preliminary study on the adhesion and proliferation of human osteoblasts
TRANSCRIPT
Preliminary study on the adhesion and proliferation of human osteoblasts
on starch-based scaffolds
A.J. Salgado a,b,*, M.E. Gomes a, A. Chou b, O.P. Coutinho c, R.L. Reis a, D.W. Hutmacher b,d
aDepartment of Polymer Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, PortugalbDivision of Bioengineering, Faculty of Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore
cDepartment of Biology, University of Minho, Campus de Gualtar, Braga, PortugaldDepartment of Orthopaedic Surgery, Faculty of Medicine, National University of Singapore, Singapore 119260, Singapore
Abstract
Up to today, several techniques have been used to produce biodegradable porous scaffolds for tissue engineering. In this work, a new
technique based on extrusion by using blowing agents in combination with a 50:50 (wt.%) blend of starch/cellulose acetate (SCA) was
studied. The results show that by using this technique it was possible to obtain scaffolds with 70% of porosity and a fully interconnected
network of pores, with sizes ranging from 200 to 500 Am. After their production, the mechanical properties of these scaffolds were tested,
presenting a compressive modulus of 124.6F 27.2 MPa and a compressive strength of 8.0F 0.9 MPa. These values are within the best found
in the literature and show that by using this technique, it is possible to produce scaffolds that, from a mechanical standpoint, may be suitable
for bone tissue engineering. Cell culturing experiments showed that cells were viable and that there were no signs of cellular death after 3
weeks of culture. Finally, biochemical assays demonstrate that cells maintained the osteogenic phenotype throughout the experiment and
deposition of mineralized extracellular matrix could be detected. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Starch-based scaffolds; Degradable polymers; Tissue engineering; Cell culture; Osteoblast
1. Introduction
Current tissue engineering techniques require the use of a
porous biodegradable/bioresorbable scaffold, which serves
as three-dimensional template for initial cell attachment and
subsequent tissue formation, both in vitro and in vivo [1–5].
Ideally, such scaffolds should have several properties that
allow them to be used as organs or tissue substitutes. Firstly
and most importantly, they should be biocompatible, that is,
they must not trigger strong immunological responses nor
cytotoxicity phenomena [6]. Moreover, these materials
should disclose mechanical properties that match those of
the tissue that is going to be substituted and at the same time
allow for the sequential growth of the tissue while the
scaffold slowly degrades [6–8]. Porosity is also a very
important issue because of the need for nutrient delivery,
gas exchanges and the three-dimensional cell and tissue
growth within the scaffold structure [9,10]. Finally, these
materials should have a suitable surface chemistry in order
to allow good cell attachment, proliferation and differentia-
tion [3,11], and at the same time should be easily sterilizable
[7].
During the last few years, several materials, as well as
distinct original processing techniques, have been proposed
for being used in tissue engineering. However, the ideal
biomaterials/scaffolds are yet to be found. In the last years,
natural polymers have been presented as an alternative to the
current biomaterials used in tissue engineering. One of the
advantages of these materials is their low cost as a result of
an almost unfailing source of raw material. Biodegradable
starch-based polymeric blends have recently been suggested
for a wide range of biomedical applications such as tissue
engineering scaffolds [12], bone cements [13], devices and
hydrogels for controlled release of drugs [13,14] and to be
used in bone fixation/filling of bone defects in the orthopae-
dic field [15]. Such blends have shown to be biodegradable
and biocompatible [16–18], being lower molecular weight
starch chains, fructose and maltose, their typical degradation
products [16].
The present work puts forward a new scaffold based on a
blend of starch with cellulose acetate (SCA) processed by a
novel extrusion technique based on the use of the raw
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0928 -4931 (02 )00009 -7
* Corresponding author. Department of Polymer Engineering, Univer-
sity of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
E-mail address: [email protected] (A.J. Salgado).
www.elsevier.com/locate/msec
Materials Science and Engineering C 20 (2002) 27–33
polymeric material mixed with a certain percentage of blo-
wing agents. The resulting morphological and mechanical
properties as well as the behaviour and proliferative capa-
bilities of human osteoblasts when seeded on these scaffolds
were also characterized.
2. Materials and methods
2.1. Scaffold manufacturing
The polymer used in this study was a 50:50 (wt.%) corn
starch/cellulose acetate biodegradable blend (SCA, Nova-
mont, Italy). The scaffolds were obtained by using a
technology based on extrusion with blowing agents. This
methodology allows an accurate control of pore size and
distribution through the control of the processing parameters
and by changing the amount and type of blowing agent
used.
