Article pubs.acs.org/crystal
Graphene-like Zinc Substituted Hydroxyapatite Jun Ma*,†,‡ and Jinli Qin† †
Advanced Biomaterials and Tissue Engineering Center and ‡Department of Biomedical Engineering, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China ABSTRACT: The biological bone minerals form within a complex microenvironment that consists of various ions and template molecules. Silk fibroin provides an attractive template for biomimetic hydroxyapatite synthesis. In this study, graphene-like zinc substituted hydroxyapatite crystals were prepared using silk fibroin and sodium alginate as template molecules, and the resulting products were investigated. The alginate ions interacted with silk fibroin, and the additional zinc ions also influenced the crystal formation of hydroxyapatite. The graphene-like sheets with hydroxyapatite phase were approximately 3 nm in thickness, and the size was more than 100 nm. The structure of the zinc substituted hydroxyapatite was studied by transmission electron microscopy, and the results revealed that small crystallites of several nanometers were assembled into large size sheets. The synergy effect of zinc and alginate ions led to the preferred growth along (002) and (211) orientations. Accordingly, the mechanisms of crystal growth regulation have been demonstrated.
■
of bi-/multitemplate regulation during apatite formation.5,15,16 The assembly of silk fibroin and collagen was found to tune mineral structures, leading to smaller particles and narrower size distributions.17 The use of two template molecules in the synthesis is considered to provide more precise control over crystal morphology through the interaction with the templates and produce different crystal nanoparticles. It is known that some cationic ions (e.g., Zn2+, Mg2+, and Sr2+) can easily enter the apatite crystal structure, and they are found to play important roles in biological functions. In this study, our aim is to reveal the influence of zinc substitution on the mineralization process of silk fibroin. Herein, silk fibroin is used as the primary template because (1) the fibroin molecule is able to regulate the growth of apatite and (2) the mineralized silk fibroin is a biocompatible biomaterial. Using cationic substitution, it is possible for us to tune the properties of the obtained minerals, such as morphology, structure and crystallinity. Furthermore, the incorporation of zinc ions into hydroxyapatite could add more biological functions to the nanoparticles, such as immunological modulation,18 antibacterial property19,20 and osteoblast response.21 The calcium phosphate precipitates could maintain the apatite phase when Zn/(Zn + Ca) reached 15−20 mol %.22 In this study, the highest Zn/(Zn + Ca) molar ratio in the synthesis was 10%. In addition, acidic molecules and/or carboxyl groups were suggested to play vital roles in the nucleation and formation of apatite crystals.23 We also added sodium alginate into the reaction in order to tune the crystallization process. It was demonstrated that the acidic molecules could stabilize the size and morphology of biomimetic
INTRODUCTION Hydroxyapatite is an important biomineral which forms the main constituent of bone and teeth. Hydroxyapatite and its apatite analogues are studied to explore fundamental aspects of biomineralization, and they are also used as functional biomaterials in the fields of tissue engineering and drug delivery.1−3 The biomineralization process, the formation of nanocrystal hydroxyapatite regulated by template molecules (e.g., collagen), and the resultant complicated hierarchical structures have been extensively investigated for several decades.2,4,5 However, it is still difficult to mimic the mineral deposition in the biological systems for fabricating hard tissues in vitro. Until now, not only the mechanisms of biological apatite deposition remain unclear but also the synthetic apatite from the biomimetic approaches using template regulation methods cannot reach the levels of biological apatite. It triggers us to investigate the underlying mechanisms of complex template molecules in the apatite deposition process. A number of template molecules have been investigated, such as proteins,6,7 enzymes,8 polymers,9 DNA,10 polypeptides, and other molecules.11 Besides collagen, silk fibroin has been used to regulate the synthesis of hydroxyapatite crystals in the past decade. Nanocrystals with diameters of 2−3 nm were obtained, and the preferred (002) orientation was observed.12 Another study demonstrated that crystals with a rod-like shape of 20−60 nm in length and 10−20 nm in diameter were obtained using silk fibroin as template.13 The blue shift of amide II peaks indicated strong chemical interactions between minerals and fibroin molecules, and fibroin was suggested to accelerate the phase transition from amorphous calcium phosphate to hydroxyapatite.13,14 In the biological systems, many template molecules are involved in the regulation of mineral formation at the same time. Researchers have possessed a lot of interest in the synergy effects © XXXX American Chemical Society
Received: November 12, 2014 Revised: January 11, 2015
A
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
Article
Crystal Growth & Design
Figure 1. TEM images of typical hydroxyapatite nanoparticles with different zinc substitution concentrations produced by the regulation of silk fibroin: (a, b) 0% Zn; (c) Zn/(Zn + Ca) = 5%; (d) Zn/(Zn + Ca) = 10%. “Zn/(Zn + Ca)” means the molar percentage of Zn/(Zn + Ca) used in the synthesis.
