Chitosan-apatite composites: synthesis and properties
The aim of this short review was to discuss applications of a unique biopolymer chitosan in practical medicine, especially for bone tissue engineering. The article highlights the preparation and properties of innovative chitosan-based biomaterials such as CaP-chitosan (CS-CP)-composites and chitosan...
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Sukhodub, L.F. Sukhodub, L.B. Chorna, I.V. 2019-06-13T07:29:40Z 2019-06-13T07:29:40Z 2016 Chitosan-apatite composites: synthesis and properties / L.F. Sukhodub, L.B. Sukhodub, I.V. Chorna // Вiopolymers and Cell. — 2016. — Т. 32, № 2. — С. 83-97. — Бібліогр.: 78 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000910 https://nasplib.isofts.kiev.ua/handle/123456789/152817 615.31:541.64:539.6:617-089.844:615.28 The aim of this short review was to discuss applications of a unique biopolymer chitosan in practical medicine, especially for bone tissue engineering. The article highlights the preparation and properties of innovative chitosan-based biomaterials such as CaP-chitosan (CS-CP)-composites and chitosan-alginate (CS-AG)-scaffolds. This paper takes a closer look at the physicochemical properties, spectral characteristics and chemical modifications of the chitosan molecule. The obtained chitosan-apatite composites were analysed using X-ray diffraction to verify the crystalline nature of their structures. It was observed that the addition of chitosan to the composite material reduces apatite crystallinity. Besides, an accent was made on antibacterial properties of chitosan, the use of chitosan nanoparticles to produce nanofibers and controlled drug delivery systems. Keywords: chitosan, hydroxyapatite, biocomposites, X-ray diffraction. Мета цього короткого огляду – розгляд застосування унікального біополімеру хітозану в практичній медицині, особливо для інженерії кісткової тканини. Основне місце в статті відведено синтезу та властивостям інноваційних біоматеріалів на основі хітозану, таким як CaP-хітозан (CS-CP)-композити і хітозан-альгінатні (CS-AG)-скаффолди. У статті висвітлено фізико-хімічні властивості, спектральні характеристики і хімічні модифікації молекули хітозану. Отримані хітозан-апатитні композитні матеріали були проаналізовані з використанням методу рентгенівської дифракції для аналізу кристалічної природи їх структур. Було виявлено, що додавання хітозану до композитного матеріалу призводить до зниження кристалічності вихідного апатиту. Крім того, акцентовано увагу на антибактеріальні властивості хітозану, використанні наночастинок хітозану для отримання нановолокон і створенні системи контрольованої доставки ліків. Цель этого краткого обзора – рассмотреть применения уникального биополимера хитозана в практической медицине, особенно для инженерии костной ткани. Основное место в статье отведено синтезу и свойствам инновационных биоматериалов на основе хитозана, таким как CaP-хитозан (CS-CP)-композиты и хитозан-альгинатные (CS-AG)-скаффолды. В статье освещены физико-химические свойства, спектральные характеристики и химические модификации молекулы хитозана. Полученные хитозан-апатитные композитные материалы были проанализированы с использованием метода рентгеновской дифракции для анализа кристаллической природы их структур. Было обнаружено, что добавление хитозана к композитному материалу приводило к снижению кристалличности исходного апатита. Кроме того, акцентировано внимание на антибактериальных свойствах хитозана, использовании наночастиц хитозана для получения нановолокон и создании систем контролируемой доставки лекарств. We thank the Ukrainian Fundamental Foundation (project NU/7-2013) for funding our researches, Dr. V.G. Lugin from State technological university of Belarus for IR-studies of the apatite-biopolymer samples. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Reviews Chitosan-apatite composites: synthesis and properties Хітозан-апатитні композити: синтез і властивості Хитозан-апатитные композиты: синтез и свойства Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Chitosan-apatite composites: synthesis and properties |
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Chitosan-apatite composites: synthesis and properties Sukhodub, L.F. Sukhodub, L.B. Chorna, I.V. Reviews |
| title_short |
Chitosan-apatite composites: synthesis and properties |
| title_full |
Chitosan-apatite composites: synthesis and properties |
| title_fullStr |
Chitosan-apatite composites: synthesis and properties |
| title_full_unstemmed |
Chitosan-apatite composites: synthesis and properties |
| title_sort |
chitosan-apatite composites: synthesis and properties |
| author |
Sukhodub, L.F. Sukhodub, L.B. Chorna, I.V. |
| author_facet |
Sukhodub, L.F. Sukhodub, L.B. Chorna, I.V. |
| topic |
Reviews |
| topic_facet |
Reviews |
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2016 |
| language |
English |
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Вiopolymers and Cell |
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Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Хітозан-апатитні композити: синтез і властивості Хитозан-апатитные композиты: синтез и свойства |
| description |
The aim of this short review was to discuss applications of a unique biopolymer chitosan in practical medicine, especially for bone tissue engineering. The article highlights the preparation and properties of innovative chitosan-based biomaterials such as CaP-chitosan (CS-CP)-composites and chitosan-alginate (CS-AG)-scaffolds. This paper takes a closer look at the physicochemical properties, spectral characteristics and chemical modifications of the chitosan molecule. The obtained chitosan-apatite composites were analysed using X-ray diffraction to verify the crystalline nature of their structures. It was observed that the addition of chitosan to the composite material reduces apatite crystallinity. Besides, an accent was made on antibacterial properties of chitosan, the use of chitosan nanoparticles to produce nanofibers and controlled drug delivery systems. Keywords: chitosan, hydroxyapatite, biocomposites, X-ray diffraction.
