Synthesis and Characterization of Hydroxyapatite-chitosan Nanocomposite Materials for Medical Engineering Applications

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Synthesis and characterization of hydroxyapatite/chitosan nanocomposite

materials for medical engineering applications

M.R. Nikpour a,b, S.M. Rabiee a,c, M. Jahanshahi a,b,⇑

a Nanotechnology Research Institute, Babol University of Technology, Babol, Iranb Faculty of Chemical Engineering, Babol University of Technology, Babol, Iranc Faculty of Mechanical Engineering, Babol University of Technology, Babol, Iran

a r t i c l e i n f o

Article history:

Received 9 July 2011Received in revised form 7 October 2011Accepted 13 January 2012Available online 2 February 2012

Keywords:

A. Nano-structuresA. Polymer–matrix compositesA. Mechanical propertiesD. Electron microscopy

a b s t r a c t

Incorporationof hydroxyapatite(HA) withorganicpolymerin favorof compositeswouldbe usedin bioma-terial engineering. Accordingto prior researches, because of itschemical similarity to natural boneand den-tal,thisproduct could improvebioactivity and bonebondingability. In thisresearch, nano-hydroxyapatite/chitosan compositematerial was prepared via in situ Hybridization route. The surface chemical character-ization on the nanocomposite was evaluated by Fourier transformed infrared (FTIR) and X-ray diffraction(XRD). Surface topography, roughness and morphology of the samples were observed by atomic forcemicroscopy (AFM) and scanning electron microscopy (SEM). The characterizationresults confirmedhomo-geneity, interaction and integration between the HA and chitosan matrix. It was indicated that compositesamples consist of homogeneous aggregations around 40–100 nm, in which many HA nanocrystals alignalong the chitosan molecules. HA grain gradually decreased in size when amount of chitosan increasedfrom0 to 6 g into 100 cc solution. It can be seen that by increasing chitosan, the aggregation of nanoparti-cles enhance and subsequently, improve the expected compatibility among HA filler and chitosan matrix.Furthermore, the mechanical compressive testing indicated that the synthesized composites have accept-ablemechanical behaviorfor tissue substitution. Themechanistic of the biodegradablenanocompositesys-

tems, their preparation and characterization for medical usage are strongly discussed. 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Hydroxyapatite (HA,Ca10(PO4)6(OH)2) is an interesting biomate-rialwithpotential orthopedic, dental, andmaxillofacial applicationsdue to its excellent biocompatibility, bioactivity, and osteoconduc-tivity [1]. HA powders, used for the treatment of bone defects, havethe problem that they easily migrate from the implanted sites. Also,in Compared to pure HA, polymeric nanocomposites are exhibitedimproved properties, such as modulus, strength, and stiffness[2,3]. Therefore, novel composites of HA and organic polymers thatcancompensate for the weak mechanical properties of HA have be-come of great interest [4,5]. Chitosan, (C6H11O4N)n is an N-deacety-lation product of chitin and is a unique polysaccharide basedbiopolymerthat shares a numberof chemicaland structuralsimilar-ities with collagen and has been used as a skin grafting template,hemostatic agent, DNA and drug delivery vehicle, and as a woundhealing material [6–11]. Also, chitosan (CTS) films support thegrowth, function, and cellular activity of osteoblasts and chondro-

cytes [12]. Inorganic–organic composite materials are increasinglyimportant due to their extraordinary properties, which arise fromthe synergism between the properties of the components. TheseHA/chitosan nanocomposite has been developed as a good candi-datefor tissue engineering, particularly boneand cartilage scaffolds[13,14]. HA particles have been incorporatedinto chitosan matricesto enhance the bioactivity of tissue engineering scaffolds for hardtissue regeneration [15–21]. In addition, biomimetic inspirationfrom natural chitin composites has been used to study biominerali-zation phenomena in the formationof chitin based nanocompositeswith silica, calcium carbonate, and HA [17,22]. Incorporation of HAwith chitosan, the mineral component of bone, could improve thebioactivity andthe bonebonding abilityof the HA/chitosancompos-ites [23]. Biomaterial scientists had paid their attention to investi-gate HA/chitosan composites with different HA content ranging,such as quick hardening past for bone repair [24,25], porous HA/chitosan scaffoldfor tissue engineering[26]. Moreover, results fromvarious papers exhibited that chitosan vast applied for HA surfacemodify and have an excellent benefit [27,28].

