G Model COLSUB-6103; No of Pages ARTICLE IN PRESS Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering Nguyen Kim Nga a,∗ , Luu Truong Giang a , Tran Quang Huy b , Pham Hung Viet c , Claudio Migliaresi d a School of Chemical Engineering, Hanoi University of Science and Technology, Dai Co Viet Road, Hanoi, Viet Nam National Institute of Hygiene and Epidemiology, Yersin Street, Hanoi, Viet Nam c Research Center for Environmental Technology and Sustainable Development, Hanoi University of Science, 334 Nguyen Trai Street, Hanoi, Viet Nam d Department of Industrial Engineering and BIOtech Research Centre, University of Trento, Via Mesiano, 77, I-38123 Trento, Italy b a r t i c l e i n f o Article history: Received 29 July 2013 Received in revised form 26 October 2013 Accepted November 2013 Available online xxx Keywords: Hydroxyapatite nanorods Pluronic SBF Bioactivity Bone tissue engineering a b s t r a c t This study presents the physicochemical characterization of the pluronic surfactant-assisted size control of hydroxyapatite (HAp) nanorods for bone tissue engineering (BTE) Rod-shaped HAp nanoparticles were synthesized via a simple route by hydrothermal treatment and with the assistance of the triblock co-polymer PEO20 -PPO70 -PEO20 (P123) The films of poly (d, l) lactic acid (PDLLA) were prepared as a substrate to spread synthesized HAp nanorods Powder X-ray diffraction (XRD), field electron scanning microscopy, Fourier transform infrared spectroscopy, nitrogen adsorption isotherms, and energy-dispersive X-ray spectroscopy were used to characterize the structure and composition of the HAp samples Results showed that regular rod-shaped HAp nanoparticles (with a mean length of 120 nm and a mean width of 28 nm) were successfully produced Moreover, synthesized HAp nanorods revealed the rapid formation of bone-like apatite with a distinctive morphology, similar to flower-like apatite; the formation was observed as early as days after incubation in stimulated body fluids This study is a positive addition to the ongoing research on the preparation of HAp nanostructures toward the development of biocompatible composite scaffolds for BTE applications © 2013 Elsevier B.V All rights reserved Introduction Bone tissue engineering (BTE) has attracted considerable scientific attention in the fields of nanomedicine and biotechnology because it offers a more promising method for bone repair and regeneration than traditional methods (e.g., allografts and autografts) [1] BTE uses scaffolding materials as template for cell interactions and formation of extracellular matrix, thereby providing structural support to the newly formed tissue [2] Till date, a number of biomaterials have been investigated for possible use in BTE scaffolds, and these biomaterials can be classified into the following categories according to their composition: biodegradable polymers [3–9], bioactive inorganics [10,11], and polymer/bioactive ceramic composites [12–20] Calcium phosphates with Ca/P ratios of 1.5–2.0 and belonging to the group of bioactive inorganics have three main compounds namely, tetraphosphate Ca4 P2 O9 , hydroxyapatite Ca10 (PO4 )6 (OH)2 , and tricalcium phosphate Ca3 (PO4 )2 Among them, hydroxyapatite (HAp) is thermodynamically the most stable calcium phosphate ∗ Corresponding author Tel.: +84 38680 110; fax: +84 38680 070 E-mail address: nga.nguyenkim@hust.edu.vn (N.K Nga) in physiological environments [10] and a major component of bones and teeth HAp has been widely studied alone or as filler in polymeric composites for BTE applications because of its chemical similarity to inorganic components of bone matrix, strong affinity for host hard tissues, and osteoconductivity [6] A number of studies have focused on investigating the formation of nanoscale HAp for additional applications, such as producing restorable scaffolds that can be replaced by endogenous hard tissues over time [21] Several wet chemistry methods (e.g., co-precipitation, hydrothermal, and ultrasonic) assisted by organic molecules (e.g., sodium dodecylbenzene sulfonate, sodium dodecyl sulfate [22], block copolymer poly(lactide-co-glycolide)-blockmonomethoxy (polyethylene glycol) and polyvinyl pyrrolidone (PVP) [23], cetyltrimethylammonium bromide (CTAB) [24], polyvinyl alcohol [25], and polyethylene glycol 400/CTAB [26], CTAB/n-pentanol/n-hexane/water [27], double-hydrophilic block copolymer