The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro

G Model BCP 12448 No. of Pages 10

Biochemical Pharmacology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro Xiaohong Wanga,* , Jian Huangb , Kui Wangb , Meik Neufurtha , Heinz C. Schrödera , Shunfeng Wanga , Werner E.G. Müllera,* a ERC Advanced Investigator Grant Research Group at Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, D-55128 Mainz, Germany b Department of Chemical Biology School of Pharmaceutical Sciences, Peking University, Xueyuan Road 38, Beijing 100191, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 November 2015 Accepted 14 December 2015 Available online xxx

Inorganic polyphosphate (polyP) is a physiological polymer composed of tens to hundreds of phosphate units linked together via phosphoanhydride bonds. Here we compared the biological activity of polyP (chain length of 40 phosphate units), complexed with Gd3+ (polyP
Gd), with the one caused by polyP (as calcium salt) and by GdCl3 alone, regarding their potencies to induce hydroxyapatite (HA) formation in SaOS-2 cells in vitro. The three compounds, GdCl3, polyP and polyP
Gd were found to be non-toxic at concentrations up to at least 30 mM. Selecting a low, 5 mM, concentration it was found that polyP
Gd significantly induced HA formation, as determined by Alizarin Red S staining and by quantitative determinations using that dye. Under those conditions polyP
Gd and to a smaller extent also polyP or GdCl3 (5 mM each) caused HA crystal formation arranged in a nest-like pattern. Exposure of cells to polyP
Gd resulted in a strong increase in alkaline phosphatase activity; this enzyme did not cause a distinct degradation of polyP but of polyP
Gd which was extensively hydrolyzed. The morphogenetic activity of gadolinium, in the form of polyP
Gd, is underscored by the finding that this polymer causes a strong upregulation of the genes encoding morphogenetic protein-2 (BMP2) as well as collagen type I. It is concluded that polyP
Gd is not an inert polymer but acts as a morphogenetically active polymer and induces HA formation in vitro. ã 2015 Elsevier Inc. All rights reserved.

Keywords: Inorganic polyphosphate Gadolinium Hydroxyapatite formation Bone Osteoblasts SaOS-2 cells

1. Introduction Bone formation (osteogenesis) and maintenance (homeostasis) is differentially controlled in a balanced interplay of bone-forming cells (osteoblasts) and bone-degrading cells (osteoclasts) (see Ref. [1]). A disorder of this balance, osteoblastic bone formation and osteoclastic bone resorption, results in the clinical pictures of osteoporosis (hyperactivity of the osteoclasts) and osteopetrosis (hyperfunction of the osteoblasts); reviewed in: Lazner et al. [2]. The tuned interaction between the activities of these anabolically active and catabolically active cell types is under the control of cell adhesion molecules (integrins), and soluble/diffusible intra- and extracellular organic factors and their corresponding receptors [3]. In addition, inorganic mineralic deposits, e.g., hydroxyapatite (HA)

* Corresponding authors. Fax: +49 6131 39 25243. E-mail addresses: [email protected] (X. Wang), [email protected] (W.E.G. Müller).

or calcium carbonate, induce inductive substances of organic nature and, by that, modulate the differentiation of the bone precursor cells to functionally active osteoblasts and osteoclasts [4,5]. Besides these organic mediators, inorganic polymers, e.g., biosilica/silicate and polyphosphate, strongly influence bone metabolism (reviewed in Ref. [6–10]). Inorganic polyphosphate (polyP) is a biopolymer which is synthesized from ATP in enzymatic reactions (bio-polyP) by some microorganisms and multicellular animals (see Ref. [6,11–14]). Depending on the counter-ion bio-polyP occurs in biological systems either in the soluble, amorphous or the crystalline state. Previously, we reported that polyP modulates HA synthesis in the in vitro SaOS-2 cell system [15]. Detailed studies on cellular level that had been performed showed that after exposure of SaOS2 with polyP of different chain lengths, and complexed with Ca2+, the expression of the bone-cell specific alkaline phosphatase (ALP) is induced [16]. This enzyme had been implicated in phosphate metabolism in bone, an assumption that is supported by the observation that phosphate ions accumulate in areas of highest

http://dx.doi.org/10.1016/j.bcp.2015.12.011 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

2

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

ossification [17]; (see Ref. [16]). Some data suggest that the biological function of ALP is to generate inorganic phosphate (Pi) during bone metabolism, while others assume that this enzyme hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi) accumulating during the ATP-driven mineral deposition processes [18]. Our studies [16] indicate that in SaOS-2 cells ALP becomes upregulated allowing a facilitated hydrolysis of polyP at the spot, where Pi is used as a substrate for HA crystal formation. In this context it must be stressed that besides Pi anions, Ca2+ cations are required for HA synthesis. After exposure of SaOS-2 to polyP, the cells release Ca2+ from their intracellular stores into the cytosol [16]. In turn, and to assure that polyP does not chelate Ca2+ out of the culture medium, polyP has been administered as polyP (Ca2+ salt) to the culture. The potential osteogenic influence of polyP is deduced from the finding that in SaOS-2 cells, incubated with polyP, not only an increased expression of bone morphogenetic protein-2 (BMP2) occurs but also an inhibition of phosphorylation of factor IkBa that is supposed to abolish RANKL-mediated NF-kB activation in RAW 264.7 cells [19]. PolyP is not a biologically inert polymer. Compelling evidence could be elaborated in in vitro cell culture experiments with SaOS2 cells, revealing that polyP causes an increased activity/synthesis of ALP [16]. Even more, polyP was shown to increase the intracellular Ca2+ level, suggesting that this polymer modulates intracellular signal transduction pathways. This assumption has been decidedly corroborated by the observation that SaOS-2 cells that had been incubated with polyP showed an increased steadystate expression of BMP2 [19]. This mediator is crucially important for the development of osteoblast precursor cells to mature HAforming osteoblasts; BMP2 controls the expression and the activation of the transcription factor Runx2 [20]. This inducer factor causes a stage-correlated and increasing expression of a series of bone-specific proteins, e.g., of bone-specific alkaline phosphatase, collagen type I, osteopontin and at a later stage of RANKL, asialoprotein, bone sialoprotein and osteocalcin (reviewed in Refs. [7,21]). The recent finding that polyP modulates the function of key pathways involved in extracellular HA deposition (ALP) and in intracellular signaling processes (via BMP2) indicates that polyP acts as an inorganic, morphogenetically-active polymer. Related to Ca2+ are the ions from the lanthanides, since they have very similar ionic radii (Ca2+ with 0.106 nm and e.g., gadolinium (Gd3+) with 0.105 nm) and preferentially form ionic complexes with oxygen donor groups [22,23]. In turn, lanthanide cations have been used, as transition state analogues, for investigations to elucidate Ca2+ binding sites within ion channels (reviewed in Ref. [24]). Gadolinium, in the form of GdCl3, promotes mineral deposition in capillaries as well as mineralization of gastric mucosa, and prolongs the prothrombin time. In addition, GdCl3 blocks K-type Ca2+-channels through a competitive interaction with Ca2+. Furthermore gadolinium ions (Gd3+) cause an increased expression of hepatic cytokines and of several cytokineregulated transcription factors, e.g., c-JUN, C/EBP-b (a transcription factor maintaining homeostasis of cell growth in liver, of insulindependent pathways and controls innate immune system directed against infection to a large number of pathogens) and C/EBP-d (likewise a transcriptional regulator for innate responsive reactions against Gram-negative bacteria) [25]. Finally, the effect of Gd3 + interacts with protein kinase C inhibitors, known to suppress the activity of drug metabolizing enzymes [26]. In the present study we show that polyP, in a complex with gadolinium, as polyP
Gd, causes a superior biological effect, compared to polyP and GdCl3, given as single components separately, and causes a significant induction of HA crystal formation on SaOS-2 cells as well as a distinct gene induction of BMP2 and of collagen type I. These results show that polyP
Gd retains a morphogenetic potential. Since polyP has also been