The polymer was previously mixed with 2% (wt.%) of a
solid blowing agent based on citric acid (trade name BIH40
from Clariant, USA) in a biaxial rotating drum. The mixture
was then extruded in a Carvex twin-screw extruder, with
a die of 12 mm in diameter. During the processing, the
blowing agent reacts by heating, releasing CO2 and H2O
and therefore creating the pores within the structures. The
resulting materials were then cut in cuboids measuring
5� 5� 4 mm3.
2.2. Porosity calculation
The porosity was calculated through the following meth-
od: (1) measuring the weight and volume of each sample,
(2) from these measurements, the apparent density of the
SCA scaffolds was calculated using the following formula:
q* =m (g)/V (cm3) and (3) finally, the porosity was obtained
using the formula porosity = e = 1� q*/q� 100% (qSCA=1.28 g/cm3).
2.3. Cell culture experiments
2.3.1. Human osteoblast cultures and cell seeding on starch-
based scaffolds
Human osteoblasts were isolated from calvarian bone
explants obtained from 30–35 year old patients who had
undergone routine surgery. All patients gave their written
consent and experiments were approved by hospital
authorities. After being removed, the calvarian bone pieces
were rinsed in a 70% ethanol solution and washed three
times in PBS medium (137 mM NaCl, 2.7 mM KCl, 10
mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Cell culture
flasks were then filled with five to six explants of calvarian
bone and incubated with M199 medium (Gibco, USA)
supplemented with 10% FBS (Hyclone, USA) and 1%
antibiotics (Gibco) in a humidified atmosphere at 37 jCand 5% CO2, with medium replacement every 4 days. Cell
growth from explant cultures was observed after 3 weeks.
Primary cultures were maintained until they reached 80–
90% of confluence and were passaged three times. At this
stage, cells were trypsinized with 0.25% trypsin/EDTA
(Hyclone), centrifuged, resuspended in culture medium
and counted in a hemocytometer. The cell suspension
was then mixed in a 3:1 ratio with fibrin glue (Tisseel
Kit, Immuno, Austria) and aliquots of 20 Al containing
3� 105 cells were seeded onto the top of the porous
structures, which were previously placed in 24-well culture
trays. After cell seeding, 1 ml of fresh culture medium was
added to each well and cells were incubated in a humidi-
fied atmosphere at 37 jC and 5% CO2 for 3 weeks with
medium changes every 3 days. On the second week, the
osteoblastic phenotype as well as the production of bone
extracellular matrix were stimulated by supplementing the
culture medium with 10 mM h-glycerophosphate (Sigma,
USA), 100 AM ascorbic acid (Sigma) and 10�7 M dex-
amethasone (Sigma). All experiments related with osteo-
blast behaviour while seeded on starch-based scaffolds
were repeated.
2.3.2. Cellular viability and proliferation assays
Cellular viability and proliferation were assayed by
confocal laser microscopy (CLM) and a biochemical assay,
the MTS test. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
(Promega, USA) test is an assay in which the substrate—
MTS—is bioreduced into a brown formazan product by
NADPH or NADP produced by mitochondrial enzymes,
which are active in living cells [19,20]. This assay or similar
to it (MTT, WST-1) has been widely used to measure
cellular viability and proliferation [20–23]. In this assay,
intensity of the colour is directly related to the number of
viable/living cells. So an increase in the colour will mean
that the number of viable cells increased and hence, cellular
proliferation occurred. Scaffolds/cells constructs (n = 5)
were placed in culture medium containing MTS in a 5:1
ratio and incubated in a humidified atmosphere at 37 jC and
5% CO2. After 3 h of incubation, 100 Al of solution from
each scaffold were transferred to 96-well plates and the
optical density was determined at 490 nm with a reference
filter at 620 nm.
For CLM, samples were stained with two fluorescent
dyes: fluorescein diacetate (Molecular Probes, USA), which
stains viable cells green, and propidium iodide (Molecular
Probes), which stains necrotic and secondary apoptotic
cells. The scaffolds/cells constructs were first incubated
with 2 Ag/ml FDA (Molecular Probes) for 30 min at 37 jC.The samples were then rinsed three times in PBS medium,
placed for 2 min in a 100 Ag/ml solution of propidium
iodide (Molecular Probes), rinsed again in PBS once and
viewed under a confocal laser microscope (Olympus IX70,
HLSH100 Fluoview). Depth projection images were con-
structed from 50 horizontal image sections through the
scaffolds/cells constructs.
