3 Dental Anthropology 2021 │ Volume 34│ Issue 01 Growth Rates of Accessory Human Enamel: A Histological Case Study of a Modern-Day Incisor from Northern England Christopher Aris 1* and Emma Street 2 1 University of Kent, Canterbury, UK 2 No affiliations The study of modern human enamel growth rates via histological analysis is common within the study of biological anthropology and bioarchaeolo- gy, commonly focusing on the variation between cusps of the same tooth (e.g. Mahoney, 2008), with- in single populations (e.g. Schwartz et al., 2001), and between populations (e.g. Smith et al., 2007; Aris et al., 2020a, 2020b). A common trend between these lines of research is the exclusive use of what are deemed as dental samples containing no evi- dence of pathology, stress markers, or growth of accessory enamel (defined here as: growth of enamel outside of the features typically used to define and identify human tooth types). While past research has touched on how some human enamel growth features vary between individuals suffer- ing from stress and those not suffering from stress resulting in dental morphologies, these typically concern the accuracy of making certain calcula- tions relating to enamel growth (Lukacs & Guatelli -Steinberg, 1994; Guatelli-Steinberg & Lukacs, 1999), and the development of non-accessory enamel (defined here as: growth of the enamel fea- tures which define how human tooth types are identified and classified) in individuals presenting evidence of stress on their dental morphology (e.g. Fitzgerald & Saunders, 2005). Comparison of enamel growth rates collected from teeth present- ing accessory enamel to those with no evidence of stress markers or non-metric traits from the same population, and comparison of accessory enamel growth to the growth of non-accessory enamel within the same tooth, have yet to be conducted. This project aims to begin to address these issues and widen our understanding of accessory enamel growth in modern-day humans through the case study of a modern-day upper first incisor. Background Amelogenesis and daily enamel growth Amelogenesis is the process of secretion and min- eralization of protein matrix by ameloblast cells (Boyde, 1989; Nanci & Smith, 1992; Smith & Nanci, 1995). During the secretory stage of amelogenesis, ABSTRACT This study investigates enamel growth of a modern-day human upper first incisor (S197) possessing a talon cusp (accessory cusp). Growth rates collected from the accessory enamel are com- pared to data collected from the primary cusp and cusps of a standard incisor sample from the same population. Upper first incisors (n=12) and S197 were analysed using histological methods. Daily secre- tion rates (DSRs) were calculated for inner, mid, and outer regions of cuspal and lateral sites. Additional DSRs were calculated for equivalent regions of S197’s accessory cusp. S197’s primary cusp DSRs were significantly faster than the accessory cusp for all lateral regions, but significantly slower in the inner and mid cuspal regions. S197’s primary cusp DSRs were also significantly slower than the standard inci- sor sample for all regions except the lateral cuspal. The DSRs of the standard sample were significantly faster than those of S197’s accessory cusp for all lateral regions, but significantly slower in the inner cus- pal region. This case study displays that human teeth possessing accessory cusps can present varying DSRs to teeth lacking accessory enamel from the same population, and that accessory enamel growth may not follow the same pattern of increasing DSRs along the length of enamel prisms. *Correspondence to: Christopher Aris Department of Archaeology Ella Armitage Building University of Sheffield Sheffield, UK c.aris@sheffield.ac.uk Keywords: incisor, enamel, daily secretion rates 4 Dental Anthropology 2021 │ Volume 34│ Issue 01 ameloblast secretion is altered according to a daily circadian rhythm, producing short-period markers along the length of enamel prisms (e.g., Asper, 1916; Gysi, 1931; Massler & Schour, 1946; Okada, 1943; Kajiyama, 1965; Dean et al., 1993; Smith & Nanci, 2003). These daily forming markers are known as cross striations (e.g. Boyde, 1963; 1990; Kajiyama, 1965; Bromage, 1991; Dean, 1995; Fitz- gerald, 1995, 1998; Antoine, 2000; Antoine et al., 2009). The formation of cross striations