shown to act as a suitable matrix for bone formation and for drug delivery [27] polyP
Gd might be tested in the future for a potential application as a functional scaffold applicable for some bone tissue engineering approaches provided with – at least – osteoconductive properties. 2. Materials and methods 2.1. Materials Na-polyP (average chain of approximately 40 phosphate units) was obtained from Chemische Fabrik Budenheim (Budenheim; Germany); GdCl3 from Sigma–Aldrich (Taufkirchen; Germany). The sources of all other chemicals has been given previously [16,28]. 2.2. Cells and incubation conditions Human osteogenic sarcoma SaOS-2 cells [29] were cultured in McCoy’s medium (Biochrom, Berlin; Germany) (containing of 5 mM Na-phosphate and 1 mM CaCl2), supplemented with 15% heat inactivated fetal calf serum (FCS; Biochrom), Na-pyruvate (1 mM; Sigma), Ca(NO3)2 (0.5 mM; Sigma), penicillin (100 U/ml; Sigma), and streptomycin (100 mg/ml; Sigma) in 6-well plates (surface area 9.46 cm2; Orange Scientifique, Braine-l’Alleud; Belgium) in a humidified incubator at 37  C and 5% CO2 as described [30]. Routinely, 2  104 cells were added per well (total volume 3 ml). Details are given previously [28]. Different concentrations of GdCl3 were added from a stock solution of 200 mM; polyP was added as Ca2+ salt, complexed by addition of the Na-salt of polyP with Ca2+ at a stoichiometric molar ratio of 2:1 (based on phosphate; [16]). Separately, polyPGd was prepared by mixing of the Na-salt of polyP with GdCl3 in a 3:1 stoichiometric ratio. 2.3. Cell proliferation/viability assays SaOS-2 cells were seeded at a density of 2  104 cells per 3-ml well in a 24-multi-well plate (Orange Scientifique) and cultured for 3 days in McCoy’s medium/15% FCS. Increasing concentrations of GdCl3, polyP or polyP
Gd were added to the cultures. After incubation, cell proliferation was determined applying the colorimetric method based on the tetrazolium salt XTT (Cell Proliferation Kit II; Roche, Mannheim; Germany) as described [31]. 2.4. Induced mineralization in SaOS-2 cells SaOS-2 cells were seeded in multi-well plates. After an incubation period of 2 days mineralization was induced with an activation cocktail, composed of 50 mM ascorbic acid, 10 nM dexamethasone, and 1 mM b-glycerophosphate [16]. Immediately or after an incubation period of up to 7 days the extent of mineralization was assessed by staining with 10% Alizarin Red S (A3757 Sigma) [32]. A quantitative determination of HA was likewise achieved with Alizarin Red S as a probing dye and by performing the reaction in solution [16,33]. The moles of Alizarin Red S bound were determined after generating a calibration curve; the values were normalized to the total DNA amount that had been measured in parallel cultures, using the PicoGreen assay (P0990 Sigma) [34]. 2.5. Digital light microscopy The cell layers were photographed with a KEYENCE BZ8000 epifluorescence microscope (KEYENCE, Neu-Isenburg; Germany) using a S-Plan-Fluor 20 lens.

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

2.6. Scanning electron microscopy and energy-dispersive X-ray spectroscopy Scanning electron microscopic (SEM) analysis was performed with a SU 8000 microscope (Hitachi High-Technologies Europe, Krefeld; Germany) and employed at low voltage (1 kV; suitable for analysis of inorganic morphological structures), as described [34]. Details of the application of energy-dispersive X-ray spectroscopy (EDX) were given previously [16]. The beam-deceleration mode was used to improve the scanning quality, as described [35]. The SEM was coupled to an XFlash 5010 detector, an X-ray detector allowing a simultaneous EDX-based elemental analyses [16]. The mapping was performed by using the HyperMap technique, as described [36]. Prior to the analyses the samples had been thoroughly washed with 50 mM Tris–HCl (pH 7.4; supplemented with 100 mM NaCl). 2.7. Alkaline phosphatase assay Alkaline phosphatase (ALP) was determined in extracts from SaOS-2 cells using a photometric assay (APF Sigma) [16,37]. After incubation, the cells were washed with phosphate-buffered saline (PBS) and then homogenized in a 12 mM Tris/NaHCO3 buffer (pH 6.8; with 1 vol.% of Triton X-100). After centrifugation (15,000  g, 5 min, 4  C) the supernatant was collected for determination of protein and DNA concentration and of ALP activity, as described [37]. The enzyme assay (200 ml) was composed of 0.1 M 2-amino2-methyl-1-propanol (pH 10.5; A9199 Sigma), 2 mM MgCI2 and the reagent 2 mM 4-nitrophenylphosphate (N9389 Sigma); aliquots of 20 ml of cell extract each were added to the assays. After termination of the assay (10 min), the absorbance was measured at 410 nm. After establishment of a calibration curve (p-nitrophenol) the enzyme activity was quantified. Six parallel assays were performed and the mean values (SD) were calculated.

