Quantification of calcium content in bone by using ToF-SIMS–a first approach
© Henss et al.; licensee Springer. 2013
Received: 2 October 2013
Accepted: 4 November 2013
Published: 14 November 2013
The determination of the spatially resolved calcium distribution and concentration in bone is essential for the assessment of bone quality. It enables the diagnosis and elucidation of bone diseases, the course of bone remodelling and the assessment of bone quality at interfaces to implants. With time-of-flight secondary ion mass spectrometry (ToF-SIMS) the calcium distribution in bone cross sections is mapped semi-quantitatively with a lateral resolution of up to 1 μm. As standards for the calibration of the ToF-SIMS data calcium hydroxyapatite collagen scaffolds with different compositions were synthesized. The standards were characterised by loss of ignition, x-ray diffractometry (XRD) and x-ray photoelectron spectroscopy (XPS). The secondary ion count rate for calcium and the calcium content of the standards show a linear dependence. The obtained calibration curve is used for the quantification of the calcium content in the bone of rats. The calcium concentration within an animal model for osteoporosis induction is monitored. Exemplarily the calcium content of the bones was quantified by XPS for validation of the results. Furthermore a calcium mass image is compared with an XPS image to demonstrate the better lateral resolution of ToF-SIMS which advances the locally resolved quantification of the calcium content.
KeywordsCalcium quantification bone Time of flight secondary ion mass spectrometry Osteoporosis Bone imaging
Quantification of the mineral content in osseous tissue is important for assessing bone quality–especially in case of bone diseases like such as osteoporosis. Dual energy X-ray absorptiometry (DEXA) and quantitative computed tomography (Q-CT) are widely used for the clinical diagnosis of osteoporosis [1, 2]. With DEXA X-ray absorption of the whole body is measured to determine the bone mineral density (BMD). However, DEXA does not sufficiently include bone thickness and bone volume when determining the BMD. Thus in clinical research micro-and nano-computed tomography (μ-CT and n-CT) with a spatial resolution of 10 μm or 10 nm, respectively, are often used for the complementary 3D analysis of bone architecture. Although DEXA and Q-CT are essential, these x-ray based methods are not chemically selective and do not allow the exclusive evaluation of the local Ca content.
Therefore, in bone research additional spectrometric and spectroscopic methods are applied to determine the elemental composition of bone samples. These include atom absorption spectroscopy  or inductively coupled plasma optical emission spectroscopy (ICP-OES) of ashed and dissolved bone samples, mostly applied to prove and quantify elements like Pb, Na, K, Al, Mg as well as Ca and P in bone [4, 5]. Also laser ablation combined with inductively coupled plasma mass spectrometry (LA-ICP-MS) allows quantitative analysis and imaging in the lower μm regions . Zoehrer et al. used x-ray photoelectron spectroscopy (XPS) to evaluate the calcium and phosphorous content as well as the Ca/P ratio to assess bone quality in case of fragility fractures of male patients . In this study the well-established bone mineral density distribution (BMDD) was applied to characterise the degree and distribution of bone mineralisation. BMDD is calculated from backscattered electron images and is a validated method for the clinical use .
For the development of modified therapies and implants for patients with osteoporosis a profound knowledge about the local calcium distribution and content, especially in the most stressed and most fractured regions of the skeleton, is highly eligible. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) as a surface sensitive technique is chemically specific and offers high spatial resolution–its applicability for the investigation of biological samples and biomaterials interfaces has already been demonstrated in several cases so far [9, 10]. In our previous studies ToF-SIMS was successfully applied for the analysis of osteogenic differentiated hMSCs, for tracking pharmaceuticals in a biomaterial from in vitro to in vivo experiments, as well as for the investigation and imaging of osteoporotic bone in an animal model [11–13]. ToF-SIMS provided detailed chemical information of bone with high sensitivity and a lateral resolution of up to 300 nm. Mass images revealed the distribution of the organic compounds as well as the localisation of mineralised parts of the bone. Assuming the secondary ion count of calcium to be proportional to its surface concentration, a semi-quantitative comparison of healthy and osteoporotic bone confirmed the reduction of the calcium content in diseased tissue, as proven additionally by DEXA measurements .
In the present paper a first step toward the quantitative evaluation of the Ca content of bone is reported, thereby expanding the use of ToF-SIMS for the analysis of osseous tissue. Generally, quantification of concentrations using ToF-SIMS is challenging, as the ionisation process and hence the ion yields strongly depend on the chemical environment of the elements and molecules at the surface . Therefore appropriate standards with an almost identical chemical composition as the analysed material are required, and the relative fragment mass intensity of the analyte of interest should follow a linear relationship with its concentration in the standard. For quantification in biological systems internal standards are often used to derive a calibration curve, or sensitivity factors are estimated from a model matrix . For the quantification of Ca in healthy trabecular bone the standards should consist of collagen with 60–70 wt% of mineralised hydroxyapatite (HAP) or correspondingly less for osteoporotic bone. Once the quantification with ToF-SIMS is reliable, distinct additional benefit is gained. ToF-SIMS has a higher lateral resolution in contrast to XPS, and in comparison with BMDD from SEM images, ToF-SIMS allows to distinguish unequivocally between different elements (e.g. differentiation of Sr and Ca).
