G. Wei 2004)

ARTICLE IN PRESS
Biomaterials 25 (2004) 4749–4757
Structure and properties of nano-hydroxyapatite/polymer composite
scaffolds for bone tissue engineering
Guobao Wei a , Peter X. Ma a,b,c, *
a Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109-2009, USA
b Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109-1078, USA
c Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor, MI 48109-1055, USA
Received 9 June 2003; accepted 11 November 2003
Abstract
To better mimic the mineral component and the microstructure of natural bone, novel nano-hydroxyapatite (NHAP)/polymer
composite scaffolds with high porosity and well-controlled pore architectures were prepared using thermally induced phase
separation (TIPS) techniques. The morphologies, mechanical properties and protein adsorption capacities of the composite scaffolds
were investigated. The high porosity (90% and above) was easily achieved and the pore size was adjusted by varying phase
separation parameters. The NHAP particles were dispersed in the pore walls of the scaffolds and bound to the polymer very well.
NHAP/polymer scaffolds prepared using pure solvent system had a regular anisotropic but open 3D pore structure similar to plain
polymer scaffolds while micro-hydroxyapatite (MHAP)/polymer scaffolds had a random irregular pore structure. The introduction
of HAP greatly increased the mechanical properties and improved the protein adsorption capacity. In a dioxane/water mixture
solvent system, NHAP-incorporated poly(l-lactic acid) (PLLA) scaffolds developed a fibrous morphology which in turn increased
the protein adsorption three fold over non fibrous scaffolds. The results suggest that the newly developed NHAP/polymer composite
scaffolds may serve as an excellent 3D substrate for cell attachment and migration in bone tissue engineering.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Nano; Hydroxyapatite; Phase separation; Scaffold; Protein adsorption
1. Introduction
There is a growing need for bone regeneration due to
various clinical bone diseases such as bone infections,
bone tumors and bone loss by trauma [1]. Current
therapies for bone defects include autografts, allografts,
xenografts and other artificial substitutes such as metals,
synthetic cements and bioceramics [2,3]. However, these
substitutes are far from ideal and each has its specific
problems and limitations. For example, autografts are
associated with donor shortage and donor site morbidity
whereas allografts and xenografts have the risk of
disease transmission and immune response [4], and
synthetic materials wear and do not behave like true
bone. As a result, recent research has been devoted to
bone tissue engineering in which a 3D porous scaffold is
loaded with specific living cells and/or tissue-inducing
*Tel.: +1-734-764-2209; fax: +1-734-647-2110.
E-mail address: mapx@umich.edu (P.X. Ma).
factors to launch a tissue regeneration or replacement in
a natural way [5,6].
In the tissue engineering approach, the temporary 3D
scaffold serves an important role in the manipulation of
the functions of osteoblasts and a central role in the
guidance of new bone formation into desired shapes. In
principle, a biodegradable matrix with sufficient mechanical
strength, optimized architecture and suitable
degradation rate, which could finally be replaced by
newly formed bone, is most desirable. The scaffolding
materials for bone tissue engineering should also be
osteoconductive so that osteoprogenitor cells can adhere
and migrate on the scaffolds, differentiate, and finally
form new bone [7,8]. Biodegradable polymers, mainly
polyesters such as poly(lactic acid) (PLA), poly(glycolic
acid) (PGA) and their copolymers (PLGA), have been
widely used to develop porous 3-D scaffolds using
various fabrication techniques [8–14]. These synthetic
materials have been demonstrated to be biocompatible
and degrade into non-toxic components with a
0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2003.12.005

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G. Wei, P.X. Ma / Biomaterials 25 (2004) 4749–4757
controllable degradation rate in vivo [15]. Another
major class of biomaterials for bone repair is ceramics
such as hydroxyapatite (HAP) and tricalcium phosphate
(TCP) [16,17]. Being similar to the mineral component
of natural bone, they showed good osteoconductivity
and bone bonding ability [18]. However, the main
limitation for the use of HAP ceramics was their
inherent brittleness and difficulty for processing [19].
