International Journal of Biological Macromolecules 73 (2015) 170–181

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Chitosan–nanohydroxyapatite composites: Mechanical, thermal and bio-compatibility studies Pratik Roy, R.R.N. Sailaja ∗ The Energy and Resources Institute (TERI), SRC, Bangalore 560071, India

a r t i c l e

i n f o

Article history: Received 11 September 2014 Received in revised form 4 November 2014 Accepted 5 November 2014 Available online 2 December 2014 Keywords: Bionanocomposite Nanohydroxyapatite Mechanical properties Cytocompatibility Apatite formation

a b s t r a c t Bionanocomposites of chitosan were prepared with nanohydroxyapatite (nHA) using 2-hydroxyethyl methacrylate (HEMA) as coupling agent. The tensile and flexural properties for 8% nHA loading showed optimal values. Compressive modulus also considerably increased from 525.16 MPa (0% nHA) to 1326.5 MPa with 10% nHA. Surface functionalization of fillers along with the addition of HEMA as coupling agent led to enhanced mechanical properties similar to human bone. The mechanical properties were further analyzed using micromechanical theories which indicated good interfacial adhesion between the matrix and fillers. The composites showed cytocompatibility. Multiple layers of apatite formation have been observed when the nanocomposites were soaked in simulated body fluid (SBF). Hence, these composites showed potential for bone substitute applications. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The need for a benign bone substitute is becoming a major need especially for elderly population with various bone defects. This requirement needs: (a) material matching in chemical composition with natural bone; (b) biocompatibility and mechanical strength similar to human bone. Extensive studies on hydroxyapatite are being carried out as it is a well-known material for biocompatibility and osteoconductivity with a chemical structure and composition matching the human bone and hence can mimic the same [1]. However, hydroxyapatite alone has certain shortcomings such as poor load bearing properties, difficulty to cast into the desired shape and its tendency to migrate from the implanted sites [2,3]. Hence, another biocompatible, natural and abundant biopolymer such as chitosan has been combined along with hydroxyapatite to develop bone substitute composites. Chitosan is a linear polysaccharide derived from partial deacetylation of chitin [4,5]. The ability of chitosan to support cell attachment and proliferation is attributed to its chemical properties. The polysaccharide backbone of chitosan is structurally similar to glycosaminoglycans, the major component of the extracellular matrix of bone and cartilage [6]. Chitosan is considered as an appropriate functional material for biomedical applications because of high biocompatibility, biodegradability, non-antigenicity and adsorption properties [7,8]. Further, anti-inflammatory or allergic reactions have not been

∗ Corresponding author. Tel.: +91 80 25356590. E-mail address: [email protected] (R.R.N. Sailaja). http://dx.doi.org/10.1016/j.ijbiomac.2014.11.023 0141-8130/© 2014 Elsevier B.V. All rights reserved.

observed in human subjects following topical application, implantation, injection and ingestion [7,8]. An updated review article by Pighinelli et al. [9] suggested the need and importance of natural biopolymer such as chitosan–hydroxyapatite composites which can play a vital role in skeletal reconstruction. However, natural biopolymers such as chitosan have poor load bearing characteristics with rapid degradability. Mechanical properties especially compressive strength is important to tolerate the internal stress till tissue regeneration takes place. Han et al. [10] suggested that alginate–chitosan–hydroxyapatite composite exhibited enhanced mechanical strength due to strong ionic interactions. Further efforts to enhance mechanical strength using biocompatible materials like titania was carried out by Kavitha et al. [11]. It was found that the nanocomposites thus developed showed large surface area with good antibacterial activity. An improvement in compressive strength and Young’s modulus was observed by adding a small amount of citric acid due to salting out effect [12]. It has also been envisaged that enhanced chemical bonding with the inorganic material such as nHA will restrict its migration and also reduce tissue damage. Thus, nHA has been blended with chitosan and gelatin to improve mechanical properties [13]. A drastic reduction in mechanical properties was observed for chitosan/hydroxyapatite composites as nHA loading increased [14]. Chitosan is a brittle material and hydroxyapatite is also brittle, thus a combination of the two further reduces the mechanical properties. Ai et al. [15] studied the effect of micro and nanosized hydroxyapatite particles in chitosan–starch composites. The composites loaded with nanosized particles showed increased modulus values.

