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Preparation of a novel three-dimensional hydroxyapatite microstructure and its application in tissue engineering

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https://www.eduzhai.net American Journal of M aterials Science 2013, 3(5): 130-135 DOI: 10.5923/j.materials.20130305.04 The Manufacture of a Novel 3D Hydroxyapatite Microstructure Derived from Cuttlefish Bones for Potential Tissue Engineering Applications Gérrard Eddy Jai Poinern*, Derek Fawcett M urdoch Applied Nanotechnology Research Group. Department of Physics, Energy Studies and Nanotechnology School of Engineering and Energy, M urdoch University, M urdoch, Western Australia 6150, Australia Abstract Porous three-dimensional hydroxyapatite scaffolds were synthesised from aragonitic cuttlefish bones via a micro wave based technique. Due to its close similarity to the mineral phase found in the natural hu man bone matrix, synthetically produced hydroxyapatite is considered a viable apatite substitute for natural bone in a number of bio medical applications. The X-ray diffract ion data confirms the conversion of crystalline formations of calciu m carbonate, the main component of the cuttlefish, to hydro xyapatite. The synthesis process is straightforward, efficient and creates a highly porous 3 dimensional microstructure that has the potential to be used in a wide range of hard tissue engineering applications such as bone repair, filling materia l and bone augmentation. Keywords Hydro xyapatite, M icrostructure, Micro wave Irradiation, Regenerative Medicine 1. Introduction The human skeletal system is co mposed of a hard tissue called bone and more flexib le cartilaginous materials which provide the structural support and movement capability of the body[1]. In the event of an accident, where the bone is fractured or damaged due to diseases, bone substitutes are needed to repair the fracture or replacement the infected bone tissue[2]. The demand for natural bone and bone substitutes is high. In Australia alone, the Australian Orthopaedic Association’s National Joint Replacement Registry recorded mo re than 75,000 hip and knee replacements being carried out annually[3]. Th is health issue is not unique to Australia and has been identified by World Health Organization as one of the most serious health problems facing the world today[4]. In spite of being the most preferred option, with its excellen t b io co mp at ib ilit y and ost eo gen ic capacity , autog rafts have p rob lems associated with dono r s ite mo rb idity and limited supply[5, 6]. Given an adequate blood supply and no infection, autographs are successful donor t issues due to th eir ident ical nature. Ho wever, following the bone replacement procedure, patients receive immunosuppressive therapy which involves taking drugs designed to supp ress the immun e system. During the * Corresponding author: G.Poinern@murdoch.edu.au (Gérrard Eddy Jai Poinern) Published online at https://www.eduzhai.net Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved therapy, many of these drugs used tend to kill rapid ly dividing cells and cause serious side effects. Therefore, to be a successful autograft procedure, right balance of immunosuppressive drugs and antibiotics to keep infection under control are needed to prevent imp lant site and donor morb idity. The autograft bone is usually harvested from the patient’s iliac crest in the form of cancellous bone. This means that the patient has only a limited nu mber of potential harvest sites capable of supplying a suitable autograft. It is due to this limited supply that has led to the search for alternative sources of bone materials such as xenogenic and allogenic bone grafts. However, both of these replacement bone sources do solicit a significant detrimental response from the immune system. In spite of this allogenic bone grafts are routinely used due to the high clin ical demand[7, 8]. Nevertheless, concerns regarding the immune response and the possibility o f pathogen infection fro m bone sources have resulted in the search for alternative synthetic biocompatible materials have been investigated [9]. Inorganic materials such as calcium phosphate ceramics, porous coralline, and calciu m sulphate’s have all been investigated in the past and demonstrated both good biocompatibility and bioactiv ity properties[10-12]. One particular material that has been extensively studied for a variety of bone repair and replacement procedures is hydroxyapatite (HAP), wh ich is a member of the calciu m phosphate family[13-15]. The stoichiometric chemical formula of HAP is[Ca10(OH)2(PO4)6] and is highly biocompatible due to its close chemical similarity to the American Journal of M aterials Science 2013, 3(5): 130-135 131 mineral co mponent of natural bone[16]. There are four significant advantages of using HAP for bone t issue engineering: 1) it has been found to have good biocompatibility with surrounding body tissues; 2) it is highly stable under a variety of physiological conditions and its biodegradability in situ is slow; 3) it p