causes al- terations in the refractive index of enamel prisms, making them observable in thin sections under transmitted light (e.g. Berkovitz et al., 2002; Zheng et al., 2013). Daily secretion rates (DSRs) can be calculated from cross striations. These rates accelerate from inner enamel regions proximal to the enamel den- tine junction towards the outer enamel surface (e.g. Beynon et al., 1991 Beynon et al., 1998; Reid et al., 1998; Lacruz & Bromage, 2006; Mahoney, 2008; Aris et al., 2020a, 2020b). Daily secretion rates are also faster relative to their proximity to the dentine horn (Beynon et al., 1991). Due to DSRs varying within a tooth, analysis of these rates are undertak- en for specific regions (e.g. Dean, 1998) where the crown is divided into cuspal, lateral, and cervical enamel, and then further subdivided into inner, mid, and outer regions. Typically, DSRs are broad- ly similar when equivalent regions are compared between cusps within a molar (Mahoney, 2008). Analysis of DSRs for human samples have ex- amined variations within individual teeth (Mahoney, 2008), differences between biologically male and female groups (Schwartz et al., 2001), and more recently variations between populations (Aris et al., 2020a, 2020b). Despite the breadth of these studies, they have universally used teeth ab- sent of evidence of stress, pathology, and accessory enamel growth. Thus, our understanding of how human DSRs vary in accessory enamel in compari- son to non-accessory enamel is limited. Enamel growth patterns within pathological cases While the DSRs of accessory enamel have not yet been analysed, certain features of enamel growth have been analysed for individuals presenting signs of stress on their dentition. These studies have focused on the possible changes in amelogen- esis, which leads to the formation of enamel growth defects observable from internal and exter- nal analysis. Lukacs and colleagues have published a series of papers explaining the pattern and ex- pression of enamel defects in modern humans. These can vary due to diet, geographic location, and climate. In particular, these papers present evidence of longer crown formation times (CFTs) in stressed individuals (Lukacs et al., 1989; Lukacs, 1991, 1992, 1999; Lukacs & Joshi, 1992; Lukacs & Pal, 1993; Lukacs & Guatelli-Steinberg, 1994; Luck- as & Walimbe, 1998; Guatelli-Steinberg & Lukacs, 1999). As CFTs are directly related to the products of daily enamel growth (e.g. Massler & Schour, 1946) there is potential that accessory enamel pos- sesses growth rates which vary from non-accessory enamel. Fitzgerald and Saunders (2005) investigated the possibility of using enamel defects to predict the age at which stress was incurred and thus improve the way in which we interpret the influence of stress on enamel growth patterns. This concept was based on the ability to age through examining interior enamel structures, and that these struc- tures would be notably altered during stressful events. Through the use of a large sample size (274 teeth from 127 Roman subadults), they concluded that enamel formation patterns are more highly impacted according to the severity of the cause of stress, and that there is no minimum requirement of stress level for enamel to be effected (Fitzgerald & Saunders, 2005). Multiple papers have since been published on this topic, all conclusively stating that stress impacts enamel structures, significantly in- creases CFTs, and reduces the reliability of DSR calculations (Reid & Dean, 2006; Holt et al., 2012; Birch & Dean, 2014; Primeau et al., 2015). As a re- sult of these studies, we can reliably say that non- accessory enamel grows differentially in individu- als presenting evidence of stress. It is therefore im- portant to expand our understanding of how acces- sory enamel grows in relation to non-accessory enamel. Material and methods Dental sample Upper permanent first incisors (n=13) were select- ed from a modern-day collection consisting of teeth extracted between 1964 and 1973 at dental surgeries in northern England and southern Scot- land. All 13 samples originated from Newcastle- Upon-Tyne, including an incisor presenting an ac- cessory enamel cusp (S197). The accessory cusp of S197 has developed on the cingulum and reached beyond half the distance to the incisal edge (Figure 1), as such