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Biomimetic Growth and Self-Assembly of Fluorapatite Aggregates by Diffusion into Denatured Collagen Matrices.

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Angew. Chem. Int. Ed.Engl. 1996,108, No. 22
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Biomimetic Growth and Self-Assembly of
Fluorapatite Aggregates by Diffusion into
Denatured Collagen Matrices**
Rudiger Kniep” and Susanne Busch
Nature produces inorganic solids with fascinating outer
shapes well suited to their function. The mechanisms of formation of these biogenic solids involve the combination of organic
and inorganic components. Organic matrices control the mass
transfer, define the location and number of nucleation centers,
determine the orientation of the (crystal) growth, and/or act as
templates for the final morphology of the inorganic solids.
In our studies on mimicking the processes involved in bone or
tooth formation,[’] we have investigated the biomimetic growth
of fluorapatite in gelatin matrices. A simple (static) diffusion cell
was employed, like that known, in principle, for solid-state reactionsC2]
and that already used in more complex form in dynamic
collagen systems.[31The experimental conditionsr4]were chosen
such that at constant temperature (25 “C) solutions of calcium
and of phosphate/fluoride diffuse into a gelatin plug from opposite ends (the so-called double diffusion techniq~e[~I).
In the
course of the diffusion, discrete crystallization zones form,
which are denoted as Liesegang bands in analogy to the classical
work of Liesegang (Liesegang rings) .[61 The time-dependent
growth of the fluorapatite aggregates can be particularly well
monitored in the middle band on the phosphate side; the largest
aggregates were also obtained in this band. The crystallization
products in all stages of development correspond to the fluorine-rich boundary region of the solid solution series hydroxyapatite/fluorapatite (Fig. I).”,
Although the gelatin141used here as the organic matrix is of
rather poorly defined s t r u c t ~ r e , [ ~it. ’ is
~ ~responsible for a
specific function in the control of the growth of the fluorapatite
aggregates. The use of other organic matrices (e.g., polysaccha400
j501
300
100
50
20
25
30
35
201”
40
45
50
55
Fig. 1. X-ray powder diagram (Cu,,, radiation) of a sphere fraction and calculated
diagram of fluorapatite (P6,lm; a = 936.6(3), c = 685.3(3) pm) [S]. Refined lattice
constants of the sphere fraction: a = 937.6(3), c = 688.6(2) pm (hydroxyapatite 171:
a = 942.4(4), c = 687.9(4) pm). Remarkably, the diffraction peaks of the sphere
fraction are comparatively sharp for biomimetic apatite, the X-ray diagram indicates a single phase, and the background is relatively low (small amounts of X-ray
amorphous components). N = count rate.
[*I
[**I
Prof. Dr. R. Kniep, Dip1.-Ing. S. Busch
Eduard-Zintl-Institut der Technischen Hochschule
Hochschulstrasse 10, D-64289 Darmstadt (Germany)
Fax: Int. code +(6151)166029
This work was supported by the Fonds der Chemischen Industrie
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~~
rides, agar, and carragenan) under otherwise identical experimental conditionsr4] leads to fundamentally different morphologies of the fluorapatite (irregular intergrowth of short
hexagonal prisms or xenomorphic precipitates). When gelatin is
used as the organic matrix, “anisotropic” spheres are formed as
the end product, the centers of which contain an elongated
hexagonal-prismatic seed crystal.
For unchanged morphology of the aggregates, the rates of
diffusion of the solutions and of formation of the Liesegang
bands, and hence the rate of growth of the fluorapatite are
determined by the initial pH of the gelatin. Thus, in a gelatin
plug that has not been acidified (PH 5.5, length 3 cm), apatite
spheres with diameters of 100 pm are first observed after six
weeks in the middle Liesegang band on the phosphate side.
If the pH of the gelatin is adjusted to 2.5-3.5, apatite spheres
with a diameter of 100 pm are formed after only two weeks.