In this study, the preparation of hydroxyapatite/chitosannanocomposites using in situ hybridization method is attempted.However, evaluation and characterization of the products includingmorphology roughness, the effect of enhancement various

1359-8368/$ - see front matter 2012 Elsevier Ltd. All rights reserved.doi:10.1016/j.compositesb.2012.01.056

⇑ Corresponding author at: Nanotechnology Research Institute, Babol Universityof Technology, Babol, P.O.BOX: 484, Iran. Tel./fax: +98 111 3220342.

E-mail addresses: [email protected], [email protected] (M. Jahanshahi).URL: http://nano.nit.ac.ir/IndexEn.aspx (M. Jahanshahi).

Composites: Part B 43 (2012) 1881–1886

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Composites: Part B

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component of chitosan on surface, dispersion, structural and parti-cle size nanoparticles-polymer matrix are investigated. In addition,mechanical behaviors of products are considered as well.

2. Materials and methods

2.1. Chemicals materials

Chitosan from crab shells with a degree of deacetylation >85%,were purchased from Sigma–Aldrich Co. Acetic acid (HAc, 99.8%)obtained from Merck. Poly (ethylene glycol) (PEG) with moderatemolecular weight of 400 that, was used as a agent in calcium ni-trate tetrahidrate (Ca(NO3)24H2O) solution to control the particlegrowth during precipitation, Cetyltrimethylammonium bromide(CTAB) that, has been used as a moisturing agent, Calcium nitratetetrahidrate (Ca (NO3)24H2O), diammonium hydrogen phosphate((NH4)2HPO4) and sodium hydroxide (NaOH), were supplied byMerck, respectively.

2.2. Preparation of hydroxyapatite/chitosan nanocomposites

Chitosan was dissolved in 2% (v/v) acetic acid solution to obtaina polymer solution. Different composition of chitosan aqueoussolution was prepared by dissolving the different amount chitosanpowder into 100 cc distilled water containing acetic acid and stir-red for 24 h until a good suspension was achieved. Samples of thisexperiment consist of 0, 2, 4, 6 g of chitosan which are named asHC0, HC2, HC4, and HC6. All solutions were taken in such amountthat Ca/P molar ratio was maintained at 1.67. Then Ca(NO3)24H2Oof 0.5 M solution added in chitosan solution and vigorously stirredat room temperature with a mechanical stirrer (2500 rpm). Subse-quently, (NH4)2HPO4 suspension of 0.5 M were added drop wiseinto the prior mixture under extreme stirred until the better sus-pension was achieved. Stirring was continued up for another24 h to obtain a homogeneous polymer solution. The resulting

solution was held unstirred for another night to remove the airbubbles trapped in viscous liquid. In whole process, the pH valuewas adjustedto about 11 by using 2 M NaOHsolution and the reac-tion temperature was kept at 25 C. Finally the white gelatinousprecipitate was separated by centrifuging the suspension at3000 rpmfor 30 min (Z-36HK Hermle, Germany). Then the mixturewas dried in a freeze-dryer (FDE-0350, Korea) at 50 C for 48 h tomake powder. The schematic diagram of the experimental setup isillustrated in Fig. 1.

