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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01

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Preparation and Characterization of GelatinHydroxyapatite Composite for Bone Tissue Engineering Md. Jakir Hossana, M. A Gafurb, M. R Kadirb and Mohammad Mainul Karima,* a

Department of Applied Chemistry and Chemical engineering, University of Dhaka, Dhaka-1000, Bangladesh b PP & PDC, Bangladesh Council of Scientific and Industrial Research (BCSIR ), Dhaka, Bangladesh *To whom correspondence should be addressed: Mohammad Mainul Karim E-mail: [email protected] E-mail: [email protected] or [email protected] Web Site: http://www.ijens.org

Abstract--

In biomedical research, fabrication of porous scaffolds from advanced biomaterial for healing bone defects represents a new approach for tissue engineering. Hydroxyapatite ceramics have been recognized as substitute material for bone and teeth in orthopeadic and dentistry field respectively due to their chemical and biological similarity to human hard tissue. In this study, to mimic the mineral and organic component of natural bone, hydroxyapatite (HAp) and gelatin (GEL) scaffolds were prepared. The raw material was first compounded and resulting composite were molded into the petridishes. Using Solvent casting process, it is possible to produce scaffolds with mechanical and structural properties close to natural trabecular bone. The chemical and thermal properties of composites were investigated by Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analyzer (TGA), Differential Thermal Analyzer (DTA) and Thermomechanical Analyzer (TMA). Crystallographic characterization by X-Ray diffraction and morphological characterization by SEM revealed the formation of a micro porous hydroxyapatite gelatin composite. It was observed that the pores in the scaffolds are interconnected and their sizes range from 80 to 400μm. Since one osteoblast occupies an area of approximately 700 μm, hence the pore size of 500 μm (diameter of a spherical pore) is compatible with osteoconduction, however the optimum pore size for osteoconduction is 150 μm. These results demonstrate that the prepared composite scaffold is a potential candidate for bone tissues engineering.

1. INTRODUCTION Tissue engineering is an interdisciplinary and multidisciplinary field that aims at the development of biological substitutes that restore, 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. 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]. Therefore, the chemical composition, physical structure, and biologically functional moieties are all important attributes to biomaterials for tissue engineering. The most important advances in the field of biomaterials over the past few years have been in bioactive biomaterials. Tissue

engineering presents an alternative approach for the healing of diseased, damaged and traumatized bone tissue. In a typical tissue engineering application, osteogenic cells would be harvested from the patient and seeded on a synthetic porous (structure having a large surface area for a more efficient cell interaction) scaffold that acts as a guide for tissue growth creating a living biocomposite. The biocomposite system would then be implanted back into the patient. Eventually, the scaffold will be absorbed by the body as non-toxic degradation products at the same rate that the cells produce their own extracellular matrix. One of the major challenges of tissue engineering is to develop a suitable bone scaffold. The primary concerns are biocompatibility, biodegradability, pore size, pore connectivity and an adequate mechanical strength. Bone tissue engineering has the potential to reach millions annually to repair the bone defects caused by diseases, trauma or congenital defects. In 2003 the potential market for tissue engineered products for musculoskeletal applications totaled 23.3 billion in the US and is expected to rise to 39 billion by the year of 2013[3]. In 2004 alone there were 1.3 million bone grafts procedures [4]. In the united states alone, at least eight million surgical operations were carried out annually, requiring a total national healthcare cost exceeding 400 billion per year [5,6]. Autografts (from the patient) were considered the gold model for bone defects and allografts ( from the donor were also commonly used). Commonly used materials for this purpose are biodegradable polymers such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and their copolymers (PLGA).[7–10] For example, PLA degrades within the human body to form lactic acid, a naturally occurring substance which is easily removed from the body material. Scaffolds based on these two polymers have been used in numerous tissue engineering applications [5]. However, most polymers have relatively poor mechanical strength which is required for many applications [11–14]. A common way to improve the mechanical properties of polymers is to make use of filler particles. Thus, in order to obtain a better combination of biocompatibility, biodegradation and mechanical strength, composites of polymer and bioactive ceramics have been considered for bone tissue engineering [15–18]. Hydroxyapatite (HAp), owing to its excellent bioactivity,

