Materials Science and Engineering C 20 (2002) 101 – 109

www.elsevier.com/locate/msec

In vitro evaluation of hydroxyapatite reinforced

polyhydroxybutyrate composite
J. Ni, M. Wang*
School of Mechanical and Production Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore

Abstract

Particulate hydroxyapatite (HA) was incorporated into polyhydroxybutyrate (PHB) to form a bioactive and biodegradable composite for
applications in hard tissue replacement and regeneration. HA/PHB composite containing 10, 20, and 30 vol.% of HA was made for in vitro
evaluation. In vitro studies were conducted using an acellular simulated body fluid (SBF). Composite specimens were immersed in SBF at 37
jC for various periods of time prior to surface analysis and mechanical testing. Results obtained from scanning electron microscopic (SEM)
examination, thin film X-ray diffraction (TF-XRD) analysis, and Fourier transform infrared (FTIR) spectroscopy showed that a layer of bone-
like apatite formed within a short period on HA/PHB composite after its immersion in SBF, demonstrating high in vitro bioactivity of the
composite. The bioactivity and mechanical properties of the composite could be changed by varying the amount of HA in the composite.
Dynamic mechanical analysis (DMA) revealed that the storage modulus (EV) of HA/PHB composite increased initially with immersion time
in SBF, due to apatite formation on composite surface and decreased after prolonged immersion in SBF, indicating degradation of the
composite in a simulated body environment. HA/PHB composite thus has the potential for its intended applications. D 2002 Elsevier Science
B.V. All rights reserved.

Keywords: Hydroxyapatite; Polyhydroxybutyrate; Composite; In vitro behaviour; Bioactivity; Biodegradation

1. Introduction environment to materials that can in some way alter their

The original concept of using a bioceramic to reinforce a
polymer was introduced by Bonfield et al. [1] in the early
1980s. Various particulate bioceramics as the reinforcement
(and also as the bioactive phase) have been incorporated
into biocompatible and ‘‘bio-stable’’ polymers to produce
bone substitutes [1– 6], considering the fact that human
cortical bone at the ultrastructure level is a composite
consisting of nanometer-size apatite crystals and collagen
fibrils [7]. The mechanical as well as biological performance
of bioactive ceramic/polymer composites can be controlled
through using different particulate bioceramics for the
composites and also through varying the amounts of bio-
ceramic particles in the composite [8]. Hydroxyapatite (HA)
reinforced high-density polyethylene (HDPE), the first bio-
ceramic/polymer composite, has been used clinically for
orbital floor reconstruction and orbital volume augmentation
[9], and in middle ear implants [10].
In recent years, emphasis in biomaterial engineering has
moved from materials that remain stable in the biological
*
Corresponding author. Tel.: +65-790-5151; fax: +65-791-1859.
E-mail address: mmwang@ntu.edu.sg (M. Wang).
properties (i.e., ‘‘biodegrade’’) in response to the cellular
and extracellular environment. Biodegradable materials are
designed to degrade gradually in the body and will be
replaced eventually by newly formed tissues. After being
implanted in the body, a biodegradable bone substitute
material will have gradual decreases in strength and stiffness
over a clinically determined optimal period. As bone repairs
itself, the external load will be transferred from the biode-
grading implant to bone. This approach provides the best
biomaterials solution to tissue replacement and regeneration,
if requirements for the initial strength and stiffness and other
short-term performance can be met. Following this philos-
ophy and using established composite technology, biode-
gradable composites consisting of particulate bioceramics
and polyhydroxybutyrate (PHB), which is a natural polymer
and can degrade in the human body environment, were
manufactured for potential applications in hard tissue repair
[11,12]. These composites should be fully characterised and
assessed before they can be used clinically.
In vivo studies are essential for biomaterials that are
currently developed. However, results obtained from in vivo
experiments are often difficult to interpret due to the com-
plexity of various cellular responses. On the other hand, in

