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Biomaterials 34 (2013) 8671e8680

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Biomaterials

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Induction of carbonic anhydrase in SaOS-2 cells, exposed tobicarbonate and consequences for calcium phosphate crystalformation

Werner E.G. Müller a,*, Heinz C. Schröder a, Ute Schlossmacher a, Vlad A. Grebenjuk a,Hiroshi Ushijima b, Xiaohong Wang a,*

a ERC Advanced Investigator Grant Research Group at Institute for Physiological Chemistry,University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, D-55128 Mainz, GermanybDepartment of Obstetrics and Gynaecology, Nihon University, 4-8-24 Kudanminami, Chiyoda-ku, Tokyo 102-8275, Japan

a r t i c l e i n f o

Article history:Received 11 July 2013Accepted 29 July 2013Available online 14 August 2013

Keywords:Bone metabolismCarbonic anhydraseOsteoblastsSaOS-2 cellsCa-carbonateHydroxyapatite

* Corresponding authors. Tel.: þ49 6131 39 25910;E-mail addresses: wmueller@mail.uni-mainz.de (W

mainz.de (X. Wang).

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.07.096

a b s t r a c t

Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified inlower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown thatafter exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mM, a significantincrease of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cellsbecome denser and larger in the presence of bicarbonate if simultaneously added together with themineralization activation cocktail (b-glycerophosphate/ascorbic acid/dexamethasone). In parallel, withthe increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomesupregulated. This effect, measured on transcriptional level is also substantiated by immunohistologicalstudies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonicanhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-depositproducing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray anal-ysis revealed an accumulation of the signals for the element carbon and, as expected, also for phos-phorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-IIactivity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and asubsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest thatCa-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We proposethat activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiencydiseases.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Bone, a biomineral, is composed of a mineral phase (Ca-de-posits; w60e70% w/w) and an organic matrix (mainly collagen;w20e30% w/w) and 10% of water (reviewed in Refs. [1,2]).The process of mineralization in bone is highly regulated by atuned interplay between the bone-forming cells (osteoblasts) andthe bone-resorbing cells (osteoclasts) in concert with complexorganic extracellular (fibrillar) macromolecules, forming a three-dimensional porous scaffold. During bone formation the

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extracellular matrix undergoes mineralization primarily aroundcollagen fibrils, functioning as the basic building block of the bone.In addition to those fibrillar proteins, non-collagenous proteins actas second components in a regulatory way to the mineralizationprocess. Among those is the dentin matrix phosphoprotein 1(DMP1), non-collagenous, acidic extracellular matrix protein that isinvolved in regulating cellular morphogenesis and differentiation[3]. DMP1, an extracellular matrix protein and a member of thesmall integrin-binding ligands, is crucial for the initial mineralcrystal formation. This protein has a high affinity for Ca2þ and in-duces mineralization in vitro. Hydroxyapatite crystals are depositedonto DMP1 if a Ca2þ and phosphate buffer is supplied [4]. This Ca2þ

deposit formation in vitro is a sequential and stepwise processwhich starts with a rapid nucleation phase during which Ca2þ be-comes bound to DMP1, while the subsequent hydroxyapatite

Fig. 1. Effect Na-bicarbonate on the process of biomineralization in SaOS-2 cells. Afteran incubation of 3 d the medium (RPMI-1640 medium/10% FCS) was replaced andsubstituted by medium lacking (filled bars; open bars) or supplemented with 20 mM

Na-bicarbonate (downwards hatched; upwards hatched). The cells were continued togrow for 1 d, 3 d, or 5 d in the absence (filled) or presence of MAC (open, downwards orupwards hatched). At the indicated incubation period the extent of Ca-biomineralization was determined by spectrophotometric assay with Alizarin Red S.Standard errors of the means are shown (n ¼ 10 experiments per time point).*p < 0.05.

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crystal formation takes days or weeks. Around the DMP1 matrixthe osteoclasts, or odontoblasts, are localized to maintain a dy-namic intracellular Ca2þ balance, controlled by various trans-membraneous Ca2þ transport mechanisms, e.g. Ca-ATPase, Naþ/Ca2þ exchangers and intracellular Ca2þ-binding proteins (see Ref.[5]). In addition to DMP1, other Ca2þ-binding proteins, belonging tothe small integrin-binding ligand N-linked glycoproteins, exist thatare known to play a regulatory role in Ca-phosphate deposition inbone [6].

