Key Engineering Materials Vol 587 (2014) pp 86-92 © (2014) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/KEM.587.86
Online: 2013-11-15
Synthesis of nano calcium phosphate via biomimetic method for bone tissue engineering scaffolds and investigation of its phase transformation in simulated body fluid Majid Raz1, a, Fathollah Moztarzadeh1, b Mohammad A. Shokrgozar2,c Mohammadreza Tahriri 1, 3, d 1
Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran 2 3
Cell Bank, Pasteur Institute of Iran, Tehran, Iran
Dental Materials Department, School of Dentistry, Tehran University of Medical Sciences (TUMS), Tehran, Iran
[email protected],
[email protected],
[email protected],
a
d
[email protected]
(Corresponding author: Majid Raz) Keywords: Calcium Phosphate, Biomimetic,Ttissue engineering, Simulated body fluid.
Abstract. In this study the formation of calcium phosphate phase via double diffusion method into a hydrogel matrix was investigated and its phase transformation in simulated body fluid was studied. White precipitate was formed within the hydrogel, due to the diffusion of calcium and phosphate ions through the hydrogel matrix in similar pH to human body. Phase composition, microstructure and structural groups in the composite samples were also characterized by X-Ray Diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infra-red (FTIR) analyses. Microstructure of precipitates formed within middle hydrogel, showed that detected materials are composed of carbonated hydroxyapatite and dicalcium phosphate dihydrate (DCPD, brushite). The particle size was about 10 nm .Analysis results showed that after incubation in simulated body fluid, dicalcium phosphate dehydrate phase transformed into crystalline hydroxy apatite. 1. Introduction Tissue engineering is an interdisciplinary field which combines the knowledge and technology of cells, engineering materials, and suitable biochemical factors to create artificial organs and tissues, or to repair damaged tissues [1]. So the use of a scaffold to serve as temporary matrix and provide structural support for the cells to attach, proliferate and differentiate is an approach in tissue engineering. Therefore, an ideal scaffold should mimic the advantageous properties of the natural ECM [1-3] and have porous structure, biocompatibility with the cells, appropriate mechanical strength and biodegradation rate[4]. To achieve these desired properties it is vital to use biomaterials which can be divided into broad categories of synthetic or naturally derived. Natural bone ECM is a nanocomposite of organic phase deposited within an organic matrix[5]. The inorganic phase of bone is carbonated hydroxyapatite (CHA), and the organic matrix is mainly collagen type I[6]. therefore a large number of researchers has been worked on collagen, and on its derivative, gelatin. Collagen has antigenicity because of its animal source, but gelatin has relatively low antigenicity compared to collagen and promotes cell adhesion, proliferation and differentiation[7]. Chitosan is a natural polymer and is the partially deacetylated form (poly-b(1,4)-2- amino-2deoxy-D-glucose) of chitin which is used in biomedical application due to its biological properties like biocompatibility, biodegradability, hydrophilicity, good adhesion and non-toxicity [8]. But, there is a need to improve its mechanical properties, especially for bone tissue engineering All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, www.ttp.net. (ID: 130.88.90.140, The University of Manchester, Manchester, United Kingdom-03/04/15,19:21:39)
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applications. This can be achieved with addition of biomaterials like gelatin and hydroxyapatite (HAp) [9].HAP has shown well osteoconductivity and bioactivity [5, 10]. Although, the use of HAP alone is confined because of its brittleness and difficulty to process into complex shapes. Thus the combination of HAP and a polymer like gelatin and chitosan, would give us a composite with favorable properties of both HAP and gelatin/chitosan [9, 11, 12]. In order to prepare biomimetic nanophase scaffolds and to mimic the natural bone structure, some biomimetic method like in situ mineralization of calcium phosphate phase within polymer matrix has been recently considered[12]. The objectives of this project were to prepare calcium phosphate via in situ mineralization within gelatin/chitosan hydrogel and to study phase conversion of dicalcium phosphate dihydrate to hydroxyapatite after soaking in simulated body fluid in biological pH and temperature. 2. Experimental procedure 2.1. Materials Gelatin (Merck, microbiology grade, Catalogue No.104070), acetic acid(Merck, Catalogue No100062),Disodium hydroxide phosphate (1 M) (Merck, Catalogue No. 106573), calcium chloride (0.6 M) (Merck, Catalogue No. 2380), Tris buffer (Merck, Catalogue No. 8382), hydrochloric acid (HCl) (Merck, Catalogue No. 280211),sodium hydroxide (Merck, Catalogue No. 109890) were purchased from Merck chemical Co. Chitosan (85% DD medium molecular weight and product number of 448877)was provided from Aldrich-Sigma Co. All commercially available solvents and reagents were of analytical grade. 