A robust, semi-automated approach for counting cementum increments imaged with synchrotron X-ray computed tomography

Cementum, the tissue attaching mammal tooth roots to the periodontal ligament, grows appositionally throughout life, displaying a series of circum-annual incremental features. These have been studied for decades as a direct record of chronological lifespan. The majority of previous studies on cementum have used traditional thin-section histological methods to image and analyse increments. However, several caveats have been raised in terms of studying cementum increments in thin-sections. Firstly, the limited number of thin-sections and the two-dimensional perspective they impart provide an incomplete interpretation of cementum structure, and studies often struggle or fail to overcome complications in increment patterns that complicate or inhibit increment counting. Increments have been repeatedly shown to both split and coalesce, creating accessory increments that can bias increment counts. Secondly, identification and counting of cementum increments using human vision is subjective, and it has led to inaccurate readings in several experiments studying individuals of known age. Here, we have attempted to optimise a recently introduced imaging modality for cementum imaging; X-ray propagation-based phase-contrast imaging (PPCI). X-ray PPCI was performed for a sample of rhesus macaque (Macaca mulatta) lower first molars (n = 10) from a laboratory population of known age. PPCI allowed the qualitative identification of primary/annual versus intermittent secondary increments formed by splitting/coalescence. A new method for semi-automatic increment counting was then integrated into a purpose-built software package for studying cementum increments, to count increments in regions with minimal complications. Qualitative comparison with data from conventional cementochronology, based on histological examination of tissue thin-sections, confirmed that X-ray PPCI reliably and non-destructively records cementum increments (given the appropriate preparation of specimens prior to X-ray imaging). Validation of the increment counting algorithm suggests that it is robust and provides accurate estimates of increment counts. In summary, we show that our new increment counting method has the potential to overcome caveats of conventional cementochronology approaches, when used to analyse three-dimensional images provided by X-ray PPCI.