it is diagnosed as a talon cusp (Edgar et al., 2016). The remaining 12 incisors made up a standard sample, with each tooth presenting no evidence of stress, pathology, or accessory enamel growth. Right teeth were selected unless it was 5 Dental Anthropology 2021 │ Volume 34│ Issue 01 unavailable or the left was better preserved. The collection itself is curated at the Skeletal Biology Research Centre, University of Kent, as part of the UCL/Kent Collection. Ethical approval for the his- tological analysis of this dental sample was ob- tained from the UK National Health Service re- search ethics committee (REC reference: 16/ SC/0166; project ID: 203541). Sample preparation Resin casts were produced for each incisor prior to any destructive analysis, and were produced using standard methods (Aris, 2020). The casts repro- duced the surface morphology of the tooth crown allowing for future study of microwear, crown morphology, and enamel surface features includ- ing linear enamel hypoplasia and perikymata. Thin sections were produced using standard histological procedures (e.g. Schwartz et al., 2005; Mahoney, 2008; Aris, 2020). The incisors were em- bedded in an epoxy resin and hardener mixture (Buehler®) to minimise the chance of the teeth frac- turing during sectioning. Embedded samples were then cut at a low speed using a diamond-edged wafering blade (Buehler® IsoMet 1000 Precision Cutter) at a longitudinal angle through the apex of the incisal crowns. The samples were then mount- ed on glass microscope slides and lapped using progressively finer grinding pads (Buehler®) until around 120µm in thickness. Ground samples were polished using 0.3µm aluminium oxide powder until evidence of lapping was removed from the mounted dental samples. Polished samples were then placed within an ultrasonic bath for two minutes in order to remove any remaining debris before being dehydrated using 90% and 100% etha- nol-based solutions (Fisher scientific®). The dehy- drated sections were finally cleared using Histocle- ar® and mounted with a glass cover slip using a mounting medium (DPX®). All sections were ex- amined using polarised light microscopy (Olympus BX53 Upright Microscope). Analysis and image capture was conducted using micro im- aging software (cellSens) (see below for detail). Daily secretion rates The DSRs for the incisors were calculated for the inner, mid, and outer areas of the lateral and cus- pal enamel sites of each tooth using standard methods (e.g. Beynon et al., 1991a; Schwartz et al., 2001; Mahoney, 2008; Aris et al., 2020a, 2020b). Each region within the cuspal and lateral sites was determined by dividing the length of the enamel regions into three equidistant portions, following the longitudinal axis of local enamel prisms (Figure 2). The lateral enamel areas were determined with- in the section of imbricational enamel equidistant between the dental cervix and dentine horn. Re- gions of cuspal enamel were determined within the appositional enamel starting near the dentine horn. Additional DSRs were calculated for isolated re- gions of S197’s accessory cusp (see Figure 2). These regions were selected in a fashion as to mirror the cuspal and lateral regions of the primary cusp. Within each enamel region a measurement was made of five consecutive cross striations along the length of an enamel prism. This measurement was subsequently divided by five, giving a mean daily rate of matrix secretion (µm/day). This process was repeated to produce six mean DSRs for each region. For the standard incisor sample these re- sults were then similarly divided to give a grand mean and standard deviation, following the stand- ard statistical and methodological approaches of studying human enamel growth rates (e.g. Beynon et al., 1991 Beynon et al., 1998; Reid et al., 1998; Lacruz & Bromage, 2006; Mahoney, 2008; Aris et al., 2020a, 2020b). For S197 the six mean DSRs for each region were kept separate for future analysis. All cross striation measurements were taken be- tween 20x and 40x magnification (Figure 3). Statistical analysis Independent sample T-tests were used to compare mean equivalent regional DSRs between the select- ed samples. First, the same DSRs of the primary cusp and accessory cusp of S197 were compared. Figure 1. Depictions of upper first permanent incisor S197 prior to sectioning highlighting the regions defined as accessory and non-accessory enamel. Moving left to right the images display the tooth from the labial, lingual, and mesial directions. 