To combine the osteoconductivity of calcium phosphates
and good biodegradability of polyesters, polymer/ceramic
composite scaffolds have been developed
for bone tissue engineering either by direct mixing or by
a biomimetic approach [7,8,20]. Compared to plain
polymer scaffolds in which neo tissue matrix was formed
only in the surface layer (o240 mm) [21], the composite
scaffolds supported cells growth and neo tissue formation
throughout the scaffold including in the very center
of the scaffold [6].
Polymer/ceramic composite scaffolds mimic the natural
bone to some extent. Natural bone is composed of
inorganic compound (mainly partially carbonated
HAP on the nanometer scale) and organic compounds
(mainly collagen). The nanometer size of the inorganic
component (mainly bone-like apatite) in natural
bone is considered to be important for the mechanical
properties of the bone [22]. Recent research in this
field also suggested that better osteoconductivity
would be achieved if synthetic HAP could resemble
bone minerals more in composition, size and
morphology [23,24]. In addition, nano-sized HAP may
have other special properties due to its small size and
huge specific surface area. Webster et al. [25,26]
have shown significant increase in protein adsorption
and osteoblast adhesion on the nano-sized ceramic
materials compared to traditional micron-sized ceramic
materials.
In previous works, the Ma group has developed a
variety of scaffolds using thermally induced phase
separation (TIPS) [8–10]. Both pore structure and pore
wall morphology can be controlled by phase separation
parameters. They have also demonstrated that the
addition of micron-sized hydroxyapatite (MHAP) increase
the adsorption of proteins and extracellular
matrix (ECM) components [27]. The cell seeding
uniformity into the scaffold was improved substantially
due to the introduction of osteoconductive hydroxyapatite
[6]. The MHAP/PLLA composite scaffolds were
shown to suppress apoptosis of osteoblasts by a possible
mechanism of enhanced adsorption of serum proteins
such as vitronectin and fibronectin onto the scaffolds
[27, 28]. In this paper, we further mimicked the size scale
of hydroxyapatite in natural bone and aim to fabricate
novel and improved composite scaffolds. The pore
structure, pore wall morphology, mechanical properties
and protein adsorption capacity were systematically
investigated.
2. Materials and methods
2.1. Materials
Poly(l-lactic acid) (PLLA) with an inherent viscosity
of 1.4–1.8 dl/g was purchased from Boehringer Ingelheim
(Ingelheim, Germany) and was used as received.
PLGA85 (Medisorb s , LA:GA=85:15) with an inherent
viscosity of 0.6 was obtained from Alkermes Inc.
(Wilmington, OH). NHAP were purchased from Berkeley
Advanced Biomaterials Inc. (San Leandro, CA).
Conventional HAP particles (MHAP, particle size on
the micrometer scale), tetrahydrofuran (THF), dioxane
and benzene were obtained from aldrich chemical
company (Milwaukee, WI).
2.2. Fabrication method
NHAP/PLLA composite scaffolds were fabricated
using a phase separation technique similar to that
reported previously [8]. Briefly, the HAP nanocrystal
powder was dispersed in a solvent by sonication for 30 s
at 15 W (Virsonic 100, Cardiner, NY). After that, PLLA
was dissolved in the NHAP suspended solvent at about
60 C to make homogeneous solutions with desired
concentrations. 2.5 ml polymer/NHAP mixture was
added into a Teflon vial, sonicated again and then
transferred into a freezer at a preset temperature to
induce solid–liquid or liquid–liquid phase separation.
The solidified mixture was maintained at that temperature
overnight and then transferred into a freeze-drying
container which was maintained at a temperature
between 5 C and 10 C by salt–ice bath for freezedrying.
The samples were freeze-dried at 0.5 mmHg for 7
days to remove solvent. The final composition of the
composite foam was determined by the concentration of
the polymer solution and NHAP content in the mixture.
2.3. SEM characterization
The porous morphologies of the scaffold were
examined with scanning electron microscopy (SEM)
(S-3200N, Hitachi, Japan) at 20 or 15 kV. The samples
were cut by a razor blade or fractured after being frozen
in liquid nitrogen for several minutes and then were
coated with gold for 200 s using a sputter coater (Desk-
II, Denton Vacuum Inc.).
