Direct Deposited Porous Scaffolds of Calcium Phosphate Cement With Alginate

ACTBIO 1725
No. of Pages 9, Model 5G
30 April 2011
Acta Biomaterialia xxx (2011) xxxxxx
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Contents lists available at ScienceDirect
Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat
Direct deposited porous scaffolds of calcium phosphate cement with alginate

for drug delivery and bone tissue engineering
Gil-Su Lee a,b, Jeong-Hui Park a,b, Ueon Sang Shin b, Hae-Won Kim a,b,c,
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Department of Nanobiomedical Science and WCU Research Center, Dankook University Graduate School, Yongin, South Korea
Institute of Tissue Regeneration Engineering, Dankook University, Yongin, South Korea
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Department of Biomaterials Science, School of Dentistry, Dankook University, Yongin, South Korea
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a r t i c l e
i n f o
Article history:
Received 23 November 2010
Received in revised form 7 April 2011
Accepted 12 April 2011
Available online xxxx
Keywords:
Porous scaffolds
Self-setting cements
Calcium phosphates
Protein delivery
Bone regeneration
a b s t r a c t
This study reports the preparation of novel porous scaffolds of calcium phosphate cement (CPC) combined with alginate, and their potential usefulness as a three-dimensional (3-D) matrix for drug delivery
and tissue engineering of bone. An a-tricalcium phosphate-based powder was mixed with sodium alginate solution and then directly injected into a brous structure in a Ca-containing bath. A rapid hardening
reaction of the alginate with Ca2+ helps to shape the composite into a brous form with diameters of hundreds of micrometers, and subsequent pressing in a mold allows the formation of 3-D porous scaffolds
with different porosity levels. After transformation of the CPC into a calcium-decient hydroxyapatite
phase in simulated biological uid the scaffold was shown to retain its mechanical stability. During
the process biological proteins, such as bovine serum albumin and lysozyme, used as model proteins,
were observed to be effectively loaded onto and released from the scaffolds for up to more than a month,
proving the efcacy of the scaffolds as a drug delivering matrix. Mesenchymal stem cells (MSC) were isolated from rat bone marrow and then cultured on the CPCalginate porous scaffolds to investigate the
ability to be populated by cells and their subsequent differentiation along the osteogenic lineage. It
was shown that MSC increasingly actively populated and also permeated into the porous network with
time of culture. In particular, cells cultured within a scaffold with a relatively high porosity level showed
favorable proliferation and osteogenic differentiation. An in vivo pilot study of the CPCalginate porous
scaffolds after implantation into the rat calvarium for 6 weeks revealed the formation of new bone tissue
within the scaffold, closing the defect almost completely. Based on these results, the newly developed
CPCalginate porous scaffolds could be potentially useful as a 3-D matrix for drug delivery and tissue
engineering of bone.
2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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Rapid setting cements are very useful in bone tissue regeneration, as either a direct lling or an injectable material. Calcium
phosphate cements (CPCs) have been one of the most widely studied bioactive ceramics for this purpose [1,2]. Although some challenges, such as the mechanical properties, the introduction of
macropores and control of the dissolution rate, remain to be improved, many fascinating properties of CPCs make them a useful
choice in the treatment of bone defects [2,3]. CPCs have been found
to be cell and tissue compatible, and they self-set, making them
useful as an injectable material requiring minimally invasive sur-
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Corresponding author at: Department of Nanobiomedical Science and WCU

Research Center, Dankook University Graduate School, Yongin, South Korea. Tel.:
+82 41 550 1926.
E-mail address: kimhw@dku.edu (H.-W. Kim).
gery, and, in addition, they can carry therapeutic molecules within

the formulation [4,5].
Scaffolds with a three-dimensional (3-D) porous network provide effective matrix conditions for bone tissue engineering [68].
Tissue cells are ex vivo cultured with the scaffolds to better mimic
the structure and function of native tissues than the materials or
cells alone [8,9]. In the course of ex vivo engineering of tissues the
controlled release of therapeutic molecules such as growth factors
is favored, to modulate cellular function and speed up bone
formation. CPC-based materials have also been considered good
candidates for the delivery of therapeutics carried within their
structure, because they self-hard under mild conditions, with the
therapeutics being safely incorporated, and retain a sustainable release prole [4]. To apply CPC-based materials to bone tissue engineering their development as 3-D scaffolds which support cell
proliferation and cellmaterial composite construction is necessary.
To this end we here aim to develop a novel cell scaffolding
material made of CPCs in combination with sodium alginate. In
1742-7061/$ - see front matter 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2011.04.008
Please cite this article in press as: Lee G-S et al. Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone
tissue engineering. Acta Biomater (2011), doi:10.1016/j.actbio.2011.04.008
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particular, a brous network was formulated by directly depositing

