Abstract— The scaffold for bone tissue engineering should be

porous to allow bone cell in growth along with mechanical property in par with bone structure to ensure integrity of neo-tissue. Studying the effect of compositional variation on the microstructure and mechanical strength of gelatin-chitosan-hydroxyapatite (HAp) based scaffold is the main the focus of the current research. HAp-chitosan nanopowders with variable composition from 20:80 to 40:60 wt% were synthesized using coprecipiation and characterized using XRD, TEM, FTIR and thermogravimetric (TG) analysis. The porous composite scaffold containing HAp- chitosan nanopowders and gelatin having 3D network of interconnected pores with an average pore size varying between 50-200 µm was fabricated using freeze drying method. Glutaraldehyde was used as a cross linker to conjugate inter gelatin and chitosan- gelatin network and characteristic IR band for (-C=N-) group in the scaffold revealed that fact. The highest porosity of 70 vol% was observed in a scaffold having composition of gel: chi: HAp of 10:67:23 and a similar composition of 20:50:30 in teh scaffold exhibited a total porosity of 57 vol%. With increase in HA content from 17 to 35 wt% in the scaffold both compressive strength and Young’s modulus values increased from 1 MPa to 3.2 MPa and 17 MPa to 132 MPa respectively, the values were very similar to that of human spongy bone. These types of composite scaffolds find application as efficient biomaterial for natural bone tissue regeneration and bone tissue engineering.

Keywords—Composite Scaffold, Mechanical strength, Porosity,

Spongy bone.

I. INTRODUCTION ESEARCH on artificial bone substitute material for bone replacement and bone healing has expanded considerably

over the last four decades [1, 2]. Natural bone in mammals is a hierarchically structured inorganic-organic composite material. The inorganic phase of bone tissue is constituted of hydroxyapatite[Ca10(PO4)6(OH)2, HAp] crystals embedded in a collagenous matrix, which imparts high strength and toughness to the bone [3,4]. In recent years, more efforts have been directed towards the synthesis of new materials which mimic

Sudip Dasgupta is with the Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha 769008 INDIA (corresponding author’s phone: 919937565248; e-mail:dasguptas@nitrkl.ac.in).

Kanchan Maji is with the Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha 769008 INDIA (e-mail: 512cr105@nitrkl.ac.in).

natural process to repair or replace the injured bone [5,6].The combination of inorganic (hydroxyapatite) and organic (polymer) networks offers a direction to design new biomaterials with excellent properties for bone tissue engineering [7].

Hydroxyapatite (HAp), being osteoconductive in nature, has been successfully used as bone fillers, coating of orthopaedic implant, filler of inorganic/polymer composites, cell culture carrier and so on but the application of pure HAp is being limited, due to its brittle nature. Moreover, HAp in sintered form exhibits lower bioactivity as compared to its poorly crystalline particulate counterpart. In fact, natural bone is composed of poorly crystalline nano-hydroxyapatite(n-HAp) dispersed in collagen matrix. Therefore, because of their analogy to bone matrix as well as good biological and mechanical performances, development of nano HAp –biopolymer based composites has received much more attention recently.

Among various biopolymers, natural biopolymers find important applications in the field of orthopaedic and other biomedical fields, due to their excellent biocompatibility and biodegradability. Various kinds of polymer/ceramic composite system such as HAp/ collagen [8, 9], HAp/chitosan [10], HAp/collagen/poly (lacticacid) [11], HAp/alginate/collagen [12], HAp/gelatin [13-16] were employed for preparing scaffolds for bone tissue engineering. A combination of HAp- chitosan- gelatin seems to be a very interesting bone substitute material because of its resemblance with natural bone matrix. Chitosan (poly-1, 4-D-glucosamine), a partially deacetylated form of chitin, is structurally similar to glycosamino glycan present in the extracellular bone matrix, and exhibits good wound healing property [17]. Gelatin, a derivative of collagen, seems to be very attractive as a binding agent or matrix, because it contains a lot of biological functional groups, such as amino acids within its backbone which are conducive to cell growth and proliferation. Further, gelatine exhibits superior plasticity, and adhesiveness that is essential for developing mechanical strength in the scaffold and its biodegradation rate can be regulated by the degree of cross-linking with suitable cross linker such as glutaraldehyde, genipin etc.

