Mechanical modelling of tooth wear rsif.royalsocietypublishing.org

Research Cite this article: Karme A, Rannikko J, Kallonen A, Clauss M, Fortelius M. 2016 Mechanical modelling of tooth wear. J. R. Soc. Interface 13: 20160399. http://dx.doi.org/10.1098/rsif.2016.0399

Received: 20 May 2016 Accepted: 20 June 2016

Subject Category: Life Sciences – Earth Science interface Subject Areas: biomechanics, bioengineering Keywords: chewing machine, microwear, plant material, phytoliths, grit, tooth wear

Authors for correspondence: Aleksis Karme e-mail: [email protected] Janina Rannikko e-mail: [email protected]



These co-first authors contributed equally to this study. Electronic supplementary material is available at http://dx.doi.org/10.1098/rsif.2016.0399 or via http://rsif.royalsocietypublishing.org.

Aleksis Karme1,†, Janina Rannikko1,†, Aki Kallonen2, Marcus Clauss3 and Mikael Fortelius1 1 Department of Geosciences and Geography, Division of Biogeosciences, and 2Department of Physics, Division of Materials Physics, University of Helsinki, Helsinki, Finland 3 Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

AK, 0000-0003-4852-2636; JR, 0000-0002-5542-4201; MC, 0000-0003-3841-6207; MF, 0000-0002-4851-783X Different diets wear teeth in different ways and generate distinguishable wear and microwear patterns that have long been the basis of palaeodiet reconstructions. Little experimental research has been performed to study them together. Here, we show that an artificial mechanical masticator, a chewing machine, occluding real horse teeth in continuous simulated chewing (of 100 000 chewing cycles) is capable of replicating microscopic wear features and gross wear on teeth that resemble wear in specimens collected from nature. Simulating pure attrition (chewing without food) and four plant material diets of different abrasives content (at n ¼ 5 tooth pairs per group), we detected differences in microscopic wear features by stereomicroscopy of the chewing surface in the number and quality of pits and scratches that were not always as expected. Using computed tomography scanning in one tooth per diet, absolute wear was quantified as the mean height change after the simulated chewing. Absolute wear increased with diet abrasiveness, originating from phytoliths and grit. In combination, our findings highlight that differences in actual dental tissue loss can occur at similar microwear patterns, cautioning against a direct transformation of microwear results into predictions about diet or tooth wear rate.

1. Introduction There has been considerable debate regarding the relative role of extrinsic ‘grit’ and internal minerogenic plant parts ( phytoliths) as drivers of tooth wear. Hardness of enamel, opal phytoliths and external grit have been in the centre of the debate. Although opal phytoliths in pasture and fodder plants were measured harder than enamel [1], the role and hardness of phytoliths have been repeatedly questioned, partly on the basis of modern experimental work [2– 5]. Despite the conclusion that phytoliths cause plastic deformation rather than removal of enamel, a recent ground-breaking study shows that phytoliths do in fact impact enamel [5]. Very recently, particles softer than enamel were reported to generate grooves, resulting from tissue loss and not plastic deformation, on enamel in macro- (aluminium and brass spheres) and nanoscale (amorphous silicon dioxide spheres) [6]. There is now growing recognition that an experimental study of dental wear is needed to resolve the long-running debate, and more experimental work is now being published. In pioneering work, Maas [7,8] constructed two experimental studies for microwear focusing on species differences in enamel microstructure, direction of shearing forces relative to enamel prisms, size of the abrasive particles and magnitude of the force. The effect of individual particles on dental enamel has also been studied experimentally, but not in a manner that would have generated cumulative patterns similar to those caused by long-term chewing [4,9]. The effect of different diets was explored in rabbits (Oryctolagus cuniculus) with respect to three-dimensional texture analysis and microwear, where grass diets resulted in scratch-dominated microwear patterns and lucerne diets resulted in more pitted patterns [10]. Low silica content in the diet also resulted in more variable surface textures and microwear patterns. Exposing rabbits and guinea pigs (Cavia porcellus) to diets designed to reflect

& 2016 The Author(s) Published by the Royal Society. All rights reserved.