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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01 osteoconductivity and chemical similarity to the mineral component of natural bone, has been a preferred bioceramic in the fabrication of composite scaffolds [7,19,20]. It should be added that polymer ceramic system resembles the natural bone structure which itself is a composite composed of nanosized hydroxyapatite embedded in collagen matrix. The biological response to the scaffold material is influenced by a number of factors including the size and morphology of the pores within the fabricated scaffold [21,22]. The optimum pore size is different depending on the cell type. However, generally an interconnected pore structure with a pore size in the range of 100–500 mm is considered to be optimal for osteoconduction and nutrient transfer for optimal tissue growth [23,24]. Various processing methods have been adopted in the preparation of porous scaffolds including gas foaming [7], freeze drying [9,25], casting methods and porogen particulate leaching [26–28]. Among these techniques, casting methods seems to be a rather simple and economical approach in the preparation of porous scaffold materials. In this casting method, the scaffold’s porosity can be controlled by the amount of a water soluble leaching agents such as sodium chloride, while the pore size can be manipulated by the size of the salt crystals [29,30]. There are some recent reports on the use of this procedure in the preparation of porous (PLGA) and HAp–PLA scaffolds. This method has also been applied in the fabrication of porous gelatin-HAp scaffold [29]. Due to its biodegradability, biocompatibility and cost efficiency, gelatin, a natural polymer can be used as a scaffold for tissue engineering. However, as it was mentioned previously, scaffolds based upon polymeric material alone such as gelatin are not ideal in terms of their mechanical strength [29]. To address this issue and to keep a proper balance between the biological and the mechanical strength, the addition of bioactive HAp particles within a gelatin matrix has been reported in the literature [25]. The purpose of this study was therefore to extend into the preparation of hydroxyapatite–gelatin composites and to investigate the structure. 2.

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white coloration during preparation is an indication for the formation of hydroxyapatite[32]. After reaching this point, the suspension was stirred for 2 h, before it was left undisturbed for 24 h at room temperature. The HAp precipitates were then separated from suspension by filtration. At some times the wet cakes were washed with hot distilled water and dried in a dryer at 1000C. Then it was powdered in a mortar with pestle. 2.2 Preparation of Gelatin – HAp Composite The slurry composite was prepared using solvent casting method. As dry GEL is essentially intractable material, it can readily become castable or shapeable when transformed into a sol-GEL state by dissolution in water up to about 5-30 wt% . In order to have a homogenous and strong composite, the HAp particles finer than 150 μm were obtained using Ultrasonicator. Definite GEL content 12.33 wt% was dissolved in dionized water at temperature of 45°C. Then the 5 wt%, 10 wt%,15 wt% and 20wt% HAp contents were added to prepare four different composites. The reinforced slurry composite was then heat treated on magnet stirrer under constant mixing at 40°C for 1 h. The slurry was deagglomerated by magnet stirring. The temperature was monitored continuously. It should be noted that to make a well distributed homogenous composite, the heat must not be applied directly to the composite. By using an interface water bath beaker, the heat treatment process can be homogenously applied to the reaction vessel. Some air bubbled were generated which was removed by glass rod. The slurry immediately was transferred into definite size of petridishes. The molds were frozen at - 40°C and then dried in a commercial freeze-dryer for 3 h for solvent removal. After that, the white composites were removed and placed in room temperature for 24 h. For statistical analysis in all assays, five samples of each type were investigated and the average was reported. 3.

RESULTS AND D ISCUSSION

3.1 TGA/DTA/ DTG analysis for Gelatin –HAp Composite

MATERIALS AND METHOD

2.1 Synthesis of HAp Powder Preparation of gelatin–hydroxyapatite composite scaffold started by preparing hydroxyapatite crystallites using a chemical precipitation method [31]. At first 1M Ca(OH) 2 (96% pure) was taken with 100 ml distilled water in a beaker. On the other hand 0.6 M H3PO4 was taken with 100ml distilled water in another beaker. Appropriate amounts (Ca/P ratio of 1.67) of orthophosphoric acid solution was added gradually (2 drops per second) from burette in the beaker with magnetic stirrer at room temperature. The appearance of milky