3

(NM_001200.2 [38]), Fwd: 50 -ACCCTTTGTACGTGGACTTC-30 (nt1681 to nt1700) and Rev: 50 -GTGGAGTTCAGATGATCAGC-30 (nt1785 to nt1804, 124 bp) and human collagen type I (NM_000088 [39]) Fwd: 50 -ATGCCTGGTGAACGTGGT-30 [nt2311 to nt2328] and Rev: 50 -AGGAGAGCCATCAGCACCT-30 [nt2397 to nt2379] (87 bp). The threshold position was set to 50.0 relative fluorescence units above PCR subtracted baseline for all runs. Expression levels were normalized to the reference gene GAPDH, essentially as described [34]. 2.10. Statistical analysis After finding that the respective values follow a standard normal Gaussian distribution and that the variances of the respective groups are equal, the results were statistically evaluated using paired Student’s t-test [40]. All given statistical P-values are based on the comparison between individual test groups (polyP plus/minus GdCl3) and the respective control (absence of the test compound[s]—or time 1 day of the incubation period). 3. Results 3.1. Effect of polyP
Gd, GdCl3 or polyP on proliferation/viability of SaOS-2 cells The effects of polyP
Gd as well as of the single components polyP and GdCl3 on proliferation/viability of SaOS-2 cells were determined by applying the MMT colorimetric assay. The data revealed that the viability/proliferation of SaOS-2 cells, deduced on the values of the extent of development of the dye formazan, did not change within the tested concentration range of 0.3–30 mM for GdCl3, polyP or polyP
Gd (Fig. 1). 3.2. Gadolinium-induced mineralization in SaOS-2 cells: Alizarin Red staining

2.8. Degradation of polyP and incubation conditions The medium was collected from SaOS-2 cells, incubated with 5 mM polyP
Gd per 1 ml for 5 days at 37  C. Aliquots of this medium were added to 20 mg of polyP (Na+ salt) or 20 mg polyP
Gd per 1 ml–150 ml culture medium. The duration of the incubation was either 1 h (taken as a control), or 72/96 h; then the samples were subjected to gel electrophoresis. The size of the chain length of polyP was determined by gel electrophoresis using 7 M urea/16.5% polyacrylamide gels [15]. The gels were stained with toluidine blue.

The influence of gadolinium on the extent of mineralization of SaOS-2 cells was determined in vitro using McCoy’s medium/10% FCS and applying Alizarin Red S as a dye to monitor HA formation. For the experiments shown here the concentrations of the test

2.9. Quantitative real-time RT-PCR analysis Quantitative real-time PCR (qRT-PCR) determination of the expression of BMP2 and collagen type I was performed as described [28,34]. In brief, SaOS-2 cells were incubated as described; then the cells were harvested, total RNA was extracted and cleaned of possible DNA contamination by DNAse I treatment. After firststrand cDNA synthesis, using the M-MLV reverse transcriptase (RT) (Promega, Mannheim; Germany), approximately 5 mg of total RNA was used for qRT-PCR in a 40 ml reaction mixture in an iCycler (BioRad, Hercules, CA). The reactions were run in triplicate using 1/10 serial dilutions. Then the samples were supplemented with the SYBR Green master mixture (ABgene, Hamburg; Germany) and 5 pmol of each primer pair for the following three transcripts: for the house keeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase, GenBank accession number NM_002046.3) forward primer Fwd: 50 -ACTTTGTGAAGCTCATTTCCTGGTA-30 [nt1019 to nt1043] and reverse primer Rev: 50 -TTGCTGGGGCTGGTGGTCCA30 (nt1117 to nt1136) (product size 118 bp); as well as for BMP2

Fig. 1. The MTT colorimetric assay was applied to assess the viability/proliferation of SaOS-2 cells in vitro. Addition of GdCl3 alone (open bars) did not change the viability over the concentration range between 0.3 and 30 mM; likewise polyP did not affects growth (cross-hatched bars). If the cells are exposed to polyP
Gd the viability changed insignificant around the 100% viability (striped bars). The recordings of developed formazan dye in the controls (filled bars) were set to 100%. The results are expressed as means (n = 10 experiments each)  SEM.

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

4

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

compounds (GdCl3 or polyP) and polyP
Gd were set to 5 mM. In the absence of a cocktail to activate mineralization the staining intensity is low (Fig. 2A-a). Addition of the activation cocktail, consisting of b-glycerophosphate/ascorbic acid/dexamethasone, to the cells increased the staining intensity significantly (Fig. 2A-b). Addition of GdCl3 or polyP(Ca2+ salt) intensified the staining intensity of the SaOS-2 cells in the presence of the activation cocktail (Fig. 2A-c and A-d). Even more, if the two agents were applied together, as polyP
Gd, a stronger boost was observed (Fig. 2A-e). The same reagent, Alizarin Red S was also applied to monitor quantitatively the effect of the compounds on HA in the liquid phase (Fig. 2B). In the absence of an activation cocktail the amount of Alizarin Red S complex formed was lower than 0.03 nmol/mg DNA (not shown in Fig. 2B) and increased in the presence of the cocktail during the 5 days incubation to 0.31  0.06 nmol/mg (Fig. 2B). This extent of mineralization substantially increased in activated SaOS-2 cells during an incubation period of 7 days if the test compounds were added simultaneously with the activation cocktail. The increase was already significant after a 3-days incubation period (correlated to the controls [activated SaOS-2 cells], without polyP and GdCl3 at day 5)—and was even more pronounced after 7 days. If compared to polyP
Gd (1.23  0.11

nmol/mg, 7 days after addition of the activation cocktail), the enhancement was less pronounced if the two components are added separately as GdCl3 (0.61  0.08 nmol/mg) or polyP (0.86  0.08 nmol/mg). The significant increase of the AR-reacting material in SaOS-2 cells, exposed to polyP
Gd versus polyP and GdCl3 alone is clearly seen 5 days and 7 days after addition of the complex (Fig. 2B). 3.3. Fine structure of HA crystals on SaOS-2 cells The HA crystal formation onto SaOS-2 cells, after an incubation period of 5 days, had been inspected by SEM analysis. In the absence of the activation cocktail no nodules could be identified on the cells (Fig. 3A) . In contrast, if the cells were incubated with the mineralization activating cocktail distinct HA nodules could be identified (Fig. 3B). In the presence of the activation cocktail the cultures were incubated with GdCl3 (Fig. 3C), polyP (Fig. 3D), or both components together, as polyP
Gd (Fig. 3E). It becomes apparent that the density of the nodules in activated cultures incubated with GdCl3 or with polyP alone is lower, compared to polyP
Gd. At higher magnification it can be observed that the nodules consist of irregularly arranged prism-like nanorods (Fig. 3F–H). The average sizes of the crystallite nodules is larger