The experiment was conducted with four different diets and pure attrition with 25 pairs of horse molar teeth. Horse teeth were used because they are relatively easy to obtain, large and the three dental tissues, enamel, dentine and cement, are all visible on the surface at the same time. The third and fourth premolars, as well as the first and second molars, were used, as their shape is more symmetrical than teeth on either end of the cheek tooth row. Five pairs of teeth were made to chew in each diet and a set of five teeth were also made to chew in water only, simulating pure attrition (ATTR) without a buffering layer of food. Chewing was conducted with a mechanical masticator (figure 1) built at the University of Helsinki, Department of Geosciences and Geography and Department of Physics [27], and the methods were developed in pilot studies [27,28]. The pelleted diets were based on lucerne, L (or alfalfa, Medicago sativa), which by nature contains very low levels of internal abrasives [29], grass, G, which contains higher levels of

2

(b)

moving part

25 mm

arm with a lower tooth arm with an upper tooth

Figure 1. Mechanical masticator. Panel (a) shows the machine and teeth attached. Panel (b) shows a scheme of the machine. (Online version in colour.)

internal abrasives, and grass with the addition of rice hulls, GR, which contains again higher levels [29]. The fourth pelleted diet included grass and rice hulls (internal abrasives) and added sand (external abrasives), GRS. These diets were chosen because of the aforementioned attributes, and because the exact same diets had been used in previous studies with live animals, where they had been demonstrated to cause differences in tooth wear [11,12]. Abrasives, measured as acid detergent insoluble ash (ADIA) in dry matter, increased in diets without sand from L (5 g kg21), to G (16 g kg21) and to GR (24 g kg21). A basic assumption was that most of the abrasives are silica phytoliths [30]. Lucerne, which is a flowering plant in the pea family, is essentially phytolith-free and is similar to browse in this respect. GRS contained sand grains (mean diameter of 233 mm) as an external abrasive, which increased the ADIA value and abrasiveness (ADIA 77 g kg21). A complete ingredient list and nutrient composition of diets can be found elsewhere [11]. Teeth were submerged in a relatively thick food bolus to achieve a cushion effect during chewing, avoiding pure non-buffered attrition. In total, 1 l of dry pellets was mixed with 3 l of water, which had 3 g salt per 1 l to avoid the expansion of the plant cells. Before the chewing procedure, teeth were prepared to fit the machine (figure 2a). After the roots had been cut off, the teeth were glued into three-dimensionally printed polylactic acid rings with epoxy, in order to fit them in the mechanical masticator. Preparations were finished by cutting the occlusal surfaces at an angle of 338. Before chewing, the surfaces of the teeth were polished with an abrasive disc grinding machine (grain size 68 mm, CAMI 220, ISO 6344 P220) (figure 2b). Teeth were positioned in the mechanical masticator so that scratches due to chewing ( parallel to chewing direction) could be differentiated from the initial polishing striation ( perpendicular to chewing direction) (figure 2b,c). Fading of initial polishing striations was used as a rough indicator of wear rate. All tooth pairs were made to chew submerged in the diet– water mixture for 6 h and 30 min, which equals roughly 100 000 chews (260 chews per minute) resulting in a speed of 108 mm s21, comparable to real chewing speeds of horses [31]. The mechanical masticator simulates a repeated, full-occlusion single stroke movement between a pair of upper and lower teeth with surfaces flattened to a single plane [27,28]. A motor moves the other arm of the machine, which has a lower tooth

J. R. Soc. Interface 13: 20160399

2. Material and methods

(a)

