Biomaterials 21 (2000) 1129}1134

Thin "lm of low-crystalline calcium phosphate apatite formed

at low temperature夽
Hyun-Man Kim *, Yoonji Kim , Su-Jin Park , Christian Rey
, HyunMi Lee ,
Melvin J. Glimcher, Jea Seung Ko
Laboratory of Hard Tissue Engineering, Department of Oral Anatomy and Dental Research Institute, College of Dentistry, Seoul National University, 28-22,
YeonKun-Dong, ChongRo-Ku, Seoul 110-749, South Korea

Laboratoire de Physico-Chimie des Solides, Institut Polytechnique de Toulouse, C.N.R.S. 38, rue des 36 Ponts, Toulouse 31400, France
Laboratory for the Study of Skeletal Disease and Rehabilitation and Department of Orthopedic Surgery, Children's Hospital and Harvard Medical School,
300 Longwood Ave., Boston, MA 02115, USA
Received 24 September 1999; accepted 3 December 1999

Abstract

Surface modi"cation of biomaterials to improve biocompatibility without changing their bulk properties is desired for many
clinical applications and has become an emerging technology in biomaterial research and industry. In the present study, a simple
method of coating the solid surfaces of metals, organic tissue matrices, glasses, inorganic ceramics as well as organic polymers with
a thin "lm of low-crystalline apatite crystals (LCA) was developed. Acidic solution containing calcium and phosphate ions was
neutralized with alkaline solution to form calcium phosphate precipitates at low temperature. Precipitates of solid calcium phosphate
particles were, then, removed by "ltration. Concentration of free ions in the "ltered ion solution which were not involved in the
formation of calcium phosphate precipitate was high enough to induce the heterogeneous nucleation on the solid surfaces at low
temperature. Thin layers of calcium phosphate crystals were formed on the surfaces of metals, glasses, inorganic ceramics, organic
polymers including hydrophobic ones, and biological tissue matrices with this solution. The thin layer of crystals consisted of poorly
crystalline calcium phosphate apatite crystals which contain high amount of labile ions like bone crystals and did not dissolve in the
physiologic solutions. Various cells attached to this crystal layer and proliferated well.  2000 Elsevier Science Ltd. All rights
reserved.

Keywords: Low-crystalline apatite; Thin "lm; Biomaterial; Low temperature; Cell culture

1. Introduction Calcium phosphate apatite is the only type of calcium

Calcium phosphate (Ca-P) apatite has been well known
to have good properties as a biomaterial for bone repair,
augmentation, substitution, and surface coating [1}3].
Coating the surfaces of dental and orthopedic materials
with biocompatible calcium phosphate apatite can elicit
favorable biological and chemical responses on the surfa-
ces and this can enable us to mimic the reactions occur-
ring in the natural calci"ed tissues without losing bulk
properties of materials such as durability and inertness.
夽
Part of this paper was presented at the sixth International Confer-
ence of the Chemistry and Biology of Mineralized Tissues, Vittel,
France, 1}6 November 1998.
* Corresponding author.
phosphate crystal in calci"ed tissues such as bones, teeth,
calci"ed cartilage, and cultured matrix produced by os-
teoblasts [4}8]. It has biocompatibility with most cell
types such as osteoblasts, osteoclasts, "broblasts, and
periodontal ligament cells found in the calci"ed tissues
[9}13] and has osteoconductivity allowing the formation
of bone on its surface by attachment, migration, prolifer-
ation, and di!erentiation of bone-forming cells [14}16].
All apatites, however, are not similar in their crystallo-
graphic properties. Pure hydroxy apatite crystals consist-
ing of calcium, phosphate, and hydroxyl ions are
stoichiometric crystals of rods with high crystallinity
[17], while biocrystals isolated from bone or calci"ed
cartilage are non-stoichiometric apatites of low crystal-
linity [6}8]. Biocrystals are small thin plates [6}8]
in shape of extensive speci"c surface that makes them