2.1 Preparation of the mineralised collagen standards
Scaffolds from mineralised collagen I  were prepared by synchronous mineralisation of a collagen type I solution according to a method developed by Bradt et al. . The procedure was published in detail elsewhere .
To describe the procedure briefly, acid-soluble collagen type I isolated from calf skin (Collaplex 1.0, GfN, Wald-Michelbach, Germany) was dissolved in 10 mM HCl and mixed with a CaCl2 solution. The pH was adjusted to 7 by addition of TRIS and phosphate buffer and the mixture then warmed to 37°C for 12 h. Under these conditions collagen fibril reassembly and formation of nano-crystalline HAP occurs simultaneously. The product–homogeneously mineralised collagen fibrils–was collected by centrifugation. By variation of the collagen to calcium and phosphate ratio in the precursor mixture the collagen to mineral ratio in the final product could be adjusted.
To prepare the standards, thoroughly resuspended mineralised collagen was condensed by vacuum filtration using a porous G4 glass filter frit (Schott, Germany) and then cross-linked with an aqueous solution of 1% N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC; Merck, Germany) for 1 h. Finally, the scaffolds were rinsed in distilled water, in 1% glycine solution, once again in water, and freeze dried.
2.2 XRD of standards
XRD analysis was carried out in scanning mode with Cu Kα and Cu Kβ radiation on a Panalytical X’Pert PRO instrument. The Cu anode was operated at 40 kV and 40 mA. Samples were rotated during measurement. For the data analysis only Cu Kα lines were used, and the XRD pattern was compared with entries in the ICSD database.
2.3 X-ray photoelectron spectroscopy (XPS)
XPS measurements were carried out with a PHI 5000 Versaprobe Scanning ESCA Microprobe (Physical Electronics) using a monochromatic Al Kα X-ray source (hv = 1486.6 eV).
2.3.1 Analysis of the standards
Quantitative analysis of the standard samples was performed by recording detailed spectra for the elements of interest. An x-ray spot size of 100 μm diameter was used and the analyser pass energy was set to 23.5 eV. After subtraction of a Shirley-type background function, elemental concentrations were calculated from the peak areas by applying the appropriate sensitivity factors (provided by the instrument manufacturer). Assuming that all Ca originates from HAP (formula Ca5(PO4)3OH), the HAP content is evaluated on the basis of the Ca content in at% and wt%.
2.3.2 Analysis of bone cross sections
Elemental mapping was carried out for C 1s (for energy calibration) O 1s, N 1s, P 2p and Ca 2p lines with a pass energy of 93.9 eV and a spot size of the focussed x-ray beam of 15 μm in diameter at 2.5 W. The energy interval of each signal was partitioned into sixteen intervals, each of them was than assigned to one specific channel of the multi-channel detector to reduce measurement time. The analysed area was 500 × 500 μm2 divided in 128 × 128 pixels.
In addition to the XPS imaging detailed point spectra were recorded on the same areas that were investigated by mapping. We therefore selected three spots of interest on every sample, which were a) on the trabecular structure, b) on the edge of the trabecular and c) on an area far away from the trabecular. Here, we recorded detail spectra for C 1s (for energy calibration) O 1s, N 1s, P 2p and Ca 2p with a spot size of 15 μm in diameter at 2.5 W. The pass energy was set to 23.5 eV. Elemental concentrations were calculated as described above.
For evaluation of the HAP content of the bone samples we used a calibration factor F (F = 1.15), which was determined by the ratio of the measured and the nominal Ca content obtained by the analysis of pure hydroxyapatite as reference material. Further details of the XPS analysis of calcium phosphate phases and systematic errors of calculated Ca and P concentrations are described elsewhere (Kleine-Boymann M, Rohnke M, Sann J, Henss A, Janek J, Differentiation of biologically relevant calcium phosphate phases by surface-sensitive techniques, submitted to Appl Surf Sci).
2.4 Time-of-flight secondary ion mass spectrometry (ToF-SIMS)
The mass image was taken in the low current bunched mode (lc-bu), where good mass resolution is combined with an optimized lateral resolution of about 1 μm. The applied primary ion current was 0.13 pA.
2.5 Sample preparation of bone cross sections
For the application and testing of the calibration curve bone samples from a long-term small animal model for osteoporosis induction were used. Osteoporosis was induced in female Sprague–Dawley rats by ovariectomy (OVX) combined with a special multi-deficiency diet. The animal study was performed in full compliance to the German animal protection laws and was approved by the ethical commission of the local governmental institution (“Regierungspräsidium” Giessen, Germany, permit number: 89/2009 & 20/10-No. A31/2009). The protocol and procedures employed in the animal experiment were reported previously in detail .