Figure 2. TEM images of typical hydroxyapatite nanoparticles with different zinc substitution concentrations produced by the regulation of silk fibroin and sodium alginate: (a) Zn/(Zn + Ca) = 5%; (b) SAED pattern of the area indicated by an asterisk in a; (c) Zn/(Zn + Ca) = 5%, magnification; (d) Zn/ (Zn + Ca) = 10%.
apatite.5,24−26 The alginate molecules could strongly interact with calcium ions due to electrostatic attraction. The presence of alginate molecules could change the speed of transformation of amorphous phase to crystalline apatite and control the crystal morphology.
Methods. The biomimetic mineralization was carried out and the products were aged at 37 °C for 3 days. All of the precipitates were washed and lyophilized. The products collected were white powders with good flowability. From the transmission electron microscopy (TEM) images in Figure 1, the resultant structures changed from rectangle flake shape to large pieces of graphene-like sheets after zinc ions were added in the co-precipitation synthesis. For hydroxyapatite nanoparticles, the particle length was 97 ± 19 nm and the width was 19 ± 7 nm. The thickness was measured around 5−10 nm.
■
RESULTS AND DISCUSSION Hydroxyapatite nanoparticles with different concentrations of zinc substitution were obtained using chemical co-precipitation method. Silk fibroin was added in the reaction as described in B
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
Article
Crystal Growth & Design
Figure 3. High resolution TEM images of the hydroxyapatite nanoparticles: (a, b) 0% Zn, silk fibroin; (c, d) Zn/(Zn + Ca) = 10%, silk fibroin and alginate.
Figure 4. FESEM images of the hydroxyapatite nanoparticles: (a) Zn/(Zn + Ca) = 5%, silk fibroin; (b) Zn/(Zn + Ca) = 10%, silk fibroin; (c) Zn/(Zn + Ca) = 5%, silk fibroin and alginate; (d) Zn/(Zn + Ca) = 10%, silk fibroin and alginate.
Most of the particles had a sharp end, as shown in Figure 1b. The morphology of the hydroxyapatite nanoparticles seemed similar to that in the previous results.13 From Figure 1c, it was measured that the sheets were about 3 nm in thickness and the size was more than 100 nm. The folds on the sheet seemed darker; thus the whole sheet looked like wrinkled paper. When more zinc ions were used, the morphology did not change obviously. Sodium alginate was added to replace half of the silk fibroin used. It was found that the graphene-like structure was maintained and the crystallinity became higher. From the
selected area electron diffraction (SAED) pattern, it was shown that the mineral sheet was hydroxyapatite phase. The diffraction from the (002) (d ∼ 3.44 Å) plane of crystalline hydroxyapatite appeared as a pair of arcs, indicating that the c-axis of hydroxyapatite was preferred in the growth direction. According to the magnified images in Figure 2c,d, the graphene-like sheets had irregular shape and some areas were folded such as that without the alginate template. The high resolution TEM images showed that the lattice belonged to hydroxyapatite crystal, as shown in Figure 3. In the C
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
Article
Crystal Growth & Design
Figure 5. XRD patterns of the hydroxyapatite nanoparticles: (a) different Zn/(Zn + Ca) ratios, silk fibroin; (b) different templates. SF means silk fibroin, and Alg means alginate.