rovides good osteoconductivity, and 4) it has good osteoinductivity capabilit ies[17-22]. It is due to these favourable advantages that has resulted in HAP being used as the major co mponent in bone cements and is routinely used to coat titanium bas ed med ical imp lants[23]. In the case of metallic imp lants, the HAP coating improves bone fixat ion, bone in-growth and increases the life span of the implant in the body. This paper reports the rapid chemical conversion of a 3-D porous CaCO3 micro metre scale structure found in cuttlefish bone to a HAP structure. A wet chemical technique, incorporating microwave heating was used during the conversion process. The advantage of using micro wave heating in this process arises fro m the volumetric d istribution of energy throughout the material. This is fundamentally different to the conventional thermal conduction mechanism normally found in ovens and furnaces[24]. Unlike other hydrothermal based conversions of cuttlefish bone this microwave based conversion process is rapid in co mparison[25, 26]. Thus, allo wing a g reater throughput of material and imp roving the overall efficiency of the micro wave based conversion process. Advanced characterisation techniques such as x-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM) were used to investigate the effectiveness of the conversion process. XRD was used to verify the chemical conversion of CaCO3 to HAP, while FESEM microscopy was used to confirm the integrity of the 3-D micro metre scale structure after processing. The results of the XRD and FESEM analysis indicate that a 3-D micro metre scale HAP structure is formed at the end of the conversion process. 2. Materials and Methods 2.1. Materials All analytical grade reagents used in this work were supplied by Chem-Supply (Australia) and the cuttlefish bone samples were collected fro m a local beach. The main reactants used to synthesis the nanometre sized HAP powder used for comparative analysis consisted of calciu m nitrate tetrahydrate[Ca(NO3)2.4H2O] and potassium di-hydrogen phosphate[KH2PO4]. During the synthesis process the solution pH was maintained by the addition of ammon iu m hydroxide[NH4OH]. All aqueous solutions used throughout this study were made using Milli-Q® water (18.3 MΩ cm-1) produced by an ultrapure water system (Barnstead Ultrapure Water System D11931; Thermo Scientific, Dubuque, IA). 2.2. Preparati on of HAP Powder Used for Comparison Pur pos es The synthesis of the HAP powder begins by adjusting the pH of a 40 mL solution of 0.32M calciu m n itrate tetrahydrate to a value of 9.0 by adding and mixing approximately 2.5 mL of ammoniu m hydro xide. The solution was then exposed to ultrasound irradiat ion for 1 h using an UP50H Ultrasound Processor[50 W, 30 kHz, MS7 Sonotrode (7mm d iameter, 80 mm length)] supplied by Hielscher Ult rasound Technology. During the second hour of ultrasonic irrad iation a 60 mL solution of 0.19 M potassium d i-hydrogen phosphate was slowly added drop-wise into the reactive mixtu re. Throughout the second hour the calciu m:phosphate[Ca:P] rat io was maintained at 1.67 and the solution pH maintained at 9.0. At the end of the second hour, the solution was filtered using centrifugation (15,000g) for 20 minutes at room temperature. The resultant white precipitate was then transferred to a fused silica crucible ready for thermal treatment. The thermal treat ment consisted of p lacing the crucible into an LG® (Australia) household microwave rated at 1100 W (2450 MHz) for 40 minutes. After cooling, the resulting white agglo merated mass was ground to form a fine powder and then characterised using XRD. A schematic presentation of the procedure is presented in Figure 1. Figure 1. Schematic of the synthesis procedure used to produce nanometre scale HAP powders used for comparative analysis 132 Gérrard Eddy Jai Poinern et al.: The M anufacture of a Novel 3D Hydroxyapatite M icrostructure Derived from Cuttlefish Bones for Potential Tissue Engineering Applications Fi gure 2. Schemat ic of cuttlefish conversion process to produce a 3-D HAP micrometre scale scaffold struct ure 2.3. Microwave B ased Conversion of Cuttlefish Samples The conversion process begins with a raw cuttlefish bone being washed and scrubbed to removal all traces of organic matter. In the next stage, the cuttlefish bone was soaked in a series of M illi-Q® water solutions to progressively remove any sea salt (Na Cl) content. After the salt had been re moved, the cuttlefish bone was given a final wash in M illi-Q® water before being allowed to air dry. The dried cuttlefish bone was then cut up into 1 cm2 portions, with the thickness of the portions being 1 cm fo r samp les being used for conversion. Each sample then underwent a pre-treat ment procedure that consisted of two steps. In the first step each sample was placed into a 30% solution of ammon ia (Chem-Supply, Australia) and permitted to soak fo r 30 minutes. Following the initial soaking, a 0.19 M solution of K2H3PO4 was added to the solution. Then the solution containing the cuttlefish bone was then placed into a micro wave bomb (45 ml, 4782 Parr Instrument Co mpany) and sealed. The bo mb was then placed into an LG® (Australia) household microwave oven rated at 1100 W (2450 M Hz). The micro wave was set to full power and the thermal t reatment was carried out over 10 minutes. The conversion procedure is presented schematically in Figure 2 and images of the sample during processing are shown in Figure 3. After p rocessing samples were examined using XRD and FESEM. 