TGA tell the physical properties of the polymer used in the scaffold preparation. TGA shows the change in mass with the increase of temperature. TGA/DTA and DTG studies was carried out by TG/DTA 6300, SII Nano Technology, Japan, system controlled by an EXSTAR 6300 controller. TGA and DTA studies have been carried out on Gelatin-HAp composite sample in different weight. Experiments have been performed using simultaneous TGA-DTA analysis by heating the sample at 20 Cel/min in the temperature range 0˚C and 600˚C in nitrogen atmosphere and a typical plots has been shown in figures 1-5.


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1.4%

111.2Cel 125.9Cel 97.6% 94.6%

100.0

25.00 250.0

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294.1Cel 89.8%

126.7Cel 139.8Cel 6.4%94.4% 91.2%

90.0

80.0 20.00

332.8Cel 67.7%

200.0

70.0 44.1% 326.3Cel 71.3%

60.0

50.0 371.2Cel 45.6%

10.00 100.0

TG %

150.0

DTA uV

DTG ug/min

15.00

40.0

320.8Cel 8.69uV

30.0

5.00 20.0

326.5Cel 53.2ug/min

50.0 126.0Cel 19.5ug/min

10.0

0.00 0.0

0.0

-10.0 100.0

200.0

300.0 Temp Cel

400.0

500.0

Fig. 1. TG, DTA and DTG curves for Pure Gelatin.

TG (figure-1) shows the 1.4% initial loss due to moisture. The first onset temp, 2nd onset temperature, first 50% degradation temperature, 2nd 50% degradation temperature,1st maximum slope, 2nd maximum slope are 111.20C, 294.10C, 125.90C, 332.80C, 126.7oC and 326.3oC respectively. The total

700.0

100.0

291.1Cel 93.1%

25.00 127.0Cel 97.1%

800.0

degradation loss is 44.1%. DTA curves shows endothermic peaks at 320.80C are due to thermal degradation. The DTG curves show the two peaks at 126.00C and 326.50C. There is two steps degradation, the initial degradation is due to moisture and the 2nd degradation is due to composite.

330.1Cel 68.3%

80.0

20.00 48.8%

600.0

329.6Cel 68.7%

60.0 15.00 368.2Cel 44.3%

40.0 547.7Cel 41.0%

400.0

TG %

DTA uV

DTG ug/min

500.0

10.00 323.0Cel 9.85uV

300.0

20.0

5.00 200.0 0.0 328.6Cel 182.0ug/min

100.0 0.00

-20.0 0.0

100.0

200.0

300.0 Temp Cel

400.0

500.0

600.0

Fig. 2. TG, DTA and DTG curves for gelatin +5% HAp Composite. 145501-7272-IJET-IJENS © February 2014 IJENS

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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01 The onset temperature (figure-2), 50% degradation temperature, maximum slope are 291.10C, 330.10C, and 329.6oC respectively. The total degradation loss is 48.8%. DTA curves shows endothermic peaks at 323.0 0C are due to

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thermal degradation. The DTG curves shows the one peak at 328.60C . There is one step degradation is due to thermal degradation of composite.

128.2Cel 95.3%

100.0 800.0

40.00 4.3%

158.0Cel 94.1%

287.1Cel 93.6% 331.1Cel 69.5%

700.0

80.0

30.00 331.3Cel 48.2% 69.4%

600.0

60.0

373.3Cel 45.3%

544.2Cel 41.5%

400.0

40.0

TG %

20.00

DTA uV

DTG ug/min

500.0

10.00 300.0

200.0

20.0

329.4Cel 198.1ug/min

0.00

0.0

100.0 -10.00

-20.0

0.0

100.0

200.0

300.0 Temp Cel

400.0

500.0

600.0

Fig. 3. TG, DTA and DTG curves for gelatin +10% HAp Composite.

