Osteopontin Stabilizes Metastable States Prior to ... - ACS Publications

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Osteopontin Stabilizes Metastable States Prior to Nucleation during Apatite Formation Casper Jon Steenberg Ibsen,† Denis Gebauer,‡ and Henrik Birkedal*,† †

iNANO and Department of Chemistry, Aarhus University, 14 Gustav Wieds Vej, 8000 Aarhus, Denmark Department of Chemistry, University of Konstanz, Universitätsstr. 10, Box 714, D-78457 Konstanz, Germany

‡

S Supporting Information *

ABSTRACT: Osteopontin, which is a phosphoprotein with strong ties to in vivo bone mineralization, is shown to change the precipitation pathway of calcium phosphate. We show that the presence of the phosphoprotein, even in minute concentrations, can stabilize an otherwise oversaturated mixture against precipitation. At moderate concentrations, we find that the protein introduces a new intermediate state into the reaction pathway leading to apatite formation. This new intermediate was found to share many characteristics of a coacervate or polymer-induced liquid-like precursor (PILP) phase. Our results show that these types of complex phases should be considered when discussing the mechanisms of bone mineralization on a subcellular level.

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INTRODUCTION In the past decade, the field of crystallization has experienced rejuvenation. Numerous experimental observations have surfaced that cannot be satisfactorily explained within the framework of the classical theories.1−12 The mounting evidence have changed our perception of crystal formation from strictly adhering to the classical concept of crystallization by monomer addition to now recognizing more complex pathways, collectively crystallization by particle attachment.3 One such instance is the observation of prenucleation clusters, liquid-like nanoscale hydrated clusters, as a prelude to the formation of amorphous calcium carbonate.6,7,13 In biomineralization, the pathways to crystallization are often highly complex and many organisms make use of amorphous precursor strategies to shape their complex microstructures and macrostructures.14,15 One of the more important biominerals in this respect is apatite, which is found in mammalian bone and teeth. Although there is proof that apatite biomineralization may traverse an amorphous precursor pathway,16,17 the exact steps beyond that are still debated in the literature, markedly whether or not it also involves a crystalline octacalcium phosphate intermediate.18 Prenucleation species have also been implicated in apatite formation,4 although it has been suggested that, in this system, they may be ion association complexes.19 However, because of the drastic changes in speciation of phosphate with pH caused by its acid/base equilibria, the prenucleation behavior can be expected to be highly pHdependent. Mineralization in biological systems is further influenced by the presence of a multitude of specialized proteins and small molecules. A prominent emerging concept is that of the polymer-induced liquid precursor (PILP).20,21 This concept is © 2016 American Chemical Society

based on charged macro-anions being able to form liquid coacervate-like but metastable phases with mineralization solutions, which have favorable properties, with respect to controlled mineralization. These include improved wetting and infiltration abilities, combined with high saturation of the ions used to mineralize the tissue.14,21 Similar effects have been observed previously in assemblies of charged nanoparticles with polyelectrolytes22 and for polyelectrolytes in combination with small molecule polyions.23,24 Herein, we investigate the early stages of the path leading to apatite formation at pH 8, using the constant pH titration approach of Gebauer et al.6,7,13,25 We investigate the influence of a biomineralization-associated protein, namely, osteopontin (OPN). This protein is one of a family of phosphorylated sialoproteins associated with the extracellular organic matrix of mammalian bone and teeth. It is thought to be actively involved in the bone mineralization process,26−29 because of its preferential accumulation along growth fronts28 and at healing interfaces.30,31 The protein contains a large amount of aspartic acid, glutamic acid, and post-phosphorylated serine residues, making it highly negatively charged.29 This makes it ideal for binding to Ca ions or apatite surfaces. OPN is found in many other tissues, including milk, which is the source of the OPN used in this study. OPN has been shown to have an inhibitive effect on apatite formation, while some related proteins of the family are mineralization promoters.26,28,32 The degree of OPN phosphorylation varies with site in the body and is important for the degree to which the protein impacts mineralization.32 Received: August 1, 2016 Revised: November 11, 2016 Published: November 14, 2016 8550