A.J. Salgado et al. / Materials Science and Engineering C 20 (2002) 27–3328
2.3.3. Cellular adhesion
Cellular adhesion and extracellular matrix production
were assessed by fluorescence microscopy and environ-
mental scanning electron microscopy (ESEM).
For the ESEM experiments, cells were previously fixed
in glutaraldheyde for 4 h, dried and examined with a JEOL
JSM-5800LV scanning electron microscope at 15 kV.
The fluorescence microscopy experiments were per-
formed using phalloidin conjugated with Alexa Flour 488
(Molecular Probes). Samples were first fixed in paraformal-
dehyde for 30 min at room temperature. Scaffolds/cells
constructs were then washed in PBS, incubated for 30 min
in a 200 Ag/ml RNase solution, rinsed in PBS, incubated in
phalloidin (Molecular Probes) for 45min at room temperature
in the dark, washed in PBS, counterstained with 5 Ag/ml of PI
and finally washed in PBS. The samples were then dried and
viewed under a fluorescence microscope (Olympus IX70).
2.3.4. Alkaline phosphatase activity assay
Alkaline phosphatase (ALP) activity from the scaffolds/
cells constructs was quantified by the specific conversion of
p-nitrophenyl phosphate ( pNPP) into p-nitrophenol ( pNP).
Supernatants were collected weekly (n= 5) and frozen at
� 20 jC and then thawed prior to analysis. The enzyme
reaction was set up by mixing 50 Al of the sample with 150
Al of substrate buffer containing 1 M diethanolamine HCl
(pH 9.8) and 2 mg/ml of pNPP. The solution was incubated
at 37 jC for 1 h and the reaction was then stopped by a
solution containing 2 M NaOH and 0.2 mM EDTA in
distilled water. The optical density was determined at 405
nm with a reference filter at 620 nm. A standard curve was
made using pNP values ranging from 0 to 200 nmol/ml. The
results are expressed in nmol of pNP produced/ml/h.
2.3.5. Osteocalcin assay
Osteocalcin production was assessed using an ELISA kit
(IBL, Germany). Supernatants were weekly collected (n = 5)
and frozen at � 20 jC and then thawed prior to analysis. A
25 Al amount of each sample was used and the assay was
performed under the instructions of the manufacturer.
Results are presented in nmol/ml.
2.4. Western blot
After 3 weeks, cells seeded on the scaffolds were washed
in PBS medium, placed in 800 Al of lysis buffer (20 mM
Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, pH
8.0) supplemented with a cocktail of protease inhibitors
(100 AM phenylmethylsulfonyl fluoride (PMSF), 1 mM
benzamidine, 50 AM leupeptin, 50 AM antipain and 1 AMpepstatin) and finally sonicated at 40 V for about 10–20 s.
After sonication, supernatants were collected and total
protein was quantified using the Bradford assay (Biorad,
USA). Proteins were then submitted to an SDS-PAGE
electrophoresis and then electro-transferred to a previously
activated Hybond C nitrocellulose membrane. This mem-
brane was first blocked with 5% (w/v) skim dry milk for 1 h
in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.4)
with 0.1% Tween 20 (TBS-T). Blots were further incubated
with the primary antibody, mouse anti-bovine cross-reacted
with human osteonectin obtained from Developmental
Studies Hybridoma Bank in a 1:1000 working dilution,
washed in 1% milk TBS-T solution and incubated overnight
at 4 jC with the secondary antibody, rabbit anti-mouse
conjugated with horseradish peroxidase (Pierce, USA). The
immune complex was detected by ECL system (Amersham)
and visualized by chemiluminescence.
3. Results and discussion
3.1. Scaffold characterization
The processing method described in detail by Gomes et
al. [24], i.e. extrusion with blowing agents, allows to obtain a
porous structure resulting from the release of the gases (CO2
and H2O) upon the decomposition of the blowing agent.
The structure of the porous scaffolds consists of a fully
interconnected network of pores with around 70% of
porosity (Fig. 1), presenting a size between 200 and 500
Am. These values are within the range of those believed to
be ideal for tissue engineering of bone [9]. The mechanical
tests showed that these materials had a compressive mod-
ulus of 124.6F 27.2 MPa and a compressive strength of
8.0F 0.9 MPa. The stated values can be further increased if
the materials are reinforced with hydroxyapatite [24]. Fur-
ther details on the degradation behaviour as well as on other
aspects of these scaffolds can be found in the literature [24].
