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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 15  |  Issue : 3  |  Page : 358-363

Comparison of the chemical composition and the structural characteristics of normal enamel from the teeth affected with early childhood caries in pre-term and full-term born children: An In vitro study


Department of Pedodontics and Preventive Dentistry, Sharad Pawar Dental College, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi(M), Wardha, Maharashtra, India

Date of Submission17-Apr-2020
Date of Decision30-May-2020
Date of Acceptance20-Jun-2020
Date of Web Publication1-Feb-2021

Correspondence Address:
Dr. Suruchi Gupta
Department of Pedodontics and Preventive Dentistry, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (Deemed To Be University), Sawangi (Meghe), Wardha - 442 004, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_116_20

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  Abstract 


Background: Early childhood caries (ECC) is considered as one of the most concerned topics of investigation in order to prevent its occurrence by targeting multiple causative factors. Time of birth of a child has an effect on the development of tooth and might show differences in the structure and composition of enamel. Aim: To evaluate the chemical composition and structural characteristics of enamel in primary teeth of pre-and full-term born children affected with ECC. Material and Methods: Twenty extracted primary teeth from pre-term (n = 10) and full-term (n = 10) born children affected with ECC were selected. Unaffected portions of enamel were subjected to polarized light microscope (POLMI), scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDS). Results: The thickness of prenatally formed enamel was less in pre-term born children under POLMI. The concentrations of calcium and phosphorus were higher in full-term born children on EDS analysis. The surfaces of enamel in primary teeth from full-term group were smooth whereas pitted and eroded along with a larger distribution of hypoplasia in pre-term group when observed under SEM. Conclusion: Primary teeth enamel of pre-term born children was found to be defective in both qualitative and quantitative aspects and could be a possible risk factor in the causation of ECC.

Keywords: Early childhood caries, enamel, neonatal line, primary teeth


How to cite this article:
Bhansali P, Baliga S, Thosar N, Gupta S. Comparison of the chemical composition and the structural characteristics of normal enamel from the teeth affected with early childhood caries in pre-term and full-term born children: An In vitro study. J Datta Meghe Inst Med Sci Univ 2020;15:358-63

How to cite this URL:
Bhansali P, Baliga S, Thosar N, Gupta S. Comparison of the chemical composition and the structural characteristics of normal enamel from the teeth affected with early childhood caries in pre-term and full-term born children: An In vitro study. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2021 Feb 25];15:358-63. Available from: http://www.journaldmims.com/text.asp?2020/15/3/358/308537




  Introduction Top


Early childhood caries (ECC) is a chronic preventable disease affecting children worldwide. The term of ECC was introduced in an attempt to focus on its multifactorial causation rather than a single well defined etiology. It has a diverse pattern of occurrence and affects the developing dentition leading to premature tooth loss, speech disorders, and psychological trauma. Literature suggests that children born pre-term and/or low-birth-weight children are at higher risk to develop ECC.[1]

Children born prior to 37 weeks of gestation are known as pre-term infants. Unlike full-term infants, they might have low birth-weight, affected physical and psychological growth, and increased susceptibility to infections as well as renal, respiratory, gastrointestinal, immunological, metabolic and hematological disturbances.[2] Premature birth of a child also affects the development of enamel of the deciduous and permanent teeth.

During the process of initiation of caries, enamel is the first tissue to get affected. It consists of about 96% inorganic material, constituting biological hydroxyapatite crystals with a hexagonal lattice structure packed and arranged in prisms.[3] The organization of enamel might vary in relation to the birth of a child. Enamel deposition occurs in increments represented by incremental lines. Neonatal line (NNL) is a distinct incremental line related to the time of birth that demarcates the prenatally formed enamel from the postnatally formed enamel. The NNL is found in all primary teeth denoting a growth pause in the enamel formation and is seen as a biological landmark of birth.[4] Location of NNL differs in teeth due to tooth germs being at different developmental stages when child is born.