2.3. Characterization

2.3.1. X-ray diffraction and FTIR analysis

The crystal structure and the phase present in resulting pow-

ders were analyzed with X-ray diffraction (XRD). This instrument(Philips PW 3710) works with voltage and current settings of 35 kV and 28.40 mA respectively, and uses Cu Ka radiation(1.5405 Å). For qualitative analysis, XRD diagrams were recordedin the interval 20 6 2h 6 45 at scan speed of 2/min. The meancrystallite sizes ‘‘D’’ were determined according to the Scherrerequation that is, D = 0.9k / b cosh, where D is the average crystallitesize in A, b is the peak broadening of the diffraction line measuredat half of its maximum intensity in ‘‘radian,’’ k is the wavelength of X-rays, and h is the Bragg’s diffraction angle [23]. The structure of obtained product was determined by FTIR spectrum. These prod-ucts were analyzed by FTIR analysis (Perkin Elmer, USA) withinscanning range of 400–4000 cm1. The FTIR spectroscopy of thisdevice has provided valuable information regarding the formation

of HA powder and HA/chitosan nanocomposites. FTIR analysis hasbeen done to identify the characteristic peaks of product.

2.3.2. Scanning electron microscopy

Scanning electron microscopy (SEM, Hitachi S-4160) was usedto characterize the morphology and particle size of nanocompos-ites materials. Prior to imaging, the samples mounted on alumi-num stubs were gold coated for better conductivity.

2.3.3. Atomic force microscopy

Atomic force microscopy (AFM) images show the surface rough-ness, morphology and dispersion of powders were obtained with a JEOL 6330F field emission atomic force microscopy.

2.3.4. Mechanical testing

Compression tests were performed at room temperature by auniversal testing machine (zwich/roell 2005) at a crosshead speedof 0.03 mm s1 in the pressure of 10 Bar. The specimens wereshaped into circular discs of approximately 5 mm diameter and

Fig. 1. Scheme of nanocomposite preparation by in situ hybridization.

Fig. 2. X-ray diffraction of synthesized composites with different amount of chitosan ratio.

1882 M.R. Nikpour et al. / Composites: Part B 43 (2012) 1881–1886



5 mm height. Elastic modulus was calculated as the slope of theinitial linear portion of the stress–strain curve. The specimens ateach sample were tested, and the data represented the average va-lue of three measurements.

3. Results and discussion

3.1. Crystal structure analysis and FTIR absorbance of nanoparticles on

HA

Fig. 2 shows the X-ray diffraction pattern of the pure HA andHA/chitosan nanocomposite samples. The use of synthesizedn-HA powder with the CTS polymer as a matrix thus provides an

effective means to produce nanocomposites. For HA nanoparticles,the existence of 2h peaks at approximately 23.2, 26, 29.3 and32.2 corresponding to the diffraction planes (211), (002), (210)and (30 0) of the HA crystallites, respectively, that confirms the for-mation and presence of HA in products. These peaks refer to the b-TCP, HA, none reacted Ca(NO3)2 and b-TCP dual with HA together,respectively. It can be seen that crystalline structure refers in pre-pared products by in situ hybridization method. After compositesformation, crystallization of the n-HA particles still existed, mean-while has decreased as shown in Fig. 2 that indicating the crystal-line characteristic peaks of HA were not alteration significantly,which could be resulted from the interface binding between parti-cles and matrix. It is also noted that peaks in the precipitated HA/chitosan nanocomposites became slightly broader and weaker ascompared to the pure HA nanoparticles. The XRD peaks, (211),(002), (210) and (300) have shifted to higher 2h values in caseof HA/CTS composites as compared to pure HAp (i.e., from2h = 23.2–23.8, from 26 to 26.4, from 29.3 to 30.2 and from 32 to32.8 in composite samples resp.), which these are possibly due tocompression from the contracting polymeric matrix through inter-facial bonding. It seems the shift and decrease in crystallinity of each peak of the polymer as well as HA after composite formationclearly indicates the presence of bonding between HA particles andpolymer matrix. The peak at 32.2 (300) of n-HA is weakened aftercomposite formation, which also indicates the participation of n-HA in bonding with the polymer. It could be due to molecularinteractions between HA and chitosan as well. Taking into accountthe broadening of each peak in XRD, mean crystallite size has beencalculated using Scherrer’s equation that the approximate crystal-lite sizes of HC0, HC2, HC4, and HC6 samples are found to be aboutless than 50 nm.