Figure 6. Different zinc substitution concentrations in hydroxyapatite using silk fibroin as a template: (a) thermal gravimetric analysis; (b) differential thermal analysis.
area marked by an asterisk, (002) and (211) planes were identified. Such areas were also observed when silk fibroin was used as template and no zinc was added. It was found that the formation of graphene-like structure was not seen when either zinc ions or silk fibroin was used alone. It was considered that zinc ions had interaction with silk fibroin and promoted the crystal growth along both (211) and (002) directions. The zinc content in the minerals was also checked using in situ energy dispersive X-ray spectroscopy (EDS) of TEM. The sample prepared using silk fibroin and sodium alginate as bitemplate and Zn/(Zn + Ca) = 10% was examined. From the EDS results, it was found that the Zn/(Zn + Ca) ratio was 11.5% and the Ca/P ratio was about 1.62. It was interesting that the (Zn + Ca)/P ratio (1.83) was higher than 1.67. It was usually considered that zinc ions could replace calcium ions and the (Zn + Ca)/P ratio should be 1.67 or less.19,27 The increased zinc content in the final products was suggested to be related to the additional templates. The utilization of silk fibroin and sodium alginate could promote the deposition of zinc in the apatite mineral sheets. As shown in Figure 4, the morphology observed by field emission scanning electron microscopy (FESEM) revealed that the zinc substituted hydroxyapatite nanoparticles looked like wrinkled flowers. Some large pieces of sheets could be seen for Zn/(Zn + Ca) = 5% when sodium alginate was used with silk fibroin (Figure 4c). These large sheets were not observed by
TEM because only small ones in the suspension were attached on the copper grids when preparing the TEM samples. The mineral structure was characterized using X-ray diffraction (XRD) characterization (Figure 5). After adding zinc ions, the diffraction peaks became weaker and wider, indicating that the crystallinity decreased. The diffraction patterns confirmed that the main hydroxyapatite phase existed in the products. The hydroxyapatite diffraction intensities decreased upon increasing zinc ions. When 10% Zn/(Zn + Ca) was used, it was found that another phase appeared in the XRD patterns. The peak at 21° (indicated by an asterisk) was attributed to (021) of brushite. After silk fibroin and alginate were used together, it was found that the peak at 21° disappeared. However, this peak existed when sodium alginate was used alone. It was inferred that the combination of silk fibroin and sodium alginate facilitated the formation of hydroxyapatite crystals. The mineral contents also decreased when zinc was added, as confirmed by thermal gravimetric analysis (TGA, Figure 6a). The mineral contents were estimated to be 92.2, 86.8, and 80.6 wt % when zinc substituted 0%, 5%, and 10% calcium, respectively. The zinc substitutions caused more weight loss. The weight loss was suggested to be attributed to by the partial decomposition of silk fibroin and release of water from the minerals, which was also approved by the differential thermal analysis (DTA), as shown in Figure 6b.28 Because the usage of silk fibroin was the same for each sample, the zinc substitution could increase the content of D
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
Article
Crystal Growth & Design
Figure 7. FT-IR results of the hydroxyapatite nanoparticles: (a) different Zn/Ca ratios, silk fibroin; (b) different templates.