The chemical as well as mineral contents varies along the thickness of enamel.[2] Morphological studies carried out on primary teeth of pre-term infants revealed higher frequency of hypomineralized enamel, broadened neonatal lines and regular manifestation of distinct incremental lines in postnatally formed enamel compared with enamel of primary teeth in full-term infants.[4]

Enamel is an exceptional hard tissue of the tooth that does not alter and thus the changes in its structure consequential to any insult are indefinitely recorded. Moreover, any variations in the chemical composition of enamel render tooth susceptible for early colonization of cariogenic bacteria leading to ECC.[3] Therefore, the purpose of the study was to evaluate the chemical composition and to analyze the structural organization of enamel in pre-term and full-term born children affected with ECC.


  Materials and Methods Top


The study proposal was submitted to and approved by institutional ethical committee of Datta Meghe University of Medical Sciences (Deemed to be University), Sawangi, Wardha. Data were analyzed using N master software.

Children reporting to the Department of Paedodontics and Preventive Dentistry, Sharad Pawar Dental College, Sawangi (Meghe), Wardha were examined and their case histories were recorded. The children up to 6 years old affected with ECC were included in the study. However, children with systemic diseases were excluded from the study.

In the children diagnosed with ECC, the teeth indicated for extraction like grossly carious and non-restorable as well as the teeth near to exfoliation were extracted under local anesthesia.

Twenty primary teeth were collected from 20 children in which Group 1 included 10 primary teeth from children affected with ECC born pre-term and Group 2 included 10 primary teeth from children affected with ECC born full-term.

All the extracted teeth from both the groups were washed using normal saline and cleaned of any visible blood or gross debris by means of a handpiece fitted with hard bristle brush and pumice. Areas of carious enamel and dentin were removed with the use of round diamond burs. All the teeth were cut using hard tissue saw microtome to separate the roots and obtain a flat surface that ensures stability of the samples. Samples were then sectioned mesiodistally in two equal halves using diamond disc and were stored dry.

One half of the 20 sectioned samples were subjected to polarized light microscopy (POLMI). However, the other half was subjected to scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis.

Polarized light microscopy

20 samples from both the groups were further sectioned faciolingually. The individual ground sections were prepared and viewed under a polarized light microscope at ×10 magnification. Firstly, the neonatal line (NNL) was located. The distance of NNL from cementoenamel junction (CEJ) at the buccal aspect was measured using a measuring grid. The values for the widths of prenatally and postnatally formed enamel and maximum thickness of enamel were obtained. For all the measurements, mean and standard deviation was calculated.

SEM-EDS analysis

The remaining halves of 20 samples from both the groups were subjected to three cycles of gold sputter-coating and were then transferred to Scanning Electron Microscope. They were viewed under ×100, ×500, ×1000, and ×3000 magnification. The obtained photomicrographs were examined for surface texture of enamel, presence or absence of cracks, pattern of enamel prisms and enamel rods as suggested by Azinovic Z et al.[5]

The elemental compositions of Calcium (Ca), Phosphorus (P), Oxygen (O) and Carbon© were quantified using a scanning electron microscope equipped with an EDAX X-MAX detector. The individual concentration gradients of Ca, P, O and C were measured in weight percentage. The ratios of Ca/P and Ca/C were derived. The mean and standard deviation of all the measurements was calculated.

Ethical clearance

The Institutional Ethics Committee of DMIMSDU has approved the Research work proposed to be carried out at Sharad Pawar Dental College, Sawangi(M), Wardha. Date: 21st March 2019 with Reference no DMIMS(DU)/IEC/2019/140.


  Results Top


Statistical analysis was done by using Chi square test and student's unpaired t-test and software used in the analysis was SPSS 22.0 version and Graph Pad Prism 6.0 version and P < 0.05 is considered as level of significance.