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 1 9 - X
102 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109
vitro studies can provide information that indicates the in
vivo performance of materials [13,14]. A common feature of
bioactive materials is that their surfaces develop a biologi-
cally active hydroxy carbonate apatite (HCA) layer after im-
plantation, which is essential for establishing bonding with
bone [15]. For a material to be bioactive in vivo, it must have
the ability to induce HCA formation on its surface in vitro. A
bioactive material designed to be degradable in the human
body should also exhibit degradation in a simulated body
environment. In this investigation, the surface structure of
hydroxyapatite reinforced polyhydroxybutyrate (HA/PHB)
composite after its immersion in a simulated body fluid
(SBF) was studied. The mechanical performance of the com-
posite was also evaluated before and after immersion in SBF.
2. Materials and methods
2.1. Composite and test specimens
Commercially available HA (Taihei Chemicals, Japan)
and PHB (ICI, UK) were raw materials for making the
composite, with both HA and PHB being used in their as-
received state without further treatment. The raw materials
were fully characterised prior to composite production. The
median particle size of as-received HA was 24.5 Am. X-ray
diffraction (XRD) analysis of the ceramic powder showed
that it was phase-pure HA. XRD patterns of PHB only
exhibited peaks of the HB unit. (PHB is a semicrystalline
polymer and, hence, shows diffraction peaks.) Manufacture
of the HA/PHB composite followed a standardised proce-
dure, which consisted of compounding, milling, and com-
pression molding [12]. Composite containing 0, 10, 20, and
30 vol.% of HA was used in this investigation.
Test specimens were cut from compression molded
plates, ground, and polished using up to #1000 sandpaper
to remove any defects. They were then cleaned in an
ultrasonic bath containing distilled water and dried in an
oven before they were used in various tests.
2.2. Immersion in simulated body fluid (SBF)
An acellular simulated body fluid (SBF) was used for in
vitro experiments. The SBF was prepared by dissolving
reagent-grade chemicals of NaCl, NaHCO3, KCl, K2HPO4

3H2O, MgCl2
6H2O, CaCl2
2H2O, Na2SO4, and (CH2
OH)3CNH2 into distilled and deionised water and buffered
with HCl to pH 7.4 at 37 jC. It had ion concentrations that
were nearly the same as those in human blood plasma [16].
Specimens with a surface area of 1520 mm were
immersed in SBF at 37 jC for various periods of time.
100 ml of SBF was used for each specimen. Changes of the
surface structure of immersed specimens were analysed after
the specimens had been removed from SBF, washed with
distilled water, and dried. Furthermore, after drying, some
immersed specimens of the composite containing 20 vol.%
of HA were embedded in an acrylic resin, and ground and
polished for examining their cross-sections.
Rectangular specimens of the composite with dimensions
of 2051 mm were also immersed in SBF at 37 jC for 2
weeks, 1, 2, and 4 months, respectively. Mechanical proper-
ties of these rectangular specimens were evaluated using
dynamic mechanical analysis (DMA) after their preset
immersion time had been reached.
2.3. Scanning electron microscopy (SEM)
Before SEM examination, the surface and polished cross-
sections of immersed specimens were coated with a thin layer
of platinum. These specimens were then examined using a
JEOL JSM5600LV SEM at an accelerating voltage of 20 kV.
2.4. Thin-film X-ray diffraction analysis (TF-XRD)
TF-XRD analysis of immersed specimens was performed
on a Rigaku DMAX 22001 X-ray diffractometer with a thin-
film analyzer. Cu Ka radiation was used for the diffraction
with a voltage of 40 kV and a current of 30 mA. Specimens
were aligned at 1j to the incident beam. A step size of 0.02j
(2h) and a scan speed of 3j/min were used and the dif-
fraction data were collected from 20j to 50j (2h).
2.5. Fourier transform infrared spectroscopy (FTIR)
FTIR spectra of mineral crystals formed in vitro were
obtained using a Perkin Elmer System 2000 Fourier trans-
form infrared spectrometer. A small amount of the mineral
crystals was gathered from the surface of each immersed
specimen, milled with KBr and pressed into a transparent
film for FTIR analysis. FTIR spectra were collected over the
range of 4000– 400 cm1.
2.6. Dynamic mechanical analysis (DMA)
Mechanical properties of HA/PHB composite before and
after immersion in SBF were evaluated using a Perkin Elmer
DMA 7 system. A 15 mm knife edge, three-point bending
platform with a 5 mm probe tip was used. DMA tests were
performed in a temperature range of 30 –100 jC and at a
heating rate of 4 jC/min. Assuming that the physiological
frequency was 1 Hz, all tests were conducted at this fre-
quency. From DMA tests, three properties, namely, storage
modulus (EV), loss modulus (EW), and loss tangent (tand),
could be determined. For polymers and their composites, the
following equations exist [17]:
E ¼ EV þ iEW ð1Þ
tand ¼ EW=EV ð2Þ
where E is the dynamic modulus. The storage modulus
represents the capability of a material to store mechanical
energy and resist deformation. The higher the storage mod-
ulus, the stiffer the material.
 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109 103