poly-(vinylpyrrolidone)-b-poly(vinylpyrrolidone-altmaleic anhydride)-b-poly-(vinylpyrrolidone) [28]) have been studied to produce rod-shaped (or needle-like) HAp nanoparticles A study has suggested that better osteoconductivity can be achieved if the synthetic HAp resembles bone minerals in composition, size, and morphology [29] In a previous study, we successfully prepared HAp nanorods via hydrothermal technique 0927-7765/$ – see front matter © 2013 Elsevier B.V All rights reserved http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx assisted by CTAB; the HAp nanorods exhibited high cellular affinity but with wide size distributions (mean diameter of 17–58 nm and length of 85–439 nm) [24] Surfactants (e.g., CTAB) are efficient chemicals in modifying the desired size and shape of nanostructures because of their self-assembly into rod-like micelles or lamellar structures at high concentrations to improve their properties [24,30] As reported, the wide size distributions of synthetic HAp can reduce the mechanical properties of scaffolds [31] Despite recent advances in the synthesis of HAp for BTE applications, the products still face major drawbacks related to bioactivities, fracture toughness, and other mechanical properties [21] In this study, we focus on the synthesis and characterization of HAp nanoparticles that resemble bone minerals with narrow size distributions to develop biocompatible composite scaffolds for BTE applications Rod-shaped HAp nanoparticles were synthesized by triblock co-polymer PEO20 -PPO70 -PEO20 (P123) to control their sizes P123 is a non-ionic chemical that has been used to assist the synthesis of rod-like nanostructures [32] A possible mechanism for the formation of rod-shaped HAp nanoparticles was also proposed to identify the effect of P123 on the formation of HAp nanorods The films of poly (d, l) lactic acid (PDLLA) were prepared as substrates to spread HAp nanorods and test their bioactivity Bioactivity was determined by assessing the formation of apatite on the surface of HAp/PDLLA films upon incubation in stimulated body fluids (SBF) after 1, 3, and days Experimental 2.1 Chemicals All reagents were of analytical grade and used as received without further purification Calcium chloride dihydrate (CaCl2 ·2H2 O), sodium monophosphate dihydrate (Na2 HPO4 ·2H2 O), NaOH, C2 H5 OH, CHCl3 , hydrochloric acid (HCl), the pluronic co-polymer PEO20 -PPO70 -PEO20 (P123), NaCl, NaHCO3 , KCl, K2 HPO4 ·3H2 O, MgCl2 ·6H2 O, Na2 SO4 , and tris-hydroxymethyl aminomethane ((HOCH2 )3 CNH2 ) were obtained from Sigma–Aldrich PDLLA was purchased from Boehringer Ingelheim (Ingelheim, Germany) Buffer solutions (pH 4, 7, and 9) were purchased from Merck Deionized water was used to prepare all solutions and reagents 2.2 Synthesis and characterization of HAp nanoparticles For a typical experiment and in the absence of P123, 2.53 g CaCl2 ·2H2 O was dissolved in 86 mL deionized water and brought to a pH ranging from 9.5 to 11 by adding a 0.4 weight percent (wt%) NaOH solution 0.2 M Na2 HPO4 was added into the solution by drops to a Ca/P molar ratio of 1.67 The resulting mixture was vigorously stirred at 40 ◦ C for h to form white suspension The suspension was then transferred to a 200 mL teflon-lined stainless steel autoclave, and the suspension was maintained at 180 ◦ C for 24 h White precipitate was collected and washed several times with deionized water before being dried at 60 ◦ C overnight and, finally, calcinated at 500 ◦ C for h In a typical reaction in the presence of P123, a mixture of a specific amount of P123 (either 1, 2, or g) and 2.53 g CaCl2 ·2H2 O were dissolved in 86 mL deionized water by stirring for 30 to produce clear gel The following steps are the same as those for the synthesis without P123 Finally, a series of samples was produced and denoted 0P123, 1P123, 2P123, and 3P123 based on the amount of P123 used Powder X-ray diffraction patterns of synthesized samples were recorded on a Siemens D5005 diffractometer through CuK␣ radiation ( = 0.15406 nm) Powder morphology was examined by field emission scanning microscopy (S4800, Hitachi, Japan) The average diameter and length of nanorods were measured through scanning electron microscopic (SEM) images Fourier transform infrared spectra (FTIR) were recorded on a Nicolet 6700 spectrometer by KBr pellet technique in the range of 4000–400 cm−1 with a resolution of cm−1 All measurements