Among 20 children (10 in each group), males and females were equally distributed with the mean age of 5.01 ± 0.99 years and 5.02 ± 0.98 years in a Group A and Group B, respectively.[Table 1]
Table 1: Distribution of gender and age amongst two study groups

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On comparing the distance between cementoenamel junction and neonatal line using POLMI; the mean distance between CEJ and NNL of enamel in Group A (0.265 ± 0.272 mm) was significantly lesser than in Group B (1.365 ± 0.360 mm) as shown in [Table 2]. The comparison between thickness of pre- and post-natally formed enamel under POLMI [Table 3] showed that the thickness of prenatally formed enamel in preterm born children (0.023 ± 0.028 mm) was lesser than in full-term born children (0.215 ± 0.021 mm). Whereas, the postnatally formed enamel was thick in the pre-term children (0.296 ± 0.052 mm) compared to the enamel of full-term children (0.172 ± 0.035 mm). The mean maximum facial thickness of enamel from the pre-term incisors (0.420 ± 0.34 mm) was significantly less than that for the full-term children (0.5091 ± 0.058 mm).
Table 2: Comparison of the distance between cementoenamel junction and neonatal line in enamel of preterm and full-term primary teeth

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Table 3: Comparison of the thickness of pre-and postnatally formed enamel in pre-term and full-term primary teeth

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[Table 4] compares the values of composition of elements using EDS analysis in weight percentage in primary teeth enamel of pre-term and full-term born children. The weight percentage of Calcium (Ca) was found to be significantly lower in pre-term children (32.87 ± 1.73) than full-term (34.71 ± 2.48). Similarly, weight percentage of Phosphorus (P) was found to be lower in pre-term children (14.20 ± 1.33) than full-term children (13.99 ± 1.50). But the difference was statistically not significant. The weight percentages of Carbon© in full-term children (30.23 ± 2.18) were lower than pre-term children (32.23 ± 1.23). Similarly, the weight percentage of Oxygen (O) in pre-term children (27.41 ± 1.25) was higher than full-term (26.07 ± 1.08) children. The difference between the values of C and O were found to be statistically significant.
Table 4: Energy dispersive X-ray spectroscopy analysis values in weight percentages in pre-term and full-term enamel

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[Table 5] compares the ratios of Calcium/Carbon (Ca/C) and Calcium/Phosphorus (Ca/P) in both groups. The difference between the ratios of Ca/C was found to be statistically significant where enamel of pre-term children had lower value (1.02 ± 0.07) than full-term children (2.34 ± 0.30). The Ca/P ratio revealed no statistically significant difference between the two groups though enamel of full-term children (2.34 ± 0.07) had higher values than pre-term children (2.50 ± 0.13)
Table 5: Comparison of the ratios of Calcium/Carbon and Calcium/Phosphorus in pre-term and full-term enamel

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[Table 6] depicts the distribution of the appearance of surface texture, the pattern of prism, presence of cracks and distribution of enamel hypoplasia in primary enamel from both the groups under SEM Amongst the group of pre-term children the surface texture was found to be serrated in 3 teeth, eroded in 2 teeth and pitted in 5 teeth. 50% of teeth showed presence of cracks. Higher magnification showed 70% evidence of enamel hypoplasia with irregular arrangement of prism pattern. By contrast, on comparing it with full-term group children, showed remarkably smooth surface texture except small serrations in 3 teeth and pits in 3 teeth. 30% of teeth showed the presence of cracks. Higher magnification usually showed prism free enamel whereas, evidence of hypoplastic enamel was found to be in 30% in enamel of primary teeth in full-term born children.
Table 6: Distribution of structural characteristics in pre-term and full-term enamel

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  Discussion Top


ECC remains difficult to manage in spite of its well-known causative factors. ECC affects teeth but the outcomes of this ailment lead to extensive health issues.[1] The commonly involved etiologies for the causation of ECC include lack of oral hygiene, colonization with mutans streptococci, frequent sugar consumption, maternal psychosocial and behavioral factors, and perinatal disturbances.[2]

Worldwide, an estimated 15 million babies are born pre-term every year.[6] These babies are always at a risk of developing complications. Recent literatures prove that prematurely born individuals have deprived mineralization of the teeth and inferior oral health indicators.[7],[8] Enamel is the outermost structure of the crown of the tooth and there are variations throughout the thickness of the enamel in the chemical and mineral content.[3]

Considering the multifactorial causation of ECC and varying nature of enamel, there might be differences in the mode of initiation of carious lesion with respect to the birth of a child. Therefore, the primary aim of the study was to assess chemical constitution and structure of enamel and its association with the pre-term and full-term birth of a child affected with ECC.