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different proportions of internal and external abrasives (sand), and measuring the resulting effects manually and by computed tomography, differences in wear and in the responding tooth growth could be demonstrated [11,12]. Most recently, the effects of external grit on the microwear signature were demonstrated in live sheep [13] and angles of approach in chewing were shown to affect microwear [14]. Microscopic study and analysis of worn tooth surfaces [15,16] (microwear) have become a standard tool for identifying the diet preferences of fossil and living vertebrates. Microwear analysis has been widely used to study diet among ungulates [17 –19] and other animals [20–23]. Among ungulates, individuals consuming browse (dicotelydonous plants) can be distinguished by their microwear patterns from those consuming graze (monocotelydonous plants). The main pattern, reconfirmed in a large number of studies, is that browsers have pit-dominated microwear while grazers have scratch-dominated microwear [15,17,24]. Among another widely studied group, the primates, hard food consumers have a mainly pit-dominated microwear pattern and tough food consumers a more scratch-dominated microwear pattern [25,26]. Here, we present the microwear analysis results and actual dental tissue loss (i.e. tooth wear rates) of a chewing experiment with the standardized pelleted foods used in two previous live animal studies [11,12]. The experiment was conducted under controlled laboratory conditions, using mechanical modelling (chewing machine) [27] to occlude real horse teeth submerged in food in a simple chewing cycle. In designing the mechanical masticator, it was not intended to develop a system that mimics the conditions in the actual animal mastication with detailed precision; for example, we did not intend to emulate the consistency and properties of saliva, or to exactly replicate the direction and detailed movement of antagonistic dental contact. Instead, the aim was a simplified system that would allow changing various factors in mastication, for example different standardized feeds, to test whether expectations based on a simplistic interpretation of microscopic and macroscopic wear could be confirmed. The aim of this study was to produce microwear features seen in nature, i.e. pits and scratches, and to quantify macroscopic wear with real teeth and diets, in order to achieve a detailed picture of the wear process and its components.

3

(a) roots

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lingual side

buccal side

PLA ring

33º

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(c)

(b)

0.4 mm

0.4 mm

Figure 2. (a) Schematic of the preparation of a tooth (modified from Bertin et al. [28]). First, the roots are cut off, then the tooth is placed inside a polylactic acid (PLA) ring with epoxy and finally the occlusal surface is cut. (b) Polished occlusal surface where striation from the polishing procedure goes from the upper left corner to lower right corner. (c) Occlusal surface after 6 h and 30 min of chewing. Microwear counting area (0.4  0.4 mm2) is marked with a black square. Scale bars, 0.4 mm.

attached, in a horizontal back and forth movement. An upper tooth is attached to another arm, which is flexible. As the lower tooth contacts the upper tooth, the upper tooth gives way slightly, and the lower tooth slides along it, simulating occlusal contact during the power stroke of natural chewing. To avoid the food particles and sand particles from settling to the bottom of the food container, we used a mechanical mixer and compressed air conducted to the bottom of the container to keep the food circulating. Silicon moulds (President Plus w regular body, Colte`ne/ Whaledent AG, Altsta¨tten, Switzerland) were made from a tooth surface before and after chewing. Transparent epoxy casts (EpoFixw, Struers Inc., Cleveland, Ohio, USA, base liquid and hardener) were made out of the moulds and examined with a stereomicroscope (SZX 10, Olympus Industrial, Essex, UK). The casts were orientated so that the chewing scratches were 458 from the centre line and illuminated from below in such a way that a totally reflecting surface could be viewed through the microscope. We decided to use a light microscope instead of scanning electron microscope (SEM), because of easier access, affordability and reduced time. Light microscopes have been used successfully in other microwear studies [18,19,24,32]. Note that light microscope results cannot be compared with SEM results directly due to different magnification. Images from each tooth were taken from two standard locations in the enamel band in the middle of the teeth with a Colorview III camera (Soft Imaging System GmbH, Mu¨nster, Germany) attached to the microscope. All images were taken with 32 magnification. Images were processed with Corel Paint Shop Prow X5 (Corel Corporation, Ottawa, Canada) to improve the visibility of wear features. Images were made