2.2. Mineralization of calcium phosphate phase The chitosan/gelatin 5 wt. % solution was prepared by dissolving of gelatin and chitosan in ratio of (80/20) in acetic acid solution (1% v/v) and stirring at 60°C for 1 h to gain a homogenous polymer solution. Afterwards the solution was poured in the middle part of a chamber and the system was placed at 4°C for 1 day to form a hydrogel. Two solutions were prepared as sources of calcium and phosphate ion with concentration of 0.1 M and 0.04 M by dissolving of calcium chloride and disodium hydrogen phosphate in distilled water respectively. These concentrations were selected due to similarity with hydroxyapatite of natural bone. The pH of these solutions was set at 7.4 by addition of Tris buffer and hydrochloric acid. Then, solutions were poured in the opposite sides of the prepared hydrogel and the system was kept in 4°C for a week. Diffusion of ions within the hydrogel formed composite with white precipitation in the prepared hydrogel matrix (Fig. 1). By freeze drying of prepared composite a porous structure was obtained. Eventually, in order to neutralize the acetate group in the structure of spongy composites, samples were soaked in sodium hydroxide solution and freeze dried.
Figure 1. Formation of white precipitate (calcium phosphate) within gelatin-chitosan hydrogel matrix via double diffusion method
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2.3. Preparation of SBF SBF solution was prepared based on Tadashi Kokubo method[13] and the prepared samples were incubated in SBF for 1, 3, 7 and 14 days and their bioactivity investigated. Furthermore the phase conversion of the calcium phosphate phases in biological pH and temperature. 2.4. Characterization of scaffold 2.4.1. X-ray diffraction analysis Crystal structure characterization of the prepared composites was performed by means of powder X-ray diffraction analysis, using an INEL Equinox3000 (Cu Kα radiation) operating at a voltage of 40 kV and current of 30 mA. Scanning range (2θ angle) was selected between 0–118°. Furthermore, the average crystallites size was calculated from XRD data using the Scherrer equation [Eq. (1)]: t = 0.9λ/βcosθ
(1)
Where t is the crystallite size, λ is the wavelength of Cu-Kα radiation (1.540560 Å), and β is the full width at half maximum intensity. 2.4.2. FTIR analysis FT-IR spectra of the composite scaffolds were obtained using FTIR NEXUS 670 USA operating at the range of 400-4000 cm-1 wavenumber and used in the absorption mode. 2.4.3. Scanning electron microscopic examination The microstructure and morphology of the prepared scaffolds was examined before and after immersion in SBF, by scanning electron microscopy (SEM) using a Philips XL30 microscope. 3. Results and Discussion 3.1. XRD analysis XRD analysis was successfully applied in order to evaluate the precipitated phase within the hydrogel matrix before and after soaking in SBF. The XRD pattern obtained from the samples before and after incubation in SBF solution is represented in Fig.2 and 3, respectively. These diffraction patterns were compared with calcium phosphate compounds cards present in ICDD database and the phases present in each sample were indexed. In the XRD pattern of the samples mineralized in 4°C, low intensity and broad diffraction peaks are present. These peaks are indicative of hydrogel matrix and brushite (CaHPO4•2H2O) (JCPDS # 2-0085) precipitates that have amorphous or semi-crystalline nature. The diffraction pattern of samples incubated in SBF solution reveals sharp peaks that are mainly related to the presence of Hydroxyapatite (JCPDS # 09-432), as well as the crystalline nature of the precipitates. It should be taken into consideration that since matrix phase has amorphous structure and very low intensity peak compared to the Hydroxyapatite, therefore, this phase is not detectable. As a result, the amorphous precipitates in the samples after incubation in SBF solution at 37°C transformed into the hydroxyapatite with well crystalline structure. This phase transformation mimics the mineralization of natural bone in the body. Furthermore, with increasing incubation time in SBF, the peaks intensity related to hydroxyapatite increases and become narrower which can be attributed to crystallization increase. In addition, by applying Sherrer equation the crystallite size was calculated. Before incubation in SBF, the average crystallite size was calculated as 10 nm and for incubated samples in SBF, was determined 15 to 25 nm that this phenomenon showed the increase of crystallite size after incubation in SBF.