Fig. 2. Influence of GdCl3, polyP, and polyP
Gd on HA mineralization by SaOS-2 cells. The cells were incubated with 5 mM of each of the compounds. (A) The cells were grown for 7 days in the (a) absence or (b) presence of the activation cocktail. In separate experiments the cultures were incubated with the activation cocktail and additionally with (c) GdCl3, (d) polyP, or (e) polyP
Gd. After incubation, the cell assays were stained with Alizarin Red S. to assess the degree of magnification, the diameters of the wells measure 15 mm. (B) To determine quantitatively the HA mineralization in SaOS-2 cells, the cultures were assayed at the end of the incubation with Alizarin Red S, using the spectrophotometric assay. Standard errors of the means are shown (n = 5 experiments per time point). Significant differences between the control group [without polyP and GdCl3] and the experimental group [polyP  GdCl3] are marked (*);*P < 0.05. The significant increase of mineralization of SaOS-2 cells exposed to polyP
Gd in comparison with polyP or GdCl3, is separately marked (**).

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

5

Fig. 3. Effect of GdCl3, polyP, and polyP
Gd on the formation of HA nodules by SaOS-2 cells; 5 mM of each compound were added to the cells. SEM analysis. The cultures were incubated for 5 days either (A) in the absence or (B) in the presence of the activation cocktail. (C and F) Incubation of the activated cells with GdCl3, (D and G) with polyP, or (E and H) with polyP
Gd. Some nodules (no) and cell layers (c) are marked.

in cultures treated with the two components together as polyP
Gd (Fig. 3H), than those which are formed during exposure of the single components (Fig. 3F–G). 3.4. Element analyses of the nodules formed The element analyses of the nodules formed by SaOS-2 cells were performed for two reasons; first, to identify that the nodules consist of organic material, of Ca2+ and phosphate. The SaOS-2 cultures were incubated for 7 days in the presence of 5 mM polyP
Gd (Fig. 4A and B) . The EDX analysis of an area, including a part of a nodule, shows signal peaks for C, N, O and Na and also P and Ca, indicative for HA. A strong signal for Si is seen that originates from the Si wafer. It should be mentioned that under the conditions used, no signals for Gd could be detected, which should appear at 1.185 keV (for Gd Ma; [41]). Line-scan analysis was performed through a HA nodule region (Fig. 4C and D) and revealed that the nodules are indeed composed of high levels of P and Ca, and also C. Again no Gd signals could be recorded. 3.5. Induction of ALP activity Along the experiments reported previously [16], the level of ALP increased in SaOS-2 cells after exposure to 5 mM GdCl3, polyP or, as shown in the presented series of experiments, also to polyP
Gd (Fig. 5). The analytical data show that already 3 days after exposure of SaOS-2 cells, activated with the mineralization activating cocktail with all three compounds, a significant increase in the intracellular ALP activity occurs. The effect of GdCl3 was lowest (7.7  1.0 mmol/min/mg protein) but still significantly higher than the activity seen in the controls (3.2  0.7 mmol/min/mg); higher are the activities in cells incubated with polyP (10.3  1.5 mmol/ min/mg) or with polyP
Gd (12.9  1.4 mmol/min/mg). As already

described recently [16], the peak of compound-caused increase of ALP is seen at day 5, again with the highest activity for polyP
Gd (22.1  4.1 mmol/min/mg), compared to polyP alone (14.3  1.7 mmol/min/mg), or to GdCl3 (7.7  1.1 mmol/min/mg). At this stage the effect of polyP
Gd is significantly higher, if compared with polyP or GdCl3 alone; likewise the increase of ALP activity in response to polyP to GdCl3 is significantly higher than the one of GdCl3 alone. After that stage the activities decline to levels seen at day 3. 3.6. Degradation of polyP by exposure to culture medium The experiments in the previous paragraph show that cells that had been exposed to polyP
Gd show intracellularly an elevated level of ALP. In order to asses, if also in the incubation medium this enzyme exists, such a medium samples was incubated with polyP and also polyP
Gd. The medium was collected from SaOS-2 cells that had been incubated for 5 days with 5 mM polyP
Gd and then added to polyP or polyP
Gd. The gel electrophoretic analyses revealed that polyP was not significantly degraded by the medium during a 72 h incubation period (Fig. 6). In contrast, the polymer polyP
Gd was partially degraded after 72/96 h (Fig. 6); after staining with toluidine blue it becomes visible that polyP samples did not degrade markedly, while complexed polyP
Gd showed a higher degradation to oligo-phosphate after 72 h or 96 h, respectively. 3.7. Expression of BMP2 and collagen type I genes Recently we identified that high concentrations (100 mM) of polyP cause a slight, but significant, increase of the steady-state expression of the BMP2 [19] and also of collagen type I (unpublished). In continuation, we determined here, if GdCl3,

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

6

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

Considering published data [42,43] indicating that in SaOS2 cells BMP2 and collagen type I genes are expressed and induced, in parallel or sequentially, the qRT-PCR experiments were extended for collagen type I. The qRT-PCR results showed that again polyP
Gd caused a strong induction of gene expression already at day 5, while a longer incubation period was required to observe a significant upregulation of collagen type I if the cells are exposed to GdCl3 or polyP (Fig. 7B). Again, the expression level of collagen type I in polyP
Gd is significantly higher, if compared with the level in cells, treated with polyP or GdCl3 alone. 4. Discussion

Fig. 4. Element distribution in a layer of SaOS-2 cells that had been incubated with polyP
Gd. (A) Cells were grown for 7 days in the activation cocktail together with 5 mM polyP
Gd and then analyzed by SEM. The circle marks the area with a nodule (no) which had been analyzed by EDX. (B) The area, selected in (A), was analysed by EDX; the spectrum highlights the biogenic elements and also P and Ca. (C) Magnification of an area along a nodule (no) by SEM imaging. The analysis was performed along the indicated line. (D) EDX line-scan profiles showing the relative intensities of C, P and Ca as a function of position. The nodule area (no) and the cellular surroundings (c) are marked.

either administered alone, or in complex with polyP is superior to polyP. Therefore, activated SaOS-2 cells were incubated with these compounds for 1 up to 7 days, using the lower concentration of 5 mM for those compounds each (Fig. 7). Applying the technique of qRT-PCR it was found that already at day 3 a significant upregulation (1.6-fold; with respect to the expression as day 1) of the steady-state level for BMP2 is measured in those cells which had been treated with polyP
Gd (Fig. 7A). At day 5 in all assays shown here, a significant increase in the steady-state expression was seen, both for GdCl3 alone (1.4-fold), polyP (1.4-fold) and also for polyP
Gd (2.1-fold). The stimulatory effect of polyP
Gd on the expression of BMP2 in activated cells is significant if compared with the steady-state level of the respective transcript in cells exposed to polyP or GdCl3 alone.