Besides of the hydroxyapatite-composed bones, biomineralizedotoliths exist that are found in the vestibular labyrinth of thevertebrate ear. They consist besides of organic matrix proteins to90e95% of Ca-carbonate in the aragonite form (see Ref. [7,8]).Otolin, a collagenous protein had been identified as an importantcomponent for the growth and function of otolith structures [9,10].In addition, otoconins, calcium-binding proteins exist in thevestibular system, which initiate Ca-deposition [11]. The detailedmechanism of the biogenic formation of Ca-deposits in the ear isnot yet known. However, since the studies of Pellegrino and Biltz[12] it is known that Ca-carbonate also exists in vertebrate bones.

Under physiological conditions biomineralization of bones,teeth and otoconia in vertebrates mainly proceeds extracellularly,while pathological calcification of soft tissues predominantly oc-curs intracellularly [13]. It has been proposed that bone formation,based on a tightly controlled process between osteoblasts andfibrillar organic structures, starts from collagen fibrils aroundwhich poorly crystalline carbonated apatite crystals, carbonate-apatite aggregates are deposited (see Ref. [14,15]). The analyses,based upon X-ray and electron diffraction, Fourier transforminfrared spectroscopy, as well as on the determination of thechemical composition revealed that e at least under in vitro con-dition e osteoblasts contain low concentrations of carbonate ionsin their Ca2þ/phosphate mineralic phase.

Spectroscopic studies suggested that Ca-deposition in osteo-blasts starts intracellularly in calcium-containing vesicles andsubstantially contributes to the bone apatite formation [15].Amorphous Ca-phosphate particles formed are then released fromthe cells, associate with collagen fibrils and undergo crystallization.In contrast it has been suggested that the extracellular fluid issufficiently saturated with respect to Ca2þ and phosphate, perhapsthere stored as Ca2þ-polyphosphate (Ca-polyP) [16], to allow Ca-phosphate deposition [17].

Ca-phosphate formation [17], like Ca-carbonate deposition [18],is an exergonic process. Like most reactions in the intermediarymetabolism of a biological system, the reactions forming thoseskeletal elements are controlled by enzymes either directly orindirectly, by modulating metabolite or ion transport systems. Thebio-silica spicules of the siliceous sponges have been proven, as afirst skeletal system, to be enzymatically synthesized. It is theprotein silicatein, discovered by Shimizu et al. [19] and Cha et al.[20] that had been shown to be providedwith enzymatic propertiesthat facilitate and accelerate polycondensation of ortho-silicate toproceed [21,22]. With respect to Ca-deposit formation the primeenzymic candidate for the formation of those inorganic nano-micro-particles is the carbonic anhydrase (CA). CAs comprise afamily of enzymes which reversibly catalyze hydration and dehy-dration reactions of CO2/H2CO3 [23,24] and in turn Ca-carbonateprecipitation [25]. Four of the seven metazoan isoenzymes arecytosolic, CA-I, -II, -III, and -VII. Among those, the CA-II is the moststudied and most widely distributed [26]. Experimental evidencehas been presented revealing that the CAs are very likely involvedbone formation [27]. The mammalian CA-II, a cytosolic enzyme, istargeted in intact cell system to the cell membrane [28,29] and wasfound to be upregulated after exposure of SaOS-2 to bicarbonate.Those cells, expressing CA-II in response to bicarbonate, were found

to strongly synthesize Ca2þ-rich deposits, composed of the ele-ments O, Ca, C and P. Furthermore, the synthesis of those depositscan be inhibited by the specific CA-II inhibitor acetazolamide [30].

In the present study we elucidate the effect of bicarbonate on theexpression of the CA-II gene in Ca-phosphate synthesizing SaOS-2cells and measure the effect of this anion on the formation of Ca-phosphate by these cells in the absence or presence of the CA inhib-itor acetazolamide. Then, we analyzed the Ca-phosphate deposits,that are formed on the SaOS-2, by energy-dispersive X-ray (EDX)spectroscopy for an accumulation of carbon, besides of phosphorousand calcium. Finally we tested the inhibitory potential of phosphateon the CA-II activity in order to get an insight into the postulatedfeedback regulatory system between the CA-driven Ca-carbonatedeposition, the subsequent lowering of this process by ortho-phosphate that might be followed by Ca-phosphate deposition.

2. Material and methods

2.1. Cultivation of SaOS-2 cells

SaOS-2 cells (human osteogenic sarcoma cells Ref. [31]) were cultured in RPMI-1640 medium (R6504; Sigma, Taufkirchen; Germany), lacking Na-bicarbonate butcontaining 2 mM L-glutamine [32] and 10% heat-inactivated fetal calf serum (FCS).Themediumwas supplementedwith 1mM CaCl2,100 units/ml penicillin and 100 mg/ml streptomycin, and, where indicated, with 20 mM Na-bicarbonate. The cells wereincubated in 25 cm2

flasks or in six-well plates (surface area 9.46 cm2; OrangeScientifique, Braine-l’Alleud; Belgium) in a humidified incubator at 37 �C [33,34].Routinely, 3�105 cells/well were added (total volume 3 ml). Where indicated thecultures were supplemented with the mineralization activation cocktail (MAC),composed of 5 mM b-glycerophosphate, 50 mM ascorbic acid and 10 nM dexameth-asone [35]. Routinely, the mineralization activation cocktail was added 3 days afterstarting the experiments. During each medium change new MAC was added.

