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Identification of dicalcium phosphate dihydrate deposited during osteoblast mineralization in vitro
Journal of Inorganic Biochemistry 131 (2014) 109–114
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Identification of dicalcium phosphate dihydrate deposited during osteoblast mineralization in vitro Zheng-Lai Zhang, Xiao-Rong Chen, Sha Bian, Jian Huang ⁎, Tian-Lan Zhang ⁎, Kui Wang Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, PR China
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Article history: Received 20 April 2013 Received in revised form 15 November 2013 Accepted 17 November 2013 Available online 23 November 2013 Keywords: Osteoblasts Mineralization Dicalcium phosphate dehydrate Octacalcium phosphate
a b s t r a c t The hydroxyapatite (HAP) with variable chemical substitutions has been considered as the major component in the mineralized part of bones. Various metastable crystalline phases have been suggested as transitory precursors of HAP in bone, but there are no consensuses as to the nature of these phases and their temporal evolution. In the present study, we cultured rat calvarial osteoblasts with ascorbate and β-glycerophosphate to explore which calcium phosphate precursor phases comprise the initial mineral in the process of osteoblast mineralization in vitro. At the indicated time points, the deposited calcium phosphate was analyzed after removing organic substances from the extracellular matrix with hydrazine. The features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP), in addition to HAP, were detected in the mineral phases by high resolution transmission electron microscopy. And there was a trend of conversion from DCPD- and OCP-like phases to HAP in the course of mineralization, as indicated by Fourier-transform infrared microspectroscopy, energydispersive X-ray spectroscopy and synchrotron X-ray powder diffraction analyses. Besides, biochemical assay showed a progressive decrease in the ratio of mineral-associated proteins to calcium with time. These findings suggest that DCPD- and OCP-like phases are likely to occur on the course of osteoblast mineralization, and the mineral-associated proteins might be involved in modulating the mineral phase transformation. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Bone biomineralization is a complex process modulated by organic macromolecules under cellular control. In the form of nonstoichiometric calcium phosphate, carbonated hydroxyapatite (HAP) has been described as the most thermodynamically stable mineral phase in bone [1]. The crystals of HAP are initially observed in the gap regions of aligned type I collagen fibrils and grow in the shape of nanometer-size irregular platelets with their c-axes oriented along the long axis of the collagen fibril [2]. Mineral formation of bone is understood to be a sequential process, but a clear understanding of the mechanism(s) in the early stages of mineralization has been elusive [3–5]. The proposed precursors of bone mineral are amorphous calcium phosphate (ACP) [6,7], dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) [8], and octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) [9]. However, the identification of a precursor phase in bone mineralization is a challenging task, because these minerals may transform during specimen preparation [10–13]. A recent study with cryogenic transmission electron microscopy assay demonstrated that apatite formation in zebra
⁎ Corresponding authors. Tel.: +86 10 82801539; fax: +86 10 62015584. E-mail addresses: [email protected] (J. Huang), [email protected] (T.-L. Zhang). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.11.006
fish bone did not occur directly by the association of ions from solution but proceeded through an ACP precursor phase [14,15]. Evidence from Raman microspectroscopy further pointed out that an OCP-like phase presented prior to the formation of carbonated HAP at the suture boundaries of mice calvaria [16]. The identification of mineral precursors has provided valuable clues to a full understanding of the process of bone mineralization. Osteoblasts are the primary cells contributing to bone formation. Osteoblasts can be obtained by enzymatic digestion of fetal rat calvaria and cultured in the presence of ascorbic acid (AA) and β-glycerophosphate (β-Gp). These cells can undergo a sequence of differentiating events restricted in time, such as the expression of alkaline phosphatase, osteopontin, and osteocalcin, as well as nodule formation and mineral deposition [17,18]. Therefore, a timing study of osteoblast culture would be useful to the identification of calcium phosphate precursors deposited at the different stages of mineralization. One of the principal issues hindering the study of mineral phase is that the high levels of matrix obscure the signals arising from small amounts of mineral. To eliminate the interference effects of proteins, hydrazine has commonly been used in bone mineral research to isolate calcium phosphate crystals from the extracellular matrix [19–21]. As claimed by previous investigators, hydrazine treatment either caused only insignificant alterations to bone mineral or did not alter bone mineral in any way [22–27]. In the present investigation we exploited the mineralizing capabilities of
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osteoblast cultures to monitor what mineral was formed at the different stages of mineralization. At the indicated time points, the organic matrix was removed by hydrazine and the deposited calcium phosphate was analyzed. By combined analyses of the high resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared microspectroscopy (FT-IRM) and synchrotron X-ray powder diffraction (XRD), we have demonstrated that HAP yields, at least in part, from the DCPD- and OCP-like phases in the mineral deposition by osteoblast cultures. 2. Materials and methods 2.1. Cell culture Osteoblasts were obtained by sequential enzyme digestion of excised calvarial bones from 2-day-old neonatal Sprague–Dawley rats (1% trypsin in phosphate buffered saline for 30 min; 0.2% collagenase type II in Dulbecco's modified Eagle medium (DMEM) for 30 min; 0.2% collagenase type II in DMEM for 90 min), as previously described [28]. The first two digests were discarded. Cells from the third digest were collected by centrifugation and resuspended in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (termed complete media) at 37 °C in 95% humidified air containing 5% CO2. All procedures were performed in accordance with the regulations laid down by the ethical guidelines of Peking University. 