The growth of the fluorapatite spheres begins with elongated
hexagonal-prismatic seed crystals, which reach a critical length
of 5-10 pm. The progressive stages of the self-assembled
growth of the needlelike seed crystals to give spherical aggregates are shown in Figure 2, and require approximately one
week. First, oriented upgrowths occur on the prism faces at both
ends of the seed crystal simultaneously and roughly symmetrically (mirror symmetry perpendicular to the prism axis), leading
to the formation of dumbbell-shaped aggregates. The orientation of these upgrowths does not necessarily indicate the existence of strict crystallographic relations; nevertheless, the maximum angle of divergence (between the prism axes of the
growing crystals and the prism axis of the seed crystal) appears
to be fixed at approximately 45”. The dumbbell region is extended through further growing generations according to the principles of self-similarity;[’’] however, the “bar” of the dumbbell
(seed crystal) initially remains visible. This situation can be
simulated in a two-dimensional form which mirrors reality very
well in a simplified way, as shown in Figure 3.r’21 Starting
from a needlelike seed crystal, in the first generation similar
needlelike crystals are formed at both ends, which have a
maximum angle of 45” to the long axis of the seed crystal. The
length of the crystals of each successive generation is shorter
than the length of the crystals of the preceding generation by a
factor of about 0.68 (estimated mean value from scanning electron microscope (SEM) images, error = ca. f10%). Each
“new” end of a crystal generation leads to self-similar multiplication. The result is illustrated in Figure 3, which shows the
simulation of five successive self-similar crystal generations at
each end of the seed crystal. The fact that the crystals interpenetrate in the simulation is certainly not realistic, but this has no
great effect on the morphology of the aggregate. It is more
important that the bar of the dumbbell (the seed crystal) remains visible in this growth state and correspondingly in the
simulation, that the surface curvature of the dumbbell in the
direction of the seed crystal is already indicative of the “closing”
of the aggregate, and that the extent of the aggregate, growing
from the two ends of the seed crystal, increases by a factor of
approximately 2 relative to the length of the seed crystal. The
further development of the aggregate-going from the dumbbell to a sphere--can again be seen in Figure 2. Figure 4 shows
the SEM image of the surface of a just completed (notched)
surface of a sphere. This reveals that also in this last generation
needlelike crystals are formed. Their prism cross section is only
about 0.05 pm, which means that the closed aggregate has a very
large surface area.
The (notched) spheres formed in the gelatin matrix continue
their growth by adding radial shells of material and forming
almost perfect spheres. This is a second (probably more conven-
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Fig. 4. SEM image of the surface of a just closed
sphere aggregate. In accordance with the self-similar branching patterns [12] described in the text and
shown in Figure 3, the dumbbell-shaped aggregates
close to give notched spheres after the tenth generation.
tionally spherolitic) growth mechanism, which will not be described further here. After about four weeks,
sphere diameters of up to 400 pm are
found. Our observations indicate that
the important limiting factor in the
growth of the spheres is the gelatin matrix, which, under the given experimental conditions, is stable for only about
six weeks.
The self-assembled hierarchical
growth of the fluorapatite aggregates
shows immediate parallels to the topological branching criteria of the macromolecular starburst dendrimer~."~'
However, the dimensionality-and
Fig. 2. SEM images of progressive stages of the self-assembled (hierarchical) growth of the fluorapatite aggrethus the information transfer-in the
gates: from the elongated hexagonal-prismatic seed (top left) through dumbbell shapes to the sphere (bottom
aggregates described here extends to
right).
the macroscopic range.
In accordance with their microcrystalline structure, the spherical aggregates are opalescent. A fraction of spheres with diameters of up to 300 pm is shown in Figure 5. On a flat support, the
Fig. 5. Optical micrograph of a fluorapatite sphere fraction. The long edge of the
figure corresponds to about 4 mm.
Fig. 3. SEM image of a dumbbell-shaped fluorapatite aggregate (top), Two-dimensional simulation [I21 of self-similar branching in five successive generations (bottom). The simulation shows branching (splitting) into only four crystals (with divergence angles of 3 0 ) of the new generation, which is a simplified representation. In
fact, orders of branching are higher (see also Fig. 2), and are, in principle, only
limited by the maximum angle of divergence (ca. +4Y) formed by the long axis of
the new crystals and the long axis of the preceding seed crystal. The factor of
shortening of preceding generations is discussed in the text.