3. Results

3.1. Scanning electron microscopy (SEM)

Mineral crystals were observed to grow on all HA/PHB

composite specimens only after 1-day immersion in SBF. Fig.
1a shows the crystals formed on the surface of the composite
containing 10 vol.% of HA. The crystals grew locally on the
specimen surface following the contour of the specimen.
After 3 days immersion, mineral crystals covered nearly the
whole surface of the specimen, but the mineral layer formed
was very thin and, hence, the original contour of the specimen
could still be seen (Fig. 1b). After 7 days immersion, the
mineral layer grew thicker and the surface of the mineral layer
was considerably less rough (Fig. 1c). The morphology of the
mineral layer formed after immersion in SBF for 2, 4, and 8
weeks was similar to that of the mineral layer formed after
immersion in SBF for 7 days. In addition, nucleation of new
mineral particles could be seen on the mineral layer already
formed after 4 weeks immersion in SBF.
The formation and growth of mineral crystals on HA/
PHB composite of other compositions was similar to that on
10 vol.% HA/PHB (Figs. 2 and 3), but the mineral crystals
grew more rapidly on 20 and 30 vol.% HA/PHB than on 10
vol.% HA/PHB. For 20 vol.% HA/PHB, a thin and uniform
mineral layer was formed after 3 days immersion (Fig. 2b)
and new mineral particles were already present after 7 days
immersion (Fig. 2c). The size of these new near-spherical
crystals was around 3 – 4 Am. For 30% HA/PHB, the mineral
layer was formed only after 1 day immersion (Fig. 3a) and
more near-spherical crystals were observed on the specimen
after 7 days immersion (Fig. 3c). These observations indi-
cated that the mineral layer was formed layer by layer on the
surface of the composite and that the composite containing
larger amount of HA had greater ability to induce the
formation of minerals in vitro. Fig. 4 shows at a high
magnification the morphology of the mineral particles,
revealing that the mineral particles were composed of small
flake-like crystallites, which is similar in morphology to
crystals formed on other biomaterials [16]. A cross-section
of the mineral layer formed in vitro is shown in Fig. 5,
displaying a compact structure.
Mineral crystals were also found on unfilled PHB after
its immersion in SBF for 14 days (Fig. 6a), but the growth
of these crystals was very slow. After 28 days immersion,
the mineral layer formed was still very thin and rough (Fig.
6b). Only after 56 days immersion was a uniform mineral
layer observed (Fig. 6c).

3.2. Thin-film X-ray diffraction (TF-XRD) Fig. 1. SEM micrographs of 10 vol.% HA/PHB composite after immersion
in SBF for (a) 1 day, (b) 3 days, (c) 7 days.

TF-XRD was used to determine the phase(s) of mineral

crystals (or layer) formed on the composite surface. Fig. 7 observed after 7 days immersion, with one peak at 26j (2h)
shows TF-XRD patterns of 10, 20, and 30 vol.% HA/PHB and another peak between 31j and 33j (Fig. 7a and b). The
composite before and after immersion in SBF. For both 10 broad apatite peaks indicated low crystallinity of apatite
and 20 vol.% HA/PHB composite, apatite peaks were first formed in vitro at this early stage of immersion. The HA
104 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109

and PHB peaks, which appeared in the XRD patterns of HA/ immersion time, indicating the growth of an apatite layer
PHB composite before immersion in SBF, were suppressed on the composite surface in SBF and another peak at 49j
in XRD patterns of the composite after 7 days immersion. appeared after 28 days immersion. For 30 vol.% HA/PHB
The intensity of apatite peaks increased gradually with composite, apatite peaks appeared in XRD patterns only

Fig. 2. SEM micrographs of 20 vol.% HA/PHB composite after immersion Fig. 3. SEM micrographs of 30 vol.% HA/PHB composite after immersion
in SBF for (a) 1 day, (b) 3 days, (c) 7 days. in SBF for (a) 1 day, (b) 3 days, (c) 7 days.
 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109 105

Fig. 4. Morphology of mineral crystals formed in vitro.