2.2. Mineralization assay For the mineralization experiments,osteoblasts were plated at 50,000 cells/cm2 in 100 mm tissue culture dishes and allowed to adhere for 24 h at 37 °C/5% CO2 in a humidified incubator before treatment with osteogenic media (complete media supplemented with 50 μg/ml AA and 10 mM β-GP (Sigma, St. Louis, MO)). The cell cultures were maintained for up to 7, 14, 21 and 28 days respectively and the culture medium was replaced every 3 days. Mineralization of the cultures was visualized directly in the culture dishes by 2.5% silver nitrate staining (von Kossa staining) which stains mineralized areas in black. 2.3. Mineral-associated protein assay According to the procedure described by Grynpas et al. [29], the mineral-associated proteins were extracted twice with 0.50 M EDTA at 4 °C for a total of 48 h. The protein content was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as a standard. Calcium concentrations were measured from mineral-dissolving, hydrochloric acid extracts of cell layers (also containing mineralized extracellular matrix) using a calcium kit (Diagnostic Chemicals). The protein expression was normalized to the total calcium content in the mineralized matrix. 2.4. Isolation and characterization of mineral phase Calcium phosphate crystals were isolated from the organic matrix synthesized by osteoblasts by a modified methods described by Kuhn et al. [20]. Briefly, cells and matrix layers were harvested from the culture dishes after the culture medium was removed, rinsed quickly three times with neutral pH, 0.01 M Tris buffer, and lyophilized. The samples were then incubated with anhydrous hydrazine (10 mg/10 ml) at 4 °C under rotary mixing for 24 h and intermittent ultrasonication for about 5 min each hour to remove the organic matrix as much as possible. After 24 h the solvent was removed and the residue was washed twice with 100% ethanol. The residue was assayed as described below. As controls for mineralization analysis, we also cultured synthetic HAP nanopowder of
b200 nm particle size (Sigma-Aldrich) with osteogenic medium and went through every procedure used for cell sample preparation. HRTEM observations were carried out using a Hitachi H-9000 NAR electron microscope with electron acceleration energy of 300 kV. The fast Fourier transforms (FFT) of the HRTEM images were created by software named Digital Micrograph (version 3.5.2; Gatan Inc., USA). EDX analysis was performed using a Hitachi S-5200 field emission SEM equipped with EDX spectroscopy (ISIS, Link Analytical, Oxford Instruments) Synchrotron XRD spectra are collected using beam line BL14B1 at the Shanghai Synchrotron Radiation Facility, China. The X-ray wavelength was 1.2398 Å with the energy of 10 keV. We obtained the diffraction patterns by performing integration from the two-dimensional images with FIT2D software. The diffraction angle 2θ was converted to the condition of Cu Kα radiation wavelength (λ = 1.54 Å) through the Bragg's relation: λ = 2d∙sinθ. Phase identification was performed with the XRD analytical software Jade 5.0 (MDI, Livermore, CA) using the powder diffraction file (PDF). PDF card 09-0077 was used as the reference for DCPD, card 09-0432 for HAP, and card 26-1056 for OCP. FT-IR spectra were recorded on a Nicolet Magna 750-II spectrometer equipped with a Nic-Plan TM IR microscope. Data were collected in transmission mode, 128 scans per point at 4 cm− 1 resolution. The measurement was in the range of 4000–450 cm−1 and performed at random positions in the samples. 3. Results 3.1. Osteoblast mineralization At the end of the experimental period, culture dishes were stained by the von Kossa reagent. As shown in Fig. 1, the calcium deposition stained in black was detected first in the cultures of osteoblasts at day 7, and the extent of mineralization increased up to the furthest time point at day 28. 3.2. HRTEM, EDX, synchrotron XRD and FT-IRM results HRTEM was performed on deposited calcium phosphate in osteoblast cultures to observe changes in crystalline phases after removal of the organic components at different time intervals. As shown in the crystallized domains in Fig. 2, the measured interplanar spaces of 0.410, 0.522 and 0.824 nm correspond respectively to the (200), (101), and (100) planes of HAP. And those of 0.360 nm and of 0.798 nm correspond to the (220) face of OCP and the (020) plan of DCPD, respectively. From HRTEM analysis, DCPD phase was clearly identifiable at day 7, and OCP phase appeared usually at day 14 and 21, while HAP phase was the dominant phase at day 28. The probability of the three crystalline phases appeared in HRTEM images (n = 10) for each time point (7, 14, 21 and 28 days) implied that the thermodynamically metastable phases like DCPD and OCP appeared at the initial stage of the mineralization process of osteoblasts in vitro, and then partially transformed to HAP over time. Further evidence for the notion is from the EDX data shown in Fig. 3. The molar ratios between calcium and phosphorus (Ca/P) of the mineral phases, as calculated from the EDX analysis (n = 3) at day 7, 14, 21 and 28, are 1.10 ± 0.42, 1.37 ± 0.08, 1.72 ± 0.08 and 1.53 ± 0.03, respectively. The first three Ca/P ratios were found to be in agreement with the theoretical stoichiometric molar ratio for DCPD (Ca/P = 1.00), OCP (Ca/P = 1.33) and HAP (Ca/P = 1.67), respectively. The Ca/P ratio around 1.53 at day 28 is lower than the stoichiometric value of 1.67 for HAP, but is consistent with nonstoichiometry of calcium deficient HAP. The EDX data showed a change in the Ca/P ratio from 1.10 to 1.53 reflecting the temporal transformation of the mineral phases. The synchrotron XRD profiles of mineral phases are shown in Fig. 4. The peaks were substantially broadened because of the smaller crystal
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Fig. 1. The mineral formation by osteoblasts as detected using von Kossa at various times. (A) Day 7. (B) Day 14. (C) Day 21. (D) Day 28. Magnification, 200×.
