1 INTRODUCTION

Domestic pigs (Sus scrofa f. domestica), both farm breeds and minipigs, are frequently used as model organisms in biomedical research (Giunta et al., 1993; Lunney et al., 2021; Štembirek et al., 2012; Swindle et al., 2012; Vodička et al., 2005; Wang et al., 2007). A major reason for using pigs as experimental animals is that their gross anatomy and physiology are more similar to that of humans than are those of the mouse, rat or rabbit. This overall greater similarity between Homo sapiens and Sus scrofa also pertains to the development and morphology of the dentition (Bivin & Mc Clure, 1976; Davies, 1990; Fejerskov, 1979; Ide et al., 2013; Štembirek et al., 2010; Štembirek et al., 2012; Wang et al., 2014; Weaver et al., 1962). Both humans and pigs have a diphyodont dentition, four types of teeth (incisors, canines, premolars, and molars) in both upper and lower jaws, and low crowned (brachydont), bunodont molars that possess thick enamel. Accordingly, many studies have used pigs as model organisms in various fields of dental research. These studies either focused on the normal process of enamel mineralization (Depalle et al., 2023; Gil-Bona et al., 2023; Kirkham et al., 1988; Robinson et al., 1987, 1988; Sova et al., 2018) or on the effects of undernutrition, exposure to excess levels of drugs or toxicants, or other kinds of developmental stress on dental hard tissue formation (Dobney et al., 2007; Dobney & Ervynck, 2000; Ervynck & Dobney, 1999, 2002; Giunta et al., 1993; Kierdorf et al., 2004; McCance et al., 1961; Richards et al., 1986; Skinner & Byra, 2019; Tonge & McCance, 1973).

Mammalian dental hard tissues are formed in an incremental mode and, as they are not remodeled, keep a permanent record of their formation in the form of regular incremental markings in their microstructure (Boyde, 1989; Dean, 2000; Hillson, 2014; Hogg, 2018; Klevezal, 1996; Naji, 2022; Risnes, 1998; Smith, 2008). In order to reconstruct dental growth processes, the periodicity of these markings must be known. The best approach to unambiguously characterize the periodicity of incremental markings and to determine dental growth parameters is in vivo labeling of forming dental hard tissues with substances that are incorporated at the growth (mineralization) front of the respective tissue, thereby producing a signal that can be identified on microscopic inspection (Bromage, 1991; Bromage et al., 2009; Emken et al., 2021; Iinuma et al., 2004; Kahle et al., 2018; Kawasaki & Fearnhead, 1975; Kierdorf et al., 2013; Ohtsuka & Shinoda, 1995; Okada & Mimura, 1938, 1940; Papakyrikos et al., 2020; Schour & Poncher, 1937; Smith, 2006; Yilmaz et al., 1977).

Establishing dental growth parameters is a crucial part of comparative biological research aiming at elucidating life history traits of both, extant and extinct mammals. This approach can provide insights into adaptations of developmental processes that have occurred during the evolutionary history of a species, including changes in the course of domestication (Dean, 2010; Dean et al., 2020; Dirks et al., 2009; Emken et al., 2023; Funston et al., 2022; Jordana et al., 2014; Le Cabec et al., 2017; Modesto-Mata et al., 2020; Nacarino-Meneses et al., 2017; Nacarino-Meneses & Chinsamy, 2021; O'Meara et al., 2018). A knowledge of the growth rates of dental hard tissues is also decisive for the reconstruction of the timing of stress events affecting tooth formation. Factors studied in this context include husbandry practices, nutritional, climatic and social influences, and the exposure to environmental toxicants (Austin et al., 2016; Beaumont & Montgomery, 2016; Dobney et al., 2007; Dobney & Ervynck, 2000; Ervynck & Dobney, 1999, 2002; Kierdorf et al., 2012, 2016; Niven et al., 2004; Richter et al., 2010, 2011; Shepherd et al., 2012).