the composite suspension under a Ca-containing solution.
The deposited suspension rapidly sets to form a gelled network
in the presence of alginate and is cross-linked by Ca2+ ions
[10,11]. The hardened porous scaffold is considered to be cell compatible and useful for bone tissue engineering. Moreover, the scaffold is considered to be able to load and deliver bioactive molecules
contained within the structure. The processing techniques to develop the CPCalginate porous scaffolds are described and the
in vitro cellular responses of mesenchymal stem cells (MSC) from
rat bone marrow to them have been investigated, prior to their
application in bone tissue engineering. An in vivo pilot study was
also performed to evaluate tissue compatibility, and the drug delivery potential of the scaffold was assessed using two different model proteins.
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2. Materials and methods
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2.1. Preparation of the composite suspension
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The experimental a-tricalcium phosphate (a-TCP)-based cement powder was prepared as described in a previous report
[12]. Commercial calcium carbonate and anhydrous dicalcium
phosphate (both from Aldrich) were mixed and thermally reacted
at 1400 C for 3 h, then air quenched, which resulted in complete
reaction to form the a-TCP phase [12]. The powders were ball
milled and sieved down to 45 lm, and then kept under vacuum
for further use. The average particle size of the a-TCP was
4.79 lm, measured using a particle size analyzer (Saturn DigiSizer
5200, Micromeritics, USA). Sodium alginate (Aldrich) solution was
prepared in 5% Na2HPO4 (in distilled, deionized water) at a concentration of 2 wt.%. The cement powder was mixed with the alginate
solution at an appropriate ratio to prepare the composite suspension for dispensation through a nozzle.
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2.2. Direct dispensation into scaffolds
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The possibility of the direct deposition of the composite suspensions was examined by varying the mixing ratio of cement powder/
alginate solution (from 1.0 to 2.5 by weight). Above a ratio of 2.0
the suspension was too viscous to dispense through the nozzle,
therefore ratios of 1.02.0 were used for the experiments. The
mixed suspension was placed in a syringe and then dispensed into
a Ca-containing bath (150 mM CaCl2) in order to rapidly solidify
the deposit, as schematically illustrated in Fig. 1. The dispensation
pressure was adjusted to 500 kPa using a regulator (IEI, AD2000C).
The size was controlled by means of different needle gauges (23
27 G). After dispensing the materials into 10 ml of Ca-containing
solution within a cylindrical mold (u = 10 mm) the brous deposits were further pressed down manually to produce a disc-shaped
scaffold of specic height. The process of depositing scaffolds within the Ca-containing bath took 1 min. The height of the scaffold
was varied in order to obtain different levels of porosity (1.2 mm
for low, 1.5 mm for medium and 2.0 mm for high porosity). Likewise, the amount (weight) of scaffold material to be dispensed
was varied (0.5 g for low, 0.4 g for medium and 0.3 g for high
porosity) while the height of the scaffolds was kept constant
(3 mm) to give different levels of porosity. The as-hardened scaffolds were used for further in vitro cell assays and in vivo animal
studies without further treatment, such as soaking in water.
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2.3. Characterization of 3-D porous scaffolds
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The composite scaffolds obtained were thoroughly washed in

distilled water and then immersed in simulated body uid (SBF
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Fig. 1. Schematic drawing of the processing set-up to prepare calcium phosphate

cement (CPC)/alginate composite (CPA) brous network for use as a 3-D tissue
engineering scaffold. CPCalginate suspension was injected through a needle using
an air pump regulator into a cylindrical mold containing CaCl2 (150 mM) which
promotes rapid setting of the composite suspension.
containing 284.0 mM Na+, 10 mM K+, 3.0 mM Mg2+, 5.0 mM Ca2+,

