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TISSUE ENGINEERED BONE AND ADIPOSE TISSUE – AN IN VITRO STUDY
Annie John1, Yoshito Ikada2 and Yasuhiko Tabata3
1Division of Implant Biology
Biomedical Technology Wing,
Sree Chitra Tirunal Institute for Medical Sciences & Technology
Thiruvananthapuram 695012
2Faculty of Medical Engineering,
Suzuka University of Medical Sciences,
1001-1 Kishioka-cho
Mie 510-0293, Japan.
3Institute for Frontier Medical Sciences,
Kyoto University
Kyoto 606-8507, Japan
Bone marrow is a complex tissue composed of hematopoietic and stromal mesenchyme stem cells (MSCs) with a
differentiation potential to adipogenic, fibroblastic, reticular, osteogenic and chondrogenic lineages. However, the number
of multipotent MSCs of bone marrow cells rapidly decreases with donor age and thereby causes only limited tissue repair
in adults. Therefore, considering the clinical application to hard and soft tissues, it is necessary to efficiently proliferate the
MSCs without damaging their differentiation potentials. If osteogenesis and adipogenesis can be controlled from MSCs, it
will be a therapeutic way to break through the problems involving breast and bone plastic and reconstructive surgeries. In
the present study, we have studied the proliferation and differentiation of rat MSCs on substrates like polystyrene and
polystyrene coated with - Type I and IV collagen and ProNectin F. We evaluated the addition effect of basic fibroblast
growth factor (bFGF) on the proliferation of MSCs and that of dexamethasone (dex) and rh bone morphogenetic protein -
2 (rhBMP-2) on their osteogenesis. MSCs were also induced for adipogenesis by incubation in different adipogenic media
containing insulin, methylisobutylxanthine and dex. The adhesion and proliferation of MSCs were enhanced in the
presence of bFGF in the primary culture, and was significantly higher for the Type IV collagen, followed by ProNectin F
and then the Type 1 collagen-coated substrate. The ALPase activity was high at the dex concentrations of 10-9 and 10-8
M for Type 1 collagen-coated and Type IV collagen-coated or polystyrene substrates and 10-7 M for ProNectin-coated
plates. After 4 weeks of cells in culture, ALPase activity and calcium deposited on the different substrates were high on
Type IV collagen compared to ProNectin F and Type 1 collagen-coated substrates. von Kossa stained mineralized
plaques were visible. Increase in osteocalcin and ALPase levels were also high in Type IV collagen compared to
ProNectin F and Type I-coated plates. On the other hand, adipogenesis was induced in MSCs at the dex concentration of
10-6 and 10-7 M. This change was more pronounced for Type IV collagen-coated substrates than the Type 1 collagen-
coated substrates. However, ALPase activity was low in such adipogenic differentiated cultures. Adipocytes could be
clearly identified by the presence of lipid vacuoles, as multilocular with small lipid inclusions or as unilocular adipocytes in
foci containing single cells or few cells.
INTRODUCTION
Current therapies such as autografts,
allografts, xenograft, or metal prosthesis for
regeneration and replacement of bone defects
resulting from tumors, surgical resections,
trauma, ageing, etc poses several health
problems in their own way. Similarly, the use
of pieces of adipose tissue and artificial tissue
substitutes such as silicon, collagen and
hydroxyapatite for soft tissue augmentation in
facial hemiatrophy, hypomastia,
mammoplasty and other deformities again
face safety issues. So bone and fat grafting
mediated via tissue engineering of stem cells
for repairing defects, represent a new
direction towards bone and adipose
regeneration in this millennium. Now, the goal
is to provide stem cells an appropriate
environment to proliferate and differentiate to
the specific lineage for ultimate regeneration
of the lost or damaged tissue.
Bone marrow is a complex tissue
composed of hematopoietic and stromal
mesenchyme stem cells (MSCs) with a
differentiation potential to adipogenic,
fibroblastic, reticular, osteogenic and
Trends Biomater. Artif. Organs. Vol. 16 (1) pp 28-33 (2002) http://www.sbaoi.org
chondrogenic lineages. However, the number
of multipotent MSCs of bone marrow cells
rapidly decreases with donor age and thereby
causes only limited tissue repair in adults.