Мета цього короткого огляду – розгляд застосування унікального біополімеру хітозану в практичній медицині, особливо для інженерії кісткової тканини. Основне місце в статті відведено синтезу та властивостям інноваційних біоматеріалів на основі хітозану, таким як CaP-хітозан (CS-CP)-композити і хітозан-альгінатні (CS-AG)-скаффолди. У статті висвітлено фізико-хімічні властивості, спектральні характеристики і хімічні модифікації молекули хітозану. Отримані хітозан-апатитні композитні матеріали були проаналізовані з використанням методу рентгенівської дифракції для аналізу кристалічної природи їх структур. Було виявлено, що додавання хітозану до композитного матеріалу призводить до зниження кристалічності вихідного апатиту. Крім того, акцентовано увагу на антибактеріальні властивості хітозану, використанні наночастинок хітозану для отримання нановолокон і створенні системи контрольованої доставки ліків.
Цель этого краткого обзора – рассмотреть применения уникального биополимера хитозана в практической медицине, особенно для инженерии костной ткани. Основное место в статье отведено синтезу и свойствам инновационных биоматериалов на основе хитозана, таким как CaP-хитозан (CS-CP)-композиты и хитозан-альгинатные (CS-AG)-скаффолды. В статье освещены физико-химические свойства, спектральные характеристики и химические модификации молекулы хитозана. Полученные хитозан-апатитные композитные материалы были проанализированы с использованием метода рентгеновской дифракции для анализа кристаллической природы их структур. Было обнаружено, что добавление хитозана к композитному материалу приводило к снижению кристалличности исходного апатита. Кроме того, акцентировано внимание на антибактериальных свойствах хитозана, использовании наночастиц хитозана для получения нановолокон и создании систем контролируемой доставки лекарств.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152817 |
| citation_txt |
Chitosan-apatite composites: synthesis and properties / L.F. Sukhodub, L.B. Sukhodub, I.V. Chorna // Вiopolymers and Cell. — 2016. — Т. 32, № 2. — С. 83-97. — Бібліогр.: 78 назв. — англ. |
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83
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
© 2016 L. F. Sukhodub et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
UDC 615.31:541.64:539.6:617-089.844:615.28
Chitosan-apatite composites: synthesis and properties
L. F. Sukhodub1, L. B. Sukhodub2, I. V. Chorna1
1 Sumy State University
2, Rymskogo-Korsakova., Sumy, Ukraine, 40007
2 Mechnikov Institute of Microbiology and Immunology NAMS of Ukraine
14, Pushkinska Str., Kharkiv, Ukraine, 61057
l_sukhodub@yahoo.com
The aim of this short review was to discuss applications of a unique biopolymer chitosan in practical medicine,
especially for bone tissue engineering. The article highlights the preparation and properties of innovative chi-
tosan-based biomaterials such as CaP-chitosan (CS-CP)-composites and chitosan-alginate (CS-AG)-scaffolds.
This paper takes a closer look at the physicochemical properties, spectral characteristics and chemical modifi-
cations of the chitosan molecule. The obtained chitosan-apatite composites were analysed using X-ray diffrac-
tion to verify the crystalline nature of their structures. It was observed that the addition of chitosan to the
composite material reduces apatite crystallinity. Besides, an accent was made on antibacterial properties of
chitosan, the use of chitosan nanoparticles to produce nanofibers and controlled drug delivery systems.
K e y w o r d s: chitosan, hydroxyapatite, biocomposites, X-ray diffraction.
Introduction
Over the last years, chitosan (CS) has attracted a
great interest of scientists as functional polymeric
material because of its remarkable intrinsic proper-
ties: biodegradability, biocompatibility, nontoxicity,
antibacterial activity, mucoadhesive, analgesic and
haemostatic properties [1–3]. Chitosan is a linear,
semi-crystalline polysaccharide composed of
(1→4)-2-acetamido-2-deoxy-β-D-glucan (N-acetyl
D-glucosamine) and (1→4)-2-amino-2-deoxy-β-D-
glucan (D-glucosamine) units [1, 4–6]. Chitosan can
be easily obtained from a natural polymer chitin af-
ter its partial deacetylation by chemical hydrolysis
under severe alkaline conditions or by enzymatic
hydrolysis [7, 8]. Chitosan is well tolerated by living
tissues, including the skin, ocular membranes, the
nasal epithelium. A low or no toxicity of chitosan
compared with other natural polysaccharides has
been demonstrated by in vivo toxicity studies [9].
Some studies showed that chitosan, as an immune
adjuvant, could effectively promote the local im-
mune response and enhance antigen presentation
[10]. The combination of CS with different materi-
als, such as hydroxyapatite (HA), is very promising,
especially for orthopedics and traumatology [11].
All these features make chitosan an outstanding can-
didate for biomedical applications.
Physical and chemical properties of chitosan
Chitosan as the product of partial deacetylation of
chitin contains five types of active functional groups:
primary amine groups at the C-2 position of each
deacetylated structural unit, secondary amide groups,
ether groups of polysaccharide main chain as well as
both primary and secondary hydroxyl groups at the
C-6 and C-3 positions, respectively (Fig. 1).
The molecular weight (MW) of CS is in the range
of 300 to 1 000 kDа and it depends on the source and
the method used for obtaining CS. Degree of CS
Reviews ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2016. Vol. 32. N 2. P 83–97
doi: http://dx.doi.org/10.7124/bc.000910
mailto:l_sukhodub@yahoo.com
84
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
deacetylation (DD) (N- acetylglucosamine / glucos-
amine) may vary from 30 to 95 %. The higher DD of
CS, the greater the quantity of protonated amino
groups in the polymer and, correspondingly, the
higher the amount of charge on the macromolecule.