295.6 mM Cl, 8.4 mM HCO3 , 2.0 mM HPO24 , 1.0 mM SO24 ) at
37 C for periods of up to 7 days. Samples were washed and dried
under vacuum and the morphology was examined by scanning
electron microscopy (SEM) (Hitachi S-3000H). Composition change
was monitored by energy dispersive spectroscopy (EDS) (Bruker
SNE-3000 M) in a scanning electron microscope. An X-ray diffractometer (Rigaku Ultima IV) was used to detect changes in the crystalline phases of the scaffolds after them. The pore structure of
scaffolds with different porosities was analyzed by micro-computed tomography (lCT) (Skyscan model 1172). A disc (u
10 3 mm) of each sample was placed with the top and bottom
surfaces parallel to the scanning plane. Scanning was with a
11 Mp X-ray camera and 758 les were acquired with an image
pixel size of 19.92 lm. The surface charge of the a-TCP particles
was determined by measurement of the zeta potential (Zetasizer
ZEN3600, Malvern Instruments). The a-TCP particles were sieved
(40 lm) and dispersed in distilled water at 1 mg ml1 and the zeta
potential measured at room temperature and pH 7.0 using a disposable capillary cell (DTS1060C) and Zetasizer software (v.
6.20). The measurement was repeated on three different samples.
The elastic modulus of the scaffolds was measured by means of
dynamic mechanical analysis (DMA) (DMA25, Metravib, France).
Samples with three different porosities were prepared with the
dimensions 5 mm diameter 10 mm height to which a dynamic
compression load was applied. The storage modulus of the samples
was recorded. Three samples were tested for each group.
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2.4. Assas of protein delivery capacity
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Protein release from the CPCalginate porous scaffold was assessed using bovine serum albumin (BSA) and lysozyme as the
model proteins. Loading of each protein was carried out in two different ways: one was to add the protein to the alginate solution
and then mix this with CPC powder, which was subsequently
deposited in a protein-containing porous scaffold (loading I);
the other was to add the protein to the CPC suspension, which
was incubated for 1 h with gentle agitation, and then the solution
was mixed with alginate solution which was then deposited in a
porous scaffold (loading II). Protein content in each scaffold sample was set at 33.3 lg mg scaffold1. 1 g of the protein-containing
porous scaffold was used for the protein release test. This was
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based on a pilot study that showed that the deposition of 1.4 g of

the suspension resulted in the production of 1 g ( 0.039 g) of nal
scaffold sample. Each sample was immersed in 10 ml of phosphate
buffered saline (PBS), pH 7, and 37 C for up to 28 days. At each
measurement time (1, 2, 3, 6 and 24 h, and 2, 3, 7, 10, 14, 21 and
28 days) the scaffold was removed and the remaining medium assessed by the BCA (bicinchoninic acid) method. At each measurement point the medium was refreshed.
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2.5. In vitro osteoblast culture and proliferation
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For the cell response tests CPCalginate scaffolds having three

different levels of porosity (low 13.6%, medium 34.0%, and high
53.7%) were prepared. Mesenchymal stem cells (MSC) derived
from rat bone marrow were harvested from the femora and tibiae
of 5-week-old male rats [13]. The femora and tibiae were rapidly
dissected and placed in a-minimal essential medium (a-MEM).
The sectioned bone was treated with collagenase and dispase solution for 30 min and then the bone marrow was ushed out and
centrifuged at 1500 r.p.m. The pellet was disrupted and cultured
under normal culture conditions, in a-MEM supplemented with
10% fetal bovine serum, containing antibiotic/antimycotic solution
(10,000 U penicillin, 10,000 lg streptomycin, and 25 lg amphotericin B/m, Gibco) at 37 C in an atmosphere of 5% CO2/95% air. After
5 days of culture, non-adherent cells were removed and supplemented with new medium. Cells were maintained under normal
culture conditions and underwent three passages before use for
further in vitro assays.
A scaffold sample was placed into each well of 24-well plate and
a suspension of 1 105 cells was seeded on each sample. Cells
were incubated for up to 14 days under the inuence of osteogenic
factors (50 lg ml1 ascorbic acid, 100 nM dexamethasone and
10 mM b-glycerophosphate). Cell proliferation was then measured
using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. When the
MTS reagent (tetrazolium salt) is applied to living cells it is reduced
by the cells to a colored formazan product which is soluble in culture medium. The quantity of formazan product, which is directly
proportional to the number of living cells, was measured at an
absorbance of 490 nm using an ELISA plate reader (iMARK, BioRad).
Three replicate samples were tested by MTS assay.
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2.6. Alkaline phosphatase determination
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As an a priori index for in vitro osteogenic differentiation of the

