Spatial Organization of Hydroxyapatite Nanorods on a Substrate via a

Aug 13, 2013 - The growth of uniform single-crystal HAp (hydroxyapatite) nanorods on ... In human teeth, ameloblasts secrete organic matrix during the...
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Spatial Organization of Hydroxyapatite Nanorods on a Substrate via a Biomimetic Approach Yao-Hung Tseng, Mie Elholm Birkbak, and Henrik Birkedal* Department of Chemistry and iNANO, Aarhus University, 140 Langelandsgade, DK-8000 Aarhus, Denmark S Supporting Information *

ABSTRACT: The growth of uniform single-crystal HAp (hydroxyapatite) nanorods on specific sites on a substrate is achieved. HAp nanorods were synthesized under hydrothermal conditions in a solution containing calcium and phosphate ions, urea as a source of base, and with gelatin as a structure directing agent. The nanorods are hundreds of micrometers in length and 50−200 nm in width. Without gelatin, platelike HAp crystals were formed. To grow these uniform HAp nanorods on specific sites on a substrate, the surface of a gold-coated glass slide was coated by a self-assembled monolayer of 11-mercaptoundecanoic acid by stamping with a patterned PDMS (polydimethylsiloxane) stamp. The HAp nanorods were grown exclusively in the carboxylate functionalized regions. Selective area electron diffraction analyses show that the single-crystal HAp nanorods formed on the substrate grow along the crystallographic c-axis. In comparison to the nonpatterned sample, an increase of the X-ray diffraction intensity ratio of (00l)/(h00) peaks in the patterned sample indicates that nanorods are preferentially oriented with their c-axes perpendicular to the substrate, which was confirmed by SEM cross-sectional images. In this strategy, both the morphology and the deposition sites of HAp nanorods are controlled.

H

location where synthesized minerals are deposited or formed on surfaces.23−29 Among these methods, a substrate patterned by a SAM (self-assembled monolayer) is frequently employed to mimic the insoluble organic matrix in biology to induce or inhibit crystal growth.23,24,28 Thereby patterned distributions of crystals can be formed on substrates. This approach is able to control the crystal growth in a micrometer-scale region. However, it is not straightforward to manipulate the morphologies, properties, and sizes of nanoparticles through this method. Many efforts have been focused on either preparing oriented HAp structures30−33 or patterning HAp on a substrate25,29 for mimicking sophisticated structures of bone and teeth. Here we describe a simple wet chemical method for controlling morphology and orientation of HAp crystals grown on specific sites of a substrate. To achieve the construction of complex structures, we herein introduced a substrate patterned by a SAM in a mineral growth solution with or without the presence of the organic additive gelatin. Uniform HAp nanorods were found to form on specific sites of the substrate. In this one-pot reaction, the crystal morphology and growth site can be controlled at the same time. Additionally, we used gelatin as a structure-modifying agent to emulate the action of biomolecules on crystal growth. Different from other methods for the fabrication of complex structures, our approach provides a route for the preparation of a hierarchical structure encompassing length scales from the nano

ierarchical structures play a vital role in mineralized tissues, such as teeth, bone, and shells.1 These sophisticated structures are composite materials containing an organic matrix and nano- or microscale amorphous or crystalline minerals. It is believed that the organic matrix governs the formation of these sophisticated structures. However, the influence of the organic matrix on mineralization is poorly understood. Also the selfassembly strategy adopted by biology for constructing its exquisite structures is not yet mastered by scientists. An insoluble organic matrix may act as a scaffold to guide the mineral formation on a macroscopic scale.2−6 Additionally, soluble matrix molecules (e.g., proteins) and small molecule additives also play a role for controlling mineral polymorph and morphology.7−13 Therefore, these two factors are important for the formation of complex structures. The biological, hierarchical structures are commonly observed to be bulk architectures constructed by microscale building blocks further made of primary units (nanoparticles).14−17 To build synthetic hierarchical structures, researchers have successfully used organic additives to control the formation of primary particles and their aggregates.18−20 However, it remains challenging to organize primary units in the specific positions for the construction of large scale hierarchical structures encompassing many length scales. In human teeth, ameloblasts secrete organic matrix during the formation of enamel structure. Since the diameters of enamel prisms and ameloblasts are identical and on the order 4−6 μm,21 it has been suggested that the ameloblasts could act as organic templates to form the arrays of enamel prisms.17,22 Inspired by nature, scientists have developed several strategies to control the © 2013 American Chemical Society