0142-9612/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 6 5 - 3
1130 H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134
metabolically active with high surface reactivity [18].
Their surfaces are assumed to be covered with non-apa-
titic highly reactive labile ions of CO\, PO\, HPO\,
  
where organic constituents or cells can be attached to
form a cell}material interface [19,20]. These properties
are not expected from highly crystalline apatite crystals
widely used clinically so far, but only from bone-like
low-crystalline apatite crystals (LCAs). Apatite coatings
formed at high temperature by plasma spraying have
been shown to be heterogeneous and to contain large
oxy}hydroxy apatite crystals [21] as well as a high pro-
portion of amorphous phases [22]. Thus, there is a de-
mand to develop a method to form an apatite layer with
properties similar to those of bone crystals on solid
surfaces such as metals, polymers, and ceramics to en-
hance bioreactivity as well as biocompatibility when en-
gineering implant materials.
In the present study, a simple method to get a thin coat
of LCAs was developed. Apatite crystals were induced to
form and grow directly on solid surfaces including the
surfaces of low interfacial energy by increasing the solu-
tion supersaturation in the solution. The crystallographic
analysis of the apatite crystals in the thin coat showed
a close similarity to that of bone crystals in their crystal
habit, crystallinity, and the amount of labile domains of
surface ions.
2. Materials and methods
2.1. Preparation of thin xlm of apatite
In the classical Ostwald's nucleation theory, the free
energy for nucleation depends on the supersaturation of
solution (S), the net interfacial energy for nucleation (p),
the temperature (¹), and the particle surface area (A):
*G"!R¹ ln S#pA.
This nucleation theory indicates that increasing the solu-
tion supersaturation S and reducing the net interfacial
energy p can induce the heterogeneous nucleation. In the
present study, the solution supersaturation S was greatly
increased to reduce the free energy for nucleation *G.
This increase was high enough to induce heterogeneous
nucleation even on the solid surface of low surface energy
without any treatment to decrease the net interfacial
energy p. Homogeneous precipitation occurring in highly
supersaturated solutions was prevented by using a low-
temperature system.
Following this strategy, highly supersaturated stable
calcium and phosphate ion solution was prepared at low
temperature. Synthetic calcium phosphate apatite crys-
tals [23] were dissolved in 0.2 N HCl (1 mg/ml). 1 ml of
this acidic ion solution was mixed with 1.35 ml of 0.2 M
tris[hydroxymethyl]amino methane (Tris) solution to
form Ca-P precipitate at 43C. Then, Ca-P precipitate was
removed by "ltration (pore size: 0.22 lM) to get a meta-
stable calcium and phosphate ion solution (pH 7.3). Ma-
terials to be coated were left in this Ca-P ion solution at
83C for 24 h to induce the nucleation of Ca-P on the
surfaces, then the temperature was maintained or in-
creased (up to 603C) to form a thin "lm coat of LCAs by
the growth of apatite crystals. Thicker thin "lm was
obtained by increasing the time of growth phase.
This method was used to coat the surfaces of the
following materials; metals, glasses, inorganic ceramics,
organic polymers including hydrophobic organic poly-
mers such as poly lactic-gluconic acid co-polymer sponge
(PLGA), polystyrene, polypropylene, silicone, polytetra-
#uorethylene, and organic biological tissue matrix like
decalci"ed membranes of crab (consisting of chitin), col-
lagens, and "bers of silk.
To exclude the possibility of gravitational precipitates
of amorphous calcium phosphates (ACPs) formed by
homogeneous nucleation in the solution as the source of
crystals on the surfaces, the surfaces of materials such as
disks of titanium or polypropylene were positioned verti-
cally and the crystals formed on the lateral surfaces were
examined.
2.2. Analysis of the thin xlm of apatites
2.2.1. Electron microscopic analysis
The phase and habit of Ca-P crystals were examined
in the thin "lm formed on carbon-coated formvar-
reinforced grids using transmission electron microscopy
(TEM) (1200 IIX, JEOL, Japan), or on a solid surface
using scanning electron microscopy (SEM) (840A, JEOL,
Japan) after sputter-coating with gold}palladium. The
electron di!raction patterns were also obtained.
2.2.2. X-ray diwraction analysis
The long-range orders of crystals which were scraped
o! from the surface of culture dishes were determined by
powder X-ray di!raction [24] using di!ractometer
(MXP 18, Mac Science, Japan) at 100 mA and 50 kV.
Bone crystals isolated from bovine femur [7] were also
analyzed for comparison.
2.2.3. Fourier-transformed infrared spectroscopy
(FTIR) analysis
The FTIR spectra of crystals were recorded on
a Perkin Elmer Fourier transform spectrometer 1700,
after embedding the crystals removed from the culture
dishes and bone crystals isolated from bovine femur [7]
in KBr pellets. Resolution-enhanced deconvolution
of the FTIR spectra was performed with constructor
software. The sensitivity coe$cients and the band-
widths are reported in the "gure legends. Integral in-
tensities of deconvoluted spectra in the l4 PO\ were