Fig. 3 displays the FTIR spectra of various HA/chitosan compos-ites and pure HA samples. The absorption bands observed around

Fig. 3. FTIR spectra of (a) HC0, (b) HC2, (c) HC4 and (d) HC6 samples.

Fig. 4. SEM micrograph of (a) HC0, (b) HC2, (c) HC4 and (d) HC6 composites.

M.R. Nikpour et al./ Composites: Part B 43 (2012) 1881–1886 1883



2917, 1642, 1456, and 1552 cm1 were assigned to methylene(CH2), amide I (C@O), amino (NH2), and amide II (ANH) in nano-composite, respectively. The absorption bands observed aroundat 1099, 952, 839, and 563 cm1 correspond to a phosphate groupin HA while the stretching band (approximately between 2900 to3700 cm1) were assigned to OH group in HA. As carbonate bandsaround at 1457, 1433 and 840 cm1 also was observed. A compar-ison of FTIR analysis for different samples was confirmed the effec-tive existence of HA nanoparticles surrounded at nanocomposite.The feature bands for HPO42 were assigned at 1131 cm1,

1062 cm1, 988 cm1, 876 cm1, 576 cm1, 528 cm1. It seemsthe magnitude of these bands becomes weaker and finally disap-pears with the development of in situ hybridization. Furthermorecharacteristic bands for PO4

3 appeared at 963 cm1.

3.2. Morphological studies

Fig. 4a–d indicate the SEM micrographs of fracture surface of the pure HA and composite samples with different contents of chitosan. The composite samples approach exhibited same

Fig. 5. AFM images of (a) HC0, (b) HC2, (c) HC4 and (d) HC6 samples.




morphology and homogenous distribution of such kind of HA par-ticles within the chitosan matrix that visible in this Fig. 4b–d. Themicrographs show that with increase in amount of chitosan load-ing; the surface roughness for the composites is almost increased.As can be seen in the SEM image Fig. 4b–d, many elliptic aggrega-tions are found in the composite. Furthermore, in the respectiveaggregate, there exist many small HA crystallites of 40–100 nm.On the other hand, SEM clearly showed that the nanoparticles ex-hibit a uniform size distribution and spherical inorganic mineralsare dispersed within polymeric matrices.

Obviously, the particle sizes in these composites are quite uni-form that may significant surface effective consociation or cover-ation chitosan. Maybe could gathered these harvest recognized tothe surface improvement that modifies the solubility of the nano-particles in the organic solvents, and prevent from augment parti-cles agglomeration. Fig. 5a–d were used to determine surfacetopography and illustrate the surface properties of pure HA andnanocomposites in 3D and 2D topographies. The figures exhibitdispersed nanoparticles in the polymer matrix. As can be seen,pure HA has a relatively smooth and uniform surface. In thisFig. 5b–d, HA/chitosan composites presents some different mor-phology as compared with that of pure HA. It can be seen; theHA particles are surrounded and embedded into the polymer chainof chitosan. Indicating the HA particles has a core effect on thepolymerization of composites. It seems penetration of HA particlesinto the chitosan matrix leads to similar formations of aggregatesand agglomerates on the surface of composites. In the other hand,the existence of these particles increases surface roughness negli-gibly along with the AFM images. The micrograph shows the pres-ence of agglomerations among the particles, which can be resultedfrom the interface binding between HA particles and the matrix,that it seems due to high specific surface energy of n-HA particlesresulting in their aggregations too. Nanosized HA particles withhomogeneous dispersion are well identified in case of HA/CTScomposites. It give the impression the particle size of HA is alsocontrolled by the biopolymer matrix in composite as depicted in

the AFM micrograph. According to the obtained results, to add var-ious content of chitosan at the surface roughness of synthesizedproducts make no diversity and from this achieved results it wouldbe understood that the place which maximum value of chitosanexisted, the most present of aggregation occur on the nanocompos-ite surface as well. we can see the HA particles in triple chitosannanocomposites are covered with a continuous smooth chitosanlayer and the particle sizes of composites are almost smaller dueto the increasing values of chitosan.