In crystalline form, CS is insoluble in aqueous solu-
tions with pH > 7 whereas in dilute acids (pH < 6) it
becomes soluble due to protonation of NH2-
groups [5]. Chitosan’s primary amines confer impor-
tant material properties. At low pH, the amines are
protonated making chitosan a cationic polyelectro-
lyte. In protonated form, the amines allow connection
through electrostatic interactions [12]. Due to its
amino groups, chitosan can form bonds with a wide
variety of organic and inorganic molecules, such as
lipids, proteins, DNA and some negatively charged
synthetic polymers [13–16]. At high pH, the amines
are deprotonated and chitosan undergoes a transition
from a soluble cationic polyelectrolyte to an insolu-
ble polymer. Insolubility of chitosan in water means
that the intermolecular interactions between macro-
molecules exceed the interactions in the system of
“chitosan-water molecules”. Importantly, this pH-re-
sponsive switch is near neutrality (chitosan’s appar-
ent pKa has been reported to range between 6 and 7)
[17, 18] suggesting chitosan as a biologically-derived
A
B
Fig. 1. The structural formula of chitin (A) and chitosan (B)
Fig. 2. Schematic representation of the metal-chitosan complexation [22]
85
Chitosan-apatite composites: synthesis and properties
stimuli-responsive polymer for medical applications
(e.g., injectable matrices) [19]. There are well known
the nucleophilic properties of the amines that allow
connections through covalent linkages that can be
formed through a range of coupling chemistries [20].
Chitosan’s metal binding properties [21] allow con-
nections through chelation mecha nisms. Fig. 2 illus-
trates the structural chemical formula of two strands
of chitosan and the “cross-linking” mechanism
caused by the divalent metal ion [22].
Micro/nanoparticles and hydrogels are widely
used in the design of chitosan-based therapeutic sys-
tems [4]. The cationic nature of CS provides the
electrostatic interaction with quantivalent linear an-
ionic polysaccharides (glycosaminoglycans (GAG),
proteoglycans etc.). This factor is very important be-
cause of a large number of growth factors and other
proteins bound to GAG, that is why the formation of
GAG-CS complexes provides maintenance and ac-
cumulation of these necessary biopolymers in vivo.
Thus, chitosan based scaffolds deliver growth fac-
tors in a controlled fashion to promote the in-growth
and biosynthetic ability of chondrocytes [4]. Under
the action of cellular enzymes, especially lysozyme,
CS degraded depending on the degree of crystallini-
ty and deacetylation [6]. Hence these properties
must be necessarily taken into account at creating
chitosan based implants. Also it should be noted that
the CS hydrogel composite is able to form a porous
structure using, for example, lyophilization techno-
logy (“freeze-drying”). A pore size in the CS hydro-
gel composite depends on speed of freezing. The
degree of porosity and pore orientation significantly
affect the mechanical properties of the implant.
Another property of CS is its internal antibacterial
ability [23–25].
Chitosan in bone tissue engineering
Tissue engineering (TE) is an interdisciplinary area
that contains both a basic knowledge of life sciences
and engineering to create biologically compatible,
biodegradable scaffolds (matrices) in different forms
(powder, microcapsules, gels, films, etc.) for a wide
use in nanomedicine. Systems for the controlled
drug delivery by using chitosan and its derivatives
are of huge interest [4]. Chitosan is widely used in
bone tissue engineering because of its ability to pro-
mote a cell growth and the formation of mineral ma-
trix by osteoblasts [26]. Biocompatibility of chitosan
minimizes the local inflammation, and its conver-
sion into a porous structure contributes to osteocon-
ductivity [27]. The Chitosan - Calcium Phosphate
composites (CS-CP) were the subject of intensive
study in the world [28–31].
CS-CP composites
Biomaterials that mimic the structure and composi-
tion of bone tissues at the nanoscale level are ex-
tremely important for the development of bone tis-
sue engineering applications [32]. CS-CP compo si-
tes have certain advantages compared to other simi-
lar structures. Thus, during the resorption, the degra-
dation products of chitosan and calcium phosphate
(calcium ions, phosphates, glycosamines, etc.) are
naturally metabolized and do not induce the increas-
ing calcium and phosphorus concentrations in urine,
serum or internal organs. A composite material con-
tains both the macro- and micropores and nano-sized
crystals of HA. This promotes an increase in the re-
active surface and material osteoconductive activity.
Similar 3D-macroporous ceramics pierced by chito-
san grid have better mechanical properties [33, 34];
therefore, there is a perspective of the CS-CP com-
posites future use in the clinic. The main advantages
of CS-CP biomaterials are: structural organization,
which is close to the structure of natural bone, bio-
compatibility, biodegradation, macro- and micropo-
rosity, regulation of resorption rate, immobilization
of drugs, antibacterial action, simplicity of flowsheet
synthesis, low cost.
Among a wide range of calcium phosphates hy-
droxyapatite (HA), Ca10(PO4)6(OH)2 is a widely
used material for biomedical application in dentistry
and orthopedy due to the excellent bioactivity, bio-
compatibility and osteoconductivity [31, 35].
However, the migration of HA powder from im-
planted sites and bone defects is really a big prob-
lem. Therefore, organic compounds in nanocompo-
86
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
sites are promising for improving weak mechanical
properties of HA [36].
The CS-HA composites have been recently syn-
thesized in our laboratory. For this aim the chitosan
macromolecules ( MW 500 kDa, 200 kDa, 39 kDa)
with DD 85 % were supplied by the “Bioprogress”
(Moskow, Russia). Calcium acetate Ca(CH3COO)2,
sodium dihydrogen phosphate NaH2PO4, and sodi-
um hydroxide (NaOH) were of analytical grade and
supplied by “Merck”. An influence of the chitosan’s
molecular weight and various ways of the synthesis
on obtaining the CS-HA composite structure have
been studied. Calcium acetate was used as the
0.167 M solution in 1 % CH3COOH (first way of
synthesis) or was added in solid state to chitosan so-
lution in 1 % CH3COOH (second way of synthesis).
By the first way of synthesis 0.8 g of chitosan
powder with certain MW were added to 20 mL of the
0.167 M (CH3COO)2Ca stock solution in 1 w %
CH3COOH. The final chitosan concentration in the
reaction mixture was 0.4 w %. The calcium acetate–
chitosan solution was stirred in a shaker (160 rpm)
for 1.5 h at 37 ºC, then 20 mL of 0.1 M NaH2PO4
solution were added gradually to the above mixture.