Therefore, considering the clinical application
to hard tissue and soft tissues, it is necessary
to efficiently proliferate the MSCs without
damaging their differentiation potentials. If
adipogenesis and osteogenesis can be
controlled from MSCs, it will be a therapeutic
way to break through the problems involving
breast and bone, plastic and reconstructive
surgeries. In clinical applications, the pre-
coating of biomaterials with extracellular
matrix (ECM) proteins would promote cell
adhesion and enhance defect healing. This
study reports the proliferation and
differentiation of rat MSCs towards
osteogenesis and adipogenesis, on
substrates coated with ECM proteins such as
Type I & IV collagen and ProNectin F, using
growth factors.
EXPERIMENTAL
Type I and Type IV collagen aqueous
solution (10% in 1 mM HCl, Nitta Gelatin Co.
Ltd, Osaka, Japan) and ProNectin F (a unique
protein polymer that incorporates multiple
copies of the RGD cell attachment ligand of
human fibronectin interspersed between
repeated structural peptide segments, Sanyo
Chemical Industries, Japan, - 10µg/ml) diluted
with phosphate-buffered saline (PBS-) was
poured into each well of the 6 multi-well tissue
culture plates for 5 min at room temperature
(RT). The excess solution was then removed
and wells dried at RT for 1-2 hours.
Thereafter, the wells were rinsed twice with
PBS prior to cell seeding to obtain collagen
and ProNectin F-coated wells.
Whole marrow plugs were obtained
from the femur and tibia of adult male Fischer
rats (2 - 3 weeks old) by a syringe aspiration
method with PBS-. The marrow plugs were
centrifuged and re-suspended in Minimum
Essential Medium (MEM) Alpha Medium
supplemented with 10% Foetal Calf Serum
(FCS), kanamycin sulphate and sodium
bicarbonate. The prepared cell suspension
(100 µl) was seeded into each uncoated and
coated well of the 6 multi-well tissue culture
plates containing 2 ml of MEM Alpha Medium
and incubated at 5 % CO2, 95 % air at 37 oC
for 9 days. For studies on adhesion and
proliferation of cells on different substrates,
basic fibroblastic growth factor (bFGF) was
not used, but to induce cell proliferation,
different concentrations of bFGF was added
into each of the uncoated wells in the primary
culture. The cell number was counted based
on fluorimetric determination of DNA with a
dye bisbenzimidazole Hoechst H 33258.
Next, when MSCs were cultivated in
the MEM Alpha Medium and expanded to 80-
90 % confluence in the primary culture, they
were passaged for their alkaline phosphatase
(ALPase) activity which is a marker for
osteoblastic phenotype and thereafter the
extent of mineralization. For this, cells (1 x 105
cells/ml) of the first passage were seeded into
each uncoated and coated well of the 6 multi-
well tissue culture plates containing the
osteogenic medium - MEM Alpha Medium
with 15 % FCS supplemented with L-ascorbic
acid (50 µg/ml), β-glycerophosphate (10 mM)
and different concentrations of
dexamethasone (Dex- 10-6,-7,-8 and –9 M) [1] for
14 days and the lysed cell extract was
estimated for ALPase activity (p-nitrophenyl
phosphate assay and for protein – Lowry
method). Thirdly, rh BMP-2 (bone
morphogenetic protein - 100 ng/ml) was
added onto bFGF treated (bFGF treated cells
of the primary culture) and non-treated cells of
the first passage, for MSCs differentiation and
maintained in culture for 14 days and stained
for ALPase (Sigma kit 85L-3R) and mineral
(von Kossa stain). Area percentage of mineral
plaques were quantified using Image analysis-
ProPlus 4.0 soft ware program. Osteocalcin
(rat ELISA system) and ALPase activity were
detected in the lysed cell extract. Finally, cells
were also subjected in the osteogenic medium
for 28 days and detected for calcium (O-
complexone (OCPC) method) deposition.