MSC during culture on the CPCalginate composite scaffolds alkaline phosphatase (ALP) activity was determined. After culture for 7
and 14 days in osteogenic medium the cell layer was harvested and
treated with 0.1% Triton X-100 cell lysis medium and further disrupted by sequential freezing and thawing. The total protein content was assayed using a commercial DC protein assay kit
(BioRad), and the aliquot of the reaction sample was determined
after normalization to the total protein content. The ALP activity
of the cells was determined using an ALP assay kit (procedure
No. ALP-10, Sigma) [13]. The p-nitrophenol produced in the presence of ALP was determined by its absorbance at 405 nm. Three
replicate samples were tested for ALP activity.
Cell test data are represented as means standard deviation
(SD), and statistical analysis was carried out by one-way analysis
of variance (ANOVA). Statistical signicance was considered at
P < 0.05.
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2.7. In vivo pilot study on bone compatibility
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10-week-old male SpragueDawley rats were used for the

in vivo study. The surgical protocol was in accordance with the
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guidelines of the Animal Care and Use Committee of Dankook University, South Korea. Animals were anesthetized by means of intramuscular injection using ketamine (80 mg kg1) and xylazine
(10 mg kg1). An incision was made in the anterior region of the
calvarium and a 5 mm diameter critical sized full thickness bone
defect was prepared using a trephine drill under continuous sterile
saline irrigation. For the in vivo test scaffolds with the dimensions
5 mm diameter 2 mm height were prepared using a different
sized mold and the amount of composite deposited was also adjusted to produce scaffolds with high porosity (53.7%). The prepared scaffolds were implanted within the calvarium defect.
Defects without implanted scaffolds were used as negative controls. Soft tissues were sutured to achieve primary closure. Six
weeks after implantation the animals were killed. The area of the
original surgical defect and the surrounding tissues were removed
en bloc and xed in 10% neutral formalin solution and then decalcied. Tissues were embedded in a parafn block and then serial
sectioned using a microtome (Leica). The 46 lm thickness sections were mounted on microscope slides. Slides with tissue sections were deparafnized and hydrated through series of xylene
and alcohol. The tissue slides were stained with hematoxylin and
eosin (H&E) and Massons trichrome (MT) and viewed under an
optical microscope for histological observation.
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3. Results and discussion
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3.1. Scaffold fabrication and morphology
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Upon deposition of the mixture suspension into a Ca-containing

bath rapid hardening occurred due to the reaction of sodium alginate with Ca2+ ions. Therefore, the as-deposited shape could be
maintained during the process, resulting in a 3-D network. In fact,
without alginate in the CPC composite rapid hardening was not
possible, resulting in complete disintegration of the scaffolds during dispensation. With the powder to liquid ratios used here (1.0
2.0), necessary to allow injectability through the nozzle, the alginate-free CPC suspensions were observed not to harden, even after
several hours. However, this hurdle could be overcome by the use
of alginate, due to its rapid crosslinking reaction with Ca2+ ions in
the deposition bath. Therefore, the addition of alginate to the CPC
composite and the use of an appropriate powder to liquid ratio are
the essential processing considerations in terms of obtaining both
injectability and hardenability.
The diameter of bers could be tuned by changing the needle
gauge and the composition of the suspension, as shown in
Fig. 2a. Here we could obtain ber diameters in the range 200
600 lm with different gauge needles (23, 25, 26 and 27 G) and
compositions (CPA20 and CPA15). Fiber diameter decreased as
the ratio of CPC powder to alginate liquid was increased (from
1.5 to 2.0) and the needle gauge increased (from 23 to 27 G, corresponding to a decrease in needle inner diameter from 0.32 to
0.16 mm). The piled up brous network was further shaped into
a 3-D scaffold by applying a compressive load. By varying the level
of compression the porosity of the scaffolds was easily controlled.
Here we varied the porosity of the composite scaffolds at low,
medium and high levels.
In fact, compared with other types of bioceramics, there has
been little development of porous scaffolds based on CPC composites. A recent review highlighted the importance of CPC-based scaffolds as potential drug delivering systems because of their selfhardening property [14,15]. Some studies on scaffold fabrication
incorporated biopolymer phases such as chitosan and poly(lactic
acid)/alginate within CPCs and applied conventional scaffolding
methods [6,16]. Compared with those previous works, the scaffolds
developed here are produced by a novel methodology, direct depo-
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Fig. 2. (a) Fiber diameter variations (from 200600 lm) with change in needle gauge (23, 25, 26 and 27 G) and the composition of the suspension (CPA20 and CPA15).
Diameter decreases with increasing needle gauge (decreasing needle diameter) and increasing the ratio of CPC powder to alginate liquid. Further compression of the piled up
brous network facilitated shaping into a 3-D scaffold, the porosity of which, depending on the compression level, was easily controlled. (b) Pore structure of the CPCalginate
3-D porous scaffolds revealed by micro-computed tomography (lCT). 3-D reconstructed and 2-D cross-section images are shown. The measured porosities were 13.6%, 34.0%,
and 53.7%, respectively, for the low, medium and high porosity scaffolds. Porosity data are presented as means 1 standard deviation for three different samples.
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sition of suspensions and formation of 3-D structures. Using this