calculated [25].
 H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134 1131

2.2.4. Calcium and phosphate analysis 3. Results

The concentration of calcium was determined by
atomic absorption spectroscopy [26] (Perkin Elmer,
USA). Concentration of phosphate was determined by
a colorimetric method [27].
2.3. Culture of cells
L929 "broblasts (ATCC NCTC clone 929) and
MC3T3 osteoblasts were cultured on thin "lms of
apatite formed on various substrates such as culture
plates (Corning, USA), without any other treatment of
the surfaces. Cells were cultured in CO incubator

supplied with 95% air and 5% CO at pH 7.4 in

a-MEM supplemented with 10% fetal bovine serum
(FBS). Attachment and proliferation of cells were examin-
ed directly under phase contrast microscope and/or SEM.
With this simple method, thin "lm layers of LCAs were
formed on the surfaces of metals, glasses, inorganic cera-
mics, organic polymers including hydrophobic organic
polymers such as PLGA, polystylene, polypropylene, sili-
cone, polytetra#uorethylene, and organic biological tis-
sue matrix like decalci"ed membranes of crab (consisting
of chitin), collagens, and "bers of silk (Fig. 1). The "lms of
LCAs formed on both hydrophilic and hydrophobic sur-
faces were not disintegrated or detached from the surface
when immersed in the physiological solution for over
1 month.
The "rst Ca-P formed on the surface comprised par-
ticles of nano-size, which was studied by examining the
"rst precipitation formed on the carbon-coated formvar
"lm using TEM (Fig. 2a). Formation of "lms of LCAs on

Fig. 1. Sequential observation of the formation of thin layer of LCAs on the surface of the culture dishes. (a) Non-discrete particles are scattered in the
initial stage. (b}c) Then, discrete crystals form and grow on the surface, and proliferate and interconnect to cover all surfaces. (d) Apatite coating of
LCAs is covering porous PLGA co-polymer sponge. Scanning electron microscope (SEM), scale bar: (a}c) 1 lm, (d) 10 lm.

Fig. 2. TEM observation of the formation of thin layer of LCAs. Non-stained examination. (a) The "rst Ca-P depositions on the carbon-coated
formvar "lm under TEM do not give any di!raction maxima of Ca-P crystals (inset), which indicates ACP as a seeding substance of heterogeneous
nucleation on the surface. Scale bar: 500 nm. Electron di!raction: 100 kV, 80 cm. (b) Cross-sectional view of the thin "lm shows closely packed
nano-sized crystals of thin plates in the thin "lm. Scale bar: 100 nm.
1132 H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134