The pH value was adjusted to 11.8 by using 10 M
NaOH solution. The obtained suspension was aged
for 5 days at 22 ºC, then washed thoroughly with
deionized water to pH 7.4. Finally, the precipitate
was separated by centrifuging the suspension. For
the analysis the product was dried at 37 oC and an-
nealed for 1 h at 900 oC. The second way of synthesis
was provided by addition of solid (CH3COO)2Ca
(0.526 g) to the 20 mL of the 0.4 w % chitosan solu-
tion in 1 % CH3COOH. The calcium acetate–chito-
san solution was stirred in a shaker (160 rpm) for
1.5 h at 37 ºC, after which 20 mL of 0.1 M NaH2PO4
solution were added gradually to the above mixture.
The pH value of solution (about 11.0) was corrected
by using 10 M NaOH solution. A white precipitate
was observed after the phosphate solution addition
at both ways of synthesis. It was associated with the
hydroxyapatite formation. Both suspensions were
aged for 5 days at 22 ºC, then washed thoroughly
with deionized water to obtain solution of pH 7.4.
Finally, the precipitate was separated by centrifug-
ing the suspension. For the analysis the product was
dried at 37 oC and annealed for 1 h at 900 oC. The
Obtained composites consisted of 80 w % of HA and
20 w % of CS.
The crystallinity and structure of precipitates were
examined using an X-ray diffractometer DRON-3
(“Burevestnik”, Russia) connected to a computer-
aided system for the experiment control and data
processing. The Ni-filtered CuKα radiation (wave-
length 0.154 nm) with a conventional Bragg–
Brentano θ-2θ geometry was used. The current and
voltage of X-ray tube were 20 mA and 40 kV respec-
tively. The samples were tested in a continuous mode
in the range 10º to 60º at a rate 2.0 º/min with
2θ-angles. The samples phase composition was de-
termined and crystallite size calculated. The average
crystallite size (L) and strain (ε) were calculated in
the [0 0 c] crystal direction using XRD data by
Scherrer equation [37]. The crystalline phases were
identified by comparing the experimental XRD pat-
terns to the standards compiled by the Joint
Committee on Powder Diffraction and Standards
(JCPDS cards). The formation of crystalline HA
phase occurs in both the presence and the absence of
polymer, independently from chitosan MW and syn-
thesis way. The existence of 2θ peaks at approxi-
mately 26º, and 31.9º corresponding to the (002) and
(211) diffraction planes confirms the formation of
HA phase in products [36]. Taking into account the
broadening of each peak in XRD spectra, mean crys-
tallite sizes for examined samples were calculated
using Scherrer equation and approximation method
(Table 1).
As can be assumed from the Table 1, 20 w % of
chitosan in material composition did not decrease
significantly crystallite sizes of HA-CS samples
compared to HA sample. The changes in the polymer
molecular weight and the way of synthesis had no
observable effect on the peaks positions and/or their
intensity. However, the strain in [0 0 c] direction in-
creased for all HA-CS composite samples indicating
significant defects in the hydroxyapatite crystal struc-
ture caused by the presence of the polymer.
87
Chitosan-apatite composites: synthesis and properties
To evaluate the functional groups of CS, HA and
synthesized composite HA-CS (Fig. 3), the FTIR
analysis was performed by the instrument “Thermo
Nicolet Nexus 470 ESP” (Minsk Technological
University, Belarus). The sample for spectral analy-
sis was prepared in a traditional way: a small amount
of the preparation (synthesized composite powder)
was mixed with KBr in the ratio 1:200 in an agate
mortar while grinding. From the obtained mixtures
corresponding tablets were prepared in press-forms
of stainless steel under hydraulic pressure.
Transmission spectrum was obtained in the frequen-
cy range of 400–5000 cm–1 with a spectral resolution
of 0.125 cm–1.
FTIR-spectrum of CS(c) shows characteristic
peaks of amide I at 1651 cm−1 (–C=O stretching),
amide III at 1378 cm–1 (C– N stretching coupled
with NH in plane deformation), CH2 wagging cou-
pled with OH in plane deformation at 1324 cm−1.
The peak at 1598 cm–1 belongs to the bending vibra-
Table 1. Structure characteristics of hydroxyapatite
nanocrystals in dried at 37 °C HA-CS nanocomposites
Sample Miller
Indices
Crystallite
sizes by
Scherrer, nm
Approximation method
Crystallite
sizes, nm
Strain,
∙103
HA
(0 0 2) 22.8
22.8 0.003
(0 0 4) 22.8
HA / CS
500 (1)
(0 0 2) 21.7
21.5 0.097
(0 0 4) 22
HA / CS
500 (2)
(0 0 2) 19.6
21.4 0.71
(0 0 4) 18.2
HA / CS
200 (1)
(0 0 2) 18.3
17.8 0.27
(0 0 4) 19
HA / CS
200 (2)
(0 0 2) 19
18.4 0.32
(0 0 4) 19.7
HA / CS
39 (1)
(0 0 2) 18.2
21.7 1.53
(0 0 4) 15.6
HA / CS
39 (2)
(0 0 2) 21.6
20.7 0.342
(0 0 4) 22.6
Fig. 3. FTIR-spectra of a) HA-CS nanocomposite (down), b) HA, sintered at 900 ºC (middle), c) CS with MW 500 kDa (top)
88
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
tions of the N–H and C–N (amide II band). Peaks at
1430 belong to the N–H stretching of the amide and
ether bonds [38]. The vibration band about
1029 cm−1 – 1077 cm−1 indicates the C–O stretching
vibration in the primary and secondary hydroxyl
groups in chitosan [39, 40], wide band (3429 cm–1)
is attributed to –OH stretching and –NH2 asymmet-
ric stretching vibrations [41], the peak at 2868 cm–1
is caused by –CH2-stretching vibrations [42].
FTIR-spectrum of CS (c) shows characteristic
bands of amide I (the main contribution of C=O
stretching) at 1651 cm–1 and amide II (the main con-
tribution of N–H bending) together with N–H bend-
ing vibration of primary amine groups at 1598 cm–1.