During the last decade, our group has studied the nature and periodicity of regular incremental markings in enamel of both, domestic pigs and wild boar (Emken et al., 2021, 2023; Kierdorf et al., 2014, 2019). To establish the periodicity of enamel incremental markings, we initially compared the number of these markings in postnatal enamel with known eruption dates of the respective teeth (Kierdorf et al., 2014, 2019). Subsequently, we performed in vivo fluorochrome labeling of forming enamel and counted the incremental markings between fluorescent labels separated by a known time interval (Emken et al., 2021, 2023). These studies proved that so-called laminations, which exhibit a daily periodicity, are the dominant incremental markings in porcine enamel. In addition, long-period enamel incremental markings (striae of Retzius) with a periodicity of either 2 or 3 days were observed in pig molars (Emken et al., 2021; Kierdorf et al., 2019). The former study also revealed the presence of dentin incremental markings with a daily (von Ebner lines) or a two day (Andresen lines) periodicity in pig M₂.

Based on lamination counts in enamel it was possible to precisely reconstruct and compare crown growth parameters of mandibular second molars in domestic pigs (Linderöd breed) and wild boar (Emken et al., 2023). This study revealed that mean crown formation time (CFT) of this tooth was considerably shorter in the domestic pig (162 days) than in the wild boar (205 days). The difference in CFT was mainly due to a higher enamel extension rate (EER) in the domestic pigs. In contrast, no marked difference in daily enamel secretion rate (DSR) was recorded between domestic pigs and wild boar. The more rapid recruitment of secretory ameloblasts during M₂ crown formation in the domestic pig, reflected by a higher EER compared to wild boar, was considered a side-effect of the selection for rapid body growth in the course of pig domestication (Emken et al., 2023).

So far, no detailed information on dentinal growth rates in pig teeth has been reported in the scientific literature. A single value of about 3 μm/day for the apposition rate of root dentin near the dentin-cementum junction (DCJ) in a domestic pig canine was communicated by Dean (1998). Giunta et al. (1993) reported a dentin apposition rate (DAR) of 3.81 μm/day for a permanent mandibular first incisor from a minipig. However, such single values are insufficient to characterize the complex growth dynamics of dentin in mammalian teeth, as is for instance evidenced by the high spatiotemporal variation of DAR previously recorded in coronal dentin of sheep mandibular first molars by Kahle et al. (2018).

As dentin is deposited throughout the life of a tooth, it provides the potential to record in its structure or elemental composition the exposure of an individual to environmental influences such as pollutants (Blanz et al., 2018; Kierdorf et al., 2016; Richter et al., 2010, 2011; Shepherd et al., 2012; Suckling et al., 1995) over a much longer period than would be possible by analyzing the enamel, formation of which ceases with crown completion. Dentin is produced by neural crest-derived cells, the odontoblasts, in a two-step process (Nanci, 2018a). First, a collagen-rich organic matrix (predentin) is secreted that is subsequently mineralized. As mineral deposition lags behind matrix formation, a seam of predentin is located between the odontoblast and the dentin mineralization front.

To broaden the data base for dentin growth parameters of pig teeth in general, and to provide further insight into the influence of the domestication process on dental hard tissue formation in Sus scrofa, in the present study we reconstructed two important dentinal growth parameters. Dentin apposition (DAR) and extension rates (DER) were determined in mandibular first molars of domestic pigs and wild boar using teeth from a previously reported fluorochrome labeling experiment (Emken et al., 2021, 2023). The first molar was chosen for study because its postnatal crown development was largely and its root formation completely covered by the period of fluorochrome injections. Furthermore, the presence of a neonatal line (NNL) in the crown of this tooth provides an additional landmark for the analysis of growth processes.

2 MATERIALS AND METHODS

The mandibular first molars analyzed in the present study originated from a female and a male wild boar and a female and a male domestic pig of the Linderöd breed. The two individuals in each group were littermates whose mandibular second molars had already been used in the studies by Emken et al. (2021, 2023). The Swedish Linderöd pig is a largely unimproved breed whose development is considered to more closely resemble that of early forms of domesticated pigs than that of more improved modern breeds (Hansson, 2008; Kierkegaard et al., 2020; Ollivier, 2009).