DOI: 10.1021/acs.chemmater.6b01088 Chem. Mater. 2016, 28, 8550−8555

Article

Chemistry of Materials

consuming free ions. This is consistent with the formation of a transient amorphous calcium phosphate phase, which, shortly afterward, transforms to a less-soluble and more-stable apatite phase. When OPN is introduced into the reaction mixture, the behavior of the system changes drastically. Where the first stage was terminated by a sharp decrease in the measured amount of free calcium in the additive-free experiment, instead, an abrupt change of slope is observed, leading into a new OPN-induced metastable plateau (labeled MP on figure). The duration of this segment or the capacity for assimilating calcium is directly proportional to the protein concentration (see the Supporting Information). Furthermore, the time between the first and the second drop in free calcium is greatly extended, indicating that the protein is capable of stabilizing the intermediate. To gain insight into the nature of this new transient state induced by the protein, aliquots of the reaction mixture were subjected to analytical ultracentrifugation (for details, see the Experimental Section in the Supporting Information). Since detection was performed using UV absorption, we do not directly measure all calcium phosphate entities in solution but only those with bound protein. Figure 2 shows the AUC data

Lenton et al. very recently used high concentrations of OPN to stabilize amorphous calcium phosphate at pH 7.0.33 In other recent work, OPN has been used by Rodrigues et al. as a process direction agent in PILP-like mineralization of collagen sponges at pH 7.4.34 However, they did not investigate the reaction pathway, which is the focus of the present work.

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RESULTS AND DISCUSSION We investigated the crystallization behavior leading to apatite at pH 8.00 by slowly titrating dilute calcium chloride into a volume of phosphate buffer (pH 8.00) while monitoring the activity of calcium in the solution and keeping the pH constant (see the Supporting Information for detailed description).This was done for a series of buffer solutions each with different amounts of OPN added, giving a set of curves that depict the measured amount of free calcium as a function of added calcium, as shown in Figure 1. The additive free experiment

Figure 1. Titration data showing the measured free calcium as a function of added calcium. Note that there is a small offset in free calcium when adding OPN, because of the presence of small amounts of calcium in the OPN sample. The additive free system displays three distinct features. First, a prenucleation stage, where the measured amount of free calcium increases linearly with added calcium. This is terminated by two consecutive decreases in the measured amount of free calcium as solids precipitate from the supersaturated solution. OPN acts on this latter part, by introducing a new segment to the titration curve, labeled MP, and by stabilizing the first intermediate in a dose-dependent manner.

Figure 2. AUC data recorded for titration with 0.30 mg/mL OPN added. Only species bound to the protein are detected. Inset marks when the 300 μL samples were drawn from the reaction volume. The sedimentation coefficient is given in units of Svedberg (S) and is proportional to the mass of whatever is sedimenting. Therefore, the data can be interpreted as a distribution of sizes, assuming that all detected species have similar densities. Prior to the MP stage (grays and black), only one signal is observed, corresponding to the monomeric protein. After the transition, multiple absorptions appear at higher sedimentation coefficients, which is consistent with larger species forming in solution. The magnitude of these absorptions increases with the amount of added calcium.

(gray line) displays three distinct features. Initially, the measured amount of free calcium increases approximately linearly with the amount of added calcium. However, the measured amount of free calcium does not coincide with the amount of calcium that has been added (black line). Instead, there is a growing deficit of calcium. In the work by Gebauer et al. on calcium carbonate, a similar behavior was observed, which was concluded to be due to the formation of stable prenucleation clusters in solution, based on complementary analytical ultracentrifugation data.7 In the calcium phosphate system, some indications of cluster formation prior to nucleation of a solid phase have been reported by Dey et al.,1 although is not unequivocally clear if they qualify as prenucleation clusters.4−6 The linear trend continues until the solution reaches a certain level of supersaturation, culminating in two consecutive decreases in the measured amount of free calcium. These events are caused by the precipitation of solids from the supersaturated solution,

obtained from a titration experiment with 0.30 mg/mL OPN. The insert indicates when the samples were drawn. The first three samples (black and grays) were all drawn prior to the transition into the MP- stage. They all featured a single absorption centered at ∼2 S corresponding to the native protein. The absence of other absorptions means that the protein formed only monomers in solution. After the transition to the MP stage, additional absorptions from larger species with bound protein were also detected. Based on the magnitude of the absorptions, these species appear to increase in number as the titration continued. Furthermore, the absorptions span a broad range of sedimentation coefficients meaning the distribution of sizes (or densities) is far from uniform. Considering that the transition to the MP stage occurred via a change of slope instead of a sudden drop in free calcium, we believe that this signals the formation of droplets of a PILP-like 8551


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Figure 3. (A) Fourier transform infrared (FTIR) spectra of samples isolated from titration solutions by preparative ultracentrifugation. The approximate time points are indicated in panel (B). Notably, this is merely a representative titration curve, because the sample preparation consumed the entire reaction volume. (C) Magnified view of the ν3(PO4) domain. The absorption profiles of both intermediates are similar to ACP (black line, synthetic ACP control) and differ significantly from apatite (orange line). (D) Synchrotron powder diffractogram of material isolated during the MP stage. Other than some minor impurities (marked by an asterisk (*)), the data are consistent with an amorphous calcium phosphate phase.