6 Dental Anthropology 2021 │ Volume 34│ Issue 01 Figure 2. Cross section of Sample 197 displaying the regions from which DSRs were collected. Right superimpo- sitions show the cuspal (top) and lateral (bottom) sites of the primary cusp. Left superimpositions show the cus- pal (top) and lateral (bottom) sites of the accessory enamel. White squares represent the inner, mid, and outer regions of each site respectively moving from the enamel dentine junction towards the outer enamel surface. Daily secretion rates were collected from healthy clinical teeth from equivalent cuspal and lateral sites to the right superimpositions. Figure 3. Cross section of the cuspal enamel site of the primary cusp of Sample 197. The right superimposition displays a portion of the mid cuspal region, and the white arrows indicate individual cross striations. 7 Dental Anthropology 2021 │ Volume 34│ Issue 01 Second, the DSRs collected from the primary cusp enamel of S197 were compared to those of the standard clinical sample. Third, the DSRs of the accessory enamel of S197 were compared to those of the standard clinical sample. All statistical anal- yses were conducted using SPSS 24.0. Results Accessory enamel DSRs compared to primary cusp DSRs Table 1 displays the results of comparing the mean DSRs of the primary cusp enamel to those of the accessory cusp enamel, all collected from S197. For the inner and mid regions of the lateral enamel the primary cusp enamel presented significantly faster DSRs. These were faster by a mean rate of 0.53µm/ day (p<0.00) in the inner region, and 0.47µm/day (p=0.01) in the mid region. Conversely, accessory enamel presented significantly faster DSRs for the inner and mid cuspal enamel regions. These were faster by a mean rate of 2.14µm/day (p<0.00) in the inner region, and 1.02µm/day (p<0.00) in the mid region. Non-accessory enamel DSRs compared to rest of popula- tion Table 2 displays the results of comparing the mean DSRs of the primary cusp enamel of S197 to those of the standard clinical sample. For all regions of the lateral enamel, the standard sample presented significantly faster DSRs. These were faster by a mean rate of 0.27µm/day (p=0.01) in the inner re- gion, 0.51µm/day (p<0.00) in the mid region, and 0.37µm/day (p=0.02) in the outer region. The standard sample also presented significantly faster DSRs for the inner and mid cuspal enamel regions. These were faster by a mean rate of 0.69µm/day (p<0.00) in the inner region, and 0.65µm/day (p<0.00) in the mid region. Accessory enamel DSRs compared to rest of population Table 3 displays the results of comparing the mean DSRs of the accessory enamel of S197 to those of the standard clinical sample. For all regions of the lateral enamel, the standard sample presented sig- nificantly faster DSRs. These were faster by a mean rate of 0.80µm/day (p<0.00) in the inner region, 0.98µm/day (p<0.00) in the mid region, and 0.59µm/day (p<0.00) in the outer region. Converse- ly, the accessory enamel sample presented signifi- cantly faster DSRs for the inner cuspal enamel re- gion by a mean rate of 1.45µm/day (p<0.00). Discussion Inter-regional enamel growth of S197 The lateral enamel DSRs of the primary cusp were significantly faster than those of the accessory enamel in the inner and mid regions. Conversely, the accessory enamel cuspal DSRs were significant- ly faster than those of the primary cusp for the in- ner and mid regions (see Table 1). This finding goes against those of past research, which found Table 1. Results of the independent samples T-tests comparing the mean regional DSRs (µm/day) of the accessory enamel of Sample 197 to the primary cusp enamel of Sample 197. Significant results are marked in bold, *p<0.00. Enamel Region Sample N Mean Min Max S.D. Sig. Lateral Enamel Inner Accessory 6 2.24 2.02 2.37 0.14 0.00* Primary cusp 6 2.77 2.48 2.98 0.19 Mid Accessory 6 2.51 2.37 2.81 0.16 0.01 Primary cusp 6 2.98 2.67 3.35 0.24 Outer Accessory 6 3.13 2.89 3.78 0.33 0.42 Primary cusp 6 3.35 2.88 3.77 0.34 Cuspal Enamel Inner Accessory 6 4.65 4.30 4.98 0.21 0.00* Primary cusp 6 2.51 2.14 2.78 0.12 Mid Accessory 6 3.91 3.37 4.41 0.37 0.00* Primary