Chitosan-apatite composites: synthesis and properties

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


Introduction
Over the last years, chitosan (CS) has attracted a great interest of scientists as functional polymeric material because of its remarkable intrinsic properties: biodegradability, biocompatibility, nontoxicity, antibacterial activity, mucoadhesive, analgesic and haemostatic properties [1][2][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-Dglucan (D-glucosamine) units [1,[4][5][6].Chitosan can be easily obtained from a natural polymer chitin after 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 immune response and enhance antigen presentation [10].The combination of CS with different materials, such as hydroxyapatite (HA), is very promising, especially for orthopedics and traumatology [11].All these features make chitosan an outstanding candidate 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.000910deacetylation (DD) (N-acetylglucosamine / glucosamine) 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 solutions with pH > 7 whereas in dilute acids (pH < 6) it becomes soluble due to protonation of NH 2groups [5].Chitosan's primary amines confer important material properties.At low pH, the amines are protonated making chitosan a cationic polyelectrolyte.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][14][15][16].At high pH, the amines are deprotonated and chitosan undergoes a transition from a soluble cationic polyelectrolyte to an insoluble polymer.Insolubility of chitosan in water means that the intermolecular interactions between macromolecules exceed the interactions in the system of "chitosan-water molecules".Importantly, this pH-responsive switch is near neutrality (chitosan's apparent 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] 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 connections through chelation mecha nisms.Fig. 2 illustrates 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 systems [4].The cationic nature of CS provides the electrostatic interaction with quantivalent linear anionic polysaccharides (glycosaminoglycans (GAG), proteoglycans etc.).This factor is very important because 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 accumulation of these necessary biopolymers in vivo.Thus, chitosan based scaffolds deliver growth factors 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 crystallinity 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 technology ("freeze-drying").A pore size in the CS hydrogel 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][24][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 promote a cell growth and the formation of mineral matrix by osteoblasts [26].Biocompatibility of chitosan minimizes the local inflammation, and its conversion into a porous structure contributes to osteoconductivity [27].The Chitosan -Calcium Phosphate composites (CS-CP) were the subject of intensive study in the world [28][29][30][31].

CS-CP composites
Biomaterials that mimic the structure and composition of bone tissues at the nanoscale level are extremely important for the development of bone tissue engineering applications [32].CS-CP compo sites have certain advantages compared to other similar structures.Thus, during the resorption, the degradation products of chitosan and calcium phosphate (calcium ions, phosphates, glycosamines, etc.) are naturally metabolized and do not induce the increasing calcium and phosphorus concentrations in urine, serum or internal organs.A composite material contains both the macro-and micropores and nano-sized crystals of HA.This promotes an increase in the reactive surface and material osteoconductive activity.Similar 3D-macroporous ceramics pierced by chitosan grid have better mechanical properties [33,34]; therefore, there is a perspective of the CS-CP composites future use in the clinic.The main advantages of CS-CP biomaterials are: structural organization, which is close to the structure of natural bone, biocompatibility, biodegradation, macro-and microporosity, regulation of resorption rate, immobilization of drugs, antibacterial action, simplicity of flowsheet synthesis, low cost.
Among a wide range of calcium phosphates hydroxyapatite (HA), Ca 10 (PO 4 ) 6 (OH) 2 is a widely used material for biomedical application in dentistry and orthopedy due to the excellent bioactivity, biocompatibility and osteoconductivity [31,35].However, the migration of HA powder from implanted sites and bone defects is really a big problem.Therefore, organic compounds in nanocompo-sites are promising for improving weak mechanical properties of HA [36].
The CS-HA composites have been recently synthesized 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(CH 3 COO) 2 , sodium dihydrogen phosphate NaH 2 PO 4 , and sodium 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 % CH 3 COOH (first way of synthesis) or was added in solid state to chitosan solution in 1 % CH 3 COOH (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 (CH 3 COO) 2 Ca stock solution in 1 w % CH 3 COOH.The final chitosan concentration in the reaction mixture was 0.4 w %.The calcium acetatechitosan solution was stirred in a shaker (160 rpm) for 1.5 h at 37 ºC, then 20 mL of 0.1 M NaH 2 PO 4 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 o C and annealed for 1 h at 900 o C. The second way of synthesis was provided by addition of solid (CH 3 COO) 2 Ca (0.526 g) to the 20 mL of the 0.4 w % chitosan solution in 1 % CH 3 COOH.The calcium acetate-chitosan solution was stirred in a shaker (160 rpm) for 1.5 h at 37 ºC, after which 20 mL of 0.1 M NaH 2 PO 4 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 centrifuging the suspension.For the analysis the product was dried at 37 o C and annealed for 1 h at 900 o C. 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 computeraided system for the experiment control and data processing.The Ni-filtered CuK α radiation (wavelength 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 respectively.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 determined 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 patterns 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 synthesis way.The existence of 2θ peaks at approximately 26º, and 31.9ºcorresponding 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 crystallite 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 increased for all HA-CS composite samples indicating significant defects in the hydroxyapatite crystal structure caused by the presence of the polymer.
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 analysis 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 frequency 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), CH 2 wagging coupled with OH in plane deformation at 1324 cm −1 .The peak at 1598 cm -1 belongs to the bending vibra-  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 -NH 2 asymmetric stretching vibrations [41], the peak at 2868 cm -1 is caused by -CH 2 -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 contribution of N-H bending) together with N-H bending 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 -CH 2wagging coupled with -OH in plane deformation appears 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 -CH 2 -and -CH 3 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 -1 and 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 chitosan amine functionalities is suggested by the presence of the band, attributed to NH 3 + 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 phosphates.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 hydrogen bonds with participation of ether and/or -OHgroups 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 absent in the HA-CS spectrum.All these facts confirm molecular interactions between CS and HA in the HA-CS composite.