2.4. Characterisation of Materials Analysis of the XRD spectra was used to identify the HAP peaks in synthesised powders and examine the cuttlefish sample before and after conversion. The HAP powder pattern and the converted cuttlefish pattern were then compared and peak identification was carried out. XRD spectra were recorded at roo m temperature, using a Siemens D500 series diffractometer[Cu Kα = 1.5406 Å radiation source] operating at 40 kV and 30 mA. The diffraction patterns were collected over a 2θ range fro m 20° to 60° with an incremental step size of 0.04° using flat p lane geometry. The acquisition time was set at 2 seconds for each scan. The mo rphological features of the converted cuttlefish samples were investigated using FESEM. All micrographs were taken using a high resolution FESEM[Zeiss 1555 VP-FESEM] at 3 kV with a 30 µm aperture operating under a pressure of 1×10-10 Torr. In addition, the FESEM micrographs were also used to estimate the size of major structure features seen in the imag es . Figure 3. Conversion process: (a) ammonia soaking of cuttlefish bone for 30 min; (b) addition of K2H3PO4 and placing solution into microwave bomb; (c) sealed bomb placed in Microwave oven for thermal treatment, and (d) cuttlefish bone converted to 3-D HAP micrometre scale scaffold st ruct ure 3. Results and Discussions The XRD technique was used to identify and confirm the presence of crystalline HAP both in the synthesised powder used for co mparative purposes and the converted cuttlefish samples. A typical XRD pattern of the synthesised ultra fine HAP powder is presented in Figure 4 and reveals the main (h k l) indices associated with HAP: (002), (211), (300), American Journal of M aterials Science 2013, 3(5): 130-135 133 (202), (130), (002), (222) and (213). The (002) reflection peak fro m the XRD pattern was used to calculate the crystallite size of the powder using the Debye-Scherrer equation (Insert Figure 4)[27, 28]. The crystallite size was estimated to have a mean value of 30 ± 5 n m. The HAP XRD spectra as then used to in a co mparative study between the pre-treated cuttlefish and the converted cu ttlefis h . The cuttlefish sample spectrum (b) prior to treat ment is presented in Figure 5 and reveals an XRD pattern with numerous peaks indicat ing the presence of a CaCO3 based structure. The post-treatment sample spectrum (c) reveals a pattern that is similar to the synthesized HAP samp le spectra (a). The peaks in the left hand side of the pattern are dominant, can be easily seen and clearly indicate the presence of the (002), (211), (300) and (202) indices. In the right hand side of the spectrum the peaks are not as prominent, but can still be identified as the (130), (222), (213) and (004) indices. In the pre-treated sample the CaCO3 peaks on the right hand side of the spectrum were quite dominant. This is not the case after treatment where the peaks are significantly s maller. The results of the XRD study have clearly shown that the cuttlefish sample has been effectively converted into HAP during the treat ment p ro cess . Figure 4. A typical experimental XRD spectrum of the synthesized HAP powder used for comparative purposes Figure 5. Comparisons between the XRD spectra of synthesised HAP powder (a), pre-treated cuttlefish bone sample (b) and converted cuttlefish bone sample (c) 134 Gérrard Eddy Jai Poinern et al.: The M anufacture of a Novel 3D Hydroxyapatite M icrostructure Derived from Cuttlefish Bones for Potential Tissue Engineering Applications After the conversion process the sample was still soft and retains the 3-D micro metre scale structure seen in the original cuttlefish sample as seen in Figure 6. Inspection of Figure 6 reveals that there is some surface damage to the 3-D structure resulting from the in itial removal of o rganic material. A mong the parallel longitudinal wall structures, many of the smaller walls between the individual chambers have been damaged to varying degrees. However, the overall structure is still intact and the damage is minimal since it is not widespread across the surface of the samp le (typically, around 20% max). Despite the minor surface damage to the sample, cells would still be able to enter and move freely within the structure due to their s mall size (generally in between 10 to 20 µm)[29]. biomed ical and tissue engineering applications. The results of this study have shown that the conversion of CaCO3 based cuttlefish bones to a 3-D HAP micro metre scale structure is simp le and straightforward. The resultant HAP material resembles the mineral phase found in natural bone tissue, with the advantage of being modelled into a definite