process the pore conguration, including stem size and porosity,
are controllable, and the scaffolds can be formulated into complex
shapes by packing appropriate amounts within a designed mold.
Although here we randomly piled up the deposits to form a 3-D
structure improvements to the process such as direct writing will
be possible to produce well-dened 3-D congurations. This remains a further interesting research area.
The pore structure of the 3-D composite scaffolds was revealed
by lCT, as shown in Fig. 2b. In the case of the low porosity scaffold
(porosity 14%) some pores appeared to be clogged due to compression. The medium porosity scaffold (porosity 34%) had greater pore space and better pore interconnection. The pores in the
high porosity scaffold (porosity 54%) were highly spaced and
interconnected, providing suitable 3-D pore channels for cell
migration and tissue perfusion [17,18].
The CPCalginate 3-D porous scaffolds were immersed in simulated body uid and the phase transformation investigated in
terms of surface microstructure and phase analysis. The microstructure of the scaffolds after varying immersion times is shown
in Fig. 3a. Before immersion (0d) the surface was dense, with
CPC particles embedded within the alginate matrix. After immersion for 1 day (1d) some tiny crystallites started to form on the
surface. By day 3 (3d) the crystalline phase grew to form an even
network of platelet-like crystallites. After 7 days (7d) the formation of nanocrystals was even greater, with micron sized islands
having formed, which become layered and merged together. The
phase changes of CPC and the CPCalginate scaffold were monitored by XRD during the immersion test. Initially only a-TCP peaks
were noted (closed circles). With immersion HA appeared as a new
phase (asterisks), the intensity of HA peaks increasing with
increasing immersion time, suggesting phase transformation of
a-TCP to HA [19]. By day 7 only HA phase was observed, suggesting
complete transformation. The EDS analysis supported the phase
conversion of a-TCP to HA, as deduced from the change in Ca/P ratio, which increased from 1.517 (similar to a-TCP) to 1.659 (similar
to HA). Based on this phase evolution of the CPCalginate under
simulated body uid conditions, the scaffold developed here is
considered to retain good bioactivity and to provide favorable substrate conditions for bone-associated cells to grow and develop
into tissue, as the scaffold in body uid will transform into bone
mineral like HA phase, which should play a signicant role in biological reactions [20,21].
While the CPCalginate scaffolds showed pore congurations
and compositions that are favorable for use in tissue engineering,
the mechanical properties of the scaffolds need to be improved
for load-bearing applications. Although the scaffolds were shown
to be relatively brittle, the elastic moduli of the scaffolds measured
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Fig. 3. Immersion tests of the CPCalginate 3-D porous scaffolds in simulated body uid for different times. (a) SEM microstructure change before (0d) and after immersion
for days 1, 3 and 7 (days are noted in each image). Tiny crystallites started to appear on day 1, which grew considerably over day 3 and 7, with development of highly faceted
nanocrystallines. (b) XRD patterns of the scaffolds during the immersion tests. The initial a-TCP phase started to transform into HA phase on day 1, which continued to
develop with immersion time to reach almost complete transformation into HA at day 7. (c) EDS analysis of the Ca and P atomic composition of the transformed phase,
showing the increment in Ca/P ratio from 1.517 to 1.659, supporting the phase transformation of a-TCP to HA.
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by dynamic mechanical analysis were in the range similar to that

of trabecular bone (96 63 MPa for high, 398 63 MPa for medium
and 573 87 MPa for low porosity scaffolds versus 50500 MPa for
trabecular bone) [22], suggesting possible application for bone
regeneration, however, mainly in non-load-bearing areas. However, considering the porosity of our scaffolds the values observed
are relatively lower than those reported for HA-based bioceramic
scaffolds [2325]. This is mainly due to the intrinsic poor mechanical properties of the CPC-based materials. Further improvements
in the mechanical properties are thus needed for load-bearing
applications, which will be further study.
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3.2. MSC culture and osteogenic differentiation
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To address the possibility of using the CPCalginate porous scaffolds for bone tissue engineering we rst investigated the responses of MSC derived from rat bone marrow to scaffolds with
different levels of porosity. 1 105 MSC were seeded on the scaffolds and cultured for up to 14 days in osteogenic medium containing ascorbic acid, dexamethasone and b-glycerophosphate. Cell
growth was measured as mitochondrial activity of the cells (MTS
assay), as shown in Fig. 4. MSC proliferated well on all three types
of scaffolds, showing an ongoing increase in MTS level with culture
time. At the initial time point of 3 days cell proliferation was significantly higher in the high porosity scaffold compared with the low
and medium porosity scaffolds (P < 0.05), and this was maintained
for up to 7 days. By day 14 the difference between the scaffolds
with medium and high porosity was reduced.
The cell morphology on the brous scaffolds was observed by
SEM at different magnications during culture for 7 and 14 days,
as shown in Fig. 5 (low Fig. 5a, medium Fig. 5b and high porosity
Fig. 5c). Cells on day 7 showed an elongated morphology with good
adherence to the underlying ber stems. By day 14 cells showed
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Fig. 4. Proliferation of mesenchymal stem cells (MSC) derived from rat bone
marrow upon the 3-D porous scaffolds with different porosity levels (low, medium
and high) during culture for 3, 7 and 14 days. Signicant differences were noticed
between the scaffolds at each culture time (P < 0.05 vs. low porosity and #P < 0.05
vs. medium porosity, ANOVA, n = 4).
more extensive cytoskeletal processes covering the stem surface