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In the present study, an attempt to enhance the mechanical properties of chitosan–starch composite reinforced with nHA and very small amount of nano carbon fiber (NCF) has been carried out. Biocompatible material i.e. HEMA has been added as a coupling agent to enhance interfacial bonding. Enhanced interfacial adhesion due to addition of HEMA will lead to better interaction between aminosilane functionalized nHA and chitosan, thereby leading to better mechanical strength. The bio-nanocomposites thus developed have been characterized for mechanical, thermal, water absorption and in vitro biocompatibility and osteogenicity properties. 2. Experimental

171

thus prepared were kept in zip-locked plastic packets and labeled for further processing. 2.5. Compression molding The nanocomposites were prepared by using compression molding machine (Compression Molding Press, Santec, India). Thoroughly mixed nanocomposites powder were placed in mold covered with polished stainless steel plates and then compression molded at 200 ◦ C under the pressure of 18 MPa. The heating time was kept at 10 min and the curing time at 5 min. After curing, the molds were cooled to room temperature before releasing the pressure for demolding. Sheets of dimensions 15 cm × 15 cm × 0.4 cm were obtained.

2.1. Materials Chitosan powder used in this study was obtained from Marine Chemicals, Cochin (India) with 85% deacetylation. nHA was procured from J.K. Impex, Mumbai (India). Tapioca starch used in this study was purchased from Natsyn Catalysts, Bangalore (India). HEMA monomer was purchased from M/s. Tech Dry India Pvt Ltd., Bangalore, India. NCF and 3-aminopropyl triethoxy silane (APTS) was purchased from Global Nanotech, Mumbai (India). All other chemicals were purchased from S.d.Fine Chem, Bangalore (India) and were used as received.

3. Characterization

2.2. Silane treatment of nHA and NCF

3.2. X-ray diffraction (XRD)

10 g of nHA powder along with 300 ml of DMF were introduced into a two necked round bottom flask. The mixture was stirred at reflux temperature (153 ◦ C) under nitrogen atmosphere for 45 min, followed by drop wise addition of 5 ml of APTS. The reaction was carried out in a locally fabricated microwave reactor at 80 ◦ C (Enerzi Microwave Systems, India) for 1 h under reflux. After reaction, the modified nHA was separated by centrifugation (8000 rpm, 5 min) and washed with chloroform and absolute alcohol. The resulting modified nHA powder was dried in a vacuum oven at 60 ◦ C for at least 24 h before use. Similar silane surface treatment was given to NCF to enhance dispersion and compatibility with the other components of the nanocomposites.

X-ray diffraction measurements (XRD) for the composites have been performed using advanced diffractometer [PANalytical, XPERT-PRO] equipped with Cu-K␣ radiation source (X = 0.154 nm). The diffraction data were collected in the range of 2 = 2–60◦ using a fixed time mode with a step interval of 0.05◦ .

2.3. Preparation of crosslinked thermoplastic starch

3.1. Fourier transform infrared spectroscopy (FTIR) The Fourier transform infrared spectroscopy (FTIR) spectra of nanocomposites were recorded between 600 and 4000 cm−1 using Bruker ALPHA FT-IR spectrometer. The samples were coated on a potassium bromide (KBr) plate and dried in a vacuum oven at 120 ◦ C before it was tested.

3.3. Scanning Electron Microscopy (SEM) Scanning Electron Microscopy [SEM] (JEOL, JSM-840A microscope) has been used to study the morphology of the fractured specimens. To study the surface morphology of the nanocomposites the specimens were soaked in hot water for 1 h. These specimens were then dried and gold sputtered prior to microscopy (JEOL, SM1100E).

In order to improve starch processability, it has been plasticized and crosslinked using glycerol and glutaraldehyde, respectively. Thermoplastic tapioca starch was prepared following the method described by Sailaja and Chanda [16]. In this method, 48% of starch, 33% of glycerol and 19% of water were initially mixed for 15 min and left it to stand for 1 h in order to allow starch to swell. Further, blending of starch mixture was carried out using a high speed mechanical mixer with a hydro foil blade impeller at 1500 rpm by heating the mixture at 70 ◦ C for 30 min followed by slow addition of 25% (w/w) glutaraldehyde crosslinking agent. The crosslinking reaction was allowed to continue for 30 min with constant stirring. The crosslinked thermoplastic starch (CTS) thus obtained was cut into small pieces, oven dried and ground into fine powders.

Transmission electron microscopy (TEM) for nanocomposites has been performed using a JEOL, Model 782, operating at 200 kV. TEM specimens were prepared by dispersing the composite powders in methanol by ultrasonication. A drop of the suspension was put on a TEM support grid (300 mesh copper grid coated with carbon). After drying in air, the composite powder remained attached to the grid and was viewed under the transmission electron microscope.