Tissue Engineered Bone and Adipose Tissue – An In Vitro Study
29
Now, to generate adipocytes, the
MSCs of the first passage were incubated in
Dulbecco’s Modified Eagle Medium (DMEM)
low glucose (GIBCO BRL, Life Technologies),
10 % Fetal Bovine Serum (FBS) and
penicillin-streptomycin mixture (1x) GIBCO
BRL, Life Technologies) in different coated
and uncoated wells and cells were maintained
at 37 oC, 5 % CO2 and 90 % humidity for 14
days. The differentiation into adipocytes were
initiated by switching the media to adipogenic
induction medium (DMEM with 10% FBS
containing 10 µg/ml insulin, O.5 mM
methylisobutylxanthine (MIX) (Sigma) and 1
µM Dex for 48 h and later to adipogenic
maintenance media (DMEM containing 10 %
FBS and 10 µg/ml insulin) for 14 days [2].
Adipogenesis was assessed by viewing the
accumulation of lipid droplets in the cells
associated with cell differentiation, by the Oil
Red O stain.
RESULTS
The adhesion and proliferation of cells
depended on the substrate type for cells and
was significantly higher for the Type IV
collagen, followed by ProNectin F and then
the Type 1 collagen-coated substrate (Figure
1). The MSCs isolated were of fibroblastic
shape and proliferated with time until the 9th
day (Figure 2). Proliferation of cells were
enhanced in the presence of bFGF (3 ng/ml)
in the primary culture, and the ALPase activity
was greatly influenced by the concentration of
Dex and the substrate type for cells. The
activity was high at the Dex concentrations of
10-9 and 10-8 M for type 1 collagen-coated and
Type IV collagen-coated or uncoated
substrates and 10-7 M for ProNectin F-coated
plates. Increase in osteocalcin and ALPase
levels were high on Type IV collagen
compared to ProNectin F and Type I-coated
plates. After 4 weeks of cells in culture,
calcium deposited on the different substrates
were high on Type IV collagen compared to
ProNectin F and Type 1 collagen-coated
substrates. Cellular nodules stained positive
for von Kossa and cells in association with the
nodules for ALPase (Figure 3 a & b) under
light microscope (Olympus U – MCB). Per
area von Kossa stained mineralized plaques
were high for Type IV collagen and ProNectin
F-coated plates. Adipocytes could be clearly
identified as multilocular with many small lipid
inclusions or as unilocular, in foci containing
single cells or few cells and was
comparatively conspicuous on Type IV
coated-wells (Figure 4 a, b & c). In rh BMP-2
cultures, co-existence of cellular nodules and
foci of multilocular inclusions of lipid droplets
were observed (Figure 5).
DISCUSSION
The ability of cells to adhere to a
biomaterial is an early effect of tissue
regeneration, which is imperative for
engineering essentially every type of
functional tissue and maintaining healthy
homeostasis throughout the life of a tissue.
Various materials used as scaffolds in defect
healing provide only a structural component
that will physically sustain tissue regeneration,
which is not enough to improve cell adhesion
properties. ECM molecules that may affect
cell adhesion on clinical applications include
collagen and fibronectin [3]. They possess
RGD (arginine-glycine-aspartic acid) tripeptide
sequences, which are recognised by integrins
[4-6]. In turn enhanced biocompatibility and
integration of material surfaces exhibiting
such signals may possibly promote interaction
of the materials with the host tissue and
potentially enhance tissue remodelling.
The complex process of bone
fabrication is directed by osteoblasts, which
are responsible for the secretion of osteoid
and its subsequent mineralization [7, 8].
Ascorbic acid and β-glycerophosphate [9, 10]
regulates the synthesis of osteogenesis. The
key factor in providing bone-like nodules in
the culture is the presence of dexamethasone
[11, 12]. Since there is no single definitive
marker for bone, the mineralized nodules
observed in our study were identified as being
‘bone-like’ by cells associated with the
30
Annie John et. al.
