DOI: 10.1021/cg1009303
Silk Fibroin Hydrogels Coupled with the n16N-β-Chitin Complex: An in Vitro Organic Matrix for Controlling Calcium Carbonate Mineralization
2010, Vol. 10 5169–5175
Ellen C. Keene,† John S. Evans,‡ and Lara A. Estroff*,† †
Department of Materials Science and Engineering, Cornell University, Ithaca, New York, United States, and ‡Laboratory for Chemical Physics, New York University, New York, New York, United States Received July 14, 2010; Revised Manuscript Received October 19, 2010
ABSTRACT: Previous results have shown that the nacre specific peptide, n16N, from the Japanese pearl oyster Pinctada fucata has a binding affinity for β-chitin. As a result, the n16N-chitin assembly is able to selectivity nucleate aragonite. Here, we have added silk fibroin hydrogels to the in vitro assay to more fully represent the in vivo matrix. Crystallization, with a silk fibroin hydrogel and n16N on β-chitin, results in metastable vaterite and amorphous calcium carbonate, which form as flat deposits with hemispherical centers. Acidic peptide controls (p-Asp/p-Glu) were also tested in the silk-chitin assay and result in flat calcite that grows into the β-chitin substrate. Fluorescence imaging of that matrix, made with labeled n16N, shows that n16N binds to β-chitin in the presence of silk gel. These results demonstrate that the addition of a silk hydrogel to the n16N-β-chitin assembly changes the microenvironment for mineralization. This work contributes to our understanding of the roles of individual nacre matrix components (and their assemblies) in controlling crystal growth. Introduction Mollusk shells are hierarchical nanocomposites of minerals grown within an organic matrix. Several species of mollusk bivalves have shells with a bilayer structure composed of two different polymorphs of calcium carbonate: the outer calcitic prismatic layer and the inner nacreous aragonite layer. Nacre, or mother-of-pearl, has received a lot of attention due to its polymorphic control,1,2 exceptional toughness,3-6 and bioactivity (i.e., as bone implants to encourage bone regeneration).7-9 The nacre organic matrix is composed of three major components: β-chitin, a silk fibroin-like protein hydrogel, and an assembly of acidic proteins.2,10 Utilizing this organic matrix, the mollusk is able to control, simultaneously, crystal polymorph, morphology, and orientation. Studying the effects on mineralization of individual components from the nacre organic matrix can miss synergistic effects at play in the matrix. Systematically studying combinations of multiple components, as presented here, can lead to a better understanding of the role of the macromolecular assemblies in biomineral formation, as well as lead to the design of new materials. The peptide n16N, from the Japanese pearl oyster Pinctada fucata, is a 30 amino acid, intrinsically disordered peptide that can fold upon interaction with a target (Table 1).11,12 Previous studies have shown that n16N interacts with calcium carbonate13,14 as well as β-chitin,15 and is able to promote the formation of lamellar aragonite.16 The parent protein, n16, is believed to participate in the formation of a nacre protein complex that is involved in regulating polymorph control via aragonite and chitin binding motifs,17 and, in the presence of magnesium, has been shown to induce the formation of platy aragonite.18 Previously, we developed a synthetic, organic matrix by combining n16N and chitin (R and β) and found that only the combination of n16N with β-chitin resulted in aragonite formation.15 Here, we increase the complexity of
Table 1. Nacre n16 Polypeptide Sequences
a For clarity, cationic amino acid residues are highlighted in blue, anionic residues are highlighted in red, and cysteine residues are highlighted in green.
*Corresponding author. Address: 214 Bard Hall, Ithaca, NY 14853. Phone: 607-254-5256. Fax: 607-255-2365. E-mail:
[email protected].
our assay with the addition of another organic matrix component, a silk fibroin hydrogel, to the n16N-β-chitin system. In particular, we are interested in identifying how silk fibroin hydrogel interacts with the other components of the matrix and influences crystal growth. Cryo-TEM and environmental SEM studies of nacre have suggested that the silk-like protein is a weakly ordered β-sheet, hydrated gel.10,19 While the exact role of this hydrogel in nacre growth is unknown, possible roles have been speculated.19 For example, diffusion rates, ion activities, and water “structure” all differ in a hydrogel as compared to solution. Hydrogels can act as site directing agents by suppressing crystallization and, therefore, preventing uncontrolled crystallization until nutrients are in contact with the nucleating site or with already formed mineral.20 While many different hydrogels have been used to study crystal growth,20 here we focus solely on a silk fibroin hydrogel since it has been identified in nacre and may have interactions with the other matrix components, such as n16N and β-chitin. Previous in vitro studies of nacre components, including the seminal work of Falini et al.2 and others,10,21-23 have not used silk gels but rather films or solutions.24 Nevertheless, Falini demonstrated that silk is a critical component for obtaining the aragonite polymorph, as well as the in-growth of crystals into β-chitin.2,25 Here, we demonstrate that with
r 2010 American Chemical Society
Published on Web 11/10/2010
pubs.acs.org/crystal
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Keene et al.