2.4. Blend preparation

4.1. Compression properties

A mixture of chitosan (47.5%), CTS (47.5%) and HEMA (5%) were taken for the preparation of composites. The loading of NCF was 0.1% and the loading of nHA was varied from 0 to 15% (w/w of chitosan, CTS and HEMA mixture). The mixture was mixed in a kitchen mixer for 10 min and then sonicated using Ultra Sonicator (Branson, 2510E/DTH) for 30 min. The nanocomposite powders

The compressive properties of the nanocomposites were performed as per ASTM: D 695 by using Zwick UTM (Zwick Roell, ZHU, 2.5) with a pre load of 4.5 kN and a test speed of 5 mm/min. The samples were having a length of 5 cm, width of 1.5 cm and a thickness of 0.4 cm. A minimum of five specimens were tested for each variation in composition of the blend and results were averaged.

3.4. Transmission electron microscopy (TEM)

4. Mechanical properties

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4.2. Flexural properties The flexural properties of the nanocomposites were measured by using Zwick UTM (Zwick Roell, ZHU, 2.5) with a test speed of 5 mm/min. The tests were performed as per ASTM: D 790-10 method. The samples were having a length of 5 cm, width of 1.5 cm and a thickness of 0.4 cm. At least five specimens were tested for each variation in composition of the blend and results were averaged. 4.3. Tensile properties The tensile properties of the nanocomposites were measured by using Zwick UTM (Zwick Roell, ZHU, 2.5) with Instron tensile flat surface grips at a cross head speed of 5 mm/min. The tensile tests were performed as per ASTM: D 638 method. The specimens tested were of rectangular shape having length, width and thickness of 8 cm, 1.5 cm and 0.4 cm, respectively. A minimum of five specimens were tested for each variation in composition of the blend and results were averaged. 5. Thermal analysis 5.1. Thermogravimetric analysis (TGA) Thermogravimetric analyses (TGA) were carried out for the nanocomposites using Perkin-Elmer Pyris Diamond 6000 analyzer (Perkin Elmer Inc., USA) in a nitrogen atmosphere. The samples were subjected to a heating rate of 10 ◦ C/min in a heating range of 30–600 ◦ C with Al2 O3 as reference material. 6. Water absorption test Water absorption of the nanocomposites was measured according to ASTM: D 570-98 with minor modification. The specimens were first dried in oven at 50 ◦ C for an hour and then cooled in desiccators and immediately weighed (W0 ). The conditioned specimens were completely immersed in a container filled with distilled water at room temperature for 2 h. After soaking, each specimen was taken off from the container and the extra water on the surface of the specimen was removed by adsorbing with filter paper and the specimen were weighed again (W1 ). The water-soluble content from the composite in the container was dried by water evaporation in an oven at 50 ◦ C for 24 h and weighed. This weight is denoted as Wsol . At least 3 specimens were tested for each variation in the nHA loading and the results were averaged. The water absorption percentage (Wa ) was then calculated by the following equation: Wa =

W1 − W0 + Wsol W0

where W1 , W0 and Wsol are the weight of the specimen containing water, the weight of the dried specimen and the weight of the water-soluble residuals, respectively.

temperature (37 ◦ C). At the end of soaking time in SBF solution, the composite samples were removed and rinsed with deionized water and dried in a hot air oven for further investigation for the formation of hydroxyapatite layer on the surface of the samples. This apatite formation was examined by Scanning Electron Microscopy. 8. In vitro cytotoxicity test 8.1. Cell line and culture medium RAW 264.7 (Mouse, Macrophages) and L-929 (Mouse, connective tissue) cell line, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 ␮g/ml) and amphotericin B (5 ␮g/ml) in a humidified atmosphere of 5% CO2 at 37 ◦ C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS) for L 929 and scrapped for RAW 264.7 with cell scraper. The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 microtitre plates. 8.2. Determination of cell viability by MTT assay The monolayer cell culture was trypsinized/scrapped with cell scrapper and the cell count was adjusted to 1.0 × 105 cells/ml using DMEM containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was discarded and the monolayer was washed once with the culture medium. 100 ␮l of different nanocomposites concentrations were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37 ◦ C for 24 h in 5% CO2 atmosphere. The microscopic examination for these samples was carried out and observations were noted every 24 h. After 24 h, the nanocomposite solutions in the wells were discarded and 50 ␮l of MTT in phosphate buffered saline (PBS) was added to each well. The plates were gently shaken and incubated for 3 h at 37 ◦ C in 5% CO2 atmosphere. The supernatant was removed and 100 ␮l of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a micro plate reader at a wavelength of 540 nm and the percentage of cell death was calculated. The cell death is directly proportional to the levels of tumor necrosis factor alpha (TNF-␣) in the cell supernatants. The percentage growth inhibition was calculated using the following formula and concentration of test composite needed to inhibit cell growth by 50% (CTC50 ) values is generated from the dose-response curves for each cell line. % Growth inhibitiin = 100 −

