
covered with a viscous substance. (Bar: 300 m); (b)
This illustration shows image of higher magnification of
Figure 2(a). Dense coating with dextran was seen on
the fibers (Bar: 100 m).
Figure 3. SEM image of dextran-coated PVF sponge
After storage in MEM for 4 weeks following immersion
in dextran solution (Bar: 100 m).
3.2. Effect of Dextran on Osteogenesis in the
PVF Sponge used as a Scaffold in vivo:
Histological Examination
Based on histological findings of PVF scaffold after a
4-week implantation in the rat subcutis, there was no bone
recognized in the sponge without immersion in dextran
solution prior to seeding of rBMCs as shown in Figure 4.
Densely arranged fibrous connective tissue infiltrated the
fibers of the sponge. In the sponge with immersion in dex-
tran solution prior to rBMCs seeding, conspicuous osteo-
genesis was recognized on fibers accompanied by infiltra-
tion of connective tissue (Figure 5).
Figure 4. Histological findings of implanted PVF
sponge without immersion in dextran solution before
implantation. There was no apparent bone in the sponge.
Between fibers of the sponge, densely arranged fibrous
connective tissue was seen. C: Fibrous tissue infiltrated
in the sponge; F: Fiber of sponge (Bar: 200 m).
Figure 5. Histological findings of implanted PVF sponge
immersed in dextran solution before implantation Con-
spicuous osteogenesis was recognized among the fibers
of sponge. Infiltrated fibrous connective tissue was also
seen. B: Bone; C: Fibrous connective tissue infiltrated in
the sponge; F: Fiber of sponge (Bar: 200 m).
M. Yoshikawa et al. / J. Biomedical Science and Engineering 3 (2010) 751-757 755
Copyright © 2010 SciRes. JBiSE
3.3. Quantity of Osteocalcin in Implanted
Sponge with and without Immersion in
Dextran Solution: Immunochemical
Quantitative Analysis
In the sponge without immersion in dextran solution
before rBMCs seeding and subcutaneous tissue implan-
tation, 9.42 5.67 ng/scaffold of osteocalcin was meas-
ured. However, 25.28 5.71 ng/scaffold of osteocalcin
was detected in the sponge immersed in dextran solution.
There was a significant difference between the sponges
with and without immersion in dextran solution (Figure
6: p < 0.01).
3.4. Quantity of Calcium in Implanted Sponge
with and without Immersion in Dextran
Solution: Biochemical Quantitative Analysis
Quantity of calcium detected in the implanted sponge
with immersion in dextran solution before rBMCs seed-
ing was 129.20 19.69 μg/scaffold (Figure 7). In im-
planted sponge without immersion in dextran solution,
detected quantity of calcium was 79.41 8.69 μg/scaf-
fold. There was a significant difference between the im-
planted sponges with and without immersion in dextran
solution prior to seeding of rBMCs (p < 0.01).
The tooth is classified into an anterior tooth, a canine, a
premolar and a molar tooth. Moreover, those are distin-
guished morphologically about a maxillary or a mandibu-
lar tooth, and left or right. Tooth configurations vary, and
there are differing origins of hard tissues and pulp that
Figure 6. Osteocalcin level of implanted sponges with or
without immersion in dextran solution prior to seeding of
rBMCs. On comparison between sponges without and with
immersion in dextran solution, there was a greater quantity of
osteocalcin in sponges with dextran than in those without dex-
tran. Values are means ± SD (n = 3). * p < 0.01 vs. sponges
with immersion in dextran solution.
Figure 7. Calcium level of implanted sponges with or
without immersion in dextran solution followed by
seeding of rBMCs. On comparison between sponges
with and without immersion in dextran solution, quantity
of calcium was greater in sponges with immersion in
dextran solution than in those without immersion in dex-
tran solution. Values are means ± SD (n = 3). * p < 0.01
vs. sponges with immersion in dextran solution.
construct a tooth. Therefore, tooth regeneration is diffi-
cult for the complicated configuration [18]. It was re-
ported that a structure similar to a mature tooth including
dentine, enamel, cementum and pulp was induced from a
tooth germ in vitro [7]. For tooth regeneration, several
steps will be required, such as restoration of the defect
by regeneration of hard tissue, realization of endothelial
and fibrous conjugation to a regenerated root, regenera-
tion of the dentine-pulp complex, and then, complete
tooth regeneration.