Comparative study on Mechanical Strength of Macroporous Hydroxyapatite-Biopolymer

Based Composite Scaffold

Sudip Dasgupta, and Kanchan Maji

R

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To match natural bone in mechanical and biological performances, synthetic bone substitute materials prepared by addition of HA into the polymeric mixture of chitosan and gelatin was reported [18]. Attachment and proliferation of osteoblasts on chitosan-gelatin based 3D structures containing sintered HA were also studied [19]. Further, Hasirci et al [20] studied the effect of sintered and non sintered HAp on the mechanical and biological properties of 1:1 chitosan- gelatine scaffold and found sintered HAp more effective in enhancing bioactivity of the scaffold. Currently, intensive research is being conducted on HAp–chitosan–gelatin based composites as films for tissue engineering applications [21–23]. For better performance as a bone substitute material, the scaffold should posses macroporous (100-150 mm) microstructure as well as superior mechanical strength. Despite none of the research efforts were directed in studying the effects of compositional variation on porosity and mechanical strength of hydroxyapatite-chitosan –gelatin based scaffold. Our objective was to enhance the mechanical strength of the composite scaffold even with the increase pore size and total interconnected porosity in the scaffold. In the present investigation, HAp–chitosan–gelatin nanocomposite scaffold was prepared by varying the ratio of synthesized HAp- chitosan nanopowder and added gelatin, in order to explore the effect of compositional variation on the structural, morphological, and mechanical properties of the composite scaffold for bone tissue engineering application.

II. MATERIALS AND METHOD

2.1Materials Chitosan [percentage deacetylation (90%), molecular weight

6000 Da] and gelatin were purchased from Sigma Aldrich (USA). Di-ammonium hydrogen phosphate (NH)2HPO4 and calcium nitrate tetrahydrate were purchased from Merk (Germany).Glacial acetic acid and ammonia solution (NH4OH) were procured from LOBA chemical (India).

Chitosan (Chi) solution was prepared by dissolving required amount of medium molecular weight chitosan in a solution containing 4 ml of acetic acid and 96 ml of deionized water with stirring for 5 h to get a perfectly transparent solution. A glutaraldehyde solution was prepared by dissolving 0.5 mL glutaraldehyde (50%) in 100 mL deionized water.

2.2Preparation of HAp/Chitosan composite nanopowder

The chitosan/HAp composite nanopowders with varying composition of HAp: Chi (20:80, 30:70, 40:60) were synthesized using coprecipitation method. First, 100 ml of (NH)2HPO4 (.019M) solution was added to required strength of 100 ml chitosan solution. The mixed chitosan/ (NH)2HPO4 solution was then dropped slowly into the solution of calcium nitrate (.031M) with vigorous stirring and the pH was adjusted with NH4OH solution to about 10. The dropping speed of chitosan/ (NH)2HPO4 solution was about 4 mL min-1

and the reaction was carried out in ambient condition at 25 oC. After titration, the stirring was continued for 24 h and the obtained slurry was aged for another 24 h. Finally, the precipitate was

filtered, and washed with deionized water and dried in a vacuum over at 60oC.

2.3Preparation of Porous HA/Chitosan-Gelatin composite scaffold

Required amount of gelatin was dissolved in deionized water and heated up to 40oC under continuous stirring for 4 h. 2 gm of HAp- chitosan composite nanopowder was then added to the solution with constant stirring for 40 min to disperse the HAp-chitosan nanoparticles. Required amount of glutaraldehyde aqueous solution was then added to the slurry and stirred for 2 h. The resulting slurry was put in PTFE cylindrical mould and then rapidly prefreezed at -20˚C to solidify the water and kept overnight. Next, the frozen samples were lyophilized using a freeze-dryer at -52˚C for 24 hrs.

TABLE-I COMPOSITION OF THE PREPARED SCAFFOLD

Sample Composition

of HAp:chi

nanopowders

(wt ratio)

Composition of

the scaffold;

Gel:Chi:HAp (wt

ratio)

1. 20:80 20:63:17

2. 30:70 10:67:23

3 30:70 20:60:20

4. 40:60 10:55:35

5. 40:60 20:50:30

2.4 Characterization 2.4.1 XRD Analysis The ground samples were characterized by X-ray diffraction

(XRD) using fully automated X- ray diffractometer (Panalytical, USA) fitted with Ni filter. The diffraction patterns were recorded with a XRD analyser using CuKα radiation (λ = 1.542 nm) at 35 kV and 10 mA. The samples were scanned from 10o to 70o in 2θ (where θ is the Bragg angle) in a continuous mode.

