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Biosurface and Biotribology 1 (2015) 263–269 www.elsevier.com/locate/bsbt

Dental development and microstructure of bamboo rat incisors

L.C. Huaa,b, P.S. Ungarb,n, Z.R. Zhoua,nn, Z.W. Ninga, J. Zhenga, L.M. Qiana, J.C. Roseb, D. Yanga

aTribology Research Institute, School of Mechanical Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, China bDepartment of Anthropology, University of Arkansas, Fayetteville, AR 72701, USA

Received 27 October 2015; accepted 3 November 2015

Abstract

Bone adapts to habitual loads by remodeling to resist stresses that would otherwise break it. The question of whether the same holds for teeth, however, remains unanswered. We might expect species with ever-growing dentitions to alter enamel histology in response to diet. In this study we fed bamboo rats (Rhizomys sinensis) different foods to assess effects on enamel microstructure. Results indicate that gnawing fracture-resistant items (i.e., bamboo) produces substantively different dental microstructures than does eating less mechanically-challenging ones (i.e., potato). Bamboo induces a structured, anisotropic pattern of rods that strengthens incisor enamel, whereas potato produces less structured, weaker enamel. Blood tests suggest that these differences are not related to nutrient variation. Rather, these ever-growing teeth evidently require a specific mechanical environment to develop normal dental microstructure. & 2015 Southwest Jiaotong University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Enamel histology; Rhizomys sinensis; Diet; Tooth wear

