DOI: 10.1002/cbic.201500057
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Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation Werner E. G. Mìller,*[a] Meik Neufurth,[a] Jian Huang,[b] Kui Wang,[b] Qingling Feng,[c] Heinz C. Schrçder,[a] Brbel Diehl-Seifert,[d] Rafael MuÇoz-Esp,[e] and Xiaohong Wang*[a] Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3¢) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen
for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, vaterite, and aragonite, is significantly accelerated in the presence of a peptide rich in aspartic acid and glutamic acid. We propose that the initial CaCO3 bioseed formation is driven by CA, and that the subsequent conversion to calcium phosphate/calcium hydroxyapatite (exchange of carbonate by phosphate) is a non-enzymatic exchange process.
Introduction The early ocean was rich in silicate anions and poor in carbonates (reviewed in ref. [1]). This physical environmental characteristic changed 750–700 million years ago (Ma)[2] with the increase in atmospheric CO2 ; in turn, the carbonate concentration of the sea water increased. Calcium carbonate (CaCO3) occurred first in an unstable amorphous phase, termed amorphous calcium carbonate (ACC), and then in three crystalline polymorphs: vaterite, aragonite, and calcite.[3] According to the kinetics and thermodynamics, the unstable amorphous phase [a] Prof. Dr. W. E. G. Mìller, Dr. M. Neufurth, Prof. Dr. Dr. H. C. Schrçder, Prof. Dr. X. Wang ERC Advanced Investigator Grant Research Group Institute for Physiological Chemistry University Medical Center of the Johannes Gutenberg University Duesbergweg 6, 55128 Mainz (Germany) E-mail: [email protected] [email protected] [b] Dr. J. Huang, Prof. Dr. K. Wang Department of Chemical Biology School of Pharmaceutical Sciences, Peking University Xueyuan Road 38, Beijing 100191 (China) [c] Prof. Dr. Q. Feng Laboratory of Advanced Materials of Ministry of Education of China School of Materials Science and Engineering, Tsinghua University Haidian District, Beijing 100084 (China) [d] Dr. B. Diehl-Seifert NanotecMARIN GmbH Duesbergweg 6, 55128 Mainz (Germany) [e] Dr. R. MuÇoz-Esp Department of Physical Chemistry of Polymers Max Planck Institute for Polymer Research Ackermannweg 10, 55128 Mainz (Germany)
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forms first, and is then transformed into the metastable phase vaterite or aragonite, followed by transformation to the stable calcite phase.[4] These changes in the crystallization steps are kinetically driven and follow a controlled series of dehydration and crystallization events. The phases are kinetically controlled and separated by activation energy (Ea) barriers. In turn, the overall process from ACC to calcite involves a series of exergonic reactions, each with a distinct Gibbs free energy (DG) and Ea level. The magnitude of the Ea barriers can be lowered by enzymes, for example, carbonic anhydrases (CAs), which considerably increase the reaction velocity.[4, 5] In addition, particular proteins can either enhance or impede the enzymemediated process by influencing the value of Ea.[6] Parallel with the abundance of silicate anion and carbonate ions in the marine environment, some of the first animals on Earth, the sponges (phylum Porifera), linked with the choanoflagellates, Placozoa, Ctenophores, used those ions as the basis for their mineral inorganic scaffolds, first silica, as in the siliceous sponges (classes Demospongiae and Hexactinellida) and subsequently carbonate (calcareous sponges, Calcarea). This split occurred about 580 Ma.[7, 8] The hard calcareous scaffold of basal sponges is calcite,[9] but other invertebrate skeletons, for example coral skeleton and the cuttlebone of the cuttlefish (genus Sepia), are predominantly composed of aragonite.[10] Later, around 510 Ma (after the Lower Tommotian), the inorganic component (“ground substance”) was primarily calcium phosphate as crystalline calcium hydroxyapatite (Ca5[PO4]3[OH]: HA).[11] One advantage of the Ca(H2PO4)2-based skeletons is their higher resistance against dissolution in vitro and in vivo.[11] It has been reported that a hexactinellid sponge (Caulo-
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Full Papers phacus sp.) uses silica with calcite as a composite material to join the spicules.[12] Likewise, in plants such as Ficus elastica,[13] cystoliths, which form in distinct cells in the epidermis and function as internal light scatterers, are composed of a mainly mineral material of ACC, with a minor component of silica. Initially the silica stalk forms, then ACC is deposited onto this.[14] Four distinct mineral phases are distinguished in these cystoliths: an (almost) pure silica phase, covered by a Mg-rich silica phase, which is overlaid by an ACC phase; around this complex biocomposite, a bulky and less-stable ACC phase is deposited.[14] Interestingly, it was reported that in the bivalve Corbicula fluminea, vateritic deformities are often found within aragonitic parts in the shell of this bivalve.[15] It was postulated that in those vateritic “islands” the biological control of aragonite formation is impaired, very likely because of environmental stress. Furthermore, the authors imply that this blocking is caused by distinct proteins in the shell, in order to prevent the switch from vaterite to aragonite. Vaterite might be stabilized by its higher content of organic material and magnesium. A similar inhibition of the steps of crystallization of CaCO3 to the different isoforms has been described for cultured pearls of the freshwater mussel Hyriopsis cumingii Lea. The progression of CaCO3 crystallization to aragonite is blocked at the vaterite stage, depending on the level of pollutants in the surrounding milieu.[16, 17] Recently we demonstrated that the crystallization of amorphous CaCO3 (ACC) to crystalline calcite is blocked in vitro at the vaterite stage, by the protein silintaphin 2 , which was isolated from the sponge Suberites domuncula.[6] This transformation process was performed in the presence of carbonic anhydrase (CA), an enzyme that accelerates CaCO3 mineralization.[5, 18] Silintaphin-2 is rich in the amino acid residues aspartic acid (D) and glutamic acid (E), the D/E peptide, within its Ca2 + -binding domain.[19] It has been proposed that the carboxyl groups of these residues absorb/dissociate Ca2 + ions on the surface of CaCO3 crystals.[20] It can be postulated that in nature the stabilization, fixation, and freezing of an intermediate stage of an overall exergonic reaction, like the transformation of ACC to calcite, is blocked by distinct organic constituents within the organic–inorganic hybrid structure, for example in shells or cuttlebone.[21] As outlined above, metazoan inorganic CaCO3 scaffold skeletal elements are evolutionary older than the Ca(H2PO4)2-based bones. Therefore, we recently asked whether CaCO3 deposits also exist during early bone formation and act as bioseeds for calcium phosphate/HA formation. We used human osteogenic SaOS-2 cells,[22] a cell line that has mature osteoblastic characteristics.[23] We reported that after exposure of SaOS-2 cells to Ca(HCO3)2 in vitro, there was a significant increase in Ca deposition.[24] This biomineralization process is enzymatically controlled by CA. Inhibitors of CA have been shown to prevent mineral deposition,[18, 24] whereas CA activators accelerate the mineralization process in SaOS-2 cells.[25] The transformation of CaCO3 deposits on these cells to Ca(H2PO4)2 is not well understood. Both inorganic[26] and organic phosphates (e.g., b-glycerophosphate)[27] have been identiChemBioChem 2015, 16, 1323 – 1332
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fied as sources of phosphate. Among the inorganic and organic phosphate donors, pyrophosphate, ATP, pyridoxal-5’-phosphate, and phoshoethanolamine are likely candidates. All must be subjected to enzymatic dephosphorylation, mediated by the tissue-nonspecific alkaline phosphatase (ALP) or the Ca2 + transport ATPase 1 or transglutaminase 2.[26, 28] It has been reported that FGF23 (fibroblast growth factor) and PHEX (phosphate-regulating endopeptidase homologue, X-linked) play vital roles in the regulation of phosphate homeostasis in human osteoblast-like bone cells, very likely by an interaction with the sodium phosphate co-transporter.[29] Increasing evidence exists that inorganic polyphosphates (polyP), which are found ubiquitously in mammalian cells and body fluids and in high levels especially in osteoblasts and platelets, play a role in bone mineral formation.[30–33] However, as polyP is a strong chelator of calcium ions, this polymer has to be co-supplied with Ca2 + to cell cultures, or polyP will inhibit the mineralization process by depletion of Ca2 + .[30, 33] How CaCO3 is converted to Ca(H2PO4)2 remains unknown.[24] Here, we exposed ACC, vaterite, aragonite, and calcite to different concentrations of sodium phosphate and monitored the appearance of phosphate in the CaCO3 deposits by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). ACC was chemically synthesized; biogenic vaterite and aragonite were prepared from freshwater lackluster pearls;[16, 17, 34, 35] aragonite cuttlebone was from the cuttlefish Sepia esculenta,[36, 37] and calcite was from the calcareous sponge Sycon raphanus.[9] Expression studies with SaOS-2 cells showed that ACC, as recently found for HCO3¢ (bicarbonate),[24] caused induction of the gene encoding CA. After five days incubation, Ca2 + mineral deposits were found on the surface of SaOS-2 cells. Those mineralic nodules were rich in carbon, as well as the expected calcium and phosphorus. We also show that ACC, vaterite, and aragonite undergo transformation into a phosphate-containing mineral when incubated in sodium phosphate buffer, but calcite cannot be converted. These data provide further indication that calcium carbonate bioseeds can undergo nonenzymatic conversion to calcium phosphate in vivo during bone HA formation.