were conducted at room temperature The chemical composition of HAp particles was determined with a JEOL JED-2300 Analysis Station with a ZAF (atomic number absorption fluorescence) quantitative analysis program Nitrogen adsorption–desorption isotherms were measured at −196 ◦ C with a Micromeritics ASAP 2020 apparatus The total surface areas were calculated by the Brunauer–Emmett–Teller (BET) method, whereas pore size distributions were determined by the Barrett–Joyner–Halenda (BJH) method 2.3 Production and characterization of HAp/PDLLA films The preparation of HAp/PDLLA films was described in our previous report [24] To prepare PDLLA films, we cast 0.3 g PDLLA dissolved in mL CHCl3 on a 55 mm aluminum plate The films were air-dried under a chemical hood for h and vacuum-dried for 24 h to remove all solvent A suspension of C2 H5 OH with 30 mg of HAp nanorods was dispersed on the resulting PDLLA films After being placed at 55–60 ◦ C for h, the composite films of HAp/PDLLA were vacuum-dried for another h and stored in a desiccator until used Three different samples of HAp nanorods (0P123, 1P123, and 2P123) were used, which resulted in three different HAp/PDLLA films denoted as F 0P123, F 1P123, and F 2P123 The morphologies of resulting films and the presence of HAp nanorods on the substrate were checked by FE-SEM imaging at low and high magnifications 2.4 In vitro bioactivity tests SBF with ion concentrations similar to those in human blood plasma was prepared according to the method reported in other studies [33] and filtered by a 0.22 m Millipore filter system to eliminate bacterial contamination The SBF was preserved in a plastic bottle in a refrigerator at ◦ C Prior to in vitro experiments, all films of HAp/PDLLA were cut into disks with mm diameter, sterilized in 70% ethanol at ◦ C overnight, and dried under a laminar airflow hood Two specimens for each kind of film (F 0P123, F 1P123, and F 2P123) were soaked in 10 mL of SBF in closed polyethylene containers at 37 ◦ C The SBF solution was replaced every 2.5 days After 1, 3, and days of soaking, the specimens were removed from the SBF, rinsed with deionized water, and dried in warm flowing air All operations were conducted in a laminar airflow hood to avoid microorganism contamination The apatite mineralization of HAp/PDLLA films was assessed through FE-SEM and energy-dispersive X-ray spectroscopy (EDXS) Results and discussion 3.1 Characterization of HAp nanoparticles Four samples were synthesized with different P123 concentrations (one, non-assisted-P123 and the other three, assisted-P123 samples) The main characteristics of representative samples are summarized in Table The XRD patterns of representative samples are presented in Fig The diffraction peaks can be indexed to the hexagonal lattice of HAp (JCPDS No 9-432), whereas the characteristic peaks are at (0 2), (1 2), (2 0), (2 1), (1 2), (3 0), (2 2), and (3 1) No characteristic peak of other phases (e.g., tricalcium phosphate and tetracalcium phosphate) was observed, which would confirm that a pure HAp phase was produced for all samples The morphology of the HAp samples was then identified by FE-SEM images Rod-shaped particles with a smooth surface Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx Table Characteristics of representative HAp samples Sample codes Mean diameter ± SD (nm) Mean length ± SD (nm) Aspect ratios Ca/P Morphology 0P123 1P123 2P123 103 ± 26 74 ± 25 28 ± 585 ± 315 383 ± 152 120 ± 32 5.68 5.18 4.29 1.55 1.61 1.66 Rod-like shape Rod-like shape Rod-like shape SD: standard deviation; n = 50 Fig XRD patterns of HAp samples at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500 ◦ C for h were produced for the synthesized samples, independent of P123 concentration (Fig and Fig S1, Supporting material) According to Fig 2a and Table 1, large and irregular rod-shaped HAp particles (mean width of 103 nm and mean length of 585 nm) were obtained for non-assisted P123 samples (0P123 samples) Obtained assisted P123 samples with increasing P123 concentrations can result in a significant decrease in the size of HAp nanoparticles 1P123 sample (Fig 2b) shows considerably smaller rod-shaped particles than 0P123 samples; the mean diameter is 74 nm and the mean length is 383 nm Furthermore, the FE-SEM image (Fig 2c) and particle size distributions of 2P123 demonstrate that these samples have the smallest and most uniform