1 Prof. Carla Vogt Field of teaching Analytical Chemistry Callinstr. 1, 30167 Hannover Offical location: building 2511 / room 214 Dr. Drazen Tadic Tel.: +49-511-762-19312 Managing Partner Fax: +49-511-762-2923 botiss dental GmbH e-mail: [email protected]Knesebeckstraße 59-61 10719 Berlin Hannover, 11 th of November 2011 Analyses of bone graft materials
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Prof. Carla Vogt
Field of teaching Analytical Chemistry Callinstr. 1, 30167 Hannover Offical location: building 2511 / room 214
1. Introduction The following report describes the examination and comparison of the bovine bone graft Bio-Oss
(Geistlich) and the botiss bone graft materials cerabone®, maxresorb® and maxgraft® by different
analytical methods. Specifications of the materials are given in table 1.
Table 1: Specifications of the examined bone graft materials
Bone graft
manufacturer Particle size/ mm material
Bio-Oss® Geistlich 0,25 - 1,0 Bovine, pure mineral bone pahse Maxresorb® botiss 0,5 - 1,0 Synthetic, 60% HA, 40% ß-TCP Cerabone® botiss 0,5 - 1,0 Bovine, pure mineral bone phase Maxgraft® botiss 0,5 - 2,0 Human bone from living donors
2. Scanning electron microscopy (SEM) Samples were sputtered with gold to increase the conductivity of the particles, thus reducing charging
effects. Varying images with different magnifications were taken and particle sizes were measured.
Figures 2.1 to 2.3 show particles of Bio-Oss®, cerabone® and maxresorb®. The particle sizes were
measured along the longest edge. While some particles bigger than 1 cm were found in all samples, in
particular the Bio-Oss® sample contained many particles smaller than the given range (0.25 – 1.0 mm).
Overviews of several particles as well as a single particle each are displayed in figure 2.4. In a higher
magnification (200x, 500x and 1000x) the specific surface structure of the particles is visible (see fig. 2.5
and 2.6). Structure and surface of the cerabone® particles are comparable to Bio-Oss®, while the
synthetic maxresorb® particles exhibit a different structure with a very rough surface.
Figure 1: SEM image of Bio-Oss® particles, 20-fold magnification, particle sizes measures along the longest edge are indicated.
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Figure 2: SEM image of maxresorb® particles, 22-fold magnification, particle sizes measures along the longest edge are indicated.
Figure 4: SEM image of cerabone® particles5, 21-fold magnification, particle sizes measures along the longest edge are indicated.
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magnification
overview (20-22x) single particles (75-85x)
Bio-Oss®
maxresorb®
cerabone®
Figure 4: SEM images of Bio-Oss®, cerabone® and maxresorb® showing an overview and a single particle at a higher magnification each
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magnification
200x 500x
Bio-Oss®
maxresorb®
cerabone®
Figure 5: SEM images of Bio-Oss®, maxresorb® and cerabone®; at a higher magnification of 200x and 500x respectively details of the particle surface are visible, particles demonstrate a rather rough than smooth surface
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magnification
1000x
Bio-Oss®
maxresorb®
cerabone®
Figure 2: SEM images of Bio-Oss®, maxresorb® and cerabone® at 1000-fold magnification showing the micro structure of the particle surface.