Results Effect of ACC on carbonic anhydrase gene expression Recently we showed that SaOS-2 cells exposed to HCO3¢ respond with increased expression of CA.[24] In addition, we found that this anion elevates cell biomineralization, with the formation of CaCO3 bioseed deposits on the cell surface. In the present study, we pursued this line of investigation and determined the inducing potency of HCO3¢ and ACC on CA expression. The experiments were performed in parallel with 10 mm HCO3¢ and 10 mm ACC. After three days of incubation, the mean expression of CA mRNA increased significantly (data normalized to expression of the housekeeping gene GAPDH: CA/ GAPDH mRNA increase from 0.003 0.001 to 0.0093 0.0014 (HCO3¢) and 0.0075 0.0017 (ACC); Figure 1). The levels in-
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Figure 1. Increase in expression of CA2 in SaOS-2 cells exposed to HCO3¢ or ACC. The cells were incubated for the indicated periods in medium/serum either without (open bars) or with 10 mm HCO3¢ (hatched), or 10 mm ACC (black) in the culture medium. RNA was isolated from cells and subjected to qRT-PCR (mean SE, n = 5; *p < 0.01).
creased further over the next two days to 0.015 0.001 (HCO3¢) and 0.013 0.001 (ACC). Subsequently expression decreased by 20 %, but still remained significantly higher than for the control (no added HCO3¢ or ACC).
Formation of biomineral deposits in response to ACC The mineral deposits in SaOS-2 cultures cannot be quantitated in vitro with Alizarin Red S, as this quinine derivative stains not only Ca(H2PO4)2 but also CaCO3.[38] Therefore, we used energy dispersive spectrometry (EDS) under low-energy secondary electron conditions allowing high spatial resolution, in order to assess the element distribution in the mineralic nodules formed on the surfaces of the cells. Cell-layer cultures formed in the absence of ACC did not show any nodule formation, as those visualized previously[39] (Figure 2 A). In contrast, cells exposed to 10 mm ACC and grown in medium containing 5.6 mm sodium phosphate formed mineralic deposits (~ 1 mm) that contained higher levels of Ca2 + than in the surrounding cell layer (Figure 2 B). In a composite net intensity map for carbon and phosphorous, the two elements accumulated at the same loci (Figure 2 C). In a subsequent line scan analysis, extracted from the HyperMap (hyperspectral mapping) across two calcium mineral deposits (diameter ~ 0.8 mm, Figure 2 D), for each the two biogenic elements C and P increase in intensity at the nodules (Figure 2 E). These data support previous results by showing accumulation of calcium and phosphorus, next to carbon, onto SaOS-2 cells after exposure to bicarbonate.[25] These analyses were performed by both SEM and EDSbased mappings. From these data we deduce that ACC induces mineral deposit formation on the surface of SaOS-2 cells; the data also underscore the view that CaCO3 deposits are involved in the transformation of Ca(H2PO4)2 nodules.
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Figure 2. Formation of biomineral nodules on SaOS-2 cells incubated in medium/serum with/without the calcium carbonate polymorph ACC. A) In the absence of ACC no nodular deposits are seen after eight days incubation (SEM image). B) and C) Cells were incubated for three days with 10 mm ACC then transferred to a medium containing additionally iMAC activation cocktail. Distinct nodules (no) formed after five days, with high levels of the elements Ca (B) and C and P (C). D) For higher element resolution a line scan analysis (green lines) was performed across two nodules. E) Line scan extracted from the HyperMap (net intensities: 189 points, 7.7 mm length) across two Ca mineral deposits (diameter ~ 0.8 mm); the distributions of the three elements are shown.
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Full Papers Transformation of ACC and calcite by incubation in a phosphate buffer
Transformation of vaterite and aragonite in phosphate buffer
Because the samples analyzed here contained organic and inorganic species, present in crystalline or amorphous state, FTIR spectroscopy is useful for molecular characterization of the inorganic components.[40] Furthermore, FTIR spectroscopy allows analysis of small amounts of sample. In our studies ACC was chemically synthesized and incubated with 50 or 500 mm sodium phosphate buffer (pH 7.2) for 24 h (Experimental Section). These concentrations are higher than physiological (intracellular phosphate can reach 30 mm;[41] extracellular up to 5 mm).[42] Both intracellularly and extracellularly, non-covalently linked phosphate groups might bind to scavenging proteins, for example, human plasma phosphate binding protein (HPBP, 38 kDa), and might allow local accumulation of free phosphate.[43] Therefore, we note that our phosphate concentration used (50 mm) is above a physiological one. In contrast, the shift of these peaks in the spectra for the transformation of ACC by sodium phosphate was not seen when calcite (here from the spicules of S. raphanus) was incubated with 50 mm sodium phosphate (Figure 3): the spectra were identical, with/without incubation with phosphate. The FTIR spectrum of freshly prepared ACC showed the characteristic peak absorptions: a double band at around 1450 cm¢1 (n3 CO32¢), a relatively broad band at around 868 cm¢1 (n2 CO32¢), the double band at around 1070 cm¢1 (v1), and the absence of the carbonate n4 band (712 cm¢1). The presence of a sharp v4 band at 712 cm¢1 is indicative of calcite;[44] (Figure 3). Incubation of ACC with 50 mm sodium phosphate resulted in PO43¢ forms (IR absorption bands at 560 and 600 cm¢1 (n4 PO43¢) and at 1000–1100 cm¢1 (n3 PO43¢)).[45]
XRD analysis of the samples from the pearls of the mussel H. cumingii showed that they were almost pure with respect to vaterite and aragonite forms of CaCO3 ;[16, 17, 46] (data not shown). Untreated vaterite pearls (Figure 4) clearly showed a vaterite-
Figure 3. Characteristic FTIR spectra for ACC and calcite, incubated with/ without 50 mm sodium phosphate (P). ACC is distinguished from calcite by the absence of the v4 band at 712 cm¢1. For chemically synthesized ACC incubated with P, the characteristic phosphate bands at 560 and 600 cm¢1 (v4) and 1000–1100 cm¢1 (v3) appear. The latter band is absent for calcite (spicules from the calcareous sponge Sycon raphanus), irrespective of an incubation with sodium phosphate, as seen from the absence of the IR absorption bands at 560 and 600 cm¢1 and at 1000–1100 cm¢1.
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Figure 4. FTIR spectra showing structural transformation of vaterite after incubation in 50 mm sodium phosphate. The characteristic phosphate bands (marked) are evident in the deposits treated with phosphate.
like FTIR spectrum: n1 (1086 cm¢1), n2 (874 cm¢1), n3 (1408/ 1433 cm¢1), and n4 (743 cm¢1) bands.[47] In the case of the untreated aragonitic pearls (Figure 5), we were able to identify all the aragonite-specific bands: n1 (1082 cm¢1), n2 (858 cm¢1), n3 (1446/1467 cm¢1), and n4 (702/712 cm¢1).[47–49] When these CaCO3 samples (vaterite in Figure 4; aragonite in Figure 5) were incubated with 50 mm sodium phosphate, the distinct principle phosphate bands (n4 at 560 and 600 cm¢1; n3 at 1000–1100 cm¢1) were evident.
Figure 5. FTIR spectra showing structural transformation of aragonite after incubation in 50 mm sodium phosphate.
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Full Papers Concentration-dependent transformation of aragonite by phosphate incubation The changes in the FTIR spectra for ACC, vaterite, and aragonite after sodium phosphate incubation depended on the concentration of the phosphate buffer (e.g., aragonite from the cuttlebone of the cuttlefish S. esculenta in Figure 6). The arago-
peaks for phosphate at 2q angles of 9.2, 31.8, and 44.38 are evident only in the phosphate-treated samples. As these signals of the formed calcium phosphate deposits (Figure 7; lower scan) match neither calcium hydrogen phosphate nor HA, we ascribe them to calcium phosphate hydrate. Acceleration of phosphate incorporation into CaCO3 in the presence of the D/E peptide As mentioned in the Introduction, the D/E peptide from spicules of the siliceous sponge S. domuncula blocks CaCO3 crystal formation at the vaterite stage.[6] As peptides rich in glutamic acid and aspartic acid are known to affect CaCO3 mineralization,[20] it was tempting to study the effect of this sponge peptide on the extent/rate of phosphate incorporation into the CaCO3 phases. The rate of incorporation of phosphate into both ACC and the crystalline phases, vaterite and aragonite, was significantly accelerated by the D/E peptide. In contrast, no effect was seen when calcite crystals were incubated in the absence or presence of D/E peptide. Two studies are presented here: the effects on ACC (Figure 8) and on cuttlebone aragonite (Figure 9). ACC was incubated in the presence of 50 or 500 mm sodium phosphate,
Figure 6. FTIR spectra showing increasing phosphate bands in aragonite from the cuttlebone (CB) of the cuttlefish S. esculenta with increasing concentration of sodium phosphate. Cephalopodan skeletal structures were incubated in 5, 50, and 500 mm phosphate at room temperature. The characteristic bands for carbonate and phosphate are marked.
nitic nature of this structure is well documented.[10, 50] We used this aragonitic material to demonstrate that incorporation of phosphate into the aragonitic mineral occurs at concentrations above 5 mm. It is obvious that the increase in phosphate bands (especially n3 at 1000–1100 cm¢1) corresponds well with the concentration of sodium phosphate: with increasing phosphate, the bands for carbonate (especially n2 at 873 cm¢1) decrease. The integration of phosphate into ACC, vaterite, and aragonite was confirmed by XRD (e.g., cuttlebone in Figure 7). XRD spectra were recorded for skeletal material untreated or incubated with 500 mm sodium phosphate. The characteristic
Figure 7. XRD pattern of aragonite from the cuttlebone: natural state (above) or incubated with 500 mm sodium phosphate (lower). &: phosphate peaks.