greyscale and negative and local tone mapping was used to adaptively enhance the contrast. The number of pits and scratches, which were generated in the chewing procedure, and initial polishing striations, which were generated in the polishing of the occlusal surfaces with the grinding machine, were recorded from a 0.4  0.4 mm2 area inside central enamel bands (figure 2c) with Microware 4.02 (P. Ungar, Fayatteville, Arkansas, USA). Pixel coordinates of the major and minor axis of each feature were extracted, which allowed us to calculate the absolute lengths and widths with known pixel size. We refer to the major axis as ‘length’ and the minor axis as ‘width’. A feature was categorized as a pit if the ratio of length and width was less than four, and a scratch if the ratio was more than four. Features were categorized to small (length less than 20 mm), large (length 20 – 50 mm) or very large (length more than 50 mm) pits [33] and thin (width less than 15 mm) or wide (width more than 15 mm) scratches [32,34]. JMPw Pro 10 (SAS Institute Inc., Cary, North Carolina, USA) was used to analyse the results statistically and for visualization. Number of pits, number of small pits, number of very large pits, number of wide scratches, pit length and pit width were log-transformed to obtain normal distribution and equal variances. Normal distribution was tested with Shapiro–Wilk test and variance equality between groups with Levene’s test. Results were analysed with analysis of variance (ANOVA) when groups were normally distributed and their variances were equal, and with Kruskal–Wallis test when normal distribution within or equal variances between groups were not obtained. Differences between groups (with normal distribution and equal variances) were analysed with Tukey–Kramer HSD test. Wilcoxon method was used to analyse the initial polishing striation count as it did

measure (unit)

L

G

GR

GRS

ATTR

24.40 + 4.36a

19.60 + 6.01a

23.50 + 5.60a

17.25 + 6.68a

pits (n) small pits (n)

24.85 + 11.18a 18.30 + 7.99

33.45 + 15.96a,b 24.80 + 12.26

16.20 + 8.12a 12.65 + 7.91

41.00 + 22.50a,b 18.55 + 12.77

83.30 + 41.93b 44.35 + 36.47

6.05 + 3.81a,b 0.50 + 0.47a

8.20 + 4.19a,b 0.45 + 0.21a

3.20 + 1.04a 0.35 + 0.22a

17.95 + 9.33b 4.50 + 2.50b

34.65 + 8.81c 4.30 + 3.08b

large pits (n) very large pits (n)

7.00 + 2.81b

thin scratches (n) wide scratches (n)

20.85 + 3.39a 3.55 + 2.97

17.15 + 5.10a,b 2.45 + 1.24

22.50 + 5.73a 1.00 + 0.59

12.50 + 4.23b,c 4.75 + 2.93

5.25 + 2.70c 1.75 + 0.73

pit length (mm)

18.20 + 2.30

14.17 + 6.83

35.59 + 27.33

23.82 + 11.20

26.86 + 8.42

20.10 + 12.88 448.60 + 82.14a

14.47 + 6.91 137.72 + 83.65b

15.55 + 4.07 180.62 + 67.80b

7.72 + 0.73 3.80 + 3.14a,b

10.00 + 5.23 0.95 + 1.02b,c

14.09 + 3.92 0.25 + 0.56c

pit width (mm) scratch length (mm) scratch width (mm) polishing striations left after chewing (n)

12.32 + 2.17 441.53 + 115.73a

9.82 + 4.86 304.97 + 149.73a,b

9.55 + 3.38 5.20 + 1.55a

7.57 + 3.67 7.75 + 2.50a

Table 2. Statistical evaluation. p-Values from ANOVA and Kruskal – Wallis test show significant differences (less than 0.05*).

measure (unit)

log-transformation

Levene’s tests p-value

normal distribution of groups

scratches (n) pits (n)

no yes

0.6143 0.9847

yes yes

0.0003* 0.003*

small pits (n) large pits (n)

yes no

0.5109 0.1758

yes yes

0.4913 ,0.0001*

very large pits (n)

yes

0.1191

yes

,0.0001*

thin scratches (n) wide scratches (n)

no yes

0.2094 0.3507

yes yes

,0.0001* 0.1018

pit length (mm) pit width (mm)

yes yes

0.0533 0.0997

no yes

0.222kw 0.4188

scratch length (mm) scratch width (mm)

no yes

0.372 0.183

yes no

0.0002* 0.058kw

polishing striations left after chewing (n)