Where indicated with the respective assay the carbonic anhydrase inhibitoracetazolamide (A177 Sigma; [36]) was added to the cultures at a concentration of100 mM.

2.2. Mineralization by SaOS-2 cells in vitro

For a quantitative assessment of the extent of biomineralization the Alizarin RedS spectrophotometric assay for Ca2þ deposits was applied [37,38]. The amount ofbound Alizarin Red S is given in nmoles. Values were normalized to total DNA in thesamples.

Fig. 2. Formation of Ca-mineralization deposits onto SaOS-2 cells. The cells were incubated in the absence of both bicarbonate and MAC (AeC), presence of bicarbonate and absenceof MAC (DeF) and presence of both bicarbonate and MAC (GeI). The cells were analyzed after 5 d in culture by SEM. Some Ca-deposit nodules are marked (no).

1 3 5 7

0.000

0.003

0.006

0.009

0.012

0.015

0.018

Incubation period (d)

CA-II / GAPDH

m

RN

A level

1 mM

3 mM

10 mM

Na-bicarbonate

0

*

* *

*

**

*

Fig. 3. Altered expression of CA-II in SaOS-2 cells in dependence of increasing con-centrations of Na-bicarbonate. After incubation in RPMI-1640 medium/10% FCS for 3 dthe cells were transferred to fresh medium/serum plus MAC, lacking bicarbonate(closed bars) or to medium containing increasing concentrations of bicarbonate; 1 mM

(downwards hatched), 3 mM (open) or 10 mM Na-bicarbonate (upwards hatched). Afteran incubation period of 1e7 d samples were taken and subjected to RNA extraction,followed by qRT-PCR analysis. After an incubation period of 4 d the medium/serumwas changed and replaced by fresh medium/serum, containing the respective bicar-bonate component. Standard errors of the means are shown (n ¼ 5 experiments/timepoint); *p < 0.01.

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In one series of experiments the cultures, growing into culture wells werestained directly on the cover slips with 10% Alizarin red S, after fixationwith ethanol[33].

2.3. Quantitative real-time qRT-PCR

The steady-state expression level of the carbonic anhydrase gene in SaOS-2cells was determined by quantitative real-time RT-polymerase chain reaction(qRT-PCR) as described [39,40]. Amplification was performed for transcripts ofthe human carbonic anhydrase II (CA2) gene (accession number NM_000067)and of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase(GAPDH; No. NM_002046.3). The latter was used as a reference. After extractionfrom SaOS-2 cells RNA was treated with DNAse to eliminate genomic DNA. Then,first strand cDNA synthesis was performed by using the SuperScript III reversetranscriptase in a reaction mixture composed of 5 mg of total RNA, dNTPs, oli-go(dT)18, and reverse transcriptase buffer (Invitrogen, Karlsruhe; Germany) at42 �C for 1 h. After inactivation of the reverse transcriptase (65 �C, 15 min) thereactions were diluted and 2 ml of those dilutions were used as a template forthe 30 ml reaction for qRT-PCR. The reactions were run in a GFX96 PCR machine(Bio-Rad, Munich; Germany) using 1/10 serial dilutions in triplicates [41]. Eachreaction contained “Absolute Blue SYBR Green” master mixture (ABgene,Hamburg; Germany) and 5 pmol each of the following CA2 primer pairs, CA2Fwd: 50-TCCTCGTGGCCTCCTTCCTGAATC-30 (nt717 to nt740) and CA2 Rev: 50-TCAACACCTGCTCGCTGCTGAC-30 (nt850 to nt829) [PCR product size is 134 bp] andthe GAPDH primer pair GAPDH Fwd: 50-CCGTCTAGAAAAACCTGCC-30 (nt843 tont861) and GAPDH Rev: 50-GCCAAATTCGTTGTCATACC-30 (nt1059 to nt1040)[product size is 217 bp]. The reactions were run with an initial denaturation at95 �C for 10 min, followed by 40 cycles each at 95 �C for 20 s, then 58 �C for 15 s,and finally 72 �C for 20 s. Mean Ct values, efficiencies and relative expressionlevels were calculated by the CFX Manager v.3.0 software (Bio-Rad, Munich;Germany).