1. Introduction tooth growth throughout life. Does enamel microstructure and tooth strength vary depending on diet in these species? Bone responds to forces that act on it by remodeling its Eating is from an evolutionary perspective like a perpetual microstructure to buttress against failure [1]. No such response death match in the mouth, with plants and animals developing has been documented for dental enamel, despite the extra- hard or tough tissues for protection, and teeth evolving ways to ordinary stresses associated with breaking food. Enamel matrix sharpen or strengthen themselves to overcome those defenses is laid out in rods (prisms) of bundled hydroxyapatite crystals [4]. How can a tooth resist the high stresses often required to organized in various patterns to resist fracture and tooth break without being broken? Vertebrates have evolved several breakage [2]. In most vertebrates, enamel formation is com- mechanisms involving the chemistry of dental tissues [5,6] and pleted before dental eruption and use, so there is no opportu- distributions of enamel and dentin across tooth crowns [7]. nity to alter microstructure. But about 2400 mammalian Many mammals also have a complex, anisotropic enamel species today (and many more fossil ones) have ever- microstructure that strengthens the tissue against fracture [2]. growing teeth potentially capable of structural change [3].In Enamel is laid down in rows of rods that form crossing or these species, epithelial cells proliferate and differentiate into decussating bands to resist the spread of cracks [8]. For ameloblasts continously, allowing for enamel extension and humans and many other species, the ameloblasts migrate outward from the enamel–dentin junction to the eventual tooth surface during development, leaving trails of enamel behind them. These ameloblasts are then sloughed off during eruption; nCorresponding author. Tel.: þ1 479 575 6361. fi nn so once the enamel layer is completed, its structure is xed. Corresponding author. Bone, in contrast, retains capacity to induce osteoblasts and E-mail addresses: [email protected] (P.S. Ungar), [email protected] (Z.R. Zhou). osteoclasts throughout life, and responds to loads by changing Peer review under responsibility of Southwest Jiaotong University. tissue distribution and microstructure to buttress against http://dx.doi.org/10.1016/j.bsbt.2015.11.001 2405-4518/& 2015 Southwest Jiaotong University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 264 L.C. Hua et al. / Biosurface and Biotribology 1 (2015) 263–269 external stresses [1]. Indeed, jaws and faces require specific became so brittle that their incisors broke prior to completion load environments during chewing to develop properly [9]. of the experiment. But what about enamel in ever-growing teeth produced by Incisors of all subject animals were embedded in epoxy continuously developing ameloblasts? Rodents, rabbits, and resin blocks. Their tips were abraded with abrasive papers of many other species have persistent growth of incisors and 800, 1500, and 2000 grit in turn, and then polished with sometimes molars to compensate for wear [10–13], and growth diamond paste with progressively finer particles (5 μm, rates vary with rate of wear to maintain occlusal relationships 3.5 μm, 1.5 μm, 0.5 μm). Finally, the exposed tip surfaces between opposing teeth [14]. In this study we examined incisor were treated with citric acid (pH¼3.2) for 3 min at 25 1C. The enamel of Chinese bamboo rats (Rhizomys sinensis) to assess end result was a flat surface perpendicular to the long axes of effects of diet on microstructure. We considered individuals each tooth, representing the incisal enamel edge, with rods fed fracture-resistant bamboo (88–156 MPa) for 60 and 180 (prisms) standing proud of their surrounding matrix. Addi- days [15], weaker raw potato (1.1–1.4 MPa) [16] for 60 days, tional enamel sections were taken at 3 mm and 6 mm from the and a diet of bamboo followed by potato (30 and 90 days for tip to assess consistency of pattern at different depths within each). We also examined enamel of an individual prior to the crown (see Appendices Fig. A.2). All images were taken at weaning (and food gnawing) to establish a baseline. homologous locations on each section, the enamel immediately adjacent to the enamel–dentin junction centered on the median labio-lingual plane of the tooth. We examined three different 2. Material and methods sections for microstructure of each tooth to: (1) assess effects of position (a proxy for time of development) on microstruc- Two sets of experiments were conducted to assess effects of ture; and (2) allow comparisons between subjects in consistent feeding on enamel microstructure of ever-growing incisor locations on rat incisors. teeth. Chinese bamboo rats (R. sinensis) were selected for this All specimens were mounted on SEM stubs and coated with study because of their dietary versatility and ability to subsist a gold-palladium alloy approximately 20 nm thick. Inner on foods with greatly varying fracture properties. The natural enamel surfaces from each section were then imaged at diet of the Chinese bamboo rat is comprised largely of bamboo 3000 and 10,000 using a Quanta 200 (FEI Corp, UK) roots and shoots, and to a lesser extent, seeds, fruits, and leafs scanning electron microscope in secondary electron mode. of other plants [18]. Likewise, those raised on farms in China for food and fur are typically fed a diet dominated by bamboo 3. Results stalks, but this is supplemented by rice, sugarcane, and bamboo shoots. The effects of food type on enamel microstructure are All feeding experiments for this study were conducted at evident in Figs. 1–3. Bamboo-eating individuals have a Southwest Jiaotong University using protocols approved by uniserial lamellar pattern typical of spalacid rodents [17]. the Ethics Committee of the Sichuan Academy of Medical The milk-fed individual from the first set of experiments Science. The first set of experiments was designed to assess evinces a less structured rod pattern in transverse section initial effects of a bamboo-dominated diet and age. Lower (Fig. 1). The boundaries between adjacent bands and rod incisors were extracted from sacrificed animals at 35, 65, and structure are indistinct; and crystallites within rods are less well 125 days after birth. No food was introduced before 35 days aligned too. In contrast, individuals that gnawed fresh bamboo (all nutrition came from mother's milk) so there was no stems for the duration of the experiments have a highly- feeding-related stress on the incisor in the first case. The other organized rod layout, with parallel prisms bundled into bands samples were derived from animals allowed to eat a farm-fed that meet consistently at an angle of 100–1101 in the plane of diet for 30 and 90 days. Bamboo feeding involves gnawing section (Figs. 1–3). Crystallites within them are also better and scraping with the incisors, with associated high wear rate aligned. Study animals sampled for each set of experiments and transmission of load through the tooth. The 35 day show the same distinct pattern of decussated enamel following postnatal sample was a single animal, but 65 and 125 day bamboo consumption. samples included three farm-fed individuals each (total n¼7). There are also evident differences in enamel microstructure The feeding experiments conducted for this study were between subjects fed different foods. Those fed only softer raw designed to assess effects of different food types. Three potatoes for 60 days have much less structured enamel rod siblings from a single litter were each fed for 60 days either patterns than those fed bamboo for the same period (Fig. 2). (1) raw whole potatoes, (2) fresh bamboo stalks (supplemented We had planned a 180 day potato-only diet experiment to with popcorn), or (3) bamboo followed by potatoes (30 days match the bamboo diet experiment, but it failed, as the rats each). Both food types were gnawed and scraped by the broke their incisors prior to completion of the study in animals prior to mastication. Another set of same-litter siblings all cases. was fed for 180 days 1) bamboo and 2) bamboo (90 days) Diet-related differences are consistent between individuals, followed by potatoes (90 days). All tests sampled individuals and at different sampled depths on the incisor crown (tip, from 3 separate litters (total n¼15) to assess variation within 3 mm, and 6 mm) (see Supplementary Fig. 2). Rod layout for the sample. A 180 day potato-diet group was also planned, but those fed bamboo then potato (both the 60 and 180 day the enamel of all three individuals included in the study experiments) have variable microstructure within a single tooth L.C. Hua et al. / Biosurface and Biotribology 1 (2015) 263–269 265