no

0.1787

no

0.0009 kw*

not meet normal distribution for each group. We used a p-value of 0.05 as a level of significance. X-ray microtomography (computed tomography or CT scanning) was used to evaluate the actual amount of wear in one specimen from each diet group by scanning before and after the chewing experiments. The equipment used was a custommade mCT device nanotomw (PhoenixjXray systems þ Services GmbH, Wunstorf, Germany). The parameters used in the imaging were as follows: voxel size 37.5 mm, tube voltage of 150 kV and current 80 mA filtered with 0.5 mm of Cu, with 0.58 angular step over 3608. Three-dimensional reconstructions were made using datosjx rec software (PhoenixjXray systems þ Services GmbH), thresholding reconstructed volumes and surface extraction using the software VGStudio MAX 1.2.1 (Volume Graphics GmbH, Heidelberg, Germany). Alignment, visualization and occlusal surface extraction in three dimensions were made with Rapidform XOS3 (3D Systems Inc., Rock Hill, South Carolina, USA) [35] and the amount of wear was quantified in ArcMap 10.0 (ESRI Inc., Redlands, California, USA) by

p-values from ANOVA and Kruskal – Wallis testkw

calculating the mean height for the whole surface in three dimensions both before and after chewing, and subtracting the worn surface height from the unworn one.

3. Results Every tooth had both macroscopic and microscopic wear marks after chewing; means, standard deviations and differences of pits and scratches can be seen in table 1 and the results of statistical tests in tables 2 and 3. Many characters distinguish ATTR from the other diet groups. It had fewer scratches and more large pits than any of the other diet groups, and it also had fewer thin scratches and higher number of very large pits than all non-sandy diets. ATTR had a significantly higher number of pits and shorter scratch length than L or GR (tables 1, 2 and 3).

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scratches (n)

4

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Table 1. Means and standard deviations of various microscopic measures of tooth wear in horse cheek teeth used in an artificial mastication system using four different diets (L, lucerne; G, grass; GR, grass and rice hulls; GRS grass, rice hulls and sand) as well as without food (ATTR attrition). All features were measured from an area of 0.16 mm2. Superscripts a– c within rows indicate significant differences between diets (Wilcoxon method for initial polishing striations remaining, and Tukey – Kramer HSD for all others).

— 0.2393

0.9643

— —

0.104 —

0.9519

— 0.0036*

0.0422* 0.2756

L G GR GRS ATTR

pits (n)

— 0.0097* — 0.0578

0.0048* 0.0011*

— — — —

,0.0001* — 0.0132* —

0.004* 0.001*

— ,0.0001* — 0.0108*

0.0007* 0.0015* 0.3619 0.1242

100

75

50

— 0.0097* — 0.0119* scratch width (mm) polishing striations left after chewing (n)

— 0.1425

— 0.2087

— 0.0117*

— 0.0095*

— 0.0947

0.3545 0.1209 0.2265 0.0061* 0.0014* 1 0.2689 scratch length (mm)

— — — — — — — — — — pit length (mm) pit width (mm)

— —

— —

0.0028* — 0.4667 — 0.3322 — 0.0001* — 0.974 — thin scratches (n) wide scratches (n)

0.6718 —

0.0473* —

0.0021* 0.0005* 1 0.0115* 0.9892 very large pits (n)

0.9813

0.0028*

— ,0.0001* — 0.1426 — 0.9503 small pits (n) large pits (n)

— 0.9819

— 0.0501

— ,0.0001*

— 0.7193

0.0092* 0.1181 0.953 0.9869 0.7679 0.2876 0.0004* 0.0237* 0.2408 0.7011 0.9987 0.73 0.6107 0.9295 scratches (n) pits (n)

G-ATTR G-GRS G-GR L-ATTR L-GRS L-GR L-G measure (unit)

Table 3. Statistical evaluation of differences between all diets. p-Values from Tukey –Kramer HSD and Wilcoxon method show significant differences (less than 0.05*).