(MP) and E and F prior to that (i.e., in the prenucleation stage). The MP stage featured 20 nm spherical particles interconnected into large agglomerates, which scattered like amorphous solids. The number of these agglomerates increased after the first decrease in the amount of free calcium. The sample also contained a multitude of smaller spherical particles (4−6 nm in diameter) found mostly as patches of chainlike networks. Both types of particles were also found prior to the transition to the MP stage, although in smaller quantities, likely as a result of drying. Regardless, the micrographs did not contribute conclusive evidence toward neither the presence nor the absence of prenucleation clusters in the calcium phosphate system. To further probe the formation of entities in solution during titration, we applied pseudo in situ dynamic light scattering (DLS), as described in the Experimental Section in the Supporting Information. The results of this experiment are summarized in Figure 4. The concentration index (Figure 4B) is a number that is representative of the concentration of scatterers. For the first 2 h, this number is effectively zero, meaning that either no scatterers are present or the concentration is below the detection limit. Once the titration crossed the 2 h mark, the concentration index started to increase. This coincides roughly with the expected time of the crossover to the MP stage, which, in this particular experiment, is drowned out the increased noise level (Figure 4A). The rate at which the loading index increased was roughly constant, accelerating only when approaching the second decrease in the amount of free calcium. The accompanying average hydrodynamic radii of the scatterers are shown in Figure 4C. For some measurements, the distributions of hydrodynamic radii were bimodal, in which case more than one average radius was determined. To illustrate this, the symbol sizes are weighted by the volume fraction of each characteristic size within the individual measurements, i.e., small dots correspond to minute fractions. The symbol color, gray scale, is a representative of corresponding concentration index, with dark colors representing high numbers. For the first 2 h, the data are dominated by noise, because the concentration of scatterers is too low to provide a reasonable signal. This changes once the titration enters the MP stage, throughout which we observe a single stable size distribution of scatterers, hydrodynamic diameter

intermediate, rather than the precipitation of a second type of amorphous solid.20 The transient stages were investigated in greater detail by isolating material through preparative ultracentrifugation. In the undisturbed state, prior to any rinsing, the material formed in the MP stage was dense, gel-like, and highly transparent. These traits are characteristic of the PILP phenomenon, which, in many ways, is reminiscent of coacervates known from polymer science35,36 albeit the obtained phases are only metastable. Fourier transform infrared (FTIR) spectra of this sample (MP, red) and a sample isolated after the first nucleation event (I, brown) are compared in Figure 3A with that of synthetic ACP (black) and the end product from an additive-free titration (apatite, orange). The approximate time points where titrations were stopped are shown in Figure 3B. The ν3(PO4) domain (magnified views, Figure 3C) of both isolated intermediates are consistent with ACP, although with a larger full width at half-maximum (fwhm) than the synthetic ACP, shown as comparison. This broadening is likely due to overlapping absorptions from bound protein or it could be a size effect. The presence of protein in these samples is evident from the C−H backbone absorptions at 3000 cm−1, as well as several bands of intermediate strength between 1550 cm−1 and 1300 cm−1. Both also feature significant contributions from water (1640 cm−1 and 3260 cm−1, stretch and bend respectively) which must be structural, considering the rinsing process employed prior to the FTIR experiment. The precipitate isolated from the MP stage was also subjected to X-ray investigations at a beamline i711 (Maxlab, Sweden). As seen in Figure 3D, the sample scattered like an amorphous material. The small sharp spikes, denoted by stars in the figure, are remnants of NaCl salt. Clearly, the intermediates are noncrystalline in nature. These results do not discount the possibility of the OPN-induced transient phase being PILP-like while in solution since compression and dehydration could easily accelerate the transition into an amorphous solid. Complementary to the FTIR investigations, aliquots of reactant solution drawn at different time points were imaged using TEM. The micrographs are shown on Figure S2 in the Supporting Information, with A−D originating from the plateau 8552