lattice water in the final products. Furthermore, the temperature of the phase transition of hydroxyapatite to tricalcium phosphate decreased to about 900 °C for Zn/(Zn + Ca) = 10%. Meanwhile, no such phase transition was found for 0% and 5% Zn/(Zn + Ca) below 1000 °C. The crystallization of minerals was also characterized using Fourier transform infrared spectroscopy (FT-IR), as shown in Figure 7. The broad band between 550 and 610 cm−1, associated with phosphate groups, splits into two bands at 562 and 602 cm−1 upon hydroxyapatite crystallization, consistent with the XRD results. The bands at about 3570 cm−1 were also attributed by hydroxyl groups. The bands at around 876 cm−1 can be assigned to CO32−, indicating that hydroxyapatite minerals formed were carbonated hydroxyapatite with hydroxyl ions substituted by carbonate ions, as described in natural bones.29,30 These bands became weaker after sodium alginate was added. The carbonyl group had a band at 1652 cm−1 when silk fibroin was used alone. When sodium alginate was added with silk fibroin, the band of the carboxyl group moved to 1637 cm−1. The red shift of carboxyl bands indicated strong interaction between the two templates. It was also observed that most of the amide bands became weaker for the combination of silk fibroin and alginate. The amide bands of the raw silk fibroin were compared (data not shown). During the mineralization process, the blue shift of amide bands belonging to the interaction between the template molecules and hydroxyapatite was found, whereas the amide I band shifted from 1638 to 1652 cm−1 and the amide II band shifted from 1514 to 1560 cm−1.13 After adding sodium alginate, the amide I band moved back to 1637 cm−1, but the amide II band was at 1560 cm−1 without any shift. In this study, the additional sodium alginate was considered to play a role of inhibitor between silk fibroin and hydroxyapatite, which limited the growth of crystals along certain directions. The formation of large pieces of graphene-like structures could benefit from the combination use of silk fibroin and sodium alginate. According to the preceding results, a growth process for bitemplate regulated mineralization was proposed. After the zinc incorporation, hydroxyapatite nanocrystals changed from thin strip-insole shape to irregular flake and smaller lattice size.22 The hydrophilic blocks on the silk fibroin molecules are nucleation sites, and zinc, calcium, and phosphate ions are attracted by the hydrophilic blocks. The precipitation occurred when the ionic interactions became strong enough, as shown in Figure 8a. The carboxyl groups on the sodium alginate have strong interactions with cationic ions in the solution. The zinc and calcium divalent
Figure 8. Graphene-like apatite formation mechanisms based on the interaction between the template molecules and ions: (a) silk fibroin; (b) sodium alginate; (c) silk fibroin and alginate.
ions could act as cross-linking agents for alginate chains, as shown in Figure 8b. When sodium alginate and silk fibroin both exist in the solution, their hydrophilic groups may have strong Hbonding and they are tangled with each other in the solution. In addition, fibroin and alginate may cover the surface of the obtained hydroxyapatite nanoparticles. According to the TEM E
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
Article
Crystal Growth & Design
apatite nanoparticles, calcium nitrate and sodium hydrogen phosphate were used. Zinc nitrate was used to introduce zinc ions in the precipitant. For example, 10 mL of 0.1 M Zn(NO3)2 solution, was mixed with 90 mL of 0.1 M Ca(NO3)2 solution in a water bath of 37 °C. The stirring speed was around 350 rpm. Subsequently, 20 mL of silk fibroin (2.5 mg/mL) was added. In another beaker, 60 mL of 0.1 M Na2HPO4 was mixed with 20 mL of sodium alginate solution (2.5 mg/mL). The phosphate solution was added slowly to the reaction. The pH value was measured using a pH meter and adjusted to pH 7−8 using 1 M NaOH solution. After stirring for 2 h, the mixture was moved to a bottle, which was placed in a water bath of 37 °C. The aging process lasted for 3 days. The obtained slurry was filtered, and the filter cake was washed again. The purified slurry was frozen under −70 °C and lyophilized under −55 °C and 10−20 Pa. The dried powders were collected and used for further characterization. Characterization. For TEM (G2, FEI, Eindhoven, The Netherlands) characterization, the freeze-dried powder was dispersed in ethanol by sonication, followed by dropping on a carbon-coated TEM copper grid. The samples were examined under the voltage of 200 kV. High resolution TEM, EDS, and SAED were done on the same machine. For FESEM (Nova NanoSEM 450, FEI) observation, the powders were placed on a conductive double-sided adhesive tape and a thin gold coating was applied to avoid any charging artifacts. For XRD measurements, the samples were pressed on the sample holder and subsequently measured on a XRD-7000 diffractormeter (Shamadzu, Kyoto, Japan) at 40 kV and 40 mA using Cu Ka radiation. For FT-IR experiments performed on VERTEX 70 (Bruker, Ettingen, Germany), the freeze-dried powders were mixed with KBr and pressed into pellets. Spectra were collected with a 4 cm−1 resolution after 64 scans. TGA and TDA were performed using a Diamond TG/DTA system (PerkinElmer, Waltham, MA, USA) purged with N2 at 100 mL/ min. Approximately 2−3 mg samples were used in the experiments to heat from room temperature to 1000 °C at 10 °C/min.