2.4.2 FTIR Spectra The characteristic peaks of the pure HAp and its composites

were analysed using Fourier transform infrared (FTIR) (Perkin Elmer, USA) spectroscopy. The scaffolds were put in a vacuum oven at 50oC for 48 h before they were ground to a suitable size for IR analysis with spectrometer. The pellet for the FTIR measurement was prepared by mixing the sample (2mg) with 200 mg of IR-grade KBr. The absorption spectra were measured using spectrum 100 spectrometer at a wavenumber of 4000-400 cm-1 with a resolution of 1 cm-1.

2.4.3 Thermo gravimetric analysis The thermogravimetric (TG) analysis of the composites was

studied on 50mg of powder samples using a TG analyser

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(Netzsch, Germany) and measurements were recorded from 50˚C - 1000˚C at 10˚C/min heating rate in air.

2.4.4 Porosity of Scaffold The porosity of composite scaffold (1×1×1 cm3) was

measured using Archimedes principle with xylene as liquid medium using the following equation.

Porosity = (W2-W1)/ (W2-W3) × 100% Where, W1 is the weight of the sample in air, W2 is the

weight of sample with liquid in pores, and W3 is the weight of the sample suspended in xylene.

2.4.5 SEM Observation The microstructure of the scaffold was observed using field

emission scanning electron microscopy (FESEM) Novanano 450, FEI, Netherland). The surface of the scaffolds was gold coated, and then placed inside the FESEM chamber.

2.4.6 Mechanical testing The mechanical properties of the composite scaffolds were

determined using a Universal Testing Machine (Tinius Olsen, UK). For compressive testing, the samples were cylinders of approximately 6 mm in diameter and 12 mm in height in accordance with the compression mechanical test guidelines set in American Standard Test and Measurement (ASTM F 451-95). The average of 4 samples was recorded and the values were expressed as the means ± standard error.

III. RESULTS AND DISCUSSION

3.1 HAp/Chitosan composite powder 3.1.1 XRD analysis

Phase analysis of the composite nanopowder was performed using XRD. The characteristic diffraction peaks for both chitosan and HAp were observed in the synthesized nanopowder. The sharp peaks of (002) and (112) plane in HAp (Fig. 1) confirmed that the synthesized powder was well crystallized. No other calcium phosphate phases were detected in XRD pattern in Fig. 1 that indicated the presence of phase pure hydroxyapatite- chitosan in the synthesized nanopowders.

Fig. 1 XRD pattern of 40 HAp/ 60 chitosan nanopowders

3.1.2 FTIR Analysis

Fig. 2 shows the FT-IR spectrum of HAp-chitosan composite nanopowder. The characteristic bands for different functional groups present in the scaffold are shown in table II. Two bands were observed at 3570 and 622 cm-1 due to the stretching of hydrogen bond in OH ions present in HAp and liberation mode of hydrogen-bonded OH ions respectively. The band at 1024 cm-1 arises from ѵ3 PO4, whereas the band at 603 and 555 cm-1 suggest the presence of ѵ4 PO4. The absorptions bands observed at 2887, 1631, 1595, and 1543 cm-1 were assigned to methylene (-CH2), amide І(C=O), amino (-NH2), and amide ІІ (-NH) groups in chitosan respectively. Carbonate bands at 1382, and 852 cm-1 appeared because of the carbonate incorporation in HAp from CO2 in air. The FT-IR spectra also indicated the shifting of characteristic bands of amine (1660 cm-1) and C-O (1592 cm-1) in chitosan to lower wavenumber in the HAp- chitosan composite nanopowder, as a result of bonding interaction between chitosan and HAp because of electrostatic interaction between –NH3

+ and PO4-3

and as well as between C-Oδ- and Ca+2.

4000 3500 3000 2500 2000 1500 1000 500

(PO4-3)

Tran

smitt

ance

(a.u

.)