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Figure 8. FTIR spectra showing accelerated phosphate incorporation into ACC after incubation with sodium phosphate (concentrations as indicated) with/without 10 mm D/E peptide. A distinct increase of the areas of the phosphate bands, with respect to the carbonate bands is seen in samples supplemented with D/E peptide. In the trace obtained from ACC, 50 mm phosphate and the D/E peptide, the phosphate signal is slightly smaller compared to the trace from ACC and 50 mm phosphate.
in the absence or presence of 10 mm D/E peptide: a pronounced increase in the phosphate bands is evident in the FTIR spectra for D/E peptide-treated materials, at the expense of the carbonate bands. The same effect was observed for aragonite. A quantitative evaluation of the effect of the D/E peptide on the rate of incorporation of phosphate into both ACC and crystalline aragonite was performed. Under standard conditions (incubation at room temperature for 24 h), the CaCO3 the minerals were incubated with 50 or 500 mm sodium phosphate 1327
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Full Papers Discussion Recent experimental evidence has indicated that Ca(H2PO4)2based mammalian calcium hydroxyapatite (HA) bone formation starts with the formation of calcium carbonate bioseeds.[18, 24] During this process, the gene encoding carbonic anhydrase (CA) is induced, thus implying that the CA plays a role during initial bone formation (Figure 11, top). These data are corroborated by the finding that activators of CA, for example quinolinic acid,[25] induce and accelerate bioseed formation in SaOS-2 cells. Here, we report that both HCO3¢ and ACC induce expression of the CA gene in SaOS-2 cells in vitro. Biogenic ACC is classified as stable or metastable, depending on its water content.[51] ACC can associate tightly with membranes, a process during which ACC undergoes loss of water Figure 9. Accelerated phosphate incorporation into cuttlebone CaCO3/ara(becomes more dehydrated). It is not known by which intracelgonite mineral is seen in the assays containing 50 or 500 mm sodium phoslular signaling pathways HCO3¢ and ACC induce CA expression; phate, in the absence or presence of D/E peptide. the NF-kB pathway is a prime candidate.[52, 53] Our recent study suggested that bone formation (HA synthesis) starts with the formation of CaCO3 bioseeds, a process that is driven enzymatically.[4] Following this bone mineral for(Figure 10). Parallel assays were performed with 10 mm D/E mation, exchange of the carbonate anions by phosphate ions peptide, followed by FTIR spectroscopy. Quantification of the can be postulated and experimentally tested. No enzyme is efficiency of incorporation of phosphate into the CaCO3 minerknown to catalyze this carbonate–phosphate exchange. However, the supply of phosphate ions by ALP during HA formation is quite well understood (Figure 11). Both organic precursors (e.g., b-glycerophosphate)[54] and inorganic polymers (e.g., polyphosphate)[26] are substrates for ALP. These enzymatic processes proceed in close vicinity to bone-forming cells (reviewed in ref. [4]). The beneficial function of Figure 10. Quantification of the incorporation of phosphate into the CaCO3 mineral for A) ACC and B) aragonite, after exposure to 50 or 500 mm sodium phosphate with (&) or without (&) 10 mm D/E peptide. Data are the ratios CaCO3-based implants/prosthetof the v3 phosphate group intensities to v2 carbonate in the FTIR data (*p < 0.01). ics is well established (as outlined in the Introduction). Such al was evaluated as the ratio between the n3 signal for the implants are bioabsorbable and apparently prone to incorporaphosphate group and n2 for carbonate. The data revealed that tion into new bone tissue.[55] Several groups have attempted to develop bone replacement materials composed of CaCO3, incorporation of phosphate into CaCO3 (at the expense of and have focused on the conversion of CaCO3 into HA.[56, 57] carbonate) increased more than fivefold in the presence of 50 mm phosphate (> tenfold for 500 mm phosphate; FigSeveral studies have reported the use of crystalline CaCO3 ure 10 A). When D/E peptide was included in the buffer, a signif(mostly natural aragonite including coralline and cuttlefish icant increase in incorporation was detected (> 15-fold for bones) as a potential bone substitute to accelerate regenera50 mm phosphate, > 20-fold for 500 mm phosphate). tion of bone material.[58, 59] Care has to be taken to purify The extent of incorporation of phosphate into aragonite was CaCO3 minerals from potentially toxic constituents.[60] Several comparatively lower: the phosphate/carbonate ratios were attempts have been made to convert natural vaterite or araonly about one-tenth of those for ACC (Figure 10 B), but again, gonite structures (from coral or nacres) to HA by a hydrotherthe presence of D/E peptide resulted in significantly higher inmal process,[61–65] including a successful attempt at room temcorporation of phosphate into the CaCO3 mineral. perature.[63] Based on these observations we tested the hypothesis that CaCO3-formed bioseeds are transformed into a mixed CaCO3 :Ca(H2PO4)2 hybrid mineral and finally to HA by exposure to phosphate ions. Basically, the reaction of CaCO3 to ChemBioChem 2015, 16, 1323 – 1332
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Full Papers small amount of CaCO3 is dissolved under these conditions. However, for the dissolution of CaCO3 under otherwise physiological conditions but with 1.0– 1.6 mm phosphate, the equilibrium may be expressed as follows [Eq. (2)]: 5 CaCO3 ðsÞ þ 3 PO4 3¢ ðaqÞ þ OH¢ ðaqÞ Ð Ca5 ðPO4 Þ3 ðOHÞ ðsÞ þ 5 CO3 2¢ ðaqÞ
ð2Þ
The equilibrium constant can be evaluated as K = 3.10 Õ 1017, and the Gibbs free energy change (of DG0) with ¢61.05 kJ mol¢1. Consequently, the substitution of the anion in CaCO3 by phosphate is an exergonic reaction (thermodynamically possible). It has been established that in some organisms phosphate incorporation into ACC takes place, and thereby stabilized for short- and long-term use in the body.[71] The concentration of sodium phosphate used in the experiments for converting CaCO3 into Ca(H2PO4)2 seems to be higher than physiological, but it should be noted that in vivo this conversion might occur over longer periods. In addition, the ALP that provides monomeric phosphate and calcium ions for HA formation has been shown to display a progressive mechanism of action.[31] Therefore this enzyme can deliver high amounts of phosphate at a spot during hydrolysis of long-chain polyP molecules; the polymer is degraded without dissociation Figure 11. Top: Proposed link between two enzymatic reactions: 1) CaCO3 bioseed forfrom the enzyme after each hydrolytic step. 3¢ mation driven by CA, which provides HCO ; this ion is transported to the site of CaCO3 Here, we demonstrate that ACC, vaterite, and araformation by the membrane-bound chloride/bicarbonate anion exchanger (AE), and/or by the sodium bicarbonate co-transporter (NBC). 2) ALP degrades polyp (present as gonite, when exposed to phosphate (sodium phosa Ca2 + salt) and thereby supplies the major components phosphate and calcium for the phate), exchange their carbonate anion, CO32¢, to synthesis of HA ((Ca5(PO4)3(OH)). These processes take place on the membrane of bonephosphate. The D/E peptide accelerates the exforming cells (osteoblasts); polyP is synthesized in platelets. The two enzymatic reactions change reaction, in line with earlier data that indicatare linked by a nonenzymatic carbonate/phosphate exchange reaction, which occurs for ACC, vaterite, and aragonite, but not for calcite. The activation energies associated with ed that peptides rich in glutamic acid and aspartic the exergonic reactions stabilize the individual isoforms. Bottom: in the presence of acid affect the CaCO3 mineralization processes (resodium phosphate both vaterite and aragonite undergo transformation to calcium phosviewed in refs. [6, 20]). This exchange (carbonate by phate. The isoforms are separated by distinct activation energy barriers that have to be phosphate) was demonstrated by FTIR and XRD exovercome for the exergonic reactions to proceed. periments. Interestingly, significant anion exchange does not occur with calcite. To the best of our knowledge, CaCO3 does not exist as calcite in mammals, even though substantial amounts of calcium carbonate had Ca(H2PO4)2 is an exergonic reaction (DG0);[66] Figure 11. The solbeen found in vertebrate bone[72] and in mammalian otoubility of calcite under physiological conditions without phosphate was calculated on the basis of the following chemical liths.[73, 74] Therefore, we propose that initial CaCO3 bioseed forequilibrium [Eq. (1)]: mation is enzymatically driven by CA, and that the subsequent carbonate/phosphate exchange reaction is a non-enzymatic ð1Þ CaCO3 ðsÞ þ H2 CO3 ðaqÞ Ð Ca2þ ðaqÞ þ 2 HCO3 ¢ ðaqÞ step in a phosphate-rich extracellular environment. This step is not enzyme driven, but the phosphate concentration in the extracellular environment is high because of the enzymatic The solubility product of calcite is 6 Õ 10¢9,[67] and the dissohydrolysis of polyP, driven by ALP (Figure 11). Platelets rich in ciation constants of carbonic acid are Ka1,H2CO3 = 4.45 Õ 10¢7 and polyP are found in high abundance surrounding of the site of Ka2,H2CO3 = 4.69 Õ 10¢11.[68] Thus, the equilibrium constant of Equabone formation and regeneration. In the platelets the dense tion (1) can be evaluated as K = 5.69 Õ 10¢5. Under physiological granules are rich in polyP.[76] Surely, in these granules and especonditions (pH 7.4, 37 8C) with pCO2 (partial pressure of CO2) of cially in the extracellular milieu, polyP is complexed and depos40 mm Hg[69, 70] and the following concentrations: 1.5–2 mm ited as an insoluble Ca2 + -polyP salt. These polymers are highly Ca2 + , 24–31 mm HCO3¢ , and 1.5 mm H2CO3 a reaction with prone to ALP hydrolysis.[30] a DG0 of 6.02 kJ mol¢1 is calculated. Accordingly, only a very ChemBioChem 2015, 16, 1323 – 1332
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Full Papers Conclusions The data presented here complement our previous studies, and reveal that two enzymes are crucial in HA/bone formation: CA during the initial bioseed formation, and ALP to supply phosphate and Ca2 + during Ca(H2PO4)2/HA synthesis. The available data suggest that the exchange of carbonate by phosphate is non-enzymatic.
