Life history parameters in acellular extrinsic fiber cementum microstructure

Life-history parameters such as pregnancies, skeletal trauma, and renal disease have previously been identified from hypomineralized growth layers (incremental lines) of acellular extrinsic fiber cementum (AEFC). The precise periodicity of these growth layers remains vaguely approximated, so causal life-history explanations using tooth cementum cannot yet be rigorously calculated or tested. On the other hand, we show how life history parameters in AEFC can be identified by two contrasting elemental detection methods. Based on our results we reject the possibility of accurate estimation of pregnancies and other life history parameters from cementum using scanning electron microscopy alone. Here, we propose a new methodological approach for cementum research, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), to measure degree and distribution of mineralization of cementum growth layers. Our results show that Tof-SIMS can significantly increase our knowledge of cementum composition and is therefore a powerful new tool for life history researchers.


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Acellular extrinsic fiber cementum (AEFC) is deposited in a regular annual rhythm in the form 43 of incremental lines around the roots of human teeth, with varying degrees of mineralization 44 [1,2]. Life-history parameters (LHP), such as pregnancies, skeletal trauma, and renal disease, 45 can be identified and precisely datable from incremental lines of AEFC in human teeth by 46 observing their visual effects [3]. These life history parameters appear to change calcium 47 metabolism [4,5], and lack of available calcium at the mineralization front of the cementum 48 causes formation of a such visually different incremental AEFC line [3]. In a study on humans 49 [3], as well as in great apes [6] "suspicious" AEFC lines were successfully detected as being 50 visibly broader and translucent in tooth ground sections (70 -80µm thick) under optical 51 magnification with transmuted polarized light. These studies also showed that some of the 52 LHPs affecting mineralization of AEFC are precisely datable from the AEFC cross-sections. 53 On the other hand, in a controlled study undertaken on goats, Lieberman [2] showed that AEFC 54 bands corresponding to a control diet low in minerals including calcium and phosphorus 55 appeared to be opaquer and relatively narrower, as observed from x-ray microradiographs of 56 thin ground sections (50µm thick). Lieberman described these bands as hypermineralized 57 (denser) due to reduced cementogeniesis. In contrast, a study undertaken by Cool Table 1). The patient was born and raised in the region of Kosovska 87 Mitrovica. At the time of the extraction she was 66 years old with no previous history of renal 88 disease, endocrinal problems, skeletal fractures or trauma. The patient was considered an 89 excellent candidate for a fertility related analysis, as she reported six pregnancies that carried 90 to full-term, starting at the age of 19 with the last one at age of 31 (Table 1). After the extraction, 91 the tooth was placed in a labelled vial containing physiological saline (solution of 0.90% w/v 92 of NaCl). The tooth was free from obvious signs of pathology.  The resin block with exposed mid-root surface represented the sample to be analysed by SEM-

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Line scans of cementum growth layers was subject to an acquisition time of 100 sec (working 109 distance 10 mm, take-off angle 35°) to obtain X-ray spectra. The X-ray spectra were used to 110 determine which minerals were present and the Ca:P atomic percent ratio.

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Mass Spectrometry Imaging 114 The sample preparation for ToF-SIMS imaging comprised several steps and it was performed 115 at University College London, Institute of Archaeology. The procedure was tailor made for this 116 research, namely the identification of Ca and HAp form AEFC growth layers using ToF-SIMS.

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After the cross section was cut out from the mid-root of the patient's tooth, the exposed root

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No 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 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. Elemental and molecular maps, as well as line scans of Ca+ and HAp+ (Ca/P ratio) are obtained 184 from the AEFC surface. We have detected a variation in the intensity of Ca+ across the 185 analyzed AEFC surface (Fig 2b). A depletion in relative Ca+ intensity can be observed from 186 12 -54μm respectively, but no depletion in intensity of HAp+ in the same line scan (Fig 2b, 187 Fig 3). This corresponds to the patient's 2 nd -5 th decade of life (Fig 3). The lowest point in Ca+ 188 intensity depletion is recorded at 32 nd μm (Fig 3) which corresponds to start of the patent's 4 th 189 decade of life (around age her age of 30). life history parameter detection in AEFC can vary between two elemental detection methods.

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Scanning electron microscope with electro-dispersive probe did not detect any significant 208 variation in Ca relative amounts across the AEFC width, nor in Ca:P ratio respectively (Fig   209   1b). The range for the Ca/P ratio (by atomic percent) varied between 1.47 to 1.73, where the 210 majority of the values (Fig 1b)  ToF-SIMS micro-image revealed clearly defined AEFC in our cross-section. Although some 238 appearance of layering within AEFC can be also observed (Fig 2a), the incremental lines of 239 AEFC were not clearly discernible from the micrograph. On the other hand, we were able to 240 estimate the approximate width of AEFC incremental lines using the micrograph itself. The 241 linescans showed obvious variation in Ca+ intensity across the AEFC width, but not for HAp+ 242 (Fig 2b, Fig 3). Ca+ intensity variation has been detected in a form of a depletion which might 243 correlate with the with patient's pregnancies, as the depletion of Ca+ intensity corresponds to 244 the patient's 2 nd -5 th life decade (Fig 3).