Morphometric, densitometric and mechanical properties of mandibular deciduous teeth in 5-month-old Polish Merino sheep

Bone, dentine, cementum and enamel are considered as four types of hard tissue. Teeth are the most durable structures of mammalian body [1]. Except for enamel, dentine and cementum represent specialized types of tissue, where type I collagen contributes significantly to the structure. Even though enamel does not arise from connective tissue and does not contain collagen, its formation follows the principles common to the formation of mineralized tissues. Hard dental tissue formation is the result of a process in which specialized cells secrete an organic matrix undergoing mineralization mainly by calcium and phosphates. Among cells, osteoblasts, odontoblasts, cementoblasts and ameloblasts play essential functions in these processes [2].

Dental disorders in sheep can lead to premature slaughter before they have attained final stage of their reproductive life and induce economic loss due to high flock replacement costs. It must be underlined that growth rate, health status and meat quality of sheep depends on tooth properties and quality determining in large extent efficiency of the masticatory apparatus and initial food break up. While incisor disorders reduce dramatically intake of forage, cheek tooth dysfunction has negative impact on food grinding [3, 4]. Thus, impaired chewing efficiency resulting from poor tooth health status leads to reduced food intake and the food eaten is digested less efficiently [5, 6]. The function of food break-up performed by teeth has particular importance in ruminants, since they have to chew highly fibrous diet to small pieces enough to aid microbial digestion in the rumen.

Etiology of tooth disorders is multifactorial. Defects of the tooth structure may result from soil ingestion and following abrasion combined with chemical attack from acids present in the ingested soil [7, 8]. Bacterial impact may lead to production of acids by hydrolysis of the food debris accumulated on the tooth surface and cause caries - demineralization of the hard tissues (enamel, dentin and cementum) and destruction of the organic matrix of the tooth. In young sheep, caries affects mainly incisors and shows up as deep holes at the neck of deciduous incisors or a snap-off at gum level [9]. It was observed that dental caries in 8–12 months old sheep occurs as the result of diet rich in soluble carbohydrates administered in young growing animals [10]. Higher susceptibility to carries may results also from demineralized enamel resulting from molar incisor hypomineralization (MIH). It is a developmental pathology mainly located in the mesio-buccal cusps, starting at the enamel-dentin-junction (EDJ) and continuing towards the enamel surface. Experimental data indicate that the ameloblasts in the hypomineralized enamel are capable of forming an enamel of normal thickness, but with a substantial reduction of their capacity for maturation of enamel [11]. Enamel hypoplasia in sheep changes enamel translucency and displays as 1–2 mm shallow pits on the surface of incisors [12]. It results from disturbed amelogenesis process during tooth development induced by dietary or disease factors in young growing sheep such as feeding grain diets with high phosphorus and low calcium, gastrointestinal parasitism and excessive fluoride ingestion [13–17]. Fluoride intoxication during tooth development also results in tooth fragility, pitting, discolouration and chalkiness of the enamel [10].

Considering lack of basic physiological data on tooth properties in sheep, the aim of the study was to evaluate morphometric, densitometric and mechanical traits of deciduous mandibular incisors, canine and the second premolar - the analyzed teeth had completely developed all their anatomical structures by the slaughter age of 5 months of life.

This experiment was conducted in accordance with national guidelines for animal experimentation and the “Guide for the Care and Use of Laboratory Animals”. The experimental procedures used in this study were approved by The Local Ethics Committee on Animal Experimentation of Medical University in Lublin, Poland (Permission number 6/2008 assigned on 15^th February 2008).

Microtomography of teeth

Microtomography (μCT) technique and SkyScan 1174 apparatus (SkyScan n.v., Kontich, Belgium) was used to determine total enamel volume (Evol), volumetric enamel mineral density (vEMD), total dentine volume (Dvol) and volumetric dentine mineral density (vDMD). Images for analysis were obtained at 50 kV and 800 μA with a 0.25 mm aluminum filter. For each tooth, 300 projection images were obtained with a rotation of 0.70°. An image pixel size corresponded to 21.72 μm. The samples were rotated over 180°. Reconstruction of the teeth was performed with the use of NRecon 1.6.1.5 software (SkyScan n.v., Kontich, Belgium). The software allows three-dimensional reconstructions of the investigated sample from the stack of two-dimensional sections (Figure 1). For each slice, the enamel and dentin regions were enclosed using a user-defined polygon. Binary images were obtained with histogram. Two regions of interest (ROI) were defined for the microtomographic images; ROI-1 for enamel and ROI-2 for dentine evaluation [18]. The volume-of-interest was defined at the values between minimum and maximum density of the investigated samples at -1000 and 3071 HU.

Standard veterinary examination of teeth in sheep would be an essential part of the clinical investigation procedures which enables age estimation or poor productive potential indication [9]. Even though simple clinical dental examination requires relatively small costs, it is less informative and provides basic information on number, size, shape and structure of teeth and their occlusion with dental pad.