2.4. Immunocytochemistry

The cells were fixed in paraformaldehyde and then incubated with the primaryantibody, the human carbonic anhydrase II (PoAb-CA2) produced in rabbits (Life-Span Biosciences, Seattle, WA) at a dilution of 1:1500 in blocking solution; incu-bation while shaking at 4 �C was for overnight. As control, the PoAb-CA2 antibodies,adsorbed with recombinant human CA2 (C6624; Sigma) were used [42]. The non-bound antibodies were removed by washing with PBS prior to the incubationwith fluorescently labeled [with Cy5 (indodicarbocyanine)] secondary antibodies(1:3000 dilution). The cell nuclei were stained with DAPI [2-(4-amidinophenyl)-1H-indole-6-carboxamidine; Sigma]. The slices were inspected with an Olympus

AHBT3 microscope under immunofluorescence light at an excitation light wave-lengths suitable for either Cy5 or DAPI.

2.5. Scanning electron microscopy and energy-dispersive X-ray analysis

For the scanning electron microscopic (SEM) analyses a HITACHI SU 8000(Hitachi High-Technologies Europe GmbH, Krefeld; Germany) was employed at lowvoltage (<1 kV; analysis of near-surface organic surfaces) [43]. The SEM microscopewas coupled to an XFlash 5010 detector, an X-ray detector that allows simultaneousenergy-dispersive X-ray (EDX)-based elemental analyses. Likewise, the same

W.E.G. Müller et al. / Biomaterials 34 (2013) 8671e86808674

combination of devices was used for higher voltage (10 kV) analysis during whichthe XFlash 5010 detector was used for element mapping of the surfaces of the de-posits. The HyperMap database was used for interpretation, as described [44]. Morethan three parallel analyses were performed both for the series of SEM photos andthe ones for EDX spectra.

2.6. Carbonic anhydrase activity assay

The recombinant human CA2 enzyme, expressed in Escherichia coli (C6624;Sigma), with a specific activity ofz5000 units/mg was used for the studies. The CO2

hydratase enzyme activity was determined as outlined [45,46]. The original phos-phate buffer was replaced by 20 mM tris(hydroxymethyl)aminomethane buffer (pH7.2). The dehydration reaction was then initiated by adding NaHCO3 to a concen-tration of 40 mM. Then the rate of the reaction was determined during the following

Fig. 4. Reactivity of anti-CA-II antibodies with SaOS-2 cells, incubated in the absence or prshown in image (B), (D), (F) and (H); in parallel the samples were stained with DAPI and theabsence of both bicarbonate and MAC. (C and D) Cultures incubated in the presence of 3 mM

together with MAC. (G and H) Cells incubated as in (E and F) but reacted with adsorbed an

60 s. The enzyme concentration was 100 ng/ml. The catalytic activity is given in mM

CO2 produced s�1.Where indicated 100 mM Na-polyP [Na-polyphosphate] with an average chain

length of approximately 40 phosphate units (Chemische Fabrik Budenheim,Budenheim; Germany) was added to the reaction assay. This polymer was subse-quently subjected to enzymatic hydrolysis using alkaline phosphatase as described[47]. The reaction product after an incubation period of 30 min (37 �C) was added tothe carbonic anhydrase reaction assay.

2.7. Additional methods

The results were statistically evaluated [48]. DNA content was determined byapplication of the PicoGreen method as described [33] using calf thymus DNA as astandard.

esence of bicarbonate for 5 d. The images of the immunostaining with PoAb-CA2 arecorresponding images are given in (A), (C), (E) and (G). (A and B) Cells incubated in theNa-bicarbonate and in the absence of MAC. (E and F) Cells incubated with bicarbonateti-CA-II antibodies. All images are in the same magnification; scale is shown in (G).

Fig. 5. Ca-deposit formation onto SaOS-2 cells in the absence or presence of bicar-bonate (minus/plus BiCa) in cultures lacking or containing MAC (minus/plus dexa-methasone [DEX], ascorbic acid [AA], b-glycerophosphate [P]). The cells werecultivated for 5 d. In addition, the cells were incubated in the absence or presence of100 mM acetazolamide (minus/plus AZ). After incubation the cells were stained withAlizarin red S.

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3. Results

3.1. Effect of bicarbonate on biomineralization of SaOS-2 cells

SaOS-2 were incubated for 3 d in the presence or absence of20 mM Na-bicarbonate; then the cultures were exposed to MAC,containing b-glycerophosphate/ascorbic acid/dexamethasone forup to 5 d as outlined in Fig. 1. After that period the extent of bio-mineralizationwas determined quantitatively with the Alizarin RedS dye. In the absence of bicarbonate and MAC the level of biomin-eralization is low at day 1 (0.032 � 0.004 nmol/mg DNA [cells]); thisamount does not change significantly during the following 5 daysof incubation. In contrast, if bicarbonate and MAC is added to thecultures the level of biomineralization strongly increases to0.092 � 0.011 (3 d) and finally to 0.192 � 0.037 (5 d). Omission ofbicarbonate from the culture medium causes a significant reduc-tion of the biomineralization to 0.068 � 0.0076 (3 d) and0.0118 � 0.023 (5 d), respectively. Addition of the carbonic anhy-drase inhibitor acetazolamide (100 mM) resulted in a total inhibitionof the biomineralization (Fig. 1).