Received: November 23, 2012 Revised: August 2, 2013 Published: August 13, 2013 4213

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Scheme 1. Setup for Patterning HAp on the Substratea

to the 100 μm scale. The successful fabrication and organization of uniform HAp nanorods on a substrate may be of relevance for the development of dental and other biomaterials. The HAp crystals were prepared through a hydrothermal reaction.34,35 In general, 2.5 mmol Ca(NO3)2·4H2O, 2.5 mmol H2NaPO4·H2O, and 5 mmol urea were dissolved in 250 mL of deionized water in a sealed polypropylene bottle. To fabricate HAp nanofibers, 0.25 g of porcine gelatin was dissolved in the water prior to addition of the other reagents. By heating the solution at 100 °C for 24 h, urea decomposes into NH3 and CO2, causing an increase of the solution pH. Consequently, calcium phosphate begins to precipitate homogenously in the solution. By introducing a functionalized substrate, heterogeneous nucleation can be expected to take place instead. To grow HAp crystals on specific sites of the substrate, microcontact printing (μ-CP) was incorporated into the synthesis. In this study, a PDMS stamp with concave square patterns (square size = 200 × 200 μm2) was used to make patterned carboxylate functionalized surfaces. The stamp was immersed in an ethanol solution containing 20 mM 11-mercaptoundecanoic acid for 30 min. After immersion, the stamp was rinsed by ethanol and blown by a N2 stream for 30 s. By stamping (30 s, pressing lightly by hand), the 11-mercaptoundecanoic acid was transferred onto the substrate. Finally, the PDMS stamp was removed gently from the substrate. The substrate was finally rinsed by ethanol before the crystallization experiment. Before growing minerals on the prepatterned SAMs substrate, ATR-FTIR was used to check if the modification of 11-mercaptoundecanoic acid on the substrate surface is successful or not. When the IR measurement was performed on bare Au film (data not shown), neither carbonyl signal nor C−H stretching absorption was observed. Different from the bare Au film, a weak C−H stretching absorption but no carbonyl signal was detected for the prepatterned SAMs substrate (data not shown). The lack of a clear carbonyl signal is probably because only a limited fraction of SAMs was detected on the prepatterned SAMs substrate, which was also revealed by poor S/N ratio in the IR spectra. Nevertheless, we believe that the modification of SAMs on the substrate is successful based on the observation of different IR results between bare Au film and prepatterned SAMs substrate. This is further supported by visual observation of the spreading of water droplets onto the patterned substrate: the SAM functionalized patches are preferentially wetted at the droplet/air front. To avoid nonspecific crystal growth on the substrate, the substrate was placed upside down in the aqueous solution (Scheme 1). The details of fabrication of the patterned PDMS (polydimethylsiloxane) are described in the Supporting Information. We follow the protocol35 reported previously for homogeneous growth of HAp crystals and nanorods in the solution. Without the patterned substrate and in the absence of gelatin, solution grown HAp crystals with lengths in the range of tens of micrometers were formed as shown in Figure 1A. Unlike typical needlelike HAp, these HAp crystals have a rectangular cross section resulting in a tapelike morphology. This result can be attributed to the solid-state transformation of OCP to HAp in the reaction, which causes the preservation of the shape of OCP single-crystal.34−36 By collecting samples at different reaction times, we confirmed that OCP appeared first and transformed to HAp gradually with or without the presence of gelatin (see Figure S2 in the Supporting Information). Because of the structural similarity of OCP and HAp, the solid-state transformation of OCP to HAp can be achieved by a hydrolysis reaction.34,36 During this process, the water layers in OCP lattice

a

The solution contains calcium ions, phosphates, urea with or without gelatin. The dark blue bars are support pillars used for mounting the substrate in the solution.