rod-shaped particles (their sizes are mainly in the range of 25–30 nm in width and 100 nm–130 nm in length) among the assisted P123 samples However, a further increase of P123 concentration produced larger and irregular rodshaped HAp As shown in Fig S1, irregular HAp particles with mean width of 82 nm and mean length of 365 nm were formed for 3P123 samples These observations confirm that the synthesis in the presence of P123 does not affect the final morphology of nanorods but significantly affects their sizes g of P123 should be optimal for the Fig FE-SEM images of representative HAp samples synthesized at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500 ◦ C for h The inset shows particle size distribution of 2P123 Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx Fig (A) FTIR spectra of representative HAp samples synthesized at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500 ◦ C for h (B) EDXS spectrum of an HAp sample (2P123) Fig (A) Nitrogen adsorption–desorption isotherms of HAp samples synthesized at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 (B) Pore size distributions of HAp samples production of the most uniform rod-shaped HAp with sizes similar to those of natural HAp Table presents Ca/P ratios for the representative HAp samples with an upward tendency by increasing the P123 concentration In our opinion, P123 concentration could not affect the chemical composition of HAp (e.g., Ca/P ratio) because P123 was used as template to control the sizes of HAp nanorods, and P123 was easily removed through solvent extraction/calcination Ca/P ratios shown in Table are the average values of three measurements The theoretical Ca/P ratios for all the samples (e.g., 0P123, 1P123, and 2P123) are 1.67 However, the experimental Ca/P values of 0P123, 1P123, and 2P123 are 1.55, 1.61, and 1.66, respectively Fig 3A presents the FTIR spectra of representative HAp samples prepared with different concentrations of P123 The spectra of samples not show significant differences, which reveal the presence of characteristic groups on HAp samples Indeed, the main vibrations of PO4 3− groups for HAp samples are detected at 1095 cm−1 , 1032 cm−1 , 960 cm−1 , 605 cm−1 , 567 cm−1 , and 474 cm−1 The peaks at 1095 cm−1 and 1032 cm−1 characterize a degenerate asymmetric-stretching vibration mode, 3 , of P O bonds, whereas the weak peak at 960 cm−1 is assigned to a nondegenerate symmetric-stretching mode, 1 [24] The bands at 605 cm−1 and 567 cm−1 are attributed to a double degenerate bending mode, 4 , of O P O bonds, and the band at 474 cm−1 is a symmetrical bending mode, 2 Apart from these characteristics, the band at 3570 cm−1 can be assigned to the stretching vibration of OH groups of HAp [34], and the broad stretching peak at 3433 cm−1 , as well as the bending peak at 1636 cm−1 , can be assigned to adsorbed water The elemental composition of the HAp samples was determined by EDXS analysis The EDXS spectrum and components for a representative sample (sample 2P123) are presented in Fig 3B and Table S1 The results revealed that elements O, P, and Ca were the major constituents of the HAp samples with 44.17 ± 4.52 wt%, 16.14 ± 1.23 wt%, and 34.64 ± 3.05 wt%, respectively A trace of Cl was detected, which could be attributed to a residue of the synthesis reaction, and incompletely removed from the sample by washing In addition, the presence of C in HAp samples is possibly due to the use of carbon tape to mount the samples Moreover, the Ca/P ratios of the HAp samples (Table 1) range from 1.55 to 1.66, which is close to the stoichiometric value of 1.67 Therefore, stoichiometric HAp was successfully produced with these samples The surface characteristics of HAp samples were continuously studied by N2 adsorption–desorption measurements The N2 adsorption–desorption isotherms of the HAp samples are shown in Fig 4A All samples exhibit a type III isotherm with an H3 hysteresis loop in the P/Po (0.9–1) [35], which suggest the formation of slit-shaped pores as secondary pores in the aggregates of nanoparticles with large pores [30,36] Pore size distributions of the samples were calculated from desorption curves through the BJH method (Fig 4B) The surface areas, pore volumes, and pore sizes are listed in Table These HAp samples not demonstrate significant discrepancies in their BET surface areas with varying P123 concentrations The BET surface areas of all