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.


Introduction
-errors in biblio -lack of precision regarding location of AEFC and CIFC.
-improve clarify regarding the use of the word "cementum": complex tissue with sub-types, indicate here clearly what is being studied.

Methods + Results
-the protocol used here is highly destructive! The crown was sectioned, and the roots separated. The end of the introduction involves a kind of blunt criticism about the pilot study of Le cabec et al 2019, yet, they did not cut the teeth, and use higher energy to avoid damaging the DNA! -Whether the teeth used here are deciduous or permanent is not clear until the discussion. This should be stated at the beginning of the "Materials".
-There are no explanation about how the thin sections were made? Any polishing involved? Slice thickness? -SR µCT acquisitions: Why choosing 1501 projections whereas SNR seems best around 3000-4000 projections? -There is a lack of explanation regarding what has been scanned: where is the FOV/ROI on the root? What size? Which root? Where along the root length? Was the ROI chosen in a standardized manner for all individuals? Explain the criteria. A figure would be helpful. -Cite more often your supplementary information.
-16 or 8 bit data? This needs to be clarified and tested as it may have an impact on the results.
- Figure 3: on the plot, there are too many curves compared to the figure legend? Also rather indicate the resolution / pixel size and not the sample-detector distances, as this is less intuitive for most non-specialist readers.
-How do the method deal with second-order increments if any? In the SOM I see they are ignored, as well as on the figure showing the I and II in light blue. But is it so frequent? I might be good to show a picture, because those structures do exist. They have been identified also by SXRF.

Discussion
L553-560: + Fig. S3: the scans generated micro-cracks: this is of concern!!! Maybe the specimen was still fresh and not totally dry? this could have induced the liberation of free radicals, especially with the low energy used.

This claim is actually going to be harmful to the synchrotron community, and a major concern!
Why? Because nonspecialists are going to be comforted in the general belief that synchrotron imaging damages specimens. So it should be made clear here why and how these micro-cracks occurred, and not let readers believe this will happen every time!

Supplementary Material
Equations 2 and 3: is the second 0 the symbol for degree (0°)? then it should be superscript to 0 or 90, here it seems to have normal size and it thus seems confusing. Or L42, explain this is for 0°, this would help non-specialists to understand the notation. L66: so why not running this on the 16 bit data? It needs to be tested if running it on 8 bit induces a loss of contrast/details that would not occur when using 16 bit data.
L78-79: I am not sure to understand how you choose by which integer you divide the 1st directional derivative image.
L104: is not this circular reasoning? the PPC SR data without any processing, thus including background noise? this needs to be clarified. L161: why "tribological" in the title??? and L187, L286: I don't understand the use of this word "tribological"? To me, this has to do when 2 surfaces are in contact and involve movement (e.g., tooth occlusal surfaces and food items). Please either define, rephrase (explain) or delete.