1st International Conference on Engineering

Available online at www.icemme.com Proceedings of the

1st International Conference on Engineering Materials and Metallurgical Engineering 22- 24 December, 2016

Bangladesh Council of Scientific and Industrial Research (BCSIR) Dhaka, Bangladesh

FABRICATION AND CHARACTERIZATION OF SYNTHESIZED NANO HYDROXYAPATITE/POLY LACTIC ACID SCAFFOLD USING FREEZE DRYING IN SITU HYBRID SUSPENSION. Md Ruhul Amin Foisala*,Ajoy Kumerb, M. A. Gafurc, Mohammad Saiful Alama, M. R. Qadirc. a

Department of Applied Chemistry and Chemical Engineering, Noakhali Science and Technology University, Sonapur, Noakhali-3802, Bangladesh. b Department of Chemistry, University of Chittagong, Hathazari, Chittagong-4331, Bangladesh. c Pilot Plant and Project Development Centre, Bangladesh Council for Scientific and Industrial Research, Dhaka-1000, Bangladesh.

Abstract Fabrication of porous scaffolds from advanced biomaterials for healing bone defects represents a new approach for tissue engineering.Hydroxyapatite ceramics have been recognized as substitute material for bone due to their chemical and biological similarity to human bone tissue.It is biocompatible and bioactive material that can be used to restore damaged human calcified tissue. HAp cannot be implant directly as a healing supplement in calcified tissue. HAp, when blended with poly lactic acid(PLA), not only improved the bending strength of the composite but also had an active role in new bone formation. Nano HAp powders were synthesized by wet precipitation technique using aqueous suspension of 0.5M calcium hydroxide[Ca(OH)2] with 0.3M orthophosphoric acid [H3PO4] added dropwise and vigorously stirred to control the crystal size at nano scale. And simultaneously temperature and pH were maintained to obtain high purity of HAp. The HAp was characterized by X-ray Diffraction (XRD) and same diffraction pattern was found with the characteristic peaks of nano HAp. Nano HAp-PLA composite scaffold blocks were producedby freezedrying in situ synthesized hybrid suspension. Different loading concentrations of nano hydroxyapatite (nHAp) particles with the Poly Lactic Acid (PLA)fabricated scaffold blocks has been studied on their mechanical, thermal and morphological properties.The chemical and thermal properties of scaffold blocks were investigated by simultaneous thermo-gravimetric analyzer (TGA), differential thermal analyzer (DTA) and thermos-mechanical analyzer (TMA). Crystallographic characterization by X-Ray diffraction and morphological characterization by scanning electron microscopereveled the formation of the micro porous hydroxyapatite and poly lactic acid scaffold block. Throughout those analyses it can evaluated that, the scaffold blocks possess low moisture content, uniform nHAp distribution through scaffold block, porosity obtained at microscale and 15% HAp contained scaffold block maximum load holding ability. Keywords:HAp, Scaffold,X-ray Diffraction Analysis, Thermo Gravimetric Analyzer, Differential Thermal Analyzer,Thermo Mechanical Analyzer.

1.

INTRODUCTION

Tissue engineering is an interdisciplinary and multidisciplinary field that aims at the enlargement of biological substitutes that renovated maintain, or improve tissue function [1]. In a typical tissue engineering approach, to control tissue formation in three dimensions (3D), a considerable porous scaffold is critical[2]. In addition to defining the 3D geometry for the tissue to be engineered, the scaffold provides the microenvironment (synthetic temporary extracellular matrix) for regenerative cells, supporting cell attachment, proliferation, differentiation, and neo tissue genesis [2-3]. Therefore, the chemical compositions, physical structure, and biologically functional moieties are all important attributes to biomaterials for tissue engineering. Hydroxyapatite (HAp), Ca10(PO4)6(OH)2, is the developing bioceramic, which is composed primarily of calcium and phosphorous with hydroxide ions that are eliminated at elevated temperatures[4]. HAp and other related calcium phosphate materials widely used in various biomedical applications due to its excellent biocompatibility and bone bonding ability and its close similarities with inorganic mineral phase of human bone tissue[5]. Synthetic HAp is similar to naturally occurring HAp in contrast of crystallographic and chemical studies. The ratio of Ca/P is to be 1.667 to promote intimate bone growth onto femoral implant [6]. The quality of a scaffold is closely dependent on the overall attributes and characteristics of the synthesized powders. Such attributes include phase composition, purity, crytsallinity, particle size, particle-size distribution, specific surface area, density and particle morphology. These important factors determine the resulting success of the HAp/PLA scaffold onto orthopedic implants[7]. Use of calcium phosphate polymer composites creates a highly biocompatible product by increasing cellmaterial interactions compared to the polymer alone [8]. Furthermore, the sustained release of calcium and phosphate from those composites, where the two ions serve as substrates in the remodeling reactions of mineralized tissues is an added benefit [9]. Due to the extensive use of PLAs in the biomedical field, their composites with HAp constitute the most common