PolyP has been identified in all higher eukaryotic organisms tested so far [44], and is deposited there intracellularly in specialized tissues and organelles [44]. The length of the biogenic polyanionic polyP varies around 40 residues (reviewed in Refs. [13,45]); it is deposited as largely insoluble polymers, complexed with basic amino acids or polycationic spermidine or with Mg2+ or Ca2+. To accomplish the biological function polyP must be present in the soluble transport form as a salt with Na+ or (to a smaller extent) with K+. Especially the Na+ and K+ salts of polyP can be considered as the transportable forms of polyP [46] (reviewed in Ref. [12]), which undergo conversion to more insoluble salts with other metals (e.g., Zn, Fe, Cu, and Cd). By that, polyP elicits their biological functions. Stimulated by the recent finding that Gd3+ promotes Ca-phosphate precipitation onto bone cells [47] we incubated Na-polyP in complex with GdCl3 (stoichiometric ratio 3:1) in order to allow the formation of polyP
Gd, a salt that is largely insoluble (as gadolinium polyP; [46]). In this form we tested the potential bi-functionality of polyP
Gd to act as both an osteoblast-stimulating polymer [via polyP] [16] and as a promoter of Ca-phosphate precipitation [Gd3+] [47]. The data obtained, and presented here, support this expectation. In the first step, the toxicity of gadolinium, as GdCl3 and as polyP
Gd, was tested. Within the concentration range of 0.3– 30 mM gadolinium turned out not to impair growth/proliferation of SaOS-2 cells. This is an expected result, since GdCl3 was already previously found to be non-toxic on cells in culture up to concentrations of 200 mM [47] and polyP did not affect cell growth up to 100 mM [19]. For the functional and molecular biological assays, concentrations for polyP and GdCl3 had been applied which allowed a (potential) higher discrimination/resolution of the data obtained and not to work at (potential) saturating/ plateau levels of the compounds. Accordingly, in this study we have chosen the low concentration of 5 mM polyP and also of 5 mM polyP
Gd. This concentration has previously been found to be the threshold value of being ineffective and started to cause a biological effect [16,19]; in those studies it was determined that 10 mM polyP displays just a slight increase in HA mineralization by SaOS-2 cells. In the presented study we could confirm that the low concentration of 5 mM polyP causes a significant deposition of HA on SaOS-2 cells, as determined by Alizarin Red S staining and by Alizarin Red S quantification of HA in solution. The effect of GdCl3 was comparably moderate, even though significant. However, if the complex polyP
Gd is used a strong increase of HA formation by the osteoblast-like cells could be measured. The effect of enhanced HA formation was twice as high for polyP
Gd, compared with polyP alone. In a first attempt to assure that the increased Alizarin Red S staining is due to a higher HA (Ca-phosphate) deposition SEM analyses had been performed. Those SEM images confirmed that the density of the Ca-phosphate nodules on the surface of the cells is distinctly higher in assay with polyP
Gd, compared to GdCl3 or polyP alone. It often appears that the HA nodules tend to cluster together in the assay with polyP
Gd, while – under the conditions

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

7

Fig. 5. Increase in ALP enzyme activity in SaOS-2 cells after treatment with GdCl3 (open bars), polyP (cross-hatched bars) and polyP
Gd (striped bars). The cells were incubated with 5 mM of either GdCl3, polyP or polyP
Gd. After an incubation period for 1–7 days the cells were broken and the extracts were measured for ALP activity. Standard errors of the means are shown (n = 6 experiments per time point). The significance (*P < 0.05) between the enzyme activities in the test samples (after 3 days, 5 days, or 7 days of incubation) and the respective base values at 1 day are marked; *P < 0.05. The significant increase of the ALP activity at day 5 caused by polyP
Gd is significantly higher compared to the ones elicited by polyP or GdCl3 (marked with **).

used – the nodules onto the cells treated with polyP or GdCl3 remain distinctly isolated. This observation might hint that in the polyP
Gd-treated cultures the cells form a fiber reinforced network between them [48], perhaps based on collagen type I. To clarify and ascertain that crystals seen by SEM are indeed composed (at least mainly) of Ca-phosphate an EDX analyses had been performed. The overall mapping of the surfaces of the nodule-forming SaOS-2 cells, incubated with polyP
Gd, revealed that those cell regions are free of detectable gadolinium; no signals for Gd Ma [40] at 1.185 keV could be detected. Only signals for calcium and phosphorus but not for gadolinium could be resolved, in addition to other “biogenic organic elements”. From the data available it is conceivable that polyP displays after neutralization with Ca2+ its biological effect, more specific (i) upregulation of intracellular Ca2+ concentration, (ii) activation of ALP and (iii) increase of the intracellular ATP level (reviewed in

Fig. 6. Degradation of polyP after incubation with medium from SaOS-2 cells that had been incubated for 5 days with 5 mM polyP
Gd. Subsequently, either polyP or complexed polyP
Gd had been incubated with the medium for 1 h or 72/96 h. Then the samples were analyzed by urea/polyacrylamide gel electrophoresis. Migrations of the polyP standards with a chain length of 80, 40, or 10 phosphate units were used as markers.

Fig. 7. Effect of polyP, polyP
Gd (polyP U+002E Me) or GdCl3 (MeCl3) on BMP2 (A) and collagen type I (COL) (B) gene expression. SaOS-2 cells were incubated with these compounds for 1, 3, 5, or 7 days; then RNA was extracted from the cultures and the steady-state levels of the respective transcripts were quantified by qRT-PCR; in parallel the transcript level of GAPDH was determined and used as reference for normalization. The expression level of the genes after exposure to Gd3+ (open bars), polyP (cross-hatched bars) and polyP
Gd ([polyP U+002E Me]striped bars) are given as a function to the one of GAPDH. n = five experiments per time point; *P < 0.05 (the significance values have been calculated between the gene expression values at time 3 days, 5 days, or 7 days and the respective base values at 1 day [*]). The significant upregulation of both the BMP2 and the COL in cells exposed to polyP
Gd with respect to polyP or GdCl3 are differently marked (**).