Vertebrae samples of three different groups were analysed: A sham group with 13 animals (time of euthanasia in months, number of animals; 3, 7; 12, 6), OVX + diet group with 14 animals (3, 7; 12, 7) and the reference group with 8 animals at the beginning of the experiment (0, 8). After euthanasia at distinct times (0, 3, and 12) the vertebrae samples were embedded in poly methyl methacrylate (Technovit 9100; Heraeus Kulzer, Hanau, Germany) and afterwards cut and ground into slides of 20 μm–50 μm thickness.
2.6 Statistical analysis
The data were checked for statistical significance by the one-way ANOVA test accompanied by Games-Howell pair wise multiple comparisons to determine the variation of the Ca content in each group, among groups at particular times and throughout the whole experiment. The asterisks indicate the significance level (* p < 0.05, ** p < 0.01, *** p < 0.001). The results were expressed as mean and standard error. The statistical analysis was done using the software IBM SPSS Statistics 20.
3 Results and discussion
HAP content of the mineralised collagen scaffolds
Bulk (Ignition) HAP (wt%)
Surface (XPS) HAP (wt%)
HAP content of rat trabeculae of various groups at different times evaluated by ToF-SIMS
Norm. Ca-Intensity (counts)
Content HAP (wt%)
The obtained mean values for the HAP content of the analysed trabeculae agree well with the realistic range of HAP concentration for healthy bone. It is known that healthy bone consists of about 70% HAP , which is in fairly good agreement with the result of 62% HAP at the beginning of the animal experiment (reference group). Due to the maturing of the animals an increase of the HAP content is expected and is documented by 76% HAP after 3 months for the sham operated, healthy animals. Beside this a remarkable and statistical significant reduction of about 50% of the HAP content for the osteoporotic animals (OVX + diet group) can be found. However, the extreme reduction revealed by ToF-SIMS measurements is plausible and a qualitative comparison with BMD evaluated by DEXA measurements shows the same tendencies although not to this extent . Due to the experimental conditions, like the Ca and Vitamin D deficient diet, wide regions of the trabeculae are not mineralised anymore, which indicates rather osteomalacia than osteoporosis. The non-mineralised collagen matrix with small remains of Ca in the centre of the trabecular has been described in more detail in our previous study . After histological and serological investigations El Khassawna et al. also conclude the development of osteomalacia in this rat model . In consequence the Ca quantification might become more complicated due to measurement of non-mineralised regions which might result in too low Ca values for the OVX and diet group.
HAP content of osteoporotic and non-osteoporotic trabeculae evaluated by XPS and ToF-SIMS
12 months sham
12 months diet
Calculated HAP (wt%)
Corrected HAP (wt%) XPS
HAP (wt%) ToF-SIMS
By calibration curve
Finally, we like to note that further improvements will be possible. Trabecular bone does not consist of pure HAP, rather it is known that HAP is only the final phase of matured bone. During the process of bone formation different Ca phosphate phases like amorphous calcium phosphate (ACP) or octa calcium phosphate (OCP) are formed . At the end of this process usually a Ca deficient HAP phase is found. Moreover, the matured HAP is not a pure hydroxyapatite mineral. Also other anions like fluoride or carbonate are part of the crystal structure . Therefore our current approach to equate the Ca content with the HAP content of the bone samples according to the formula Ca5(PO4)3OH should only be considered as a first successful step. It is a quite challenging task to synthesise and test standards, which are comparable to natural bone in composition, lattice structure of the crystalline component, density and surface roughness. However, optimized standards will help to further improve the validity of ToF-SIMS data.
Calcium quantification by ToF-SIMS is possible, however, the quality of the results is strongly affected by the surface properties of the standards. Although the standards consisted of collagen and HAP comparable to normal bone–which makes the matrix-effect differences of the standards and bone negligible–the surface roughness is not really suitable for the ToF-SIMS measurements. Despite the influence of the surface morphology, the present results are encouraging. A linear relation is obtained between the Ca secondary ion counts rate and the Ca concentration of the prepared standards. The application of the calibration data to natural rat bone samples leads to plausible results, which were cross-checked by XPS measurements. As the ToF-SIMS analysis offers information on both organic and inorganic components of osseous tissue with high spatial resolution, the quantification of the Ca content based on the ToF-SIMS measurements might become a useful and complementary method to assess bone quality and damage. Future work on improved standards will allow a more precise quantification with smaller error. The mapping of various Ca concentrations and of different calcium phosphate phases to track the mineralisation process will be further useful applications of ToF-SIMS.
We gratefully acknowledge funding and support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the collaborative research centre–SFB/TRR 79 “Materials for tissue regeneration within systemically altered bone”, projects M5, M4 and T1.
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