Preparation and Characterization of Macroporous Magnesium Phosphate Scaffold for Bone Regeneration

Much attention has been focused on the biomedical applications of magnesium phosphate cement (MPC) as bone substitution material. The use of porous scaffolds with characteristics such as high porosity along with macropores and three-dimensional interconnected pore structures is beneficial for repairing bone defects. MPC has been proven to be degradable and biocompatible, and therefore might be applied as three-dimensional scaffolds for bone regeneration. In this study, macroporous magnesium phosphate scaffolds were fabricated by the particulate leaching method using sodium chloride as porogen. The morphology, chemical composition and cellular response to the scaffolds were investigated. The obtained scaffold had a well-interconnected porous structure with pore sizes ranging from 400 to 500 μm. The highest porosity determined using the Archimedes’s Principle could reach 71%. X-ray diffraction pattern revealed the main composition of the scaffold was NH4MgPO4·6H2O. The result of MTT test demonstrated that the obtained scaffold was cytocompatible and had no negative effects on the proliferation of MG63 cells in vitro. These results suggest that the macroporous MPC scaffolds may have potential applications for bone regeneration. INTRODUCTION As for the successful repair of bone defects, the pore structure, morphology, mechanical property and biocompatibility of the porous scaffolds are very important. An ideal scaffold should have suitable porosity and well-interconnected pores to facilitate cell attachment, proliferation and transport of nutrients and metabolic waste (Yunos et al. 2008). Magnesium phosphate cement (MPC) has been widely used in civil engineering as rapid-repair materials due to the features of fast setting and high early strength. In recent years, clinic applications of MPC as bone substitution materials have attracted much attention (Wu et al. 2008). Liu (2006) first applied MPC as inorganic bone F.Wu, Y.Ngothai, C.Liu, J.Wei, B.O’Neill, R.Musgrove 2 adhesive in screw fixation, artificial joints fixation and comminuted fracture fixation. The MPC powder consists of a mixture of magnesium oxide (MgO) and ammonium dihydrogen phosphate (NH4H2PO4). The main hydration product is NH4MgPO4·6H2O. Previous research demonstrated that MPC has good in vivo degradability and biocompatibility (Wu et al. 2006; Wu et al. 2009; Yu et al. 2010). Due to these characteristics, MPC is proposed as good scaffold candidate for bone tissue engineering. However, till now, MPC is mainly used in the form of moldable cement paste. The possibility of applying MPC as porous scaffolds has never been studied. In this study, macroporous magnesium phosphate scaffolds were fabricated using the particulate leaching method. The porosity, morphology, mechanical strength, phase composition of the porous scaffolds were investigates. In vitro cell compatibility in terms of cell proliferation of the scaffolds was also studied. MATERIALS AND METHODS MPC powder The MPC powder was composed of magnesium oxide (MgO) and ammonium dihydrogen phosphate (NH4H2PO4) in a molar ratio of 3.8:1. The MgO was prepared by heating basic magnesium carbonate pentahydrate [(MgCO3)4·Mg(OH)2 ·5H2O] in a furnace at 1500°C for 6 hours. The resultant powder was first cooled to room temperature, and then grounded in a planetary ball mill for 5 minutes, followed by sieving through 200 and 300 meshes, respectively. The grains in the range of 200 and 300 meshes were kept for further experiment. All the chemicals used were purchased from Sinopham Chemical Reagent Co., Ltd. Preparation of the porous scaffolds The macroporous magnesium phosphate scaffolds were prepared via the particulate leaching method using sodium chloride (NaCl) particles as porogen (Wei et al. 2010). The MPC powder was mixed with deionized water at a powder/liquid ratio of 5g/ml using a spatula to form a paste. NaCl particles with sizes in the range of 400-500 μm were added into the cement paste. The mixture of MPC paste and NaCl particles was placed into stainless steel mold (diameter: 6 mm, height: 10 mm) and packed under a pressure of 2 MPa. The samples were stored in a 100% relative humidity environment at 37°C for 2 days, followed by immersion in deionized water for 3 days to leach out of NaCl particles. Afterward, the samples were then dried at 50°C for 6 hours to obtain the porous scaffolds. Porosity determination Porosity of the scaffolds was determined using the method based on the Archimedes’ Principle (Li et