1st International Conference on Advancements of Medicine and Health Care through Technology, MediTech2007,
27-29th September, 2007, Cluj-Napoca, ROMANIA
Bone Matrices of Different Origins Studied by
FTIR Spectroscopy
R. Grecu, V. Coman, V. Avram, M. Băciuţ, and G. Băciuţ
Abstract — The bone matrix of deer antler, human bone and pig bone are compared in order to evidence the resemblance of their
composition and structure at the level evidenced by FTIR spectroscopy. The variation of the organic/inorganic matrix content in
bones of different origins was evaluated from the area of the characteristic absorption bands observed at ~1650 cm-1 (collagen) and
~1030 cm-1 (calcium phosphate). The higher content of organic matrix resulted for the deer antler and human skull. The
deproteination of the bone matrix by heat treatment is well evidenced by the FTIR spectra. The organic component is completely
removed by the thermal treatment of bone samples at temperatures higher than 500ºC. The spectra of these samples show also the
removal of carbonate ion incorporated into the inorganic matrix and the modification of the crystallinity of hydroxyapatite. Based on
3−
the splitting of ν 4 [PO4 ] band from the 500-700 cm-1 range of infrared spectrum a crystallinity index of hydroxyapatite was
assessed. The evolution of this index is a monotone increasing for deer antler and human skull. The values of cristallinity index for
the biogenic hydroxyapatite remain under that of the synthetic hydroxyapatite. The FTIR studies of the mentioned samples are useful
for the characterization of deer antler, a potential new biomaterial for the bone reconstruction.
Keywords: bone matrices, deer antler, FTIR spectroscopy, thermal treatment, crystallinity index.
(responsible for the synthesis of bone matrix), osteocytes
(involved in the maintenance of bone matrix) and
osteoclasts (bone destroying cells). In the case of the
antlers, all cells are dead by the time when they come to
be used. The high porosity is a necessary feature for a
faster resorption of a biomaterial with possible
applications in the bone defect reconstruction by
promoting osteoconduction (bone growth from the
existing bone by stimulation of osteoblasts to form new
bone). The SEM (scanning electron microscopy) study of
deer antler [4] evidenced larger pores in comparison with
those of pig bone.
1. INTRODUCTION
The obtaining of a higher quality biomaterial for bone
surgery as an alternative of the products on the market is
the focus of many studies. Deer antler has notable
regeneration properties (high growth speed and full
regeneration every year) that recommend it as a
promising biomaterial for the bone reconstruction.
The major constituents of bones are the organic matrix
consisting in 85 to 95% of fibrous collagen and the bone
mineral part represented by some varieties of calcium
phosphate [1-3]. The poorly crystalline hydroxyapatite is
predominant in the inorganic phase of the mature bones.
The crystals are impure, ~5-6% by weight are carbonate
substituted. The amount of water present in bone is an
important determinant of its mechanical behaviour. Other
constituents are non-collagenous proteins and
polysaccharides and, in many types of bone, living cells
and blood vessels. The bone cells are osteoblasts
Infrared spectroscopy evidences patterns both for the
organic and inorganic components of the bone samples
[5] and their evolution under the thermal treatment. In the
perspective of using the mature antler of Romanian deer
to obtain a new biomaterial with applications in the bone
reconstruction, we studied the composition and the
structure of deer antler, human and pig bones revealed by
FTIR (Fourier transform infrared) spectral method and
thermal analysis.