The four study animals were born and raised in the Tierpark Arche Warder e.V. (Warder, Germany). The two wild boar were born on 11 April 2016 and slaughtered on 7 March 2017, at the age of 330 days. The two domestic pigs were born on 21 April 2016 and slaughtered on 30 March 2017, at the age of 343 days. Both males were castrated prior to the start of the experiment. Further details on handling and treatment of the experimental animals can be found in Emken et al. (2021). Each of the four experimental animals received 12 fluorochrome (calcein and oxytetracycline) injections at 2 or 3 weeks' intervals (for the injection protocol see Table 1). The experiment was conducted in accordance with all current animal care regulations in Germany and with permission (including ethical approval) of the responsible veterinary authorities of the federal state of Schleswig-Holstein (Ministerium für Landwirtschaft, Umwelt und ländliche Räume des Landes Schleswig Holstein; Az. V312-72241.123–34).

After the end of the experiment, the animals were killed using an approved humane method, and their heads were removed and macerated. The mandibular first molars were extracted from the jaws, cleaned, and embedded in epoxy resin (Biodur E12). Ground section were performed in axio-buccolingual direction through the highest point of the anterior (mesial) tooth cusps. Production of ground sections (thickness of about 50 μm) and acquisition of microscopic images followed the protocol described by Emken et al. (2021).

Microscopic analysis of the ground sections focused on two dentinal growth parameters. DARs were determined in buccal and lingual dentin of the M₁s in five zones (upper lateral, mid-lateral, lower lateral and cervical crown zones, and upper root zone) along the vertical tooth axis (Figure 1). Measurements were performed along the reconstructed course of individual dentinal tubules between the enamel-dentin junction (EDJ) or the DCJ, and the dentin-pulp interface (DPI). To calculate daily DARs, the distance between the outer borders (directed toward the EDJ or DCJ) of consecutive fluorescent labels was divided by the number of days that had elapsed between the fluorochrome injections that had caused the respective labels. In each of the five dentin zones analyzed per tooth, one dentinal tubule was chosen for data collection in buccal and lingual dentin. The first (cuspalmost) dentinal tubule of the upper lateral crown zone started at the intersection of the NNL with the EDJ (Figure 2a). The apicalmost dentinal tubule in the upper root zone started at the DCJ at the day of completion of crown elongation, that is, at day 86 in the wild boar (Figure 2b) and day 65/66 in the Linderöd pigs. The other three dentinal tubules along which DAR was recorded were placed equidistantly at the EDJ between the upper and the lowermost one (Figure 1b). Measurements of the cuspalmost dentinal tubule started at the intersection of the NNL with the EDJ. In the other four dentinal tubules, the starting point of the measurements was the intersection of the respective tubule course with the fluorochrome label located closest to the EDJ or DCJ. The endpoint of all five dentinal tubules was the DPI that in the ground sections corresponded to the position of the dentin mineralization front at the day of death. DARs are reported as arithmetic means (± standard deviations) for the respective postnatal time spans in the five analyzed zones per tooth (Tables 2 and 3).

In overview images, the fluorochrome labels located nearest to the DPI were typically not or only faintly visible (Figure 2a,b). However, using different microscopic settings, their presence could be demonstrated (Figure 3).

Dentin extension rates were determined in postnatally formed buccal and lingual crown and root dentin along the vertical tooth axis of the M₁s (Figures 4 and 5). The DER is defined as the progression of the dentin mineralization front along the EDJ or DCJ. For calculation of daily DERs we used a method described by Dean (2012a, 2012b) and Dean and Cole (2013) for hominid teeth that was modified to the conditions in the pig molars. To reconstruct the daily DER along the vertical tooth axis we first followed the path of a selected dentinal tubule from its start at the EDJ or DCJ for 65 μm into the dentin body. From this point we followed the nearest daily incremental marking (von Ebner line) in apical direction until it met the EDJ or DCJ. If von Ebner lines were not discernible in the respective dentin area, we reconstructed the course of the mineral apposition front by drawing a line that ran from the endpoint of the 65 μm long stretch of the selected dentinal tubule parallel to the nearest fluorescent label in the dentin until the intersection of this line with the EDJ or DCJ. Daily DERs were calculated by measuring the distance in apical direction between consecutive intersection points of the selected incremental markings or the reconstructed lines with the EDJ or DCJ and dividing this distance by the number of days needed for the 65 μm progression of the respective mineral apposition front from the EDJ or DCJ into the dentin body (Figure 5). The DARs necessary for these calculations were either directly measured between the intersection of the NNL with the EDJ and the first fluorescent labels or between the two consecutive fluorescent labels closest to the EDJ or DCJ in the respective tooth area.