Refs. [10,14]). Stimulated by this finding and based on earlier observations [15,49], showing that human mandibular-derived osteoblast-like cells as well as mouse tissue contain higher levels of polyP, it even appears that polyP might be taken up as polymer into the intracellular space. Even more, it is hypothesized [49] that intra- and extra-cellularly existing polyP is under a continuous remodeling process mediated by ALP, and by that, allows morphogenetic processes to occur in parallel with HA formation of the skeleton. Considering those data the question was asked here if gadolinium, complexed with polyP, displays a more potent inducer of ALP activity in comparison to GdCl3 or polyP alone. Again, using the low concentration of 5 mM polyP
Gd, in contrast to the previously applied 100 mM polyP [16], it is measured that the stimulating effect of polyP
Gd on ALP synthesis and ALP gene expression exceeds that of polyP or of GdCl3 by a factor of around two. To obtain a direct proof if the accessibility of the ALP is different to polyP
Gd, compared to polyP, in vitro incubation studies with culture medium had been applied. The medium had been collected from cultures of SaOS-2 cells, incubated for 5 d with 5 mM polyP
Gd. Using this medium it was determined that polyP is only marginally, if at all, degraded, in contrast to polyP
Gd that is substantially hydrolyzed. These data can be explained under the assumption that polyP
Gd causes a more efficient amplifying effect

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

8

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

Fig. 8. Schematic effect of polyP
Gd on development and function of osteoblasts. It is proposed that polyP
Gd is degraded by ALP (either extra- or intracellularly) to oligo- or mono-phosphate units. After dissociation of Gd3+ from the oligoP and mono-phosphate the cation stimulates the extracellular calcium-sensing receptor (ECSR) that might activate an intracellular signal transduction pathway [53], ultimately resulting in an increased expression of BMP2. This mediator is then released into the extracellular space and contributes to the development of osteoblast precursor cells to mature functional osteoblasts. In those cells the expression of the genes encoding for ALP and collagen type I is induced. Finally, deposition of HA occurs under consumption of phosphate and Ca2+, resulting in an increased bone formation.

on intra-, and perhaps also extracellular ALP activity. However, the difference between the single component polyP and the bicomponent polyP
Gd is not merely an ionic bond formation between PO43 and Gd3+ but a stereochemical distortion of the polyP chain. Complex formation with Ca2+ and – even more – with Gd3+ results either in an intramolecular twisting of the chain or a cross-linkage of different polyP chains (see Refs. [12,50]). This interpretation opens a new avenue in the understanding of the polyP complex interaction via Gd3+ linkages in the supposition that Gd3+ adds new binding affinities to heterophilic biomolecules, not existing in Na+-, K+-, and also Ca2+ polyP salts; the ALP-driven disintegration of polyP
Gd is sketched in Fig. 8 . The functional consequence of the outlines of the previous chapter could be that polyP, in the complex with Gd3+, is delivered to bone cells, more specifically to osteoblasts, where the polymer is hydrolyzed, intra- and/or extracellularly, to (at least) oligo-, or to mono-phosphate units and Gd3+. In turn, phosphate acts as a substrate for HA synthesis proceeding at the cell-surface, while Gd3+ might interact with cell surface receptor(s), e.g., with the extracellular calcium-sensing receptor a G-protein coupled receptor which senses extracellular levels of calcium ion [51]. Recently we obtained experimental evidence that polyP is taken up by the cells, very likely via an endocytosis-like mechanism [52].

Intracellularly, the natural polymer causes an increase of the number of mitochondria and, perhaps as a consequence, induces the production of ATP [53]. Therefore, we coined the term “metabolic fuel” in order to emphasize the property of polyP to cause an increased intracellular ATP formation in the mitochondria. The detailed mechanism of action of polyP to the mitochondrial metabolism remains to be studied. Based on the existing data it is most likely that polyP undergoes enzymatic hydrolysis also within the cells and alters there the cellular metabolism by means of two signalling arms, via polyP and separately via Gd3+. Based on these facts it was consequent to elucidate the effect of polyP
Gd on BMP2 gene expression in SaOS-2 cells. The data obtained clearly showed that a concentration of 5 mM polyP
Gd was enough to increase the expression of BMP2 by 2-fold during a 7 days incubation period. This finding confirms previous data, obtained with intestinal epithelial cells, that in response to Gd3+ exposure an activation of the extracellular calcium-sensing receptor follows (scheme in Fig. 8). To continue in the proposed sequence of induction steps, the question was raised if polyP
Gd causes an increased induction of the collagen type I gene [42,54,55]. The presented qRT-PCR data unequivocally show that 5 mM polyP
Gd, in contrast to free Gd3+ or polyP, considerably upregulates the expression of collagen type I.

