Key Engineering Materials Vol. 587 (2014) pp 27-32 © (2014) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/KEM.587.27
Influence of the precipitation temperature on properties of nanohydroxyapatite powder for the fabrication of highly porous bone scaffolds Cristian Parisi1,2, a, Francesca Gervaso2, Francesca Scalera2, Sanosh Kunjalukkal Padmanabhan2, Concetta Nobile3, P. Davide Cozzoli3,4, Lucy Di Silvio1, Alessandro Sannino2 1
Biomaterials, Tissue Engineering & Imaging Group, King’s College London, Dental Institute, Floor 17, Tower Wing, Guy’s Hospital, London Bridge, London SE1 9RT, UK
2
Department of Engineering for Innovation, University of Salento, via per Monteroni, 73100 Lecce, Italy
3
National Nanotechnology Laboratory (NNL), CNR Istituto Nanoscienze, c/o Distretto Tecnologico, via per Arnesano 16, 73100 Lecce, Italy
4
Department of Mathematics and Physics “E. De Giorgi”, University of Salento, via per Arnesano, 73100 Lecce, Italy a
[email protected] (corresponding author)
Keywords: Hydroxyapatite, nanohydroxyapatite, nanoparticles, synthesis, precipitation method, bone tissue engineering, bone substitute, bone scaffold.
Abstract. The aim of the present work is to study the influence of the precipitation temperature in the synthesis of nanohydroxyapatite (n-HAp) on the properties of the resulting n-HAp powder for the fabrication of highly porous scaffolds for bone tissue engineering. The n-HAp powder was obtained by a wet precipitation technique starting from calcium nitrate tetrahydrate (Ca(NO3)2*4H2O) and phosphoric acid (H3PO4) at different temperatures: 10°C, 37°C and 50°C. Highly porous scaffolds were fabricated using the three different powders by the sponge replica method and sintering at 1300°C. Combined X-ray diffraction (XRD) and transmission electron microscopy (TEM) analyses on powders indicated that on increasing the precipitation temperature the formation of pure n-HAp is accelerated, without significant changes in particles morphology and size. Scaffolds characterized by high porosity (89%) and good compressive strength (0.53 MPa for n-HAp prepared at 37°C) were obtained. XRD analyses on sintered n-HAp confirmed the thermal stability of the material. Therefore, the as-synthesized n-HAp powder can be successfully used for the fabrication of highly porous scaffolds as bone substitutes. Introduction Large bone defects due to traumatic injuries, tumour excision, critical diseases remain a clinical problem and bone reconstructive surgery is required to provide mechanical support and promote bone regeneration [1]. The current gold standard treatment is the bone graft, in particular autologous or allogeneic grafts. However these treatments suffer from several drawbacks such as the need for a second surgery, donor site morbidity and limited supply for the autograft, disease transmission and decrease in mechanical strength after the sterilization process for the allograft [1-3]. Tissue engineering plays a key role in the development of improved new strategies for bone regeneration [3,4]. Engineered three-dimensional biomedical devices, the scaffolds, can act as artificial extracellular matrices supporting, leading and promoting the new bone tissue formation. The scaffolds should have high interconnected porosity, in order to be easily colonized by the progenitor cells, good bioresorption rate, comparable with new tissue formation rate, and mechanical strength, to support mechanical loads [4-7].