Fig. 1. Baseline sampling of farm-raised individuals at weaning (age: 35 days), and following 30 days (age: 65 days) and 90 days (age: 125 days). Farm-raised rats are typically fed primarily bamboo stalks, supplemented with rice, sugarcane, and bamboo shoots.

Fig. 2. Sixty-day experiments following weaning (Age: 95 days). Left (a)¼potato, middle (b)¼bamboo 30 days followed by potato 30 days and right (c)¼ bamboo only. Enamel sections were taken at the tip, 3 mm below the tip, then 6 mm below the tip. 266 L.C. Hua et al. / Biosurface and Biotribology 1 (2015) 263–269

Fig. 3. One-hundred-eighty-day experiments following weaning. Left (a)¼bamboo 90 days followed by potato 90 days potato only, right (b)¼bamboo only. Enamel sections taken at the tip, 3 mm below the tip, then 6 mm below the tip.

(Figs. 2 and 3). Our sampling captured the transitions, such growth. We propose that rapid wear signals epithelial cells that the enamel shows a distinctive, patterned microstructure at associated with the cervical loop to proliferate and differentiate the tip and less organized rod and crystallite layout toward the into ameloblasts faster to increase the rate of enamel/tooth cervix. extension [19,20]. Individual ameloblasts must, in turn, secrete enamel at a rate high enough to keep up with that extension. 4. Discussion Robinson et al., for example, calculated that accelerating rat incisor eruption by 120% (compensation for cutting the teeth)