Polarized light microscope was utilized in the study to determine the histological structural differences in the manifestation of primary enamel from both pre-term and full-term born children. Ultrastructurally, neonatal line (NNL) seems as a localized change in the pattern of the enamel prism with a probable diminution in the crystal concentration.[9] It represents a growth pause in the enamel at the time of birth and also termed as a biological landmark of birth.[10] The distance between cementoenamel junction (CEJ) and NNL of enamel in the pre-term born children was found to be significantly lesser than in the full-term born children. This could be due to the premature cessation of activity of ameloblasts to form enamel.[11] Tooth germs are at different developmental stages when child is born which leads to differing locations of the neonatal line. Thus, the location of the NNL in the enamel is directly related to the gestational length.[12]

Prenatally formed enamel was found to be thin in primary teeth enamel of the pre-term born children when compared with thickness of prenatal enamel primary teeth in full-term born children. Reduced thickness of enamel suggested reduced gestational length and thus shortened duration in a prenatal enamel formation. However, postnatal enamel was comparatively thicker in the pre-term born children. This suggested a partial compensatory action in the formation of postnatal enamel after the birth in pre-term born children. However, the overall width of the enamel was still significantly less than that for children born full-term. This finding was supported by Seow et al., who mentioned that, there was increased thickness of postnatally formed enamel to compensate for the severely diminished prenatally formed enamel development. However, recovery of enamel formation is not sufficient to achieve full thickness enamel formation.[11] The reduced thickness of enamel could be because of the deranged ameloblasts caused by the severe insults during metabolism in pre-term born children. Moreover, reduced supply of calcium and phosphate to the developing teeth and impaired mineral metabolism might deteriorate the formation of enamel.[13],[14] Therefore, discrepancies are likely to be reflected in reduced and abnormal mitotic activity of all the tissues including enamel. This is strongly held up by the present study's observations, which showed a significant declination in overall enamel thickness of pre-term born children. Similar results were found in the study by Seow et al., where they have reported that premature birth is related with primary teeth of smaller dimensions when compared to the teeth of full-term children.[11],[15] Various authors have supported the judgment that the effects on enamel formation are more likely to be associated with prematurity than low birth weight.[16],[17] The enamel surfaces of primary teeth were examined under scanning electron microscope in order to assess the distribution of enamel hypoplasia, to analyze the surface texture and to detect the degree of presence of cracks and erosion. According to Udea DID et al., SEM is considered to be one of the best methods to study the surface of enamel.[18]

The enamel surface in primary teeth of pre-term born children had detectable irregularities. The differences in the microstructure of enamel of primary teeth in pre-term born children suggested porous and hypomineralized surface with an abrupt break in enamel formation.

The possible reason behind increased prevalence of enamel hypoplasia in pre-term born children infants is the mechanism pointing to in utero insults during tooth formation. The poor development of enamel could be because of the decreased period of prenatal enamel formation as well as perinatal systemic complications in prematurely born children. The roughness of the surface texture of enamel might cause greater attachment of mutans streptococci promoting earlier colonization. Enamel hypoplasia is considered to be a quantitative defect in the enamel surface and is the key factor for the development of ECC since defects can make enamel susceptible to more amount of cariogenic microorganisms.[19],[20] The results of the study can also be correlated with presumptive evidences that malnutrition/under-nutrition during the prenatal and perinatal periods causes hypoplasia that explains a consistent association between enamel hypoplasia and ECC.[1] These findings are supported by various authors who reported about 18% to 43% of prevalence of enamel hypoplasia in the prematurely born children.[21],[22]