size and/or lower crystallinity of the mineral particles. The peak positions (2θ) around 24.7° and 31.90° correspond to the (002) and the merged (211), (300), (202) reflections of HAP respectively, which presented in all the samples throughout the duration of the experiment. A less pronounced diffraction band appears in the region (2θ) of 16°, indicative of the formation of small amount of OCP. The peaks around 11.5° and 29.2° are the characteristic (020) and (ī12) reflections of
DCPD respectively, while the peaks around 10.4° in the samples at day14 and day 21 correspond to the merged (020) and (010) reflections of DCPD and OCP. These XRD data demonstrate that, while HAP was the major phases, DCPD and OCP also presented in the mineral deposition. In addition, an appreciable decrease in the intensity of diffraction peak of DCPD was found with the culture time, implying that there was a transition from DCPD to OCP or HAP.
Fig. 2. HRTEM lattice micrographs(each left part of panels A, B, C and D)and FFT pattern (each right part, obtained respectively from enclosed regions (a) and (b) in the left panel) of calcium phosphate extracted from mineralized osteoblast at different time intervals. (A) After 7 days. (B) After 14 days. (C) After 21 days. (D) After 28 days.
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Fig. 3. The temporal change in the mineral phase of calcium phosphate extracted from mineralized osteoblast at different time intervals. The probability of crystalline phases appeared at different time intervals in HRTEM images (n = 10) is statisticized.
FT-IRM measurement was performed to independently characterize the minerals at different culture times (Fig. 5). All samples show broader phosphate ν1, ν3 peaks around 1034 cm−1, which are characteristic of HAP. In addition, the band at 1653 cm−1 can be assigned to the merged H2O bending from DCPD and OCP-like phases. The O\H bending modes originating from the H2O of DCPD-like phases around 1219 cm−1 were observed at day 7. The bands corresponding to DCPD-like phase disappear gradually, while those corresponding to HAP become stronger with the culture time. These FT-IRM data confirmed the presence of other mineral phases, in addition to HAP, at the initial calcification stage, as well as the partial transformation of DCPD-like phases into HAP- or OCP-like phases. To examine the possible presence of DCPD or DCPA when apatite is in contact with aqueous solution, we cultured synthetic HAP crystals in osteogenic medium and analyzed the minerals at various time intervals. In the HRTEM micrographs in Fig. 6, there seems to be a little DCPD- and OCP-like crystals as judged from the d values. However, only HAP was detected by the synchrotron XRD measurement, with no other phase formed (Supplementary Fig. S1). The results from FT-IRM measurement also confirmed that HAP was the single phase presented at days 7, 14, 21, and 28 (Supplementary Fig. S2). 3.3. Quantitative determination of mineral-associated protein The proteins in the 0.5 M EDTA extracts were analyzed using the BCA assay. The EDTA sample has conventionally been the only fraction to be examined after protein extraction from hard tissues and represents those proteins trapped/bound in the mineralized matrix [29]. Data were presented as the quantities of the protein extracts,
Fig. 4. Synchrotron XRD analysis of extracted calcium phosphate substances from mineralized osteoblasts at various time intervals. The main diffraction peaks are labeled (▲, HAP; ♦, OCP; ●, DCPD).
Fig. 5. Representative FT-IRM spectra of extracted calcium phosphate substances from mineralized osteoblasts at various time intervals. The bands shown the characteristic transmission patterns are denoted by ▲ for HAP, ♦ for OCP, and ● for DCPD, respectively.