measurements, the (002) and (211) planes have been determined and they are perpendicular to the (010) plane. The exposed plane is considered to be (010), which is inhibited to grow and the graphene-like structure forms in this condition. According to the calculation results, the electrostatic attractive interactions dominate the protein adsorption on the crystal surface of hydroxyapatite. The carboxyl group of protein interacts with calcium sites, while amino groups have strong bonds with the phosphate groups.31 The (001) surface is more preferable for the adsorption of protein molecules.32 It is to say that the (001) plane is the most stable one. However, the nonstoichiometric (010) surface (both Ca- and P-rich) is terminated over the OH channels along the (010) plane.33 The combination of fibroin and alginate leads to the preferred coverage on the (010) plane; thus the crystals could grow into a large sheet. In this study, we have successfully realized a biomimetic approach to fabricating large pieces of graphene-like hydroxyapatite crystals. In addition, the combination of silk and hydroxyapatite could be adopted to fabricate composite scaffolds for bone tissue repair. Silk fibroin was simply mixed with hydroxyapatite particles for fabricating scaffolds which were found to induce in vitro formation of trabecular-like structure.34 The nonwoven silk fibroin was mineralized and showed excellent cytocompatibility and improved viability of osteoblast cells.35 The electrospun silk fibroin/hydroxyapatite composite mats were prepared from the mixed dispersion and showed improved mechanical properties.36 The previous study proved that the mineralized silk fibroin could promote osteoblast proliferation in vitro and new bone formation in vivo.37 In summary, silk fibroin has similar performance with collagen in the mineralization regulation. From this point of view, the graphene-like hydroxyapatite crystals could provide large reactive areas with other materials and maintain unique crystal structures. It is also possible to make a bone graft material using the graphene-like hydroxyapatite crystals, which has the advantage as a biomimetic apatite material. The assembling mechanisms of bone are still unclear, especially in the range of 100−1000 nm. The biomimetic precipitation using collagen and other protein templates can only produce nanosized particles or mineralized fibers. It is still far from bone tissues. The graphene-like structure is potentially a good candidate in further assembling studies, which could be used for the fabrication of bone constructs in vitro.
■
*E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (Grant No. 2012CB933601), the National Natural Science Foundation of China (Grant No. 51202075), and the Fundamental Research Funds for the Central Universities (Grant No. 2014QN121). We are also grateful to the Analytical and Testing Center (HUST).
■
CONCLUSION The novel approach presented here for the synthesis of zinc substituted hydroxyapatite has demonstrated the mechanisms of calcium phosphate precipitation regulated by silk fibroin and sodium alginate. Our results suggest that the combination of sodium alginate and silk fibroin can influence the formation of hydroxyapatite crystals. The obtained zinc substituted hydroxyapatite crystals have a sheet-like shape with a thickness of approximately 3 nm, and the addition of sodium alginate helps the stabilization of this structure. We expect that this study will contribute to the understanding of the biomineralization process in physiological systems and become a platform for fabricating novel biomaterials.
■
AUTHOR INFORMATION
Corresponding Author
■
REFERENCES
(1) Bonzani, I. C.; George, J. H.; Stevens, M. M. Novel materials for bone and cartilage regeneration. Curr. Opin. Chem. Biol. 2006, 10, 568− 575. (2) Cui, F. Z.; Li, Y.; Ge, J. Self-assembly of mineralized collagen composites. Mater. Sci. Eng., R 2007, 57, 1−27. (3) Bose, S.; Roy, M.; Bandyopadhyay, A. Recent advances in bone tissue engineering scaffolds. Trends Biotechnol. 2012, 30, 546−554. (4) Olszta, M. J.; Cheng, X. G.; Jee, S. S.; Kumar, R.; Kim, Y. Y.; Kaufman, M. J.; Douglas, E. P.; Gower, L. B. Bone structure and formation: A new perspective. Mater. Sci. Eng., R 2007, 58, 77−116. (5) Ma, J.; Wang, J.; Ai, X.; Zhang, S. Biomimetic self-assembly of apatite hybrid materials: From a single molecular template to bi-/multimolecular templates. Biotechnol. Adv. 2014, 32, 744−760. (6) George, A.; Ravindran, S. Protein templates in hard tissue engineering. Nano Today 2010, 5, 254−266.