25

0

0

5

10

15 20 scratches (n)

25

30

35

Figure 3. A bivariate plot presenting average number of pits plotted against average number of scratches in horse cheek teeth used in an artificial mastication system using four different diets (L, lucerne; G, grass; GR, grass and rice hulls; GRS, grass, rice hulls and sand) as well as without food (ATTR attrition only). Ellipses show 90% CIs. (Online version in colour.) GRS could be distinguished from other plant material diet groups by having a higher number of very large pits and from L and GR by having shorter scratches and fewer thin scratches. GRS also had significantly higher number of large pits than GR. Polishing striations left after chewing were much fewer in GRS and ATTR than in non-sandy diets (tables 1–3). There were no statistical differences between the groups in the number of small pits, number of wide scratches, pit length, pit width and scratch width. Non-sandy diets, excluding ATTR, did not show statistically significant differences between tested characters. In general, GR had numerically fewer pits in every size category than L and G, and longer features than G. L and G were very similar. G tended to have more initial polishing striations left after chewing than GR. Average numbers of pits and scratches in every tooth pair are plotted in a bivariate plot in figure 3. The results from our CT scanning experiments describing tooth crown height loss from the whole occlusal surface (difference of surface mean heights between before and after chewing) of the single teeth were 8.6 mm for ATTR, 2.7 mm for L, 60.3 mm for G, 66.5 mm for GR and 133.5 mm for GRS (figure 4).

4. Discussion and conclusion Our approach was to combine analyses on microscopic wear features and actual dental tissue loss to form a detailed view of tooth wear through mechanical modelling. The mechanical masticator is not meant to mimic natural mastication perfectly. Rather, it provides a simple and well-defined mechanical modelling of repetitious chewing events and allows easy execution of precisely controlled experiments using different foods and machine settings. Our results show that the relationships between food properties and

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GR-GRS

GR-ATTR

125

rsif.royalsocietypublishing.org

GRS-ATTR

5 150

grass + rice, GR height change 0.067 mm wear rate per year 4.5 mm lifespan (12 cm) 27 years

GR + sand, GRS height change 0.134 mm wear rate per year 9.0 mm lifespan (12 cm) 13 years

attrition, ATTR height change 0.008 mm wear rate per year 0.58 mm lifespan (12 cm) 206 years

6

wear rate GRS>> GR> G>> ATTR> L

Figure 4. Tooth crown height changes during the chewing experiments. Surface reconstructions from CT scanned teeth, where colour change from yellow to red (light grey to dark grey in greyscale version) describes the mean height loss deviation. All the dietary groups have information about the mean height change, yearly wear rate and lifespan expectation calculated with 12 cm tooth height and 5.4 days spent per 100 000 chews. (Online version in colour.)

wear patterns partially corroborate expectations derived from teeth of live animals. Some results, however, are unexpected, giving rise to questions about our understanding of diet –teeth interactions. In this study, attrition in pure water led to a microwear signal similar to that seen in browsers: a high number of pits, but only few scratches [24,36] (figure 3). Also almost all initial polishing striations were removed. This supports the hypothesis that occlusal wear facets are caused by attrition [37] and that non-abrasive diets can be recognized from the facets caused by attrition-dominated wear [38]. The extent to which such pure attrition resembles the wear that occurs in natural chewing cannot be further explored here, but differences are likely to occur because natural attrition will always be tempered to some extent by the presence of food. External grit, especially quartz in soils, has been known to wear animals’ teeth [1,39,40]. As expected, our results show that external grit, in this case sand particles with mean grain size of 233 mm, heavily damages tooth surface, detaches enamel and thus primarily causes pitting. Material loss was equally drastic, causing a reduction of tooth surface mean height of over 130 mm during the 100 000 chewing events. The fact that sand caused pitting rather than scratching shows that scratch-dominated wear cannot be explained by ‘grit’ of large grain sizes. Similar results have been obtained with living sheep, which have been fed with fine-grained (diameter of 180 –250 mm) and medium-grained (diameter of 250–425 mm) sand [25]. It is argued that external dust causes wear, as open grass areas collect relevant amounts of dust particles [2]. Our experiment did not deliberately include ‘grit’ of fine particle size (i.e. dust), although we cannot positively exclude that fine dust was included in the amount of abrasives measurable as ADIA. We therefore cannot assess the degree to which scratches could also be generated by dust. Phytoliths have long been considered as a major agent of wear [1,13] and grazing has been considered the main driver