This study presents basic morphological, densitometric and mechanical properties of completely developed deciduous teeth such as mandibular incisors, canine and the second premolar in sheep at the age of 5 months of life for the first time. The analyses performed have shown significant differences of weight of teeth between all incisors reaching the highest value for the first incisors and diminishing gradually in i₂ and i₃ to the lowest value in i₄ (canine). The differentiated weight of the teeth resulted from analogical differences of the length and total tooth volume of the incisors. Considering significant difference of weight, length and tooth volume, it is surprising that total enamel volume was comparable in i₁ and i₂ as well as in i₃ and i₄, even though i₁ and i₂ possess approximately 40 – 68% more enamel than i₃ and i₄. Gradual reduction of total dentine volume was observed between i₁ – i₂, i₂ – i₃ and i₃ – i₄, that reached 23%, 32% and 25%, respectively. However, densitometric analysis has shown similar values for all the evaluated deciduous teeth in this study when analyzing both MvTMD and vEMD. The only one difference obtained from densitometric evaluation of teeth in sheep has shown significantly higher vDMD in i₁ when compared to i₃ and i₄. Thus, the performed densitometric measurements in both compartments have not shown considerable differences of volumetric tooth mineral density between several deciduous teeth, confirming similar course of mineralization processes of hard dental tissues in all the investigated teeth. Thus, it may be postulated that proper mineral metabolism and mineralization processes during first months of life in sheep lead to comparable mineral density threshold attainment in hard dental tissues of all teeth. The results of densitometric analysis in our study are in accordance to the other reports confirming significantly higher mineral density in enamel than in dentine. It was shown that enamel consists of 95% inorganic substance, 4% water and 1% organic substance, while in dentine all these constituents represent 70%, 20% and 10%, respectively [19–21]. Analogically to results of tooth weight, length and volume, consequent reduction of Wy was observed between i₁ – i₂, i₂ – i₃ and i₃ – i₄ and it reached 23%, 20% and 34%, respectively. Similar decrease of Wf was stated evaluating deciduous incisors and the differences between i₁ – i₂, i₂ – i₃ and i₃ – i₄ reached 24%, 25% and 21%, respectively. In case of the second premolar, its large crown and multi-root anatomical structure contributed to significantly higher values of tooth weight, volume (both in enamel and dentine) and ultimate force when compared to these parameters in incisors and canine. However, tooth length, MvTMD, vEMD and vDMD of p₂ were similar when compared with the other investigated teeth. It should also be explained here that mechanical analysis of i₁ – i₄ and p₂ was performed with two different methods such as three point bending and compressive tests and thus the assessment of Wy for p₂ was not possible. Even though the current study was not performed on large animal population, the obtained values of morphological, densitometric and mechanical parameters may be deemed as reference data for deciduous teeth in sheep since the investigated teeth were completely developed and their growth was finished. In case of the other deciduous mandibular teeth that were not evaluated in this study, longer breeding procedure is required to finish their growth and development and receive meaningful data [22].

Precise evaluation of teeth properties in terms of morphology, microstructure and mechanical properties would be much more effective than simple clinical examination to provide data on whole body and calcified tissues metabolism during crucial periods of life. Considering very rapid growth rate during neonatal and postnatal development in sheep, morphometric (both on macro- and microstructural levels), densitometric and mechanical evaluation of deciduous teeth would be informative in terms of physiological, nutritional, pharmacological, toxicological and environmental conditions taking place during first months of life. Experimental studies on growing sheep have shown that advantageous nutritional manipulations during early stages of neonatal development positively influence mineral metabolism and bone tissue properties. Oral administration with alpha-ketoglutarate (AKG) during the first two weeks of life induced improved morphological traits (weight and length), volumetric bone mineral density (vBMD) and mechanical properties (maximum elastic strength and ultimate strength) of ribs in 5 month old ram lambs [23]. Similar effects of neonatal administration with AKG were observed in studies on femur properties, where vBMD of the trabecular and cortical bone compartments and ultimate strength were significantly increased when compared to the controls receiving standard diet [24]. Three week long neonatal administration with β-hydroxy-β-methylbutyrate (HMB) in lambs significantly increased serum concentrations of bone-specific alkaline phosphatase (BAP) and osteocalcin (OC) – the bone tissue formation markers reflecting osteoblastic activity. Moreover, growth hormone (GH) and insulin-like growth factor-1 (IGF-1), the hormonal factors inducing improved bone tissue metabolism and mineralization, were significantly increased as the consequence of 21-day neonatal administration with HMB. All these effects were associated with permanent changes of bone morphology, mineral density and mechanical endurance. β-hydroxy-β-methylbutyrate administered in young lambs improved bone weight, length, volume, vBMD of the cortical bone as well as geometrical and mechanical properties of femur and lumbar spine [25]. Even though teeth properties were not evaluated in all these experiments, structural and functional changes of hard dental tissues on macro- and microstructural levels would be expected, especially when one considers significant role of osteoblasts in hard dental tissues formation. Furthermore, previous studies have shown interferences between tooth development and alkaline phosphatase, osteocalcin, bone morphogenetic proteins (BMP) and bone sialoproteins involved in signaling functions and mediating hard dental tissue interactions during development [2, 26].

An advantage resulting from morphometric, densitometric and mechanical dental evaluation in animals is the fact that the methodological approach is noninvasive since the deciduous teeth undergo physiological replacement with permanent teeth, taking place at several stages and does not require substantial intervention, sedation or general anesthesia, when compared to in vivo determination of skeletal system properties. Furthermore, in vivo evaluation of bones in sheep with microtomography and mechanical tests is not possible due to limitations resulting from ability to scan small samples in microtomographic devices, as well as consideration of ethical issues [27, 28]. These limitations do not concern deciduous teeth which can be easy collected for analyses from large animal population and reflect mineral status and metabolism resulting from postnatal growth and development of the whole flock. In individual cases, evaluation of properties of deciduous teeth may serve for breeding selection and further reproduction of sheep possessing favorable traits of teeth and better masticatory system functions, leading to improved performance and economic efficiency of the flock. Thus, results from morphometric, densitometric and mechanical evaluation of the teeth may indicate optimal breeding direction of growing sheep.