3.2. Cellular adhesion, viability and proliferation
Initial osteoblast/material interactions can be conven-
iently characterized in four stages: (i) protein adsorption to
Fig. 1. SCA scaffold obtained by extrusion with 2% of blowing agent. A
fully interconnected network of pores was obtained, with a pore size
ranging from 200 to 500 Am.
A.J. Salgado et al. / Materials Science and Engineering C 20 (2002) 27–33 29
the surface, (ii) contact of rounded cells, (iii) attachment of
cell to the substrate and (iv) spreading of cells [25]. Initial
cell attachment usually occurs within 4 h after cell seeding;
however, these values are only a rough guideline and
depend mainly on the substrate characteristics and cell type
[25,26]. Osteoblast-like cells started to spread 24 hours after
cell seeding and after 7 days, cells showed a good conflu-
ency in the fibrin glue. Cells were attached by focal
adhesion points on the rods of the scaffolds (Fig. 2a) and
also on the edges of the pores (Fig. 2b). This pattern has
been previously described in the literature [20] and showed
that cell behaviour on the tested scaffolds was within the
expected.
By the end of week 1, cells started to span across the
pores (Fig. 2b) and from then on, cell-to-cell contact points,
extracellular matrix and fibrin glue acted as a template.
After 2 weeks (Fig. 3b), cells had almost completely filled
some of the pores of the scaffold and after 3 weeks (Fig. 3c),
Fig. 2. (a) Cells were stained with 2 Ag/ml FDA and 100 Ag/ml PI: after 1
week in culture, cells were occupying the rods of the scaffold and (b) were
attached to the edges of the pores, starting to span across them (phalloidin
staining).
Fig. 3. Confocal laser microscopy of human osteoblast-like cells stained
with 2 Ag/ml FDA and 100 Ag/ml PI after (a) 1, (b) 2 and (c) 3 weeks.
A.J. Salgado et al. / Materials Science and Engineering C 20 (2002) 27–3330
the examined area was covered with cells and the scaffold
matrix had been taken by them. The spread over the pores
was achieved by the bridging of cell filopodia and by the
guidance of the extracellular matrix that had been previously
produced. However, it must be highlighted that, probably
due to slight variations on the scaffold surface, this growth
was not homogenous throughout the entire scaffold, being
only detected in some areas.
By observing Fig. 3, it can be concluded that cells
remained viable after 3 weeks in culture. After week 1,
there were some necrotic cells (red-stained cells), but after 2
and 3 weeks, there were no signs of necrosis, proving that
the scaffolds are not harmful to the cells, which is not
typical of other biodegradable polymers. Moreover, it has to
be stated that the pictures presented in Fig. 3 were taken in
the central inner region of the scaffolds, showing that its
porosity was adequate for nutrient delivery and gas ex-
changes.
Cellular viability and proliferation were also assayed via
the MTS test (Fig. 4). This is a qualitative and not a
quantitative assay; nevertheless, it shows that cells are
viable and proliferating in case of initially homogeneous
cell distribution in all specimens.
The differences registered in the O.D. after only 1 day in
culture are mainly due to different cell seeding efficacies.
After week 1, the O.D. values obtained for the SCA
scaffolds were almost similar to those of the control sam-
ples, proving that cells were proliferating on the starch-
based scaffolds. On weeks 2 and 3, cellular growth was not
significant, mainly due to the stimulation with dexametha-
sone, which is known to decrease the cellular proliferation
[26,27]. Another explanation for this fact is that once cells
reached confluency on the places where they were seeded,
the rate of cellular death and proliferation was equal and
hence the proliferation stopped. This fact is further con-
firmed by the results obtained with the control samples.
3.3. Protein expression
During the course of osteoblast differentiation, both in
vitro or in vivo models, there is the expression of several
Fig. 4. Cell viability and proliferation was assayed by the MTS test. Cell
density used was 3� 105 cells/scaffold. For control, 3� 105 cells were
seeded on 24-well-plate culture trays. Cells were kept in culture for 21 days.
Between days 7 and 14, cells were stimulated with 10 mM h-glycerophos-phate (Sigma), 100 mM ascorbic acid (Sigma) and 10� 7 M dexamethasone
(Sigma) (n= 5, F S.D.).