3.3. Mechanical testing of n-HA/CTS composites

For bone tissue engineering materials, the primary mechanicalproperties are usually imperative criterion in selecting the bonesubstitute materials. Hence, the compressive strength and com-pressive modulus of these synthesized materials were given inTable 1. From the records, it could be found that the compressivestrength of the composites had a relation to the chitosan contentin composites. The reason may be the large amount of n-HA whichcould reduce the degree of mechanical properties strength so that

the structure of matrix may be inadequate to combine the higherproportion n-HA particles, resulting in reduced mechanicalproperties. The compressive strength of HC2, HC4 and HC6 are11.45 ± 1.41, 14.47 ± 1.96, and 12.25 ± 2.70 MPa, respectively andthe average values of compressive modulus of these compositeswere found to be 1470± 162, 1265± 223 and 1163± 251 MParespectively. However, the compressive strength of HC4 was themost acceptable (14.47 ± 1.96 MPa) when compared with twoother composites. This indicated amount of CTS in the compositeis optimized but compressive modulus of HC2 is more and subse-quently better than other indicated composites. It seems severalfactors can contribute to the observed mechanical response of these nanocomposites, such as particle size and particle size distri-bution of hydroxyapatite, the mechanical properties of biopolymer,the interfacial interactions between biopolymer and hydroxyapa-tite, and good distribution of n-HA in chitosan. The elastic moduliof specimens become weaker when the chitosan interaction withbioceramics and it will be good results for bone substitute materi-als. One example of the influence of an elasticity high modulus of an implant material on surrounding bone is the dramatic bone lossaround certain joint replacement prostheses. This bone loss hasbeen attributed to the stress shielding resulting from the large dis-parity between the stiffness of the implant and the host bone [29].It might be concluded that these composites can better supportnew bone tissue regeneration at the site of implantation and main-tain sufficient integrity for bone substitute.

4. Conclusions

Nanocrystalline composites with various hydroxyapatite/chito-san ratios were successfully synthesized by in situ hybridizationmethod. Structural, morphology, micrograph and chemical compo-sition of this synthesized powder were characterized by XRD, AFM,SEM and FTIR, respectively. Chitosan indicate strong adsorptioninteraction with this calcium phosphate material. We understandin the obtained composites, HA nanoparticles well pervaded inthe matrix and from SEM and AFM, it was indicated that compositesamples consist of homogeneous aggregations around 40–100nm,in which many HA nanocrystals align along the chitosan molecules.This homogeneous distribution of inorganic nanoparticles revealsstrong interfacial interaction between the nanoparticles and thepolymer matrix. With the indicated results in compressivestrength in discussion above, it can be concluded that with incor-poration of HA nanoparticles in biocompatible matrix, mechanicalstrength of composite products are increased as well. To the best of our knowledge, the current paper is one of the first discussing po-tential biodegradable nano-hydroxyapatite/chitosan composite formedical engineering usage and deserves further study. Optimiza-tion of this fabrication method for manufacturing such nanocom-posite and adding the third component for creating the better

nanoproduct will be the subject of our next publication.

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Table 1

Compressive strength and compressive modulus of synthesized determined from

compression experiments.

Sample Compressive strength (MPa) Compressive modulus

HC2 11.45 ± 1.41 1470 ± 162HC4 14.47 ± 1.96 1265 ± 223

HC6 12.5 ± 2.70 1163 ± 251

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Synthesis and Characterization of Hydroxyapatite-chitosan Nanocomposite Materials for Medical Engineering Applications