 Mean OD of individual test group Mean OD of control group



× 100

7. Bioactivity

9. Results and discussion

7.1. In vitro bioactivity evaluation in simulated body fluid

9.1. Fourier transform infrared spectroscopy (FTIR)

In vitro bioactivity of the composites was determined by soaking them in SBF. This solution was prepared by dissolving NaCl, NaHCO3 , KCl, K2 HPO4 ·3H2 O, MgCl2 ·6H2 O and CaCl2 in deionized water and buffered with (CH2 OH)3 CNH2 and HCl (1 N) to adjust the pH value at 7.4, following the method described by Kokubo [17]. The composition of SBF solution is similar to that of human blood plasma. Chitosan composites with different loadings of nHA were immersed in SBF solution for 1, 3 and 7 days at human body

The FTIR spectra of modified starch, silane treated nHA and the nanocomposites are shown in Fig. 1. FTIR of pure components are also shown in this figure for comparison. The FTIR spectrum of pure chitosan reveals characteristic peaks at 3430 cm−1 which represents the stretching vibration of O H, while 1650 cm−1 peak shows the characteristic peak of (C O) amide I carbonyl stretching and the peaks from 1425 cm−1 correspond to the C H symmetrical deformation. The peaks at 1070 and 2885 cm−1 are related to

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173

Fig. 1. FTIR spectrum of silane treated nHA, pure chitosan, crosslinked starch, silane treated NCF and composite with 5% and 8% nHA.

(C O C) stretching vibration and aliphatic C H stretching vibration respectively [18,19]. In the starch spectrum, the peak at 3310 cm−1 is attributed to the O H stretching of the starch the band at 2937 cm−1 is attributed to the asymmetric stretching of C H, while the peak at 1654 cm−1 is due to the adsorbed water. The peaks at 1418 and 1356 cm−1 correspond to the angular deformation of (C H) bonds in starch molecule [20]. The existence of APTS molecule on the surface of modified nHA particle was confirmed by the presence of asymmetric stretching vibration absorption of CH2 group at 2943 cm−1 , the bending vibration at 1676 cm−1 of N H bond and stretching vibration at 3429 cm−1 of amine group [21]. All the above mentioned peaks do not exist in untreated nHA. The characteristic peaks for nHA are observed around at 1042, 608 and 570 cm−1 corresponds to a phosphate group (PO4 3− ) [22]. The peak at 2943 cm−1 for CH stretching

of propyl group for silane treated nHA is not seen in the composites loaded with 5% and 8% nHA. This indicates that the silane group participated in the interfacial interactions which assisted in enhancing the dispersion of nanofillers in the composite. 9.2. X-ray diffraction studies (XRD) Fig. 2 shows the XRD diffractograms of individual components as well as the nanocomposites. Neat chitosan show a crystalline peak at 17.37◦ owing to its semi-crystalline nature while starch shows three major crystalline peaks at 2 values of 25.8◦ , 31.6◦ and 39.8◦ [23]. However, when chitosan is blended with starch, two peaks at 9.41◦ and 19.36◦ can be seen indicating a shift in the peak due to interactions between them. Neat nHA has a peak at a 2 value of 25.6◦ while NCF shows two broad peaks owing to its predominantly amorphous nature at 9.55◦ and 19.36◦ respectively

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Fig. 2. X-ray diffractograms of starch, nHA, NCF, chitosan and chitosan/starch/nHA/NCF composites.

[24]. Addition of 1.5% nHA to chitosan–CTS blend (control 1), show crystalline peaks at 10.02◦ and 19.64◦ . Further, NCF is added to this blend of chitosan/CTS/nHA (control 2), the peaks overlap with that of NCF. However, in all the nHA loaded nanocomposites, the broad peak at 25.56◦ disappears indicating that nHA has exfoliated due to strong interactions. Similar observations have been reported by Nikpour et al. [2] and Zhang et al. [25] for chitosan based nanocomposites.

9.3. Compression properties 9.3.1. Relative compressive strength (RCS) Fig. 3 shows the relative compressive strength (relative to composite without nHA) versus nHA loading. The RCS values increases as nHA loading increases and reaches an optimal with 10% nHA reinforcement. Higher loadings of nHA reduce the compressive

strength as excess nHA tends to form a separate phase. Hence, it behaves like a three phase system leading to lowering of strength values. The compressive strength values increased from 16.42 MPa (without nHA) to 71.84 MPa (with 10% nHA). In this study, crosslinked thermoplastic starch has been added to chitosan to enhance mechanical strength. Further, the nHA particles have been surface modified with aminosilane to further enhance the interfacial interactions with starch, chitosan and NCF. In order to further assess, the extent of interfacial adhesion between the blend components, two theoretical models have been used to analyze the obtained RCS values. The first is the Nicolais and Narkis model [26] which assumes no adhesion between matrix and filler particles and is given below as follows