cusp 6 2.89 3.44 2.40 0.25 Outer Accessory 6 3.71 3.09 4.14 0.46 0.81 Primary cusp 6 3.84 3.48 4.24 0.27 8 Dental Anthropology 2021 │ Volume 34│ Issue 01 Table 2. Results of the independent samples T-tests comparing the mean regional DSRs (µm/day) of the healthy samples to those collected from the primary cusp enamel of Sample 197. Significant results are marked in bold, *p<0.00. Enamel Region Sample N Mean Min Max S.D Sig. Lateral Enamel Inner Primary cusp 6 2.77 2.48 2.98 0.19 0.01 Healthy 12 3.04 2.56 3.32 0.21 Mid Primary cusp 6 2.98 2.67 3.35 0.24 0.00* Healthy 12 3.49 2.86 3.80 0.27 Outer Primary cusp 6 3.35 2.88 3.77 0.34 0.02 Healthy 12 3.72 3.14 4.06 0.25 Cuspal Enamel Inner Primary cusp 6 2.51 2.14 2.78 0.12 0.00* Healthy 8 3.20 2.84 3.43 0.23 Mid Primary cusp 6 2.89 3.44 2.40 0.25 0.00* Healthy 8 3.54 3.16 3.86 0.22 Outer Primary cusp 6 3.84 3.48 4.24 0.27 0.69 Healthy 8 3.89 3.36 4.09 0.23 Table 3. Results of the independent samples T-tests comparing the mean regional DSRs (µm/day) of the healthy samples to those collected from the accessory enamel of Sample 197. Significant results are marked in bold, *p<0.00. Enamel Region Sample N Mean Min Max S.D Sig. Lateral Enamel Inner Accessory 6 2.24 2.02 2.37 0.14 0.00* Healthy 12 3.04 2.56 3.32 0.21 Mid Accessory 6 2.51 2.37 2.81 0.16 0.00* Healthy 12 3.49 2.86 3.80 0.27 Outer Accessory 6 3.13 2.89 3.78 0.33 0.00* Healthy 12 3.72 3.14 4.06 0.25 Cuspal Enamel Inner Accessory 6 4.65 4.30 4.98 0.21 0.00* Healthy 8 3.20 2.84 3.43 0.23 Mid Accessory 6 3.91 3.37 4.41 0.37 0.05 Healthy 8 3.54 3.16 3.86 0.22 Outer Accessory 6 3.71 3.09 4.14 0.46 0.74 Healthy 8 3.89 3.36 4.09 0.23 9 Dental Anthropology 2021 │ Volume 34│ Issue 01 DSRs to remain similar between equivalent regions of different non-accessory cusps in typically multi- cusped teeth (Mahoney, 2008). This unusual varia- tion in DSR differences between the cusps is the product of the cuspal DSRs of the accessory cusp slowing with distance from the enamel dentine junction (EDJ) along the enamel prism pathway. This trend also differs to that seen in past research, which has shown permanent enamel growth rates of non-accessory enamel to always accelerate with distance from the EDJ (e.g. Beynon et al., 1991, 1998; Reid et al., 1998; Lacruz & Bromage, 2006; Mahoney, 2008; Aris et al., 2020a, 2020b). This finding, in particular, demands further in- vestigation, primarily to identify if the reversed growth pattern in cuspal DSRs of accessory enamel growth is consistent in other human samples. Should this be the case then the expected principle notion of enamel growth rates increasing with dis- tance, a principle formulated on teeth not present- ing accessory enamel growth from the EDJ, would need to be addressed. It is plausible that this prin- ciple, highly supported by the data of past research (e.g. Beynon et al., 1991; Beynon et al., 1998; Reid et al., 1998; Lacruz & Bromage, 2006; Mahoney, 2008; Aris et al., 2020a, 2020b) can only accurately be ap- plied to growth of non-accessory enamel. Further research on the growth rates of accessory enamel is therefore required in order to create an equivalent growth principle for non-accessory enamel. Primary cusp enamel growth compared to standard sample Despite being the primary cusp of S197 and dis- playing standard morphology for an upper perma- nent first incisor, the regional enamel DSRs varied significantly from the mean DSRs of the standard sample (Table 2). Mean DSRs of all lateral enamel regions, and the inner and mid cuspal regions, were significantly slower in S197. However, outer cuspal DSRs were slower by only a mean rate of 0.05µm/day in S197. Overall, while this research only presents a preliminary case study, the data suggests that such enamel will grow slower than the standard sample cohort of the same tooth type within the same population. This finding primarily supports the use of teeth possessing no abnormal or excess enamel in past growth rate studies (e.g. Beynon et al., 1991; Beynon et al., 1998; Reid et al., 1998; Lacruz & Bro- mage, 2006; Mahoney, 2008; Aris et al., 2020a, 2020b), as there is now clear potential for signifi- cant differences between teeth that do and do not present accessory enamel growth as defined here. Perhaps more importantly, there is new incentive for future research to continue