Proceedings of the 1st ICEMME, 22-24, Dec, 2016, Dhaka, Bangladesh

materials in the literature about biodegradable hard tissue implants. HAp, when blended with PLA, not only improved the bending strength of the composite but also had an active role in new bone formation [10]. The HAp surface was found to be highly reactive and led to favorable attachment to tissue and bioactivity in bone repair [11-12]. Due to this bioactivity it is preferred in bone fixation applications where tissue bonding to implant is desirable. The most important advances in the field of biomaterials over the past few years have been in bioactive biomaterials[11]. Materials have been developed to incorporate bioactivity through biological recognition, including incorporation of adhesion factors, polyanionic sites that mimic the electrostatics of biological regulatory polysaccharides, and cleavage sites for enzymes involved in cell migration. Materials have also been developed to be active in biological environments by undergoing phase changes in situ, including transformations from liquid precursors to solids and from soluble materials that are immobilized on tissue surfaces. 2.METHODOLOGY Calcium Hydroxide (74.09 g/mol, density: 3.16 gm/cm3); used in this study was manufactured by Unitika Co. Ltd Japan, and dried under vacuum at 750C before synthesis of nHAp. Phosphoric Acid (85% solution, 97.995 g/mol, density: 1.685 g/ml) reacts with Calcium Hydroxide at room temperature and to maintain the pH above 10.0 Ammonium Hydroxide ( 28-30% solution) was added drop wise[6, 13]. NH4OH (25%)

0.3 M H3PO4 (75%)

0.5 M Ca(OH)2

pH Meter

Magnetic Stirrer

Temperature Meter

FIG 1: SCHEMATIC DRAWING OF APPARATUS FOR HAP POWDERS SYNTHESIS. nHAp powders were prepared via, precipitation technique. In brief, 50 ml aqueous suspension of 0.5 M calcium hydroxide [Ca(OH)2] was prepared and vigorously stirred for 30 minutes. Then 50 ml of 0.3M orthophosphoric acid [H3PO4] was slowly added drop wise (0.5 ml/min) into the Ca(OH)2 suspension. And simultaneously temperature and pH is also observed and the pH (10.5) is adjusted by adding drop wise 1 M ammonium hydroxide [NH4(OH)] solution. The suspension was well stirred (1000 rpm) using magnetic stirrer for 2 hours and aged for overnight at room temperature (as shown in figure 1. In the second day the suspension is heated at 1500C for 2 hours and stirred at 1000 rpm then cooled at room temperature. After that the suspension is sonicated at ultarsonication bath with 30 minutes’ period for 6 times and finally high-speed stirring at 1500 rpm for 6 hours and milled at ball mill for 12 hours, and then aging for 24.0 hour. On the fourth day the precipitates were subjected vacuum filtrating using Buchner funnel, repeatedly washed with deionized water and filtered again until the washed water pH become 7. The precipitates were dried at 800C for 48 hours. Dried lumps of powders were ground by clean pestles and mortars. Table 1: Weighing of the composition for scaffold preparation. Sample Name 5%nHAP/PLA 10%nHAP/PLA 15%nHAP/PLA 20%nHAP/PLA

nHAp (gm) 0.2002 0.4008 0.6022 0.8019

PLA (gm) 4.048 4.043 4.019 4.032

1,4-Dioxane (ml) 15 15 15 15

The required quantity (given at the table) of nHAp powder was dispersed in 1,4-Dioxane (88.11 g/mol, density 1.003 g/cm3); by sonication, then following the table required amount of Poly Lactic Acid (density 1.29 g/cm3) was added to the mixtures, and stirred at 500C until complete dissolution. Four solution with different content in nHAp were prepared, 5%, 10%, 15%, 20% particle by weight with regard to PLA. The nHAp/PLA solution were poured aluminum