In all teeth, measurement of daily DERs started with the first extension segment along the EDJ beginning at the intersection of the NNL with the EDJ. The number of extension segments that could be reconstructed in the root area differed between the analyzed teeth as in some ground sections the root course was not completely preserved in the section plane due to bending of the root or slight obliqueness of its course against the section plane. For a direct comparison of mean daily DERs during the first 86 days of postnatal M₁ formation of wild boar and domestic pigs, we determined the distance between the intersection of the NNL with the EDJ and the position of the dentin mineralization front along the DCJ at day 86. Due to the presence of a fluorescent label from an injection at that day, this intersection could be directly defined in the ground sections of the wild boar M₁s (Figure 4a). In the domestic pig M₁s, this point of intersection had to be reconstructed between the positions at the DCJ of the fluorescent labels resulting from the first calcein (Ca1, day 76) and the first oxytetracycline (T1, day 90) injection (Figure 4b).

Crown formation times of the first molars were determined by counting the number of laminations present in their enamel. Due to the steep inclination of the laminations in prenatal enamel, it was possible to perform lamination counts (based on their persisting cervical portions) in domestic pig M1 with worn cusps. Comparison with the less worn wild boar M1 revealed that only two or three laminations had to be added to this number to achieve a total lamination count for prenatal enamel in the M1 of the domestic pigs.

3 RESULTS

3.1 Dentin apposition rate

Mean daily DARs exhibited marked variation in the studied teeth, depending on the position of the analyzed dentin zone along the vertical tooth axis and the postnatal time span of dentin formation for which values were recorded (Tables 2 and 3). Generally, DARs showed a concordant trend in the first molars of wild boar and domestic pigs, dependent on the duration of secretory activity of the odontoblasts and the corresponding increase in the distance of the dentin formation front from, respectively, the EDJ or DCJ. Mean values increased during the first 4 to 4.5 months of postnatal dentin formation and then gradually declined toward the DPI, reaching lowest values in the last recorded time span prior to death. Highest mean daily DARs (combined values for the male and the female in each group) were recorded in the dentin of the upper-lateral crown zone. Maximum mean values for the wild boar (21.7 μm/day buccally, 23.3 μm/day lingually) were recorded in the postnatal age span of 114 to 135 days (Table 2, Figure 6a). In the domestic pigs, maximum mean values (19.6 μm/day buccally, 22.2 μm/day lingually) were recorded in the postnatal age spans of 90 to 104 days (buccally) and of 104 to 125 days (lingually) (Table 3, Figure 6b). The maximum individual DAR in a wild boar M₁ (23.8 μm/day) was recorded in the upper-lateral dentin zone formed between postnatal days 114 and 135. In the domestic pig M₁, the individual maximum DAR (22.9 μm/day) was recorded in the mid-lateral dentin zone formed between postnatal days 125 and 146.

A second trend observed in the M₁s was a decrease of DAR along the vertical tooth axis during the first 5 months of postnatal dentin formation. During this period, more cervically or apically located dentin zones mostly showed lower mean DARs compared to synchronously formed more cuspally located ones (Tables 2 and 3, Figure 6).