almost completely. When we observed the cross-sectioned internal
surface large number of cells were found deep in the pore channels,
particularly in the scaffolds with high porosity. Based on these results for cell proliferation and morphology the MSCs were demonstrated to favor the underlying CPCalginate matrices with good
adherence, active lopodial protrusions and an increase in number
with prolonged culture.
Stem cell differentiation along the osteogenic lineage upon the
composite scaffolds was investigated by determining ALP activity.
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Fig. 6. Specic alkaline phosphatase activity of cells cultured upon the 3-D porous
scaffolds with different porosity levels (low, medium and high) during culture for 7,
14 and 21 days. Signicant differences were noticed between the scaffolds at each
culture time (P < 0.05 vs. low porosity and #P < 0.05 vs. medium porosity, ANOVA,
n = 4).
Fig. 5. Morphology observed by SEM of cells grown on the CPCalginate porous

scaffolds with different levels of porosity during culture for 7 and 14 days: (a) low
porosity; (b) medium porosity; (c) high porosity. Cells on day 7 adhered well to the
underlying brous stems, showing a highly elongated cell shape with many
lopodia. By day 14 the cells had proliferated, were in contact with each other and
covered the surface of stems almost completely. This cell growth and morphology
was similarly observed for all porous scaffolds. Moreover, many cells were found
inside the scaffold (deep in the pore channels), particularly in the samples with high
porosity. Bar scale 100 lm.
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Cells were cultured on the scaffolds with different levels of porosity for up to 21 days in osteogenic medium and the ALP level measured, as shown in Fig. 6. The ALP activity showed ongoing
increases with culture time up to 21 days for all scaffolds. This
increment was greater for the scaffold with high porosity than it
was for the others. The results demonstrate that the MSC cultured
upon the 3-D porous scaffolds were stimulated to differentiate
along the osteogenic lineage and it was greater in the scaffold with
high porosity. The cell proliferation and ALP activity results suggest
the use of a scaffold with high porosity to obtain better in vitro
MSC function upon the 3-D brous matrix, which will ultimately
be useful for the production of ex vivo tissue engineered constructs
for bone tissue engineering.
The scaffold with high porosity is believed to provide good
spaces and substrate conditions for cells to migrate and proliferate
three-dimensionally, making it easier for cell movement and function through the open spaces [17,18]. However, clear reasons why
the cells favor more open scaffolds cannot be elucidated from our
experiments and results. The conditions used here are somewhat
limited in design, i.e. 2-D static culture conditions, so that the geometrical effects of the scaffolds cannot be fully considered. This
ultimately warrants further study on 3-D dynamic cultures, such
as ow perfusion systems. At least from these in vitro results on
MSC behavior, the CPCalginate porous scaffolds with high porosity are considered to have potential as a 3-D scaffold for bone tissue engineering. Based on the cellular compatibility tests we
briey performed an experiment on in vivo tissue responses to
the scaffolds, such as bone in-growth, as a pilot study.
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3.3. In vivo pilot study
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The CPCalginate scaffold with high porosity was implanted in