Seow WK in his study quoted the location of the majority of defects at the middle and incisor regions of the teeth further suggesting that the time period of the insult to enamel matches the events of the premature births.[11] Literature has found 52-96% of developmental defects of enamel in pre-term born, very low birth weight children that is in accordance with the results of the present study.[23],[24],[25] These findings thus suggested that enamel quality is affected in prematurely born children and this makes children more susceptible for the causation of early childhood caries. In contrast, Nelson et al. found similar occurrence of hypoplastic enamel between both pre-term and full-term born children [Figure 1] and [Figure 2].[26]
Figure 1: Images of primary tooth enamel in pre-term born children under scanning electron microscopy

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Figure 2: Images of primary tooth enamel in full-term born children under scanning electron microscopy

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Enamel structure might collapse if the minerals are removed because of their chief involvement in the configuration of enamel. Electron dispersive X-ray spectroscopy analyzed the percentages of major elements Calcium (Ca), Phosphorus (P), Carbon©, and Oxygen (O), as these elements form a major part of the composition of enamel prism.

The reduced percentage of Ca in enamel from pre-term children could be attributed to the disturbances in the calcium metabolism and mineralization.[27] Higher percentages of Carbon© and Oxygen (O) in primary teeth enamel of pre-term born children reflects organic matter indicating more porous enamel in teeth from the pre-term born children. This finding was in concordance with a previous histological study of enamel in primary teeth from low-birth-weight infants where primary teeth from the pre-term children contained more carbon.[28]

The ratios of Ca/C and Ca/P determined the relative composition of an individual element. Lower ratio of Ca/C in enamel of pre-term born children suggests the relatively higher amount of C that advocates more porous enamel. This could be due to a higher content of carbonate in enamel. The carbonate concentration decreases with increasing mineralization.[7] The similar results were found by Rythen et al. where Ca/C ratio was significantly lower in the enamel in primary teeth from pre-term children.[7]

The disturbances in the elemental composition of enamel in pre-term born children can be understood with the process of amelogenesis. During maturation phase of formation of enamel, the amount of C in developing enamel decreases.[29] Whereas, due to premature birth of a child, they may lack completion of maturation phase and thus increased values of C were observed. The porous enamel in the primary teeth of the pre-term born children may represent an increased risk for development of ECC.

Lower Ca/P ratio in enamel from preterm born children indicates higher P values and thus increased organic material but it does not necessarily reflect disturbances caused in by pre-term birth. Different studies have found different values for the Ca/P ratio in enamel.[30],[31],[32]

In the present study, the chemical and structural characteristics of enamel of primary teeth from pre-term and full-term born children were appraised. The findings were suggestive of hypoplastic, rough textured, less mineralized and thin enamel in primary teeth of pre-term born children. All these characteristics of enamel in pre-term born children represent quantitative as well as qualitative defects. Hence, it provides suitable sites for the adhesion and colonization of cariogenic bacteria, and may cause the bacteria to be retained at the base of the defect. Consequently, dental caries on the altered tooth surfaces would develop more rapidly than on sound tooth surfaces.[33]

Thus, the results of the study provided evidence toward pre-term birth of a child being predictable cause of ECC. In view of that, providing oral health education to the parents or caregivers, and controlling factors responsible in increasing the risk for caries should be targeted. Interventions directed at developing the environment of oral cavity can reduce the risk of ECC.[33],[34],[35],[36],[37],[38],[39]

Why this paper is important to pediatric dentists:

  • This study explains pre-term birth of a child to be one of the causes for the occurrence of ECC in children
  • The enamel of primary teeth in pre-term born children has a damaged structure and composition which makes it susceptible for initiation of ECC
  • In pre-term born infants including those with early signs of ECC, poor oral hygiene would be targeted with a professional preventive program that involves oral hygiene instructions for the mother and child, fluoride application, and diet counseling.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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