2.4. Porosity
The melting behavior of the matrices was characterized
with a differential scanning calorimeter (DSC-7, Perkin-
Elmer, Norwalk, CT) as detailed previously [9]. The
degree of crystallinity, X c , of a sample was calculated as
X c ¼ DH m =DH 0 m ;

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G. Wei, P.X. Ma / Biomaterials 25 (2004) 4749–4757 4751
where DH m was the measured enthalpy of melting and
DHm 0 was the enthalpy of melting for 100% crystalline
polymer. For PLLA, DHm 0 ¼ 203:4 J=g: The estimated
density and porosity of the matrix were obtained as
follows. The diameter and height of the matrix were
measured to calculate the volume. The mass of the matrix
was measured with an analytical balance. The density was
calculated from the volume and mass. The porosity, e;
was calculated from the measured overall densities D m of
the matrix and the skeletal density D s . For a composite
scaffold, the skeletal density was determined using the
densities of the polymer and HAP powder. The porosity
was given by
e ¼ D s D m
;
D s
where D s was calculated from the following formula:
1
D s ¼
;
ð1 X h Þ=D p þ X h =D h
where D h is the density of the HAP powder with a value
of 3.16 g/ml and X h is the percentage of HAP in the
composite while D p is the density of the polymer. D p was
calculated from
1
D p ¼
;
ð1 X c Þ=D a þ X c =D c
where X c was the degree of crystallinity of the polymer.
The density of amorphous PLLA (D a ) is 1.248 g/ml and
the density of 100% crystalline PLLA (D c ) is 1.290 g/ml.
2.5. Mechanical properties
The compressive mechanical properties of the scaffolds
were tested using a MTS Synergie 200 mechanical
tester (MTS systems Co. Eden Prairie, Minnesota). The
scaffolds have dimensions of about 16 mm in diameter
and 3 mm in thickness. A crosshead speed of 0.5 mm/
min was used. The compressive modulus was defined as
the initial linear modulus. At least five specimens were
tested for each sample. The averages and standard
deviations were graphed. A two-tail Student’s t-test
(assuming equal variances) was performed to determine
the statistical significance (po0.05) of the differences in
mechanical properties.
2.6. Protein adsorption
Protein adsorption was performed by incubating the
scaffolds in phosphate buffered saline (PBS, 0.1 m,
pH=7.4) containing 2.5% or 5% fetal bovine serum
(FBS). The disk-like specimens with dimensions of 15.5–
16.5 mm in diameter and 370.1 mm in thickness were
used. Before incubation in the medium containing
proteins, the specimens were pretreated by ethanol for
30 min and then washed by PBS three times overnight
under gentle shaking. Specimens were then put into 24-
well culture plates (one specimen for each well) and
1.5 ml FBS/PBS solution was added into each well. The
scaffolds were incubated at 37 C for a chosen incubation
time. The concentration of the protein in the FBS/
PBS solution was then quantified with a commercial
protein assay kit, BCA (Pierce, Rockford, IL), using
bovine serum albumin (BSA) standards. The amount of
absorbed proteins was determined by subtracting the
amount of proteins left in the FBS/PBS solution after
adsorption from the amount of proteins in control FBS/
PBS solution (without specimen) under the same
incubation conditions.
3. Results
3.1. Porosity
NHAP/polymer composite scaffolds with high porosity
have been fabricated using thermally induced phase
separation techniques (Tables 1 and 2). Two solvent
systems were used to obtain composite scaffolds with
different pore structures. One is a mono-solvent system
of pure dioxane or benzene and the other, a solvent/nonsolvent
mixture system of dioxane and water in different
proportions. The densities and porosities of scaffolds are
listed in Tables 1 and 2. Incorporation of nano-sized
HAP only decreased the porosity slightly. This was
especially true when NHAP content was low. Regardless
of detailed morphology and composition, all scaffolds
have a high porosity of at least 89%, which was
considered to be beneficial for cell ingrowth and
survival. The solvent system did not have much
effect on the density and porosity. The quenching
temperature did not affect the porosity significantly
either (p=0.12).