analysing the growth rates throughout all regions and types of enamel from all tooth types. Such research will serve to expand our knowledge of the growth rate patterns common in human dentition, by identify- ing if non-accessory enamel growth rates slow in the presence of accessory enamel on the same tooth, or if S197 is a unique case. Future research should also examine the growth rates of less ex- treme non-accessory enamel growth than that of S197. This would help ascertain whether the ex- tremity of accessory enamel growth is related to the slowing growth rates of the non-accessory enamel. Accessory cusp enamel growth compared to standard sample The lateral enamel DSRs of the accessory cusp of S197 presented significantly slower rates compared to those of the standard sample (Table 3). Con- versely, the inner cuspal DSRs of the accessory cusp were significantly faster. The mid cuspal re- gion was also faster by a mean rate of 0.37µm/day, but the outer cuspal region presented minimal var- iation to the standard sample (Table 3). These re- sults demonstrate the erratic and inconsistent growth patterns of the accessory enamel of S197. It is particularly unusual that the cuspal accessory enamel growth slowed from inner to outer regions, and that the outer region mean DSR climaxed at a similar rate to equivalent DSRs of the standard sample. Further research is required to ascertain whether this is a unique phenomenon or the stand- ard growth pattern for accessory enamel. However, it should be noted that accessory enamel manifestations differ between different dental non-metric traits whose etiology includes excess enamel formation. Future research investi- gating the growth of accessory enamel should therefore consider analysing growth rates of teeth grouped according to their diagnosed traits and tooth types, as it should not be assumed that acces- sory enamel grows at similar rates between these groups. This principle should be applied to all fu- ture research advised here to avoid inaccurately grouping the growth patterns of all non-accessory enamel types. Conclusions The inter-regional differences in the growth rates collected from S197 were erratic, and in some enamel regions in direct contradiction with those expected of human incisors and multi-cusped teeth. Firstly, the differences between the equiva- lent regional DSRs of the primary and secondary 10 Dental Anthropology 2021 │ Volume 34│ Issue 01 cusp of S197 vary from the similarities observed in past research comparing non-accessory cusps of the same teeth. Secondly, the presence of extreme accessory enamel formation appeared to slow the growth rates of the non-accessory enamel when compared to the growth rates of a standard sample of teeth lacking accessory enamel growth. Finally, the DSRs from the accessory cusp of S197 highlight how accessory enamel growth rates will not neces- sarily follow the trend of increasing rates with dis- tance from the EDJ. The lack of additional research greatly limits our understanding of these findings. Overall, it is clear that more research into the growth rates of accessory enamel, as well as non- accessory enamel of the same teeth, is needed. Ide- ally such research will analyse different tooth types, and teeth with different diagnosed non- metric traits, independently. Acknowledgments We would like to thank the University of Kent for granting permission to sample teeth from their clinical collection. Further thanks also go to Annie Robertson for her assistance and artistic talents. Thanks also go to the anonymous reviewer and editor for their invaluable feedback. REFERENCES Antoine, D. (2000). Evaluating the periodicity of incre- mental structures in dental enamel as a means of studying growth in children from past human pop- ulations (Doctoral dissertation, University Col- lege London). Antoine, D., Hillson, S., & Dean, M. C. (2009). The developmental clock of dental enamel: a test for the periodicity of prism cross‐striations in modern humans and an evaluation of the most likely sources of error in histological studies of this kind. Journal of Anatomy, 214(1), 45-55. Aris, C. (2020). The Histological Paradox: Method- ology and efficacy of dental sectioning. Papers from the Institute of Archaeology, 29(1), 1-16. Aris, C., Mahoney, P., & Deter, C. (2020a). Enamel thickness and