Three bands at ~1450, 1430 and 1378 cm–1 belong to
C-H asymmetric and symmetric bending vibrations
of methyl and methylene groups. The band of –CH2-
wagging coupled with -OH in plane deformation ap-
pears at 1324 cm–1. Some vibration bands in a region
of 1200–1000 cm–1 with the main peak at 1161 cm–1
indicate asymmetric and symmetric C–O–C and
C–O stretching vibrations of CS ether and hydroxyl
groups. The O–H and N–H stretching vibrations of
hydroxyl, amide and amine groups of CS appear in
FTIR spectrum by a wide band with maximum at
3429 cm–1, whereas C-H stretching of –CH2- and –
CH3 groups is revealed by two overlapped bands at
2920 and 2862 cm–1.
Pure HA (b) shows a vibration band around
3427 cm–1 and peak at 630 cm–1 corresponding to
stretching vibration of the hydroxyl group [43]. The
characteristic peaks at 576 cm–1, 604 cm–1, 961 cm–1,
1048 cm–1and 1090 cm–1 are due to the bending and
stretching modes of P–O vibrations in the phosphate
network [44, 45].
In the HA-CS spectrum (a) the protonation of chi-
tosan amine functionalities is suggested by the pres-
ence of the band, attributed to NH3
+ groups, namely
the bending vibration at 1475 cm−1. This is evidently
conditioned by the formation of electrostatic bonds
with HA. Also the low-frequency shift of the amide
I band up to 1638 cm–1 is observed, thus pointing out
the participation of carbonyls of CS amide groups in
hydrogen bonding with hydroxyl groups of HA. It
was observed the disappearance of the vibration
band in HA-CS spectrum (a) at 1324 cm−1 that may
indicate partial phosphorylation of hydroxyl groups
in CS with the feather formation of calcium phos-
phates. The wide band indicated the C–O–C and
C–O stretching vibrations in the ether; the primary
and secondary hydroxyl groups in CS spectrum (c)
were present as the vibration band with peak at
1032 cm–1 in the spectrum of HA–CS (a). These
changes may be attributed to the formation of hydro-
gen bonds with participation of ether and/or –OH-
groups in HA and CS. Additionally, the vibration
bands at 960 cm–1 and 630 cm–1, belonging to the
phosphate- and hydroxyl- groups in HA (b) were ab-
sent in the HA–CS spectrum. All these facts confirm
molecular interactions between CS and HA in the
HA–CS composite.
Chitosan-alginate-hydroxyapatite polyelec-
trolyte composites
The chitosan- and sodium alginate (AG)-based scaf-
folds have been widely used as biomaterials in tissue
engineering. Freeze-drying, also known as lyophili-
zation, has been used for the fabrication of polymer-
based hydrogels. The porous scaffolds composed of
AG and CS have been fabricated by the formation of
a polyelectrolyte complex (PEC) between macro-
molecules of both polymers [46]. They perform a
3D-grid with uniformly distributed and intercon-
nected pores. Two technologies of the scaffold for-
mation are proposed in this paper. According to the
first, the uncrosslinked alginate scaffold is formed
from 4 w % AG solution using the “freeze-drying”
technology. Next, it undergoes some crosslinking
using 1 w % CaCl2. By the second technology, the
alginate scaffold was immerged into 2 w % CS solu-
tion or the CS-HA composite acetic acid aqueous
solution. In this case the pores of the alginate scaf-
folds were filled with chitosan solution. These sam-
ples were frozen at –40 °C and lyophilized. Finally,
the samples were additionally crosslinked with
CaCl2 solution.
Chitosan-alginate (CS-AG)-scaffolds exhibit bet-
ter mechanical properties and thermostability than
89
Chitosan-apatite composites: synthesis and properties
AG-scaffold, crosslinked with calcium ions only.
The main reasons for the mechanical strength im-
provement of crosslinked scaffold could be strong
ionic interactions. In the Ca2+ crosslinked AG scaf-
fold the mechanical strength is enhanced because
Ca2+ is characterized by strong ionic interaction with
COO- in an alginate chain. For CS-AG PEC scaffold
the strong interaction exists between NH3
+ groups in
CS and COO- groups in alginate (Fig. 4). Additionally,
a decrease of porosity for crosslinked scaffold is an-
other factor enhancing the mechanical strength.
Considering the participation of chitosan in PEC,
it should be noted a series of studies of chitosan
complexes with DNA, glycosaminoglycans, chon-
droitin sulfate, hyaluronic acid, heparin, carboxy-
methyl cellulose, pectin and proteins such as gelatin,
albumin, collagen and keratin [47–52]. The stability
of such complexes depends on the charge density,
solvent, ionic strength, pH and temperature [46].
The Chitosan-, alginate- and hydroxyapatite-based
scaffolds (with a ratio of 1:1:1) were recently syn-
thesized in our laboratory using our own flowsheet.
Such precursors were used for the synthesis: chito-
san (MW 500 kDa, DD 80%, “Bioprogress”,
Moscow), sodium alginate (E401), sodium hydro-
gen phosphate (Na2HPO4) and calcium acetate
(CH3COO)2Ca∙H2O (China). 500 ml of chitosan so-
lution (2 g/l in 1 w % acetic acid) were gradually
added to 0.1 M calcium acetate (100 ml); 1 g of so-
dium alginate powder was dissolved in 0.1 M sodi-
um hydrogen phosphate (60 ml). Two obtained solu-
tions were mixed and stirred in a shaker (160 rpm) at
37 ºC for 5 h. pH was adjusted with 10 M NaOH
solution to 11.0. Additionally the mixture was stirred
by ultrasound, heated at 80 ºC for 10 min and aged
for 48 h for the HA nucleation. The obtained product
was washed with the deionized water to pH 7.0-7.4
with subsequent freezing and vacuum drying
(“freeze-drying”) at –150 °C during 16 h. As a result,
the porous HA-CS-AG-scaffold with a ratio of com-
ponents 1:1:1 was obtained. The polymer amounts
for the synthesis might be calculated to obtain the
product with required component proportions. The
cation-anionic interactions between macromolecules
of CS and AG are the main driving force in the cre-
ation and stabilization of the biopolymer scaf-
folds [31].
