The thermal behavior of bone tissue has been investigated in order to assess how it is affected struc-turally by incineration. The need of the development of such technology is caused by big number of pa-tients with oncology and dystrophic bone tissue diseases which use the artificial bone tissue replacement. The samples like small plates that cut from local area of bone fragment, were exposed to annealing in tem-perature range 560-720 C and were characterized by XRD, SEM, EPMA, XPS and method of differential dissolution.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3547

Danilchenko, S.N.

Kalinichenko, T.G.

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 2 No 4, 04NABM21(2pp) (2013)   2304-1862/2013/2(4)04NABM21(2) 04NABM21-1  2013 Sumy State University Nanoscale Apatite Biomaterials for Osteointegration  T.G. Kalinichenko*, S.N. Danilchenko†  Institute of Applied Physics NAS of Ukraine, 58, Petropavlovskaya Str., 40000 Sumy, Ukraine  (Received 11 June 2013; published online 03 September 2013)  The thermal behavior of bone tissue has been investigated in order to assess how it is affected struc-turally by incineration. The need of the development of such technology is caused by big number of pa-tients with oncology and dystrophic bone tissue diseases which use the artificial bone tissue replacement. The samples like small plates that cut from local area of bone fragment, were exposed to annealing in tem-perature range 560-720 C and were characterized by XRD, SEM, EPMA, XPS and method of differential dissolution.  Keywords: Bone Tissue, Thermal Decomposition, SEM, EPMA, XRD, XPS, Differential Dissolution.    PACS numbers: 61.46. – w, 65.80.+ n, 73.22. – f                                                           * t_kalinichenko@yahoo.com † danil50@hotbox.ru 1. INTRODUCTION  Nanoscale crystals of biogenic apatite from bone tis-sue have complicated structure that due to their func-tionality. The synthesis such structure in artificial materials for osteoplastics is impossible. This circum-stances is stimulated the investigation of natural bone tissue apatite as source of biomaterials. The organic component of donor bone tissue may be removed by pyrolysis or chemically treatment or by their combina-tion for the absence of immune reaction. For today the problem of optimization of treatment process for full organic components removement at minimal changes of biomineral structures is very important. Nanoscale bioapatite crystals with saved natural properties could be the basic material for development of artificial min-eral organic composites for osteointegration. The need of the development of such technology is caused by big number of patients with oncology and dystrophic bone tissue diseases which use the artificial bone tissue replacement.  In this work the thermal behavior of bone tissue has been investigated in order to assess how it is af-fected structurally by incineration. As a result, new technology for the production of nanostructured materials based on natural bioapatite were developed.  2. MATERIALS AND METHODS  The objects of investigation were bioapatite samples obtained from cortical (dense) bone from different ani-mals. During preliminary preparation, the samples were mechanically cleaned, washed in distilled water and dried in air. In order to obtain temperature series, the samples (in the form of small plates cut from one local region of the bone fragment) were annealed in temperature range corresponding to recrystalization of apatite (560 C-720 C) in 40 C steps. At each fixed tem-perature, the samples were held for 1h and then were slowly cooled down to room temperature. The samples were characterized using physical approaches that available as a result of conjunction the next methods: XRD, SEM, EPMA, XPS and differential dissolution. 3. RESULTS AND DISCUSSION  According to the literature [1] bone annealing in-cludes the following processes:  a) ~ 120 C - the loss of surface-bound water;  b) 400-500 C - the loss of crystal water. This loss is accompanied by a decrease of the lattice parameter a ~ 0.02 Å for Na-bearing carbonate apatite, and a ~ 0.003 Å - in the absence of ions Na; c) ~ 600 C - the loss of water and ions HPO42-;  d) for a well-crystallized carbonate apatite CO2 emissions starting at 550 C, and usually ends at 900 ... 