TG (figure-3) shows the 4.3% initial loss due to moisture The onset temperature, 50% degradation temperature, maximum slope are 287.10C, 331.10C, and 331.3oC respectively. The total degradation loss is 48.2%. DTA curves shows 140.4Cel 95.0%

40.00 800.0

35.00

4.4%

700.0

endothermic peaks at 338.10C are due to thermal degradation. The DTG curves shows the one peak at 329.40C . There is one step degradation is due to thermal degradation of composite.

100.0

289.3Cel 93.7%

167.5Cel 94.2%

90.0 333.5Cel 71.3%

80.0

30.00 600.0

342.2Cel 44.9% 66.9%

25.00

70.0

60.0

50.0

378.7Cel 48.8%

400.0

575.5Cel 47.8%

15.00

TG %

20.00

DTA uV

DTG ug/min

500.0

40.0 300.0

10.00

200.0

5.00

100.0

0.00

30.0

375.1Cel 10.62uV

20.0

10.0 331.3Cel 167.4ug/min

0.0 -5.00 0.0

-10.0 100.0

200.0

300.0 Temp Cel

400.0

500.0

600.0

Fig. 4. TG, DTA and DTG curves for gelatin +15% HAp composite.


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endothermic peaks at 375.10C are due to thermal degradation. The DTG curves shows the one peak at 331.3 0C. There is one step degradation is due to thermal degradation of composite.

TG (figure-4) shows the 4.4% initial loss due to moisture The onset temperature, 50% degradation temperature, maximum slope are 289.30C, 333.50C, and 342.20C respectively. The total degradation loss is 44.9%. DTA curves shows 129.1Cel 94.9%

700.0 30.00

100.0

284.1Cel 97.2%

157.2Cel 94.8%

90.0

330.6Cel 75.5%

4.5%

600.0

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25.00

80.0 43.4%

500.0

331.8Cel 74.9%

70.0

20.00

15.00

372.3Cel 53.8%

526.4Cel 51.1%

50.0

TG %

400.0

DTA uV

DTG ug/min

60.0

40.0

300.0 10.00

322.7Cel 10.34uV

376.0Cel 10.93uV

30.0

200.0 5.00

100.0

20.0

10.0

332.8Cel 185.6ug/min

0.00

0.0 0.0

-10.0

-5.00 100.0

200.0

300.0 Temp Cel

400.0

500.0

600.0

Fig. 5. TG, DTA and DTG curves for gelatin +20% HAp composite.

TG (figure-5) shows the 4.5% initial loss due to moisture The onset temperature, 50% degradation temperature, maximum slope are 284.10C, 330.60C, and 331.8oC respectively. The total degradation loss is 43.4%. DTA curves shows endothermic two peaks at 322.70C and 376.00C are due to initial and final thermal degradation respectively. The DTG curves shows the one peak at 332.80C . There is one step degradation is due to thermal degradation of composite.

are due to thermal degradation. From the TG/DTA data it is observed that HA-GEL composite is highly stable. The stability of composite increases with increasing % of HA.

TGA and DTGA show that all the samples exhibited three ⁰ ⁰ distinct weight loss stages at 40 C-250 C (5% weight loss of ⁰ ⁰ weakly physioabsorbed water), 250 C-500 C (decomposition of main chain of gelatin). Nevertheless major weight losses ⁰ ⁰ are observed about 50wt% in the range of 250 C-500 C for all the samples, which are corresponding to the structural decomposition of gelatin. First order derivative of TGA curves reveals the temperature at which the maximum decrease of mass occur. The ⁰ temperature at the maximum loss rate is 326.5 C for the pure ⁰ ⁰ Gelatin, 328.5 C for the 5wt% HA-GEL composite, 329.4 C o for the 10wt% HA-GEL composite, 331.3 C for the 15wt% Ha- GEL composite and 332.8oC for the 20wt% HA- GEL composite. DTGA data clearly show that endothermic peaks 145501-7272-IJET-IJENS © February 2014 IJENS

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3.2 X-Ray diffraction analysis:

Fig. 6. X-Ray diffraction spectra of pure HAp, Pure Gelatin, 5wt%, 10wt%, 15wt%, and 20wt% of HAp respectively (from top).