Chitosan-alginate-hydroxyapatite polyelectrolyte composites
The chitosan-and sodium alginate (AG)-based scaffolds have been widely used as biomaterials in tissue engineering.Freeze-drying, also known as lyophilization, has been used for the fabrication of polymerbased hydrogels.The porous scaffolds composed of AG and CS have been fabricated by the formation of a polyelectrolyte complex (PEC) between macromolecules of both polymers [46].They perform a 3D-grid with uniformly distributed and interconnected pores.Two technologies of the scaffold formation 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 % CaCl 2. By the second technology, the alginate scaffold was immerged into 2 w % CS solution or the CS-HA composite acetic acid aqueous solution.In this case the pores of the alginate scaffolds were filled with chitosan solution.These samples were frozen at -40 °C and lyophilized.Finally, the samples were additionally crosslinked with CaCl 2 solution.
Chitosan-alginate (CS-AG)-scaffolds exhibit better mechanical properties and thermostability than Chitosan-apatite composites: synthesis and properties AG-scaffold, crosslinked with calcium ions only.The main reasons for the mechanical strength improvement of crosslinked scaffold could be strong ionic interactions.In the Ca 2+ crosslinked AG scaffold the mechanical strength is enhanced because Ca 2+ is characterized by strong ionic interaction with COO -in an alginate chain.For CS-AG PEC scaffold the strong interaction exists between NH 3 + groups in CS and COO -groups in alginate (Fig. 4).Additionally, a decrease of porosity for crosslinked scaffold is another 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, chondroitin sulfate, hyaluronic acid, heparin, carboxymethyl cellulose, pectin and proteins such as gelatin, albumin, collagen and keratin [47][48][49][50][51][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 synthesized in our laboratory using our own flowsheet.Such precursors were used for the synthesis: chitosan (MW 500 kDa, DD 80%, "Bioprogress", Moscow), sodium alginate (E401), sodium hydrogen phosphate (Na 2 HPO 4 ) and calcium acetate (CH 3 COO) 2 Ca•H 2 O (China).500 ml of chitosan solution (2 g/l in 1 w % acetic acid) were gradually added to 0.1 M calcium acetate (100 ml); 1 g of sodium alginate powder was dissolved in 0.1 M sodi-um hydrogen phosphate (60 ml).Two obtained solutions 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 7.0-7.4with 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 components 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 creation and stabilization of the biopolymer scaffolds [31].

X-ray structure analysis of apatite-biopolymer composites
Recently the usage of materials in the form of hydrogels has become increasingly popular.The structure of the polymer chains that form a three-dimensional net of gel enables immobilization and sufficient content 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 alginate -in the sample 2.

