Applicability of ToF-SIMS for monitoring compositional changes in bone in a long-term animal model

Abstract

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a wellestablished technique in material sciences but has not yet been widely explored for implementation in life sciences. Here, we demonstrate the applicability and advantages of ToF-SIMS analysis for the study of minerals and biomolecules in osseous tissue. The locally resolved analysis of fragment ions deriving from the sample surface enables imaging and differentiation of bone tissue and facilitates histology on non-stained cross sections. In a rat model, bilateral ovariectomy combined with either a multi-deficiency diet or steroid treatment was carried out to create osteoporotic conditions. We focused our study on the Ca content of the mineralized tissue and monitored its decline. Calcium mass images of cross sections show the progressive degenerative changes in the bone. We observed a decreased Ca concentration in the edge region of the trabeculae and a decline in the Ca/P ratio. Additionally, we focused on the non-mineralized matrix and identified fragment ions that are characteristic for the collagen matrix. We observed trabeculae with wide ranges of nonmineralized collagen for the diet group owing to an impaired mineralization process. Here, the advantage of coeval monitoring of collagen and minerals indicated an osteomalacic model rather than an osteoporotic one.

Quantification of calcium content in bone by using ToF-SIMS–a first approach

“…The osteoporotic induction applied over several months allows a tracking of the degradation of the bone which comes along with a reduction of the bone mineral content and density [18]. In a previous study these results were confirmed by a semi quantitative analysis of the Ca content [11]. Applying the linear equation (1) improved to become a truly quantitative analysis of the HAP content as shown in Figure 3d.…”

“…The comparison with mass spectra of mineralised tissue reveals almost identical mass spectra with characteristic peaks and fragmentation patterns of HAP and collagen, which are well known from our study of bony tissue amongst others [11,24]. The plot of the obtained signal intensities of Ca versus the surface concentration obtained from XPS measurements results in a linear relationship, see Figure 3c.…”

“…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 [11].…”

“…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][12][13]. ToF-SIMS provided detailed chemical information of bone with high sensitivity and a lateral resolution of up to 300 nm.…”

Mg-corrosion, hydroxyapatite, and bone healing

“…Calcium phosphates, including hydroxyapatite (HA), precipitate on the surface of corroding Mg. 6,7 To gain a better understanding of the corrosion process and how it might facilitate mineralization in vivo, we applied energy dispersive x-ray spectroscopy (ESEM/EDX), ToF-SIMS, and XPS for chemical and elemental analyses of bone mineralization. [8][9][10][11] The importance of the MgO phase in HA formation has been reported. 9,12,13 Hence, Mg corrosion products may function as substrates facilitating the formation of low crystalline, carbonated HA (CHA), where the high Mg ion concentration inhibits its transition to crystalline HA.…”