collapse, resulting in the appearance of some slits and splits36 in the platelike HAp crystals (Figure 1b, arrow). In the presence of gelatin, longer HAp nanorods with lengths in the range of hundreds of micrometers were produced, Figure 1c. By confocal microscopy, the gelatin epiflouresence of HAp-gelatin nanorods was observed (Figure S1 in the Supporting Information), which indicates that the gelatin binds on/in the HAp nanorods. Additionally, SAXS (small-angle X-ray scattering) experiments were performed on HAp and HAp-gelatin samples, respectively (Supporting Information Figure S3). The scattering curves are dominated by Porod-type interfacial scattering. Power law fits obtained exponents of −3.737(6) and −3.960(6) for HAp and HAp-gelatin, respectively. These values are relatively close to the values for sharp interfaces, that is, −4. The small but significant increase in exponent for the HAp-gelatin sample may result from increased sharpness of the crystal surfaces possibly originating from the action of gelatin. It has been shown that carboxylate rich polymers such as polyaspartic acids can adsorb on the surfaces of OCP.37 Therefore it is not unexpected that gelatin, which is rich in glutamic and aspartic acid, can also interact with OCP crystals. During the phase transformation from OCP to HAp, we speculate that the gelatin also adsorbs on/in OCP when it was formed. As a result of the structural similarity between OCP and HAp, the gelatin still binds on/in the HAp nanorods after the phase transformation. High magnification SEM images, Figure 1d, show that the width of the HAp nanorods is about 50−200 nm. Note that both HAp crystals and nanorods have a strong tendency to align parallel to the substrate during deposition of material for SEM because the strongly anisotropic particles align during sedimentation (Figure 1). To grow HAp crystals on a substrate by heterogeneous nucleation and growth, a prepatterned SAMs substrate was immersed in the mineral growth solution before starting the crystallization experiment. We first consider the case without added gelatin. After the reaction finished, the patterned SAM squares were completely decorated by HAp crystals (Figure 2a). The HAp crystals formed exclusively in the carboxylate functionalized regions. Many attempts have been made to grow crystals on specific sites of a substrate by introducing prepatterned SAMs substrates in the aqueous solution.23,24,28,38 In these studies, surface roughness24 and functional groups28,39 of SAMs on the surface of substrates were proposed to be the factors governing the patterned crystal growth. In our experi4214

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Figure 1. SEM images of HAp crystals synthesized in solution without (a, b) and with (c, d) gelatin. In the absence of gelatin (a, b) large plate-shaped crystals are obtained as seen from the overview in panel a and the higher magnification image in panel b, where the arrows indicate the slits and splits of the platelike crystal. In the presence of gelatin (overview in panel c), higher magnification in panel d) reduced crystal cross-sectional areas are obtained.

Figure 2. SEM images of HAp crystals grown on the prepatterned SAM substrate in the absence of gelatin (a) overview and (b) higher magnification.

ment, the SAM carboxylate groups (pKa = 5.4)40 carry negative charges, as the solution pH increases to 7.8 after the reaction, and they thus govern the formation of calcium phosphates to occur only in the SAM functionalized regions. The shape of the HAp crystals remains platelike, Figure 2b, as seen in solution growth even though the HAp crystals were grown on the SAMs. However, the size of the HAp crystals seems to become smaller (crystal width = 300 ± 170 nm, 162 crystals counted), most likely

because of the rapid depletion of reagents around the nucleation sites. Introducing gelatin into the HAp patterning experiment was found to have a profound effect on HAp morphology, size and orientation. Figure 3a displays the formed patterned HAp nanorods on the substrate. The HAp crystals were grown densely on the patterned SAMs regions of the substrate (Figure 3b). In contrast to the solution growth experiments, there is a strong tendency for the nanorod axis to be preferentially perpendicular 4215

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Figure 3. SEM images of HAp nanorods grown on the prepatterned SAM substrate in the presence of gelatin (a) overview, (b) view of a single unpatterned square, (c) top view of the HAp growth region, and (d) magnification of the edge of the patterned region.