Fig. 5. Cross-sectional view of the apatite layer formed on 20 vol.% HA/

PHB composite after 28 days immersion in SBF.

after the specimen had been immersed in SBF for 3 days

(Fig. 7c). Again, HA and PHB peaks of constituent materials
of the composite were suppressed by apatite peaks in the
XRD pattern after 3 days immersion and the intensity of
apatite peaks in XRD patterns increased gradually with
immersion time.

3.3. Fourier transform infrared spectroscopy (FTIR)

FTIR was used to detect the presence of functional

groups such as phosphate and carbonate groups, thus
providing more information on the apatite formed on the
surface of composite after its immersion in SBF. Fig. 8
displays FTIR spectra of apatite formed in vitro on 20 vol.%
HA/PHB composite. There was no major difference
between the spectrum obtained at 7 days immersion time
(Fig. 8a) and the spectrum obtained at 14 days or longer
immersion time (Fig. 8b). The characteristic absorption
bands of phosphate appearing at 565, 604, and 962 cm1
Fig. 6. SEM micrographs of unfilled PHB after immersion in SBF for (a) 14
days, (b) 28 days, (c) 56 days.
were observed for all immersed specimens. The FTIR
spectra of apatite formed in vitro also had a strong absorp-
tion band at 873 cm1 which corresponded to the vibration
mode of carbonate, indicating the incorporation of carbonate
groups in the structure of apatite. Other carbonate peaks at
106 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109