X-ray structure analysis of apatite-biopoly-
mer composites
Recently the usage of materials in the form of hydro-
gels has become increasingly popular. The structure
of the polymer chains that form a three-dimensional
net of gel enables immobilization and sufficient con-
tent of water, biological fluids or drugs [53, 54]. We
investigated the composite materials that consisted
of the polymer matrix and inorganic filler and to
some extent modeled a bone. As the polymer matrix,
chitosan was used in the sample 1 and sodium algi-
nate - in the sample 2.
Fig. 4. Chemical structures of CS (top) and AG (down) and the scheme of their interaction
90
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
Sample N 1: HA–CS composite. The HA–CS
composite was synthesized by wet chemistry. Two
solutions were prepared for the synthesis: 1) 100 mL
[of] 0.1 M CaCl2, pH was adjusted up to 11.0 by ad-
dition of 10 M NaOH solution; 2) chitosan (MW 39
kDa) was dissolved in 100 ml of 0.06 M H3PO4 in
the amount, which provides chitosan concentration
in the final material from 10 to 40 w %. The second
solution was added drop by drop to the first one fol-
lowed with mixing and heating at 60 °C during
10 min. The pH value was adjusted by NaOH addi-
tion to 7.4. After aging for 24 h the resulting suspen-
sion was washed with distilled water and centri-
fuged. The degree of moisture in the obtained gel
ranged from 70 to 88 w %. The composition of sol-
ids was: HA from 60 to 90 w %, CS from 10 to
40 w %. For analysis, the composites were dried at
37 °C and annealed for 1 h at 900 ºC .
Sample N 2: HA–AG composite. Orthophosphoric
acid H3PO4 (0.06 M), anhydrous calcium chloride
CaCl2 (0.1 M), aqueous solution of sodium hydrox-
ide NaOH (10 M) and sodium alginate (E401, MW
15 kDa, China) were used as starting materials for the
HA-AG composite preparation. The composite mate-
rial was synthesized by the “wet chemistry” method:
1) sodium alginate was dissolved in 100 ml of 0.06 M
H3PO4 in the amount, which provides its content in
the final material from 10 to 40 w % at 37 °C. This
solution was added drop by drop to the calcium chlo-
ride solution with vigorous stirring. After mixing the
reactants, the suspension was treated with ultrasound.
Then, pH value was adjusted with 10 M NaOH to
10.6 and the suspension was heated at 60 °C during
10 min. After aging within 10 days the precipitate
was thoroughly washed with the deionized water;
solids were separated by centrifugation and steril-
ized. The degree of moisture in the obtained gel
ranged from 70 to 88 w %. The composition of solids
was: HA from 60 to 90 w %, AG from 10 to 40 w %.
The obtained samples were dried at 37 °C and an-
nealed for 1 h at 900 °C for further research.
To evaluate the structure of samples – the lattice
options (a, c), the average crystallite size (L, D) and
microstrain ε – two methods were used and com-
pared: X-Ray diffraction (XRD) and transmission
electron microscopy (TEM; SELMI, Sumy, Ukraine)
with the electron diffraction (ED). The obtained data
are shown in Table 2.
Both instrumental methods confirm the effect of
the polymer component on the structural properties
of HA crystallites. Thus, under the condition of syn-
thesis of HA nanoparticles in the presence of poly-
mer the size of crystallites decreased compared to
pure HA in samples dried at 37 °C. An average size
of crystallite increased after annealing and combus-
tion of the polymer component in the HA–CS and
HA–AG samples.
Fig. 5 shows the XRD patterns for the HA–CS
and HA–AG samples dried at 37 °C and annealed at
900 °C.
According to the XRD analysis, the sample 1 after
annealing at 900 °C included two phases: HA
(JCDPS 9-432, with 1.67 Ca / P ratio, concentration
97 w %) and CaO (JCDPS 37-1497, concentration
3 w %). The presence of another phase after the tem-
perature test indicates the nonstoichiometric output
of apatite. The main factor causing a small size of
crystallites and high level of microdeformations is
an addition of chitosan, which, as has been shown in
previous studies [55], reduces crystallinity of apatite
and distorts its crystal lattice.
In the sample № 2 after annealing at 900 °C only
one phase HA (JCPDS 9-432, with 1.67 Ca/P ratio)
was found which indicated the stoichiometry of the
initial apatite.
The nanostructures of the HA–CS and HA-AG
composites were examined using TEM, as shown in
our previous studies [56].
Table 2. Data of TEM and XRD analysis of structure of HA
(pure), HA-CS and HA-AG composites
Sample
Dried at 37 oC Annealed
at 900 ºC
TEM with ED XRD XRD
a, nm c, nm D, nm L, nm L, nm
HA-CS 0.949 0.688 ~80 14.3 55.4
HA-AG 0.945 0.688 ~80 24.6 58.6
HA 0.933 0.684 ~120 33.2 49.41
91
Chitosan-apatite composites: synthesis and properties
Antibacterial properties of silver-doped
HA-CS composites
One of the most important problems in modern im-
plantology is bacterial infection. The inflammation in
the implant surrounding tissue eventually leads to the
loss of the implant. The use of antimicrobial films or
coatings is a way for preventing inflammation.
Chitosan is one of the natural polysaccharides that can
form a film with antibacterial properties. There are
several mechanisms of antibacterial activity of chito-
san [47]. First, chitosan as a polycation forms electro-
static bonds with anionic molecules on the cell sur-
face and thereby affects their penetrating ability [57,
58]. Second, chitosan binds to the negatively charged
groups of DNA and thus inhibits the RNA synthesis
[24, 59]. Finally, the antibacterial activity of chitosan
can include both mechanisms, depending on the
charge density in interacting components [24, 60].
Due to a large number of OH- and NH2-groups chito-
san can easily form chelates with metal ions [21, 61].