Wavenumber (cm-1)

(OH)

(C-O)

(NH2)

(CO3-2)

(PO4-3)

HAP/Chitosan

Chitosan1760 1680 1600

1660cm-1

1634cm-11592cm-1

1558cm-1

Fig. 2 IR spectra of HAp/ Chitosan composite powder

TABLE II

PEAKS OF INFRARED SPECTRA ASSIGNED TO SYNTHESIZED HAP-CHI NANOPOWDERS Compound Functional

group Infrared

frequency (cm-1)

HA PO43-

bend ѵ4 555

HA PO43-

bend ѵ3 1024 HA Carbonate

CO3-2

1382

Chitosan Amine (-NH2)

1544

Chitosan C-O 1634 Chitosan Methylene

(-CH2) 2887

HA Structural OH 3431

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Fig. 3 TEM micrograph of HAp20-Chi 80 composite nanopowder The synthesized HAp- chitosan composite powder showed a

particle size distribution between 40 -70 nm with aspect ratio of 1.94±0.15 and found to be in agglomerated state as in TEM micrographs in Fig 3.

0 200 400 600 800 1000

0

20

40

60

80

100 92.77%

9.15%Chi

HAp

28.72%

Wei

ght (

%)

Temperature°C

HAp-Chi

(a)

0 200 400 600 800 10000

102030405060708090

100110

9.15%

42.15%

92.77%

Chi

HAp-Chi

Mas

s(W

t%)

Time°C

HAp

(b)

Fig. 4 Thermogravimetric analysis of pure HAp, pure chitosan and

HAp- chitosan nanopowders (a) HAp30: Chi70 (b) HAp40: Chi60.

The TG thermogram of the synthesized nanopowder is shown in Fig 4. The observed weight loss of 7 wt% between 70˚C to 150˚C was due to the loss of water molecules from nano-Hap and chitosan. Another 21-35 wt% weight loss between 310-600˚C is attributed to the thermal decomposition of chitosan. Negligible or no weight loss occurred after 620˚C indicating the fact that all the organic macromolecules were decomposed within 620˚C. The calculated composition of HAp- chitosan composite nanopowders from TG data closely

resembled the theoretical composition of the powder as in table III.

TABLE ІІI THEORETICAL AND ACTUAL COMPOSITIONS OF CHITOSAN/N-HAP

NANOPOWDERS AS DETERMINED BY TG ANALYSIS AT 1000˚C

3.2 HAp-Chitosan/Gelatin Composite Scaffold The composite scaffold with HAp-chitosan to gelatin

composition of 60 to 40 wt% and 25 % solids loading was prepared by freeze drying method using glutaraldehyde as a cross linker. The prepared scaffold was examined using FTIR spectroscopy to study bonding interactions between different functional groups in the scaffold. The microstructure of the scaffold was investigated using FESEM and UTM was used to study its mechanical behaviour.

3.2.1 FTIR Analysis

4000 3500 3000 2500 2000 1500 1000 500

Tran

smitt

ance

(%)

wavenumber cm-1

562 cm-1

1027 cm-1

1662 cm-1

2878 cm-1

3079 cm-1

2363 cm-1

1232 cm-1

1330 cm-1

3406cm-1

1550150014501400

1552cm-1 1446cm-1

Fig. 5 IR spectra of HAp / Chitosan-Gelatin composite scaffold

Fig 5 shows the FTIR spectrum of scaffold and different IR band or peaks are shown in table IV. Apart from PO4

3- (1027, 562 cm-1), CO3

2- (875 cm-1), OH- (3406, 605 cm-1) bands in HAp and amide I (1662 cm-1), amide III (1232 cm-1) and carboxylate (1446 cm-1) bands in gelatin, a distinct band at 1552 cm-1 was detected, which signifies the formation of –C=N- bond due to intergelatin and chitosan and gelatin interaction to develop 3D interconnected network in the scaffold. The band at 1330 cm-1 is attributed to the interaction between carboxylate group in gelatin and Ca+2 ion in HAp to

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bind the particulate reinforced composite scaffold together, a fact that is well established in the earlier studies also [14, 15].