The different degree of Ca-deposit formation in dependence onbicarbonate andMAC can also be visualized by SEM analysis (Fig. 2).In the absence of bicarbonate and MAC in the culture medium nomineralic nodules can be observed after an incubation period of 5 d(Fig. 2AeC). However, if both components, bicarbonate and MAC,are added to the SaOS-2 cells a dense accumulation of Ca-depositnodules can be seen (Fig. 2DeF). The nodules reach diameters be-tween 1 and 2 mm. Some nodules are still embedded in cellularextensions/processes that can cover the mineralic deposits byabout 30% (Fig. 2F). If the cells are incubated e under otherwiseidentical conditions e in the absence of bicarbonate but in thepresence of MAC the density of the mineralic deposits is substan-tially lower (Fig. 2G) and the diameters of the crystallites reachrarely sizes of >1 mm (Fig. 2H and I).

3.2. Induction of carbonic anhydrase in SaOS-2 cells bybicarbonate: qRT-PCR

In previous studies an alteration of the expression of the car-bonic anhydrase in response to exposure of mammalian cells tobicarbonate could measured [27,49]. We checked the expressionlevels of the CA-I, -II, -III, and -VII in SaOS-2 cells by qRT-PCR.Among them only transcripts of the gene encoding CA-II (acces-sion number NM_000067; [50]) could be detected, while no tran-scripts for the other isoenzymes could be identified (data notshown).

3.3. Increased expression of carbonic anhydrase II in SaOS-2 cellsafter incubation with bicarbonate

After incubation of SaOS-2 cells in RPMI-1640 medium/10% FCSfor 3 d followed by a subsequent transfer into medium additionallycontaining MAC a concentration-dependent increase of the CA-IIexpression level could be measured by qRT-PCR (Fig. 3). In theabsence of bicarbonate in the medium the expression level of CA-IIis low and amounts to approximately 0.003 (day 1), with respect tothe expression of the housekeeping gene (GAPDH). This steady-state expression remains almost unchanged for the following sixdays of incubation. However, if the cultures were exposed to 1 mM

bicarbonate, a significant expression level is seen after 5 d (from0.0031 � 0.0008 to 0.0073 � 0.0016). After exposure of the SaOS-2cells to bicarbonate at concentrations of 3 mM or 10 mM theexpression levels of CA-II even increases. At 3 mM bicarbonate amaximal expression level is seen at day 5 with a level of0.0126 � 0.0031. After the same incubation period of 5 d the CA-II

steady-state expression level increases to 0.0092 � 0.0021, if thecultures were incubated with 10 mM bicarbonate.

3.4. Induction of CA in SaOS-2 cells: immunostaining

The level of immunoreactive CA-II in SaOS-2 cells was deter-mined with a polyclonal antibody raised against CA-II, asdescribed [51]. After fixation and exposure to those primary an-tibodies PoAb-CA2, the immunocomplexes were detected by flu-orescently labeled anti-rabbit secondary antibodies (Fig. 4). Incultures incubated for 5 d in the absence of bicarbonate and MACthe level of immunoreactive carbonic anhydrase is low (Fig. 4B).The density of cells can be assessed by the staining pattern of thenuclei with DAPI (Fig. 4A). If the cells were incubated with 3 mM

Na-bicarbonate in the absence of MAC for 5 d the intensity of thered fluorescence is strongly increased, reflecting a higher level ofCA-II in those cells (Fig. 4D). The red fluorescence is outshined bythe DAPI-emitted fluorescence (Fig. 4C). The intensity of theimmunocomplexes, visualized by anti-CA-II in combination withthe labeled secondary antibodies, further increases if the culturesare incubated with 3 mM Na-bicarbonate in the presence of MAC(Fig. 4F). In one series of control the CA-II antibodies wereadsorbed with recombinant human CA2 and then applied forimmunostaining. Such an antiserum sample showed a stronglyreduced staining pattern (Fig. 4H) for the CA-II; while the intensityof the DAPI-reacting nuclear matter remained unchanged(Fig. 4G).