Rate of I1 wear and compensatory growth for bamboo- results in an increase in enamel secretion rate of 90% [19]. feeding subjects averaged 1.0 mm per day, whereas that for While the relationship appears not to be 1:1, individual potato feeders averaged 0.5 mm per day (see Appendices Fig. ameloblasts do respond to rapid compensatory growth by A.1). Fresh bamboo stalks are more fracture resistant than raw increasing enamel secretion rate. We believe that this results in potato, and require more gnawing prior to mastication. This alignment of individual crystallites during deposition, and leads to a faster rate of incisor wear and compensatory growth. ultimately leads to structured prismatic enamel with uniserial We suggest that the enamel secretion rate resulting from lamellar microstructure [21,22] seen in Fig. 3b. bamboo feeding led to the formation of a highly structured Raw potatoes are weaker and require less gnawing. A lower enamel rod layout, whereas the lower rate associated with wear rate must signal a slowdown of epithelial cell differentia- potato feeding did not. Envision a squeezed tube of toothpaste tion into ameloblasts, because the compensatory rate of moving in the direction opposite the forming paste trail. A enamel/tooth extension decreases to keep pace. Individual straight trail forms when the rate of tube movement matches ameloblasts likely also slow secretion to match the lower the rate of paste discharge. But when movement slows, the trail extension rate. We propose here that this leads to less becomes irregular. This phenomenon seems to hold for organized crystallites, and ultimately to enamel without the ameloblasts as they secrete rows of adjacent rods, and appropriate rod patterning. This hypothesis is based on evidently explains why potato feeding results in less structured observations of hypoplastic enamel growth defects caused by enamel rod patterning. slowed enamel secretion related to metabolic stress [23].In These results imply an epigenetic component to microstruc- such cases, rod layout is disrupted in a manner much like that tural patterning of ever-growing enamel. Bamboo rats regu- of newly weaned and potato-eating bamboo rats, resulting in larly eat mature bamboo roots and shoots in the wild [18], and bands of disorganized enamel. This pattern renders the teeth have evidently evolved to consume this fracture-resistant food, weaker, as is evident from the fact that incisors broke before which leads to a specific rate of incisor wear and compensatory completion of the 180 day potato feeding experiments in all L.C. Hua et al. / Biosurface and Biotribology 1 (2015) 263–269 267 cases, despite the lower stresses associated with gnawing this Table A.1 more compliant food. Blood tests (plasma and serum). The routine blood test (a) and total protein in This raises the question, “could the difference in microstruc- blood serum test (b) show the nutrition are similar. The microelements such as Calcium, iron and Phosphorus are very similar. ture between bamboo-fed and potato-fed rats be due to metabolic stress associated with nutrient differences between Name Abbr Bamboo diet Potato diet the two diets rather than to wear rate per se”? Results from supplemental studies of subject weight and blood chemistry (A) Plasma (routine blood test) Whilte blood cell count WBC 5.600 109/L 5.310 109/L show this to be unlikely (see Appendices Tables A.1 and A.2). Neutrophil count NEU 3.578 109/L 3.234 109/L First, individuals fed potato retain their weight during experi- Lymphocyte count LYM 1.792 109/L 1.758 109/L mental periods better than do bamboo-fed subjects, suggesting Monocytes MONO 0.162 109/L 0.207 109/L Eosinophils EOS 0.017 109/L 0.112 109/L that overall growth is not affected negatively by a lack of 9 9 bamboo in the diet. Further, blood tests on subject animals with Basophils BASO 0.050 10 /L 0.000 10 /L Neutrophils ratio NEU-R 0.639 0.609 the different diets showed similar amounts of total protein, Lymphocyte rate LYM-R 0.320 0.331 glucose, and levels of key elements known to be important to Monocytes rate MONO-R 0.029 0.039 enamel formation (e.g., calcium, iron, phosphorous, and mag- Eosinophils rate EOS-R 0.003 0.021 Basophils rate BASO-R 0.009 0.000 nesium) [6,24,25]. Thus, lower enamel secretion rate and 12 12 associated