1000 C;  e) if the molar ratio of Ca/P  1.667, the β-TCP is al-so presents.  f) In biological apatite carbonate CO2 loss can occur at lower temperatures than in well crystallized apatite. Bone extracellular matrix has a complex multi-level organization of the uneven distribution of the micro and macro elements in the osteon-lamellar structures [1, 3] (see Fig. 1).    Fig. 1 – SEM image of bovine bone tissue osteon-lamellar structure.  Initially composed of intergrown collagen and hy-droxyapatite (HAP), combustion of the organic compo-nent is complete by 560 C, with most mass loss (50-55 ) occurring by 500 C. Combustion of collagen is accompanied by an increase in HAP mean crystallite  T.G. KALINICHENKO, S.N. DANILCHENKO PROC. NAP 2, 04NABM21 (2013)   04NABM21-2 size at temperatures greater than 600 C, from 28 nm to a constant value of 190 nm at 700 C [2]. The tem-perature range of 600-700 C is characterized by signif-icant structural and micro-structural rearrangements of bioapatite. Energy Dispersive X-ray Analysis (EDX) along and across the osteon of bone tissue samples and EDX com-positional mapping were perfomed.  It was found that Mg in biopatite can be both on the surface of the nanocrystals and embedded in-crystal lattice, replacing calcium. That is in good agreement with literature data [4, 5]. It has been found that the source of systematic er-ror may be an option of a reference sample. Surface chemical composition was studied by XPS technique. The XPS measurements indicate that the samples have lines typical for phosphorous, carbon, calcium, oxygen, sodium and magnesium. Intensities of photoelectron lines from other elements were on the noise level and within the bounds of XPS method sensi-tivity are not detected. The survey photoelectron spec-trums of investigated samples are represented on Fig. 2.    Fig. 2 – XPS survey spectrums from annealed at different temperatures (560 C, 600 C, 720 C) bone samples. Analysis of recorded with high resolution lines of calcium - BE(Са2p)=347.3 eV and phosphorous - BE(P2p)=133.3 eV indicate that calcium is in state of Ca2+ and phosphorous is in state of PO4-, that cor-responds to the next chemical compounds, for exam-ple, calcium phosphat Ca3(PO4)2 (CaP) or hydroxy-apatite Ca10(PO4)6(OH)2 [6, 7]. According to differential dissolution analysis the composition of main phase of bone tissue can be fragmentary represented (without oxygen and hy-drogen) by the formula:    Ca1.66Mg0.054Sr0.001Na0.06P1.00    The content of the phase in the sample is 99.9%. Sample also contains small amounts of phase - prob-ably phosphates - Mg, Sr, Na and K. Ratios of these elements in the small phases are Mg 1.4 rel.%, Sr 4.4 rel.%, Na 3.3 rel.% - from the total in the sample content of each of these elements. These elements are almost completely covered in the main phase. At the same time in the main phase is not potassium, which either forms its own phase or concentrated in the surface layers of the main phase. The received results accomplish a modern per-formances about ultrastructural organization of nanoscaled apatite of bone tissue biominerals.  AKNOWLEDGEMENTS  Authors are grateful to the Dr. A. Sorokin and Dr. I. Prosvirin from Boreskoff Institute of catalysis SD RAS, Novosibirsk, Russia for XPS measurements and his valuable comments. Also authors thank to Dr. V. Malakhov from Boreskoff Institute of catalysis SD RAS, Novosibirsk, Russia for application of the differential dissolution analysis to native bone.   REFERENCES  1. J.C. Elliott, Structure and Chemistry of the Apatites and Other Calcium Orthophosphates. Studies in Inorganic Chemistry (Amsterdam. Elsevier. 1994). 2. F. Betts, N.C. Blumenthal, A.S. Posner, J.Cryst.Growth 53, 63 (1981). 3. S.N. Danilchenko, A.V. Koropov, I.Yu. Protsenko et al., Cryst. Res. Technol. 41 No3, 268 (2006). 4. A. Bigi, E. Foresti, R. Gregorini, Calcif. Tissue Int. 50, 439 (1992). 5. A. Bigi, G. Falini, E. Foresti, Acta Cryst. 52, 87 (1996). 6. C.C. Chusuei, D.W. Goodman, M.J. Van Stipdonk, D.R. Justers, Schweikert, Anal. Chem. 71, 149 (1999). 7. S. Kaciulis, G. Mattogno, L. Pandolfi, L. Cavalli, G. Gnappi, Appl. Surf. Sci. 151, 1 (1999).  