a rat calvarium with a critical size defect of u = 5 mm [25,26]. At
6 weeks post-implantation tissue samples were harvested and
examined by lCT, as shown in Fig. 7. A rst look at the implanted
samples with X-rays showed a weak image of a 2-D brous network in the scaffold compared with an almost clear image in the
blank (negative control) (Fig. 7a). The 2-D lCT image showed that
the defect region was fully lled in the scaffold sample, but remained almost unlled in the control (Fig. 7b). The reconstructed
3-D lCT images showed the 3-D structure of the porous scaffold
and bone in-growth within the defect region. Bone regeneration
hardly occurred in the negative control, suggesting a critical size
bone defect [2527]. On the other hand, in the scaffold sample
new bone in-growth could not be clearly differentiated from the
remaining material.
Histological staining of explants containing the porous scaffold
at 6 weeks post-operation revealed the quality of tissue and bone
formation. Fig. 8 shows (Fig. 8a and b) H&E and (Fig. 8c and d)
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ACTBIO 1725
30 April 2011
Fig. 7. (a) X-ray image and (b, c) micro-computed tomography (lCT) of the calvarium defect recovered 6 weeks after implantation of the CPCalginate scaffold with high
porosity, and a blank defect used as a negative control. (b) 2-D and (c, d) 3-D constructed images. The scaffold was implanted in a rat calvarium with a critical size defect of /
= 5 mm, and at 6 weeks post-implantation tissue samples were harvested and examined by lCT. The defect region showed 100% coverage, associated with both the scaffold
and newly formed bone. Dotted bar scale 1 mm.
Fig. 8. Histological staining of the tissues generated by the scaffold with high porosity at 6 weeks post-operation: (a, b) hematoxylin and eosin (HE) stain; (c, d) Massons
trichrome (MT) stain at different magnications. Arrows indicate defect margins (a). OB, old bone; NB, new bone; S, scaffold (b). Connective bone tissue lled the pore channel
of the scaffold throughout the defect region (a), and newly formed tissue lined the ber stems of the scaffold (b). Pale or dark blue areas stained with MT demonstrate the
formation of bony tissue, the major bone extracellular matrix.
ACTBIO 1725
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8
468
MT staining at different magnications. No observable inammatory responses to or tissue rejection of the implanted scaffolds
were found. Connective bone tissue was shown to ll the pore
channel of the scaffold throughout the defect region (Fig. 8a). A
magnied image showed newly formed tissue (dark red) lining
the ber stems of the scaffold (pale red) (Fig. 8b). MT staining demonstrated the formation of bony tissue, with the extracellular matrix appearing pale or dark blue (Fig. 8c and d).
In the histological images MT staining was even found within
the CPCalginate scaffold framework, demonstrating possible
replacement of the scaffold by cells and tissues. Although a large
portion of the scaffolds appeared not to be biodegraded during
the 6 weeks of implantation in rat calvarium, the results support
possible in vivo degradation of the composite, either CPC or alginate or both. In general, CPC derived from a-TCP have HA as the
major phase in vivo, so they are degraded very slowly due to the
transformed HA, remaining present sometimes over years in vivo
[28]. On the other hand, alginate degradation is known to occur
relatively quickly, possibly in weeks to months even in vitro,
depending on the alginate concentration and degree of cross-linking [29]. At least from this study, the CPCalginate scaffolds developed are considered to maintain their structural integrity for
6 weeks in rat calvarium, although there were indications of possible biodegradation. Although biodegradation should be controlled
in concert with the rate of tissue regeneration, the use of CPC based
on brushite instead of HA may be favored when faster degradation
is required [30,31].
The in vivo tissue responses in rat calvarium showed that the
CPCalginate porous scaffolds developed here have good tissue
compatibility and even a level of regenerative potential of bone tissue, suggesting potential as an implantable material for bone
regeneration. Based on the cell and tissue compatibility, further
study is needed into preparing constructs of stem cells in this novel
porous scaffold and in vivo implantation of the constructs for bone
tissue engineering. To obtain further stimulation of cellular responses within the scaffolds we performed a feasibility study of
the protein delivery potential of the scaffolds.
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3.4. Protein delivery potential
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BSA and lysozyme, which are negatively and positively charged,