samples are 14–27 m2 g−1 , Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx Table Surface characteristics of representative HAp samples Sample codes SBET a (m2 g−1 ) VBJH b (cm3 g−1 ) Dp c (nm) 0P123 1P123 2P123 21 14 27 0.095 0.039 0.16 3; 10; 31 42 12 a b c BET surface area Total pore volume determined using desorption branch of the isotherms Peak pore sizes from the pore size distributions but the samples differ in the structures of their pores Among three samples studied, sample 2P123 has narrow pore size distribution with a maximum peak of 12 nm, whereas sample 0P123 shows a disordered pore size distribution with three maximum peaks of approximately 3, 10, and 31 nm Sample 1P123 has a broad pore size distribution, which ranges from 20 nm to 80 nm and concentrated at approximately 42 nm 3.2 Possible mechanism for HAp particle formation A possible mechanism for the formation of HAp nanorods may be proposed based on the results obtained (Fig 5) Without P123 (Fig 5A) and at high pH, the reaction between the solutions of CaCl2 and Na2 HPO4 yields HAp via the following reaction: 10Ca2+ + 6PO4 3− + 2OH− → Ca10 (PO4 )6 (OH)2 The resulting HAp was hydrothermally treated at 180 ◦ C, calcinated at 500 ◦ C, and produced large irregularly rod-shaped HAp particles (with mean width of 103 nm and mean length of 585 nm) as shown in Fig 2a and Table Without hydrothermal treatment after calcination at 500 ◦ C, the aggregated spherical HAp particles with average diameters of 350 nm were obtained instead of the rodshaped particles (Fig S2) These results confirm that hydrothermal treatment at 180 ◦ C provides energy for the HAp nuclei to grow and produce elongated HAp crystals This crystal growth is possibly due to the intrinsic crystal habit caused by the difference in lattice energy between the different crystal planes of HAp [37] Without any control, the obtained HAp crystals are extremely long and have irregular size Theoretically, P123, a non-ionic surfactant can be selfassembled into polymeric micelles with various morphologies (e.g., spherical, cylindrical, or lamellar structures) at high concentrations above the critical micelle concentration [36] The size and shape of micelles determine the morphology of the synthesized HAp particles In aqueous solution, P123 forms nano-sized cylindrical micelles that function as “nanoreactors” Ca2+ can preferably bind first with the hydrophilic head groups on the inner surface of the resulting micelles through hydrogen bonds and then, with PO4 3− /OH− to form the P123-HAp complex that provides the necessary topology that in turns directs mineral growth and leads to the formation of elongated HAp nanocrystals (Fig 5B) At low concentrations of P123 (e.g., 1P123 samples), a number of HAp nuclei that are unbound to micelles, later grow uncontrollably to produce irregularly shaped and large HAp crystals Meanwhile, the sizes of micelles increase as the P123 concentration increases The increase in the size of micelles results in an increase in the sizes of HAp particles The most uniform HAp particles that resemble bone minerals were obtained with the suitable concentration of g 3.3 In vitro bioactivity of synthesized HAp/PDLLA films in SBF An essential characteristic of biomaterials is their ability to bind to living bone by forming a bone-like apatite layer on its surface both in vitro and in vivo The powder form of the HAp particles is unfavorable when working with SBF Thus, the bioactivity of the HAp samples was evaluated through in vitro tests by soaking films of HAp/PDLLA in SBF Fig S3 represents the SEM images of HAp/PDLLA composite films with low and high magnifications before immersion into SBF Generally, the surfaces of the composite films are rough The images with high magnification confirm a full cover of rod-shaped HAp particles with smooth morphology on the surface of the substrate The formation of a bone-like apatite layer on the HAp/PDLLA films was investigated through FE-SEM measurements after 1, 3, and days of soaking in SBF Fig S4 shows representative FE-SEM images of F 0P123 films after soaking in SBF for and days and that of F 1P123 films after soaking in SBF for and days Compared with films examined before soaking, these composite films did not show significant changes in their morphologies during the initial stage of soaking (1–3 days) However, a few bundles of HAp particles were found because