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

The data presented here revealed that exposure of SaOS-2 cells to polyP
Gd, a salt complex formed between polyP and Gd3+, causes a distinct increase in HA crystal formation. The data favor the idea that ALP, expressed by SaOS-2 cells, becomes activated if the cells are incubated with polyP or polyP
Gd. ALP degrades polyP
Gd at the location around those osteoblast-like cells. The resulting oligo- or mono-phosphate units, as well as the Gd3+ cations, now separated from the polymer, then display their biological function in the vicinity of the SaOS-2 cells. While polyP, and it hydrolysis products, serves as substrate for HA formation onto bone cells, Gd3+ (perhaps together with polyP) causes an initiation of a differentiation pathway for osteoblasts, with a sequential expression of BMP2, ALP, and collagen type I. A schematic representation of the different (proposed) steps initiated by polyP-Gd3+ is given in Fig. 8. To conclude, polyP is not an inert polymer but acts as a morphogenetically active polymer. This function is especially seen in in vitro studies using polyP
Gd. Having this new set of data it is indicated to test in a next step the potential influence of polyP
Gd on the differentiation of mesenchymal stem cells into osteoblasts in vitro, as outlined [56], and subsequently in in vivo model(s) the potential effect of a polyP
Gd as a matrix and simultaneously also as a stimulator of morphogenetic processes, providing a favorable microenvironment for bone formation. Acknowledgements W.E.G.M. is a holder of an ERC Advanced Investigator Grant (No. 268476 “BIOSILICA”). This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schr 277/10-3), the European Commission (“Bio-Scaffolds”: No. 604036; “MarBioTec*EU-CN*”: No. 268476; and “BlueGenics”: No. 311848), and the International Human Frontier Science Program. References [1] Y. Tanaka, Y. Okada, T. Nakamura, Inter- and intracellular signaling in secondary osteoporosis, J. Bone Miner. Metab. 21 (2003) 61–66. [2] F. Lazner, M. Gowen, D. Pavasovic, I. Kola, Osteopetrosis and osteoporosis: two sides of the same coin, Hum. Mol. Genet. 8 (1999) 1839–1846. [3] J.P. Bilezikian, L.G. Raisz, T.J. Martin, Principles of Bone Biology, Academic Press/Elsevier Science Publishing Co., Inc., New York, 2008. [4] H. Ohgushi, M. Okumura, T. Yoshikawa, K. Inoue, N. Senpuku, S. Tamai, E.C. Shors, Bone formation process in porous calcium carbonate and hydroxyapatite, J. Biomed. Mater. Res. 26 (1992) 885–895. [5] L. Lin, K.L. Chow, Y. Leng, Study of hydroxyapatite osteoinductivity with an osteogenic differentiation of mesenchymal stem cells, J. Biomed. Mater. Res. 89A (2009) 326–335. [6] H.C. Schröder, W.E.G. Müller, Inorganic Polyphosphates: Biochemistry, Biology, Biotechnology, Springer Press, Berlin, 1999. [7] X.H. Wang, H.C. Schröder, M. Wiens, H. Ushijima, W.E.G. Müller, Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation), Curr. Opin. Biotechnol. 23 (2012) 570–578. [8] X.H. Wang, H.C. Schröder, W.E.G. Müller, Enzyme-based biosilica and biocalcite: biomaterials for the future in regenerative medicine, Trends Biotechnol. 32 (2014) 441–447. [9] X.H. Wang, H.C. Schröder, W.E.G. Müller, Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine, Int. Rev. Cell Mol. Biol. 313 (2014) 27–77. [10] X.H. Wang, H.C. Schröder, W.E.G. Müller, Polyphosphate as a metabolic fuel in Metazoa: a foundational breakthrough invention for biomedical applications, Biotechnol. J. (2015) , doi:http://dx.doi.org/10.1002/biot.201500168. [11] B. Lorenz, J. Münkner, M.P. Oliveira, A. Kuusksalu, J.M. Leitão, W.E.G. Müller, H. C. Schröder, Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis, Biochim. Biophys. Acta 1335 (1997) 51–60. [12] I.S. Kulaev, V. Vagabov, T. Kulakovskaya, The Biochemistry of Inorganic Polyphosphates, John Wiley & Sons Inc., New York, 2004. [13] N.N. Rao, M.R. Gómez-Garcıa, A. Kornberg, Inorganic polyphosphate: essential for growth and survival, Annu. Rev. Biochem. 78 (2009) 605–647. [14] W.E.G. Müller, E. Tolba, H.C. Schröder, X.H. Wang, Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration, Macromol. Biosci. 15 (2015) 1182–1197. [15] G. Leyhausen, B. Lorenz, H. Zhu, W. Geurtsen, R. Bohnensack, W.E.G. Müller, H. C. Schröder, Inorganic polyphosphate in human osteoblast-like cells, J. Bone Miner. Res. 13 (1998) 803–812.

9

[16] W.E.G. Müller, X.H. Wang, B. Diehl-Seifert, K. Kropf, U. Schloßmacher, I. Lieberwirth, G. Glasser, M. Wiens, H.C. Schröder, Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro, Acta Biomater. 7 (2011) 2661–2671. [17] I.J. Lorch, Alkaline phosphatase and the mechanism of ossification, J. Bone Joint Surg. 31 (1949) 94–99. [18] J.L. Millan, Alkaline phosphatases structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes, Purinergic Signal. 2 (2006) 335–341. [19] X.H. Wang, H.C. Schröder, B. Diehl-Seifert, K. Kropf, U. Schlosmacher, M. Wiens, W.E.G. Müller, Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro, J. Tissue Eng. Regener. Med. 7 (2013) 767–776. [20] S.J. Lee, S.W. Kang, H.J. Do, I. Han, D.A. Shin, J.H. Kim, S.H. Lee, Enhancement of bone regeneration by gene delivery of BMP2/Runx2 bicistronic vector into adipose-derived stromal cells, Biomaterials 31 (2010) 5652–5659. [21] J. Sodek, M.D. McKee, Molecular and cellular biology of alveolar bone, Periodontology 24 (2000) 99–126. [22] E. Nieboer, The lanthanide ions as structural probes in biological and model systems, Struct. Bond. 22 (1975) 1–47. [23] R.D. Shannon, Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides, Acta Crystallogr. A 32 (1976) 751–767. [24] A. Franco, B.D. Winegar, J.B. Lansman, Open channel block by gadolinium ion of the stretch-inactivated ion channel in mdx myotubes, Biophys. J. 59 (1991) 1164–1170. [25] R. Rai, S. Yang, C. McClain, C. Karp, A. Klein, M. Diehl, Kupffer cell depletion by gadolinium chloride enhances liver regeneration after partial hepatectomy in rats, Am. J. Physiol. 270 (1996) G909–G918. [26] S. Kim, J. Cho, Y. Chung, E. Ahn, K. Lee, Y. Han, Suppression of xenobioticmetabolizing enzyme expression in rats by acriflavine a protein kinase C inhibitor. Effects on epoxide hydrolase, glutathione S-transferases, and cytochromes P450, Drug Metab. Dispos. 26 (1998) 66–72. [27] S.C. Schofield, B. Berno, M. Langman, G. Hall, M.J. Filiaggi, Gelled calcium polyphosphate matrices delay antibiotic release, J. Dent. Res. 85 (2006) 643–647. [28] M. Wiens, X.H. Wang, H.C. Schröder, U. Kolb, U. Schloßmacher, H. Ushijima, W. E.G. Müller, The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells, Biomaterials 31 (2010) 7716–7725. [29] J. Fogh, J.M. Fogh, T. Orfeo, One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice, J. Natl. Cancer Inst. 59 (1977) 221–226. [30] W.E.G. Müller, A. Boreiko, X.H. Wang, A. Krasko, W. Geurtsen, M.R. Custódio, T. Winkler, L. Luki c-Bilela, T. Link, H.C. Schröder, Morphogenetic activity of silica and bio-silica on the expression of genes, controlling biomineralization using SaOS-2 cells, Calcif. Tissue Int. 81 (2007) 382–393. [31] K. Mori, M. Berreur, F. Blanchard, C. Chevalier, I. Guisle-Marsollier, M. Masson, F. Rédini, D. Heymann, Receptor activator of nuclear factor-kB ligand (RANKL) directly modulates the gene expression profile of RANK-positive Saos2 human osteosarcoma cells, Oncol. Rep. 18 (2007) 1365–1371. [32] H.C. Schröder, A. Borejko, A. Krasko, A. Reiber, H. Schwertner, W.E.G. Müller, Mineralization of SaOS-2 cells on enzymatically (Silicatein) modified bioactive osteoblast-stimulating surfaces, J. Biomed. Mater. Res. B: Appl. Biomater 75B (2005) 387–392. [33] C.A. Gregory, W.G. Gunn, A. Peister, D.J. Prockop, An alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction, Anal. Biochem. 329 (2004) 77–84. [34] M. Wiens, X.H. Wang, U. Schloßmacher, I. Lieberwirth, G. Glasser, H. Ushijima, H.C. Schröder, W.E.G. Müller, Osteogenic potential of bio-silica on human osteoblast-like (SaOS-2) cells, Calcif. Tissue Int. 87 (2010) 513–524. [35] A. Endo, M. Yamada, S. Kataoka, T. Sano, Y. Inagi, A. Miyaki, Direct observation of surface structure of mesoporous silica with low acceleration voltage FESEM, Colloids Surf. A: Physicochem. Eng. Aspects 357 (2010) 11–16. [36] T. Salge, R. Terborg, EDS microanalysis with the silicon drift detector (CDD): innovative analysis options for mineralogical and material science application, Anadolu Univ. J. Sci. Technol. 10 (2009) 45–55. [37] R.J. Majeska, G.A. Rodan, Alkaline phosphatase inhibition by para thyroid hormone and isoproterenol in a clonal rat osteosarcoma cell line: possible mediation by cAMP, Calcif. Tissue Int. 34 (1982) 59–66. [38] I. Kochanowska, S. Chaberek, A. Wojtowicz, B. Marczynski, K. Włodarski, M. Dytko, K. Ostrowski, Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton, BMC Musculoskelet. Disord. 8 (2007) 128. [39] S. Marlovits, M. Hombauer, M. Truppe, V. Vècsei, W. Schlegel, Changes in the ratio of type-I and type-II collagen expression during monolayer culture of human chondrocytes, J. Bone Joint Surg. Br. 86 (2004) 286–295. [40] A. Petrie, P. Watson, Statistics for Veterinary and Animal Science, WileyBlackwell, Oxford, 2013, pp. 85–99. [41] W.A. High, R.A. Ayers, J. Chandler, G. Zito, S.E. Cowper, Gadolinium is detectable within the tissue of patients with nephrogenic systemic fibrosis, J. Am. Acad. Dermatol. 56 (2007) 21–26. [42] H.C. Anderson, H.H.T. Hsu, P. Raval, P.R. Reynold, D.J. Gurley, M.X. Aguilera, L.S. Davis, P.E. Moylan, Bone-inducing agent in Saos-2 cell extracts and secretions, Cells Mater. 8 (1998) 89–98.