A)
Fig. 5. A) X-ray diffraction patterns of
HA–CS nanocomposites dried at 37 °C
and annealed for 1 h at 900 °С. ♦ – as-
signed CaO phase; B) X-ray diffraction
patterns of HA-AG nanocomposites
dried at 37 °C and annealed for 1 h at
900 °С
B)
92
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
Most silver-containing antimicrobial biomaterials
consist of either Ag+ ions (silver salts or silver com-
plexes) or elemental silver (Ag-nanoparticles) incor-
porated into organic (polymers) or inorganic (bio-
glasses and HA) matrices [62, 63]. The silver-loaded
HA composites are obtained by ion-exchange meth-
ods (sol-gel or coprecipitation) that involve the silver
substitution for calcium, resulting in a Ca-deficient
hydroxyapatite. The antimicrobial response of these
materials is good, but there is pH-dependent negative
rapid release of silver [64]. Silver nanoparticles have
the antibacterial properties, delaying the growth of
Gram-positive and Gram-negative bacteria. It is also
well known that the antibacterial activity of Ag
nanoparticles is caused not only by free Ag+ ions pro-
duced from nanoparticle surface but also by the inter-
action of small active nanoparticles with the microor-
ganisms cells and destruction of their membranes
[65–67]. Chitosan-nanosilver-based films exhibit the
excellent antibacterial activity against Escherichia
coli [61]. Therefore, in the recent research [68], our
group has used the termodeposition method [31] for
obtaining antimicrobial Ag+-doped hydroxyapatite
coatings under physiological conditions with various
concentrations of silver ions and therefore different
antibacterial activity. Thus, HA-Ag+-coatings were
created on both the chitosan- modified and non-mod-
ified Ti-6Al-4V substrates. Antibacterial properties
were studied by the optical density evaluation of the
E.coli ATCC 25922 bacterial suspensions (Institute of
Micro bio logy and Immunology, National Academy
of Medical Sciences of Ukraine) before and after im-
mersing the experimental samples into these suspen-
sions. The optical density was measured by spectro-
photometry at l=540 nm 2, 24 and 48 h after the sam-
ple immersion. The bacterial cell amount was evalu-
ated from the optical density measurements of the
bacteria suspension with known concentration in
CFU mL-1 (Colony Forming Units). It was found that
the inclusion of Ag+-ions into the coating significantly
reduced the number of bacteria in a sample. Even a
stronger effect occurred for the Ag+-ions doped HA
coatings, formed at a surface of the substrate with CS
layer (Fig. 6).
The proposed approach can be taken as a promis-
ing method to create antimicrobial coatings for tita-
nium implants [68].
Chitosan-biopolymer composites for drug
delivery
Regenerative medicine plays an important role in the
restoring of damaged organs in vivo by the activation
of stimulating factors of the organism. One of the
advantages of chitosan is the ability of controlling
the release of active compounds without the use of
toxic organic solutions, because of its good solubili-
ty in weakly acid solutions. These circumstances led
to the use of CS for the creation of drug delivery
systems. For example, CS was once used for dis-
solving poorly soluble pharmaceuticals in the syn-
thesis of mucoadhesive mixtures [69–71], and for
enhancement of the peptides absorption] [72–74].
Nowadays, various types of systems (tablets, cap-
sules, microspheres, nanoparticles, films, gels) are
created using different methods (coating matrix, a
capsule shell, emulsive cross-linking, coacervation/
precipitation, spray drying, ionic gelation, sieving
method) [3]. The chosen method must be specific to
the determined component in the particular case
studied (particle size, thermal and chemical proper-
ties of active agents, the stability of the final pro-
Fig. 6. Dependence of E.coli growth on the time after immersion
of HA–CS (modified substrate), HA–Ag (unmodified substrate)
and HA–Ag (modified substrate) coatings in comparison with
the HA coating
93
Chitosan-apatite composites: synthesis and properties
duct, etc.). The main problem in the drug delivery
therapy is to provide the necessary drug concentra-
tion at the destination [75]. One of the modern ap-
proaches is the use of CaP-biopolymer scaffolds,
including polysaccharides of chitosan and alginate
in combination with hydroxyapatite, as above dis-
cussed. It was shown that chitosan coatings on algi-
nate scaffolds enhance osteoblast adhesion and pro-
liferation [46]. Additionally, improvements in the
composite mechanical properties were achieved in
the scaffolds containing both polymers compared to
those containing separate polysaccharides [76]. For
example, the effectiveness of chitosan and alginate
has been shown for more prolonged and controlled
release of lidocaine (C14H22N2O), which is a local
anesthetic drug, into physiological solution with
phosphate buffer (PBS) [75]. According to FTIR
spectroscopy, no chemical reactions have been reg-
istered between hydroxyapatite, chitosan, alginate
and lidocaine. Instead, it has been shown that each
polysaccharide affected crystal morphology of the
drug: needle-shaped crystals of lidocaine occurred
when chitosan was used as a coating, whereas the
using alginate as a coating induced the formation of
rectangular crystals [75].
Chitosan-metal complexes
As noted above, one of the important properties of
chitosan is the formation of chelate with metal ions.
Some researches prove a higher antimicrobial activ-
ity of chitosan-metal complexes compared with pure
chitosan [77]. Such complexes were obtained in two
stages. First, the nanoparticles of chitosan were pro-
duced by ionic gelation between chitosan and sodi-
um tripolyphosphate (Na5P3O10), then the obtained
nanoparticles were connected (“loaded”) with metal
ions (Ag+, Cu2+, Zn2+, Mn2+, Fe2+) [77]. Antibacterial
activities of the metal ions doped chitosan nanopar-
ticles, pure chitosan nanoparticles, metal ions and
1 w % acetic acid solution ( used as a solvent for
chitosan) were evaluated by determination of the
minimum inhibitory concentration (MIC) and mini-
mum bactericidal concentration (MBC). MIC was
determined by the broth dilution method as an equiv-
alent to the minimum sample concentration that did
not cause the visible bacteria growth in the tube,
containing the bacteria suspension and experimental
sample. To evaluate MBC, the bacteria suspension
aliquot was transferred from each tube without visi-
ble growth on a Muller–Hinton (MH) agar plate
(MH was used as a growth media) and incubated at
37 oC for 24 h. MBC was determined as the mini-
mum sample concentration that did not cause any
bacterial growth. Antibacterial properties of the chi-
tosan nanoparticles have been significantly improved
by the metal ions addition. For example, for the Cu2+
doped CS-nanoparticles, MIC and MBC against
E.coli ATCC25922, S.choleraesuis ATCC 50020 and
S. aureus 25923 were 21-42 times lower than for
pure Cu2+ ions [77]. The report states that Gram-
negative bacteria are more sensitive to chitosan-met-
al nanoparticles, due to a high negative charge at a
surface of the respective cells, on the one hand, and
a positive charge of amino groups reinforced by the
interaction of macromolecules with metal ions, on
the other hand. However, this hypothesis must be
confirmed by new experimental and theoretical
quantum-chemical researches.
