28Life-history parameters such as pregnancies, skeletal trauma, and renal disease have previously 29 been identified from hypomineralized growth layers (incremental lines) of acellular extrinsic 30 fiber cementum (AEFC). The precise periodicity of these growth layers remains vaguely 31 approximated, so causal life-history explanations using tooth cementum cannot yet be 32 rigorously calculated or tested. On the other hand, we show how life history parameters in 33 AEFC can be identified by two contrasting elemental detection methods. Based on our results 34 we reject the possibility of accurate estimation of pregnancies and other life history parameters 35 from cementum using scanning electron microscopy alone. Here, we propose a new 36 methodological approach for cementum research, Time-of-Flight Secondary Ion Mass 37 Spectrometry (ToF-SIMS), to measure degree and distribution of mineralization of cementum 38 growth layers. Our results show that Tof-SIMS can significantly increase our knowledge of 39 cementum composition and is therefore a powerful new tool for life history researchers. 40 41 131 Cluster ion sources, such as Bi 3 , are used to identify larger HAp fragment ions at for example 132 m/z 485, 541, 597, and 653, identified as Ca 5 P 3 O 12 + , Ca 6 P 3 O 13 + , Ca 7 P 3 O 14 + , and Ca 8 P 3 O 15 + , 133 respectively. Ionic species sputtered from the surface under the bismuth bombardment are 134 steered into a reflectron time-of-flight mass analyzer. Before mass spectrometry was 135 performed, an Ar n + cluster ion beam was used to remove any surface organic contaminants. 136 Identified peaks strongly localized to cementum growth layers were mapped on single ion 137 maps. Positive-ion spectra were acquired from two different 100×100 μm regions of tooth 138 encompassing the entire cementum width from mesio-buccal and disto-buccal side of the tooth, 139 respectively to localize of HAp and identification of different CaP phases within cementum 140 layers.
Results
147
SEM-EDS
148No visual evidence of cementum growth layering was found SEM micrograph using Philips 149 XL30 FEG-SEM (Hillsboro, OR, USA) equipped with an Oxford instruments energy 150 dispersive x-ray analysis detector (Fig 1a). The Ca:P ratio (by atomic percent) ranged from 151 1.47 to 1.73, with 1.59 as average value. Line scan for Ca showed no significant change in its 152 relative amounts across the width of the cementum (Fig 1b), except in the case of line spectrum 153 (8) which exhibits the lowest relative amount of Ca as of 0.13(atomic %), as well as the lowest 154 Ca:Pa ratio as of 1.29 by atomic % (Fig.1a). However, these low readings for the line spectrum 155 (8) are due to an intruding artefact deposited in cementum, which can be clearly observed on 156 electron photomicrograph of a transverse section of midroot cementum (Fig 1a), and should 157 not be taken into account when interpreting mineral distribution across the AEFC width of our 158 sample.