Fig. 5. Osteocalcin assay: supernatants were weekly collected and frozen.
After 3 weeks, supernatants were thawed. The results are shown in
osteocalcin (nmol/ml) as a function of days. On day 7, cells were stimulated
with 10 mM h-glycerophosphate (Sigma), 100 mM ascorbic acid (Sigma)
and 10�7 M dexamethasone (Sigma). Osteocalcin levels increase (14 days
in culture) 7 days after stimulation, reaching the lowest values after 3 weeks
in culture (n= 5, F S.D.).
Fig. 6. ALP activity assay: supernatants were weekly collected and frozen.
After 3 weeks, supernatants were thawed. The results are shown in p-
nitrophenol (nmol/ml/h) as a function of days. On day 7, cells were
stimulated with 10 mM h-glycerophosphate (Sigma), 100 mM ascorbic acid
(Sigma) and 10� 7 M dexamethasone (Sigma). The ALP levels decrease
(14 days in culture) 7 days after stimulation, reaching the highest values
after 3 weeks in culture (n= 5, F S.D.).
Fig. 7. Western blot analysis of osteonectin expression in human osteoblasts
seeded on starch-based scaffolds (S). A control with human osteoblasts (C)
(3� 105 cells/well) on 24-well culture plates was used. The immune complex
was detected by ECL system and visualized by chemiluminescence. Standard
proteins (PP) were used as molecular weight markers (Biorad).
A.J. Salgado et al. / Materials Science and Engineering C 20 (2002) 27–33 31
proteins typical of bone formation and extracellular matrix
mineralization. Within these can be found ALP, an early
marker from the osteogenic phenotype [26–29], osteocal-
cin, usually involved in the deposition of new extracellular
matrix [27,30], and finally, osteonectin, which is usually
related with bone morphogenesis [27].
In Figs. 5 and 6, the progression of osteocalcin and ALP
activity is shown. Osteocalcin reached its highest values and
ALP its lowest on week 2 of the experiment. During this
week and due to the stimulation with ascorbic acid [26], the
deposition of extracellular matrix proteins such as osteocal-
cin was favoured when compared to mineralization, which
is usually associated with increases in ALP activity [26].
After 3 weeks, the reverse situation happened and ALP
reached its highest values while osteocalcin its lowest
because, besides the fact that the stimulation was stopped,
it has been stated that usually, there is first the deposition of
extracellular matrix and only after mineralization occurs
[30].
In order to confirm the hypotheses of extracellular matrix
deposition, a Western blotting was applied to determine
whether or not osteoblasts were expressing osteonectin. The
results (Fig. 7) confirm that osteonectin was being
expressed, reinforcing the theory of extracellular matrix
deposition. Results from ESEM (Fig. 8) further support
these findings, as a skeletal structure typical of bone extra-
cellular matrix was found [4]. Finally, the expression of the
referred proteins showed that human osteoblasts maintained
their osteogenic phenotype throughout the whole experi-
ment. However, in future experiments, the data and pre-
liminary conclusions presented in this paper should be
confirmed by means of immunohistochemistry and conven-
tional histological techniques.
4. Conclusions
With the present work, it was possible to show that the
scaffolds based on a 50:50 (wt.%) blend of starch with
cellulose acetate present a range of properties that make
them adequate to be used in tissue engineering. Cells remain
viable after 3 weeks in culture. Most importantly, the cells in
the centre of the scaffolds remained viable after 3 weeks,
showing that the scaffolds have the adequate porosity. Cells
were also able to proliferate within the scaffolds, filling
some pores and producing a skeletal structure typical of
bone extracellular matrix. This work also shows that starch-
based scaffolds, processed by extrusion in combination with
blowing agents, might be an alternative to the current
biomaterials and processing techniques used in tissue engi-
neering and may be very useful for bone tissue engineering
in the near future.
Acknowledgements
This work was supported by a grant from the Portuguese
research council Fundac� ao para a Ciencia e a Tecnologia
(FCT).
We would like to thank Baxter (Vienna, Austria) for pro-
viding the Tisseel Kit samples.
The monoclonal antibody, AON-1 (mouse anti-bovine
osteonectin), developed by John D. Termine, was obtained
from the Developmental Studies Hybridoma Bank devel-
oped under the auspices of the NICHD and maintained by
The University of Iowa, Department of Biological Sciences,
Iowa City, IA 52242, USA.
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