Experimental Section Animal specimens and their skeletal elements: Specimen of S. raphanus were collected in the Northern Adriatic near Rovinj (Croatia) at depths of between 2 and 7 m, as epibionts on the mussel Mytilus galloprovincialis. Spicules diactines (~ 300 mm long) and triactines and tetractines (~ 300 mm) were isolated from specimens of this mussel in NaOCl (5 %, v/v) as described.[9] Specimens and pearls of the freshwater pearl mussel H. cumingii were obtained from Tangxi Pearl Farm (Jinhua, Zhejiang Province, China). Cuttlebone of the cuttlefish Sepia esculenta was a gift of the Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology (CAS, Guangzhou, China). Synthesis of amorphous calcium carbonate (ACC): ACC was synthesized according to Radha et al:[77] Na2CO3 (424 mg; #A135.2, Carl Roth) was dissolved in NaOH (2 m, 20 mL; #P031.1, Carl Roth) and then made up to 200 mL with doubly distilled water containing Na2CO3 (20 mm). CaCl2 (20 mm) was prepared by adding, CaCl2·2 H2O (294 mg) to double-distilled water (100 mL). The solutions were stored at 4 8C. For the synthesis of ACC, equal volumes of the solutions were mixed rapidly in a pre-chilled beaker and stirred constantly for one minute. The solution was filtered under vacuum and washed three times with absolute ethanol (#5054.3, Carl Roth). The final ACC was dried for 15 min at 50 8C and stored at 4 8C. Cells and cell culture conditions: SaOS-2 cells (human osteoblastlike sarcoma cells)[22] were cultured in RPMI-1640 medium (#R7388; Sigma–Aldrich), which lacks NaHCO3 but contains l-glutamine (2 mm) and heat-inactivated fetal calf serum (10 %, FCS), as described.[24] The NaH2PO4 content of the medium was 5.6 mm. The medium was supplemented with CaCl2 (1 mm), penicillin (100 U mL¢1, Sigma–Aldrich) and streptomycin (100 mg mL¢1; Sigma–Aldrich). For routine cultures the cells were incubated in 25 cm2 flasks (Corning cell culture flasks, surface area 25 cm2, CLS430639 Sigma–Aldrich) in a humidified incubator at 37 8C; routinely, 3 Õ 105 cells were added to 3 mL medium. Medium/FCS was exchanged every three days. The experiments were performed in 24-well plates (Greiner CELLSTAR; #M8812, Sigma–Aldrich), and 2 Õ 105 cells were added per well. Where indicated, either NaHCO3 (10 mm) or ACC (10 mm) was added. If not mentioned otherwise, the cultures were supplemented, after an initial incubation period of three days, with incomplete mineralization activation cocktail (iMAC: ascorbic acid (50 mm) and dexamethasone (10 nm; #D4902, Sigma–Aldrich)).[78] b-Glycerophosphate was excluded in order to study the initial phases of mineralization and under limited phosphate concentration; hence, the cells initiate biomineralization with Na3PO4 (5.6 mm) in the medium. Quantitative real-time RT-PCR: The expression of CA mRNA (134 bp of carbonic anhydrase II (CA2); accession NM_000067) in SaOS-2 cells was determined by qRT-PCR, essentially as described ChemBioChem 2015, 16, 1323 – 1332
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previously.[24] After harvesting cells, RNA was isolated and subjected to qRT-PCR with primers for CA2: CA2 Fwd: 5’-TCCTC GTGGC CTCCT TCCTG AATC-3’ (nt717–740) and CA2 Rev: 5’-TCAAC ACCTG CTCGC TGCTG AC-3’ (nt850–829). The expression was quantified in parallel with a control transcript, glyceraldehydes 3-phosphate dehydrogenase (GAPDH; accession NM_002046.3; 217 bp) with the primer pair GAPDH Fwd: 5’-CCGTC TAGAA AAACC TGCC-3’ (nt843– nt861) and GAPDH Rev: 5’-GCCAA ATTCG TTGTC ATACC-3’ (nt1059– 1040). Microscope and EDS analyses: Scanning electron microscopy (SEM) was performed with an SU 8000 (Hitachi) detector at low voltage (< 1 kV; analysis of near-surface organic surfaces). The SEM mapping experiments (EDS at sub-micrometer resolution) were performed with an Xflash FlatQUAD (Bruker): 5.5 kV, 260 pA, 120 kcps, 640 Õ 480 pixels, 33 nm pixels, 62 min.[79] EDS line scan analysis was performed with the same technique. Sample preparation and incubation with phosphate buffer: Powder was prepared from ACC and from the crystalline CaCO3 forms of vaterite, aragonite, and calcite (biological origin). ACC was chemically prepared; aragonite and vaterite were purified from the freshwater pearl mussel H. cumingii, as previously described.[16, 17] Cuttlebone from the cuttlefish S. esculenta was the source for aragonite. Natural calcite was from the calcareous spicules of S. raphanus.[9] Sample material (150 mg) was gently washed with distilled water, then incubated with NaOCl (8 %; #425044, Sigma–Aldrich) for 10 h at room temperature. The material was washed again with distilled water, then dried with ethanol. Small blocks (~ 1 mm3) were powdered and incubated with sodium phosphate buffer (50 or 500 mm, pH 7.2) for 24 h at room temperature with shaking. Then the samples were washed three times with distilled water and finally once with 99.8 % ethanol and air dried. Dried samples and negative controls (untreated) were analyzed by FTIR spectroscopy as described below. Where mentioned in the Results and Discussion sections, D/E peptide (10 mm, 30 mg mL¢1: N-DDDSQGEIQSDMAEEEDDDNVD-C, MW 2.47 kDa) synthesized by PANATecs (Heilbronn, Germany) and added to the incubation assay mixture. Fourier transformed infrared (FTIR) spectroscopy: Air-dried samples were micro-milled with an agate mortar and pestle, and analyzed by ATR-FTIR spectroscopy by using a Varian 660-IR spectrometer (Agilent), equipped with a Golden Gate ATR unit (Specac, Orpington, UK). Each spectrum represents the average of 200 scans with a spectral resolution of 4 cm¢1 (typically 550–1800 cm¢1). Baseline correction, smoothing, and analysis of the spectra were performed with the Varian 660-IR software package 5.2.0 (Agilent Technologies). Graphical display and annotation of the spectra were performed with Origin Pro (version 8.5.1; OriginLab, Northampton, MA). X-ray diffraction analyses (XRD): XRD experiments were performed as previously described.[50, 80] The patterns of dried powders were registered on a Philips PW 1820 diffractometer with CuKa radiation (l = 1.5418 æ, 40 kV, 30 mA) in the range 2q = 5–658 (D2q = 0.02, Dt = 5 s). Data analysis: For quantitative evaluation of incorporation of phosphate into the carbonate minerals, the ratio between the heights of the bands corresponding to the n3 phosphate group and the v2 carbonate band was calculated. Mean values ( SD, n = 7) of the spectra (each representing the average of 200 scans) were calculated. Significant differences were identified with a twotailed paired Student’s t-test.[81]
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Full Papers Acknowledgements W.E.G.M. is a holder of an ERC Advanced Investigator Grant (268476 BIOSILICA). This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schr 277/10–2), the European Commission (“Bio-Scaffolds”: 604036; “MarBioTec*EU-CN*”: 268476; and “BlueGenics”: 311848) and the International Human Frontier Science Program. Keywords: biological activity · biomineral skeleton · bone formation · calcium phosphate · calcium · D/E peptide
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Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation Werner E. G. Mìller,*[a] Meik Neufurth,[a] Jian Huang,[b] Kui Wang,[b] Qingling Feng,[c] Heinz C. Schrçder,[a] Brbel Diehl-Seifert,[d] Rafael MuÇoz-Esp,[e] and Xiaohong Wang*[a] Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3¢) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen
for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, vaterite, and aragonite, is significantly accelerated in the presence of a peptide rich in aspartic acid and glutamic acid. We propose that the initial CaCO3 bioseed formation is driven by CA, and that the subsequent conversion to calcium phosphate/calcium hydroxyapatite (exchange of carbonate by phosphate) is a non-enzymatic exchange process.