Fig. 4. Chemical structures of CS (top) and AG (down) and the scheme of their interaction
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 CaCl 2 , pH was adjusted up to 11.0 by addition of 10 M NaOH solution; 2) chitosan (MW 39 kDa) was dissolved in 100 ml of 0.06 M H 3 PO 4 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 followed with mixing and heating at 60 °C during 10 min.The pH value was adjusted by NaOH addition to 7.4.After aging for 24 h the resulting suspension was washed with distilled water and centrifuged.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 %, 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 H 3 PO 4 (0.06 M), anhydrous calcium chloride CaCl 2 (0.1 M), aqueous solution of sodium hydroxide NaOH (10 M) and sodium alginate (E401, MW 15 kDa, China) were used as starting materials for the HA-AG composite preparation.The composite material was synthesized by the "wet chemistry" method: 1) sodium alginate was dissolved in 100 ml of 0.06 M H 3 PO 4 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 chloride 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 sterilized.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 annealed 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 synthesis of HA nanoparticles in the presence of polymer the size of crystallites decreased compared to pure HA in samples dried at 37 °C.An average size of crystallite increased after annealing and combustion 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 temperature 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].

Antibacterial properties of silver-doped HA-CS composites
One of the most important problems in modern implantology 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 electrostatic bonds with anionic molecules on the cell surface 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 NH 2 -groups chitosan 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.♦ -assigned CaO phase; B) X-ray diffraction patterns of HA-AG nanocomposites dried at 37 °C and annealed for 1 h at 900 °С

B)
Most silver-containing antimicrobial biomaterials consist of either Ag + ions (silver salts or silver complexes) or elemental silver (Ag-nanoparticles) incorporated into organic (polymers) or inorganic (bioglasses and HA) matrices [62,63].The silver-loaded HA composites are obtained by ion-exchange methods (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 produced from nanoparticle surface but also by the interaction of small active nanoparticles with the microorganisms cells and destruction of their membranes [65][66][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-modified 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 immersing the experimental samples into these suspensions.The optical density was measured by spectrophotometry at l=540 nm 2, 24 and 48 h after the sample immersion.The bacterial cell amount was evaluated 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 promising method to create antimicrobial coatings for titanium 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 solubility 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 dissolving poorly soluble pharmaceuticals in the synthesis of mucoadhesive mixtures [69][70][71], and for enhancement of the peptides absorption] [72][73][74].Nowadays, various types of systems (tablets, capsules, 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 properties 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 duct, etc.).The main problem in the drug delivery therapy is to provide the necessary drug concentration at the destination [75].One of the modern approaches is the use of CaP-biopolymer scaffolds, including polysaccharides of chitosan and alginate in combination with hydroxyapatite, as above discussed.It was shown that chitosan coatings on alginate scaffolds enhance osteoblast adhesion and proliferation [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 (C 14 H 22 N 2 O), which is a local anesthetic drug, into physiological solution with phosphate buffer (PBS) [75].According to FTIR spectroscopy, no chemical reactions have been registered 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 activity of chitosan-metal complexes compared with pure chitosan [77].Such complexes were obtained in two stages.First, the nanoparticles of chitosan were produced by ionic gelation between chitosan and sodium tripolyphosphate (Na 5 P 3 O 10 ), then the obtained nanoparticles were connected ("loaded") with metal ions (Ag + , Cu 2+ , Zn 2+ , Mn 2+ , Fe 2+ ) [77].Antibacterial activities of the metal ions doped chitosan nanoparticles, 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 minimum 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 visible growth on a Muller-Hinton (MH) agar plate (MH was used as a growth media) and incubated at 37 o C for 24 h.MBC was determined as the minimum sample concentration that did not cause any bacterial growth.Antibacterial properties of the chitosan nanoparticles have been significantly improved by the metal ions addition.For example, for the Cu 2+ 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 Cu 2+ ions [77].The report states that Gramnegative bacteria are more sensitive to chitosan-metal 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 medicine is the development of natural methods for obtaining 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 negatively charged base plate material (any substance, a metal substrate, etc.).
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 electrical 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 perspective in the effective treatment of burns and similar injuries [78].

Conclusion
Chitosan characteristics considered in this review indicate a significant potential for its use as a biomaterial 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 molecular weight, viscosity, should be further examined to exploit the potential of this natural polysaccharide in nanomedicine.дено 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.