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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: c.vogt@acc.uni-hannover.de

Knesebeckstraße 59-61

10719 Berlin Hannover, 11th of November 2011

Analyses of bone graft materials

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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)

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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.

sample

mass / g volume / cm³

standard deviation of

volume / cm³

density1/

g cm-³

stanard deviation

of density/ g cm-³

density2/

g cm-³

Bio-Oss® 0,4773 0,1545 ± 0,0001 3,0888 ± 0,0015 1,77 maxresorb® 0,3792 0,1128 ± 0,0001 3,3613 ± 0,0016 2,64 cerabone® 0,7780 0,2467 ± 0,0001 3,1540 ± 0,0009 1,42 maxgraft® 0,3123 0,1424 ± 0,0001 2,1931 ± 0,0019 0,56

1: density obtained by displacement of helium

2: density obtained by displacement in water (values only give a rough estimation, because of measuring errors.

These results show that the synthetic material maxresorb® possesses the highest densities with 3.36

g*cm-3 and 3.49 g*cm-3 respectively. Densities of Bio-Oss® and cerabone® are settled in a similar range,

between 3.09 to 3.21 g*cm-3. Maxgraft® exhibits a much lower density of 2.19 g*cm-3.

Pure hydroxyapatite has a density of 3.16 g*cm-3 corresponding most closely to the density of

cerabone®.

Densities measured by the displacement of water are also presented in table 4. These values can only

give a general idea of the real densities, because the volumes of the displaced water cannot exactly be

read on the cylinder. Furthermore, the values could be affected by the adsorption of air bubbles to the

particle surface.

Density values measured by displacement of water are markedly lower, because helium can reach the

pores more easily than water. Nevertheless, measured values of both methods match in that

maxresorb® shows the largest density and maxgraf®t by far the lowest density.

9. X-ray fluorescence analysis (RFA) The contents of further elements of Bio-Oss®, cerabone® and maxresorb® were determined by micro-x-

ray fluorescence analysis (µ-RFA).

Beside calcium and phosphor the elements magnesium, sulfur, sodium and strontium were detected in

all samples. Other elements could not be detected without doubt, because of the very small peaks.

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The micro-x-ray fluorescence analysis is unsuitable for measurements at trace level. Furthermore,

elements with atomic numbers <11 cannot be detected. Likewise, heavy elements such as lead or

mercury cannot be detected because of the insufficient excitation energy of the x-rays.

10. Experiments with bovine blood

Experiments with bovine blood were performed to simulate the behavior of the bone grafts under

naturel conditions, especially to determine the wettability of the particle surface with blood. Therefore,

fresh blood was dripped from a buret onto the samples placed in plastic dishes and an applicator was

used to mingle the particles. The whole experiment was recorded with a video camera.

Following, single shots from the recordings of the experiments are shown. Samples can be grouped by

their wettability. Bio-Oss® and maxgraft® exhibit a poor wettability. Blood droplets that contact the

particles firstly remain their shape (see fig. 10.1 to 10.3, left side). Even after mixing with the applicator a

poor penetration of particles with blood was observed. Only areas of the particles with direct blood

contact were colored red.

Figure 10.1: Determination of the wettability of Bio-Oss® with bovine blood

Figure 10.2: Determination of the wettability of maxgraft® with bovine blood

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Recordings of maxresorb® and cerabone® demonstrate another behavior following blood contact.

Directly after the contact the blood penetrated the pores of the particles and moistened them. Bigger

particles were completely colored too, even if they only had direct blood contact with one side, showing

that the particles were soaked with blood. Altogether, the wettability of these samples is considerably

more pronounced than that of the Bio-Oss® and maxgraft®.

Figure 10.3: Determination of the wettability of maxresorb® with bovine blood.

Figure 10.4: Determination of the wettability of cerabone® with bovine blood