Figure 4. XRD profiles of (a) HAp crystals fabricated without gelatin, (b) HAp nanorods prepared with gelatin, (c) HAp crystals grown without gelatin on the prepatterned SAMs substrate, and (d) HAp nanorods grown with gelatin on the prepatterned SAMs substrate. Asterisk (*) denotes the diffraction peaks from Au film on the substrate.

to the substrate (Figure 3c). From the magnified view (Figure 3d), it is found that the deposited HAp crystals (crystal width = 63 ± 35 nm, 91 crystals counted) are as thin as HAp nanorods

synthesized with gelatin in the solution. These results indicate that both mineral deposition sites, crystal morphology and orientation are controlled in this one-pot reaction. 4216

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Figure 4a and 4b shows XRD profiles of the samples prepared with and without gelatin in the absence of the substrate. The solution cast samples were drop cast onto a substrate and all measurements are done in Bragg−Brentano geometry, that is, with the substrate plane oriented horizontally. All samples are confirmed to be HAp (JCPDS 24-0033). A significant preferred orientation of the HAp crystals was detected. In the XRD profiles of the solution grown samples (Figure 4a), the high intensity ratio of (h00) to (00l) peaks led us to conclude that the solution grown HAp crystals mainly lie down on the sample holder suggesting that the plates (without gelatin, Figure 4a) and rods (with gelatin, Figure 4b) are single crystals and that the crystal long axis coincides with the crystallographic c-axis. The patterned HAp crystals on substrates were measured using grazing incidence XRD to minimize scattering interference from the substrate. Figure 4c and 4d shows the XRD profiles of the patterned samples prepared without and with gelatin, respectively. To quantify the preferred orientation of HAp crystals, XRD data of HAp powder samples and HAp patterned substrates were refined by Rietveld methods. By using March− Dollase model in the refinements, the value of “ratio”, r, in the March−Dollase formula was refined for determining the degree of crystal preferential orientation for a designated direction (hkl). In case crystals are preferentially oriented, r is smaller than one. When crystals exhibit stronger preferred orientation, r is approaching to zero. Two preferred orientation planes were included in the refinement, (100) and (002), and their contribution is weighted. The results are summarized in the Table 1 where the dominant planes have been highlighted in bold face.

The results show that the powder samples (Figure 4a and 4b) are dominated by (100) preferred orientation, in particular for the sample prepared without gelatin. In this case, the preferred orientation originates from the alignment of the needle crystals when positioned on the flat sample holder and it is natural that (100) preferred orientation dominates as the needle long axis is parallel to the c-axis. For the patterned samples (Figure 4c and 4d), the (002) plane is more oriented; especially in the gelatin case (Figure 4d). Showing that, indeed, the HAp crystals grown in the presence of gelatin on the prepatterned SAMs substrate tend to stand vertically with their c-axes perpendicular to the substrate in comparison to the powder sample. This is further confirmed using SEM cross sectional view of the patterned HAp nanorods (Figure 5), indeed, most HAp nanorods are found to stand on the SAMs modified substrate. In the absence of gelatin (Figure 4c), HAp crystals patterned on the substrate also have the tendency with their c-axes aligned vertically to the substrate in comparison to the powder sample. However, the degree of the vertical alignments of HAp crystals with their c-axes perpendicular to the substrate is less obvious than the patterned HAp synthesized in the presence of gelatin. This difference is probably because the long HAp-gelatin nanorods were growing so densely in a limited area that they were forced to support each other to keep standing vertically on the substrate. On the other hand, the growing density of HAp platelike crystals on the substrate is smaller so the self-support effect of crystals is less apparent. Of course, we cannot exclude that the dehydration process may help the vertical alignments of HAp crystals patterned on the substrates. However, this factor alone cannot explain the different degrees of preferential orientation observed for HAp patterned substrates prepared with or without the presence of gelatin, respectively. To confirm the single crystal nature and the identity of the crystal planes of the crystals, TEM and selective area electron diffraction (SAED) were used to analyze crystal and nanorods obtained with the patterned substrates. For the HAp crystals prepared without gelatin, the aforementioned split ending of the platelike HAp crystal was confirmed (arrow, Figure 6a). By SAED analysis, the single crystal nature of the platelike crystal is confirmed. The long axis of the HAp single crystal is parallel to the crystallographic c-axis and the (100) plane is the largest exposed plane of the platelike crystal (Figure 6b). The analysis was also performed on the HAp nanorod fabricated with gelatin.