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Figure 2. The XRD pattern of prepared scaffold containing hydrogel and precipitated minerals formed at 4 ºC
Figure 3. The XRD pattern of prepared scaffold containing hydrogel and precipitated minerals after soaking in SBF (1, 3, 7 and 14 days) 3.2. FTIR analysis The FTIR spectra of the prepared scaffolds before and after soaking in SBF solution is shown in Fig.4. All bands in the FTIR spectra of the samples and their relevant chemical bonds are given in table 1. All bands can be classified in three categories as gelatin/chitosan specific wavenumbers, precipitate phase and the wavenumber related to the chemical bonding between hydrogel matrix and precipitated phase. The bands at 1075, 1240, 1541, 1647 and 2927 cm-1 and 530, 600, 872 and 1455 cm-1are related to hydrogel matrix and calcium phosphate precipitate phase, respectively. Also, a band detected at 1337 cm-1 indicates the formation of a chemical bond between Ca2+ ion from calcium phosphate phase and carboxyl group from hydrogel matrix. It can be clearly inferred from the FTIR bands for the samples before and after soaking in SBF solution that after incubation in SBF and also by increasing the incubation time, the bands that are related to calcium phosphate formation became sharper that could be the result of the high bioactivity of prepared samples. Moreover, the band at 1460 cm-1 is attributed to the carbonate group (CO3)2-which is due to the replacement of (OH)- with (CO3)2- in the calcium phosphate structure or absorbance of carbonate from the environment of the prepared samples.
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Figure 4. The FTIR spectra of prepared scaffold containing hydrogel and precipitated minerals after soaking in SBF Table 1. Infrared assignment of prepared biomimetic hydrogel/calcium phosphate nanocomposites scaffold Wavenumber (cm-1)
bond
Compound
3410
O-H Stretch
Water (humidity)
2927
C-H
Amide B
1647
C=O Stretch
Amide I
1541
N-H bend
Amide II
1455
COO
Hydrogel
1337
Ca-COO
Hydrogel/HA
1240
N-H
Amide III
1075
C-N
Hydrogel
872
CO3
Carbonated HA
600
Liberational OH
HA
530
ν2 PO4 bend
Calcium phosphate
3.3. SEM observations Precipitate particles were dispersed on the surface and within the pores of the samples before soaking in SBF. Particles shape was spherical and their mean size was estimated as125nm (Fig 5).
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Figure 5. SEM micrographs of the surface of the synthesized biomimetic nanocomposite scaffold showing precipitate-phase nanocrystals on the surface of pore walls
Figure 6. SEM micrographs of the surface of the synthesized biomimetic nanocomposite scaffold after soaking in SBF 4. Conclusions In this study, a biomimetic system using double diffusion mechanism within gelatin/chitosan hydrogel with ability of calcium phosphate formation in pH and temperature similar to human body was designed. The obtained experimental results ascertained that the formed precipitation phase within middle hydrogel was composed of carbonated hydroxyapatite and dicalcium phosphate dihydrate (DCPD, brushite). After soaking of the prepared scaffolds in simulated body fluid, the amorphous calcium phosphate phase converted to crystalline hydroxyapatite.
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Synthesis of Nano Calcium Phosphate via Biomimetic Method for Bone Tissue Engineering Scaffolds and Investigation of its Phase Transformation in Simulated Body Fluid 10.4028/www.scientific.net/KEM.587.86