3.2. Morphology
The morphology and microstructure of the scaffolds
were examined using SEM (Fig. 1): micrographs of
PLLA scaffolds (a,b), MHAP/PLLA (c,d) and NHAP/
PLLA (e–h) composite scaffolds fabricated with pure
Table 1
Densities and porosities of NHAP/PLLA composite scaffolds prepared
from dioxane solutions, phase separated at 18 C
Scaffolds
PLLA:HAP
(w/w)
D p
(g/cm 3 )
D s
(g/cm 3 )
Porosity
(%)
PLLA 100:0 1.2577 0.08812 93.0
PLLA–NHAP 90:10 1.3383 0.09708 92.8
PLLA–NHAP 70:30 1.5394 0.1176 92.3
PLLA–NHAP 50:50 1.7993 0.1475 91.8
PLLA–NHAP 30:70 2.1737 0.2304 89.4
PLLA–MHAP 50:50 1.7993 0.1505 91.6

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Table 2
Porosities of NHAP/PLLA scaffolds fabricated using dioxane/water mixture solvent system and phase separated at various temperatures
Scaffolds PLLA:HAP (w/w) Dioxane:H 2 O (v/v) Quenching temp. ( C) Porosity (%)
PLLA–NHAP 70:30 100:0 18 92.3
PLLA–NHAP 70:30 95:5 18 91.6
PLLA–NHAP 70:30 90:10 18 93.1
PLLA–NHAP 70:30 87:13 4 94.5
PLLA–NHAP 70:30 87:13 18 93.1
PLLA–NHAP 70:30 87:13 70 92.8
PLLA–NHAP 70:30 87:13 196 (LN 2 ) 92.5
dioxane or benzene. NHAP/PLLA composite scaffolds
maintained a regular internal ladder-like pore structure
similar to plain PLLA scaffolds, a typical morphology
formed by solid–liquid phase separation [8,10]. By
controlling the heat transfer direction, NHAP/PLLA
scaffolds with tubular pore architecture were also
obtained using benzene as a solvent. Nano-sized HAP
particles were distributed within the pore walls of the
scaffolds and no large aggregates appeared in pores. In
MHAP/PLLA scaffolds, the HAP platelets with size
ranging from 10 to 50 mm were found randomly
distributed in the PLLA matrix. Some were embedded
in the pore wall and some others were piled together
between pores or in the pores. The pore size of all these
scaffolds ranged from 50 to 100 mm. NHAP/PLGA85
composite scaffolds prepared with pure dioxane had
leaf-like morphology and well-interconnected pore
structures (Fig. 2).
Very different morphologies appeared in composite
scaffolds fabricated with dioxane and water mixture
solvent system (Fig. 3). The proportion of water in the
solvent systems had a significant effect on the pore size
and overall morphology of the scaffold. Even a small
amount of water (less than 5%) in the solvent system
significantly reduced the pore size from 100 mm to about
10 mm and random pore structure replaced the regular
ladder-like pore structure (Figs. 3a, band c). Another
obvious change was the pore wall surface texture.
Unlike relative solid and smooth pore wall in NHAP/
PLLA scaffold prepared from mono-solvent system,
fibrous and loose network became the characteristics of
the pore structure of the scaffolds prepared from the
mixed solvent system. When the water volume was 13%
in the mixture solvent, the scaffolds consisted of three
ranges of pore sizes. Large macropores were developed
in a range of 200–500 mm while much smaller micropores
(tens of microns) between the pore walls of the
large pores. The pore wall surface was not smooth, and
appeared as loose fibrous structure with several microns
in between. NHAP particles were entrapped in the
fibrous network of the porous structures and had little
effect on the changes in morphology with different water
proportions since plain PLLA scaffolds had the same
morphological changes (data not shown).
3.3. Mechanical properties
The compressive modulus of NHAP/PLLA scaffolds
increased with NHAP content (Fig. 4). The plain PLLA
scaffolds prepared using solid–liquid phase separation
techniques had a compressive modulus of 4.3 MPa. The
addition of NHAP into the scaffold improved the
mechanical properties. The modulus increased significantly
when the NHAP proportion reached 30% of the
composite and reached 8.3 MPa when the ratio of
NHAP to PLLA was 50:50. The solvent system had a
large effect on the mechanical properties of the porous
scaffolds (Fig. 5). The introduction of a non-solvent
component (in this case, water) in the polymer/HAP/
solvent mixture likely induced liquid–liquid phase
separation instead of solid–liquid phase separation.