Anww
Chem. Int. Ed. E n d . 1996, 35,No. 22
spheres orient themselves preferentially to form chains (electrostatic effects), and can be lifted out in the form of "strings of
beads" by using the point Of a
Thermoanalyticalinvestigations (thermogravimetry (TG) and differential thermal analysis (DTA)) on spherical aggregates dried at
temperature
were carried out at temperatures up to a maximum of 1000 "C
(heating rate
min-l; atmosphere: air).[141The X-ray Powder diagram of the sample after thermal treatment is identical to
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the diagram shown in Figure 1 except that the profiles of the
diffraction peaks are sharper (refined lattice constants:
a = 937.4(1) pm, c = 688.7(1) pm). The mass loss (total ca.
6.5%) takes place in two stages: stage 1 (80-200°C) l.Swt%,
and stage 2 (200-860 “C) 5wt %. In accord with DTA experiments on human dentine,[15]the second stage is associated with
an exothermic reaction (pyrolysis of the protein fibers).
Figure 6 shows the interference image of a just closed fluorapatite aggregate with a diameter of 125 pm, which was obtained
with an optical microscope with cross polarizers, as viewed along the optical axis of the hexagonal central seed. The
Fig. 6. Image from a polarization microscope (cross polarizers) of a just closed
fluorapatite aggregate of diameter 125 pm.Viewed along the hexagonal-prismatic
seed.
hexagonal cross section of the seed crystal can be seen in the
center, its diameter increases approximately twofold during the
course of the growth of the spherical aggregate. The interference
pattern of the total aggregate is characterized by the “Brewster
polarization cross” and the polarization colors within the cross
segments (increasing thickness of the spheres from the surface to
their centers, and variable inclination between the optical axes
of the individual crystallites and the incident polarized light).
We conclude by mentioning some possible interpretations of
the growth phenomenon described, although detailed investigations have either not been started or are not yet complete.[”- 16, 17]There are two basic possibilities, which, individually or taken together, could be responsible for the selfassembled control of the aggregate growth:
The aggregates correspond to organized composite systems
in which the growth of generations is controlled by an epitaxially linked composite of fluorapatite and the organic component.
The individual crystallites or composite units contain a permanent dipole, so that intrinsic electrical lines of force take
over control of the growth of the aggregates. The polarity of
collagen, the piezoelectric properties of calcified bone tissue,[’’] and the structural peculiarity of the apatite family,
fluctuating between centrosymmetric and acentric distribution of the X species (X = F, C1, OH),[’91support these ideas,
also bearing in mind the extremely mild biomimetic growth
conditions, which should favor a high degree of order of the
atomic arrangements, and the fact that partial exchange of
F- by OH- causes an asymmetric displacement of the two
sDecies from their normal positions.[201
In summary, a wide interdisciplinary area is emerging, which
encompasses chemistry, physics, materials science, and medicine.
2626
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Received: April 29, 1996 [Z90771E]
German version: Angew. Chem. 1996, 108, 2787-2791
Keywords: apatite
gelatin * solids
*
biomineralization
- fluorine compounds
*
[I] L. T. Kuhn, D. J. Fink, A. H. Heuer in Biomimetic Materials Chemistry (Ed.:
S. Mann), VCH, New York, 1996.
[2] H. Schmalzried, T. Frick, Ber. Bunsenges. Phys. Chem. 1995, Y9, 914.
[3] A. L. Boskey, J. Phys. Chem. 1989,93, 1628.