respectively, at pH 7 were used as model proteins [32]. The initial
protein loading was carried out by two different methods. One was
to add the protein in alginate solution and then mixed with CPC
powder, which was deposited into a porous scaffold. The other
method was to add the protein to the CPC suspension which was
incubated for 1 h with gentle agitation and then the proteinCPC
suspension was mixed with alginate solution which was subsequently deposited into a porous scaffold. The protein initially
added to each 1 g of scaffold sample was set at 33.3 mg.
Fig. 9 shows the release proles of the proteins in PBS measured
for up to 28 days. Initially (within 12 h) up to about 2030% of the
lysozyme and BSA was released for all loading conditions, which
may be proteins loosely bound on the surface and in direct contact
with the solution. After this initial burst the release of both proteins was sustained at a reduced rate with time for up to 28 days.
The release proles of BSA did not vary much between the different
methods of loading (closed squares vs. open squares). However, in
the case of lysozyme the release rate was greatly reduced when the
lysozyme was rst added to the CPC powder (loading II) compared with when the protein was rst added to the alginate (loading I). While the release of lysozyme was higher than that of BSA
for the loading I method, the trend was reversed for the loading
II method. Therefore, it is considered that interaction of the proteins with the components of scaffold, particularly CPC, is different,
i.e. CPC may better retard the release of lysozyme than that of BSA
Fig. 9. Release proles of the proteins BSA and lysozyme from the CPCalginate
porous scaffold. The protein loading method was varied in two ways: one was to
add the protein to the alginate solution and then mix this with CPC powder, which
was subsequently deposited in a protein-containing porous scaffold (loading I);
the other was to add the protein to the CPC suspension, which was incubated for 1 h
with gentle agitation, and then the solution was mixed with alginate solution which
was then deposited in a porous scaffold (loading II). The protein content in each
scaffold was set at 33.3 lg mg scaffold1. Release tests were carried out in
phosphate-buffered saline, pH 7 (PBS), for up to 14 days. The protein-containing
scaffolds were immersed in PBS. At each measurement time point the scaffold was
removed and the remaining medium assessed by the BCA method. In the case of
BSA there was no signicant difference in the release prole dependent on the
method of drug loading, however, the release rate of lysozyme was signicantly
reduced after loading II compared with loading I. P < 0.05 for lysozyme loading II
vs. lysozyme loading I and #P < 0.05 for lysozyme loading II vs. BSA loading II,
ANOVA, n = 3).
due to a strong afnity or chemical bonding [33,34]. To elucidate

one possible reason on this behavior we measured the surface
charge of the a-TCP powder and the proteins by means of zeta potential measurement, shown in Table 1. While a-TCP and BSA were
negatively charged, lysozyme was highly positively charged at pH
7. Based on this, it is considered that electrostatic attraction to the
CPC powder was higher for lysozyme than BSA. Although alginate
is also negatively charged and thus has some ionic interaction with
lysozyme, its hydrogel nature may mean it has a more open structure than CPC, allowing water permeation and providing paths for
protein release from the structure. Moreover, the different degradation behaviors of alginate and CPC, discussed above, could also
affect the rate of release of lysozyme. Based on this result it is
anticipated that this CPCalginate scaffold may be effective in
the delivery of positively charged growth factors, such as basic
broblast growth factor [35]. Further investigation is needed on
this. These results on in vitro protein release demonstrate that biological molecules are easily and safely loaded within the scaffold
and the potential role of the porous scaffolds for sustainable protein release, particularly positively charged ones, for up to at least
a month.
The results shown here conrm that the CPCalginate porous
scaffolds are cell compatible and bioactive. The rapid setting
hydrogel-like structure, together with its moldability to defective
bone sites, supports the benet of CPCalginate scaffolds as 3-D
Table 1
Zeta potential measurement of the CPC powder and the proteins used in the study.
Sample
Zeta potential at pH 7
CPC powder
18.14
Lysozyme
2.53
BSA
16.84
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ACTBIO 1725
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matrices for stem cells, recruiting them into osteogenic development. Ongoing studies on the use of CPCalginate composite scaffolds as cell delivery devices for bone tissue engineering are
underway, in combination with tissue cells and stimulatory proteins within the structure.
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4. Conclusions
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540
CPC combined with alginate solution was effectively formed

into a porous scaffold by means of ber deposition into a Ca-containing solution. The CPCalginate scaffolds self-hardened, were
moldable into various shapes and the porosity was tunable. The
scaffolds were shown to have favorable 3-D matrix characteristics
for pre-osteoblastic cell adherence and proliferation, as well as
their differentiation into osteoblasts. Implantation of a scaffold
into a rat calvarium defect provided evidence of tissue compatibility and the ability for bone regeneration through the pore geometry. Moreover, the scaffolds showed an ability to safely load
biological proteins (BSA and lysozyme) during preparation and to
release them in vitro for over a month. CPCalginate scaffolds
can be further developed into tissue engineered constructs delivering biological molecules stimulating bone regeneration.
541
Acknowledgements
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This work was supported by a Priority Research Centers Program (no. 2009-0093829) and a WCU Program (no. R31-10069)
through the National Research Foundation funded by the Ministry
of Education, Science and Technology.
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Appendix A. Figures with essential colour discrimination
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Certain gures in this article, particularly Figures 13, 8, and 9,

are difcult to interpret in black and white. The full colour images
can be found in the on-line version, at doi: 10.1016/j.actbio.2011.
04.008.
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References
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Direct Deposited Porous Scaffolds of Calcium Phosphate Cement With Alginate

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