Introduction The early ocean was rich in silicate anions and poor in carbonates (reviewed in ref. [1]). This physical environmental characteristic changed 750–700 million years ago (Ma)[2] with the increase in atmospheric CO2 ; in turn, the carbonate concentration of the sea water increased. Calcium carbonate (CaCO3) occurred first in an unstable amorphous phase, termed amorphous calcium carbonate (ACC), and then in three crystalline polymorphs: vaterite, aragonite, and calcite.[3] According to the kinetics and thermodynamics, the unstable amorphous phase [a] Prof. Dr. W. E. G. Mìller, Dr. M. Neufurth, Prof. Dr. Dr. H. C. Schrçder, Prof. Dr. X. Wang ERC Advanced Investigator Grant Research Group Institute for Physiological Chemistry University Medical Center of the Johannes Gutenberg University Duesbergweg 6, 55128 Mainz (Germany) E-mail: [email protected] [email protected] [b] Dr. J. Huang, Prof. Dr. K. Wang Department of Chemical Biology School of Pharmaceutical Sciences, Peking University Xueyuan Road 38, Beijing 100191 (China) [c] Prof. Dr. Q. Feng Laboratory of Advanced Materials of Ministry of Education of China School of Materials Science and Engineering, Tsinghua University Haidian District, Beijing 100084 (China) [d] Dr. B. Diehl-Seifert NanotecMARIN GmbH Duesbergweg 6, 55128 Mainz (Germany) [e] Dr. R. MuÇoz-Esp Department of Physical Chemistry of Polymers Max Planck Institute for Polymer Research Ackermannweg 10, 55128 Mainz (Germany)
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forms first, and is then transformed into the metastable phase vaterite or aragonite, followed by transformation to the stable calcite phase.[4] These changes in the crystallization steps are kinetically driven and follow a controlled series of dehydration and crystallization events. The phases are kinetically controlled and separated by activation energy (Ea) barriers. In turn, the overall process from ACC to calcite involves a series of exergonic reactions, each with a distinct Gibbs free energy (DG) and Ea level. The magnitude of the Ea barriers can be lowered by enzymes, for example, carbonic anhydrases (CAs), which considerably increase the reaction velocity.[4, 5] In addition, particular proteins can either enhance or impede the enzymemediated process by influencing the value of Ea.[6] Parallel with the abundance of silicate anion and carbonate ions in the marine environment, some of the first animals on Earth, the sponges (phylum Porifera), linked with the choanoflagellates, Placozoa, Ctenophores, used those ions as the basis for their mineral inorganic scaffolds, first silica, as in the siliceous sponges (classes Demospongiae and Hexactinellida) and subsequently carbonate (calcareous sponges, Calcarea). This split occurred about 580 Ma.[7, 8] The hard calcareous scaffold of basal sponges is calcite,[9] but other invertebrate skeletons, for example coral skeleton and the cuttlebone of the cuttlefish (genus Sepia), are predominantly composed of aragonite.[10] Later, around 510 Ma (after the Lower Tommotian), the inorganic component (“ground substance”) was primarily calcium phosphate as crystalline calcium hydroxyapatite (Ca5[PO4]3[OH]: HA).[11] One advantage of the Ca(H2PO4)2-based skeletons is their higher resistance against dissolution in vitro and in vivo.[11] It has been reported that a hexactinellid sponge (Caulo-
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Full Papers phacus sp.) uses silica with calcite as a composite material to join the spicules.[12] Likewise, in plants such as Ficus elastica,[13] cystoliths, which form in distinct cells in the epidermis and function as internal light scatterers, are composed of a mainly mineral material of ACC, with a minor component of silica. Initially the silica stalk forms, then ACC is deposited onto this.[14] Four distinct mineral phases are distinguished in these cystoliths: an (almost) pure silica phase, covered by a Mg-rich silica phase, which is overlaid by an ACC phase; around this complex biocomposite, a bulky and less-stable ACC phase is deposited.[14] Interestingly, it was reported that in the bivalve Corbicula fluminea, vateritic deformities are often found within aragonitic parts in the shell of this bivalve.[15] It was postulated that in those vateritic “islands” the biological control of aragonite formation is impaired, very likely because of environmental stress. Furthermore, the authors imply that this blocking is caused by distinct proteins in the shell, in order to prevent the switch from vaterite to aragonite. Vaterite might be stabilized by its higher content of organic material and magnesium. A similar inhibition of the steps of crystallization of CaCO3 to the different isoforms has been described for cultured pearls of the freshwater mussel Hyriopsis cumingii Lea. The progression of CaCO3 crystallization to aragonite is blocked at the vaterite stage, depending on the level of pollutants in the surrounding milieu.[16, 17] Recently we demonstrated that the crystallization of amorphous CaCO3 (ACC) to crystalline calcite is blocked in vitro at the vaterite stage, by the protein silintaphin 2 , which was isolated from the sponge Suberites domuncula.[6] This transformation process was performed in the presence of carbonic anhydrase (CA), an enzyme that accelerates CaCO3 mineralization.[5, 18] Silintaphin-2 is rich in the amino acid residues aspartic acid (D) and glutamic acid (E), the D/E peptide, within its Ca2 + -binding domain.[19] It has been proposed that the carboxyl groups of these residues absorb/dissociate Ca2 + ions on the surface of CaCO3 crystals.[20] It can be postulated that in nature the stabilization, fixation, and freezing of an intermediate stage of an overall exergonic reaction, like the transformation of ACC to calcite, is blocked by distinct organic constituents within the organic–inorganic hybrid structure, for example in shells or cuttlebone.[21] As outlined above, metazoan inorganic CaCO3 scaffold skeletal elements are evolutionary older than the Ca(H2PO4)2-based bones. Therefore, we recently asked whether CaCO3 deposits also exist during early bone formation and act as bioseeds for calcium phosphate/HA formation. We used human osteogenic SaOS-2 cells,[22] a cell line that has mature osteoblastic characteristics.[23] We reported that after exposure of SaOS-2 cells to Ca(HCO3)2 in vitro, there was a significant increase in Ca deposition.[24] This biomineralization process is enzymatically controlled by CA. Inhibitors of CA have been shown to prevent mineral deposition,[18, 24] whereas CA activators accelerate the mineralization process in SaOS-2 cells.[25] The transformation of CaCO3 deposits on these cells to Ca(H2PO4)2 is not well understood. Both inorganic[26] and organic phosphates (e.g., b-glycerophosphate)[27] have been identiChemBioChem 2015, 16, 1323 – 1332
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fied as sources of phosphate. Among the inorganic and organic phosphate donors, pyrophosphate, ATP, pyridoxal-5’-phosphate, and phoshoethanolamine are likely candidates. All must be subjected to enzymatic dephosphorylation, mediated by the tissue-nonspecific alkaline phosphatase (ALP) or the Ca2 + transport ATPase 1 or transglutaminase 2.[26, 28] It has been reported that FGF23 (fibroblast growth factor) and PHEX (phosphate-regulating endopeptidase homologue, X-linked) play vital roles in the regulation of phosphate homeostasis in human osteoblast-like bone cells, very likely by an interaction with the sodium phosphate co-transporter.[29] Increasing evidence exists that inorganic polyphosphates (polyP), which are found ubiquitously in mammalian cells and body fluids and in high levels especially in osteoblasts and platelets, play a role in bone mineral formation.[30–33] However, as polyP is a strong chelator of calcium ions, this polymer has to be co-supplied with Ca2 + to cell cultures, or polyP will inhibit the mineralization process by depletion of Ca2 + .[30, 33] How CaCO3 is converted to Ca(H2PO4)2 remains unknown.[24] Here, we exposed ACC, vaterite, aragonite, and calcite to different concentrations of sodium phosphate and monitored the appearance of phosphate in the CaCO3 deposits by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). ACC was chemically synthesized; biogenic vaterite and aragonite were prepared from freshwater lackluster pearls;[16, 17, 34, 35] aragonite cuttlebone was from the cuttlefish Sepia esculenta,[36, 37] and calcite was from the calcareous sponge Sycon raphanus.[9] Expression studies with SaOS-2 cells showed that ACC, as recently found for HCO3¢ (bicarbonate),[24] caused induction of the gene encoding CA. After five days incubation, Ca2 + mineral deposits were found on the surface of SaOS-2 cells. Those mineralic nodules were rich in carbon, as well as the expected calcium and phosphorus. We also show that ACC, vaterite, and aragonite undergo transformation into a phosphate-containing mineral when incubated in sodium phosphate buffer, but calcite cannot be converted. These data provide further indication that calcium carbonate bioseeds can undergo nonenzymatic conversion to calcium phosphate in vivo during bone HA formation.