RCS = [1 − 1.21( )

2/3

]

(1)

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Experimental Values 6

3.5

Nicolais & Narkis Model

Relative Compressive Modulus

Relative Compressive Strength

(a) 7

5 4 3 2 1 0 2

0

4

6

8

10

12

14

16

3 2.5 2 Kerner Halpin-Tsai Sato-Furukawa Mori-Tanaka Experimental Data

1.5 1 0.5

Percentage of nHA in the composite

0

(b) 7

Relative Compressive Strength

175

Experimental Values

6

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Percentage of nHA in the composite

Turcsanyi Model

Fig. 4. Plot of relative compressive modulus versus percentage of nHA in the composite.

5 4 3

filler–matrix interactions improve. The model expression is given as follows [29]

2 1

RCS =

0 0

2

4

6

8

10

12

14

(4)

16

Percentage of nHA in the composite Fig. 3. (a) Plot of relative compressive strength versus percentage of nHA in the composite. (b) Plot of relative compressive strength versus percentage of nHA in the composite.

where  is the corrected volume fraction of nHA particles. In order to account for the non-linear dependence of the nano-filler fraction due to its small size and aspect ratio along with high surface area, a second order correction was assumed in place of volume fraction  in Eq. (1) as given below [27].  = ˛Af + ˇ2 A2f

(2)

In Eq. (2), ˛ and ˇ are empirical constants obtained by trial and error to match with experimental results. Af is the aspect ratio of nHA taken to be 3.1 [28] while,  is the volume fraction of nHA. The volume fraction  has been determined using the following equation. i =

1 −  exp(B ) 1 + 2.5

w /i

i

(3)

wi /i

In Eq. (3), wi and i are the weight fraction and density of component i respectively. The densities of chitosan, starch, nHA and NCF are respectively measured to be 0.3 g/cm3 , 1.442 g/cm3 , 3.12 g/cm3 and 2.1 g/cm3 . The theoretical values obtained from Eq. (1) are plotted in Fig. 3(a). The values of ˛ and ˇ are given in Table 1. It can be seen that the model values are lower and do not match with the experimental RCS values. This suggests that there is some extent of interfacial adhesion between the blend components. The other theory is the Turcsanyi model which includes a parameter B which is related to interfacial adhesion. The value of B increases as Table 1 Values of constants for relative compressive strength models. Model

˛

ˇ

Nicolais–Narkis Turcsanyi

15 12.4

1 5.3

The constants ˛ and ˇ has been determined by trial and error to match with the experimental results and these are tabulated in Table 1. The RCS values from the Turcsanyi model are plotted in Fig. 3(b). It can be seen from the figure that the theoretical values show a good fit with the experimental RCS values. The value of B has been found to be 5.4 which indicate strong interfacial bonding. A similar observation has been reported by Habeych et al. [30] wherein B ≥ 3 indicates good bonding. Bliznakov et al. [31] also found an increased B value on surface treatment of glass beads as compared to composites loaded with untreated glass beads. Thus, surface treatment of the fillers and the use of HEMA compatibilizing agent led to the enhancement of bonding strength in these nanocomposites. 9.3.2. Relative compressive modulus (RCM) Fig. 4 shows the relative compressive modulus (relative to composite without nHA) for the chitosan/CTS/nHA composites. The compressive modulus increases from 525.16 MPa (without nHA) to an optimal value of 1326.5 MPa with 10% nHA. The main factor which leads to enhancement of compressive modulus and strength is the bonding between the inorganic bio-ceramic nHA and the organic biopolymers. Thus, silane treatment of nHA introduces amine groups on its surface which can interact effectively with chitosan, CTS and NCF. Further, the HEMA coupling agent also assists in further enhancing the blend interactions. The extent of these interactions has been further assessed using theoretical models. The first is the modified Kerner’s model which assumes good adhesion with rigid particles [32] and is given below.



RCS = 1 +



1 −  ; 1 + 2.5

i =

w /m

m

wi /i

(5)

where m , m and  are the volume fraction of the matrix, Poisson’s ratio of the matrix and filler respectively. wm and m is the weight fraction and density of the matrix without nHA. The Poisson ratio of the matrix and nHA filler has been taken to be 0.4 and 0.3 respectively [33,34]. The values of ˛ and ˇ for the corrected filler volume fraction are given in Table 2. The predicted values from modified Kerner’s model are plotted in Fig. 4. Halpin–Tsai model is another most widely used

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Table 2 Values of constants for relative compressive modulus models.

where



Model

˛

ˇ

Kerner Halpin–Tsai Sato–Furukawa Mori–Tanaka

14 15 17.95 1

1 −154 −152 1

=

micromechanical model and been used to evaluate the modulus of these nanocomposites [35,36]. RCM =