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011

G Model BCP 12448 No. of Pages 10

10

X. Wang et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

[43] R.J. Fernandes, M.A. Harkey, M. Weis, J.W. Askew, D.R. Eyre, The posttranslational phenotype of collagen synthesized by Saos-2 osteosarcoma cells, Bone 40 (2007) 1343–1351. [44] K.D. Kumble, A. Kornberg, Inorganic polyphosphate in mammalian cells and tissues, J. Biol. Chem. 270 (1995) 5818–5822. [45] A. Kornberg, N.N. Rao, D. Ault-Riché, Inorganic polyphosphate: a molecule of many functions, Annu. Rev. Biochem. 68 (1999) 89–125. [46] C.F.C. Bonting, G.J.J. Kortstee, A. Boekestein, A.J.B. Zehnder, The elemental composition dynamics of large polyphosphate granules in Acinetobacter strain 210A, Arch. Microbiol. 159 (1993) 428–434. [47] B.D. Gou, S. Bian, T.L. Zhang, K. Wang, Gadolinium-promoted precipitation of calcium phosphate is associated with profibrotic activation of RAW 264.7 macrophages, Toxicol. In Vitro 24 (2010) 1743–1749. [48] M.F. Baslé, F. Grizon, C. Pascaretti, M. Lesourd, D. Chappard, Shape and orientation of osteoblast-like cells (Saos-2) are influenced by collagen fibers in xenogenic bone biomaterial, J. Biomed. Mater. Res. 40 (1998) 350–357. [49] S. Omelon, J. Georgiou, Z.J. Henneman, L.M. Wise, B. Sukhu, T. Hunt, C. Wynnyckyj, D. Holmyard, R. Bielecki, M.D. Grynpas, Control of vertebrate skeletal mineralization by polyphosphates, PLoS One 4 (2009) e5634. [50] F.M. Harold, Inorganic polyphosphates in biology: structure, metabolism, and function, Bacteriol. Rev. 30 (1966) 772–794. [51] G.N. Hendy, L. D'Souza-Li, B. Yang, L. Canaff, D.E.C. Cole, Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia,

[52]

[53]

[54]

[55]

[56]

neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemi, Hum. Mutat. 16 (2000) 281–296. W.E.G. Müller, E. Tolba, Q. Feng, H.C. Schröder, J.S. Markl, M. Kokkinopoulou, X. H. Wang, Amorphous Ca2+ polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells, J. Cell Sci. 128 (2015) 2202–2207. D. Peiris, I. Pacheco, C. Spencer, R.J. MacLeod, The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts, Am. J. Physiol. Gastrointest. Liver Physiol. 292 (2007) G753–G766. S.E. Harris, L.F. Bonewald, M.A. Harris, M. Sabatini, S. Dallas, J.Q. Feng, N. Ghosh-Choudhury, J. Wozney, G.R. Mundy, Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts, J. Bone Miner. Res. 9 (1994) 855–863. T. Katagiri, A. Yamaguchi, M. Komaki, E. Abe, N. Takahashi, T. Ikeda, V. Rosen, J. M. Wozney, A. Fujisawa-Sehara, T. Suda, Bone morphogenetic protein2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage, J. Cell Biol. 127 (1994) 1755–1766. J. George, Y. Kuboki, T. Miyata, Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds, Biotechnol. Bioeng. 95 (2006) 404–411.

Please cite this article in press as: X. Wang, et al., The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.12.011