normalized relative to the calcium in the mineral at each time point. It should be noted that the level of proteins in the 0.5 M EDTA extracts declined rapidly with time (Fig. 7), consistent with the changes in the amount of noncollagenous protein in rat bone [25]. 4. Discussion Hydroxyapatite is generally known as the mineral phase of natural bones, but precipitation of Ca2+ and PO3− does not directly lead to for4 mation of HAP in vitro or in vivo [14–16]. Employing HRTEM, synchrotron XRD, FT-IRM and EDX analyses, we demonstrated the occurrence of DCPD- and OCP-like precursors, in addition to HAP, during osteoblast mineralization in vitro. The formation of DCPD, unlike the thermodynamically stable HAP, has rarely been observed under physiological conditions. Previously, a solid-state NMR study on completely dried bone samples indicated the presence of HPO2− ions, which exhibit a chemical shift similar to 4 that of OCP, but a chemical shift anisotropy closer to that of DCPD [30]. Although DCPD has generally been considered stable only at acidic pH [31], we detected DCPD at pH ~7.4 in the present study. As noted by Wuthier et al., the high P/Ca molar ratio in the “intracellular” precipitation reaction is in favor of the ACP-to-DCPD conversion [32]. Cheng and Pritzker have reported DCPD formation at high P/Ca mixing ratio at pH 7.0 [33]. Similarly, LeGeros and coworkers have demonstrated the presence of DCPD at pH 7.0 in the depths of collagen gels containing phosphate and calcium solutions [34]. As indicated by these findings, the high P/Ca ratio is a key factor for the formation of DCPD. Recently, Lu et al. analyzed the driving force and nucleation rate of calcium phosphate precipitation in simulated body fluid (SBF) based on the classical crystallization theory [35]. They demonstrated that DCPD precipitation was thermodynamically impossible in normal SBF, unless calcium and phosphate ion concentrations of SBF increased. In such case, DCPD precipitation was the most likely one because of its highest nucleation rates among calcium phosphate phases. In the present experiments, 10 mM β-GP was routinely added to the culture media of osteoblast to promote mineral deposition. The mechanism by which β-GP induces mineralization is closely linked to the high ALP activity of bone cell cultures. The osteoblast-secreted ALP can efficiently hydrolyze the added β-GP at 10 mM and increase medium phosphate concentration to 9–10 mM during the first 8 h of osteoblast culture [36,37]. Therefore, the inorganic phosphate released from β-GP could result in high P/Ca ratio in solution and provide the chemical potential for DCPD formation in the early stage of mineralization. It should be noted that some researchers have suggested the possible presence of DCPD or DCPA when HAP is in contact with aqueous solutions [38–41]. In the sample with added synthetic HAP in the culture media, however, we did not observe DCPD or DCPA by the synchrotron XRD and FT-IRM techniques.
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Fig. 6. HRTEM lattice micrographs (each left part of panels A, B, C and D) and FFT patterns (each right part, obtained respectively from enclosed regions (a) and (b) in the left panel) of precipitates after incubation of synthetic HAP in osteogenic medium at various time intervals.
In addition to DCPD, we also detected OCP-like phase in the precipitates of the cell culture. OCP has been proposed as a precursor phase in the formation of biological apatite [42]. The involvement of OCP in initial intramembranous bone formation has been demonstrated in a spectroscopic analysis [16]. Recent studies have showed that OCP is present as a transient phase during biological apatite formation in human dentin, porcine enamel and murine bone [43]. The presence of more HPO24 − found in younger, more actively mineralizing hard tissues is consistent with the role of OCP as a template for the growth of biological apatite [44]. Under physiological conditions, the higher nucleation rate of OCP than that of HAP could be a cause for the occurrence of an OCP-like phase in the mineral deposition [35]. Moreover, the transformation of DCPD may also contribute to OCP formation. Actually, our XRD measurement revealed a slight decrease in the intensity of DCPD peaks and the coexistence of OCP and DCPD over the observation period.
Fig. 7. The temporal changes in the contents of mineral associated proteins extracted from mineralized osteoblasts. Data are presented as the quantities of the protein extracts normalized relative to the calcium in the mineral at each time point.
Previously, Mandel et al. reported the similar transformation when soaking DCPD in DMEM solution at 36.5 °C within 1 week [45]. In a study on the maturation process of dental calculus by synchrotron X-ray fluorescence, Abraham et al. [46] reported a pathway of phase evolution which involved the DCPD-to-HAP transformation via OCP. It should be noted that we did not obtain any evidence for the presence of highly metastable ACP by any techniques used in this study, although ACP has been proven present in several mineralized tissues such as newly formed (outer) murine tooth enamel, fin bones of zebrafish and intra-membranes of murine calvaria tissues [14–16,47]. Differently, in chick bud mesenchymal and osteoblast mineralizing cell cultures, the only mineral found was a poorly crystalline apatite [20]. This inconsistency might arise from the difference in cell type and culture medium (Fitton-Jackson modified BGJb Medium vs. DMEM) and in the techniques employed to detect the mineral phase. In the present study, it is likely that the amorphous mineral phase was dissolved or transformed into a more mature phase during the sample preparation process. In the biochemical analysis of the mineral deposition, we found that the relative amount of mineral-associated proteins progressively decreased with culture time, consistent with the changes previously reported during bone maturation [25,48]. The temporal variation of the composition and amount of those mineral-associated proteins might be essential to the mediation of calcium phosphate phase transformations. In summary, we have followed the temporal changes in calcium phosphate phases and the mineral-associated proteins on the course of osteoblast mineralization over a 28-day period. Two precursors, the DCPD- and OCP-like phases, presented at the initial stage of mineral precipitation and subsequently transformed into thermodynamically stable HAP. Concomitantly, the ratio of the mineral-associated proteins to calcium progressively decreased with time. These findings may shed new light on the mechanism of osteoblast mineralization and provide a basis for further understanding of bone metabolism.