R. Grecu. Author is with the Analytical Chemistry Group, “Raluca
Ripan” Institute for Research in Chemistry, Cluj-Napoca, Romania,
phone:
+40-364-405-974;
fax:
+40-264-420-441;
e-mail:
[email protected]
V. Coman. Author is with the Analytical Chemistry Group, “Raluca
Ripan” Institute for Research in Chemistry, Cluj-Napoca, Romania,
phone:
+40-364-405-974;
fax:
+40-264-420-441;
e-mail:
[email protected]
V. Avram. Author is with the Analytical Chemistry Group, “Raluca
Ripan” Institute for Research in Chemistry, Cluj-Napoca, Romania,
phone:
+40-364-405-974;
fax:
+40-264-420-441;
e-mail:
[email protected]
M. Băciuţ. Author is with the Cranio-Maxilofacial Surgery, “Iuliu
Haţieganu” University of Medicine and Pharmacy of Cluj-Napoca,
phone/fax: +40-264-450-300; e-mail: [email protected]
G. Băciuţ. Author is with the Cranio-Maxilofacial Surgery, “Iuliu
Haţieganu” University of Medicine and Pharmacy of Cluj-Napoca ,
phone/fax: +40-264-450-300; e-mail: [email protected]
2. EXPERIMENTAL
The bone samples were prepared for FTIR investigation
following some stages: the removal of the excess of soft
tissue and marrow, the washing with physiological
solution and the drying at 40°C. After the grinding into a
fine powder the bones were degreased by washing with
acetone till the disappearance of the 1740 cm-1 band from
the infrared spectrum. The investigated samples were
antler tissue from the red deer (Cervus elaphus
carpaticus), human skull and pig bone. In the case of deer
antler, the studied samples consisted in a mixture of
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1st International Conference on Advancements of Medicine and Health Care through Technology, MediTech2007,
27-29th September, 2007, Cluj-Napoca, ROMANIA
cancellous and compact parts of the bone tissue,
excepting the case of a special mention. A commercial
sample of hydroxyapatite was used as reference material.
mode (resolved into well defined peaks at 601 and 561
cm-1). The bands of medium intensity observed between
1400-1500 cm-1 range as well as the band at 866 cm-1
confirm the carbonation of all bone samples.
FTIR spectra were recorded both on untreated and heattreated powdered samples compressed in KBr disks using
a Fourier transform infrared spectrometer JASCO 610.
Thermal treatment of powdered bones was done in air,
for 2 hours, in an electric furnace at 150, 600, 900 and
1200°C.
A quantitative evaluation of the relative content of the
organic matrix in bones of different origins was done
based on the ratio between area of the amide I band
centered at 1646 cm-1 and that of the complex band from
900-1200 cm-1 range assigned to calcium phosphate.
The differences between the composition of cancellous
and compact parts of the antler are well illustrated by the
FTIR spectra (Figure 2) and also by the thermal analysis
(Figures 3 and 4). In this case the ratio of
organic/inorganic matrix evidenced by the FTIR spectra
is clear greater for the sample prelevated from the
cancellous part of antler tissue.
The thermal analysis (TG, DTG, DTA) was performed
with a Derivatograph MOM OD 102 (Hungary). The
sample quantity was 200 mg, the rate of increase of oven
temperature 10°C min-1, and the sensitivity 100 mg TG,
1/5 DTG, 1/5 DTA. Measurements were conducted in air
in the temperature range of 30-1100°C.
3. RESULTS AND DISCUSSION
cancellous part
compact part
Abs
Vibrational spectroscopic methods (FTIR and Raman
spectroscopy [6,7]) are powerful methods for the
investigation of major components of bone samples, the
protein (type I collagen) and hydroxyapatite. From
infrared spectra of studied samples presented in Figure 1
one can note that deer antler, human skull and pig bone
have the same absorption bands with small differences in
their intensity.
1028
500
1500
2000
2500
3000
3500
4000
wavenumber (cm )
3420
1646
1239
1446
561
-1
Figure 2. FTIR spectra of two samples of deer antler
(three years old).
deer antler
Abs
866
1000
human skull
pig bone
500
1000
1500
2000
2500
3000
3500
4000
-1
wavenumber (cm )
Figure 1. FTIR spectra of bones of different origins:
deer antler, human skull and pig bone.
The main absorption bands from infrared spectrum have
been assigned [5] for deer antler. Thus, the strong band
centered around 3420 cm-1 is due to the water content of
bone samples. The bands characteristic to ν(CH)
vibrations of CH3 and CH2 groups from the 2800-3000
cm-1 range have a medium intensity and a low analytical
value. More intense are the bands assigned to amide
groups of organic matrix: 1646, 1535, 1239 cm-1. The
inorganic bone phase has PO4 structural units
characterized by the ν3 antisymmetric stretching (P-O)
vibrational mode (the very intense absorption band
observed at 1028 cm-1 and a shoulder around 1100 cm-1)
and the ν4 antisymmetric bending (OPO) vibrational
Figure 3. TG, DTG and DTA curves of a sample of
the compact part of deer antler (three years old).