al. 2004). In brief, the porosity (P) was calculated according to the following equation: P= (W2-W3-WS)/ (W1-W3) ×100%. WS was the weight of the scaffold sample. The specific gravity bottle filled with ethanol was weighed as W1. The scaffold was immersed in ethanol completely to allow the infiltration of ethanol into the pore structure of the scaffold. The weight of the specific gravity bottle containing ethanol and scaffold was recorded as W2. The ethanol-infiltrated scaffold F.Wu, Y.Ngothai, C.Liu, J.Wei, B.O’Neill, R.Musgrove 3 was then removed from the bottle. W3 was the weight of the specific gravity bottle with the residual ethanol. Mechanical testing The surface of the samples was slightly polished before tests. The compressive strength of the as-prepared scaffold was measured on a universal testing machine (AG-2000A, Shimadzu Autograph, Shimadzu Co., Ltd, Japan) at a loading rate of 1 mm/min. Three samples were tested for each group and the results were expressed as mean ± standard deviation (mean ± SD). Phase composition and structure characterisation The phase composition of the scaffold was characterized by X-ray diffraction (XRD; Rigaku Co., Japan) with Cu Kα radiation and Ni filter (λ =1.5406A, 100mA, 40kV) in a continuous scan mode. The 2θ range was from 10° to 80° at a scanning speed of 10°/min. The fracture surface morphology of the scaffold was observed with scanning electron microscopy (SEM; JSM6360, JEOL, Japan). Cell proliferation The proliferation of MG63 osteoblast-like cells cultured on the scaffold samples was assessed quantitatively using methyl thiazoly tetrazolium (MTT) assay. Prior to cell seeding, the scaffold samples (Φ10 × 3 mm) were sterilized by autoclaving at 120°C for 20 minutes. The tissue culture polystyrene (TCP) was taken as the control. The scaffold samples were placed in a 24-well plate and the MG63 cells were seeded at a density of 5 × 10 cells/well. The cell-seeded scaffolds were incubated at 37°C and 100% humidity with 5% CO2 in a DMEM-BFS medium. The culture medium was replaced every two days. After culturing for 3 and 5 days, 100 μL MTT solution was added into each well of the plate. The plate was then incubated for further 4 hours. The supernatant of each well was then removed and 200 μl dimethyl sulfoxide (DMSO) added. After shaking for 10 minutes, the optical density (OD) at 490 nm was measured with an enzyme-linked immunoadsorbent assay plate reader. Six samples were tested for each culture time and each test was performed in triplicate. A one-way analysis of variance (ANOVA) test was performed to detect significant effects. A p value < 0.05 was considered to be statistically significant. RESULTS AND DISCUSSIONS In this study, we prepared macroporous magnesium phosphate scaffolds using the particulate leaching method. So far, several methods have been utilized to fabricate porous scaffolds, such as gas foaming, phase separation, emulsion freeze-drying, three-dimensional printing, etc (Yunos et al. 2008). The particulate leaching method was proved to provide easy control of the pore structure (Chen et al. 2001). Fig.1 presents the macroscopic graph of the as-prepared magnesium phosphate scaffold. The scaffold showed an obvious macroporous structure. The formation of the macropores was led by the leaching of the NaCl particles. F.Wu, Y.Ngothai, C.Liu, J.Wei, B.O’Neill, R.Musgrove 4 Fig.1 The macroscopic image of the magnesium phosphate scaffold The variation of porosity with the particulate/MPC powder ratio was shown in Fig.2. The porosity of the scaffold obviously increased with the increasing particulate/MPC powder ratio. The result was in accordance with the previous research that the porogen content had significant influence on the scaffold porosity (Wang et al. 2007). Therefore, the magnesium phosphate scaffold porosity could be adjusted by the control of particulate/cement powder ratio. 1.4 1.6 1.8 2.0 2.2 2.4 2.6 20 40 60 80 Particulate / MPC powder ratio P or os it y (% ) Fig.2 The effect of particulate/MPC powder ratio on porosity of porous magnesium phosphate scaffold Fig.3 presented the variation of compressive strength with porosity of the as-prepared magnesium phosphate scaffold. The compressive strength of the scaffolds decreased with the increasing porosity, ranging from 0.8 to 2.2 MPa. The compressive strength of the magnesium phosphate scaffolds was close to the strength of spongy bone (0.24 MPa) (Callcut & Knowles, 2002). F.Wu, Y.Ngothai, C.Liu, J.Wei, B.O’Neill, R.Musgrove 5 40 45 50 55 60 65 70 75 0.0 0.5 1.0 1.5 2.0 2.5 3.0 C om pr es si ve S tr en gt h (M Pa )