Since regular and orientated pore architectures were
replaced by random pore structures in scaffolds
fabricated using mixed solvent system, the compressive
modulus decreased significantly. However, the modulus
recovered substantially when dioxane/water (87:13, v/v)
system was used. This might have resulted from the
uniform structure of the scaffolds (Figs. 3 d–f).
3.4. Protein adsorption
NHAP/PLLA composite scaffolds with different
amounts of NHAP were incubated in FBS/PBS solution
to investigate protein adsorption. For all NHAP/PLLA
composite scaffolds, the protein adsorption reached
equilibrium in 25 h and there was no significant increase
of protein adsorption during further incubation
(Fig. 6a). The addition of NHAP increased the protein
adsorption. NHAP/PLLA 50:50 and NHAP/PLLA
70:30 composite scaffolds absorbed 2.4- and 3.2-fold,
respectively, more serum proteins than plain PLLA
scaffold (Fig. 6b). The size of HAP particles also
affected protein adsorption onto the scaffolds containing
a high percentage of HAP. Serum protein adsorption
was significantly greater on NHAP/PLLA scaffolds
than on MHAP/PLLA scaffolds when the HAP/PLLA
was 50:50 or higher (po0.05, Fig. 6c). However, when
the ratio of HAP in the composite was lower than 50%,
the differences in the protein adsorption between NHAP

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G. Wei, P.X. Ma / Biomaterials 25 (2004) 4749–4757 4753
Fig. 1. SEM micrographs of plain PLLA, NHAP/PLLA and MHAP/PLLA scaffolds fabricated from 5% dioxane (a–e) and benzene (f,g) solution.
(a,b) Pure PLLA scaffold, 50, 400; (c,d) MHAP/PLLA 50:50 scaffold, 100, 500; (e,f) NHAP/PLLA 50:50 scaffold, 100, 1000;
(g) Tubular NHAP/PLLA scaffold, cross-section, 200; (h) Tubular NHAP/PLLA scaffold, longitudinal section, 100.

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Fig. 2. SEM micrographs of NHAP/PLGA85 (30:70) scaffolds fabricated from 10% dioxane solution quenched at 70 C. (a) 150 and (b) 500.
and MHAP incorporated scaffolds were not statistically
significant. With the dramatic changes in morphology of
scaffolds prepared using different solvent systems, the
protein adsorption also changed greatly. Scaffolds with
fibrous texture absorbed about four times greater
amount of protein than those with smooth pore wall
surface. These results suggested that the protein
adsorption on the scaffolds could be regulated by the
content of HAP, its size scale, and the pore wall
morphology of the scaffolds.
4. Discussion
The present studies described a phase separation
technique to fabricate polymer/NHAP-composite scaffolds
with high porosity and controlled pore architecture.
Different solvent systems were used to obtain
scaffolds with different microarchitectures and properties.
When dioxane was used alone, the porous structure
resulted from a solid–liquid phase separation of the
polymer solution. During quenching, the solvent crystallized
and the polymer was expelled from the solvent
crystallization front. Solvent crystals became pores after
subsequent sublimation. The characteristics of the pores
were determined by the morphologies of solvent crystals
under such quenching conditions. The temperature
gradient along the solvent crystallization direction led
to anisotropic structure. The introduction of HAP
particles into the polymer solution perturbed the solvent
crystallization to some extent and thereby made the pore
structure more irregular and isotropic. As expected,
MHAP/PLLA composite scaffold exhibited an isotropic
and irregular pore structure. The perturbation by
NHAP particles, however, was small even in high
proportion up to 50% due to their nanometer size scale
and uniform distribution. As a result, NHAP/PLLA
composite scaffolds maintained the main characteristic
pore architecture of solid–liquid phase separation which
was anisotropic and regular. In contrast to NHAP/
PLLA, the regular anisotropic pore structure was
obtained only when the HAP content was very low in
MHAP/PLLA scaffolds, e.g., MHAP/PLLAo10/90
(images not shown). In this case, low content of HAP
did not affect the solvent crystallization significantly
enough to alter the pore structure.