The phase of the gelatin and gelatin-HA composite was analyzed with XRD patterns, as shown in Figure 6. In pure gelatin, a broad peak at 2θ ≈ 14°, the characteristic of gelatin,

was observed. When HA was added, typical HA peaks were observed, and with increasing HA amount, the peak intensities increased and the gelatin peak decreased correspondingly.

3.3 Fourier Transform Infrared (FTIR) analysis:

Fig. 7. FTIR spectrum of Pure Gelatin


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Fig. 8. FTIR spectrum of HAp- Gelatin composite at containing 5% HAp

Fig. 9. FTIR spectrum of HAp- Gelatin composite at containing 10% HAp. 145501-7272-IJET-IJENS © February 2014 IJENS

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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01 The FTIR spectrum of the Gelatin and 5 wt%, 10 wt% HApGelatin composites are shown in Figure-7-9. The band at 1328 cm-1 in GEL is attributed predominantly to the so-called wagging vibration of proline side chains. The 1328 cm-1 band in GEL does not simply represent the carboxyl group, but it is one of a number of bands in the range of 1400- 1260 cm-1 which are attributed to the presence of type-I GEL [1, 2]. The amide A band arising from N-H stretching was distributed at 3270-3370 cm-1 relative to the degree of cross-linking, C-H stretching at ~2947 cm-1 for the amide B, C = O stretching at 1637 cm-1 for the amide I, N-H deformation at 1500-1550 cm-1 for the amide II [1,2]. The appearance of an amide I mode indicated that HA-GEL composites adopt a predominantly αhelical configuration and this is confirmed by the appearance of amide II at ~1540 cm-1 [3,4]. As HA related bands, there

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are hydroxyl group (-OH) stretching (4000-3200 cm-1) and liberational bands, and phosphate contours. There are CO 3 V3 bands at 1540-1400 cm-1 and 1530-1320 cm-1. The phosphate band is between 900 and 1200 cm-1. The shift of the 1328 cm1 band in GEL has been effectively used to confirm the chemical bond formation between carboxyl ions in GEL and HAp phases [1,3]. During the process of HA-GEL composite, the Ca2+ ions will make a covalent bond with R-COO- ions of GEL molecules. Moreover the cross-linking induces the shortening of the distance between HAp-GEL fibrils within the critical length and more amount of Ca2+ ions on HAp will have a chance to bind with R-COO- ions of GEL molecules [2-4]. 3.4. Scanning electron microscope analysis:

(a) (b) (c) Fig. 10. SEM image of microstructure of Gelatin-HAP composite with 5% HAp. (a) at 140 magnification. (b) at 330 magnifications. (c) at 4300 magnification.




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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01 Porosity characterization is based on the presence of open pores which are related to properties such as permeability and surface area of the porous structure. It was found from SEM image analysis as shown in figure 10-12 that the addition of HAp results in more dense and thicker pore walls with lower porosity, therefore the addition of HAp content improves the mechanical properties [5, 6]. Since a higher density of a scaffold usually leads to higher mechanical strength while a high porosity provides a favorable biological environment, a balance between the porosity and density for a scaffold must be established for the specific application.

[7]

CONCLUSION Gelatin and Hydroxyapatite (HAp) is one of the components most frequently used to prepare calcium phosphate composites, because of its biocompatibility, biodegradation and innocuousness. In this work we try to prepare and characterize gelatin & hydroxyapatite using TG/ DTA, SEM & FTIR analysis and X-Ray diffraction (XRD). Morphological investigation showed that the HAp particles exhibit micro-porous morphology, which provides enlarged interfaces being a prerequisite for physiological and biological responses and remodeling to integrate with the surrounding native tissue. XRD analysis revealed the exact crystalline structure of the three samples is one of the calcium-phosphate polymorph with Ca/P = 1.65 indicating the formation of calcium-hydroxyapatite phase. All the four samples exhibited almost similar diffraction pattern with characteristic peaks of HAp. The FTIR spectrum for the crosslinked composite indicates chemical bond formation between gelatin and hydroxyapatite. From the TG/DTA data it is observed that gelatin – HAp is highly stable. The degradation temperature of gelatin nearly 3000C of composite. This study indicates that, gelatin and HAp can be used as a bone replacement material.

[16]

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