3 1 + 2e  5 1 + 2T  + 8 1 − 2e  8 1 − 2T 

(6)

where e =

ı−1 , ı + 2Af

T =

ı−1 , ı+2

and ı =

Ep Em

(7)

In Eq. (7), Ep is the modulus of nHA (modulus of nHA is 54,000 MPa) [37] while Em is the modulus of the matrix without nHA (measured to be 525.16 MPa). The values obtained from Eq. (6) have also been plotted in Fig. 4. Sato and Furukawa have developed a model expression which includes an adhesion parameter . The value of  varies from 0 to 1 from perfect adhesion to no adhesion respectively. The model expression is given below [38]. RCM = 1 +

( )

2/3

1/3 2 − 2( )

(1 −

) −

( )

2/3



1/3 1 − (( ) )

1/3

2/3

 1 + ( ) − ( ) 3 1 − ( )1/3 + ( )2/3

 (8)

The value from this model has been plotted in Fig. 4 and  value has been obtained to be 0.17. From the figure it can be seen that the modified Kerner’s model gives a good fit in the entire composition range. The obtained optimal experimental value is lower than that predicted by the Halpin–Tsai model. Sato–Furukawa model also shows a good fit with the experimental value and  value of 0.17 indicates good adhesion between filler and matrix. However, all the models are able to predict the trend for these nanocomposites. The surface treatment of filler introduces the polar amine group which is able to interact effectively with the other blend components. This enhances the dispersion of the filler and this leads to better stress transfer from matrix to filler. The Mori–Tanaka model based on average stress theory and the analytical expressions for the same have been developed by Tandon and Weng [39]. The expression for this model is given as RCM =



2A +  [−2A3

2A + (1 − )A4 + (1 + )A5 A]



(9)

The detailed expressions for the constants A, A3 , A4 and A5 are given in the paper by Tandon and Weng [39]. The values evaluated for the Mori–Tanaka shown in Fig. 4 show a good fit with the experimental value although the predicted optimal value is higher. This model assumes that both filler and matrix are elastic. Further, it also assumes that the filler is perfectly aligned in the matrix. Hence, this shows a higher predicted optimal value than the experimental value as the loaded filler was rigid by nature. Fig. 5(a) shows SEM surface morphology of hot water etched nanocomposites loaded with 0.5% and 3% nHA. The matrix shows

Fig. 5. SEM micrograph showing surface morphology of hot water etched composite containing 0.5% nHA (a) and 3% nHA (b).

Fig. 6. SEM micrograph showing compressive fracture of composite containing 1% nHA (a) and 3% nHA (b).

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177

Fig. 7. TEM micrograph of nanocomposite containing 0.5% nHA (a) and 3% nHA (b).

9.4. Thermal analysis 9.4.1. Thermogravimetric analysis (TGA) Fig. 11 shows the TGA thermograms of the nanocomposites. The thermogram for pure nHA (curve a) and the composite without nHA is also shown in the figure for the sake of comparison. Pure nHA being a ceramic material is more thermally stable than the nanocomposites. The composite loaded with 3% (curve c) and 10% (curve d) nHA has improved thermal stability as compared to nanocomposite without nHA i.e. 0% nHA (curve b). Further, char formation also increases as the loading of nHA is increased. All the thermograms show single stage degradation. A weight loss of 60% for composite without nHA is observed at 385.3 ◦ C while the same weight loss occurs at 403.5 ◦ C for 3% nHA loaded composite. At higher i.e. 10% nHA composite, 60% weight loss occurs at a still higher temperature of 436.2 ◦ C indicating increased thermal stability due to the addition of inorganic ceramic nHA. The degradation of starch and chitosan at 70% weight loss above 500 ◦ C

35 1000 30 800

25 20

600

15 Flexural Modulus

400

Flexural Strength

10

200

Flexural Strength (MPa)

9.3.3. Flexural properties The flexural properties of the nanocomposites are shown in Fig. 8. The flexural strength increased by more than four and half times on adding 8% nHA (6.5 MPa (without nHA) to 30.5 MPa with 8% nHA loading). Flexural modulus also increased considerably to an optimal value of 1045 MPa (with 8% nHA) from 293.3 MPa (without nHA). Thus, the modulus value increased more than three times as compared to the composite without nHA. SEM image for 0.5% nHA loaded flexurally fractured surface shows a quasi-brittle failure with layered plastic deformation of the surface along with cavitation of the filler particles. This is also clearly seen at higher magnification of the fractured surface shown in Fig. 9(a). For 3% nHA loading the figure shows overall brittle failure wherein severe deformation of the matrix and particle pullout has taken place. A higher magnification shows a disorderly aligned but multiple folds of the matrix surface [Fig. 9(b)].