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Abbreviations HAP hydroxyapatite DCPD dicalcium phosphate dihydrate OCP octacalcium phosphate ACP amorphous calcium phosphate HRTEM high resolution transmission electron microscopy FFT fast Fourier transforms EDX energy-dispersive X-ray spectroscopy FT-IRM Fourier transform infrared microspectroscopy Synchrotron XRD synchrotron X-Ray powder diffraction DMEM Dulbecco's modified Eagle medium BCA bicinchoninic acid AA ascorbic acid β-Gp β-glycerophosphate
Acknowledgments This work was supported by National Natural Science Foundation of China (grant no. 21071007), National Natural Science Foundation of China for young (grant no. 21101008) and the provision of beam time by the Shanghai Synchrotron Radiation Facility at BL14B1. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2013.11.006. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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Identification of dicalcium phosphate dihydrate deposited during osteoblast mineralization in vitro Zheng-Lai Zhang, Xiao-Rong Chen, Sha Bian, Jian Huang ⁎, Tian-Lan Zhang ⁎, Kui Wang Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, PR China
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Article history: Received 20 April 2013 Received in revised form 15 November 2013 Accepted 17 November 2013 Available online 23 November 2013 Keywords: Osteoblasts Mineralization Dicalcium phosphate dehydrate Octacalcium phosphate
a b s t r a c t The hydroxyapatite (HAP) with variable chemical substitutions has been considered as the major component in the mineralized part of bones. Various metastable crystalline phases have been suggested as transitory precursors of HAP in bone, but there are no consensuses as to the nature of these phases and their temporal evolution. In the present study, we cultured rat calvarial osteoblasts with ascorbate and β-glycerophosphate to explore which calcium phosphate precursor phases comprise the initial mineral in the process of osteoblast mineralization in vitro. At the indicated time points, the deposited calcium phosphate was analyzed after removing organic substances from the extracellular matrix with hydrazine. The features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP), in addition to HAP, were detected in the mineral phases by high resolution transmission electron microscopy. And there was a trend of conversion from DCPD- and OCP-like phases to HAP in the course of mineralization, as indicated by Fourier-transform infrared microspectroscopy, energydispersive X-ray spectroscopy and synchrotron X-ray powder diffraction analyses. Besides, biochemical assay showed a progressive decrease in the ratio of mineral-associated proteins to calcium with time. These findings suggest that DCPD- and OCP-like phases are likely to occur on the course of osteoblast mineralization, and the mineral-associated proteins might be involved in modulating the mineral phase transformation. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Bone biomineralization is a complex process modulated by organic macromolecules under cellular control. In the form of nonstoichiometric calcium phosphate, carbonated hydroxyapatite (HAP) has been described as the most thermodynamically stable mineral phase in bone [1]. The crystals of HAP are initially observed in the gap regions of aligned type I collagen fibrils and grow in the shape of nanometer-size irregular platelets with their c-axes oriented along the long axis of the collagen fibril [2]. Mineral formation of bone is understood to be a sequential process, but a clear understanding of the mechanism(s) in the early stages of mineralization has been elusive [3–5]. The proposed precursors of bone mineral are amorphous calcium phosphate (ACP) [6,7], dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) [8], and octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) [9]. However, the identification of a precursor phase in bone mineralization is a challenging task, because these minerals may transform during specimen preparation [10–13]. A recent study with cryogenic transmission electron microscopy assay demonstrated that apatite formation in zebra
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fish bone did not occur directly by the association of ions from solution but proceeded through an ACP precursor phase [14,15]. Evidence from Raman microspectroscopy further pointed out that an OCP-like phase presented prior to the formation of carbonated HAP at the suture boundaries of mice calvaria [16]. The identification of mineral precursors has provided valuable clues to a full understanding of the process of bone mineralization. Osteoblasts are the primary cells contributing to bone formation. Osteoblasts can be obtained by enzymatic digestion of fetal rat calvaria and cultured in the presence of ascorbic acid (AA) and β-glycerophosphate (β-Gp). These cells can undergo a sequence of differentiating events restricted in time, such as the expression of alkaline phosphatase, osteopontin, and osteocalcin, as well as nodule formation and mineral deposition [17,18]. Therefore, a timing study of osteoblast culture would be useful to the identification of calcium phosphate precursors deposited at the different stages of mineralization. One of the principal issues hindering the study of mineral phase is that the high levels of matrix obscure the signals arising from small amounts of mineral. To eliminate the interference effects of proteins, hydrazine has commonly been used in bone mineral research to isolate calcium phosphate crystals from the extracellular matrix [19–21]. As claimed by previous investigators, hydrazine treatment either caused only insignificant alterations to bone mineral or did not alter bone mineral in any way [22–27]. In the present investigation we exploited the mineralizing capabilities of