Table 2. Summary of the Mineralization Experiments and the Resulting Crystal Polymorph and Morphology a
peptideb
gel
substrate
polymorph
β-chitin glass β-chitin glass β-chitin β-chitin
calcite calcite aragonite calcite calcite ACC/vaterite
silk hydrogel n16N (100 μM)
β-chitin
ACC/vaterite
silk hydrogel n16N (5 wt %) silk hydrogel n16NN
β-chitin
calcite
β-chitin
ACC/vaterite
none none none silk hydrogel silk hydrogel silk hydrogel
silk hydrogel silk hydrogel silk hydrogel silk hydrogel silk hydrogel none
none n16N n16N none none n16N
morphology rhombohedra modified rhombohedra poly crystalline poly crystalline poly crystalline flat region with rounded center flat region with rounded center poly crystalline
flat region with rounded center n16Ns β-chitin ACC/vaterite flat region with rounded center n16N none (bulk) calcite þ vaterite rhombohedra (C) þ rosettes (V) p-Asp (0.25 mg/mL) β-chitin calcite flat p-Glu (0.25 mg/mL) β-chitin calcite flat BSA β-chitin calcite poly crystalline n16N β-chitin with adsorbed calcite poly crystalline silk fibroin
peptide bindingc,d
location of results
N.D. N.D. strong fluorescence N.D. none strong fluorescence
ref 15 ref 15 ref 15 Figure S2 Figure 1B Figure 1A
N.D.
Figure 1C,D
N.D.
Figure S1
strong fluorescence
Figure 6A
strong fluorescence
Figure 6B
N.D.
Figure S3
N.D. N.D. moderate fluorescence strong fluorescence
Figure 6D Figure 6C Figure S11 Figure S9A
a Unless otherwise specified, gel concentrations are 2.5 wt %. b Unless otherwise specified, peptide concentrations are 10 μM. c Peptide binding was assessed by fluorescent labeling studies with a BODIPY maleimide dye. Fluorescence levels are described by pixel brightness levels: strong >200, moderate 100-200, and weak 99% purity) overnight59 and subsequently filtered using a standard funnel filter with Whatman filter paper. The silk solution (30 mL) was finally dialyzed (Slide-a-Lyzer dialysis cassettes, Pierce, MWCO 3,500) against DI water (3 L) for 3 days changing the water twice daily.59 The resulting silk solutions are approximately 5 wt %, which was determined by weighing the remaining solid after air drying. Fresh solutions of silk fibroin are random coil (as determined by CD spectroscopy; see below). Silk hydrogels were formed by placing silk solutions (2.5 wt %) in a 60 °C hot water bath in capped glass test tubes.59 A silk solution is a gel when it has increased its viscosity (silk does not fall after 30 s in an inverted vial) and has a β-sheet structure. All silk hydrogels were formed and cooled to room temperature prior to addition of any poly peptides (n16N, variants, or p-Asp/p-Glu). Protein secondary structure was verified by a model 400 Aviv circular dichroism (CD) spectrometer (data not shown). Chitin Purification/Preparation. Squid pen (β-chitin), from the Loligo species, was purified by refluxing the pen in 1 M sodium hydroxide solution for 3 days, changing the NaOH purifying solution daily.2,25,60 The polysaccharide was then extensively rinsed in DI water, air-dried, and stored dry until use. Chitin pieces were rehydrated in 10 mM calcium chloride (CaCl2 dihydrate, Sigma Aldrich, >99% purity) for a minimum of 2 h. Crystallization Experiments. Crystallization experiments were carried out via the vapor diffusion method (as previously described15) for 24 h. Shorter crystallization times were also tested (4 and 6 h). A substrate (β-chitin or glass coverslip) was put in the bottom of three wells of a 24-well plate. Silk hydrogels (cooled to room temperature) were mixed with a poly peptide and CaCl2 (10 mM, dihydrate, Sigma Aldrich) and then pipetted on top of the prepared substrate in the bottom of each well. After crystallization, the substrates were removed and rinsed with DI water prior to characterization. Bulk gel-grown crystals (nonsubstrate nucleated crystals) were isolated by mixing bleach (sodium hypochlorite, Fisher) with the silk gel. The bleach-silk mixture was then centrifuged (Eppendorf Centrifuge 5415C, 5 min, 8000g), the supernatant was removed, and the precipitate was rinsed with bleach followed by DI water. After