9.3.4. Tensile properties Fig. 10 shows the tensile strength and modulus of the bionanocomposites. Addition of silane treated nHA improved the tensile strength from 1.8 MPa (0% nHA) to 7.36 MPa with 8% nHA. A higher loading beyond 8% is detrimental to the tensile strength of the nanocomposites. Tensile modulus also increased significantly by the addition of nHA and reaches an optimal value of 117 MPa with 8% nHA loading as addition of rigid filler causes stiffness in nanocomposites. The surface modification of the reinforcing fillers along with the use of biocompatible HEMA as coupling agent helps to enhance stress transfer from matrix to filler, thereby effectively improving the interfacial interactions. It can be seen from Fig. 10, the modulus increased by four times as compared to composite without nHA.

Flexural Modulus (MPa)

that it has undergone large plastic deformation indicating the difficulty in debonding of nHA particles due to improved interfacial adhesion. For higher loading of 3% nHA [Fig. 5(b)] large pockets of deformed matrix prior to pullout of hydroxyapatite nanoparticles can also be seen. For 1% nHA loading Fig. 6(a) shows a more densely packed buckled quasi-brittle surface with voids left by the debonded nHA particles throughout the compressive fractured surface. For 3% nHA loading, the fracture surface predominantly shows brittle failure along with mild deformation of the matrix [Fig. 6(b)]. The presence of minute voids throughout the fracture surface shows enhanced dispersion of nHA particles and NCF owing to improved interfacial adhesion between the silane treated nanoparticles and the matrix which has also been facilitated by the addition of biocompatible HEMA coupling agent. The dispersion of nHA in the composite is seen clearly in the TEM micrographs shown in Fig. 7. Fig. 7(a) and (b) shows the TEM micrographs of nanocomposites loaded with 0.5% and 3% nHA respectively. Fig. 7(a) shows that the nanoparticles are well dispersed and there is a combination of both exfoliation and agglomeration in the matrix. The dark particles of nHA are seen to be well dispersed throughout the matrix although some regions are seen where clustering of nanoparticles have taken place. Fig. 7(b) clearly shows the rod-like nHA particles similar to ones observed by Im et al. [40]. In all the micrographs, the nanoparticles appear to be embedded in chitosan sheath. The dark regions are the nHA particles while the lighter regions correspond to starch and chitosan. The bulging of the sheath can be attributed to the agglomerated nHA particles and this gives an overall grain-like look for the morphology of the nanocomposite. However, as suggested by Dorigato et al. [41] such long streams of agglomerated particles also helps in stress transfer from matrix to filler, thus leading to enhanced mechanical strength.

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Percentage of nHA in the composite Fig. 8. Plot of flexural strength and modulus versus percentage of nHA in the composite.

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Fig. 9. SEM micrograph showing flexural fracture of the composite containing 0.5% nHA (a) and 3% nHA (b).

mainly occurs by thermal decomposition of glucosamine residues [42]. 9.5. Water absorption characteristics Water absorption has been examined in order to evaluate the water resistance characteristics of the nanocomposites in the water

250

9 8 7 6

150 5 4 100 3 2

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Peercentage of nHA in the composite

9.6. Bioactivity 9.6.1. In vitro bioactivity evaluation in simulated body fluid Fig. 13 shows the SEM micrographs for 5%, 8% and 10% nHA loaded nanocomposites immersed in SBF for 1, 3 and 7 days respectively. For 5% nHA loading [Fig. 13(A)], a larger number of nucleated sites can be seen after 1 day while the entire surface is covered with a thick and porous apatite layer with multiple cracks can be seen in 3 days. The thickness of the apatite layer increases in seven days. For 8% nHA loading [Fig. 13(B)], a large number of both single and agglomerated sites can be seen in 3 days, the grain boundaries disappears and by the end of 7 days, a dense apatite layer along with minute multiple cracks can also be seen. For higher i.e. 10% nHA loaded composite, large agglomerated apatite particles can be seen [Fig. 13(C)]. The particles at higher magnification shows

Percentage of water absorption

Fig. 10. Plot of tensile strength and modulus versus percentage of nHA in the composite.

environment. The effect of silane treated nHA on water absorption characteristics of the chitosan/CTS/nHA nanocomposites have been shown in Fig. 12. From the figure, it is evident that water absorption percentage of nanocomposites has been influenced by the incorporation of silane treated nHA. Addition of silane treated nHA reduces the water uptake of the nanocomposites significantly. The water absorption percentage steadily reduced with increase in nHA content and leveled off by 10% nHA loading. The water absorption percentage of nanocomposites decreased by 93% for 10 wt% nHA loading when compared to that of chitosan/CTS composites without nHA. Incorporation of nHA into the chitosan/CTS composite form a temporary barrier preventing water permeation into the composite matrix, owing to formation of a tortuous path for water permeation. Similar report on reduction of water absorption for chitosan/hydroxyapatite composite was reported earlier by Hu et al. [43].