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osteoblast cultures to monitor what mineral was formed at the different stages of mineralization. At the indicated time points, the organic matrix was removed by hydrazine and the deposited calcium phosphate was analyzed. By combined analyses of the high resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared microspectroscopy (FT-IRM) and synchrotron X-ray powder diffraction (XRD), we have demonstrated that HAP yields, at least in part, from the DCPD- and OCP-like phases in the mineral deposition by osteoblast cultures. 2. Materials and methods 2.1. Cell culture Osteoblasts were obtained by sequential enzyme digestion of excised calvarial bones from 2-day-old neonatal Sprague–Dawley rats (1% trypsin in phosphate buffered saline for 30 min; 0.2% collagenase type II in Dulbecco's modified Eagle medium (DMEM) for 30 min; 0.2% collagenase type II in DMEM for 90 min), as previously described [28]. The first two digests were discarded. Cells from the third digest were collected by centrifugation and resuspended in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (termed complete media) at 37 °C in 95% humidified air containing 5% CO2. All procedures were performed in accordance with the regulations laid down by the ethical guidelines of Peking University. 2.2. Mineralization assay For the mineralization experiments,osteoblasts were plated at 50,000 cells/cm2 in 100 mm tissue culture dishes and allowed to adhere for 24 h at 37 °C/5% CO2 in a humidified incubator before treatment with osteogenic media (complete media supplemented with 50 μg/ml AA and 10 mM β-GP (Sigma, St. Louis, MO)). The cell cultures were maintained for up to 7, 14, 21 and 28 days respectively and the culture medium was replaced every 3 days. Mineralization of the cultures was visualized directly in the culture dishes by 2.5% silver nitrate staining (von Kossa staining) which stains mineralized areas in black. 2.3. Mineral-associated protein assay According to the procedure described by Grynpas et al. [29], the mineral-associated proteins were extracted twice with 0.50 M EDTA at 4 °C for a total of 48 h. The protein content was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as a standard. Calcium concentrations were measured from mineral-dissolving, hydrochloric acid extracts of cell layers (also containing mineralized extracellular matrix) using a calcium kit (Diagnostic Chemicals). The protein expression was normalized to the total calcium content in the mineralized matrix. 2.4. Isolation and characterization of mineral phase Calcium phosphate crystals were isolated from the organic matrix synthesized by osteoblasts by a modified methods described by Kuhn et al. [20]. Briefly, cells and matrix layers were harvested from the culture dishes after the culture medium was removed, rinsed quickly three times with neutral pH, 0.01 M Tris buffer, and lyophilized. The samples were then incubated with anhydrous hydrazine (10 mg/10 ml) at 4 °C under rotary mixing for 24 h and intermittent ultrasonication for about 5 min each hour to remove the organic matrix as much as possible. After 24 h the solvent was removed and the residue was washed twice with 100% ethanol. The residue was assayed as described below. As controls for mineralization analysis, we also cultured synthetic HAP nanopowder of
b200 nm particle size (Sigma-Aldrich) with osteogenic medium and went through every procedure used for cell sample preparation. HRTEM observations were carried out using a Hitachi H-9000 NAR electron microscope with electron acceleration energy of 300 kV. The fast Fourier transforms (FFT) of the HRTEM images were created by software named Digital Micrograph (version 3.5.2; Gatan Inc., USA). EDX analysis was performed using a Hitachi S-5200 field emission SEM equipped with EDX spectroscopy (ISIS, Link Analytical, Oxford Instruments) Synchrotron XRD spectra are collected using beam line BL14B1 at the Shanghai Synchrotron Radiation Facility, China. The X-ray wavelength was 1.2398 Å with the energy of 10 keV. We obtained the diffraction patterns by performing integration from the two-dimensional images with FIT2D software. The diffraction angle 2θ was converted to the condition of Cu Kα radiation wavelength (λ = 1.54 Å) through the Bragg's relation: λ = 2d∙sinθ. Phase identification was performed with the XRD analytical software Jade 5.0 (MDI, Livermore, CA) using the powder diffraction file (PDF). PDF card 09-0077 was used as the reference for DCPD, card 09-0432 for HAP, and card 26-1056 for OCP. FT-IR spectra were recorded on a Nicolet Magna 750-II spectrometer equipped with a Nic-Plan TM IR microscope. Data were collected in transmission mode, 128 scans per point at 4 cm− 1 resolution. The measurement was in the range of 4000–450 cm−1 and performed at random positions in the samples. 3. Results 3.1. Osteoblast mineralization At the end of the experimental period, culture dishes were stained by the von Kossa reagent. As shown in Fig. 1, the calcium deposition stained in black was detected first in the cultures of osteoblasts at day 7, and the extent of mineralization increased up to the furthest time point at day 28. 3.2. HRTEM, EDX, synchrotron XRD and FT-IRM results HRTEM was performed on deposited calcium phosphate in osteoblast cultures to observe changes in crystalline phases after removal of the organic components at different time intervals. As shown in the crystallized domains in Fig. 2, the measured interplanar spaces of 0.410, 0.522 and 0.824 nm correspond respectively to the (200), (101), and (100) planes of HAP. And those of 0.360 nm and of 0.798 nm correspond to the (220) face of OCP and the (020) plan of DCPD, respectively. From HRTEM analysis, DCPD phase was clearly identifiable at day 7, and OCP phase appeared usually at day 14 and 21, while HAP phase was the dominant phase at day 28. The probability of the three crystalline phases appeared in HRTEM images (n = 10) for each time point (7, 14, 21 and 28 days) implied that the thermodynamically metastable phases like DCPD and OCP appeared at the initial stage of the mineralization process of osteoblasts in vitro, and then partially transformed to HAP over time. Further evidence for the notion is from the EDX data shown in Fig. 3. The molar ratios between calcium and phosphorus (Ca/P) of the mineral phases, as calculated from the EDX analysis (n = 3) at day 7, 14, 21 and 28, are 1.10 ± 0.42, 1.37 ± 0.08, 1.72 ± 0.08 and 1.53 ± 0.03, respectively. The first three Ca/P ratios were found to be in agreement with the theoretical stoichiometric molar ratio for DCPD (Ca/P = 1.00), OCP (Ca/P = 1.33) and HAP (Ca/P = 1.67), respectively. The Ca/P ratio around 1.53 at day 28 is lower than the stoichiometric value of 1.67 for HAP, but is consistent with nonstoichiometry of calcium deficient HAP. The EDX data showed a change in the Ca/P ratio from 1.10 to 1.53 reflecting the temporal transformation of the mineral phases. The synchrotron XRD profiles of mineral phases are shown in Fig. 4. The peaks were substantially broadened because of the smaller crystal
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Fig. 1. The mineral formation by osteoblasts as detected using von Kossa at various times. (A) Day 7. (B) Day 14. (C) Day 21. (D) Day 28. Magnification, 200×.