the vertically positioned solid surface or the underneath crystals of thin plates through secondary crystallization
surface excluded the possibility of gravitational precipita- [28] around pre-formed apatite crystals. By varying the
tion of particles from the ion solution as the source of time of growth, "lms of di!erent thicknesses from a
calcium phosphate particles on the surface. Particles of few nm up to about a few lm were formed and this was
ACPs on the surface were transformed to apatite, then determined by examining the cross section of thin "lm
a thin "lm layer was formed by multiplication of more using TEM (Fig. 2b).
XRD showed that the thin "lm of LCA consisted of
Table 1 poorly crystalline calcium phosphate apatite like bone
XRD of LCAs in thin "lm coating compared to bone crystals crystals (Table 1). FTIR analysis (Fig. 3a) also indicated
their poor crystallinity. Resolution enhancement [25] of
hkl 2h d (As ) FWHM

l2 CO\ (Fig. 3b) and l4 PO\ (Fig. 3c) showed peaks of
 
non-apatitic labile domains of PO\, HPO\, and
111 22.70 (22.82) 3.91 (3.89) 0.58 (0.53)  
002 25.98 (25.86) 3.43 (3.44) 0.44 (0.33) CO\, and type B CO\ substituting PO\ in addition
  
102 27.84 (28.16) 3.20 (3.17) 0.32 (0.39) to apatitic ions which were also found in bone crystals
211 31.72 (31.84) 2.82 (2.81) 0.38 (0.86) (Table 2).
112 32.16 (32.65) 2.78 (2.74) 0.44 (0.42)
300 33.14 (33.97) 2.70 (2.63) 0.20 (0.45)
Fibroblasts attached and proliferated well on the thin
"lm of LCAs formed on polyglactin 910 (vicryl) "bers
Values in parentheses are data of bone crystals isolated from bovine (Fig. 4a). Osteoblasts also adhered well to the substrate of
femur.

FWHM: full-width at half-maximum value. Table 2
The relative intensities in the l4 PO\ domains of LCAs in thin "lm

compared to bone crystals

IR band (cm\) 534 550 560 575 600 617

Domains [31] Labile HPO PO\ PO\ PO\ Labile
   
HPO PO
 
LCAs 8.17 2.50 8.89 9.27 8.66 3.24
Bone crystals 7.74 1.65 9.34 9.16 9.28 3.1

Fig. 3. FTIR spectrum of thin layer of LCAs. Crystals were obtained by

scraping the surface of polypropylene o! thin layer of LCAs. (a)
l4 PO\, l3 PO\, l2 CO\, and l4 CO\ are seen. (b) Deconvoluted
   
FTIR spectrum in the l2 CO\ domain shows that carbonate ions are

located in anionic sites of the apatitic structure indicating type B car-
bonate}apatite (CO\ substituted for PO\ groups). In addition, a la-
 
bile non-apatitic CO\ environment is also present. Deconvolution

parameters: bandwidth 10 cm\; sensitivity coe$cient 2.25. (c) Decon- Fig. 4. Cells cultured on thin "lm of LCAs. Scale bar: 10 lm. (a) SEM of
voluted FTIR spectrum in the l4 PO\ domain shows that labile MC3T3 osteoblasts cultured on thin "lm substrate of LCAs formed on

nonapatitic environment is present in addition to apatitic PO\ envi- PGLA for 2 d. (b) SEM of L929 "broblasts cultured on thin "lm of