Results Effect of ACC on carbonic anhydrase gene expression Recently we showed that SaOS-2 cells exposed to HCO3¢ respond with increased expression of CA.[24] In addition, we found that this anion elevates cell biomineralization, with the formation of CaCO3 bioseed deposits on the cell surface. In the present study, we pursued this line of investigation and determined the inducing potency of HCO3¢ and ACC on CA expression. The experiments were performed in parallel with 10 mm HCO3¢ and 10 mm ACC. After three days of incubation, the mean expression of CA mRNA increased significantly (data normalized to expression of the housekeeping gene GAPDH: CA/ GAPDH mRNA increase from 0.003 0.001 to 0.0093 0.0014 (HCO3¢) and 0.0075 0.0017 (ACC); Figure 1). The levels in-
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Figure 1. Increase in expression of CA2 in SaOS-2 cells exposed to HCO3¢ or ACC. The cells were incubated for the indicated periods in medium/serum either without (open bars) or with 10 mm HCO3¢ (hatched), or 10 mm ACC (black) in the culture medium. RNA was isolated from cells and subjected to qRT-PCR (mean SE, n = 5; *p < 0.01).
creased further over the next two days to 0.015 0.001 (HCO3¢) and 0.013 0.001 (ACC). Subsequently expression decreased by 20 %, but still remained significantly higher than for the control (no added HCO3¢ or ACC).
Formation of biomineral deposits in response to ACC The mineral deposits in SaOS-2 cultures cannot be quantitated in vitro with Alizarin Red S, as this quinine derivative stains not only Ca(H2PO4)2 but also CaCO3.[38] Therefore, we used energy dispersive spectrometry (EDS) under low-energy secondary electron conditions allowing high spatial resolution, in order to assess the element distribution in the mineralic nodules formed on the surfaces of the cells. Cell-layer cultures formed in the absence of ACC did not show any nodule formation, as those visualized previously[39] (Figure 2 A). In contrast, cells exposed to 10 mm ACC and grown in medium containing 5.6 mm sodium phosphate formed mineralic deposits (~ 1 mm) that contained higher levels of Ca2 + than in the surrounding cell layer (Figure 2 B). In a composite net intensity map for carbon and phosphorous, the two elements accumulated at the same loci (Figure 2 C). In a subsequent line scan analysis, extracted from the HyperMap (hyperspectral mapping) across two calcium mineral deposits (diameter ~ 0.8 mm, Figure 2 D), for each the two biogenic elements C and P increase in intensity at the nodules (Figure 2 E). These data support previous results by showing accumulation of calcium and phosphorus, next to carbon, onto SaOS-2 cells after exposure to bicarbonate.[25] These analyses were performed by both SEM and EDSbased mappings. From these data we deduce that ACC induces mineral deposit formation on the surface of SaOS-2 cells; the data also underscore the view that CaCO3 deposits are involved in the transformation of Ca(H2PO4)2 nodules.
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Figure 2. Formation of biomineral nodules on SaOS-2 cells incubated in medium/serum with/without the calcium carbonate polymorph ACC. A) In the absence of ACC no nodular deposits are seen after eight days incubation (SEM image). B) and C) Cells were incubated for three days with 10 mm ACC then transferred to a medium containing additionally iMAC activation cocktail. Distinct nodules (no) formed after five days, with high levels of the elements Ca (B) and C and P (C). D) For higher element resolution a line scan analysis (green lines) was performed across two nodules. E) Line scan extracted from the HyperMap (net intensities: 189 points, 7.7 mm length) across two Ca mineral deposits (diameter ~ 0.8 mm); the distributions of the three elements are shown.
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Full Papers Transformation of ACC and calcite by incubation in a phosphate buffer
Transformation of vaterite and aragonite in phosphate buffer
Because the samples analyzed here contained organic and inorganic species, present in crystalline or amorphous state, FTIR spectroscopy is useful for molecular characterization of the inorganic components.[40] Furthermore, FTIR spectroscopy allows analysis of small amounts of sample. In our studies ACC was chemically synthesized and incubated with 50 or 500 mm sodium phosphate buffer (pH 7.2) for 24 h (Experimental Section). These concentrations are higher than physiological (intracellular phosphate can reach 30 mm;[41] extracellular up to 5 mm).[42] Both intracellularly and extracellularly, non-covalently linked phosphate groups might bind to scavenging proteins, for example, human plasma phosphate binding protein (HPBP, 38 kDa), and might allow local accumulation of free phosphate.[43] Therefore, we note that our phosphate concentration used (50 mm) is above a physiological one. In contrast, the shift of these peaks in the spectra for the transformation of ACC by sodium phosphate was not seen when calcite (here from the spicules of S. raphanus) was incubated with 50 mm sodium phosphate (Figure 3): the spectra were identical, with/without incubation with phosphate. The FTIR spectrum of freshly prepared ACC showed the characteristic peak absorptions: a double band at around 1450 cm¢1 (n3 CO32¢), a relatively broad band at around 868 cm¢1 (n2 CO32¢), the double band at around 1070 cm¢1 (v1), and the absence of the carbonate n4 band (712 cm¢1). The presence of a sharp v4 band at 712 cm¢1 is indicative of calcite;[44] (Figure 3). Incubation of ACC with 50 mm sodium phosphate resulted in PO43¢ forms (IR absorption bands at 560 and 600 cm¢1 (n4 PO43¢) and at 1000–1100 cm¢1 (n3 PO43¢)).[45]
XRD analysis of the samples from the pearls of the mussel H. cumingii showed that they were almost pure with respect to vaterite and aragonite forms of CaCO3 ;[16, 17, 46] (data not shown). Untreated vaterite pearls (Figure 4) clearly showed a vaterite-
Figure 3. Characteristic FTIR spectra for ACC and calcite, incubated with/ without 50 mm sodium phosphate (P). ACC is distinguished from calcite by the absence of the v4 band at 712 cm¢1. For chemically synthesized ACC incubated with P, the characteristic phosphate bands at 560 and 600 cm¢1 (v4) and 1000–1100 cm¢1 (v3) appear. The latter band is absent for calcite (spicules from the calcareous sponge Sycon raphanus), irrespective of an incubation with sodium phosphate, as seen from the absence of the IR absorption bands at 560 and 600 cm¢1 and at 1000–1100 cm¢1.
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Figure 4. FTIR spectra showing structural transformation of vaterite after incubation in 50 mm sodium phosphate. The characteristic phosphate bands (marked) are evident in the deposits treated with phosphate.
like FTIR spectrum: n1 (1086 cm¢1), n2 (874 cm¢1), n3 (1408/ 1433 cm¢1), and n4 (743 cm¢1) bands.[47] In the case of the untreated aragonitic pearls (Figure 5), we were able to identify all the aragonite-specific bands: n1 (1082 cm¢1), n2 (858 cm¢1), n3 (1446/1467 cm¢1), and n4 (702/712 cm¢1).[47–49] When these CaCO3 samples (vaterite in Figure 4; aragonite in Figure 5) were incubated with 50 mm sodium phosphate, the distinct principle phosphate bands (n4 at 560 and 600 cm¢1; n3 at 1000–1100 cm¢1) were evident.
Figure 5. FTIR spectra showing structural transformation of aragonite after incubation in 50 mm sodium phosphate.
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Full Papers Concentration-dependent transformation of aragonite by phosphate incubation The changes in the FTIR spectra for ACC, vaterite, and aragonite after sodium phosphate incubation depended on the concentration of the phosphate buffer (e.g., aragonite from the cuttlebone of the cuttlefish S. esculenta in Figure 6). The arago-
peaks for phosphate at 2q angles of 9.2, 31.8, and 44.38 are evident only in the phosphate-treated samples. As these signals of the formed calcium phosphate deposits (Figure 7; lower scan) match neither calcium hydrogen phosphate nor HA, we ascribe them to calcium phosphate hydrate. Acceleration of phosphate incorporation into CaCO3 in the presence of the D/E peptide As mentioned in the Introduction, the D/E peptide from spicules of the siliceous sponge S. domuncula blocks CaCO3 crystal formation at the vaterite stage.[6] As peptides rich in glutamic acid and aspartic acid are known to affect CaCO3 mineralization,[20] it was tempting to study the effect of this sponge peptide on the extent/rate of phosphate incorporation into the CaCO3 phases. The rate of incorporation of phosphate into both ACC and the crystalline phases, vaterite and aragonite, was significantly accelerated by the D/E peptide. In contrast, no effect was seen when calcite crystals were incubated in the absence or presence of D/E peptide. Two studies are presented here: the effects on ACC (Figure 8) and on cuttlebone aragonite (Figure 9). ACC was incubated in the presence of 50 or 500 mm sodium phosphate,
Figure 6. FTIR spectra showing increasing phosphate bands in aragonite from the cuttlebone (CB) of the cuttlefish S. esculenta with increasing concentration of sodium phosphate. Cephalopodan skeletal structures were incubated in 5, 50, and 500 mm phosphate at room temperature. The characteristic bands for carbonate and phosphate are marked.
nitic nature of this structure is well documented.[10, 50] We used this aragonitic material to demonstrate that incorporation of phosphate into the aragonitic mineral occurs at concentrations above 5 mm. It is obvious that the increase in phosphate bands (especially n3 at 1000–1100 cm¢1) corresponds well with the concentration of sodium phosphate: with increasing phosphate, the bands for carbonate (especially n2 at 873 cm¢1) decrease. The integration of phosphate into ACC, vaterite, and aragonite was confirmed by XRD (e.g., cuttlebone in Figure 7). XRD spectra were recorded for skeletal material untreated or incubated with 500 mm sodium phosphate. The characteristic
Figure 7. XRD pattern of aragonite from the cuttlebone: natural state (above) or incubated with 500 mm sodium phosphate (lower). &: phosphate peaks.