TABLE ІV PEAKS OF INFRARED SPECTRA ASSIGNED TO FABRICATED

SCAFFOLD

3.2.2. SEM Observation The SEM micrographs of the prepared composite scaffolds

are shown in Fig 6. A large number of interconnected pores with total porosity of 50-70 volume % were observed in the scaffold. Irregularly shaped interconnected pores ( Fig 6c) with pore size between 80-200 µm were observed in the scaffolds, which provides an ideal environment for attachment and growth of bone cells. The pore size and its distribution could be controlled by modifying composition of the slurry. As evident from all the micrographs in Fig 6 and table V, with increase in gelatin content from 10-20 wt% the total porosity as well as pore sizes in the scaffold decreased. As the gelatine content is increased in the scaffold, enhanced electrostatic interaction between HAp and gelatin and covalent -C=N- bond formation between chitosan and gelatin occurred which resulted in decrease in pore size and total porosity in the scaffolds.

Fig. 6 FESEM micrographs of scaffolds (a) Gel:Chi:HAp 10: 67:23(b) Gel:Chi:HAp 20: 60:20 (c) Gel:Chi:HAp 10: 55:35

TABLE V

POROSITY DATA OF THE PREPARED SCAFFOLD Sample Composition

of the scaffold;

Gel:Chi:HAp (wt

ratio)

% Porosity

1. 20:63:17 64

2. 10:67:23 70

3 20:60:20 60

4. 10:55:35 68

5. 20:50:30 57

-5 0 5 10 15 20 25 30 35 400.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

20% gel

20% gel

10% gel

Com

pres

sive

Stre

ngth

(MPa

)

HA Content (%)

10% gel

Fig. 7 Variation in compressive strength of the scaffold with HA

contant

3.2.3 Mechanical properties

Compression tests were carried out for the scaffolds and the compressive elastic modulus (E) and compressive strength of the scaffold were plotted as a function of HAp content. The results in Fig 7 indicate that the addition of HA enhanced the compressive strength of the materials from 0.4 ± 0.07 MPa for pure Chi-Gel scaffold to about 3.2 MPa for HAp added scaffold. Moreover, with increase in HAp content in the scaffold the compressive strength value of the scaffold increased steadily even though the total porosity increased with higher gelatine content in the scaffold. Dispersion of HAp nanoparticles in chitosan-gelatin network acted as

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reinforcement against crack propagation in the scaffold and increased the compressive strength.

-5 0 5 10 15 20 25 30 35 40

0

20

40

60

80

100

120

140

160

Youn

g m

odul

us(M

Pa)

HA-content(%) Fig. 8 Variation in Young’s modulus of the scaffold with HA content

Young’s modulus values were also enhanced with HAp addition and reached a highest average value of 132 MPa at 35 wt % of HAp in the scaffold as depicted in Fig 8. For cancellous bone, compressive strength and Young’s modulus values fall in the range of 2–12 MPa and 0.05–0.5 GPa, respectively [24]. Thus, HAp-incorporated chitosan–gelatin scaffold that exhibits compressive strength at around 3.2 MPa and Young’s modulus at about 0.13 GPa could be good candidates for cancellous bone tissue-engineering applications.

IV. CONCLUSIONS

In this study, bio ceramic-polymer based composite scaffolds with variable composition of nanoHAp-chitosan and gelatin was processed using lyophilisation of slurry containing HAp-chitosan nanopowders and gelatin. Gelatin acted as a network former and binder and thus with increase in gelatine content from 10 to 20 wt% the total porosity of the scaffold decreased due to enhanced electrostatic and covalent interaction between gelatin -HAp and gelatin- chitosan respectively. Dispersion of HAp nanoparticles in gelatine and chitosan network acted as a barrier for crack growth and that is why both compressive strength and Young’s modulus of the scaffold increased with increase in HAp content irrespective of porosity factor in the scaffold. The composition of gel:chi:HAp equivalent to 10:55:35 in the scaffold exhibited the maximum values of compressive strength and Young’s modulus. The effect of composition on osteoconductivity and osteoinductivity as well as biodegradability is under investigation to evaluate the ideal composition of gelatine-chitosan and HAp based materials for potential candidates for bone tissue engineering.

ACKNOWLEDGMENT

The authors acknowledge the department of Ceramic Engineering at NIT- Rourkela for providing the necessary supports to carry out above research work.

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