When mixed solvent (dioxane/water) was used to
prepare 5% PLLA solutions, a liquid–liquid phase
separation was induced and this process dominated the
final pore structure of the system [29], resulting in
random and interconnected pore architecture. The pore
size was also much smaller. With the increase of water
content in the system, larger quench depth allowed the
system to continue on a coarsening process to further
reduce the interfacial free energy between two phases
separated in earlier stage [30]. The coarsening effect
might have resulted in the formation of macropores in
addition to the micropores (Figs. 3d–f). It is therefore
possible to optimize various phase separation parameters
to achieve a macroporous structure in composite
scaffold. The mechanism of fibrous texture formation is
not fully understood. Since PLLA is a semicrystalline
polymer, the crystallization in the polymer-rich phase
may present a possible explanation for the formation of
the fibrous structure [9].
Most mammalian cells are anchorage-dependent cells
and they need a biocompatible substrate for attachment,
migration and differentiation to form new tissues.
Recent research demonstrated that cell adhesion and
survival could be modulated by protein pre-adsorption
on the substrate [25–28]. Therefore, protein adsorption
is of importance in evaluating a scaffold for tissue
engineering. In this paper, we have shown that the
addition of hydroxyapatite particles into the composite
scaffolds greatly increased protein adsorption. In addition
to the high affinity of hydroxyapatite itself for

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G. Wei, P.X. Ma / Biomaterials 25 (2004) 4749–4757 4755
Fig. 3. SEM micrographs of NHAP/PLLA (30:70) scaffolds fabricated using dioxane/water mixture solvents. (a) Dioxane:water=95:5, 500. (b,c)
Dioxane:water=90:10, 500, 8000. (d,e,f) Dioxane:water=87:13, 45, 500, 10000.
proteins, incorporation of HAP also altered the pore
surface morphology of the composite scaffolds and may
have made them more suitable for protein adsorption.
Furthermore, NHAP/PLLA scaffold absorbed significantly
more proteins than MHAP/PLLA at high HAP
content but not at low HAP content. It was likely that
most particles were embedded in the polymer pore walls
and the effect of HAP size was not evident when the
HAP content was low in the composite. With the
increase of HAP content, more and more particles were
exposed on the surfaces of the pore walls. This
phenomenon was most evident when the scaffold
exhibited fibrous morphology. Fibrous scaffolds had
much greater surface to volume ratio than scaffolds with
solid pore walls, which might have further increased the
protein adsorption capacity.
5. Conclusion
The present study investigated new NHAP/polymer
composite scaffolds developed using thermally induced
phase separation techniques. Nano-sized HAP particles
were successfully incorporated into the PLLA porous

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scaffolds. The incorporation of NHAP improved the
mechanical properties and protein adsorption of the
composite scaffolds while maintaining high porosity and
suitable microarchitecture. These results suggest that the
newly developed NHAP/polymer composite scaffolds
may be superior for bone tissue engineering.
Fig. 4. Effects of NHAP content on the compressive modulus of
NHAP/PLLA scaffolds fabricated from 5% dioxane solutions
% po0.05, w p=0.29, n=5.
Fig. 5. Effects of mixture solvent composition and quenching
temperature on the compressive moduli of NHAP/PLLA scaffolds
fabricated from 5% dioxane/water solvent systems.
Fig. 6. Protein adsorption on porous composite scaffolds incubated in
FBS/PBS solution. (a) as a function of time, 2.5% FBS/PBS, labels
indicate the w/w ratios of NHAP to PLLA; (b) as a function of NHAP
content, 2.5% FBS; (c) Comparison of MHAP and NHAP, 5.0% %
FBS/PBS. po0.05, w p=0.71, n=3. (d) Comparison of NHAP/PLLA
(30:70) scaffolds prepared using different solvent systems, 2.5% %
FBS/PBS. po0.05, n=3.

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