Table 1. Results of Rieveld Refinements of HAp Samplesa (100) plane

(002) plane

HAp sample

χ2

wRp (%)

ratio

fraction

ratio

fraction

powder, no gelatin patterned, no gelatin powder, gelatin patterned, gelatin

1.132 2.942

11.4 4.94

0.48 0.71

0.98 0.54

5.61 0.92

0.02 0.46

8.41 5.49

0.34 1.51

0.47 0.01

3.00 0.72

0.53 0.99

11.66 5.36

a Two preferred orientation planes were included in the refinement, (100) and (002), and their contribution is weighted. The dominant planes are highlighted in bold face.

Figure 5. SEM side view of HAp nanorods (a) overview, (b) higher magnification, and (c) magnification of the HAp nanorods at the edge of the prepatterned SAMs region. 4217

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Figure 6. TEM images and their corresponding SAED results of a HAp crystal grown on the prepatterned SAM scratched from the substrate are shown in (a, b) without gelatin and (c, d) with gelatin. Arrow points the split of the platelike crystal.

scale as reorganization of particles with sizes ranging from nanoto micrometers may be problematic over large length scales. In our approach, minerals are nucleated in the carboxylate functionalized regions of preorganized SAMs and in the meanwhile gelatin is acting to regulate the crystal morphology of HAp. Consequently, micropatterned HAp nanorods are formed on a substrate. The nanorods are highly aligned with the c-axis perpendicular to the substrate. The structures are thus reminiscent of the bundles of very long HAp needles observed in enamel even though the present method does not allow for the perpendicular interweaving of such bundles on an even larger length scale as is done in enamel. To the best of our knowledge, the present work is the first to pattern the morphological controlled HAP crystals on a substrate. In conclusion, we have developed a method to grow aligned HAp crystals on specific sites on a substrate. By using the microcontact printing technique, a preorganized SAMs substrate was made to control crystal deposition in the micrometer-scale domains. For manipulating crystal morphology, gelatin was added in the reaction to produce aligned uniform HAp nanorods with width of 50−200 nm. By combining these two approaches, micropatterned aligned HAp nanorods on a substrate were constructed, which demonstrates structural hierarchies from

According to TEM and SAED analyses (Figure 6c and 6d), the nanorod growing direction is also in parallel to the c-axis of HAp crystal, while the dimension of the crystal along [110] is largely decreased, which implies the effect of gelatin on the crystal growth. 35 This suggestion is further supported by the observation of gelatin epiflouresence of HAp-gelatin nanorods in confocal microscopy images (Figure S1 in the Supporting Information), which indicates the binding of gelatin on/in the HAp nanorods. Many efforts have been made to control mineral deposition by using preorganized SAMs substrates in a solution.23,24,28,38 However only few studies38 show that the crystal morphology, modified by organic additives can be preserved during the patterning process. Biomineral structures display several levels of hierarchy from nano- to micrometer-scale.14,17,41 To learn how the hierarchical structures are built in biology, scientists have used organic additives (e.g., polymers)20,42−44 to mimic the roles of proteins for producing primary units of high-level structures. In some cases, primary crystalline or amorphorous units are able to self-assemble into complex structures (e.g., mesostructures)20,43,45 depending on the properties of organic additives used in the reaction. However, it is difficult to organize the asobtained primary units or complex structures on the micrometer4218