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volume. While these results do not contradict the observations from TEM, the reverse is not necessarily true. We cannot, with confidence, conclude that the TEM images are representative of the solvated state, because of the drying involved in the TEM sample preparation. After the first nucleation event, the size of scatterers in solution starts to slowly increase, picking up speed as the titration reaches the second nucleation event. The very distinct difference in behavior, going from the MP stage across the first nucleation event, further solidifies that the two intermediates differ greatly in nature. The observed size being constant in the MP stage is consistent with a liquid−liquid decomposition, forming droplets of PILP-like or pseudocoacervate material, whereas the continuous growth following the termination of the MP stage is more akin to the agglomeration or coarsening effects of classical uncoated particles in solution. Taken together, our results can be summarized as shown in Figure 5. At low Ca concentration, prenucleation clusters form with no significant influence of OPN (e.g., in the form of coordination complexes). At a critical Ca concentration, the liquid−liquid phase separation results in a metastable PILP/ coacervate-like phase whose temporal stability increases with OPN concentration. These then precipitate to form ACP, followed by a phase transformation to apatite. It should be stressed that these results will be dependent on the initial solution speciation of phosphate and OPN and, hence, on pH.

Figure 4. Results from pseudo in situ light scattering experiment. A protein concentration of 0.10 mg/mL was used: (A) titration curve (this dataset suffered from enhanced noise, but the trends were sound and consistent with all prior results), (B) concentration index as a function of time (this is a measure of the amount of scatterers giving rise to the DLS signal; for each time point, multiple repeat measurements were made), and (C) average hydrodynamic diameter, as a function of time, as given by the DLS software. Some measurements featured more than one size distribution. To represent this, the size of the symbols on (C) were weighted by the volume fraction of particles giving rise to the signal (i.e., a small symbol means that it is likely erroneous measurements from a few dust particles or similar). Similarly, the symbol color represents the corresponding concentration index, with dark colors representing large numbers of scatterers and light color representing small numbers of scatterers. Prior to the 2 h mark, the concentration index is essentially zero and the size data are not reliably determined.

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CONCLUSIONS In this work, we have used a constant-pH titration approach to show how a milk-based phosphoprotein interacted with forming calcium phosphate. We saw that the protein had the unique property of introducing a new intermediate into the crystallization pathway. We believe this new intermediate is the formation of a PILP-like phase34 and discussed the parallels with coacervate phases, which are known from polymer chemistry. The use of pseudo-coacervate or PILP-like phases is a very neat way for biology to control the biomineralization process locally; it is semifluid but, at the same time, has a very

averaging ∼45−50 nm, with occasional sightings of larger entities, possibly agglomerates, drifting into the measured

Figure 5. Model showing the action of osteopontin on the crystallization pathway of apatite. In Segment 1, the prenucleation stage, some calcium is bound in some type of prenucleation state. This stage was unaffected by the protein. In Segment 2, the OPN-induced intermediate, a PILP-like phase forms through liquid−liquid decomposition; this segment was characterized by a single size distribution of scatterers in DLS, increasing in concentration over time. Segment 3 involves precipitation of solid ACP. This segment was initiated by a steep decrease in the amount of free calcium. The protein acted by stabilizing the amorphous phase against phase transformation. From DLS, the concentration of scatterers continued to increase, accompanied also by a continuous increase in the size of the scatterers. Segment 4 involves the formation of apatite. The ACP transforms to hydroxyapatite (HAP). DLS showed a steep increase in the size and concentration of particles in solution. 8553