Nanoscale Apatite Biomaterials for Osteointegration

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 2 No 4, 04NABM21(2pp) (2013) 
 
 
2304-1862/2013/2(4)04NABM21(2) 04NABM21-1  2013 Sumy State University 
Nanoscale Apatite Biomaterials for Osteointegration 
 
T.G. Kalinichenko*, S.N. Danilchenko† 
 
Institute of Applied Physics NAS of Ukraine, 58, Petropavlovskaya Str., 40000 Sumy, Ukraine 
 
(Received 11 June 2013; published online 03 September 2013) 
 
The thermal behavior of bone tissue has been investigated in order to assess how it is affected struc-
turally by incineration. The need of the development of such technology is caused by big number of pa-
tients with oncology and dystrophic bone tissue diseases which use the artificial bone tissue replacement. 
The samples like small plates that cut from local area of bone fragment, were exposed to annealing in tem-
perature range 560-720 C and were characterized by XRD, SEM, EPMA, XPS and method of differential 
dissolution. 
 
Keywords: Bone Tissue, Thermal Decomposition, SEM, EPMA, XRD, XPS, Differential Dissolution. 
 
  PACS numbers: 61.46. – w, 65.80.+ n, 73.22. – f 
 
 
                                                        
* t_kalinichenko@yahoo.com 
† danil50@hotbox.ru 
1. INTRODUCTION 
 
Nanoscale crystals of biogenic apatite from bone tis-
sue have complicated structure that due to their func-
tionality. The synthesis such structure in artificial 
materials for osteoplastics is impossible. This circum-
stances is stimulated the investigation of natural bone 
tissue apatite as source of biomaterials. The organic 
component of donor bone tissue may be removed by 
pyrolysis or chemically treatment or by their combina-
tion for the absence of immune reaction. For today the 
problem of optimization of treatment process for full 
organic components removement at minimal changes of 
biomineral structures is very important. Nanoscale 
bioapatite crystals with saved natural properties could 
be the basic material for development of artificial min-
eral organic composites for osteointegration. 
The need of the development of such technology is 
caused by big number of patients with oncology and 
dystrophic bone tissue diseases which use the artificial 
bone tissue replacement.  
In this work the thermal behavior of bone tissue 
has been investigated in order to assess how it is af-
fected structurally by incineration. 
As a result, new technology for the production of 
nanostructured materials based on natural bioapatite 
were developed. 
 
2. MATERIALS AND METHODS 
 
The objects of investigation were bioapatite samples 
obtained from cortical (dense) bone from different ani-
mals. During preliminary preparation, the samples 
were mechanically cleaned, washed in distilled water 
and dried in air. In order to obtain temperature series, 
the samples (in the form of small plates cut from one 
local region of the bone fragment) were annealed in 
temperature range corresponding to recrystalization of 
apatite (560 C-720 C) in 40 C steps. At each fixed tem-
perature, the samples were held for 1h and then were 
slowly cooled down to room temperature. The samples 
were characterized using physical approaches that 
available as a result of conjunction the next methods: 
XRD, SEM, EPMA, XPS and differential dissolution. 
3. RESULTS AND DISCUSSION 
 
According to the literature [1] bone annealing in-
cludes the following processes:  
a) ~ 120 C - the loss of surface-bound water;  
b) 400-500 C - the loss of crystal water. This loss is 
accompanied by a decrease of the lattice parameter a ~ 
0.02 Å for Na-bearing carbonate apatite, and a ~ 0.003 
Å - in the absence of ions Na; 
c) ~ 600 C - the loss of water and ions HPO42-;  
d) for a well-crystallized carbonate apatite CO2 
emissions starting at 550 C, and usually ends at 900 ... 
1000 C;  
e) if the molar ratio of Ca/P  1.667, the β-TCP is al-
so presents.  
f) In biological apatite carbonate CO2 loss can occur 
at lower temperatures than in well crystallized apatite. 
Bone extracellular matrix has a complex multi-level 
organization of the uneven distribution of the micro 
and macro elements in the osteon-lamellar structures 
[1, 3] (see Fig. 1). 
 
 
 
Fig. 1 – SEM image of bovine bone tissue osteon-lamellar 
structure. 
 