Fig. 7. TF-XRD patterns of HA/PHB composite before and after immersion in SBF: (a) 10 vol.% HA/PHB, (b) 20 vol.% HA/PHB, (c) 30 vol.% HA/PHB.
 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109
Fig. 8. FTIR spectra of apatite formed on 20 vol.% HA/PHB in SBF: (a) 7
days immersion, (b) 28 days immersion.
1415 and 1454 cm1 were observed as well. These results
suggested that the apatite formed on the surface of compo-
site in SBF was carbonated apatite, which is similar in
composition and structure to bone apatite [18].
Fig. 9. Variation of storage modulus with immersion time in SBF for HA/PHB composite.
107
3.4. Dynamic mechanical analysis (DMA)
The storage modulus was determined for HA/PHB com-
posite before and after immersion in SBF. The repeatability
of measurements from three specimens of each composition
was good. Therefore, only averaged data were plotted with-
out error bars in order to present results clearly (Fig. 9).
DMA results showed that the storage modulus of HA/PHB
composite increased initially with an increase in immersion
time. After immersion in SBF, the storage modulus of the
composite became higher than that of the composite before
immersion, with a larger increase in modulus for composite
having a higher HA content. The storage modulus of unfilled
PHB did not change within the first month of immersion. It
increased only slightly after 2 months immersion. With
prolonged immersion in SBF (i.e., beyond 2 months), both
unfilled PHB and HA/PHB composite exhibited decreases in
storage modulus.
4. Discussion
SEM examination and TF-XRD and FTIR analyses
showed the formation and growth of a layer of carbonated
apatite on the surface of HA/PHB composite as well as
unfilled PHB after their immersion in SBF. This apatite is
bone-like apatite, which was also found to form on various
bioactive materials when they were immersed in SBF
[16,18 –20]. The in vitro bioactivity of HA/PHB composite
has thus been demonstrated.
Apatite was found to form on HA/PHB composite
within 1 day in SBF. Such a fast rate of apatite formation
indicates that high in vitro bioactivity can be obtained for
the composite. It was also observed that the number of
nucleation sites of apatite crystals was proportional to the
HA content of the composite and that the apatite layer grew
more rapidly on composite containing greater amount of
HA. The rate of formation and growth of an apatite layer
108 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109
on the HA/PHB composite increased in the following
order:
10 vol:% HA=PHB < 20 vol:% HA=PHB
< 30 vol:% HA=PHB ð3Þ
Therefore, the composite should have a high volume per-
centage of HA if a high degree of bioactivity is required.
Apatite was also found to form on unfilled PHB after
immersion in SBF for 14 days, but by introducing HA
particles into the PHB matrix, a much higher degree of
bioactivity can be obtained and controlled for the composite,
which is an advantage of the composite approach. The
incorporation of HA particles into PHB has also resulted
in a composite having higher stiffness than PHB.
It is shown in the present investigation that the storage
modulus of HA/PHB composite increased with an increase
in the HA volume percentage. Nazhat et al. [21] have
demonstrated that the storage modulus of a bioceramic/
polymer composite is closely related to its Young’s modulus.
Therefore, as PHB has a relatively high Young’s modulus
among all biocompatible polymers, it can be deduced that
HA/PHB composite containing high percentages of HA
should have Young’s modulus values that are within the
range for cortical bone.
PHB is well known as a biocompatible and biodegradable
thermoplastic polymer [22,23]. It was found in the present
investigation that the storage moduli of PHB and its com-
posite increased with immersion time (up to 2 months) in
SBF. These increases can be attributed to the apatite layer
formed on their surfaces. The apatite layer could be more
than 10 Am thick after 28 days immersion in SBF and it was
a dense layer (Fig. 5). This apatite layer separated composite
from SBF and thus prevented the solution from attacking the
matrix polymer. It therefore protected the composite against
in vitro degradation at the initial stage of immersion. The
stiff apatite layer may have also enhanced the storage
modulus of the composite by physically being an additional
component of the composite. This phenomenon is similar to
observations made by Zhang and Ma [24] on the apatite/
poly(L-lactic acid) composite which exhibited an increase in
compressive modulus after the immersion in SBF for 2
months. With prolonged immersion in SBF (i.e., beyond 2
months), storage moduli of PHB and HA/PHB composite
decreased gradually, indicating degradation of these materi-
als in a simulated body environment. It is believed that at
this stage of immersion in SBF, liquid had penetrated the
apatite layer and diffused into the composite, thus causing
the matrix polymer to degrade.
5. Conclusions
A biologically active apatite layer forms within a short
period on HA/PHB composite after its immersion in SBF,
demonstrating high in vitro bioactivity of the composite. The
bioactivity and mechanical properties of the composite can
be tailored by varying the HA volume percentage in the
composite. The storage modulus of the composite increases
initially with immersion time in SBF, which is due to the
formation of the apatite layer on composite surface and