growth rates in modern human permanent first molars over a 2000 year period in Britain. American Journal of Physical Anthro- pology, 173, 141-157. Aris, C., Mahoney, P., O'Hara, M. C., & Deter, C. (2020b). Enamel growth rates of anterior teeth in males and females from modern and ancient British populations. American Journal of Physical Anthropology, 173, 236-249. Asper, H. (1916). Uber die ‘‘braune Retzius’sche Parallelsteifung’’ im Schmelz der menschlichen Zahne. Schweiz Vjschr. Zahnhlk, 26(1), 275-314 Berkovitz, B. K. B., Holland, G. R., & Moxham, B. J. (2002). Oral anatomy, embryology and histology. Mosby Incorporated. Beynon, A. D., Clayton, C. B., Ramirez Rozzi, F. V. R., & Reid, D. J. (1998). Radiographic and histo- logical methodologies in estimating the chro- nology of crown development in modern hu- mans and great apes: a review, with some ap- plications for studies on juvenile homi- nids. Journal of Human Evolution, 35(4), 351-370. Beynon, A. D., Dean, M. C., & Reid, D. J. (1991). Histological study on the chronology of the developing dentition in gorilla and orangu- tan. American Journal of Physical Anthropolo- gy, 86(2), 189-203. Birch, W., & Dean, M. C. (2014). A method of calcu- lating human deciduous crown formation times and of estimating the chronological ages of stressful events occurring during deciduous enamel formation. Journal of Forensic and Legal Medicine, 22, 127-144. Boyde, A. (1989). Enamel. In Teeth (pp. 309-473). Springer, Berlin, Heidelberg. Boyde, A., (1990). Developmental interpretations of dental microstructure. In DeRousseau, J.C. (Ed.), Primate Life History and Evolution (pp. 229 -267). New York: Wiley-Liss. Boyde, A. (1990). Developmental interpretations of dental microstructure. In DeRousseau, J.C. (Ed.), Primate Life History and Evolution (pp. 229 -267). New York: Wiley-Liss. Bromage, T. G. (1991). Enamel incremental perio- dicity in the pig‐tailed macaque: A polychrome fluorescent labeling study of dental hard tis- sues. American Journal of Physical Anthropolo- gy, 86(2), 205-214. Dean, M. C. (1995). The nature and periodicity of incremental lines in primate dentine and their relationship to periradicular bands in OH 16 (Homo habilis). In J. M. Cecchi (Ed.), Aspects of dental biology: Paleontology, anthropology and evolution (pp. 239–265). Florence: International Institute for the Study of Man. Dean, M. C. (1998). A comparative study of cross striation spacings in cuspal enamel and of four methods of estimating the time taken to grow molar cuspal enamel in Pan, Pongo and Ho- mo. Journal of Human Evolution, 35(4), 449-462. Dean, M. C., Beynon, A. D., Reid, D. J., & Whit- taker, D. K. (1993a). A longitudinal study of tooth growth in a single individual based on long‐and short‐period incremental markings in dentine and enamel. International Journal of Os- teoarchaeology, 3(4), 249-264. Edgar, H. J. H., Willermet, C., Ragsdale, C. S., 11 Dental Anthropology 2021 │ Volume 34│ Issue 01 O'Donnell, A., & Daneshvari, S. (2016). Fre- quencies of rare incisor variations reflect fac- tors influencing precontact population rela- tionships in Mexico and the American South- west. International Journal of Osteoarchaeology, 26 (6), 987-1000. FitzGerald, C. M. (1995). Tooth crown formation and the variation of enamel microstructural growth markers in modern humans (Doctoral disserta- tion, University of Cambridge). FitzGerald, C. M. (1998). Do enamel microstruc- tures have regular time dependency? Conclu- sions from the literature and a large-scale study. Journal of Human Evolution, 35(4-5), 371- 386. FitzGerald, C. M., & Saunders, S. R. (2005). Test of histological methods of determining chronolo- gy of accentuated striae in deciduous teeth. American Journal of Physical Anthropolo- gy, 127(3), 277-290. Guatelli‐Steinberg, D., & Lukacs, J. R. (1999). Inter- preting sex differences in enamel hypoplasia in human and non‐human primates: Develop- mental, environmental, and cultural considera- tions. American Journal of Physical Anthropolo- gy, 110(S29), 73-126. Gysi, A. (1931). Metabolism in adult enamel. Dental Dig, 37, 661-668 Holt, S. A., Reid, D. J., & Guatelli-Steinberg, D. (2012). Brief communication: premolar enamel formation: completion of figures for aging LEH defects in permanent dentition. Dental Anthro- pology Journal, 25(1), 4-7. Kajiyama, S. (1965). Total