of the evolution of hypsodonty [3]. Our grass and grass–rice diets, rich in ADIA levels and known to contain phytoliths, did cause parallel scratches on enamel. That does not necessarily mean that phytoliths are the only wear agent, as other components of the food matrix like plant fibres, or properties of the food matrix that catch detached enamel particles or dust and keep them in the ingested bolus as additional abrasives, could also be responsible. Amorphous silica, which was used as an analogue of phytoliths in a recent study [6], has a role in the formation of microscale scratches. The diet containing most phytoliths also overwrote the striation from the initial polishing more quickly than the other non-sandy diets, suggesting tissue removal. The removal of tissue hypothesis is also supported by other research [41]. To assess whether the effects observed in microwear represent actual tissue loss, we additionally quantified the amount of tissue removed during the chewing experiment in one tooth per diet by CT scanning. In the groups containing phytoliths, but not grit (G and GR), the mean tooth height loss was more than 60 mm, which represents real tissue removal and thus strongly suggests a wear effect of phytoliths. An unexpected finding of our experiment was that the diet made from lucerne, which lacks phytoliths, generated a microscopic pattern similar to that generated by grass (figure 3). We expected the lucerne diet to behave as a browse diet and therefore to cause pitted wear, but this did not occur. There are possible explanations for the lack of correspondence between the predicted and the observed results: the experiment set-up changes the signal, or pelleted lucerne is a bad representative of ‘browse’. Lucerne as used here did not have the heterogeneous structure of leaf and stem components typical for browse, as it was ground and mixed with water to form a homogeneous, pasty material. In our set-up, lucerne does not behave as it would in real mastication, because of the cushion effect caused by evenly distributed food material; this creates a barrier between the teeth preventing most of the

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grass, G height change 0.060 mm wear rate per year 4.1 mm lifespan (12 cm) 29 years

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lucerne, L height change 0.003 mm wear rate per year 0.18 mm lifespan (12 cm) 658 years

(a)

(b)

7

(c)

1 mm

(d)

1 mm

(e)

1 mm

Figure 5. Images of an enamel band from every diet group and attrition after chewing. Scale bars, 1 mm. (a) Lucerne, (b) grass, (c) grass– rice, (d ) grass– rice – sand and (e) attrition.

attrition. Natural browsing would probably be something between our pit-dominated theoretical attrition and the lucerne diet. Tooth height loss during the chewing experiments with lucerne and theoretical attrition was minimal compared with the other categories, only less than 10 mm, being more of a polishing effect than gross wear. The scratched microwear pattern in the teeth chewed in the lucerne diet indicates that also something else than phytoliths might scratch enamel. The pellet ingredient table [11] shows that the lucerne diet includes pure lignocellulose next to other components that contain acid detergent fibre and acid detergent lignin. Fibres have been suggested previously to be responsible for polishing dental surfaces [13]; our results also raise the possibility that some plant fibres might cause scratches. More comparative and experimental work is needed to explore this possibility. One major factor often mentioned in microwear analyses is the question of overwriting and surface turnover. The microwear pattern is often thought to represent foods eaten during a few weeks, though in grazers turnover of the surface can occur in days or hours [42] and in primates even in minutes [43]. Unlike other artificial chewing studies, our experiment had the teeth do about 100 000 chews. Using data on chewing per gram dry matter, as depending on diet neutral detergent fibre (NDF) [44] and the daily dry matter intake linked to a specific diet NDF [45], a 500 kg horse would reach 100 000 chews within a range of 3.4– 5.4 days. Owing to the specific arrangement of initial polishing striations to the chewing direction in the machine, we could also visualize and calculate how many of these initial polishing scratches were still observable on the 0.4  0.4 mm2 area after 100 000 chews. The mean number of polishing striations left after chewing shows that the grass and lucerne diets caused less, and the grass–rice–sand diet more overwriting than the most phytolith-containing grass–rice diet. Rabbit and guinea pig studies with the same diets [11,12] also showed that absolute wear rate increased from lucerne to grass to grass–rice and to grass–rice–sand. This was also evident in our CT scan data. Similar results can be seen in ruminants; browsers have

