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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 124.6 F 27.2 MPa and a compressive strength of 8.0 F 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
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 0 0 9 - 7
28 A.J. Salgado et al. / Materials Science and Engineering C 20 (2002) 27–33
polymeric material mixed with a certain percentage of blo- growth from explant cultures was observed after 3 weeks.
wing agents. The resulting morphological and mechanical Primary cultures were maintained until they reached 80–
properties as well as the behaviour and proliferative capa- 90% of confluence and were passaged three times. At this
bilities of human osteoblasts when seeded on these scaffolds stage, cells were trypsinized with 0.25% trypsin/EDTA
were also characterized. (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
2. Materials and methods Kit, Immuno, Austria) and aliquots of 20 Al containing
3 105 cells were seeded onto the top of the porous
2.1. Scaffold manufacturing structures, which were previously placed in 24-well culture
trays. After cell seeding, 1 ml of fresh culture medium was
The polymer used in this study was a 50:50 (wt.%) corn added to each well and cells were incubated in a humidi-
starch/cellulose acetate biodegradable blend (SCA, Nova- fied atmosphere at 37 jC and 5% CO2 for 3 weeks with
mont, Italy). The scaffolds were obtained by using a medium changes every 3 days. On the second week, the
technology based on extrusion with blowing agents. This osteoblastic phenotype as well as the production of bone
methodology allows an accurate control of pore size and extracellular matrix were stimulated by supplementing the
distribution through the control of the processing parameters culture medium with 10 mM h-glycerophosphate (Sigma,
and by changing the amount and type of blowing agent USA), 100 AM ascorbic acid (Sigma) and 10 7 M dex-
used. amethasone (Sigma). All experiments related with osteo-
The polymer was previously mixed with 2% (wt.%) of a blast behaviour while seeded on starch-based scaffolds
solid blowing agent based on citric acid (trade name BIH40 were repeated.
from Clariant, USA) in a biaxial rotating drum. The mixture
was then extruded in a Carvex twin-screw extruder, with 2.3.2. Cellular viability and proliferation assays
a die of 12 mm in diameter. During the processing, the Cellular viability and proliferation were assayed by
blowing agent reacts by heating, releasing CO2 and H2O confocal laser microscopy (CLM) and a biochemical assay,
and therefore creating the pores within the structures. The the MTS test. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
resulting materials were then cut in cuboids measuring carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
5 5 4 mm3. (Promega, USA) test is an assay in which the substrate—
MTS—is bioreduced into a brown formazan product by
2.2. Porosity calculation NADPH or NADP produced by mitochondrial enzymes,
which are active in living cells [19,20]. This assay or similar
The porosity was calculated through the following meth- to it (MTT, WST-1) has been widely used to measure
od: (1) measuring the weight and volume of each sample, cellular viability and proliferation [20 – 23]. In this assay,
(2) from these measurements, the apparent density of the intensity of the colour is directly related to the number of
SCA scaffolds was calculated using the following formula: viable/living cells. So an increase in the colour will mean
q* = m (g)/V (cm3) and (3) finally, the porosity was obtained that the number of viable cells increased and hence, cellular
using the formula porosity = e = 1 q*/q 100% (qSCA = proliferation occurred. Scaffolds/cells constructs (n = 5)
1.28 g/cm3). were placed in culture medium containing MTS in a 5:1
ratio and incubated in a humidified atmosphere at 37 jC and
2.3. Cell culture experiments 5% CO2. After 3 h of incubation, 100 Al of solution from
each scaffold were transferred to 96-well plates and the
2.3.1. Human osteoblast cultures and cell seeding on starch- optical density was determined at 490 nm with a reference
based scaffolds filter at 620 nm.
Human osteoblasts were isolated from calvarian bone For CLM, samples were stained with two fluorescent
explants obtained from 30– 35 year old patients who had dyes: fluorescein diacetate (Molecular Probes, USA), which
undergone routine surgery. All patients gave their written stains viable cells green, and propidium iodide (Molecular
consent and experiments were approved by hospital Probes), which stains necrotic and secondary apoptotic
authorities. After being removed, the calvarian bone pieces cells. The scaffolds/cells constructs were first incubated
were rinsed in a 70% ethanol solution and washed three with 2 Ag/ml FDA (Molecular Probes) for 30 min at 37 jC.
times in PBS medium (137 mM NaCl, 2.7 mM KCl, 10 The samples were then rinsed three times in PBS medium,
mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Cell culture placed for 2 min in a 100 Ag/ml solution of propidium
flasks were then filled with five to six explants of calvarian iodide (Molecular Probes), rinsed again in PBS once and
bone and incubated with M199 medium (Gibco, USA) viewed under a confocal laser microscope (Olympus IX70,
supplemented with 10% FBS (Hyclone, USA) and 1% HLSH100 Fluoview). Depth projection images were con-
antibiotics (Gibco) in a humidified atmosphere at 37 jC structed from 50 horizontal image sections through the
and 5% CO2, with medium replacement every 4 days. Cell scaffolds/cells constructs.