number of regular incre- mental lines (Regulare Parallelstreifen nach Asper) in the enamel of human permanent teeth. Journal of Nihon University School of Den- tistry, 39, 77-83. Lukacs, J. R. (1991). Localized enamel hypoplasia of human deciduous canine teeth: prevalence and pattern of expression in rural Paki- stan. Human Biology, 63(4), 513-522. Lukacs, J. R. (1992). Dental paleopathology and agricultural intensification in South Asia: new evidence from Bronze Age Harappa. American Journal of Physical Anthropology, 87(2), 133-150. Lukacs, J. R. (1999). Enamel hypoplasia in decidu- ous teeth of great apes: Do differences in defect prevalence imply differential levels of physio- logical stress?. American Journal of Physical An- thropology, 110(3), 351-363. Lacruz, R. S., & Bromage, T. G. (2006). Apposition- al enamel growth in molars of South African fossil hominids. Journal of Anatomy, 209(1), 13- 20. Lukacs, J. R., & Guatelli-Steinberg, D. (1994). Daughter neglect in India: LEH prevalence and the question of female biological superiori- ty. American Journal of Physical Anthropology, Supplement, 18, 132. Lukacs, J. R., & Joshi, M. R. (1992). Enamel hypo- plasia prevalence in three ethnic groups of northwest India: A test of daughter neglect and a framework for the past. Recent contributions to the study of enamel developmental defects. Journal of Paleopathology Monograms Publica- tions, 2, 359-372. Lukacs, J. R., & Pal, J. N. (1993). Mesolithic subsist- ence in North India: inferences from dental attributes. Current Anthropology, 34(5), 745-765. Lukacs, J. R., & Walimbe, S. R. (1998). Physiological stress in prehistoric India: new data on local- ized hypoplasia of primary canines linked to climate and subsistence change. Journal of Ar- chaeological Science, 25(6), 571-585. Mahoney, P. (2008). Intraspecific variation in M1 enamel development in modern humans: im- plications for human evolution. Journal of Hu- man Evolution, 55(1), 131-147. Massler, M., & Schour, I. (1946). Growth of the child and the calcification pattern of the teeth. American Journal of Orthodontics and Oral Surgery, 32(9), 495-517. Nanci, A. and Smith. C. E. (1992). Development and calcification of enamel. In E. Bonucci, (Ed.) Calcification in Biological Systems (pp 313-343). Boca Raton, FL: CRC Press. Okada, M. (1943). Hard tissues of animal body. Highly interesting details of Nippon studies in periodic patterns of hard tissues are described. Shanghai Evening Post. Medical Edition of Septem- ber 1943, 15-31. Primeau, C., Arge, S. O., Boyer, C., & Lynnerup, N. (2015). A test of inter-and intra-observer error for an atlas method of combined histological data for the evaluation of enamel hypo- plasia. Journal of Archaeological Science: Re- ports, 2, 384-388. Reid, D. J., Beynon, A. D., & Ramirez Rozzi, F. V. R. (1998). Histological reconstruction of dental development in four individuals from a medie- val site in Picardie, France. Journal of Human Evolution, 35(4-5), 463-477. Reid, D. J., & Dean, M. C. (2006). Variation in mod- ern human enamel formation times. Journal of Human Evolution, 50(3), 329-346. 12 Dental Anthropology 2021 │ Volume 34│ Issue 01 Schwartz, G. T., Mahoney, P., Godfrey, L. R., Cuoz- zo, F. P., Jungers, W. L., & Randria, G. F. (2005). Dental development in Megaladapis edwardsi (Primates, Lemuriformes): implica- tions for understanding life history variation in subfossil lemurs. Journal of Human Evolution, 49 (6), 702-721. Schwartz, G. T., Reid, D. J., & Dean, C. (2001). De- velopmental aspects of sexual dimorphism in hominoid canines. International Journal of Pri- matology, 22(5), 837-860. Smith, C. E., & Nanci, A. (2003). Overview of mor- phological changes in enamel organ cells asso- ciated with major events in amelogene- sis. International Journal of Developmental Biolo- gy, 39(1), 153-161. Smith, T. M., Reid, D. J., Dean, M. C., Olejniczak, A. J., & Martin, L. B. (2007). New perspectives on chimpanzee and human molar crown de- velopment. In: Dental perspectives on human evo- lution: state of the art research in dental paleoan- thropology. Springer, Dordrecht. pp. 177-192. Zheng, J., Li, Y., Shi, M. Y., Zhang, Y. F., Qian, L. M., & Zhou, Z. R. (2013). Microtribological be- haviour of human tooth enamel and artificial hydroxyapatite. Tribology International, 63, 177- 185.