shape for a specific application. This highly porous HAP structure has the potential to be used as a cell scaffold for tissue engineering applications. ACKNOWLEDGEMENTS The authors would also like to thank Mr R. Brundavanam for his assistance with the X-ray diffract ion investigation. Disclosure The authors report no conflict of interest in this work. REFERENCES [1] Weiner, S., Wagner, H.D., 1998, The M aterial Bone: Structure-mechanical function relations. Ann. Rev. M ater. Sci., 28, 271-298. [2] Hutmacher, D.W., Schantz, J.T., et al., 2007, State of the art Figure 6. A typical FESEM micrograph of the converted cuttlefish bone and future directions of scaffold-based bone engineering sample showing structural damage caused during the removal of organic from a biomaterials perspective. J. Tissue Eng. Regen. M ed., material in the pre-treatment stage of processing 1, 245-260. The advantage of the microwave based technique is that the conversion times are relatively short compared to other non-microwave based techniques[25]. In addition, the micro wave based technique has only one heating cycle and does not need a secondary independent heating cycle at higher temperatures for extended periods to achieve calcinations[26]. Th is makes the micro wave based technique more efficient and as a result cost effective in converting the cuttlefish bone to HAP co mpared to conventional hydrothermal processes that have much longer processing times. However, further work is needed to develop a less aggressive cleaning technique to reduce surface damage during the removal of organic material. Also, future cell studies are planned to thoroughly investigate the biocompatibility of novel 3-D HAP structure. The converted sample is firm and can easily be formed into a variety of shapes and profiles, which makes it suitable for a number of potential tissue engineering applications[30, 31]. However, the material would be unsuitable for large load bearing applications in a number of hard t issue engineering procedures. [3] Online data available at: http://www.aoa.org.au/ [4] L. Lars, The Bone and Joint Decade 2000 -2010. Bull World Health Organ[online]. Vol. 81, (2003), p. 629-629. [5] LaPrade, R.F., Botker, J.C., 2004, Donor site morbidity after osteochondral autograft transfer procedures, Arthroscopy, 20(7), e69-e73. 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[16] M itchell, W., M atthews, J.B., Stone, M .H., et al., 2003, Comparison of the response of human peripheral blood mononuclear cells to challenge with particles of three bone cements in vitro, Biomaterials, 24, 737-748. [17] Santavirta, S., Konttinen, Y.T., Lappalainen, R., et al., 1998, M aterials in total joint replacement, Current Orthopaedics, 12, 51-57. [18] Kalita, S.J., Bhardwaj, A., Bhatt, H.A., 2007, Nanocrystalline calcium phosphate ceramics in biomedical engineering, M aterials Science and Engineering C, 27, 441-449. [19] Habraken, W.J.E.M ., Wolke, J.G.C., Jansen, J.A., 2007, Ceramic composites as matrices and scaffolds for drug delivery in tissue engineering, Adv. Drug Deli. Rev.59, 234-248. [20] Blom, A., 2007, Which scaffold for which application, Curr. Orthop., 21, 280-287. [21] Habibovic, P., de Groot, K., 2007, Osteoinductive biomate rials—properties and relevance in bone repair, J. Tissue Eng and Regen. M ed, 1, 25-32. [22] Dorozhkin, S.V., 2009, Nanodimensional and nanocrystals sline apatites and other calcium orthophosphates in bio medical engineering, biology and medicine, M aterials, 2, 1975-2045. [23] Stoch, A., Jastrzebski, W., Dlugon, E., et al., 2005, Sol-gel derived hydroxyapatite coatings on titanium and its alloy Ti6Al4V, J. M ol. Struct., 744, 633-640. [24] Vollmer M . 2004, Physics of the microwave oven. Physics Education, 39(1): 74-81. [25] Ivankovic, H., Gallego-Ferrer, G., Tkalcec, E., Orlic, S., Ivankovic, M . 2009, Preparation of highly porous hydroxylapatite from cuttlefish bone. J. M ater. Sci. M ater. M ed. 20(5):1039-46. [26] Rocha, J.H.G., Lemos, A.F., Kannan, S., A gathopoulos, S., Ferreira, J.M .F. 2005, Hydroxyapatite scaffolds hydrother mally grown from aragonitic cuttlefish bones. J. M ater. Chem., 15: 5007-5011. [27] Danilchenko, S.M ., Kukharenko, O.G., M oseke, C., et al., 2002, Determination of the bone mineral crystallite size and lattice strain from diffraction line broadening. Cryst. Res. Technol., 37(11), 1234-1240. [28] Brundavanam, R.K., Poinern, G.E.J., Fawcett, D., 2013, M odelling the crystal structure of a 30 nm sized particle based hydroxyapatite powder synthesised under the influence of ultrasound irradiation from X-ray powder diffraction data, Am. J. M aterials Science, 3(4): 84-90. [29] Knox, B., Ladiges, P., Evans, B., 1994, Biology. M cGraw-Hill, Sydney, Australia. [30] Ward, B.B., Feinberg, S.E., Friedman, C.D., 2002, Tissue matrices for soft tissue and mucosal augmentation and replacement. Facial. Plast. Surg. 18(1): 3-11. [31] Pou, A.M ., 2003, Update on new biomaterials and their use in reconstructive surgery. Curr. Opin. Otalaryngol. Head Neck Surg. 11(4): 240-244.

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