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(6) Gebauer, D.; Kellermeier, M.; Gale, J. D.; Bergstrom, L.; Colfen, H. Pre-nucleation clusters as solute precursors in crystallisation. Chem. Soc. Rev. 2014, 43, 2348−2371. (7) Gebauer, D.; Völkel, A.; Cölfen, H. Stable Prenucleation Calcium Carbonate Clusters. Science 2008, 322, 1819−1822. (8) Niederberger, M.; Colfen, H. Oriented attachment and mesocrystals: Non-classical crystallization mechanisms based on nanoparticle assembly. Phys. Chem. Chem. Phys. 2006, 8, 3271−3287. (9) Pouget, E. M.; Bomans, P. H. H.; Goos, J. A. C. M.; Frederik, P. M.; de With, G.; Sommerdijk, N. A. J. M. The Initial Stages of Template-Controlled CaCO3 Formation Revealed by Cryo-TEM. Science 2009, 323, 1455−1458. (10) Sear, R. P. The non-classical nucleation of crystals: microscopic mechanisms and applications to molecular crystals, ice and calcium carbonate. Int. Mater. Rev. 2012, 57, 328−356. (11) Van Driessche, A. E. S.; Benning, L. G.; Rodriguez-Blanco, J. D.; Ossorio, M.; Bots, P.; García-Ruiz, J. M. The Role and Implications of Bassanite as a Stable Precursor Phase to Gypsum Precipitation. Science 2012, 336, 69−72. (12) Wallace, A. F.; Hedges, L. O.; Fernandez-Martinez, A.; Raiteri, P.; Gale, J. D.; Waychunas, G. A.; Whitelam, S.; Banfield, J. F.; De Yoreo, J. J. Microscopic Evidence for Liquid-Liquid Separation in Supersaturated CaCO3 Solutions. Science 2013, 341, 885−889. (13) Demichelis, R.; Raiteri, P.; Gale, J. D.; Quigley, D.; Gebauer, D. Stable prenucleation mineral clusters are liquid-like ionic polymers. Nat. Commun. 2011, 2, 590. (14) Gower, L. B. Biomimetic Model Systems for Investigating the Amorphous Precursor Pathway and Its Role in Biomineralization. Chem. Rev. 2008, 108, 4551−4627. (15) Navrotsky, A. Energetic clues to pathways to biomineralization: Precursors, clusters. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12096− 12101. (16) Mahamid, J.; Aichmayer, B.; Shimoni, E.; Ziblat, R.; Li, C.; Siegel, S.; Paris, O.; Fratzl, P.; Weiner, S.; Addadi, L. Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 6316−6321. (17) Mahamid, J.; Sharir, A.; Addadi, L.; Weiner, S. Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: Indications for an amorphous precursor phase. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 12748−12753. (18) Ibsen, C. J. S.; Chernyshov, D.; Birkedal, H. Apatite formation from amorphous calcium phosphate and mixed amorphous calcium phosphate/amorphous calcium carbonate. Chem.Eur. J. 2016, 22, 12347−12357. (19) Habraken, W. J. E. M.; Tao, J.; Brylka, L. J.; Friedrich, H.; Bertinetti, L.; Schenk, A. S.; Verch, A.; Dmitrovic, V.; Bomans, P. H. H.; Frederik, P. M.; Laven, J.; van der Schoot, P.; Aichmayer, B.; de With, G.; DeYoreo, J. J.; Sommerdijk, N. A. J. M. Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate. Nat. Commun. 2013, 4, 1507. (20) Amos, F. F.; Olszta, M. J.; Khan, S. R.; Gower, L. B.: Relevance of a Polymer-Induced Liquid-Precursor (PILP) Mineralization Process to Normal and Pathological Biomineralization. In Biomineralization: Medical Aspects of Solubility; John Wiley & Sons, Ltd.: Chichester, U.K., 2007; pp 125−217. (21) Gower, L. B.; Odom, D. J. Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process. J. Cryst. Growth 2000, 210, 719−734. (22) Cha, J. N.; Birkedal, H.; Euliss, L. E.; Bartl, M. H.; Wong, M. S.; Deming, T. J.; Stucky, G. D. Spontaneous formation of nanoparticle vesicles from homopolymer polyelectrolytes. J. Am. Chem. Soc. 2003, 125, 8285−8289. (23) Lawrence, P. G.; Lapitsky, Y. Ionically Cross-Linked Poly(allylamine) as a Stimulus-Responsive Underwater Adhesive: Ionic Strength and pH Effects. Langmuir 2015, 31, 1564−1574. (24) McKenna, B. J.; Birkedal, H.; Bartl, M. H.; Deming, T. J.; Stucky, G. D. Micrometer-sized spherical assemblies of polypeptides

high concentration of active ingredients for mineralization, meaning that less water needs to be transported away from the mineralization site. The ability of OPN to stabilize this transient state helps to facilitate this process, but can also ward off unwanted or untimely calcification. The proper combination of mineralization promoters and inhibiters is crucial for the success and consistency of the biomineralization process. Clearly, when biomolecules are involved, discussions of mineralization processes need to consider states prior to the formation of solids.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b01088. Experimental methods and further titration and TEM results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Henrik Birkedal: 0000-0002-4201-2179 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Part of this study was made possible by funding from COST action TD0903. The ultracentrifugation experiments and subsequent conversion of raw data to sedimentation coefficients were performed by Marius Schmid; we thank him for his kind assistance. We also want to acknowledge Dr.’s John K. Berg and Matthias Kellermeier for general assistance performing and interpreting the titration experiments. John Berg also helped record the TEM micrographs. This work is supported by the Danish Council for Independent Research/Technology and Production Sciences and by the Danish Agency for Science, Technology and Innovation (DANSCATT). We acknowledge MAXLAB, Lund, Sweden for the provision of synchrotron radiation beamtime at the I7-11 beamline.

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