430
866
1416
1446
561
601
1st International Conference on Advancements of Medicine and Health Care through Technology, MediTech2007,
27-29th September, 2007, Cluj-Napoca, ROMANIA
0
Abs
20 C
0
150 C
0
900 C
630
0
1200 C
500
1000
1500
2000
-1
wavenumber (cm )
871
Figura 4. TG, DTG and DTA curves of a sample of
the cancellous part of deer antler (three years old).
1414
1450
561
603
Figure 5. FTIR spectra of a deer antler sample thermally
treated at different temperatures.
Abs
The literature data indicate a maximum exothermal peak
for the type I collagen between 500-540°C depending on
the extraction method [8,9]. For deer antler tissue we
noticed three exothermal effects (See Figures 3, 4 and
Table 1) determined by the presence of a collagen with
different reticular degrees (maxima at 380°C and
~450°C). The third exothermal effect observed at ~560°C
is assigned to the free collagen from deer antler that is
unreticulated. The greater mass loss by these exothermal
effects is for the sample prelevated from the cancellous
part (44%) while for the compact one it is only 34%. The
endothermal effect observed at 100°C is due to the water
loss from the bone tissue.
0
20 C
0
150 C
0
900 C
632
0
1200 C
500
1000
1500
2000
-1
wavenumber (cm )
Figure 6. FTIR spectra of a human skull sample thermally
treated at different temperatures.
871
Total
Thermal Effect [°C] / Mass loss [%]
mass
Endo
Exo
loss [%]
I
II
III
Compact part
*100
380
440
565
**30-250 260-430 430-540 540-1000 30-1000
10.0
23.0
7.0
4.0
44.0
Cancellous part
*100
380
460
540
**30-240 240-440 440-520 520-1000 30-1000
10.0
28.0
10.0
6.0
54.0
1416
1448
561
603
Table 1. Thermal analysis results for the compact and
cancellous parts of deer antler (three years old)
0
Abs
20 C
0
150 C
0
900 C
632
0
1200 C
500
1000
1500
2000
-1
wavenumber (cm )
*maximum and **range of exothermal effects respectively
Figure 7. FTIR spectra of a pig bone sample thermally
treated at different temperatures.
The amount of organic component in samples of different
origins decreases in the order human skull, deer antler,
pig bone. The thermal treatment of powdered bones at
150°C produces the diminishing of the organic
component. This process is illustrated for the deer antler,
human skull and pig bone in Figures 5-7. One can notice
also the reduction of the intensity of 1239 cm-1 band
(amide III) with the increase of temperature.
At 600ºC the deproteination of bones is finished and the
spectra of samples treated at higher temperatures put in
evidence only the hydroxyapatite from the inorganic
matrix. The absorption bands characteristic to carbonate
ion are still present in spectra of samples treated at 900ºC.
From the infrared spectra we could not establish the
nature of carbonate substitution site: hydroxy or
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1st International Conference on Advancements of Medicine and Health Care through Technology, MediTech2007,
27-29th September, 2007, Cluj-Napoca, ROMANIA
phosphate. However, we can assume that the finally step
of carbonate loss at temperatures higher than 900ºC is
from the hydroxy site if we have in view the
modifications from the spectra of samples treated at
1200°C. At this temperature the bone samples consist of
more crystalline carbonate free hydroxyapatite without
any organic components.
temperature is the modification of hydroxyapatite
crystallinity assessed by the index determined from the
FTIR spectra.
In the spectra of samples treated at 1200°C the maxima at
3570 cm-1 assigned to non-hydrogen bonded OH groups
and 630 cm-1 assigned to the OH libration are well
evidenced. These two bands are also an indicative of a
higher crystallinity of hydroxyapatite after the thermal
treatment. In the less crystallized hydroxyapatite the band
assigned to OH libration appears as a shoulder on the ν4
phosphate band or just only as a broadening of this band
in the spectrum of bone sample.
5. ACKNOWLEDGMENTS
These studies are useful to understand the properties of
some new biomaterials with possible applications in bone
reconstruction.