A.J. Salgado et al. / Materials Science and Engineering C 20 (2002) 27–33 29
2.3.3. Cellular adhesion brane was first blocked with 5% (w/v) skim dry milk for 1 h
Cellular adhesion and extracellular matrix production in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.4)
were assessed by fluorescence microscopy and environ- with 0.1% Tween 20 (TBS-T). Blots were further incubated
mental scanning electron microscopy (ESEM). with the primary antibody, mouse anti-bovine cross-reacted
For the ESEM experiments, cells were previously fixed with human osteonectin obtained from Developmental
in glutaraldheyde for 4 h, dried and examined with a JEOL Studies Hybridoma Bank in a 1:1000 working dilution,
JSM-5800LV scanning electron microscope at 15 kV. washed in 1% milk TBS-T solution and incubated overnight
The fluorescence microscopy experiments were per- at 4 jC with the secondary antibody, rabbit anti-mouse
formed using phalloidin conjugated with Alexa Flour 488 conjugated with horseradish peroxidase (Pierce, USA). The
(Molecular Probes). Samples were first fixed in paraformal- immune complex was detected by ECL system (Amersham)
dehyde for 30 min at room temperature. Scaffolds/cells and visualized by chemiluminescence.
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 45 min at room temperature 3. Results and discussion
in the dark, washed in PBS, counterstained with 5 Ag/ml of PI
and finally washed in PBS. The samples were then dried and 3.1. Scaffold characterization
viewed under a fluorescence microscope (Olympus IX70).
The processing method described in detail by Gomes et
2.3.4. Alkaline phosphatase activity assay al. [24], i.e. extrusion with blowing agents, allows to obtain a
Alkaline phosphatase (ALP) activity from the scaffolds/ porous structure resulting from the release of the gases (CO2
cells constructs was quantified by the specific conversion of and H2O) upon the decomposition of the blowing agent.
p-nitrophenyl phosphate ( pNPP) into p-nitrophenol ( pNP). The structure of the porous scaffolds consists of a fully
Supernatants were collected weekly (n = 5) and frozen at interconnected network of pores with around 70% of
20 jC and then thawed prior to analysis. The enzyme porosity (Fig. 1), presenting a size between 200 and 500
reaction was set up by mixing 50 Al of the sample with 150 Am. These values are within the range of those believed to
Al of substrate buffer containing 1 M diethanolamine HCl be ideal for tissue engineering of bone [9]. The mechanical
(pH 9.8) and 2 mg/ml of pNPP. The solution was incubated tests showed that these materials had a compressive mod-
at 37 jC for 1 h and the reaction was then stopped by a ulus of 124.6 F 27.2 MPa and a compressive strength of
solution containing 2 M NaOH and 0.2 mM EDTA in 8.0 F 0.9 MPa. The stated values can be further increased if
distilled water. The optical density was determined at 405 the materials are reinforced with hydroxyapatite [24]. Fur-
nm with a reference filter at 620 nm. A standard curve was ther details on the degradation behaviour as well as on other
made using pNP values ranging from 0 to 200 nmol/ml. The aspects of these scaffolds can be found in the literature [24].
results are expressed in nmol of pNP produced/ml/h.
3.2. Cellular adhesion, viability and proliferation
2.3.5. Osteocalcin assay
Osteocalcin production was assessed using an ELISA kit Initial osteoblast/material interactions can be conven-
(IBL, Germany). Supernatants were weekly collected (n = 5) iently characterized in four stages: (i) protein adsorption to
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.
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 Fig. 3. Confocal laser microscopy of human osteoblast-like cells stained
staining). 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) 27–33 31
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
Fig. 4. Cell viability and proliferation was assayed by the MTS test. Cell
stimulated with 10 mM h-glycerophosphate (Sigma), 100 mM ascorbic acid
density used was 3 105 cells/scaffold. For control, 3 105 cells were
(Sigma) and 10 7 M dexamethasone (Sigma). The ALP levels decrease
seeded on 24-well-plate culture trays. Cells were kept in culture for 21 days.
(14 days in culture) 7 days after stimulation, reaching the highest values
Between days 7 and 14, cells were stimulated with 10 mM h-glycerophos-
after 3 weeks in culture (n = 5, F S.D.).
phate (Sigma), 100 mM ascorbic acid (Sigma) and 10 7 M dexamethasone
(Sigma) (n = 5, F S.D.).
4. Conclusions
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