Initially composed of intergrown collagen and hy-
droxyapatite (HAP), combustion of the organic compo-
nent is complete by 560 C, with most mass loss (50-
55 ) occurring by 500 C. Combustion of collagen is 
accompanied by an increase in HAP mean crystallite 
 T.G. KALINICHENKO, S.N. DANILCHENKO PROC. NAP 2, 04NABM21 (2013) 
 
 
04NABM21-2 
size at temperatures greater than 600 C, from 28 nm 
to a constant value of 190 nm at 700 C [2]. The tem-
perature range of 600-700 C is characterized by signif-
icant structural and micro-structural rearrangements 
of bioapatite. 
Energy Dispersive X-ray Analysis (EDX) along and 
across the osteon of bone tissue samples and EDX com-
positional mapping were perfomed.  
It was found that Mg in biopatite can be both on the 
surface of the nanocrystals and embedded in-crystal 
lattice, replacing calcium. That is in good agreement 
with literature data [4, 5]. 
It has been found that the source of systematic er-
ror may be an option of a reference sample. 
Surface chemical composition was studied by XPS 
technique. The XPS measurements indicate that the 
samples have lines typical for phosphorous, carbon, 
calcium, oxygen, sodium and magnesium. Intensities of 
photoelectron lines from other elements were on the 
noise level and within the bounds of XPS method sensi-
tivity are not detected. The survey photoelectron spec-
trums of investigated samples are represented on 
Fig. 2. 
 
 
 
Fig. 2 – XPS survey spectrums from annealed at different 
temperatures (560 C, 600 C, 720 C) bone samples. 
Analysis of recorded with high resolution lines of 
calcium - BE(Са2p)=347.3 eV and phosphorous - 
BE(P2p)=133.3 eV indicate that calcium is in state of 
Ca2+ and phosphorous is in state of PO4-, that cor-
responds to the next chemical compounds, for exam-
ple, calcium phosphat Ca3(PO4)2 (CaP) or hydroxy-
apatite Ca10(PO4)6(OH)2 [6, 7]. 
According to differential dissolution analysis the 
composition of main phase of bone tissue can be 
fragmentary represented (without oxygen and hy-
drogen) by the formula:  
 
 Ca1.66Mg0.054Sr0.001Na0.06P1.00   
 
The content of the phase in the sample is 99.9%. 
Sample also contains small amounts of phase - prob-
ably phosphates - Mg, Sr, Na and K. Ratios of these 
elements in the small phases are Mg 1.4 rel.%, Sr 
4.4 rel.%, Na 3.3 rel.% - from the total in the sample 
content of each of these elements. These elements 
are almost completely covered in the main phase. At 
the same time in the main phase is not potassium, 
which either forms its own phase or concentrated in 
the surface layers of the main phase. 
The received results accomplish a modern per-
formances about ultrastructural organization of 
nanoscaled apatite of bone tissue biominerals. 
 
AKNOWLEDGEMENTS 
 
Authors are grateful to the Dr. A. Sorokin and 
Dr. I. Prosvirin from Boreskoff Institute of catalysis 
SD RAS, Novosibirsk, Russia for XPS measurements 
and his valuable comments. Also authors thank to 
Dr. V. Malakhov from Boreskoff Institute of catalysis 
SD RAS, Novosibirsk, Russia for application of the 
differential dissolution analysis to native bone. 
 
 
REFERENCES 
 
1. J.C. Elliott, Structure and Chemistry of the Apatites and 
Other Calcium Orthophosphates. Studies in Inorganic 
Chemistry (Amsterdam. Elsevier. 1994). 
2. F. Betts, N.C. Blumenthal, A.S. Posner, J.Cryst.Growth 
53, 63 (1981). 
3. S.N. Danilchenko, A.V. Koropov, I.Yu. Protsenko et al., 
Cryst. Res. Technol. 41 No3, 268 (2006). 
4. A. Bigi, E. Foresti, R. Gregorini, Calcif. Tissue Int. 50, 439 
(1992). 
5. A. Bigi, G. Falini, E. Foresti, Acta Cryst. 52, 87 (1996). 
6. C.C. Chusuei, D.W. Goodman, M.J. Van Stipdonk, 
D.R. Justers, Schweikert, Anal. Chem. 71, 149 (1999). 
7. S. Kaciulis, G. Mattogno, L. Pandolfi, L. Cavalli, 
G. Gnappi, Appl. Surf. Sci. 151, 1 (1999). 
 


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