3.5. Inhibition of Ca-deposit formation by the carbonic anhydrase-inhibitor acetazolamide

The carbonic anhydrase inhibitor [CA-II] acetazolamide [52] wasfound to reduce the formation of Ca-deposits onto SaOS-2 cells.After an incubation period of 3 d in medium/serum in the absenceof bicarbonate the cells were transferred into medium, supple-mented with MAC, and containing 3 mM Na-bicarbonate. As ex-pected, those cells strongly form Ca-deposits after 5 d, whilecultures that remained in a bicarbonate-free medium/serum pro-duced only to a low extent those deposits (Fig. 5; left two wells). Incontrast, cells incubated with both Na-bicarbonate and acetazol-amide formed only very little Alizarin red S-positive deposits(Fig. 5; middle two wells). Cultures incubated in the absence ofMAC and without the inhibitor developed only very little Alizarinred S-positive deposits (Fig. 5; right two wells).

Fig. 6. Element mapping of the surface of SaOS-2 cells; one cell that comprises a very distinct nodule (no) is circled. (A) Secondary electron image showing the nodule area that hadbeen analyzed. EDX-based elemental analyses: (B) Intensity scale for the pseudocolor documentation; dark: low intensities e red/white high intensities. (C to F) Element mapping ofthe surfaces of the Ca-deposits by EDX for the elements oxygen (C), calcium (D), carbon (E), and phosphorus (F) in pseudocolor outline. The circles correspond to the one in (A). (G)EDX analysis of the circled area; the peaks corresponding to the elements C, Ca, O, Na, Si, and P are marked. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

W.E.G. Müller et al. / Biomaterials 34 (2013) 8671e86808676

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3.6. Ca2þ-rich deposits in SaOS-2 cells after bicarbonate exposure:element mapping

Two-dimensional element mapping, the SEM/EDX set-up wasused in combination with an XFlash 5010 detector to perform anarea mapping within a nodule and in its surrounding on the surfaceof SaOS-2 cells. In Fig. 6A a secondary electron image of a nodule isshown and the corresponding element signals for O, Ca, C and P aregiven in Fig. 6CeF. A strong accumulation of the signals within thenodule for O is evident (Fig. 6C). In contrast, the signal accumula-tion for Ca is likewise distinct but less prominent (Fig. 6D). Sur-prisingly, strong signal densities are also seen in the nodule for C(Fig. 6E), in comparison to the direct vicinity of the Ca-deposits.Those high values for C are also seen in other regions of the areaunder analysis where Ca-deposits are located [they can be spottedin Fig. 6A in the bright regions]. An EDX spot analysis spanning thecircled area reveals distinct signals for C, Ca, O, Na, Si and P(Fig. 6G); the signal for Si should be attributed to the grid ontowhich the cells had been layered.

3.7. Modulation of CA activity by phosphate

The human recombinant CA-II was used to determine the effectof phosphate on the activity of this enzyme. As an assay system theCO2 hydratase activity determination procedure described under“Material and Methods” was used. The experiments revealed thatthe catalytic activity of CA-II is significantly reduced in the presenceof 10 mM o-phosphate by 61% (to 21.7 � 2.1 mM CO2 s�1); Fig. 7. Incontrast, the presence 100 mM Na-polyP did not change the activityof the enzyme. However, if the polyP had been preincubated withalkaline phosphatase to produce lower polymeric/monomeric hy-drolysis products (as described under “Material and Methods”) andthen added to the CA-II reaction the enzyme activity was signifi-cantly reduced, by 39% to 35.2 � 4.1 mM CO2 s�1; Fig. 7.

4. Discussion

In the past several models have been proposed to explain theinitiating processes, involved in hydroxyapatite/bone formation in

Fig. 7. Inhibitory effect of phosphate on the activity of the recombinant CA-II. Theenzyme activity was determined by applying the CO2 hydratase/carbonic anhydraseassay system. The recombinant enzyme was added to the dehydration assay containingNaHCO3. The activity of the enzyme is given in mM CO2 produced s�1. The control re-action did not contain any phosphate component, while in the inhibition studies either10 mM o-phosphate, 100 mM Na-polyP, or 100 mM Na-polyP hydrolyzed by alkalinephosphatase was added. Six parallel experiments have been performed; the results areexpressed as means (�standard error of the mean); *p < 0.01.

mammals (reviewed in Ref. [15]), more specific, the events thatprecede mineral formation in the extracellular matrix (ECM). Basedon experimental evidences it has been concluded (i) that miner-alization is a cell-independent process proceeding on charged non-collagenous proteins in concert with collagen, (ii) that it is a cell-mediated mechanism, during which mineralic bioseeds areformed within intracellular vesicles that are released into the ECM,controlling nucleation of Ca-deposits, and (iii) that amorphousmineralic precursors are initially formed that are subsequentlytransformed into crystalline hydroxyapatite platelets. The findingthat amorphous Ca-phosphate deposits are synthesized withinvesicles of mineralizing cells that are released into the extracellularspace where those deposits are transformed into crystallinic hy-droxyapatite platelets seems to support an intracellular origin ofthe bone apatite formation [53]. However, a direct morphogeneticlink between those two forms of deposits has not yet beenpresented.