ronment. Deconvolution parameters: bandwidth 18 cm\; sensitivity apatite formed on polygalactin 910 (vicryl) "bers for 3 d. They attach
coe$cient 2.25. and proliferate well on the "lm.
 H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134
"lm formed on PLGA (Fig. 4b). Cellular behavior on the
substrate of the "lm could be clearly observed under
phase contrast microscope due to the transparency of
the "lm.
4. Discussion
Application of low temperature was considered to be
the only condition in this system for obtaining the thin
"lm of LCAs. Less calcium and phosphate ions might be
involved in the formation of calcium phosphate precipi-
tate in the neutralized ion solution due to the low temper-
ature. Then free ions in the "ltered solution high enough
to overcome the energy barrier for heterogeneous nuclea-
tion on the surface were considered to be provided to the
surfaces. The low temperature again might preclude a ho-
mogeneous precipitation within this solution. Direct ob-
servation of the formation of a thin layer of LCAs under
TEM has con"rmed that this system follows the general
sequence for the heterogeneous nucleation and growth of
crystals from a supersaturated solution [28,29]. Nano-
sized ACPs were formed on the solid surface as the "rst
phase of Ca-P. This can be ascribed to their low surface
energy which requires less net energy for their de novo
formation [28,29]. The transformation of ACPs into
LCAs was followed. Then, a thin layered coat was formed
by multiplication and growth of LCAs. However, further
study is required to explain the exact mechanism of the
formation of the thin "lm in this system.
Crystallographic properties of the crystals in this "lm,
such as thin plate shape, low crystallinity, and high
amount of labile ions indicate that they are similar to
the crystals of bones [7]. The shape of the thin plates
of the crystals grown by this method can provide a high
speci"c area [18], which in turn enables them to be
potentially highly interactive in cellular or organic envi-
ronment. In addition, crystals were rich in non-apatitic
labile domain of PO\, HPO\, and CO\ abundant in
  
bone crystals [30,31]. It was suggested that these labile
ions cover the surfaces of LCA and be highly reactive to
organic constituents to form an organic}inorganic inter-
face [19,20].
Technically, the present method has an important ad-
vantage over the previously reported methods in accessi-
bility of coating site. Ions of calcium and phosphate can
reach into the hidden surfaces inside the bulk of materials
because they are provided in a solution. The most prob-
able candidates for the application of this method are the
porous polymers like polyglycolic acid, polylactic acid,
PLGA co-polymers which have a great potential for drug
and cell delivery as well as giving a sca!old for bone
ingrowth. In addition, application of low temperature in
this method will be advantageous in reducing the chance
of losing the activity of drugs for the drug delivery sys-
tem. This technology can also be applied to make the
1133
non-biocompatibile materials with high mechanical
properties to be more biocompatible.
There has been much e!ort to form highly bioactive
bone-like Ca-P crystals on various substrates with
a simulated body #uid or some low supersaturated Ca-P
solution [32}37]. However, in such a low supersaturated
concentration, inducing the crystal formation was limited
only to speci"c solid surfaces like the strictly manipulated
one by chemical treatment and has not been possible on
hydrophobic surfaces. In addition, those coating proce-
dures needed a relatively long time and high temperature
which might limit the control of the crystallographic
nature of Ca-P apatite crystals. In the present study,
a low-temperature system was used to delay the homo-
geneous nucleation of solid Ca-P in the ionic solution
thereby leaving the concentration of free ions high (up to
[Ca>];[PO\]"20 mM). This overcame the energy

barrier in inducing heterogeneous nucleation on the sur-
faces such as hydrophobic materials whose surface enery
is very low.
In summary, we developed a method to form a thin
apatite "lm on various materials at a low temperature.
Their crystallographic property is similar to that of the
bone crystals and cells attached and proliferated well on
this "lm. This indicates that this apatite crystal "lm can
be used as a coating material over various substrates and
can modify the biocompatibility and bioreactivity of sub-
strates without losing their own bulk properties.
Acknowledgements
The authors thank Dr. Izumi Asahina (Tokyo Medical
and Dental University, Tokyo, Japan) for providing them
with porous PLGA co-polymers sponge, Ms. Seong-
Hyun Park and Ms. Su-Jin Kim for their assistance with
TEM and SEM, Mr. Chong-Hee Cho for XRD, and Ms.
Sa-Im Park for assistance with the preparation of this
manuscript. This work was partly supported by Grant
for Basic Medical Research from Korea Research Foun-
dation (H.-M. Kim).
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