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Figure 8. FTIR spectra showing accelerated phosphate incorporation into ACC after incubation with sodium phosphate (concentrations as indicated) with/without 10 mm D/E peptide. A distinct increase of the areas of the phosphate bands, with respect to the carbonate bands is seen in samples supplemented with D/E peptide. In the trace obtained from ACC, 50 mm phosphate and the D/E peptide, the phosphate signal is slightly smaller compared to the trace from ACC and 50 mm phosphate.
in the absence or presence of 10 mm D/E peptide: a pronounced increase in the phosphate bands is evident in the FTIR spectra for D/E peptide-treated materials, at the expense of the carbonate bands. The same effect was observed for aragonite. A quantitative evaluation of the effect of the D/E peptide on the rate of incorporation of phosphate into both ACC and crystalline aragonite was performed. Under standard conditions (incubation at room temperature for 24 h), the CaCO3 the minerals were incubated with 50 or 500 mm sodium phosphate 1327
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Full Papers Discussion Recent experimental evidence has indicated that Ca(H2PO4)2based mammalian calcium hydroxyapatite (HA) bone formation starts with the formation of calcium carbonate bioseeds.[18, 24] During this process, the gene encoding carbonic anhydrase (CA) is induced, thus implying that the CA plays a role during initial bone formation (Figure 11, top). These data are corroborated by the finding that activators of CA, for example quinolinic acid,[25] induce and accelerate bioseed formation in SaOS-2 cells. Here, we report that both HCO3¢ and ACC induce expression of the CA gene in SaOS-2 cells in vitro. Biogenic ACC is classified as stable or metastable, depending on its water content.[51] ACC can associate tightly with membranes, a process during which ACC undergoes loss of water Figure 9. Accelerated phosphate incorporation into cuttlebone CaCO3/ara(becomes more dehydrated). It is not known by which intracelgonite mineral is seen in the assays containing 50 or 500 mm sodium phoslular signaling pathways HCO3¢ and ACC induce CA expression; phate, in the absence or presence of D/E peptide. the NF-kB pathway is a prime candidate.[52, 53] Our recent study suggested that bone formation (HA synthesis) starts with the formation of CaCO3 bioseeds, a process that is driven enzymatically.[4] Following this bone mineral for(Figure 10). Parallel assays were performed with 10 mm D/E mation, exchange of the carbonate anions by phosphate ions peptide, followed by FTIR spectroscopy. Quantification of the can be postulated and experimentally tested. No enzyme is efficiency of incorporation of phosphate into the CaCO3 minerknown to catalyze this carbonate–phosphate exchange. However, the supply of phosphate ions by ALP during HA formation is quite well understood (Figure 11). Both organic precursors (e.g., b-glycerophosphate)[54] and inorganic polymers (e.g., polyphosphate)[26] are substrates for ALP. These enzymatic processes proceed in close vicinity to bone-forming cells (reviewed in ref. [4]). The beneficial function of Figure 10. Quantification of the incorporation of phosphate into the CaCO3 mineral for A) ACC and B) aragonite, after exposure to 50 or 500 mm sodium phosphate with (&) or without (&) 10 mm D/E peptide. Data are the ratios CaCO3-based implants/prosthetof the v3 phosphate group intensities to v2 carbonate in the FTIR data (*p < 0.01). ics is well established (as outlined in the Introduction). Such al was evaluated as the ratio between the n3 signal for the implants are bioabsorbable and apparently prone to incorporaphosphate group and n2 for carbonate. The data revealed that tion into new bone tissue.[55] Several groups have attempted to develop bone replacement materials composed of CaCO3, incorporation of phosphate into CaCO3 (at the expense of and have focused on the conversion of CaCO3 into HA.[56, 57] carbonate) increased more than fivefold in the presence of 50 mm phosphate (> tenfold for 500 mm phosphate; FigSeveral studies have reported the use of crystalline CaCO3 ure 10 A). When D/E peptide was included in the buffer, a signif(mostly natural aragonite including coralline and cuttlefish icant increase in incorporation was detected (> 15-fold for bones) as a potential bone substitute to accelerate regenera50 mm phosphate, > 20-fold for 500 mm phosphate). tion of bone material.[58, 59] Care has to be taken to purify The extent of incorporation of phosphate into aragonite was CaCO3 minerals from potentially toxic constituents.[60] Several comparatively lower: the phosphate/carbonate ratios were attempts have been made to convert natural vaterite or araonly about one-tenth of those for ACC (Figure 10 B), but again, gonite structures (from coral or nacres) to HA by a hydrotherthe presence of D/E peptide resulted in significantly higher inmal process,[61–65] including a successful attempt at room temcorporation of phosphate into the CaCO3 mineral. perature.[63] Based on these observations we tested the hypothesis that CaCO3-formed bioseeds are transformed into a mixed CaCO3 :Ca(H2PO4)2 hybrid mineral and finally to HA by exposure to phosphate ions. Basically, the reaction of CaCO3 to ChemBioChem 2015, 16, 1323 – 1332
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Full Papers small amount of CaCO3 is dissolved under these conditions. However, for the dissolution of CaCO3 under otherwise physiological conditions but with 1.0– 1.6 mm phosphate, the equilibrium may be expressed as follows [Eq. (2)]: 5 CaCO3 ðsÞ þ 3 PO4 3¢ ðaqÞ þ OH¢ ðaqÞ Ð Ca5 ðPO4 Þ3 ðOHÞ ðsÞ þ 5 CO3 2¢ ðaqÞ
ð2Þ
The equilibrium constant can be evaluated as K = 3.10 Õ 1017, and the Gibbs free energy change (of DG0) with ¢61.05 kJ mol¢1. Consequently, the substitution of the anion in CaCO3 by phosphate is an exergonic reaction (thermodynamically possible). It has been established that in some organisms phosphate incorporation into ACC takes place, and thereby stabilized for short- and long-term use in the body.[71] The concentration of sodium phosphate used in the experiments for converting CaCO3 into Ca(H2PO4)2 seems to be higher than physiological, but it should be noted that in vivo this conversion might occur over longer periods. In addition, the ALP that provides monomeric phosphate and calcium ions for HA formation has been shown to display a progressive mechanism of action.[31] Therefore this enzyme can deliver high amounts of phosphate at a spot during hydrolysis of long-chain polyP molecules; the polymer is degraded without dissociation Figure 11. Top: Proposed link between two enzymatic reactions: 1) CaCO3 bioseed forfrom the enzyme after each hydrolytic step. 3¢ mation driven by CA, which provides HCO ; this ion is transported to the site of CaCO3 Here, we demonstrate that ACC, vaterite, and araformation by the membrane-bound chloride/bicarbonate anion exchanger (AE), and/or by the sodium bicarbonate co-transporter (NBC). 2) ALP degrades polyp (present as gonite, when exposed to phosphate (sodium phosa Ca2 + salt) and thereby supplies the major components phosphate and calcium for the phate), exchange their carbonate anion, CO32¢, to synthesis of HA ((Ca5(PO4)3(OH)). These processes take place on the membrane of bonephosphate. The D/E peptide accelerates the exforming cells (osteoblasts); polyP is synthesized in platelets. The two enzymatic reactions change reaction, in line with earlier data that indicatare linked by a nonenzymatic carbonate/phosphate exchange reaction, which occurs for ACC, vaterite, and aragonite, but not for calcite. The activation energies associated with ed that peptides rich in glutamic acid and aspartic the exergonic reactions stabilize the individual isoforms. Bottom: in the presence of acid affect the CaCO3 mineralization processes (resodium phosphate both vaterite and aragonite undergo transformation to calcium phosviewed in refs. [6, 20]). This exchange (carbonate by phate. The isoforms are separated by distinct activation energy barriers that have to be phosphate) was demonstrated by FTIR and XRD exovercome for the exergonic reactions to proceed. periments. Interestingly, significant anion exchange does not occur with calcite. To the best of our knowledge, CaCO3 does not exist as calcite in mammals, even though substantial amounts of calcium carbonate had Ca(H2PO4)2 is an exergonic reaction (DG0);[66] Figure 11. The solbeen found in vertebrate bone[72] and in mammalian otoubility of calcite under physiological conditions without phosphate was calculated on the basis of the following chemical liths.[73, 74] Therefore, we propose that initial CaCO3 bioseed forequilibrium [Eq. (1)]: mation is enzymatically driven by CA, and that the subsequent carbonate/phosphate exchange reaction is a non-enzymatic ð1Þ CaCO3 ðsÞ þ H2 CO3 ðaqÞ Ð Ca2þ ðaqÞ þ 2 HCO3 ¢ ðaqÞ step in a phosphate-rich extracellular environment. This step is not enzyme driven, but the phosphate concentration in the extracellular environment is high because of the enzymatic The solubility product of calcite is 6 Õ 10¢9,[67] and the dissohydrolysis of polyP, driven by ALP (Figure 11). Platelets rich in ciation constants of carbonic acid are Ka1,H2CO3 = 4.45 Õ 10¢7 and polyP are found in high abundance surrounding of the site of Ka2,H2CO3 = 4.69 Õ 10¢11.[68] Thus, the equilibrium constant of Equabone formation and regeneration. In the platelets the dense tion (1) can be evaluated as K = 5.69 Õ 10¢5. Under physiological granules are rich in polyP.[76] Surely, in these granules and especonditions (pH 7.4, 37 8C) with pCO2 (partial pressure of CO2) of cially in the extracellular milieu, polyP is complexed and depos40 mm Hg[69, 70] and the following concentrations: 1.5–2 mm ited as an insoluble Ca2 + -polyP salt. These polymers are highly Ca2 + , 24–31 mm HCO3¢ , and 1.5 mm H2CO3 a reaction with prone to ALP hydrolysis.[30] a DG0 of 6.02 kJ mol¢1 is calculated. Accordingly, only a very ChemBioChem 2015, 16, 1323 – 1332
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Full Papers Conclusions The data presented here complement our previous studies, and reveal that two enzymes are crucial in HA/bone formation: CA during the initial bioseed formation, and ALP to supply phosphate and Ca2 + during Ca(H2PO4)2/HA synthesis. The available data suggest that the exchange of carbonate by phosphate is non-enzymatic.