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3. Determination of Ca and P contents via ICP-OES
To determine the calcium and phosphor content two samples of each material were dissolved without
any residue by microwave digestion and diluted in ultra-pure water. The calibration was carried out using
five standard solutions containing calcium and phosphor in different known
From the measurements of each spectral line the calcium and phosphor contents were determined by
the help of the calibration line and the corresponding confidence intervals were calculated to DIN 38 402
part 51 (level of significance α = 0.05). Mean values were calculated from two spectral lines each, they
are indicated in table 2
Table 2: Calcium and phosphate contents of the samples
Sample Calcium Phosphor
Ca/P mass ratio
Ca/P molar ratio
Ca content % Cofidence interval %
P content % Confidence interval %
Bio-Oss® 36.39 36.64
± 0,75 ± 0,72
16.54 16.54
± 0,29 ± 0,28
2.20 2.22
1.70 1.71
cerabone® 39.22 39.16
± 0,73 ± 0,73
18.56 18.59
± 0,28 ± 0,28
2.11 2.11
1.63 1.63
maxresorb® 38.95 39.12
± 0,74 ± 0,73
18.24 18.21
± 0,29 ± 0,28
2.14 2.15
1.65 1.66
In addition, the Ca/P mass ratio of the samples is denoted. Pure hydroxyapatite Ca10(PO4)6(OH)2 has a
Ca/P ratio of 2.157 as well as a mass fraction of calcium and phosphor of 39.89% and 18.50%
respectively. These mass fractions correspond much closely to the Ca and P contents of the synthetic
material maxresorb. In contrast, the Ca/P mass ratio of cerabone® most closely corresponds to
hydroxyapatite.
It is noticeable that Bio-Oss® exhibits the lowest calcium and phosphor contents but the highest Ca/P
mass ratio. This might be caused by calcium carbonate contained in Bio-Oss®, but not in cerabone® and
maxresorb® (comp. results of IR spectroscopy, chapter 4).
4. IR spectroscopy
The IR spectroscopic measurements were carried out on kBr-Presslingen with a FT-IR spectrometer.
Spectra were measured in a wavelength range of 400-4000 cm -1 with a resolution of 2 cm-2. For each
measurement 100 scans were summed up.
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IR spectra of all materials are shown in figures 4.1 to 4.3.
All spectra exhibit several phosphate bands in a range between 570-605 cm-1 and 970-1100 cm-1 (figure
4.1). Likewise, a broad absorption band in the range of ~3500 cm-1, resulting from the O-H-bond of the
water, is visible in all spectra. Spectra of samples from cerabone® and maxresorb® show an additional
band at around 3575 cm-1 that is caused by the O-H bonds of hydroxyapatite (fig. 4.1). Another O-H band
of the water is visible at about 1650 cm -1.
A comparison of the spectra reveals additional bands at around 1460 cm-1, 1420 cm-1 and 880 cm-1 for
Bio-Oss® and maxgraft® (figure 4.2) that result from the vibration of CO32-.maxgraft®, the only sample of
human origin, shows two more bands at 1550 cm-1 and 1245 cm-1 (Fig. 4.2). These can be explained by
the C-H and N-H bonds of the contained collagen.
Figure 4.1: Comparison of the IR-spectra of Bio-Oss®, maxresorb® and cerabone®.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
400100016002200280034004000
tran
smis
sio
n
wave number/ cm-1
Crystallinity fo Bio-Oss®, cerabone® and maxresorb®
Bio-Oss
maxresorb
cerabone
O-H
O-H
P-O
P-O
C-O
O-H
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Figure 4.3.: Comparison of the IR-spectra of the human bone graft maxgraft® with the bovine bone grafts Bio-Oss® and cerabone®.
5. X-ray powder diffractometry
The x-ray powder diffractometry was accomplished to gain information about the chemical composition
(mineral phases) and crystallinity of the materials. For the measurements all bone grafts were grinded
into powder and fixed on a carrier.
Diffractograms of all four graft materials are presented in figures 5.1 to 5.4. Markings in the figures
indicate the reference patterns of hydroxyapatite (green dots) and tricalcium phosphate (yellow
triangles). Literature data from the data bank of the International Centre for Diffraction Data (ICDD) were
used for the mapping of peaks.
0,0
0,2
0,4
0,6
0,8
1,0
1,2
400900140019002400290034003900
Crystallinity of Bio-Oss®, cerabone® and maxgraft®
Bio-Oss
cerabone
maxgraft
O-H
P-O
P-O
C-O
O-H C-O
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Figure 5.1: X-ray powder diffractometry of Bio-Oss®. Green dots indicate reflexes correlating to the literature data of the diffraction angles of hydroxyapatite.
Figure 5.2: X-ray powder diffractometry of maxresorb®. Green dots and yellow triangles indicate reflexes correlating to the literature data of the diffraction angles of hydroxyapatite and tricalcium phosphate respectively.