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foil and frozen in 00C for 24 hours. Then the scaffolds were placed in freeze dryer in order to remove the dioxane. After extraction of dioxane, scaffold was dried in atmospheric temperature and then cut into cubes (1cm x 1 cm x 1 cm). These samples were then again dried at 400C in a vacuum dryer and stored in desiccators. 2.1 POROSITY The estimated density and porosity of the matrix were obtained as follows. The volume of the matrix is calculated using suspended weight. And the mass of the matrix was measured with an analytical balance. The density was calculated from the volume and mass. The porosity, ε, was calculated from the measured overall density Dm of the matrix and the skeletal density Ds. For a composite scaffold, the skeletal density was determined using the densities of the polymer and nHAp powder. The porosity was given by, =

(1)

=

(2)

Where, Ds was calculated from the following formula:

Where Dh is the density of the nHAp powder with a value of 3.16 gm/cm3 and Xh is the percentage of nHAp in the composite scaffold while Dp is the density of the polymer. 2.2 X-RAY DIFFRACTION ANALYSIS XRD analysis was carried out with a X-ray diffractometer where the sample was directly put into the sample holder and then experiment was carried out. Gobel Mirror was the source of X-ray. Filter was used to remove CuKβ and soller slit to pass the parallel ray. The peaks obtained were used to determine the exact crystal structure of the nHAp. XRD studys have been carried out by D8 advance, Bruker AXS, Germany. The exact crystalline structure of the samples confirmed by XRD revealed the formation of the calcium-Hydroxyapatite phase and yielded reflections. 2.3SCANNING ELECTRON MICROSCOPY ANALYSIS Image analysis was carried out using a Scanning Electron Microscope (SEM) (model: JSM-6490LA) for observing surface morphology, particle size, particle distribution, porosity and pore size. It is a High-performance, SEM with an embedded energy dispersive X-ray analyzer (EDS) with allows for semless observation and EDS analysis. 2.4 THERMOGRAVIMETRIC ANALYSIS TGA/DTA and DTG studies have been done by TG/DTA 6300, SII Nano Technology Japan, system controlled by an EXSTAR 6300 controller. TGA and DTA studies have been carried out on freeze dried nHAp/PLA composite scaffold sample in different weight percentage. Experiments have been performed using simultaneous TGA-DTA analysis by heating the sample at 200 C/min in the temperature range 300C and 6000C in nitrogen atmosphere. 2.5THERMOMECHANICAL ANALYSIS TMA studies have been done by TMA/SS 6300, SII Nano Technology, Japan, system controlled by an EXSTAR controller. TMA studies have been carried out on the nHAP/PLA composite scaffold in different weight percentage. Experiments have been performed using simultaneous TMA analysis by heating thesample at 50C/min in the temperature range 300C and 1000C in nitrogen atmosphere. 3. RESULT AND DISCUSSION 3.1 POROSITY ANALYSIS nHAp/PLA composite scaffolds with high porosity have been fabricated using freeze drying in situ synthesized hybrid suspension. One solvent systems were used to obtain composite scaffolds with pore structures, is a mono-solvent system of pure 1,4-dioxane. The densities and porosities of scaffolds are found 60%, 59%, 58% and 57% for 5%, 10%, 15% and 20% nHAp/PLA composite Scaffold respectively. Incorporation of nano sized HAp only decreased the porosity slightly. Regardless of detailed morphology and composition, all scaffolds have a porosity of at least 57%, which was considered to be beneficial for cell in growth and survival. The solvent system plays the most important effect on the density and porosity. 3.2 X-RD ANALYSIS The sample nHAp exhibit almost same diffraction pattern with the characteristic peaks of HAp. The peak analysis of the XRD pattern shows 14.9 nm crystal size and the exact crystalline structure of the sample with Ca/P =1.65 including the (210), (211), (202), (311), and (213) planes.

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Intensity (Counts)

XRD analysis of HAp Powder 1800 1600 1400 1200 1000 800 600 400 200 0

31.76, 1712 32.78, 1067 25.86, 933 46.62, 507

34, 586 39.7, 412

28.9, 379

20

25

30

35

40

45

49.44, 635 53.2, 404

50

55

60

65

70

2 Theta WITH COMPARING STANDARD SAMPLE. FIG. 2: XRD PATTERN OF HYDROXYAPATITE PARTICLE XRD analysis of the nHAp/PLA composite scaffold shows that with the increasing percent of HAp content the nHAp characteristic peaks intensity increases as the nHAp content in increases in the scaffold sample.