The questionwhich has been addressed in the present study hasbeen formulated both based on an evolutionary view that Ca-carbonate is the earliest Ca-based skeletal scaffold in metazoans[54] and because of recent findings that the carbonic anhydrase, anenzyme that accelerates Ca-carbonate deposit formation, isinvolved in bone formation [27]. It is hypothesized that Ca-carbonate might be one precursor mineral for Ca-phosphate de-posits in SaOS-2 cells. To approach a solution to this question thesemineralized bone nodules had been exposed to bicarbonate, ananion that is crucial for intracellular pH buffering and that mainlyexists as a soluble salt (Ca- or Na-bicarbonate). In the presence ofcalcium ions, bicarbonate is converted to the conjugate carbonicacid and water insoluble Ca-carbonate deposits. The carbonicanhydrase (CA) is accelerating the velocity of hydration of CO2, thestarting reaction of Ca-carbonate deposit formation.

The data show that SaOS-2 cells, exposed to bicarbonate, form asignificantly increased amount of Ca-deposits, as analyzed byAlizarin Red S. This process is paralleled by an enhanced CA-II geneexpression and results in an intensified Ca-deposit formation in thepresence of MAC. MAC contains ascorbic acid, required both forcollagen synthesis and for differentiation of osteoprecusor cells, b-glycerophosphate, acting as a phosphate source for Ca-phosphateformation, and dexamethasone that stimulates cellular differenti-ation processes [55]. Importantly, the CA-II inhibitor acetazolamideabrogated significantly the Ca-phosphate deposition process. Thesedata favor the assumption that a CA-II-driven enzymatic process isinvolved in the formation of bioseeds, required for the initial Ca-phosphate deposit synthesis. CA-II is a ubiquitously presentenzyme found in the cytoplasm of almost all metazoan cells. TheCA, in particular the CA-II, is surely involved in bone resorption[56], but recent studies implicate that this enzyme is also involvedin bone formation [27]. These Janus-faced metabolic reactionscontrolled by CAs can be explained because of the reversibility ofthe CA reaction. The CA acts both as a Ca-carbonate anabolicenzyme, facilitating and accelerating bicarbonate formation, aprecursor molecule for Ca-carbonate synthesis, and also as a cata-bolic enzyme that promotes Ca-carbonate dissolution, as shown,e.g. in corals [57]. In mammalian bone tissue, Ca-phosphatedissolution and resorption is triggered in osteoclasts throughacidification by CA-II via modulating the V-ATPase [58].

During the initial phases of the controlled bone-synthesizingprocess poorly crystalline carbonated apatite is deposited [15]that contains several percents (4e6 wt%) of carbonate in theapatite crystals [59,60]. The detailed role of carbonate during boneformation is not yet understood. Recent studies suggest that anincreased carbonate content in apatite crystals affects bone for-mation towards an anabolic direction [61]. The EDX mappingstudies, shown in the present contribution also indicate that

Fig. 8. Schematic outline of the sequential deposition of Ca-carbonate and Ca-phosphate. The CA drives/accelerates the formation of bicarbonate which reacts then to carbonic acidand finally undergoes precipitation to Ca-carbonate. Bicarbonate is provided to the CA via the chloride/bicarbonate anion exchanger (AE), or by the sodium bicarbonatecotransporter (NBC). It is proposed that the Ca-carbonate crystallites are formed in the vicinity of the plasma membrane. In the second step Ca-phosphate precipitates in theextracellular space onto the Ca-carbonate bioseeds. Finally, ortho-phosphate, released form polyP, downregulates the activity of the CA and, by that, retards the Ca-carbonatecrystallite formation.

W.E.G. Müller et al. / Biomaterials 34 (2013) 8671e86808678

the regions of Ca-phosphate deposits onto SaOS-2 cells are not onlyrich in calcium and phosphorous but also in carbon. This observa-tion might be taken as an indication that carbonate and phosphatedeposits are co- or sequentially synthesized onto SaOS-2 cells. Evenmore, the CA-II has been proven to bee under certain physiologicalconditions [pH regulation] e localized at the plasma membranes ofhuman pancreatic cells [28]. There, the CA is involved in pH regu-lation and also in the secretion of bicarbonate via the Cl�/HCO�

exchanger [62] and/or via the additional HCO�3 channel [29,63].

Moreover, experimental evidence is available that the CA-II isresponsible for the production of bicarbonate [62]. The bulk ofthese data corroborate our assumption that the CA acts as abioseed-forming enzyme synthesizing Ca-carbonate deposits ontowhich Ca-phosphate is layered.