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50
100
150
200
250
300
350
400
5 15 25 35 45 55
Inte
nsi
ty /
cts
Diffraction angle/ °
Bio-Oss®
hydroxy apatite reflexes (literature data)
0
50
100
150
200
250
300
5 15 25 35 45 55
Inte
nsi
ty /
cts
diffraction angle / °
maxresorb®
hydroxy apatiet reflexes (literaturedata)
tricalcium phosphate reflexes(literature data)
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Figure 5.3: X-ray powder diffractometry of cerbone®. Green dots indicate reflexes correlating to the literature data of the diffraction angles of hydroxyapatite.
Figure 5.4: X-ray powder diffractometry of maxgraft®. Green dots indicate reflexes correlating to the literature data of the diffraction angles of hydroxyapatite.
0
20
40
60
80
100
120
140
160
5 15 25 35 45 55
Inte
nsi
ty /
cts
diffraction angle/ °
cerabone®
Hydroxyapatite reflexes (literature data)
0
20
40
60
80
100
120
140
5 15 25 35 45 55
inte
nsi
ty /
cts
diffraction angle / °
maxgraft®
hydroxylapatite reflexes (literaturedata)
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As expected, hydroxyapatite peaks could be assigned in all diffractograms. Diffractograms of maxresorb®
demonstrate additional reflexes belonging to the mineral phase β-tricalcium phosphate (Ca3(PO4)2),
because this material is a biphasic composition of 60% hydroxyapatite and 40% β-tricalcium phosphate.
A conspicuous fact is the varying width of the peaks as well as the height of the baseline. Cerabone® and
maxresorb® exhibit the narrow peaks and a low baseline, indicating a high crystallinity as well as few
components disturbing the measurement. This is obvious for the synthetic bone graft maxresorb®. For
cerabone® it can be explained by the specific production process. The bovine bone is treated several
hours in a high temperature process with temperatures up to 1200°C resulting in the production of
highly crystalline hydroxyapatite. By comparison, the diffractogram of Bio-Oss® exhibits broader peaks
suggesting a lower crystallinity. A comparison with results from the IR spectroscopy provides further
explanation; in contrast to samples cerabone® and maxresorb®, Bio-Oss® as well as maxgraft® contains
carbonate. The integration of carbonate in the crystal lattice of hydroxyapatite could broaden peaks in
the x-ray powder diffractometry (figure 5.1 and 5.4). As expected, the diffractogram of maxgraft® shows
the broadest peaks and a high baseline indicating a low crystallinity and amorphous structures.
Maxgraft® is derived from human donor bone and composed of mineralized collagen fibres.
6. Thermogravymetry
The thermogravimetric analysis (TGA) was carried out on four graft materials under air atmosphere and
from room temperature up to 1000°C with a heating rate of 5°C/min.
Table 6.1 presents the residual mass of the samples given in percentages.
Table 3: Residual masses following thermogravimetric analysis
sample Residual mass in %
Bio-Oss® 92,64 maxresorb® 98,61
cerabone® 99,52
maxgraft® 61,48
Mass reduction of Bio-Oss® following heating has two clear phases, with a reduction of 3.3% and 4%
respectively. The first reduction can be attributed to the vaporization of water, the second to
vaporization of carbon dioxide.
Mass reduction of maxresorb® and cerabone® is very similar with residual masses between 98.6% and
99.5%. Compared to samples 1 and 2, the reduction has only one phase and is much lower indicating a
lower water content and only marginal formation of carbon dioxide. These results are in accordance with
the results of the IR spectroscopy stating that Bio-Oss® and maxgraft®, but not maxresorb® and
cerabone® contain a recognizable proportion of carbonate.
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8. Density determination
Density of the seven samples was determined by displacement of helium with a pycnometer.
Additionally, a distinct mass of each probe was weighed in a measuring cylinder and filled with ultra-pure
water. The volume of the displaced water could be calculated from the weighed sample mass and the
volume read from the cylinder.
The weighed mass, measured volumes and calculated densities of each sample are given in table 8.1.
Table 4: Measuring values and results of the density determination.