Chitosan nanofibers
One of the newest applications of chitosan in medi-
cine is the development of natural methods for ob-
taining chitosan nanofibers [78]. Similar structures
are produced by the “Nanospider” technology. The
process occurs as follows. 1) In a special chamber
between the cathode and anode a voltage of 60 kV is
applied. 2) As a result, at a surface of the cathode
covered with a thin film of chitosan solution, a spiral
flow of polymer molecules is formed (Taylor cone).
3) The velocity of the particles increases with the
movement toward the negatively charged electrode;
the diameter of individual flow reduces to nanoscale.
4) Solvent molecules are evaporated and the
stretched macromolecules are drawn together, i.e. a
phase transition from liquid to solid state is observed.
5) The obtained nanofibers are adsorbed to the nega-
tively charged base plate material (any substance, a
metal substrate, etc.).
94
L. F. Sukhodub, L. B. Sukhodub, I. V. Chorna
There are different physical, chemical (the type
of solvent, the DD and MW parameters of chitosan,
the solution homogenization without formation of
macromolecular tangles, the viscosity and electri-
cal conductivity of the cathode forming solution)
and technological (the humidity in the chamber 30-
50 w %, the distance between electrodes 150-
180 mm) features of this technology to vary for
obtaining the chitosan nanofibers with a size of 70-
200 nm. The first attempts of application of the
nanostructures, obtained by this technology, in
medicine, e.g. as bandage, demonstrate a new per-
spective in the effective treatment of burns and
similar injuries [78].
Conclusion
Chitosan characteristics considered in this review in-
dicate a significant potential for its use as a biomate-
rial with the required properties (porosity, degree of
biodegradation) in the applied medicine, particularly
for bone regeneration. However, some extra efforts
are necessary to improve the mechanical properties
of chitosan-based biomaterials for the applications.
Another a very significant feature of chitosan is its
ability of interacting with anionic biomolecules such
as growth factors, glycosaminoglycans and DNA.
The binding to DNA molecules makes it possible to
obtain the material suitable for the application in
gene therapy. Taking into account a combination of
chitosan properties (biocompatibility, antibacterial
ability, the complexation with growth factors and
DNA) the conclusion can be made that chitosan is a
very promising candidate for tissue engineering
scaffolds. However, some parameters such as mo-
lecular weight, viscosity, should be further examined
to exploit the potential of this natural polysaccharide
in nanomedicine.
Acknowledgements
We thank the Ukrainian Fundamental Foundation
(project NU/7-2013) for funding our researches, Dr.
V.G. Lugin from State technological university of
Belarus for IR-studies of the apatite-biopolymer
samples.
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Хітозан-апатитні композити: синтез і властивості
Л. Ф. Суходуб, Л. Б. Суходуб, І. В. Чорна
Мета цього короткого огляду – розгляд застосування унікаль-
ного біополімеру хітозану в практичній медицині, особливо
для інженерії кісткової тканини. Основне місце в статті відве-
97
Chitosan-apatite composites: synthesis and properties
дено синтезу та властивостям інноваційних біоматеріалів на
основі хітозану, таким як CaP-хітозан (CS-CP)-композити і хі-
тозан-альгінатні (CS-AG)-скаффолди. У статті висвітлено фі-
зико-хімічні властивості, спектральні характеристики і хімічні
модифікації молекули хітозану. Отримані хітозан-апатитні
композитні матеріали були проаналізовані з використанням
методу рентгенівської дифракції для аналізу кристалічної при-
роди їх структур. Було виявлено, що додавання хітозану до
композитного матеріалу призводить до зниження кристаліч-
ності вихідного апатиту. Крім того, акцентовано увагу на анти-
бактеріальні властивості хітозану, використанні наночастинок
хітозану для отримання нановолокон і створенні системи
контрольованої доставки ліків.
К л юч ов і с л ов а: хітозан, гідроксиапатит, біокомпозити,
рентгенівська дифракція.
Хитозан-апатитные композиты: синтез и свойства
Л. Ф. Суходуб, Л. Б. Суходуб, И. В. Черная
Цель этого краткого обзора – рассмотрение применения уни-
кального биополимера хитозана в практической медицине,
особенно для инженерии костной ткани. Основное место в
стать е отведено синтезу и свойствам инновационных биомате-
риалов на основе хитозана, таким как CaP-хитозан (CS-CP)-
композиты и хитозан-альгинатные (CS-AG)-скаффолды.
В статье освещены физико-химические свойства, спектраль-
ные характеристики и химические модификации молекулы
хитозана. Полученные хитозан-апатитные композитные мате-
риалы были проанализированы с использованием метода рент-
геновской дифракции для анализа кристаллической природы
их структур. Было обнаружено, что добавление хитозана к
композитному материалу приводило к снижению кристаллич-
ности исходного апатита. Кроме того, акцентировано внима-
ние на антибактериальных свойствах хитозана, использовании
наночастиц хитозана для получения нановолокон и создании
систем контролируемой доставки лекарств.
К л юч е в ы е с л ов а: хитозан, гидроксиапатит, биокомпози-
ты, рентгеновская дифракция.
Received 21.02.2016
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