size and/or lower crystallinity of the mineral particles. The peak positions (2θ) around 24.7° and 31.90° correspond to the (002) and the merged (211), (300), (202) reflections of HAP respectively, which presented in all the samples throughout the duration of the experiment. A less pronounced diffraction band appears in the region (2θ) of 16°, indicative of the formation of small amount of OCP. The peaks around 11.5° and 29.2° are the characteristic (020) and (ī12) reflections of
DCPD respectively, while the peaks around 10.4° in the samples at day14 and day 21 correspond to the merged (020) and (010) reflections of DCPD and OCP. These XRD data demonstrate that, while HAP was the major phases, DCPD and OCP also presented in the mineral deposition. In addition, an appreciable decrease in the intensity of diffraction peak of DCPD was found with the culture time, implying that there was a transition from DCPD to OCP or HAP.
Fig. 2. HRTEM lattice micrographs(each left part of panels A, B, C and D)and FFT pattern (each right part, obtained respectively from enclosed regions (a) and (b) in the left panel) of calcium phosphate extracted from mineralized osteoblast at different time intervals. (A) After 7 days. (B) After 14 days. (C) After 21 days. (D) After 28 days.
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Fig. 3. The temporal change in the mineral phase of calcium phosphate extracted from mineralized osteoblast at different time intervals. The probability of crystalline phases appeared at different time intervals in HRTEM images (n = 10) is statisticized.
FT-IRM measurement was performed to independently characterize the minerals at different culture times (Fig. 5). All samples show broader phosphate ν1, ν3 peaks around 1034 cm−1, which are characteristic of HAP. In addition, the band at 1653 cm−1 can be assigned to the merged H2O bending from DCPD and OCP-like phases. The O\H bending modes originating from the H2O of DCPD-like phases around 1219 cm−1 were observed at day 7. The bands corresponding to DCPD-like phase disappear gradually, while those corresponding to HAP become stronger with the culture time. These FT-IRM data confirmed the presence of other mineral phases, in addition to HAP, at the initial calcification stage, as well as the partial transformation of DCPD-like phases into HAP- or OCP-like phases. To examine the possible presence of DCPD or DCPA when apatite is in contact with aqueous solution, we cultured synthetic HAP crystals in osteogenic medium and analyzed the minerals at various time intervals. In the HRTEM micrographs in Fig. 6, there seems to be a little DCPD- and OCP-like crystals as judged from the d values. However, only HAP was detected by the synchrotron XRD measurement, with no other phase formed (Supplementary Fig. S1). The results from FT-IRM measurement also confirmed that HAP was the single phase presented at days 7, 14, 21, and 28 (Supplementary Fig. S2). 3.3. Quantitative determination of mineral-associated protein The proteins in the 0.5 M EDTA extracts were analyzed using the BCA assay. The EDTA sample has conventionally been the only fraction to be examined after protein extraction from hard tissues and represents those proteins trapped/bound in the mineralized matrix [29]. Data were presented as the quantities of the protein extracts,
Fig. 4. Synchrotron XRD analysis of extracted calcium phosphate substances from mineralized osteoblasts at various time intervals. The main diffraction peaks are labeled (▲, HAP; ♦, OCP; ●, DCPD).
Fig. 5. Representative FT-IRM spectra of extracted calcium phosphate substances from mineralized osteoblasts at various time intervals. The bands shown the characteristic transmission patterns are denoted by ▲ for HAP, ♦ for OCP, and ● for DCPD, respectively.
normalized relative to the calcium in the mineral at each time point. It should be noted that the level of proteins in the 0.5 M EDTA extracts declined rapidly with time (Fig. 7), consistent with the changes in the amount of noncollagenous protein in rat bone [25]. 4. Discussion Hydroxyapatite is generally known as the mineral phase of natural bones, but precipitation of Ca2+ and PO3− does not directly lead to for4 mation of HAP in vitro or in vivo [14–16]. Employing HRTEM, synchrotron XRD, FT-IRM and EDX analyses, we demonstrated the occurrence of DCPD- and OCP-like precursors, in addition to HAP, during osteoblast mineralization in vitro. The formation of DCPD, unlike the thermodynamically stable HAP, has rarely been observed under physiological conditions. Previously, a solid-state NMR study on completely dried bone samples indicated the presence of HPO2− ions, which exhibit a chemical shift similar to 4 that of OCP, but a chemical shift anisotropy closer to that of DCPD [30]. Although DCPD has generally been considered stable only at acidic pH [31], we detected DCPD at pH ~7.4 in the present study. As noted by Wuthier et al., the high P/Ca molar ratio in the “intracellular” precipitation reaction is in favor of the ACP-to-DCPD conversion [32]. Cheng and Pritzker have reported DCPD formation at high P/Ca mixing ratio at pH 7.0 [33]. Similarly, LeGeros and coworkers have demonstrated the presence of DCPD at pH 7.0 in the depths of collagen gels containing phosphate and calcium solutions [34]. As indicated by these findings, the high P/Ca ratio is a key factor for the formation of DCPD. Recently, Lu et al. analyzed the driving force and nucleation rate of calcium phosphate precipitation in simulated body fluid (SBF) based on the classical crystallization theory [35]. They demonstrated that DCPD precipitation was thermodynamically impossible in normal SBF, unless calcium and phosphate ion concentrations of SBF increased. In such case, DCPD precipitation was the most likely one because of its highest nucleation rates among calcium phosphate phases. In the present experiments, 10 mM β-GP was routinely added to the culture media of osteoblast to promote mineral deposition. The mechanism by which β-GP induces mineralization is closely linked to the high ALP activity of bone cell cultures. The osteoblast-secreted ALP can efficiently hydrolyze the added β-GP at 10 mM and increase medium phosphate concentration to 9–10 mM during the first 8 h of osteoblast culture [36,37]. Therefore, the inorganic phosphate released from β-GP could result in high P/Ca ratio in solution and provide the chemical potential for DCPD formation in the early stage of mineralization. It should be noted that some researchers have suggested the possible presence of DCPD or DCPA when HAP is in contact with aqueous solutions [38–41]. In the sample with added synthetic HAP in the culture media, however, we did not observe DCPD or DCPA by the synchrotron XRD and FT-IRM techniques.
