Cementum ultrastructure, a comparative perspective from synchrotron x-ray scanning STEPHAN NAJI1,2,4, WILLIAM RENDU2,3, LIONEL GOURICHON4, ZHONGHOU CAI5, STUART STOCK6 1. New York University, USA; 2. CNRS, NYU-CIRHUS; 3. CNRS, PACEA – UMR 5199; 4. Université Côte d’Azur-CNRS, CEPAM; 5. Advanced Photon Source-Argonne National Laboratory, 6. Department of Cell & Molecular Biology, Northwestern University
Contact:
[email protected]
Introduction
Mammal teeth are composed of enamel, dentin, and cementum. While the first two are only developing until the end of tooth formation, cementum continues to grow throughout life in order to maintain optimal tooth attachment. All three tissues have a cyclic incremental growing pattern, daily for the dentin and enamel, annual or seasonal for the cementum. While dentin and enamel growth cycles are well understood1, cementum annual growth pattern has never been formally demonstrated, but repeatedly observed empirically in over 70 mammal species2. Furthermore, the nature of the alternating light-dark growth pattern observed in transmitted light microscopy is still unexplained3. The standing debate about these lines revolves around 2 arguments: the banding pattern is either characterized by collagen fiber orientations or differential mineral composition4. Recent analyses on beluga whale teeth using synchrotron x-ray fluorescence mapping and diffracted intensities, revealed significant variations in mineral composition for each increment5. Similarly, recent Raman analyses6 confirmed that cementum mineral crystals and collagen fibers are oriented in the same direction. They also evidenced a difference in mineralization between dark and light bands on human samples. Goal: This study investigates the structure of individual acellular increments using x-ray fluorescence mapping and x-ray diffraction mapping with microbeams of synchrotron radiation on 21 samples of reindeer, red deer, bison, and human from reference and archaeological collections. The goal is to explore mineral composition calcium, phosphorous and zinc on of individual increments of acellular cementum and test if their variations match cementum increments observed optically.
Cementum: types and
3 functions
“The Good…
Gingivae Periodontal ligament
…the Bad … Mixed
Acellular Extrinsic Fiber Cementum (AEFC)
Alveolar bone Cementum (yellow)
…the Ugly”
Taphonomy & Pathology
• Provide attachment for the periodontal ligament. •Growth rate is regular and continuous (2-3 μm/year) CIFC
2
1
1 2
Cellular Intrinsic Fiber Cementum (CIFC) :
Acellular Cementum Cellular Cementum
•Function is adaptive (mechanical stress and repair) •Growth rate is highly irregular and up to 30 times faster than AEFC
Cementum increments (black arrows); TGL = Tome’s Granular Layer
• Histological understanding of cementum growth is critical to properly select the best Region of Interest • Even when affected by taphonomic or pathologic agents, acellular cementum can be correctly assessed
Material and method 4. Synchrotron x-ray microprobe for fluorescence mapping and microdiffraction
Advance Photon Source Species
N. sample
N. sections
Rangifer tarandus (reindeer) Cervus elaphus (red deer) Bos taurus (cattle) Modern Human
8 2 2 3
7 2 3 3
Reindeer 017 -78, M2 Born 06/1960, died 04/1966 6 years and 10 months.
PL
Cementum
Dentin
2. Teeth were sectioned following standard protocols for human7, and ungulates8
1. Samples processed at the APS Argonne National Laboratory (Chicago)
50 µm
3. ROI are selected for optimum increment contrast
X-ray microprobe of ROI
Results 1. Variation in Zn fluorescent intensity is a very sensitive indicator of changing biomineralization in all species 2
3 3 Cementum
3
1
Air
4 Dentine
PDL
H-S layer
2
Cementum
PL
1
3. Peaks could be matched to optic increments
2. In all 4 species, fluorescence signals for Ca and P are always strongly correlated, and consistently rose and fell together with Zn and cAp diffraction signal
190000
PL
Cementum
Dentin
H-S layer
PDL
180000
4500
Bos 028-94261
Reindeer 017-78 2
5000
4
1
4
Dentin
170000 4000 160000
2
3
3500
Human S82
Variation of Zn across air (1), PDL (2), cementum (3) and dentin (4) in 4 species
Conclusion
66
63.5
61
58.5
56
53.5
51
46
48.5
43.5
41
38.5
36
33.5
31
28.5
26
23.5
21
Reindeer 017 -78
140000
Bos 028-94261
Variation of Ca, P, Zn and diffraction intensity across increments
In concordance with the most recent studies5,6, the acellular cementum banding light-dark pattern observed in transmitted bright-field microscopy is due to differential mineral composition. • The dark increments are hypermineralized and the light increments are hypomineralized. • This conclusion confirms the hypothesis of differential cementum growth, with a denser mineral component forming during the “bad” season (fall-winter/humid) and a lighter mineral density forming during the “good” season (spring-summer/dry). On-going analyses look at diffracted intensities data to test if crystallographic texture exists and varies across the acellular bands.
References
Ca P Zn Diff. Lumin.
1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 251 261 271 281 291 301 311 321 331
Red Deer 040-CL302
3000
16
4
1
18.5
2 Periodontal ligament
3
4
13.5
1
1
49
150000
49
Human S82: 49 dark increments
1
Human S46: 12 dark increments
Variation of luminescence profile, Ca,P and Zn
• On-going analyses are exploring diffracted intensities data to test if crystallographic texture exists and varies across the acellular bands.
X-ray Diffraction pattern for hydroxyapatite crystals
1. Zheng L, Ehardt L, McAlpin B, About I, Kim D, Papagerakis S, Papagerakis P. 2014. The tick tock of odontogenesis. Experimental Cell Research 325:83–89 / 2. Klevezal GA. 1996. Recording Structures of Mammals: Determination of Age and Reconstruction of Life History. Rotterdam: A. A. Balkema Series / 3. Yamamoto T, Hasegawa T, Yamamoto T, Hongo H, Amizuka N. 2016. Histology of human cementum: Its structure, function, and development. Japanese Dental Science Review 52:63–74 / 4. Naji S, Colard T, Blondiaux J, Bertrand B, d’Incau E, Bocquet-Appel J-P. 2016. Cementochronology, to cut or not to cut? International Journal of Paleopathology 15:113–119 / 5. Stock SR, Finney LA, Telser A, Maxey E, Vogt S, Okasinski JS. 2017. Cementum structure in Beluga whale teeth. Acta Biomaterialia 48:289–299. / 6. Colard T, Falgayrac G, Bertrand B, Naji S, Devos O, Balsack C, Delannoy Y, Penel G. 2016. New Insights on the Composition and the Structure of the Acellular Extrinsic Fiber Cementum by Raman Analysis. PLOS ONE 11:e0167316 / 7. Colard T, Bertrand B, Naji S, Delannoy Y, Bécart A. 2015. Toward the adoption of cementochronology in forensic context. Int J Legal Med 129:1–8. 8. Rendu W. 2010. Hunting behavior and Neanderthal adaptability in the Late Pleistocene site of Pech-de-l’Azé I. Journal of Archaeological Science 37:1798–1810.