FIG. 3: COMPARISON OF XRD PATTERN OF 5%, 10%, 15%, 20% NHAP/PLA COMPOSITE SCAFFOLD MATRIX WITH STANDARD HAP. 3.3 SCANNING ELECTRON MICROSCOPIC ANALYSIS SEM study of the composite blocks confirmed the formation of three dimensional porous structure with pore size in the range of 1µm-10µm.

a) PLA-HAp Scaffold block with 5% HAp.

b) PLA-HAp Scaffold block with 10% HAp

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c) PLA-HAp Scaffold block with 15% HAp

d) PLA-HAp Scaffold block with 20% HAp

FIG. 4: SEM MICROSTRUCTURE OF COMPOSITE SCAFFOLD BLOCK AT 2500 MAGNIFICATION. The prominent microstructural features manifested the effect of HAp concentration on the pore size and its distribution. The first sample comprising 5% HAp in PLA matrix exhibit relatively large crystal size and pore size of 30µm (fig. 4.a) whereas the sample with 20% HAp in PLA matrix revealed a smaller pore size of 20µm (fig. 4.d). 3.4 THERMOGRAVIMETRIC AND DIFFERENTIAL THERMAL ANALYSIS TGA and DTGA show that all the sample exhibited two distinct weight loss stages at 400C-1100C (maximum 5.0% and minimum 2%) the weakly bonded water, 3000C-4500 C (decomposition of the main chain of PLA). The major weight loss is observed in the 3000C-4500C region for the entire sample. That is corresponding to the structural decomposition of PLA. First order derivative of TGA curves revels the temperature at which the maximum decrease of mass occur. The temperature at the maximum loss rate is 378.00C, 378.10C, 378.10C and 378.70C for 5%, 10%, 15%, and 20% HAP content in PLA respectively. So it is clear that the HAp percent is not effecting the PLA breakdown.

100.0

20.00 5% HAP,95%PLA Scaffold - S11 10% HAP,90%PLA Scaffold - S12

350.0 15.00

15% HAP,85%PLA Scaffold - S13 20% HAP,80%PLA Scaffold - S14

50.0

10.00

300.0

5.00

0.0

200.0

150.0

-50.0 -5.00

-10.00 -100.0

100.0

-15.00

-150.0

-20.00 50.0 -25.00

-200.0

0.0 -30.00 100.0

200.0

300.0 Temp Cel

400.0

500.0

600.0

FIG. 5: COMPARATIVE THERMOGRAM OF 5%, 10%, 15%, 20% NHAP/PLA COMPOSITE SCAFFOLD.

237

TG %

0.00 DTA uV

DTG ug/Cel

250.0


3.5 THERMOMECHANICAL ANALYSIS TMA shows the load holding temperature 46.80C, 49.80C, 55.570C and 55.080C respective to 5%, 10%, 15% and 20% nHAp/PLA composite scaffold. Which represents with the increase of nHAp content in the scaffold the load holding temperature is increasing and after 15% the load holding temperature start to decrease.

0 .0 0

-10 0 . 0

5% H AP S11 10% HA P S12 15% HA P S13 20% HA P S14

-10 .0 0

-10 0 . 0

-2 0 .0 0

TMA um

-3 0 .0 0 -10 0 . 0

Load mN

-10 0 . 0

-4 0 .0 0 -10 0 . 0 -5 0 .0 0

-10 0 . 0 -6 0 .0 0

-10 0 . 0 -7 0 .0 0 4 0 .0 0

4 5 .0 0

5 0 .0 0

5 5 .0 0

6 0 .0 0 6 5 .0 0 Tem p C el

7 0 .0 0

7 5.00

8 0.00

8 5 .0 0

FIG. 6: COMPARISON OF THERMO MECHANICAL THERMOGRAM OF 5%, 10%, 15% AND 20% NHAP/PLA COMPOSITE SCAFFOLD. 4. CONCLUSION Various techniques, including SEM, XRD, TMA, TGA, DTA were performed to characterize the resulting nHAp/PLA composite scaffold. Morphological investigation showed that the HAp particles exhibit micro-porous morphology, which provides enlarge interfaces being a prerequisite for physiological and biological responses and remodeling to integrate with the surrounding native tissue. 5. REFERENCE

[1] [2]

[3] [4]

[5]

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