lower wear rates than grazers, and mixed-feeders are in between [42,46]. Teeth in attrition chewing had the lowest number of initial polishing scratches left after chewing; this is caused most probably by the attritional polishing effect that notably leads to little actual tissue loss. The more wear food particles cause, the quicker they overwrite existing microwear patterns. This will lead to problems if the diet is reconstructed from microwear analysis only, as already a short-term shift can change the whole microwear pattern very quickly. This is very clear when one compares microwear results and the amount of actual wear quantified in our CT scans. For the sake of future discussion, we present a possible analysis methodology for microscopic wear features in the electronic supplementary material, S1, with non-parametric density plots of all, not classified, individual features described by pure feature lengths and widths analysed by modal clustering based on density contours. This methodology would resemble texture analysis [10,21] but is made using light stereo microscopy with image and statistical analyses. Internal structures of teeth, i.e. the arrangement of enamel, dentine and cement, affect how teeth are worn down. Differential wear of dental tissues carries a dietary signal that has so far been little explored. Most studies focus on enamel wear, though in many herbivores wear of all three dental tissues together is important. For example, as dentine and cement wear away, they leave the brittle enamel exposed to fracturing. Although microwear was studied quantitatively on enamel only, we observed wear of dentine and cement. Dentine and cement were worn more than enamel by diets containing abrasives (figures 4 and 5b–d). This is evident because enamel bands begin to emerge above their surrounding area. By contrast, lucerne and attrition diets (figures 4 and 5a,e) caused very light wear on all tissues and with three tissues wearing at very similar rates, a feature typically observed on natural wear facets (figure 5e). Our microwear results demonstrate that all diets studied, including pure attrition, caused microwear features (microscopic wear features) to the tooth surface. Diet-specific microwear patterns between lucerne, grass and grass–rice could not be distinguished statistically with the characters

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[47–49]. In attrition, a horse molar would wear down in about 200 years, and even more extremely on lucerne, in more than 600 years. These ‘browsing’ diets with lucerne and pure attrition have wear rates that explain why certain animals can live with very brachydont teeth. To conclude, simplified mechanical modelling simulates mastication and tooth wear with plausible rates. Clear differences in dietary components and their corresponding wear rates were not always distinguishable by microscopic wear features. Plant material, phytoliths, grit and teeth are all agents of tooth wear.

Authors’ contributions. Al.K. designed the methods and Al.K. and Ak.K. the equipment. Al.K., M.F. and M.C. designed the experiment. J.R. and Al.K. ran the experiment and performed the analyses. Ak.K. and Al.K. performed the CT scans. J.R. and Al.K. analysed the results. J.R., Al.K., M.F., M.C. and Ak.K. prepared the manuscript. M.F. and Al.K. first had the idea of building a simple chewing machine. All authors gave final approval for publication.

Competing interests. We declare we have no competing interests. Funding. Al.K. had funding from the Finnish Cultural Foundation. Acknowledgements. We would like to thank the Finnish Cultural Foundation, Doctoral Program in Geosciences and the Academy of Finland for funding the authors. We appreciate the constructive comments from the referees that helped us to elevate the level of our article. Authors would also like to acknowledge Thomas Bertin who did some preliminary experiments with the chewing machine, Szabolcs Galambosi for assistance in the design, Vainion Teurastamo Oy for providing the horse skulls, Helena Korkka for facilitating tooth preparation and Jukka Ukkonen for manufacturing repair parts for the chewing machine. A special acknowledgement belongs to Pauli Engstro¨m, who was working with the design and assembly of the chewing machine, contributing in a very significant way.

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Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material. More specific data can be obtained from Aleksis Karme and Janina Rannikko.

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Mechanical modelling of tooth wear.

Different diets wear teeth in different ways and generate distinguishable wear and microwear patterns that have long been the basis of palaeodiet reco...
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