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Fig. 6. HRTEM lattice micrographs (each left part of panels A, B, C and D) and FFT patterns (each right part, obtained respectively from enclosed regions (a) and (b) in the left panel) of precipitates after incubation of synthetic HAP in osteogenic medium at various time intervals.
In addition to DCPD, we also detected OCP-like phase in the precipitates of the cell culture. OCP has been proposed as a precursor phase in the formation of biological apatite [42]. The involvement of OCP in initial intramembranous bone formation has been demonstrated in a spectroscopic analysis [16]. Recent studies have showed that OCP is present as a transient phase during biological apatite formation in human dentin, porcine enamel and murine bone [43]. The presence of more HPO24 − found in younger, more actively mineralizing hard tissues is consistent with the role of OCP as a template for the growth of biological apatite [44]. Under physiological conditions, the higher nucleation rate of OCP than that of HAP could be a cause for the occurrence of an OCP-like phase in the mineral deposition [35]. Moreover, the transformation of DCPD may also contribute to OCP formation. Actually, our XRD measurement revealed a slight decrease in the intensity of DCPD peaks and the coexistence of OCP and DCPD over the observation period.
Fig. 7. The temporal changes in the contents of mineral associated proteins extracted from mineralized osteoblasts. Data are presented as the quantities of the protein extracts normalized relative to the calcium in the mineral at each time point.
Previously, Mandel et al. reported the similar transformation when soaking DCPD in DMEM solution at 36.5 °C within 1 week [45]. In a study on the maturation process of dental calculus by synchrotron X-ray fluorescence, Abraham et al. [46] reported a pathway of phase evolution which involved the DCPD-to-HAP transformation via OCP. It should be noted that we did not obtain any evidence for the presence of highly metastable ACP by any techniques used in this study, although ACP has been proven present in several mineralized tissues such as newly formed (outer) murine tooth enamel, fin bones of zebrafish and intra-membranes of murine calvaria tissues [14–16,47]. Differently, in chick bud mesenchymal and osteoblast mineralizing cell cultures, the only mineral found was a poorly crystalline apatite [20]. This inconsistency might arise from the difference in cell type and culture medium (Fitton-Jackson modified BGJb Medium vs. DMEM) and in the techniques employed to detect the mineral phase. In the present study, it is likely that the amorphous mineral phase was dissolved or transformed into a more mature phase during the sample preparation process. In the biochemical analysis of the mineral deposition, we found that the relative amount of mineral-associated proteins progressively decreased with culture time, consistent with the changes previously reported during bone maturation [25,48]. The temporal variation of the composition and amount of those mineral-associated proteins might be essential to the mediation of calcium phosphate phase transformations. In summary, we have followed the temporal changes in calcium phosphate phases and the mineral-associated proteins on the course of osteoblast mineralization over a 28-day period. Two precursors, the DCPD- and OCP-like phases, presented at the initial stage of mineral precipitation and subsequently transformed into thermodynamically stable HAP. Concomitantly, the ratio of the mineral-associated proteins to calcium progressively decreased with time. These findings may shed new light on the mechanism of osteoblast mineralization and provide a basis for further understanding of bone metabolism.
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Abbreviations HAP hydroxyapatite DCPD dicalcium phosphate dihydrate OCP octacalcium phosphate ACP amorphous calcium phosphate HRTEM high resolution transmission electron microscopy FFT fast Fourier transforms EDX energy-dispersive X-ray spectroscopy FT-IRM Fourier transform infrared microspectroscopy Synchrotron XRD synchrotron X-Ray powder diffraction DMEM Dulbecco's modified Eagle medium BCA bicinchoninic acid AA ascorbic acid β-Gp β-glycerophosphate
Acknowledgments This work was supported by National Natural Science Foundation of China (grant no. 21071007), National Natural Science Foundation of China for young (grant no. 21101008) and the provision of beam time by the Shanghai Synchrotron Radiation Facility at BL14B1. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2013.11.006. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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