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Percentage of nHA in the composite Fig. 11. TGA thermograms of pure nHA and composites with different loadings of nHA.

Fig. 12. Percentage of water absorption of chitosan composites loaded with 0%, 1%, 3%, 5%, 7%, 8% and 10% nHA.

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Fig. 13. SEM morphologies of the composite A (5% nHA), B (8% nHA) and C (10% nHA) in SBF, after 1, 3 and 7 days of immersion. Higher magnification shown in the inset.

highly porous interlinked network. Such interconnected network helps promote both osteointegration and osteoconductivity [44]. By the end of 3 and 7 days, multiple layers of dense apatite along with a large number of tiny apatite crystals can be seen covering the entire surface. The above observation is also supported by the EDS spectra (Fig. 14) wherein, the intensities of P and Ca increases in 7 days as compared to that of the composite immersed in SBF for 3 days [Fig. 14(a) and (b)]. Increased intensities of P and Ca indicate enhanced apatite deposition as the time period of soaking increases from 3 days to 7 days. Similar observation on the apatite formation for silver substituted hydroxyapatite-titania composite has been reported by Kotharu et al. [45]. 9.7. In vitro cytotoxicity test 9.7.1. MTT assay Biocompatibility of the prepared nanocomposites was examined by MTT assay. This assay measures the metabolic activity of the cells which can correlate with number of viable cells and is based

on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The principle involved in the cleavage of tetrazolium salt 3-(4,5 dimethyl thiazole-2-yl)-2,5diphenyl tetrazolium bromide (MTT) into a blue colored product (formazan) by mitochondrial enzyme succinate dehydrogenase. The number of cells was found to be proportional to the extent of formazan production by the cells used [46]. In this study RAW264.7 and L-929 cell lines were examined for cytotoxicity of varied concentration of composite loaded with 10% nHA. Fig. 15 shows the percentage of cytotoxicity for both the cell lines. Cytotoxicity percentage increased by 32.3% by increasing the concentration of nanocomposite from 62.5 ␮g/ml to 125 ␮g/ml. However, for higher concentration of nanocomposite specimen i.e. 500 ␮g/ml and 1000 ␮g/ml the rise in cytotoxicity percentage is only 9.4% for RAW-264.7 cell line. However, for L-929 cell line the corresponding percentage of cytotoxicity levels have been found to increase by 41.2% and 18.9% respectively. Composite specimen with 10% nHA was found to be nontoxic against both cell lines with CTC50

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Concentration tested

% cell death

Chitosan composite with 10% nHA

1000 ␮g/ml 500 ␮g/ml

4.66 ± 1.56 4.66 ± 1.03

10. Conclusions In this study, chitosan/CTS/nHA nanobiocomposite with various loadings of nHA (0–15%) were developed by using compression molding machine. The SEM results revealed that the nHA particles were exfoliated in the matrix as confirmed by XRD analysis. The tensile, compressive and flexural strength of the composites showed considerable increase with increasing nHA loading up to 10% for tensile strength and up to 8% for compressive and flexural strength respectively. Micromechanical models clearly confirm the presence of enhanced interfacial bonding interactions between the fillers and matrix. Cytotoxicity test confirms that the developed composite is cytocompatible. The biocomposite demonstrated an excellent ability of inducing apatite formation in simulated body fluid, which indicates that these nanocomposites have a promising potential to be used as load bearing bone substitutes. Acknowledgement The authors are grateful to the Indian Council of Medical Research for kindly sanctioning funds to carry out this research work. References

Fig. 14. EDS spectra of chitosan composite containing 5% nHA after immersion in SBF for (a) 3 days and (b) 7 days.

showing more than 1000 ␮g/ml value. Percentage of cell death (RAW-264.7 cell line) was also calculated by measuring the production of TNF-␣ which showed a very negligible value (Table 3). Based on the above results it can be deduced that the nanocomposite is nontoxic.

Fig. 15. Influence of chitosan/CTS/nHA composite on RAW-264.7 and L-929 cell lines metabolic activity as measured using MTT assay (data are presented as the means ± 2 standard deviation).

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Chitosan-nanohydroxyapatite composites: mechanical, thermal and bio-compatibility studies.

Bionanocomposites of chitosan were prepared with nanohydroxyapatite (nHA) using 2-hydroxyethyl methacrylate (HEMA) as coupling agent. The tensile and ...
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