decreases after prolonged immersion in SBF, displaying
degradation of the composite in a simulated body environ-
ment. HA/PHB composite has the potential as a bioactive
and biodegradable material for applications in hard tissue
replacement and regeneration.
Acknowledgements
J. Ni thanks Nanyang Technological University (NTU)
for providing a research studentship, which enabled her to
conduct the work reported. Assistance provided by fellow
researchers and technical staff in the School of MPE, NTU
is gratefully acknowledged.
References
[1] W. Bonfield, M.D. Grynpas, A.E. Tully, J. Bowman, J. Abram, Hy-
droxyapatite reinforced polyethylene — a mechanically compatible im-
plant material for bone replacement, Biomaterials 2 (1981) 185 – 186.
[2] M. Wang, W. Bonfield, L.L. Hench, BioglassR/high density polyethy-
lene composite as a new soft tissue bonding material, in: J. Wilson,
L.L. Hench, D. Greenspan (Eds.), Bioceramics, vol. 8, Pergamon,
Oxford, 1995, pp. 383 – 388.
[3] R.L. Orefice, G.P. LaTorre, J.K. West, L.L. Hench, Processing and
characterization of bioactive polysulfone-BioglassR composites, in: J.
Wilson, L.L. Hench, D. Greenspan (Eds.), Bioceramics, vol. 8, Per-
gamon, Oxford, 1995, pp. 409 – 414.
[4] M. Wang, T. Kokubo, W. Bonfield, A-W glass – ceramic reinforced
polyethylene composite for medical applications, in: T. Kokubo, T.
Nakamura, F. Miyaji (Eds.), Bioceramics, vol. 9, Pergamon, Ox-
ford, 1996, pp. 387 – 390.
[5] R.L. Reis, I.B. Leonor, M.T. Rego, A.M. Cunha, M.H. Ferdandez,
R.N. Correia, Stiff and bioactive composites based on starch, poly-
ethylene and SiO2 – 3CaO
P2O5 – MgO glasses and glass – ceramics,
in: R.Z. LeGeros, J.P. LeGeros (Eds.), Bioceramics, vol. 11, World
Scientific, Singapore, 1998, pp. 169 – 172.
[6] M. Wang, C.Y. Yue, B. Chua, L.C. Kan, Hydroxyapatite reinforced
polysulfone as a new biomaterial for tissue replacement, in: H. Ogushi,
G.W. Hastings, T. Yoshikawa (Eds.), Bioceramics, vol. 12, World Sci-
entific, Singapore, 1999, pp. 401 – 404.
[7] J.B. Park, Biomaterials: An Introduction, Plenum, New York, 1979.
[8] W. Bonfield, M. Wang, K.E. Tanner, Interfaces in analogue biomate-
rials, Acta Materialia 46 (1998) 2509 – 2518.
[9] R.N. Downs, S. Vardy, K.E. Tanner, W. Bonfield, Hydroxyapatite-poly-
ethylene composite in orbital surgery, in: W. Bonfield, G.W. Hastings,
K.E. Tanner (Eds.), Bioceramics, vol. 4, Butterworth, Oxford, 1991, pp.
239 – 246.
[10] R.E. Swain, M. Wang, B. Beale, W. Bonfield, HAPEXk for otologic
applications, Biomedical Engineering: Applications, Basis and Com-
munications 11 (1999) 315 – 320.
[11] M. Wang, J. Weng, C.H. Goh, J. Ni, C.X. Wang, Developing trical-
cium phosphate/polyhydroxybutyrate composite as a new biodegrad-
able material for clinical applications, in: S. Giannini, A. Moroni
(Eds.), Bioceramics, vol. 13, Trans Tech Publications, Zurich, 2000,
pp. 741 – 744.
 J. Ni, M. Wang / Materials Science and Engineering C 20 (2002) 101–109
[12] M. Wang, C.X. Wang, J. Weng, J. Ni, P.Y. Quek, Production and
evaluation of biodegradable hydroxyapatite/polyhydroxybutyrate
composite, Proceedings of the 10th International Conference on Bio-
medical Engineering, Singapore, 2000, pp. 545 – 546.
[13] T. Kitsugi, T. Yamamuro, T. Nakamura, T. Kokubo, Bone bonding
behavior of MgO – CaO – SiO2 – P2O3 – CaF2 glass (mother glass of
AW-glass – ceramics), Journal of Biomedical Materials Research 23
(1989) 631 – 648.
[14] C.J. Kirkpatrick, C. Mittermayer, Theoretical and practical aspects of
testing potential biomaterials in vitro, Journal of Materials Science:
Materials in Medicine 1 (1990) 9 – 13.
[15] L.L. Hench, Bioceramics: from concept to clinic, Journal of the Amer-
ican Ceramic Society 74 (1991) 1487 – 1510.
[16] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, Solu-
tions able to reproduce in vivo surface-structure changes in bioactive
glass – ceramic A-W, Journal of Biomedical Materials Research 24
(1990) 721 – 734.
[17] I.M. Ward, Mechanical Properties of Solid Polymers, Wiley, New
York, 1985.
[18] P. Li, C. Ohtsuki, T. Kokubo, K. Nakanishi, N. Soga, K. de Groot, The
role of hydrated silica, titania, and alumina in inducing apatite on
implants, Journal of Biomedical Materials Research 28 (1994) 7 – 15.
109
[19] J. Weng, Q. Liu, J.G.C. Wolke, X. Zhang, K. de Groot, Formation and
characteristics of apatite layer on plasma-sprayed hydroxyapatite coat-
ings in simulated body fluid, Biomaterials 18 (1997) 1027 – 1035.
[20] J. Weng, M. Wang, In vitro formation of bone-like apatite on the
surface of solution-cast poorly crystallized hydroxyapatite/chitin com-
posite, Key Engineering Materials 192 – 195 (2001) 657 – 660.
[21] S.N. Nazhat, R. Joseph, M. Wang, R. Smith, K.E. Tanner, W. Bon-
field, Dynamic mechanical characterization of hydroxyapatite rein-
forced polyethylene: effect of particle size, Journal of Materials
Science: Materials in Medicine 11 (2000) 621 – 628.
[22] M. Canetti, M. Urso, P. Sadocco, Influence of the morphology and of
the supermolecular structure on the enzymatic degradation of bacterial
poly(3-hydroxybutyrate), Polymer 40 (1999) 2587 – 2594.
[23] N. Yoshie, K. Nakasato, M. Fujiwara, K. Kasuya, H. Abe, Y. Doi, Y.
Inoue, Effect of low molecular weight additives on enzymatic degra-
dation of poly(h-hydroxybutyrate), Polymer 41 (2000) 3227 – 3234.
[24] R. Zhang, P.X. Ma, Porous poly(L-lactic acid)/apatite composites cre-
ated by biomimetic process, Journal of Biomedical Materials Research
45 (1999) 285 – 293.