Figure 1
Figure 2
Figure 3-a
Figure 4-b
Figure 4-a Figure 4-b Figure 4-c
Figure 5
Figure 1: Comparison of adhesion and proliferation of rat MSCs from bone marrow on different substrates in
primary culture; Figure 2:Rat bone marrow-derived MSCs; Figure 3: Differentiated MSCs with rh-BMP-2, areas
stained positive for a) von Kossa, and b) ALPase; Figure 4: Differentiating adipocytes-multilocular/unilocular with
lipid inclusions (Oil Red O stain) in a) osteogenic medium with Dex, and b) & c) in adipogenic medium; Figure
5:Differentiated osteoblast cells (ALPase positive) and differentiated adipocytes (lipid inclusions), in osteogenic
medium with rh-BMP-2 ; Note: All Cells (Fig. 2-5) were seeded on Type IV Collagen-coated wells.
nodules showing ALPase activity, which is
regarded as typical of osteoblast activity [13].
In addition, bone Gla-protein (osteocalcin) a
low mol. wt. γ-carboxyglutamic acid-containing
protein [14] which is currently believed to be
exclusively synthesized by cells associated
with mineralized tissues [15, 16] also favoured
mineralisation. In other systems with mouse
marrow stromal cell lines and fetal rat calvarial
cells, BMP-2 (25 – 100 ng/ml) greatly
stimulated ALP activity, osteocalcin
production and bone nodule formation [17,
18]. The inductive role of 0.3 ng/ml bFGF in
the proliferation of rat bone marrow cells and
3 ng/ml bFGF in stimulation of ALPase activity
lead to bone-like mineralized tissue [19] in
contrast to 3 ng/ml bFGf used on our study.
Synergistic effect of bFGF (2.5 ng/ml) and
BMP-2 (50 ng/ml) enhanced the osteogenic
potency of bFGF in rat marrow MSC culture
as shown by osteocalcin mRNA expression,
bone nodule formation and calcium deposition
[20], but there was no adipogenic
differentiation.
On the other hand, adipogenesis was
induced in MSCs at the dex concentrations of
10-6M in the osteogenic medium as small foci
of cells with lipid inclusions which was more
pronounced for Type IV collagen-coated
substrates. Dexamethasone could have
induced adipogenesis [21, 22], but ALPase
activity was low in such adipogenic
differentiated cultures. It is reported that
collagen Type IV increases modulation during
adipocyte differentiation [23]. However, in our
adipogenic cultures, adipocytes could be
clearly identified by the presence of red lipid
vacuoles, as multilocular with small lipid
inclusions or as unilocular adipocytes in foci
containing single cells or few cells. Culture of
murine BMS2 stromal cells in hydrocortisone,
indomethacin and methylisobutylxanthine
accelerated the spontaneous adipogenesis
observed in the cell line [24, 25]. An
association between a progressive loss of
bone with age and an increase in adipose
tissue has been observed in the elderly and in
osteoporotics [26, 27]. Whether this change in
Tissue Engineered Bone and Adipose Tissue – An In Vitro Study
31
marrow tissue composition is a result of
opposing effect on the differentiation of
osteoblasts and adipocytes is yet to be
thought, because they share a common
precursor [28]. In the rat marrow culture an
inverse relationship was observed between
adipogenesis and osteogenesis [29] and the
expression of collagen IV protein is reported
to be important in the course of adipogenesis
and osteogenesis [30]. This study
demonstrated that the osteogenic medium
and adipogenic medium with the substrate
type for cell proliferation had a great influence
on the osteogenic and adipogenic
differentiation of rat MSCs.
CONCLUSION
One strategy to improve cell adhesion
on materials is the pre-coating of implant with
extracellular matrix proteins to enable specific
cell-extracellular matrix interaction in
conjunction with growth factors. Needless to
say, this would help to develop functional
biological prosthetic grafts to address both
acute and degenerative disorders in various
connective tissues, which are beyond the
capabilities of natural repair or current medical
practices. Now that techniques and conditions
that support the expansion of MSCs in culture
have been established in stem cell research,
clinical protocols of regenerating tissues in
human defects are not far away. This study
demonstrated that the dex concentration and
the substrate type for cell proliferation has a
great influence on the osteogenic and
adipogenic differentiation of rat MSCs. The
MSC proliferation was enhanced by bFGF
addition and osteogenesis promoted by rh
BMP-2.
Acknowledgement
The Director, SCTIMST,
Thiruvananthapuram and The Japan Society
for the Promotion of Science (JSPS) are
gratefully acknowledged for the support of this
work.
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