The results presented in this paper belong to the scientific
research project according to the Contract CEEX no. 73 /
2006-2008, VIASAN Programme, financially supported
by the Romanian Excellence Research Programme.
6. REFERENCES
[1] Băciuţ G. and Băciuţ M., Reconstrucţia defectelor osoase
maxilofaciale (Reconstruction of maxilofacial bone defects),
Sincron Publishing House, Cluj-Napoca, 1998.
[2] Băciuţ M., Hedeşiu M., Bran S., Băciuţ G., Dinu C., Rotaru
H., Mitre I., Câmpian R.S. and Balog C., Implantarea orală în
condiţii speciale de suport osos alveolar deficitar (Oral implant
in special conditions of bone support with alveolus deficit),
Clujul Medical, vol. LXXIX Supplement, pp. 80-88, 2006.
[3] Bran S., Băciuţ M., Băciuţ G., Hurubeanu L., Rotaru Al.,
Câmpian R.S. and Dinu C., Reconstruction of craniofacial bone
defects with alloplastic materials, European Cells and
Materials, Supplement. 1, vol. 9, pp.17-19, 2002.
[4] Băciuţ M., Băciuţ G., Simon V., Albon C., Coman V.,
Prodan P., Florian Şt.I. and Bran S., Investigation of deer antler
as a potential bone regenerating biomaterial, Journal of
Optoelectronics and Advanced Materials, 2007, in press.
[5] Coman V., Grecu R., Băciuţ M., Băciuţ G., Prodan P. and
Simon V., Investigation of some different bone matrices by
vibrational spectroscopy, Journal of Optoelectronics and
Advanced Materials, 2007, in press.
[6] Parker F.S., Applications of infrared spectroscopy in
biochemistry, biology and medicine, Adam Hilger Ltd London,
Plenum Press, New York, pp. 494-497, 1971.
[7] Smith R. and Rehman I., Fourier transform Raman
spectroscopic studies of human bone, Journal of Materials
Science: Materials in Medicine, vol. 5, pp. 775-778, 1994.
[8] Lozano L.F., Peña-Rico M.A., Heredia A., Ocotlán-Flores
J., Gómez-Cortés A., Velázquez R., Belío I.A. and Bucio L.,
Thermal analysis study of human bone, Journal of Materials
Science, vol. 38, pp. 4777-4782, 2003.
[9] Utech M., Vuono D., De Luca P. and Nastro A., Correlation
of physical-chemical properties of healthy and pathologic
human bones, Journal of Thermal Analysis and Calorimetry,
vol. 80, pp. 435-438, 2005.
[10] Termine J.D. and Posner A.S., Infrared analysis of rat
bone: age dependency of amorphous and crystalline mineral
fractions, Science (Wash. D.C.), vol. 153, pp. 1523-1525, 1966.
[11] Blumenthal N.C., Posner A.S. and Holmes J.M., Effect of
preparation conditions on the properties and transformation of
amorphous calcium phosphate, Materials Research Bulletin,
vol. 7, pp. 1181-1190, 1972.
Termine and Posner [10,11] proposed a method that
correlates the size change of hydroxyapatite crystals from
bones with the splitting of the phosphate ν4 band
observed in the 500-700 cm-1 region of the spectra. The
evolution with temperature of the crystallinity index
calculated using the Termine’s method is illustrated in
Figure 8.
Crystallinity index
5.0
deer antler
human skull
pig bone
4.5
4.0
3.5
3.0
2.5
0
200
400
600
800
1000
1200
0
Temperature ( C)
Figure 8. The cristallinity index of studied samples.
The effect of temperature on the crystallization of
hydroxyapatite from the deer antler and human skull is
more reduced comparatively to the pig bone. We mention
the value of 5.71 for the crystallinity index calculated in
the case of a commercial hydroxyapatite sample heated at
1200ºC.
4. CONCLUSIONS
FTIR spectroscopy put in evidence the main components
of studied bones, namely the organic matrix (collagen)
and the mineral component (carbonate substituted
hydroxyapatite).
The effect of thermal treatment is the complete
deproteination of bones at temperatures around 600ºC
and the removal of carbonate ion from samples treated
over 900ºC. Another effect of this treatment at high
432