METHODS
Preparation of Apatite Nanoparticles. The silk fibroin was prepared by removing silk sericin using boiling Na2CO3 solution and CaCl2/ethanol solution. The silk fibroin powder was prepared by freezedrying of purified silk fibroin solution after dialysis. For the synthesis of F
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
Article
Crystal Growth & Design (7) Shen, L. M.; Bao, N. Z.; Zhou, Z. Y.; Prevelige, P. E.; Gupta, A. Materials design using genetically engineered proteins. J. Mater. Chem. 2011, 21, 18868−18876. (8) Unuma, H.; Matsushima, Y.; Kawai, T. Enzyme-mediated synthesis of ceramic materials. J. Ceram. Soc. Jpn. 2011, 119, 623−630. (9) Junginger, M.; Kita-Tokarczyk, K.; Schuster, T.; Reiche, J.; Schacher, F.; Muller, A. H. E.; Colfen, H.; Taubert, A. Calcium Phosphate Mineralization beneath a Polycationic Monolayer at the AirWater Interface. Macromol. Biosci. 2010, 10, 1084−1092. (10) Samano, E. C.; Pilo-Pais, M.; Goldberg, S.; Vogen, B. N.; Finkelstein, G.; LaBean, T. H. Self-assembling DNA templates for programmed artificial biomineralization. Soft Mater. 2011, 7, 3240− 3245. (11) Marsi, T. C. O.; Santos, T. G.; Pacheco-Soares, C.; Corat, E. J.; Marciano, F. R.; Lobo, A. O. Biomineralization of Superhydrophilic Vertically Aligned Carbon Nanotubes. Langmuir 2012, 28, 4413−4424. (12) Kong, X. D.; Cui, F. Z.; Wang, X. M.; Zhang, M.; Zhang, W. Silk fibroin regulated mineralization of hydroxyapatite nanocrystals. J. Cryst. Growth 2004, 270, 197−202. (13) Wang, J.; Yu, F.; Qu, L.; Meng, X.; Wen, G. Study of synthesis of nano-hydroxyapatite using a silk fibroin template. Biomed. Mater. 2010, 5, No. 041002. (14) Kong, X.; Sun, X.; Cui, F.; Ma, C. Effect of solute concentration on fibroin regulated biomineralization of calcium phosphate. Mater. Sci. Eng., C 2006, 26, 639−643. (15) Colfen, H. Biomineralization: A crystal-clear view. Nat. Mater. 2010, 9, 960−961. (16) Nudelman, F.; Pieterse, K.; George, A.; Bomans, P. H.; Friedrich, H.; Brylka, L. J.; Hilbers, P. A.; de With, G.; Sommerdijk, N. A. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat. Mater. 2010, 9, 1004−1009. (17) Wang, J.; Zhou, W.; Hu, W.; Zhou, L.; Wang, S.; Zhang, S. Collagen/silk fibroin bi-template induced biomimetic bone-like substitutes. J. Biomed. Mater. Res., Part A 2011, 99, 327−334. (18) Velard, F.; Laurent-Maquin, D.; Guillaume, C.; Bouthors, S.; Jallot, E.; Nedelec, J. M.; Belaaouaj, A.; Laquerriere, P. Zinc Doped Hydroxyapatite as Immunomodulatory Biomaterial. Int. J. Artif. Organs 2009, 32, 457−458. (19) Shepherd, D.; Best, S. M. Production of zinc substituted hydroxyapatite using various precipitation routes. Biomed. Mater. 2013, 8, No. 025003. (20) Thian, E. S.; Konishi, T.; Kawanobe, Y.; Lim, P. N.; Choong, C.; Ho, B.; Aizawa, M. Zinc-substituted hydroxyapatite: A biomaterial with enhanced bioactivity and antibacterial properties. J. Mater. Sci.: Mater. Med. 2013, 24, 437−445. (21) Webster, T. J.; Massa-Schlueter, E. A.; Smith, J. L.; Slamovich, E. B. Osteoblast response to hydroxyapatite doped with divalent and trivalent cations. Biomaterials 2004, 25, 2111−2121. (22) Ren, F. Z.; Xin, R. L.; Ge, X.; Leng, Y. Characterization and structural analysis of zinc-substituted hydroxyapatites. Acta Biomater. 