Until now no biochemical data have been elaborated that couldshed light into the predicted replacement mechanism of the car-bonate anion in the Ca-carbonate by a phosphate anion underformation of Ca-phosphate. It is accepted that ortho-phosphate actsas a substrate for Ca-phosphate/hydroxyapatite synthesis, origi-nating either from an inorganic polyP polymer, or the organo-phosphate b-glycerophosphate (see Ref. [43]). The presentedconcept here proposes that Ca-carbonate deposits precede Ca-phosphate formation. A replacement of the anions in the Ca-saltsis thermodynamically possible even under ambient conditions[64]. This reaction is also possible because of the considerable highdissociation propensity of Ca-carbonate and a comparable highprecipitation tendency of Ca-phosphate. On the other hand, theinitial complete Ca-carbonate decomposition time is fairly long [65]but it can be substantially reduced by increasing the reactiontemperature [64] and e by that e by overcoming the barrier of theactivation energy for the exchange reaction. In a biochemicalenvironment, like in bone, a reduction of the activation energy ofthe reaction can be achieved by enzymes, e.g. by the CA. In acoupled biochemical reaction chain, driven by CA, Ca-carbonate canbe dissolved via the intersection molecule bicarbonate and in turnthe released Ca2þ becomes accessible to ortho-phosphate to un-dergo salt formation to the highly insoluble Ca-phosphate.

It had been suggested that the transformation of Ca-carbonateinto Ca-phosphate is facilitated by dissolution of initially formedCa-carbonate deposits through complex formation of calcium with

polyP or pyrophosphate (PP), present in bone cells [66,67]. It is wellknown that polyP and pyrophosphate are able to form reasonablysoluble complexes with calcium ions that prevent precipitation[68e70]. In turn, degradation of the polyP/pyrophosphate compo-nent of the formed Ca-polyP/Ca-PP$complexes, catalyzed by thebone alkaline phosphatase [43], results in the release of free Ca2þ

that precipitated in the presence of ortho-phosphate under for-mation of Ca-phosphate deposits. This model also implies that theinitial formation of Ca-carbonate in bone tissue supports Ca-phosphate deposition by a localized accumulation of Ca2þ in theform of Ca-carbonate deposits. The substrate for the enzymatic Ca-carbonate deposition, bicarbonate, can be provided to the extra-cellular space in close vicinity to the Cl�/HCO� exchanger (AE), orintracellularly in the plasma membrane proximity to the Na:HCO�

3cotransporter (NBC); see Ref. [71]. Subsequently, the formed solu-ble Ca-polyP/Ca-PP complexes might be transformed into Ca-phosphate deposits. This view is sketched in Fig. 8.

In the final section of the presented work we describe thatortho-phosphate, as well as the hydrolytic disintegration productsof polyP, display an inhibitor effect on the recombinant CA-II. Theinhibitory concentrations are in the range of 10 mM, with respect toortho-phosphate, a concentration which is certainly presentaround the Ca-phosphate deposition sites within the growing bone[72]. This finding offers a new feedback regulatory system betweenthe CA-driven Ca-carbonate deposition and a subsequent inacti-vation of this process by ortho-phosphate.

5. Conclusion

The data presented here support the view that the Ca-phosphate/hydroxyapatite deposition reactions in bone tissue arepreceded by Ca-carbonate precipitation, a process that is driven byan increased CA activity. The proposed hypothesis, the enzymaticsynthesis of Ca-carbonate via CA, leaves room for a future detailedlocalization of the deposits formed by poorly crystalline Ca-carbonate or by carbonated apatite in the vicinity of the plasmamembrane. The proposed process of the enzymatic formation ofCa-carbonate deposits that act as bioseeds for the synthesis of Ca-phosphate/hydroxyapatite offers also new therapeutic potentials,via modulation (inhibition/activation) of CA.

W.E.G. Müller et al. / Biomaterials 34 (2013) 8671e8680 8679

Acknowledgments

W.E.G. M. is a holder of an ERC Advanced Investigator Grant (No.268476 BIOSILICA). This work was supported by grants from theDeutsche Forschungsgemeinschaft (Schr 277/10-2), the EuropeanCommission (“Bio-Scaffolds”: Customized Rapid Prototyping ofBioactive Scaffolds, No. 604036; Industry-Academia Partnershipsand Pathways “CoreShell”: No. 286059; “MarBioTec*EU-CN*”: No.268476; and “BlueGenics”: No. 311848), the International HumanFrontier Science Program, the Public Welfare Project of Ministry ofLand and Resources of the People’s Republic of China (Grant No.201011005-06) and the International Science & Technology Coop-eration Program of China (Grant No. 2008DFA00980).

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