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(19) Oaki, Y.; Imai, H. Adv. Funct. Mater. 2005, 15, 1407. (20) Song, R.-Q.; Coelfen, H. Adv. Mater. 2010, 22, 1301. (21) Martin, L. B.; Boyde, A.; Grine, F. E. Scanning Microsc. 1988, 2, 1503. (22) White, S. N.; Luo, W.; Paine, M. L.; Fong, H.; Sarikaya, M.; Snead, M. L. J. Dent. Res. 2001, 80, 321. (23) Zhu, J. H.; Song, J. M.; Yu, S. H.; Zhang, W. Q.; Shi, J. X. CrystEngComm 2009, 11, 539. (24) Kim, Y. Y.; Douglas, E. P.; Gower, L. B. Langmuir 2007, 23, 4862. (25) Urch, H.; Franzka, S.; Dahlhaus, D.; Hartmann, N.; Hasselbrink, E.; Epple, M. J. Mater. Chem. 2006, 16, 1798. (26) Briseno, A. L.; Aizenberg, J.; Han, Y. J.; Penkala, R. A.; Moon, H.; Lovinger, A. J.; Kloc, C.; Bao, Z. A. J. Am. Chem. Soc. 2005, 127, 12164. (27) Lu, C. H.; Qi, L. M.; Ma, J. M.; Cheng, H. M.; Zhang, M. F.; Cao, W. X. Langmuir 2004, 20, 7378. (28) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495. (29) Tan, J.; Saltzman, W. M. Biomaterials 2004, 25, 3593. (30) Hayakawa, S.; Li, Y.; Tsuru, K.; Osaka, A.; Fujii, E.; Kawabata, K. Acta Biomater. 2009, 5, 2152. (31) Lu, Z. Z.; Xu, H. Y.; Xin, M. D.; Li, K. W.; Wang, H. J. Phys. Chem. C 2010, 114, 820. (32) Wei, X.; Fu, C.; Savino, K.; Yates, M. Z. Cryst. Growth Des. 2012, 12, 3474. (33) Xu, Y.; Ma, G.; Wang, X.; Wang, M. Cryst. Growth Des. 2012, 12, 3362. (34) Tseng, Y. H.; Mou, C. Y.; Chan, J. C. C. J. Am. Chem. Soc. 2006, 128, 6909. (35) Zhan, J. H.; Tseng, Y. H.; Chan, J. C. C.; Mou, C. Y. Adv. Funct. Mater. 2005, 15, 2005. (36) Iijima, M.; Kamemizu, H.; Wakamatsu, N.; Goto, T.; Doi, Y.; Moriwaki, Y. J. Cryst. Growth 1997, 181, 70. (37) Bigi, A.; Bracci, B.; Panzavolta, S.; Iliescu, M.; Plouet-Richard, M.; Werckmann, J.; Cam, D. Cryst. Growth Des. 2004, 4, 141. (38) Tseng, Y. H.; Liu, M. H.; Kuo, Y. W.; Chen, P. L.; Chen, C. T.; Chen, Y. F.; Mou, C. Y. Chem. Commun. 2012, 48, 3215. (39) Aizenberg, J.; Black, A. J.; Whitesides, G. H. J. Am. Chem. Soc. 1999, 121, 4500. (40) Smalley, J. F.; Chalfant, K.; Feldberg, S. W.; Nahir, T. M.; Bowden, E. F. J. Phys. Chem. B 1999, 103, 1676. (41) Oaki, Y.; Kotachi, A.; Miura, T.; Imai, H. Adv. Funct. Mater. 2006, 16, 1633. (42) Yu, S. H.; Cölfen, H.; Tauer, K.; Antonietti, M. Nat. Mater. 2005, 4, 51. (43) Xu, A.-W.; Antonietti, M.; Yu, S.-H.; Coelfen, H. Adv. Mater. 2008, 20, 1333. (44) Wang, T.; Antonietti, M.; Cölfen, H. Chem.Eur. J. 2006, 12, 5722. (45) Tseng, Y. H.; Lin, H. Y.; Liu, M. H.; Chen, Y. F.; Mou, C. Y. J. Phys. Chem. C 2009, 113, 18053.

nano- to micrometer-scale. The successful fabrication of these uniform HAp nanorods on the micrometer-scale shows promise for applications in dental materials.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details of the fabrication of PDMS stamp and the growth of HAp crystals on the prepatterned substrate, confocal images of gelatin patterned on a glass slide and the HAp-gelatin composite sample (Figure S1), XRD data of samples synthesized at different times with and without gelatin (Figure S2), and SAXS data of HAp and HAp-gelatin powder samples (Figure S3). This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +45 871 55936. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Jan Skov Pedersen for kind help with the SAXS measurements and for access to SAXS instrumentation. Funding from the Human Frontiers Science Program (HFSP, grant RGP0004/2010) is gratefully acknowledged.

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REFERENCES

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