microstructure are much more likely the result of Red blood cell count RBC 4.79 10 /L 5.74 10 /L fi Hemoglobin HGB 148 g/L 174 g/L compensation for slower wear than of nutrient insuf ciency. Hematokrit HCT 0.427 0.514 Mean corpuscular volume MCV 89.2 fl 89.6 fl 5. Conclusions Mean corpuscular hemoglobin MCH 30.8 pg 30.3 pg Mean corpuscular hemoglobin MCHC 345 g/L 338 g/L In sum, we propose that Rhizomys sinensis requires the wear concentration Platelet count PLT 79 109/L 135 109/L environment produced by gnawing fracture-resistant bamboo for genetic programming to effect development of normal (B) Blood Serum enamel rod structure and strong incisor teeth. This suggests an Name Abbr Bamboo diet Potato diet elegant mechanism for controlling enamel histology in mam- Glucose Glu 11.91 mol/L 10.93 mol/L mals with ever-growing teeth. If our hypothesis is correct, rate Osmotic pressure Osm 269 mOsm/L 265 mOsm/L of enamel secretion is tied to compensatory tooth growth, Total cholesterol TC 2.08 mmol/L 2.29 mmol/L which in turn reflects wear rate dictated by the diet to which a Triglyceride TG 1.48 mmol/L 0.97 mmol/L Total protein TP 42.0 g/L 51.6 g/L species with ever-growing teeth has evolved. Future studies Albumin Alb 18.4 g/L 22.3 g/L focusing specifically on enamel secretion rate and other factors Globulin Glb 23.6 g/L 29.3 g/L potentially involved in crystallite growth and layout (e.g., Albumin/globulin A/G 0.8 0.8 amelogenins in the developing enamel matrix) will help us Coper Cu 19.3 umol/L 17.1 umol/L better understand the process, but results presented here make Zinc Zn 87.1 umol/L 102.5 umol/L Calcium Ca 1.68 mmol/L 1.89 mmol/L it clear that tooth wear related to diet can affect enamel Iron Fe 8.78 mmol/L 11.30 mmol/L turnover rate, microstructure, and tooth strength. Kalium K 2.88 mmol/L 3.76 mmol/L Sodium Na 132.0 mmol/L 131.2 mmol/L Acknowledgments Chlorine Cl 86.0 mmol/L 89.4 mmol/L Magnesium Mg 1.40 mmol/L 1.48 mmol/L Phosphorus P 1.81 mmol/L 1.57 mmol/L This work was supported by National Natural Science Foundation of China (Nos. 51535010, 51222511, and 51290291), the Southwest Jiaotong University PhD Innovation Fund (No. between subjects fed the different diets (Appendices Table YH1000212471405); the China Scholarship Council (No. A.1a), as were the glucose (Glu), osmotic pressure (Osm), total 201407000023) and the University of Arkansas. protein (TP), albumin (Alb), and globulin (Glb) in the serum. These results suggest that the bamboo rats were able to obtain Appendices A the nutrients needed for normal growth, development, and physiological function regardless of diet [26,27]. Moreover, (A) Blood test results the concentrations of microelements (e.g. calcium, iron, phosphorus, magnesium) in the blood serum, which can affect A standard suite of nutrient assessment blood tests was enamel mineralization, were also similar between the rats fed conducted at the Sichuan Province People’s Hospital on different diets [6,24,25]. This suggests that the variation in bamboo rats fed bamboo and potato for 30 days each to assess enamel structure seen in the experiments was not the result of effects of these diets that might affect normal growth and differences in nutrient load. And the fact that the potato-fed development of dental enamel. The blood tests (Appendices rats actually lost less weight than the bamboo-eating ones over Table A.1) included comparisons of plasma (a) and serum the course of the study further suggests that potato-feeding (b) differences between individuals fed bamboo and potato does not have a deleterious effect on growth (Appendices only. The components in the plasma samples were similar Table A.2). 268 L.C. Hua et al. / Biosurface and Biotribology 1 (2015) 263–269

Table A.2 Independent weight test under different diets (bamboo-feeding and potato feeding).

Date (days) Weight (g)

Potato diet Sample 1 0 702 3 669 6 657 9 729 12 697 Sample 2 0 790 3 825 6 769 9 719 12 642 Sample 3 0 896 3 839 6 788 9 749 12 747 Bamboo diet Fig. A.1. Box-and-whiskers plots of rates of growth and wear of I1s of Sample 1 0 663 bamboo rats fed different diets. 3 606 6 542 9 493 12 439 Sample 2 0 681 3 646 6 739 9 529 12 Die Sample 3 0 671 3 619 6 564 9 525 12 479

Incisor growth and wear rates

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