For restoration of a tooth defect, stem cells should be
differentiated into odontoblasts from odontoblast pre-
cursor cells in a scaffold. Therefore, it would be desir-
able for the scaffold to be configurated according to the
defect. PVF sponge is considered a desirable scaffold
material because it can easily be shaped to the configu-
ration of the defect. PVF scaffold has previously been
used for three-dimensional in vivo osteogenic examina-
tion [19]. On SEM observation, pores measuring about
100-280μm in diameter were recognized in PVF sponge.
It was reported that such a pore size is suitable for os-
teogenesis in an HA scaffold [20]. This finding suggests
that osteogenesis in the PVF sponge is possible. How-
ever, there is a conspicuous difference in the internal
structures of a hydroxyapatite (HA) scaffold and those of
a PVF scaffold. Reticular structure of PVF sponge was
shown by SEM findings. Stem cells seeded in PVF
sponge may flow out of the sponge with the suspension,
while HA scaffold, stem cells may be taken into the
PVF with
PVF without
(ng/scaffold) P < 0.01
PVF with
PVF without
P < 0.01
g / scaffold)
10 0
12 0
14 0
16 0
756 M. Yoshikawa et al. / J. Biomedical Science and Engineering 3 (2010) 751-757
Copyright © 2010 SciRes. JBiSE
pores. The PVF sponge is an unfavorable substrate for
adhesion of BMCs.
Therefore, for osteogenesis in PVF sponge by subcu-
taneous tissue implantation in vivo, the sponge should be
modified so that seeded stem cells could adhere in the
sponge. In fact, it was reported that modification of PVA
by extracellular matrix is necessary for adhesion and
proliferation of stem cells in the sponge [21,22]. As a
result of proliferation and differentiation to osteoblasts
of stem cells, bone is formed. In this in vivo study of
seeding bone marrow cells in PVF sponge, there was no
osteogenesis recognized in the sponge without dextran
coating. This finding showed that rBMCs did not adhere
to the construction in the PVF scaffold. PVA having al-
dehyde groups and hydroxyl groups as functional groups
reacts with formalin and produces acetal in the in-
tramolecule of PVA. As a result, it is thought that these
functional groups were covered and lost reactivity. PVF
sponge without functional group could not be used as a
scaffold for osteogenesis by BMCs.
It was reported that intercellular adhesion involves
direct binding by cell adhesion molecule in an extracel-
lular matrix or cell membrane [21,22]. In addition, the
cells are activated by stimulus from cytokine and adhe-
sive high polymer and adhere to the surface of the scaf-
fold [23]. By coating the inner structures of PVF with a
highly cytotropic substance, adhesion of seeded stem
cells is enabled.
It is known that dextran is a major extracellular matrix
and it is a natural macromolecule polysaccharide. Fur-
thermore, dextran shows excellent biocompatibility and
adhesiveness to protein and cells [15]. It has also been
reported that dextran promotes differentiation of BMCs
to osteoblasts [24]. Therefore, rBMCs may adhere to
PVF sponge through dextran. It is evident that stable
adhesion may occur between PVF sponge and dextran
[25,26]. Based on the SEM findings in this experiment,
it seemed that the internal structures of the PVF sponge
were covered with a layer of dextran after immersion in
the solution. Osteogenesis was subsequently recognized
in the PVF scaffold that had been immersed in dextran
solution. These findings show that dextran adhered suf-
ficiently to the internal structure of the sponge and fa-
cilitated the attachment of the rBMCs. It was reported
that dextran agglutinates BMCs and then promoted ad-
herence of the cells to the pore wall in the scaffold
Greater osteogenesis was induced by 10 kDa of dex-
tran in PVF sponge than by higher molecular weight
dextran. It is necessary to retain BMCs in the PVF
sponge for effective osteogenesis. One method to
achieve the purpose appears to be using low-molecular-
weight dextran to coat the scaffold. It was suggested in
this study that coating PVF sponge with low-molecular-
weight dextran (10 kDa) effectively promoted osteogene-
sis in the sponge.
For regeneration of a tooth defect, in this study, PVF
sponge was selected as the scaffold because it could be
easily shaped and showed excellent biocompatibility. In
addition, it was considered that dextran was useful to
promote adhesion of BMCs. In vivo osteogenesis by
rBMCs in PVF sponge as a scaffold with dextran coating
was examined histologically, biochemically and immu-
The findings obtained from this study were as follows.
1) rBMCs did not adhere to untreated PVF sponge and
no bone was formed.
2) The fiber in PVF sponge should be covered with a
layer of the dextran by immersion in a solution of 10kDa
3) In PVF sponge immersed in dextran solution, the
dextran layer coating the inner structure of the sponge
persisted after 4 week immersion in MEM.
4) In PVF sponge coated with dextran, osteogenesis
by rBMCs was promoted.
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