3.2 Dentin extension rate

Mean daily DERs showed a marked variation along the vertical tooth axis in both the crowns and the roots of the wild boar and domestic pig M₁. Figure 7 demonstrates the variation of DERs (means of values from the female and male individual per group) for buccal and lingual tooth sides. In the tooth crown, values decreased from very high values at the intersection of the NNL with the EDJ (maximum value in wild boar 208.1 μm/day, maximum value in domestic pig 237.2 μm/day) to lowest values in the cervical crown area slightly cuspal to the crown-root-border (50.0 μm/day in wild boar, 67.0 μm/day in the domestic pig). After this nadir, DER values started to increase already in the cervicalmost crown area and peaked in the upper root area with maximum values of 208.0 μm/day in wild boar and 272.2 μm/day in domestic pigs. After these peaks, DER values markedly declined toward the root tip.

In individual teeth, DER values in corresponding extension segments were mostly higher on the lingual compared to the buccal tooth side (Figure 7). During postnatal crown and early root formation, the domestic pigs achieved considerably higher mean DER values than the wild boar (Figure 7). Accordingly, mean DERs for buccal and lingual tooth sides for the first 86 days of postnatal tooth development in the domestic pigs more than doubled those in the wild boar (Figure 8). Due to the more rapid progression of dentin extension in the domestic pigs, they reached highest mean DER values in their roots earlier than the wild boar. Thus, dentin root extension in the wild boar M₁s peaked after about 132 days of postnatal tooth formation and 12,400 μm of EDJ/DCJ extension (mean values for buccal and lingual tooth sides), while in the domestic pig M₁s, peak root dentin extension occurred already after about 74 days of postnatal tooth formation and 8200 μm of EDJ/DCJ extension. In the wild boar, peak rates of root dentin extension therefore occurred simultaneously with peak values of DAR in crown dentin (upper-lateral crown zone, Table 2) while in the domestic pig, peak rates of root extension occurred about 6 to 8 weeks prior to peak values of DAR (Table 3).

4 DISCUSSION

5 CONCLUSIONS

The present study, for the first time, provides comprehensive data on the spatiotemporal dynamics of dentin formation in mandibular first molars of domestic pigs and wild boar. The results demonstrate similar DARs in comparable dentin zones of domestic pigs and wild boar first molars, but marked differences in DER, with higher values in domestic pigs. These findings parallel the results of a previous study comparing crown growth parameters in mandibular second molars of domestic pigs and wild boar (Emken et al., 2023). In this study we observed that daily enamel secretion rates (DSRs) were similar in corresponding crown regions of domestic pigs and wild boar, but that the former exhibited higher enamel extension rates (EERs) and, in consequence, considerably shorter crown formation times. The findings of the present and our previous study (Emken et al., 2023) could suggest that in rapidly developing pig molars, the secretory activities of ameloblasts and odontoblasts are close to or at their physiological maximum. Further acceleration of tooth formation would therefore only be possible by increasing the number of simultaneously active ameloblasts and odontoblasts, i.e., by increasing the recruitment rate of these cells.

It has been argued that in the course of pig domestication artificial selection was associated with the development of growth trajectories that enabled more rapid body growth and weight gain (Albarella et al., 2006; Larson & Fuller, 2014; Sanchez-Villagra, 2022). Selection for rapid body weight gain and a shorter reproductive cycle during pig domestication may have also triggered effects in dental development that were not the primary focus of the artificial selection regime (Sanchez-Villagra, 2022). We assume that the observed increase in EER and DER in domestic pig molars compared to wild boar molars, which causes a considerable shorter CFT in the former, constitutes such a side effect occurring in the process of pig domestication.

AUTHOR CONTRIBUTIONS

Horst Kierdorf: Writing – original draft; writing – review and editing; investigation; formal analysis; conceptualization; methodology; resources; supervision; visualization; project administration. Simon Emken: Writing – original draft; investigation; formal analysis; visualization; data curation. Carsten Witzel: Writing – review and editing; formal analysis; conceptualization; investigation; methodology; visualization. Uwe Kierdorf: Writing – review and editing; formal analysis; conceptualization; investigation; visualization. Kai Frölich: Writing – review and editing; resources; methodology.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.