of the aggregation of such particles on the surface of the composite films after the initial (1–3 days) immersion in SBF (e.g., after day for the F 0P123 film and after days for the F 1P123 film), as shown in Fig S4(a) and (d) After days of soaking in SBF, both composite samples exhibited morphological changes, which are different from those before soaking As shown in high-resolution FE-SEM images in Fig S4(c) and (f), their morphologies were rougher with scant deposition of needlelike mineral crystals (160 nm in length and 20 nm in diameter for F 0P123; 120 nm in length and 20 nm in diameter for F 1P123) Such an observation revealed the induction of a mineral phase on the surface of these films after days in SBF FE-SEM images of F 2P123 films after 1, 3, and days of treatment in SBF are presented in Fig During incubation in SBF, the morphology of F 2P123 composite films drastically changed with the appearance of abundant deposits In particular, the intense formation of spherical mineral particles (mean diameter of m) composed of tiny needle-like crystals was already observed after day of immersion in SBF (Fig 6a and b) The same tendency was seen after days (Fig 6c and d), but the size of the spherical particles with needle-like crystallites increased with longer soaking time (their mean diameter increased to m) Thereafter (beyond days), the mineral phase was largely spread on the surface of F 2P123 films FE-SEM images with different magnifications (Fig 6e and f) reveal that a coral-like mineral layer of numerous tiny needle-like crystals was deposited on the entire surface of F 2P123 films after days in SBF FE-SEM images with higher resolution (Fig 6g and h) indicate the formation of a flower-like mineral layer composed of tiny needle-like crystallites on the entire surface of F 2P123 This interesting morphology of the mineral layer is typical in bone-like apatite [33] The given results reveal the complete formation of the mineral layer on the surface of F 2P123 even for short soaking periods, which indicates its highest in vitro bioactive response among the three composite films studied As reported in past studies, mineralization involves the nucleation and growth of bone-like apatite onto biomaterials in SBF [38], which is mainly associated with the uptake of calcium and phosphate ions from the SBF solution The composite film (F 2P123) composed of HAp nanoparticles (2P123) with the smallest sizes and the highest BET surface area among the studied HAp samples (Tables and 2) certainly resulted in its high uptake for calcium and phosphate ions, thereby exhibiting the highest bioactivity Consequently, the composite films (F 0P123 and F 1P123) consisted of the HAp nanoparticles with large dimensions and low BET surface areas (both against their uptake capacity of calcium and phosphate ions) show considerably lower bioactivity The Ca/P ratio is a key characteristic used to identify the mineral phase among biologically relevant minerals To further analyze the chemical composition of the released mineral layer, EDXS analysis was conducted for the typical composite films after SBF treatment The results indicated that the main elements (i.e., Ca, P, and O) were found in the mineral layer and C was also detected as a trace Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx Fig Possible mechanism for the formation of (A) HAp nanoparticles in the absence of P123 and (B) HAp nanorods in the presence of P123 element (not shown) Table S2 presents the Ca/P ratios of the composite films after days of soaking in SBF for the F 0P123 film and after 1, 3, and days for F 2P123 films As shown in Table S2, the Ca/P ratio for F 0P123 after days of treatment is 1.55, which reveals the formation of a new apatite phase on the surface of the F 0P123 in SBF However, the intensity of the carbon peak on EDXS patterns of F 0P123 before and after treatment in SBF increased, thereby suggesting that the apatite produced was carbonated For F 2P123 films, the Ca/P ratio showed an increasing trend with increasing soaking time; the Ca/P ratio was 1.36, 1.47, and 1.57 for 1, 3, and days of SBF, respectively As shown in the literature, a number of other calcium phosphate minerals (e.g., amorphous calcium phosphate Cax (PO4 )y ·zH2 O (ACP), dicalcium phosphate dihydrate CaHPO4 ·2H2 O (DCPD), octacalcium phosphate Ca8 H2 (PO4 )6 ·5H2 O (OCP), tricalcium phosphate ␣- and -Ca3 ·(PO4 )2 (TCP), and Mgsubstituted tricalcium phosphate (Ca,Mg)3 (PO4 )2 ·(Mg-TCP)) can also be produced under conditions similar to those that form apatite in vitro These minerals can be transformed from one type to another depending on the pH and composition of the biological micro-environment and can be distinguished through their Ca/P ratio through EDXS [37] Therefore, based on EDXS analyses and Ca/P ratios (Table S2), OCP and TCP were probably released on the surfaces of F 2P123 films after and days of soaking, respectively The highest value of 1.57 can be assigned to a bone-like apatite layer produced in F 2P123 after days Considering the increase in the intensity of the carbon peak for EDXS patterns before and after days of immersion in SBF for F 2P123, we find that the carbonated apatite layer was already produced on the surface of F 2P123 after days of immersion in SBF Table S3 compares the in vitro bioactivity of HAp nanorods as prepared in this study with those reported in previous studies [39] The formation of new bone-like mineral was already detected after day of incubation in SBF for the prepared HAp nanorods in this study; it was detected only after 45 days for HAp nanorods, HAp nanospheres, and HAp fibrous microparticles in past studies [39] Moreover, after a quick period of incubation in SBF (7 days), a highly interesting and distinctive morphology of bone-like apatite resembling the flower-like apatite was exclusively observed for the HAp nanorods prepared in this study (Fig 6g and h) The in vitro bioactivity of the prepared HAp nanorods in this study that is more efficient than the other HAp particles should be attributed to their close morphology (e.g., size and shape) to natural HAp As shown in Table S3, the HAp nanoparticles prepared in this study have a mean width of 28 nm and a mean length of 120 nm, which is extremely similar to those of bone minerals The other HAp particles possess morphologies that are considerably different Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx Fig SEM images of HAp/PDLLA composite film (F 2P123) after soaking in SBF for (a and b) day, (c and d) days and (e–h) days from those of the prepared HAp nanorods and the mineral part of bone For instance, the best HAp particles in the previous study have spherical and near-spherical shape (approximately 50 nm in size) or extremely short nanorods (average width of 60 nm and average length of 110 nm) [39] Based on the comparison of the results of the present and previous study, the size and shape of HAp particles have a crucial function in their bioactivity and biocompatibility in promoting the bone-like apatite phase The morphological similarity of synthetic HAp particles can facilitate both their in vitro and in vivo bioactivity Conclusion This study demonstrates that rod-shaped hydroxyapatite particles resembling bone minerals are easily synthesized with controllable sizes with a suitable concentration of the pluronic Please cite this article in press as: N.K Nga, et al., Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering, Colloids Surf B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.11.001 G Model COLSUB-6103; No of Pages ARTICLE IN PRESS N.K Nga et al / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx–xxx surfactant P123 The HAp nanorods revealed a uniform rod shape with sizes similar to those of bone minerals (mean width of 28 nm and mean length of 120 nm), Ca/P of 1.66, and stoichiometric HAp In vitro bioactivity tests showed that the HAp nanorods induce rapid formation of bone-like apatite after an extremely short soaking time in SBF, which makes them excellent candidates for bone repair and reconstruction This study can provide an efficient protocol for the controlled synthesis of HAp nanorods similar to bone minerals in view of developing biocompatible composite scaffolds for bone tissue engineering Acknowledgments This study was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.02-2012.42 The authors would like to thank Dr Anne-Lise Haenni, 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Fig XRD patterns of HAp samples at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500 ◦ C for h were produced for the synthesized samples, independent of P123... images of representative HAp samples synthesized at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500 ◦ C for h The inset shows particle size distribution of