2009, 5, 3141−3149. (23) Dey, A.; Bomans, P. H.; Muller, F. A.; Will, J.; Frederik, P. M.; de With, G.; Sommerdijk, N. A. The role of prenucleation clusters in surface-induced calcium phosphate crystallization. Nat. Mater. 2010, 9, 1010−1014. (24) Koutsopoulos, S.; Dalas, E. The effect of acidic amino acids on hydroxyapatite crystallization. J. Cryst. Growth 2000, 217, 410−415. (25) Sarig, S. Aspartic acid nucleates the apatite crystallites of bone: A hypothesis. Bone 2004, 35, 108−113. (26) Gomez-Morales, J.; Delgado-Lopez, J. M.; Iafisco, M.; Hernandez-Hernandez, A.; Prat, M. Amino Acidic Control of Calcium Phosphate Precipitation by Using the Vapor Diffusion Method in Microdroplets. Cryst. Growth Des. 2011, 11, 4802−4809. (27) Tang, Y. Z.; Chappell, H. F.; Dove, M. T.; Reeder, R. J.; Lee, Y. J. Zinc incorporation into hydroxylapatite. Biomaterials 2009, 30, 2864− 2872. (28) Rosseeva, E. V.; Buder, J.; Simon, P.; Schwarz, U.; FrankKamenetskaya, O. V.; Kniep, R. Synthesis, Characterization, and Morphogenesis of Carbonated Fluorapatite-Gelatine Nanocomposites:
A Complex Biomimetic Approach toward the Mineralization of Hard Tissues. Chem. Mater. 2008, 20, 6003−6013. (29) Mostafa, N. Y.; Hassan, H. M.; Abd Elkader, O. H. Preparation and Characterization of Na+, SiO44−, and CO32− Co-Substituted Hydroxyapatite. J. Am. Ceram. Soc. 2011, 94, 1584−1590. (30) Wei, X.; Fu, C.; Savino, K.; Yates, M. Z. Carbonated Hydroxyapatite Coatings with Aligned Crystal Domains. Cryst. Growth Des. 2012, 12, 3474−3480. (31) Zhou, H.; Wu, T.; Dong, X.; Wang, Q.; Shen, J. W. Adsorption mechanism of BMP-7 on hydroxyapatite (001) surfaces. Biochem. Biophys. Res. Commun. 2007, 361, 91−96. (32) Shen, J. W.; Wu, T.; Wang, Q.; Pan, H. H. Molecular simulation of protein adsorption and desorption on hydroxyapatite surfaces. Biomaterials 2008, 29, 513−532. (33) Jimenez-Izal, E.; Chiatti, F.; Corno, M.; Rimola, A.; Ugliengo, P. Glycine Adsorption at Nonstoichiometric (010) Hydroxyapatite Surfaces: A B3LYP Study. J. Phys. Chem. C 2012, 116, 14561−14567. (34) Bhumiratana, S.; Grayson, W. L.; Castaneda, A.; Rockwood, D. N.; Gil, E. S.; Kaplan, D. L.; Vunjak-Novakovic, G. Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds. Biomaterials 2011, 32, 2812−2820. (35) Zhao, Y.; Chen, J.; Chou, A. H. K.; Li, G.; LeGeros, R. Z. Nonwoven silk fibroin net/nano-hydroxyapatite scaffold: Preparation and characterization. J. Biomed. Mater. Res., Part A 2009, 91A, 1140− 1149. (36) Ming, J. F.; Zuo, B. Q. A novel electrospun silk fibroin/ hydroxyapatite hybrid nanofibers. Mater. Chem. Phys. 2012, 137, 421− 427. (37) Niu, L.; Zou, R.; Liu, Q. D.; Li, Q. L.; Chen, X. M.; Chen, Z. Q. A Novel Nanocomposite Particle of Hydroxyapatite and Silk Fibroin: Biomimetic Synthesis and Its Biocompatibility. J. Nanomater. 2010, No. 729457.
G
DOI: 10.1021/cg501659x Cryst. Growth Des. XXXX, XXX, XXX−XXX