Experimental Section Animal specimens and their skeletal elements: Specimen of S. raphanus were collected in the Northern Adriatic near Rovinj (Croatia) at depths of between 2 and 7 m, as epibionts on the mussel Mytilus galloprovincialis. Spicules diactines (~ 300 mm long) and triactines and tetractines (~ 300 mm) were isolated from specimens of this mussel in NaOCl (5 %, v/v) as described.[9] Specimens and pearls of the freshwater pearl mussel H. cumingii were obtained from Tangxi Pearl Farm (Jinhua, Zhejiang Province, China). Cuttlebone of the cuttlefish Sepia esculenta was a gift of the Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology (CAS, Guangzhou, China). Synthesis of amorphous calcium carbonate (ACC): ACC was synthesized according to Radha et al:[77] Na2CO3 (424 mg; #A135.2, Carl Roth) was dissolved in NaOH (2 m, 20 mL; #P031.1, Carl Roth) and then made up to 200 mL with doubly distilled water containing Na2CO3 (20 mm). CaCl2 (20 mm) was prepared by adding, CaCl2·2 H2O (294 mg) to double-distilled water (100 mL). The solutions were stored at 4 8C. For the synthesis of ACC, equal volumes of the solutions were mixed rapidly in a pre-chilled beaker and stirred constantly for one minute. The solution was filtered under vacuum and washed three times with absolute ethanol (#5054.3, Carl Roth). The final ACC was dried for 15 min at 50 8C and stored at 4 8C. Cells and cell culture conditions: SaOS-2 cells (human osteoblastlike sarcoma cells)[22] were cultured in RPMI-1640 medium (#R7388; Sigma–Aldrich), which lacks NaHCO3 but contains l-glutamine (2 mm) and heat-inactivated fetal calf serum (10 %, FCS), as described.[24] The NaH2PO4 content of the medium was 5.6 mm. The medium was supplemented with CaCl2 (1 mm), penicillin (100 U mL¢1, Sigma–Aldrich) and streptomycin (100 mg mL¢1; Sigma–Aldrich). For routine cultures the cells were incubated in 25 cm2 flasks (Corning cell culture flasks, surface area 25 cm2, CLS430639 Sigma–Aldrich) in a humidified incubator at 37 8C; routinely, 3 Õ 105 cells were added to 3 mL medium. Medium/FCS was exchanged every three days. The experiments were performed in 24-well plates (Greiner CELLSTAR; #M8812, Sigma–Aldrich), and 2 Õ 105 cells were added per well. Where indicated, either NaHCO3 (10 mm) or ACC (10 mm) was added. If not mentioned otherwise, the cultures were supplemented, after an initial incubation period of three days, with incomplete mineralization activation cocktail (iMAC: ascorbic acid (50 mm) and dexamethasone (10 nm; #D4902, Sigma–Aldrich)).[78] b-Glycerophosphate was excluded in order to study the initial phases of mineralization and under limited phosphate concentration; hence, the cells initiate biomineralization with Na3PO4 (5.6 mm) in the medium. Quantitative real-time RT-PCR: The expression of CA mRNA (134 bp of carbonic anhydrase II (CA2); accession NM_000067) in SaOS-2 cells was determined by qRT-PCR, essentially as described ChemBioChem 2015, 16, 1323 – 1332
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previously.[24] After harvesting cells, RNA was isolated and subjected to qRT-PCR with primers for CA2: CA2 Fwd: 5’-TCCTC GTGGC CTCCT TCCTG AATC-3’ (nt717–740) and CA2 Rev: 5’-TCAAC ACCTG CTCGC TGCTG AC-3’ (nt850–829). The expression was quantified in parallel with a control transcript, glyceraldehydes 3-phosphate dehydrogenase (GAPDH; accession NM_002046.3; 217 bp) with the primer pair GAPDH Fwd: 5’-CCGTC TAGAA AAACC TGCC-3’ (nt843– nt861) and GAPDH Rev: 5’-GCCAA ATTCG TTGTC ATACC-3’ (nt1059– 1040). Microscope and EDS analyses: Scanning electron microscopy (SEM) was performed with an SU 8000 (Hitachi) detector at low voltage (< 1 kV; analysis of near-surface organic surfaces). The SEM mapping experiments (EDS at sub-micrometer resolution) were performed with an Xflash FlatQUAD (Bruker): 5.5 kV, 260 pA, 120 kcps, 640 Õ 480 pixels, 33 nm pixels, 62 min.[79] EDS line scan analysis was performed with the same technique. Sample preparation and incubation with phosphate buffer: Powder was prepared from ACC and from the crystalline CaCO3 forms of vaterite, aragonite, and calcite (biological origin). ACC was chemically prepared; aragonite and vaterite were purified from the freshwater pearl mussel H. cumingii, as previously described.[16, 17] Cuttlebone from the cuttlefish S. esculenta was the source for aragonite. Natural calcite was from the calcareous spicules of S. raphanus.[9] Sample material (150 mg) was gently washed with distilled water, then incubated with NaOCl (8 %; #425044, Sigma–Aldrich) for 10 h at room temperature. The material was washed again with distilled water, then dried with ethanol. Small blocks (~ 1 mm3) were powdered and incubated with sodium phosphate buffer (50 or 500 mm, pH 7.2) for 24 h at room temperature with shaking. Then the samples were washed three times with distilled water and finally once with 99.8 % ethanol and air dried. Dried samples and negative controls (untreated) were analyzed by FTIR spectroscopy as described below. Where mentioned in the Results and Discussion sections, D/E peptide (10 mm, 30 mg mL¢1: N-DDDSQGEIQSDMAEEEDDDNVD-C, MW 2.47 kDa) synthesized by PANATecs (Heilbronn, Germany) and added to the incubation assay mixture. Fourier transformed infrared (FTIR) spectroscopy: Air-dried samples were micro-milled with an agate mortar and pestle, and analyzed by ATR-FTIR spectroscopy by using a Varian 660-IR spectrometer (Agilent), equipped with a Golden Gate ATR unit (Specac, Orpington, UK). Each spectrum represents the average of 200 scans with a spectral resolution of 4 cm¢1 (typically 550–1800 cm¢1). Baseline correction, smoothing, and analysis of the spectra were performed with the Varian 660-IR software package 5.2.0 (Agilent Technologies). Graphical display and annotation of the spectra were performed with Origin Pro (version 8.5.1; OriginLab, Northampton, MA). X-ray diffraction analyses (XRD): XRD experiments were performed as previously described.[50, 80] The patterns of dried powders were registered on a Philips PW 1820 diffractometer with CuKa radiation (l = 1.5418 æ, 40 kV, 30 mA) in the range 2q = 5–658 (D2q = 0.02, Dt = 5 s). Data analysis: For quantitative evaluation of incorporation of phosphate into the carbonate minerals, the ratio between the heights of the bands corresponding to the n3 phosphate group and the v2 carbonate band was calculated. Mean values ( SD, n = 7) of the spectra (each representing the average of 200 scans) were calculated. Significant differences were identified with a twotailed paired Student’s t-test.[81]
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Full Papers Acknowledgements W.E.G.M. is a holder of an ERC Advanced Investigator Grant (268476 BIOSILICA). This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schr 277/10–2), the European Commission (“Bio-Scaffolds”: 604036; “MarBioTec*EU-CN*”: 268476; and “BlueGenics”: 311848) and the International Human Frontier Science Program. Keywords: biological activity · biomineral skeleton · bone formation · calcium phosphate · calcium · D/E peptide
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