[4] Experimental Procedure: Pig skin gelatin (5.5 g, 300 Bloom, Aldrich) was
placed in water (50 mL), acidified with glutamic acid or 2N HCI to pH 2.5-3.5,
and then heated with stirring for a few minutes in a water bath. The central tube
of the diffusion cell (length 3 cm, diameter 2.5 cm, closed at one end) was filled
with the hot gelatin and left at room temperature for 24 h in a vertical position
until gelation occurred. The L-shaped tubes (vertical section: 18 cm, horizontal
section: 6 cm, standard ground joints) were then attached to the central gelatin
tube, filled with the aqueous solutions ( 0 . 1 3 3 ~CaCI, or 0 . 0 8 ~Na,HPO,/
0 . 0 2 7 ~KF), and closed with ground glass stoppers. Both solutions were previously adjusted to the physiological pH of 7.4 with cc,a,a-tris(hydroxymethyl)methylamine/HCI. The diffusion experiments were carried out at 25 “C
(thermostat). At the end of the experiment, the gelatin plug was pressed out of
the central tube and cut into sections corresponding to the Liesegang bands. To
isolate the solids, these sections were washed several times with hot water and
centrifuged.
[5] K. T. Wilke, J. Bohm, Krutullziichtung, Harri Deutsch, Frankfurt/Main, 1988.
[6] R. E. Liesegang, Z . Phys. Chem. 1914, 88, 1.
[7] K. Sudarsanan, R. A. Young, Acta Crystullogr. Sect. B 1978, 34, 1401.
[8] P. E. Mackie, R. A. Young, J. Appl. Crystullogr. 1973, 6, 26.
[9] The Science and Technology of Gelatin (Eds.: A. G. Ward, A. Courts), Academic Press, London, 1977.
[lo] W. Babel, Chem. Unserer Zeit, 1996, 30, 86.
[Ill B. B. Mandelbrot, Die,fraktale Geometrie der Natur, Birkhauser, Basel, 1991.
[I21 “Selbstahnliche Verzweigungen” und “Simulation von elektrischen Feldlinien
im Bereich selbstorganisierter polarer Aggregate”: H. Dolhaine, unpublished.
[13] D. A. Tomalia, A. M. Naylor, W. A. Goddard 111, Angew,. Chem. 1990, 102,
119; Angew. Chem. I n t . Ed. Engl. 1990,29, 138.
[14] Liberation of the constitutional water from hydroxyapatite above 1250 ”C:
P. W. Arnold, Trans. Faraday. Soc. 1950, 46, 1061.
1151 M. Okazaki, J. Takahashi, H. Kimura, J Osaka Univ.Dent. Srh. 1989,29,1989.
[I61 “Kristallfeldberechnungen an Apatiten”, J. Brickmann, S. Hauptmann, unpublished .
(171 “Messung von permanenten Dipolen an kleinen Aggregaten”, F. Laeri, unpublished.
[I81 S. Mann in Inorganic Materials, (Eds.: D. W. Bruce, D. O’Hare), Wiley,
Chichester, 1992.
[I91 M. Bauer, W. E. Klee, Z . Kristallogr. 1993, 206, 15.
[20] M. Braun. C. Jana, Chem. Phys. Lett. 1995, 245, 19.
Prototypical Reagent for the Synthesis
of Substituted Hydrazines**
Uno Maeorg, Leif Grehn, and Ulf Ragnarsson”
Dedicated to Professor Ivar Ugi
on the occasion of his 65th birthday
Substituted hydrazines are valuable precursors of heterocycles and components of many pharmaceuticals, agrochemicals, and dyestuffs. Direct alkylation of hydrazine normally
gives rise to mixtures of mono-, di-, and trisubstituted products
in which all the groups are located at the same nitrogen atoms.[’]
This often makes purification troublesome. Although many
[*] Dr. U. Ragnarsson, Dr. U. Maeorg, Dr. L. Grehn
Department of Biochemistry
University of Uppsala, Biomedical Center
P.O. Box 576, S-75123 Uppsala (Sweden)
Fax: Int code +I8552139
e-mail: urhki@bmc.uu.se
[**I We thank the Swedish Natural Science Research Council, the National Agency
for Higher Education, the National Board for Industrial and Technical Development, and Astra Draco AB for their generous support of our programs.
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