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Bioceramics 25
The aim of this study is the optimization of the synthesis of nanohydroxyapatite (n-HAp) for the fabrication of scaffolds for bone tissue engineering applications. Hydroxyapatite (HAp) is the most used bioceramic in bone tissue engineering due to its very good biocompatibility and its chemical composition very close to the mineral part of the natural bone tissue [6,7]. In particular, in this work we analysed the influence of the precipitation temperature in the synthesis of n-HAp on the properties of the resulting n-HAp powder and the highly porous scaffolds fabricated using such powders. Materials and methods Synthesis and characterization of the powder. The nanohydroxyapatite powder was synthesized using the wet precipitation technique: a 0.5 M aqueous solution of calcium nitrate tetrahydrate (Ca(NO3)2*4H2O, Sigma Aldrich) was very slowly added to a 0.3 M aqueous solution of phosphoric acid (H3PO4, Sigma Aldrich) and stirred for 2 hours (Ca/P = 1.67). Ammonium hydroxide (NH4OH, Sigma Aldrich) was added to the solution to regulate the pH between 9.5 and 10. Then, the precipitation process took place at three different temperatures (10°C, 37°C, 50°C) and was monitored at different precipitation times for each temperature to obtain pure n-HAp. At the end of the process, the precipitate was separated from the supernatant liquid by centrifugation (3000-6000 rpm for 3-9 minutes), washed twice with double-distilled water to remove NH4+ and NO3- and then dried for 24 hours at 65°C in an oven. The subsequent calcination in air at 900°C (heating rate 5°C/min) for 1 hour induced the phase transformation of the amorphous calcium phosphate precipitate into nanohydroxyapatite crystals. After the thermal treatment, the n-HAp was ground in an agate mortar and sieved (80 µm) in order to obtain a very fine powder. The composition of the powders was verified by X-ray diffraction (XRD) analyses (Rigaku D/Max Ultima, Tokyo, Japan). XRD patterns were obtained with CuKα radiation (λ = 1.5418 Å) in step scanning mode recorded in the 2θ range of 20°-80°, with a step size of 0.02° and step duration of 0.5 s. The crystallinity and the presence of secondary phases in the n-HAp powders were evaluated as reported in our previous studies [8,9]. The morphology and the size of the nanoparticles in the powders were evaluated by transmission electron microscopy (TEM) analyses, performed by a JEOL JEM 1011 microscope operating at an accelerating voltage of 100 kV. The samples were prepared by depositing a few drops of a powder suspension (in acetone) onto a carbon-coated copper TEM grid, and then allowing the solvent to evaporate. Fabrication and characterization of the scaffolds. The nanohydroxyapatite fine powders were used for the fabrication of highly porous scaffolds by the sponge replica method. A polyurethane sponge was impregnated with a ceramic slurry: 70 wt% of n-HAp powder in a 2 wt% polyvinyl alcohol binder solution. Few drops of an organic deflocculating agent (Dolapix CE-64, Zschimmer & Schwarz) were used in order to obtain a suitable viscosity for the sponge impregnation [9]. The polyurethane sponges used as a template (density of 30 kg/m3, 25 ppi, kindly provided by ORSA Foam S.p.A.) were cut in cubes of 15 mm length and gently squeezed after the impregnation into the n-HAp slurries. Then the impregnated cubes were heated up to 500°C for 1 hour (1°C/min) in order to burnout the polyurethane foam. Then, the highly porous replicating scaffolds were sintered at 1300°C for 3 hours (5°C/min). The composition and the thermal stability of the sintered scaffolds were verified by XRD analyses. The linear shrinkage of the samples was calculated by measuring the dimensions of the cubes before and after the sintering. Each scaffold was also weighted and the bulk density (ρ) was calculated as the ratio of the weight to the volume of the specimen. The porosity of the scaffolds was then calculated using the following: (1)
P = 1 - ρ/ρ0 3
where ρ0 is the density of dense HAp (3.156 g/cm ).
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The mechanical strength of the scaffolds was evaluated by compression tests. The tests were performed using a universal testing machine (Lloyd LR5K Instrument, UK) equipped with a 1 kN load cell at a crosshead speed of 0.5 mm/min. The stress at failure (σF) was calculated as the ratio of the cross-sectional area to the maximum fracture load. Scanning electron microscopy (SEM) analyses (Zeiss Evo 50 XVP, Jena, Germany) were used for evaluating the microstructural morphology of the scaffolds after the sintering process. Results and discussions Powders properties. The XRD patterns in Fig. 1 show that by increasing the precipitation time (t1