the last centrifugation, crystals were resuspended in +ethanol and the ethanol-crystal mixture was placed on glass coverslips and air-dried before characterization. All crystallization experiments were done in triplicate. Etching/Dissolution Experiments. The mineral deposits were etched in DI water for 20 h. During this time, the samples were gently agitated on a rocking table. After etching, the samples were air-dried and characterized. To selectively etch ACC, deposits were etched with 1 M KOH for 8 h.29 Morphology and Polymorph Analysis. The morphology of grown crystals was examined via polarized light microscopy (Leica) and via scanning electron microscopy (SEM, Leica Stereoscan 440, 15 kV, 900 pA) after they were coated with a thin layer of Au/Pd. Elemental composition was determined by energy dispersive X-ray analysis (EDX, detector: Kevex; analyzer and software: Evex). The polymorph of the grown crystals was determined via Raman (Renishaw InVia micro-Raman system, 785 nm excitation frequency) and via X-ray diffraction (Bruker D8 Diffractometer with a HI-STAR area detector), transmission mode, 40 mA, 40 kV). Chitin X-ray structural parameters were assigned based upon literature values.25,61,62 Adsorption Experiments. Similar to our previous work,15 chitin substrates were incubated with solutions of 5 μM n16N plus 2.5% β-sheet silk fibroin hydrogel on a rocking table at room temperature
Keene et al. for 24 h to allow the peptide/protein time to adsorb onto the chitin substrate. Chitin samples that were incubated with silk hydrogels and peptide separately were rinsed with DI water only between incubations. After 24 h, substrates were washed with DI water, saline solution (0.2 M NaCl), buffer (10 mM Tris, pH 7.2), and finally DI water again to remove any unbound protein. Substrates were immediately used for crystal growth or fluorescence experiments. Fluorescence Experiments. Substrates with adsorbed peptide were reacted with BODIPY FL N-(2-aminoethyl) maleimide (Invitrogen) according to manufacturer’s instructions with the modifications described in our previous work.15 Specimens were imaged via fluorescence microscopy (Olympus BX51 equipped with a Roper Cool Snap CCD Camera, 100 ms exposure time) using the Image Pro imaging software package. A mercury lamp with a “green” filter (λex = 460-500 nm, λem = 510-560 nm, dichroic filter = 505 nm) was used for fluorescence imaging. A neutral density filter was used to reduce the intensity of the mercury lamp by 25%. All samples were exposed to identical exposure times (100 ms) and microscope settings to validate comparison across samples. All images were recorded in grayscale (12 bit image, capture area 1394 1040 pixels, gain of 1, and 1 1 binning) and were scaled to the same intensity values using the Image Pro imaging software package. All images were processed in ImageJ (histograms).
Acknowledgment. L.A.E. acknowledges funding support from the J. D. Watson Investigator Program (NYSTAR contract # C050017) and Cornell’s Center for Materials Research (CCMR), a National Science Foundation Materials Research Science and Engineering Center (MRSEC) program (DMR 0520404). Particular acknowledgement is made of the use of the Electron and Optical Microscopy, X-ray and Diffraction Analysis, and Surface Analysis Characterization facilities of the CCMR. This work also made use of shared facilities at Cornell’s Nanobiotechnology Center (NBTC) with support from the National Science Foundation STC Program (ECS-9876771). J.S.E. acknowledges support from the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-FG02-03ER46099. Portions of this work represent Contribution 57 from the Laboratory for Chemical Physics, New York University. And special thanks to Hanying Li for the squid pen and Martha Estroff for the silkworm cocoons. Supporting Information Available: Additional SEM and optical micrographs as well as Raman spectra and fluorescence histograms (Figures S1-S11). This material is available free of charge via the Internet http://pubs.acs.org.
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